simocyclinone D8
naturalproductmech:mibig-e03313fa44 MINTED SEEDED
Grounding review: No 3-star ChEBI entry shares this structure, so the record keeps a content-hashed identifier.
What these status tags mean
Curation status describes a workflow state. SEEDED means the record entered through the source-seeding workflow; it does not imply that no later curation occurred. PROPOSED marks an addition awaiting review; REVIEWED records curator sign-off on identity, structure, filing class and producer claims; DEPRECATED retains a superseded or out-of-scope record. These are workflow decisions, not independently verified human review. Agent-assisted contributions may appear in the record history.
Grounding describes the identifier mapping: EXACT uses an ontology identity asserted by the source; MINTED keeps a local identifier when no defensible ontology identity is available; REVIEW_NEEDED flags an exact structure whose ontology identity needs adjudication. The grounding note retains any unresolved alternatives. These tags do not establish the strength of producer or activity evidence.
Structure
| Standard InChIKey | PLEGMCYXNQPJNV-TYXWGSLYSA-N |
|---|---|
| SMILES | C[C@H]1O[C@H](C[C@@H](OC(=O)\C=C\C=C\C=C\C=C\C(=O)NC2=C(O)C3=C(OC2=O)C(Cl)=C(O)C=C3)[C@@H]1OC(C)=O)C1=CC=C2C(=O)C34OC3(C(O)CC3(O)CC(C)=CC(=O)C43O)C(O)C2=C1O |
| Stereochemistry | undefined stereocentres — the InChIKey does not distinguish this compound from its stereoisomers |
| Cross-references | npatlas:NPA08898, pubchem:54736483, chembl:CHEMBL1235969 |
Filing pathway
Polyketides — computed by NPClassifier npclassifier.gnps2.org@2026-09-07, which is why it is pinned rather than recomputed. The gene cluster's asserted class is pks, saccharide, other, from MIBiG.
Producer organisms 2
A claim that this taxon makes the compound. The basis says what the evidence addressed — a knockout and a database assertion are not the same claim.
| Taxon | Basis | Cluster | Evidence |
|---|---|---|---|
| Streptomyces antibioticus NCBITaxon:1890 |
BGC_CHARACTERIZED | mibig:BGC0001072 | PMID:24594357 |
| Streptomyces antibioticus NCBITaxon:1890 |
SOURCE_ASSERTION | mibig:BGC0000270 | PMID:12115055 |
Occurrences 1
Somebody found the compound in this organism and cited it. That is not a claim that the organism makes it, and nothing here promotes one to the other.
| Taxon | Source | Reference |
|---|---|---|
| Streptomyces antibioticus NCBITaxon:1890 |
LOTUS | DOI:10.1007/S00203-002-0429-Z |
Biosynthetic gene clusters 2
| Accession | Host | Locus evidence | Genome |
|---|---|---|---|
| mibig:BGC0001072 | Streptomyces antibioticus NCBITaxon:1890 |
CLUSTER_DEMONSTRATED | genbank:AF324838.2 |
| mibig:BGC0000270 | Streptomyces antibioticus NCBITaxon:1890 |
CLUSTER_UNSTATED | genbank:AF322256.1 |
A locus claim and a taxon claim are different questions. Heterologous expression settles the first and leaves the second where the isolation report left it.
Mechanism & Biosynthesis Overview
Causal graph — biosynthesis 8 nodes, 7 edges
SimReg1 genetics in S. antibioticus Tu6040: insertional disruption, text-reported complementation and wild-type extra-copy production. The source's unresolved construct and figure discrepancies are retained (review #534). No change to the owner's incomplete stereochemistry or open shared-structure discussion.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Native simReg1 locus | encodes | Native SimReg1 regulatory protein | Exact native sequence grounding; no engineered allele is assigned the canonical protein accession. | https://www.ncbi.nlm.nih.gov/protein/AAK06808.1 |
| Tu6040 simReg1::hyg insertion mutant | disrupts | Native simReg1 locus | The hyg cassette was inserted at the unique BsaAI site within simReg1 carried on a reported 4.3-kb BamHI fragment, then transferred through pKC1139 and double crossover selection (39 C for 3 days, subsequent loss of apramycin resistance). This is an insertion, not a clean whole-gene deletion. Southern signals are reported as 4.3 kb in wild type and 6.3 kb in mutant. Results name a KpnI probe from pKCEsimR1; Figure 4 caption instead names a 1.4-kb SmaI probe. Native AF324838.2 BamHI reconstruction spans 4576 bp between 0-based cuts 67163 and 71739, with one internal BsaAI cut at 70167. This discrepancy is not silently corrected or treated as a sequenced mutant/plasmid. | DOI:10.1186/2191-0855-2-1 |
| Tu6040 simReg1::hyg insertion mutant | has_assay_phenotype | Simocyclinones and precursors not detected | Native S. antibioticus Tu6040 production assay: TSB preculture for 2 days at 30 C, 5 mL into 100 mL NL5 for 5 days at 30 C; ethyl-acetate extraction and methanol reconstitution, HPLC/TLC analysis (methods specify HPLC-MS). NL5 includes glycerol 25 g/L and L-glutamine 5.84 g/L, pH 7.3. Antibiotic amounts were normalized to dry biomass; the methods state at least three independent production experiments. These results concern source-assigned simocyclinones/precursors, not a separately quantified yield of chemically pure D8 or all possible culture conditions. Growth and morphology were reported similar to wild type. No detection is not a quantified zero or proof of an isolated catalytic step. | DOI:10.1186/2191-0855-2-1
|
| Reported mutant carrying pSSimR1-1 | reportedly_carries_intact_copy_of | Native simReg1 locus | Table 1 describes mutant plus pSSimR1-1, containing native-promoter simReg1 in integrative pSET152. Methods derive it via a 2.3-kb BamHI segment and a reported 1.4-kb SmaI segment. Native-locus SmaI cuts at 0-based 68877/70373 span 1496 bp and include full simReg1 plus 476 bp of simD2, not a second complete simD2 CDS. This reconstruction does not establish the exact cloning junctions, functional neighbor rescue or the experimental plasmid sequence. | DOI:10.1186/2191-0855-2-1 |
| Reported mutant carrying pSSimR1-1 | reportedly_restores | Text-reported wild-type-like production | Native S. antibioticus Tu6040 production assay: TSB preculture for 2 days at 30 C, 5 mL into 100 mL NL5 for 5 days at 30 C; ethyl-acetate extraction and methanol reconstitution, HPLC/TLC analysis (methods specify HPLC-MS). NL5 includes glycerol 25 g/L and L-glutamine 5.84 g/L, pH 7.3. Antibiotic amounts were normalized to dry biomass; the methods state at least three independent production experiments. These results concern source-assigned simocyclinones/precursors, not a separately quantified yield of chemically pure D8 or all possible culture conditions. The text reports mutant rescue comparable to wild type, but the same paragraph names wild type as the recipient and cites Figure 5b, whose caption identifies wild type versus wild type with an extra copy. Keep the text-reported rescue, not an independently illustrated rescue comparison or universal exclusion of polar effects. No matched empty-vector production control is shown in the inspected article and supplements. | DOI:10.1186/2191-0855-2-1
|
| Wild type carrying pSSimR1-1 | carries_additional_native_promoter_copy_of | Native simReg1 locus | Wild-type Tu6040 received integrative pSSimR1-1. This native-promoter extra-copy experiment differs from the constitutive ermE reporter construct and from the mutant complementation claim; the same rescue-fragment uncertainty applies. | DOI:10.1186/2191-0855-2-1
|
| Wild type carrying pSSimR1-1 | increases_assayed_production_to | Reported 2.5-fold simocyclinone production | Native S. antibioticus Tu6040 production assay: TSB preculture for 2 days at 30 C, 5 mL into 100 mL NL5 for 5 days at 30 C; ethyl-acetate extraction and methanol reconstitution, HPLC/TLC analysis (methods specify HPLC-MS). NL5 includes glycerol 25 g/L and L-glutamine 5.84 g/L, pH 7.3. Antibiotic amounts were normalized to dry biomass; the methods state at least three independent production experiments. These results concern source-assigned simocyclinones/precursors, not a separately quantified yield of chemically pure D8 or all possible culture conditions. The reported average is 2.5 times wild type; this is not a purified SimReg1 reaction rate, a measured intracellular SimReg1 concentration or an independently quantified effect of every sim cluster gene. | DOI:10.1186/2191-0855-2-1
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 14 nodes, 14 edges
His-SimReg1 DNA-shift assays and mixed-extract perturbation. Promoter-region labels, reported fragment lengths and native-locus reconstructions remain distinct; assay fragments are not grounded as whole genes or proteins. No pure-D8 binding, SimR competition, physical protein complex or phosphorylation-independence claim is made (review #534).
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Assayed codon-optimized His-SimReg1 | has_reported_native_parent | Native SimReg1 regulatory protein | Synthetic simReg1s from pMA-simR1 was amplified with SSR1F/R, cloned NcoI/EcoRI into pET21d as pETSR1c-15, and expressed in E. coli BL21(DE3)pLysS. His-resin purification and SDS-PAGE appear in methods and supplement 2. Full engineered DNA/protein sequence and tag junctions are not established here; canonical Q9AMH3 denotes only the native parent. The apparent band near 35 kDa is not an exact mass/identity measurement or proof of absolute purity. Predicted REC 15-123 and DNA-binding 167-239 domains, three of six conserved PhoB residues and no cluster kinase do not prove independence from phosphorylation or rule out an external kinase. | DOI:10.1186/2191-0855-2-1 |
| Assayed codon-optimized His-SimReg1 | shifts_in_emsa | PD4 simD4-region DNA | EMSA with His-SimReg1, 50 ng DNA in 20 uL, 25 min at room temperature; 20 mM Tris-HCl pH 8, 1 mM each EDTA/DTT/MgCl2, 100 mM KCl and 10% glycerol, 4.5% native gel at 4 C, ethidium-bromide readout. Figure 6 tests 1.8 and 4.5 ug protein against a no-protein lane; not every fragment shifts equally at both doses. This does not measure a footprint, Kd, transcriptional direction or native enzyme activity. The methods report 513 bp. Literal Table 2 primers yield a native coverage model of 516 bp (530 bp including unmatched 5-prime tails). SR1D4F/R; native bases 70099..70614 include 180 bp simReg1 and 14 bp simD4. This fragment differs from the GusA-reporter insert. Native-primer mappings are curator reconstructions, not sequenced assay constructs or corrected source values. | DOI:10.1186/2191-0855-2-1 |
| Assayed codon-optimized His-SimReg1 | shifts_in_emsa | PR1 simReg1-region DNA | EMSA with His-SimReg1, 50 ng DNA in 20 uL, 25 min at room temperature; 20 mM Tris-HCl pH 8, 1 mM each EDTA/DTT/MgCl2, 100 mM KCl and 10% glycerol, 4.5% native gel at 4 C, ethidium-bromide readout. Figure 6 tests 1.8 and 4.5 ug protein against a no-protein lane; not every fragment shifts equally at both doses. This does not measure a footprint, Kd, transcriptional direction or native enzyme activity. The methods report 490 bp. Literal Table 2 primers yield a native coverage model of 487 bp (498 bp including unmatched 5-prime tails). SD2R1F/R; native bases 69150..69636 include 204 bp simD2 and 114 bp simReg1. Binding to this region alone does not establish activation versus repression of its own gene. Native-primer mappings are curator reconstructions, not sequenced assay constructs or corrected source values. | DOI:10.1186/2191-0855-2-1 |
| Assayed codon-optimized His-SimReg1 | shifts_in_emsa | PD3 simD3-region DNA | EMSA with His-SimReg1, 50 ng DNA in 20 uL, 25 min at room temperature; 20 mM Tris-HCl pH 8, 1 mM each EDTA/DTT/MgCl2, 100 mM KCl and 10% glycerol, 4.5% native gel at 4 C, ethidium-bromide readout. Figure 6 tests 1.8 and 4.5 ug protein against a no-protein lane; not every fragment shifts equally at both doses. This does not measure a footprint, Kd, transcriptional direction or native enzyme activity. The methods report 300 bp. Literal Table 2 primers yield a native coverage model of 367 bp (381 bp including unmatched 5-prime tails). simX5D3F/R; native bases 61331..61697 overlap annotated simC8 and simD3. The primer label is not evidence for a separate simX5-promoter assay. Native-primer mappings are curator reconstructions, not sequenced assay constructs or corrected source values. | DOI:10.1186/2191-0855-2-1 |
| Assayed codon-optimized His-SimReg1 | shifts_in_emsa | PX4 simX4-region DNA | EMSA with His-SimReg1, 50 ng DNA in 20 uL, 25 min at room temperature; 20 mM Tris-HCl pH 8, 1 mM each EDTA/DTT/MgCl2, 100 mM KCl and 10% glycerol, 4.5% native gel at 4 C, ethidium-bromide readout. Figure 6 tests 1.8 and 4.5 ug protein against a no-protein lane; not every fragment shifts equally at both doses. This does not measure a footprint, Kd, transcriptional direction or native enzyme activity. The methods report 350 bp. Literal Table 2 primers yield a native coverage model of 406 bp (422 bp including unmatched 5-prime tails). simEXX4F/R; native bases 59598..60003 overlap simEx and simX4. Native-primer mappings are curator reconstructions, not sequenced assay constructs or corrected source values. | DOI:10.1186/2191-0855-2-1 |
| Assayed codon-optimized His-SimReg1 | shifts_in_emsa | PR2Ex simR/simX-region DNA | EMSA with His-SimReg1, 50 ng DNA in 20 uL, 25 min at room temperature; 20 mM Tris-HCl pH 8, 1 mM each EDTA/DTT/MgCl2, 100 mM KCl and 10% glycerol, 4.5% native gel at 4 C, ethidium-bromide readout. Figure 6 tests 1.8 and 4.5 ug protein against a no-protein lane; not every fragment shifts equally at both doses. This does not measure a footprint, Kd, transcriptional direction or native enzyme activity. The methods report 780 bp. Literal Table 2 primers yield a native coverage model of 883 bp (902 bp including unmatched 5-prime tails). SR2ExXF/R; native bases 57298..58180 contain the full annotated simReg2 CDS, 50 bp of simC5 and one base of simEx. The reported intergenic-region label must not be mistaken for a fragment consisting only of intergenic DNA. Native-primer mappings are curator reconstructions, not sequenced assay constructs or corrected source values. | DOI:10.1186/2191-0855-2-1 |
| Assayed codon-optimized His-SimReg1 | shifts_in_emsa | PA7 simA7-region DNA | EMSA with His-SimReg1, 50 ng DNA in 20 uL, 25 min at room temperature; 20 mM Tris-HCl pH 8, 1 mM each EDTA/DTT/MgCl2, 100 mM KCl and 10% glycerol, 4.5% native gel at 4 C, ethidium-bromide readout. Figure 6 tests 1.8 and 4.5 ug protein against a no-protein lane; not every fragment shifts equally at both doses. This does not measure a footprint, Kd, transcriptional direction or native enzyme activity. The methods report 300 bp. Literal Table 2 primers yield a native coverage model of 356 bp (376 bp including unmatched 5-prime tails). simA7F/R; native bases 41493..41848 include two bases of annotated simA7. Native-primer mappings are curator reconstructions, not sequenced assay constructs or corrected source values. | DOI:10.1186/2191-0855-2-1 |
| Assayed codon-optimized His-SimReg1 | shifts_in_emsa | PEx2 fragment as labelled in the study | EMSA with His-SimReg1, 50 ng DNA in 20 uL, 25 min at room temperature; 20 mM Tris-HCl pH 8, 1 mM each EDTA/DTT/MgCl2, 100 mM KCl and 10% glycerol, 4.5% native gel at 4 C, ethidium-bromide readout. Figure 6 tests 1.8 and 4.5 ug protein against a no-protein lane; not every fragment shifts equally at both doses. This does not measure a footprint, Kd, transcriptional direction or native enzyme activity. The methods report 550 bp. Literal PEx2F and PEx2R map at 12396..12409 and 12930..12943 on the same positive strand of AF324838.2. They do not define a facing PCR pair as written. Retain Figure 6g's reported fragment label; exact construct identity remains unresolved, and no primer is silently reverse-complemented to repair it. Native-primer mappings are curator reconstructions, not sequenced assay constructs or corrected source values. | DOI:10.1186/2191-0855-2-1 |
| Assayed codon-optimized His-SimReg1 | shifts_in_emsa | PSR3 simB7-upstream DNA | EMSA with His-SimReg1, 50 ng DNA in 20 uL, 25 min at room temperature; 20 mM Tris-HCl pH 8, 1 mM each EDTA/DTT/MgCl2, 100 mM KCl and 10% glycerol, 4.5% native gel at 4 C, ethidium-bromide readout. Figure 6 tests 1.8 and 4.5 ug protein against a no-protein lane; not every fragment shifts equally at both doses. This does not measure a footprint, Kd, transcriptional direction or native enzyme activity. The methods report 319 bp. Literal Table 2 primers yield a native coverage model of 319 bp (325 bp including unmatched 5-prime tails). PR3F/R; native bases 3908..4226 include 15 bp of simB7. The study proposes co-transcription with simB5/B4/X5/X7/Reg3; this is not a separately tested simReg3 promoter or an experimentally confirmed operon. Native-primer mappings are curator reconstructions, not sequenced assay constructs or corrected source values. | DOI:10.1186/2191-0855-2-1 |
| Assayed codon-optimized His-SimReg1 | shifts_in_emsa | PSEx1 simX-region DNA | Figure 7b has seven lanes, although the caption describes four: PSR2 with 0/1.8 ug His-SimReg1, then PSEx1 with 0.9/1.8/0/3.6/4.5 ug. The assay uses the methods' EMSA conditions. Methods report a 280-bp PSEx1 amplicon; results instead call a 69-bp region a fragment. Literal PEx1F/R map a 280-bp span at 58111..58390 containing 211 bp of annotated simEx (simX). This does not localize binding to only 69 bp or prove overlap/competition with SimR operators. | DOI:10.1186/2191-0855-2-1 |
| Assayed codon-optimized His-SimReg1 | no_shift_detected_in_emsa | PSR2 simR-region DNA | Figure 7b compares PSR2 without protein and with 1.8 ug His-SimReg1; do not generalize this negative result to all concentrations or native regulation. Methods report 300 bp; results describe a 67-bp upstream region. Literal PSR2F/R define 303 native bp (57851..58153), including 233 bp of simReg2. These are different descriptions, not a demonstrated 67-bp footprint. | DOI:10.1186/2191-0855-2-1 |
| Assayed codon-optimized His-SimReg1 | no_shift_detected_in_emsa | Internal simD4 DNA negative control | Supplement 3 shows no shifted internal-simD4 DNA band at 0, 0.9, 1.8 or 4.5 ug protein. This is the actual negative-control DNA, not an unspecified random sequence. D4For/Rev map native bases 70651..70917, 267 bp wholly inside simD4. Separate E. coli crude-extract controls are described as data not shown; they were not independently inspected as gels. | DOI:10.1186/2191-0855-2-1 |
| Simocyclinone-enriched culture extract | abolishes_his_simreg1_emsa_shift_of | PR1 simReg1-region DNA | At 5% and 10% final reaction volume, ethyl-acetate culture extract reconstituted in methanol removed the PR1 shift. Figure 8a includes matched methanol-only controls retaining the shift. Extract source is extra-copy Tu6040 cultured 72 hours; the paragraph alternates pSSimR1 and pSSimR1-1. The source reports roughly/more than 95% combined simocyclinones, not pure D8. Supplement 1 has a major simB peak and simA/C/D peaks; its caption gives SD8 retention at 24.7 min. UV peak size is not a measured mass fraction. Loss of the gel shift does not establish direct D8 binding, a ligand Kd or a uniquely identified active constituent. | DOI:10.1186/2191-0855-2-1
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| Simocyclinone-enriched culture extract | abolishes_his_simreg1_emsa_shift_of | PA7 simA7-region DNA | Figure 8b shows loss of the PA7 shift with the same mixed extract at 5% and 10% final volume. Only four lanes are displayed; unlike Figure 8a, this panel does not show methanol-only lanes even though the text makes a general solvent-control statement. The PR1 extract identity and ligand-attribution limitations also apply; this is not pure-D8 feedback. | DOI:10.1186/2191-0855-2-1
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 8 nodes, 7 edges
SimReg1-dependent activation of a simD4-region gusA reporter in S. lividans 1326. Heterologous transcriptional reporter output is distinct from native SimD4 protein abundance, ketoreductase activity and activation of every EMSA-positive region (review #534).
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| pUWLsimReg1 constitutive regulator construct | is_reported_to_express | Native SimReg1 regulatory protein | Native simReg1 PCR product was cloned HindIII/BamHI in pUWL-oriT under constitutive ermE promoter control. Literal primers cover native bases 69486..70300 (815 bp; 832 with unmatched tails), including the complete native CDS. The source calls it 0.8 kb. No His-tag or codon-optimized construct is assigned to this reporter experiment; the whole experimental plasmid sequence is not established. | DOI:10.1186/2191-0855-2-1 |
| S. lividans 1326 with reporter and regulator | carries | pUWLsimReg1 constitutive regulator construct | S. lividans 1326 is the heterologous reporter host, not the native Tu6040 simocyclinone producer. Transfer of the regulatory construct does not establish heterologous synthesis of simocyclinone D8. | DOI:10.1186/2191-0855-2-1
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| S. lividans 1326 with reporter and regulator | carries | pSimD4script promoter-gusA construct | The putative simD4 promoter fragment was cloned XbaI/KpnI upstream of gusA in pGUS. The source reports 0.5 kb; literal reporter primers map native bases 70071..70570 (500 bp, 518 with unmatched tails), including 208 bp of simReg1. This differs from the PD4 EMSA fragment and does not imply transfer of a complete additional simReg1 gene. | DOI:10.1186/2191-0855-2-1 |
| S. lividans 1326 with reporter only | carries | pSimD4script promoter-gusA construct | Reporter-only strain is the comparison for adding pUWLsimReg1; it is not a matched pUWL empty-vector strain. The pGUS controls instead address promoterless reporter background. | DOI:10.1186/2191-0855-2-1
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| S. lividans 1326 with reporter and regulator | increases_reporter_readout_to | Increased PD4-driven glucuronidase activity | Relative to reporter-only S. lividans 1326, source-reported GusA activity increases from 3.3 +/- 0.24 to 6.25 +/- 0.43 munits/mg (Figure 9 axis), approximately 1.9-fold. TSB preculture 2 days then 5 days at 30 C; lysates assayed with p-nitrophenyl-beta-D-glucuronide at 37 C, absorbance 415 nm every minute for 20 minutes. Error type and reporter replicate count are not specified here; do not transfer the production assay's replicate count. This is a promoter-reporter readout, not direct native SimD4 enzyme activity, an RNA-polymerase interaction assay or a demonstration of activation at every bound region. | DOI:10.1186/2191-0855-2-1
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| Promoterless pGUS strains with or without regulator | have_assay_phenotype | Low promoterless glucuronidase activity | Promoterless pGUS strains with and without pUWLsimReg1 show roughly 0.25 +/- 0.06 munits/mg in the same assay. These controls support promoter-dependent reporter output, not the assertion that SimReg1 binds only simD4 or that all host-mediated effects are excluded. | DOI:10.1186/2191-0855-2-1
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| Promoterless pGUS strains with or without regulator | provides_promoterless_controls_for | Increased PD4-driven glucuronidase activity | Figure 9 compares promoterless pGUS strains, with and without pUWLsimReg1 (strains 2 and 4), to PD4-gusA reporter strains without and with regulator (strains 1 and 3). This edge records the experimental comparison linking background controls to the reported activation readout; it is not a biochemical causal interaction or a matched pUWL empty-vector control. | DOI:10.1186/2191-0855-2-1
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Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 11 nodes, 23 edges
SimR DNA binding, engineered truncations and point mutants in producer-cluster regulation. Native Q9AMH9 is distinct from the 259-aa paper parent and tagged assay constructs; source residue numbering and assay boundaries are retained (review #536). This graph does not assert SimR catalysis or remeasure native export.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Assayed codon-optimized C-terminally tagged SimR | has_native_sequence_parent | Native SimR / SimReg2 protein | The paper calls its parent 259 aa. Supplement S2 exactly matches native Q9AMH9 residues 3..261, omitting the native MW start; deposited protein is those 259 residues plus LEHHHHHH (267 aa). Paper positions map to UniProt with +2. Synthetic codon-optimized simR, pIJ10499, was expressed in E. coli and purified by nickel affinity. This edge does not assert native N-terminal processing, a corrected native annotation, or equivalence of tagged and native proteins. | DOI:10.1093/nar/gkr640 |
| Assayed codon-optimized C-terminally tagged SimR | shifts_in_emsa | 138-bp simR-simX-region probe with OR and OX | The 138-bp simR-simX-region probe contains OR and OX; this is not either short isolated operator or a whole gene. EMSA: 0.1 nM radiolabelled probe in 20 uL, 20 mM Tris pH 8, 1 ug calf-thymus DNA, 100 mM NaCl, 8% glycerol, 22 C for 10 min, 5% native PAGE. Plotted protein series 0/0.8/1.6/3.1/12.5/25/50/200 nM. Fold changes are the authors' gel-based binding comparisons, not fitted Kd values or native transcription/production measurements. The source assigns lower and upper shifted complexes to occupancy of one and both sites; the gel alone is not a determination of a physiological tetramer. | DOI:10.1093/nar/gkr640
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| Assayed codon-optimized C-terminally tagged SimR | protects_regions_from_dnase_i | 138-bp simR-simX-region probe with OR and OX | Figure 8 uses 200 nM tagged WT as the full-footprint reference at both OR and OX. The readout is DNase I protection, not gene expression or a measured equilibrium constant. | DOI:10.1093/nar/gkr640
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| His-tagged SimR Delta10 (paper numbering) | is_engineered_derivative_of | Assayed codon-optimized C-terminally tagged SimR | pIJ10500: NdeI/XhoI cloning into pET20b through sequence-verified pUC18 intermediates; source reports deletion of 10 N-terminal paper residues, retaining paper 11..259 (native Q9AMH9 13..261) and a C-terminal tag. Literal upstream primer translation includes an added initiator M before that retained segment (Delta10 begins MM). This is a primer-derived reading-frame model, not an experimentally determined mature N-terminus or a deposited complete plasmid sequence. The downstream primer is consistent with the ERPALA parent tail. Supplement S1 shows purified bands, not proof of identical folding or absolute purity. | DOI:10.1093/nar/gkr640 |
| His-tagged SimR Delta10 (paper numbering) | shows_reduced_emsa_binding_to | 138-bp simR-simX-region probe with OR and OX | The 138-bp simR-simX-region probe contains OR and OX; this is not either short isolated operator or a whole gene. EMSA: 0.1 nM radiolabelled probe in 20 uL, 20 mM Tris pH 8, 1 ug calf-thymus DNA, 100 mM NaCl, 8% glycerol, 22 C for 10 min, 5% native PAGE. Plotted protein series 0/0.8/1.6/3.1/12.5/25/50/200 nM. Fold changes are the authors' gel-based binding comparisons, not fitted Kd values or native transcription/production measurements. Reported approximately 30-fold reduction relative to tagged WT. This is not complete loss of all binding, nor proof that only the deleted residues directly contact DNA; truncation can also alter arm positioning. | DOI:10.1093/nar/gkr640
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| His-tagged SimR Delta10 (paper numbering) | produces_retracted_dnase_i_footprint_on | 138-bp simR-simX-region probe with OR and OX | DNase I protection of the simR-simX-region probe, upper/lower strand separately labelled, 40 uL EMSA buffer, 22 C for 10 min, 10 U DNase I for 60 s; G+A ladder and no-protein controls. Compared with tagged WT, each truncation has the same reported retraction: OR upper strand 2 bp left and 1 bp right, OR lower unchanged; OX upper 1 bp left/right unchanged; OX lower 1 bp at each end. Boundaries are source figure orientations, not independent genomic-coordinate assignments. This derivative was assayed at 200 nM, versus 200 nM WT. The authors use saturating doses; differing doses must not be represented as equal-dose occupancy or as quantitative affinity measurements. | DOI:10.1093/nar/gkr640
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| His-tagged SimR Delta15 (paper numbering) | is_engineered_derivative_of | Assayed codon-optimized C-terminally tagged SimR | pIJ10501: NdeI/XhoI cloning into pET20b through sequence-verified pUC18 intermediates; source reports deletion of 15 N-terminal paper residues, retaining paper 16..259 (native Q9AMH9 18..261) and a C-terminal tag. Literal upstream primer translation includes an added initiator M before that retained segment (Delta10 begins MM). This is a primer-derived reading-frame model, not an experimentally determined mature N-terminus or a deposited complete plasmid sequence. The downstream primer is consistent with the ERPALA parent tail. Supplement S1 shows purified bands, not proof of identical folding or absolute purity. | DOI:10.1093/nar/gkr640 |
| His-tagged SimR Delta15 (paper numbering) | shows_reduced_emsa_binding_to | 138-bp simR-simX-region probe with OR and OX | The 138-bp simR-simX-region probe contains OR and OX; this is not either short isolated operator or a whole gene. EMSA: 0.1 nM radiolabelled probe in 20 uL, 20 mM Tris pH 8, 1 ug calf-thymus DNA, 100 mM NaCl, 8% glycerol, 22 C for 10 min, 5% native PAGE. Plotted protein series 0/0.8/1.6/3.1/12.5/25/50/200 nM. Fold changes are the authors' gel-based binding comparisons, not fitted Kd values or native transcription/production measurements. Reported approximately 30-fold reduction relative to tagged WT. This is not complete loss of all binding, nor proof that only the deleted residues directly contact DNA; truncation can also alter arm positioning. | DOI:10.1093/nar/gkr640
|
| His-tagged SimR Delta15 (paper numbering) | produces_retracted_dnase_i_footprint_on | 138-bp simR-simX-region probe with OR and OX | DNase I protection of the simR-simX-region probe, upper/lower strand separately labelled, 40 uL EMSA buffer, 22 C for 10 min, 10 U DNase I for 60 s; G+A ladder and no-protein controls. Compared with tagged WT, each truncation has the same reported retraction: OR upper strand 2 bp left and 1 bp right, OR lower unchanged; OX upper 1 bp left/right unchanged; OX lower 1 bp at each end. Boundaries are source figure orientations, not independent genomic-coordinate assignments. This derivative was assayed at 200 nM, versus 200 nM WT. The authors use saturating doses; differing doses must not be represented as equal-dose occupancy or as quantitative affinity measurements. | DOI:10.1093/nar/gkr640
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| His-tagged SimR Delta22 (paper numbering) | is_engineered_derivative_of | Assayed codon-optimized C-terminally tagged SimR | pIJ10502: NdeI/XhoI cloning into pET20b through sequence-verified pUC18 intermediates; source reports deletion of 22 N-terminal paper residues, retaining paper 23..259 (native Q9AMH9 25..261) and a C-terminal tag. Literal upstream primer translation includes an added initiator M before that retained segment (Delta10 begins MM). This is a primer-derived reading-frame model, not an experimentally determined mature N-terminus or a deposited complete plasmid sequence. The downstream primer is consistent with the ERPALA parent tail. Supplement S1 shows purified bands, not proof of identical folding or absolute purity. | DOI:10.1093/nar/gkr640 |
| His-tagged SimR Delta22 (paper numbering) | shows_reduced_emsa_binding_to | 138-bp simR-simX-region probe with OR and OX | The 138-bp simR-simX-region probe contains OR and OX; this is not either short isolated operator or a whole gene. EMSA: 0.1 nM radiolabelled probe in 20 uL, 20 mM Tris pH 8, 1 ug calf-thymus DNA, 100 mM NaCl, 8% glycerol, 22 C for 10 min, 5% native PAGE. Plotted protein series 0/0.8/1.6/3.1/12.5/25/50/200 nM. Fold changes are the authors' gel-based binding comparisons, not fitted Kd values or native transcription/production measurements. Reported at least 120-fold reduction relative to tagged WT. This is not complete loss of all binding, nor proof that only the deleted residues directly contact DNA; truncation can also alter arm positioning. | DOI:10.1093/nar/gkr640
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| His-tagged SimR Delta22 (paper numbering) | produces_retracted_dnase_i_footprint_on | 138-bp simR-simX-region probe with OR and OX | DNase I protection of the simR-simX-region probe, upper/lower strand separately labelled, 40 uL EMSA buffer, 22 C for 10 min, 10 U DNase I for 60 s; G+A ladder and no-protein controls. Compared with tagged WT, each truncation has the same reported retraction: OR upper strand 2 bp left and 1 bp right, OR lower unchanged; OX upper 1 bp left/right unchanged; OX lower 1 bp at each end. Boundaries are source figure orientations, not independent genomic-coordinate assignments. This derivative was assayed at 400 nM, versus 200 nM WT. The authors use saturating doses; differing doses must not be represented as equal-dose occupancy or as quantitative affinity measurements. | DOI:10.1093/nar/gkr640
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| His-tagged SimR Delta25 (paper numbering) | is_engineered_derivative_of | Assayed codon-optimized C-terminally tagged SimR | pIJ10503: NdeI/XhoI cloning into pET20b through sequence-verified pUC18 intermediates; source reports deletion of 25 N-terminal paper residues, retaining paper 26..259 (native Q9AMH9 28..261) and a C-terminal tag. Literal upstream primer translation includes an added initiator M before that retained segment (Delta10 begins MM). This is a primer-derived reading-frame model, not an experimentally determined mature N-terminus or a deposited complete plasmid sequence. The downstream primer is consistent with the ERPALA parent tail. Supplement S1 shows purified bands, not proof of identical folding or absolute purity. | DOI:10.1093/nar/gkr640 |
| His-tagged SimR Delta25 (paper numbering) | shows_reduced_emsa_binding_to | 138-bp simR-simX-region probe with OR and OX | The 138-bp simR-simX-region probe contains OR and OX; this is not either short isolated operator or a whole gene. EMSA: 0.1 nM radiolabelled probe in 20 uL, 20 mM Tris pH 8, 1 ug calf-thymus DNA, 100 mM NaCl, 8% glycerol, 22 C for 10 min, 5% native PAGE. Plotted protein series 0/0.8/1.6/3.1/12.5/25/50/200 nM. Fold changes are the authors' gel-based binding comparisons, not fitted Kd values or native transcription/production measurements. Reported at least 120-fold reduction relative to tagged WT. This is not complete loss of all binding, nor proof that only the deleted residues directly contact DNA; truncation can also alter arm positioning. | DOI:10.1093/nar/gkr640
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| His-tagged SimR Delta25 (paper numbering) | produces_retracted_dnase_i_footprint_on | 138-bp simR-simX-region probe with OR and OX | DNase I protection of the simR-simX-region probe, upper/lower strand separately labelled, 40 uL EMSA buffer, 22 C for 10 min, 10 U DNase I for 60 s; G+A ladder and no-protein controls. Compared with tagged WT, each truncation has the same reported retraction: OR upper strand 2 bp left and 1 bp right, OR lower unchanged; OX upper 1 bp left/right unchanged; OX lower 1 bp at each end. Boundaries are source figure orientations, not independent genomic-coordinate assignments. This derivative was assayed at 400 nM, versus 200 nM WT. The authors use saturating doses; differing doses must not be represented as equal-dose occupancy or as quantitative affinity measurements. | DOI:10.1093/nar/gkr640
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| His-tagged SimR R18A (paper numbering) | is_engineered_derivative_of | Assayed codon-optimized C-terminally tagged SimR | Modified QuikChange substitution R18A in paper numbering corresponds to native Q9AMH9 R20A; all native residue identities were checked. Supplement S7 identifies the variant, but does not deposit its complete nucleotide sequence here. The Y67A HTH mutant is a control outside the N-terminal arm. | DOI:10.1093/nar/gkr640
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| His-tagged SimR R18A (paper numbering) | has_reported_emsa_binding_to | 138-bp simR-simX-region probe with OR and OX | The 138-bp simR-simX-region probe contains OR and OX; this is not either short isolated operator or a whole gene. EMSA: 0.1 nM radiolabelled probe in 20 uL, 20 mM Tris pH 8, 1 ug calf-thymus DNA, 100 mM NaCl, 8% glycerol, 22 C for 10 min, 5% native PAGE. Plotted protein series 0/0.8/1.6/3.1/12.5/25/50/200 nM. Fold changes are the authors' gel-based binding comparisons, not fitted Kd values or native transcription/production measurements. R18A is approximately 15-fold reduced. This finding is limited to this construct, probe and assay; no universal residue dispensability is inferred. | DOI:10.1093/nar/gkr640
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| His-tagged SimR R19A (paper numbering) | is_engineered_derivative_of | Assayed codon-optimized C-terminally tagged SimR | Modified QuikChange substitution R19A in paper numbering corresponds to native Q9AMH9 R21A; all native residue identities were checked. Supplement S7 identifies the variant, but does not deposit its complete nucleotide sequence here. The Y67A HTH mutant is a control outside the N-terminal arm. | DOI:10.1093/nar/gkr640
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| His-tagged SimR R19A (paper numbering) | has_reported_emsa_binding_to | 138-bp simR-simX-region probe with OR and OX | The 138-bp simR-simX-region probe contains OR and OX; this is not either short isolated operator or a whole gene. EMSA: 0.1 nM radiolabelled probe in 20 uL, 20 mM Tris pH 8, 1 ug calf-thymus DNA, 100 mM NaCl, 8% glycerol, 22 C for 10 min, 5% native PAGE. Plotted protein series 0/0.8/1.6/3.1/12.5/25/50/200 nM. Fold changes are the authors' gel-based binding comparisons, not fitted Kd values or native transcription/production measurements. R19A is similar to tagged WT. This finding is limited to this construct, probe and assay; no universal residue dispensability is inferred. | DOI:10.1093/nar/gkr640
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| His-tagged SimR R22A (paper numbering) | is_engineered_derivative_of | Assayed codon-optimized C-terminally tagged SimR | Modified QuikChange substitution R22A in paper numbering corresponds to native Q9AMH9 R24A; all native residue identities were checked. Supplement S7 identifies the variant, but does not deposit its complete nucleotide sequence here. The Y67A HTH mutant is a control outside the N-terminal arm. | DOI:10.1093/nar/gkr640
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| His-tagged SimR R22A (paper numbering) | has_reported_emsa_binding_to | 138-bp simR-simX-region probe with OR and OX | The 138-bp simR-simX-region probe contains OR and OX; this is not either short isolated operator or a whole gene. EMSA: 0.1 nM radiolabelled probe in 20 uL, 20 mM Tris pH 8, 1 ug calf-thymus DNA, 100 mM NaCl, 8% glycerol, 22 C for 10 min, 5% native PAGE. Plotted protein series 0/0.8/1.6/3.1/12.5/25/50/200 nM. Fold changes are the authors' gel-based binding comparisons, not fitted Kd values or native transcription/production measurements. R22A is approximately 15-fold reduced. This finding is limited to this construct, probe and assay; no universal residue dispensability is inferred. | DOI:10.1093/nar/gkr640
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| His-tagged SimR Y67A (paper numbering) | is_engineered_derivative_of | Assayed codon-optimized C-terminally tagged SimR | Modified QuikChange substitution Y67A in paper numbering corresponds to native Q9AMH9 Y69A; all native residue identities were checked. Supplement S7 identifies the variant, but does not deposit its complete nucleotide sequence here. The Y67A HTH mutant is a control outside the N-terminal arm. | DOI:10.1093/nar/gkr640
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| His-tagged SimR Y67A (paper numbering) | has_reported_emsa_binding_to | 138-bp simR-simX-region probe with OR and OX | The 138-bp simR-simX-region probe contains OR and OX; this is not either short isolated operator or a whole gene. EMSA: 0.1 nM radiolabelled probe in 20 uL, 20 mM Tris pH 8, 1 ug calf-thymus DNA, 100 mM NaCl, 8% glycerol, 22 C for 10 min, 5% native PAGE. Plotted protein series 0/0.8/1.6/3.1/12.5/25/50/200 nM. Fold changes are the authors' gel-based binding comparisons, not fitted Kd values or native transcription/production measurements. Y67A is strongly impaired in the displayed lanes. No numerical fold reduction or fitted Kd is inferred for Y67A. | DOI:10.1093/nar/gkr640
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Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 11 nodes, 13 edges
DNA-length and sequence dependence of tagged SimR binding and trypsin protection. Distinct oligonucleotides, mixed proteolysis products and the S5B-versus-crystal sequence discrepancy remain explicit (review #536); no universal no-binding or native protein-processing claim.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Assayed codon-optimized C-terminally tagged SimR | has_native_sequence_parent | Native SimR / SimReg2 protein | The paper calls its parent 259 aa. Supplement S2 exactly matches native Q9AMH9 residues 3..261, omitting the native MW start; deposited protein is those 259 residues plus LEHHHHHH (267 aa). Paper positions map to UniProt with +2. Synthetic codon-optimized simR, pIJ10499, was expressed in E. coli and purified by nickel affinity. This edge does not assert native N-terminal processing, a corrected native annotation, or equivalence of tagged and native proteins. | DOI:10.1093/nar/gkr640 |
| Assayed codon-optimized C-terminally tagged SimR | yields_tryptic_products | Co-migrating tryptic SimR fragments | Trypsin assay: 1 nmol tagged SimR with 1 pmol bovine trypsin in 100 uL, 50 mM Tris pH 8, 20 mM CaCl2, 150 mM NaCl at 4 C. DNA preincubation is equimolar for 5 min; SDS-PAGE samples at 0/5/10/15 min. Band persistence is not a measured protease-rate constant or an in vivo half-life. Without operator DNA, the dominant band contains two Edman-identified species: paper Ser23 start (4 pmol) and Ser20 start (2-3 pmol), mapping to native Ser25 and Ser22. Supplement S4 also shows 30/60-min lanes. Do not represent this as one homogeneous product or physiological proteolytic activation. | DOI:10.1093/nar/gkr640
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| Assayed codon-optimized C-terminally tagged SimR | no_shift_detected_in_emsa | Wild-type OX-region 15-bp duplex | Supplement S5 uses radiolabelled duplex and 8% native PAGE, not the 5% gel for the 138-bp probe. Displayed SimR series is 0/1.5/3.1/6.2/12.5/25/50 nM. No binding detected for the displayed 15mer through 50 nM. This is a bounded negative result, not absence at every concentration or for every 15-bp sequence. | DOI:10.1093/nar/gkr640
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| Assayed codon-optimized C-terminally tagged SimR | shifts_in_emsa | Wild-type OX-region 17-bp duplex | Supplement S5 uses radiolabelled duplex and 8% native PAGE, not the 5% gel for the 138-bp probe. Displayed SimR series is 0/1.5/3.1/6.2/12.5/25/50 nM. The native OX core sequence is TTCGTACGGTGTATGAA, matching AF324838.2 58132..58148(+). This is not the mutated crystallization duplex. | DOI:10.1093/nar/gkr640 |
| Assayed codon-optimized C-terminally tagged SimR | shows_stronger_emsa_binding_to | Wild-type OX-region 23-bp duplex | Supplement S5 uses radiolabelled duplex and 8% native PAGE, not the 5% gel for the 138-bp probe. Displayed SimR series is 0/1.5/3.1/6.2/12.5/25/50 nM. Relative to the nested 17mer, the 23mer gives stronger binding in this comparison. Do not assign a fitted Kd or a numerical fold change from the picture. | DOI:10.1093/nar/gkr640
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| Wild-type OX-region 15-bp duplex | does_not_detectably_protect_from_trypsin | Assayed codon-optimized C-terminally tagged SimR | Trypsin assay: 1 nmol tagged SimR with 1 pmol bovine trypsin in 100 uL, 50 mM Tris pH 8, 20 mM CaCl2, 150 mM NaCl at 4 C. DNA preincubation is equimolar for 5 min; SDS-PAGE samples at 0/5/10/15 min. Band persistence is not a measured protease-rate constant or an in vivo half-life. Figure 3 compares the 15mer condition with no-DNA protein and longer DNA conditions. Lack of protection is not itself an independent thermodynamic binding assay. | DOI:10.1093/nar/gkr640
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| Wild-type OX-region 17-bp duplex | protects_less_well_from_trypsin | Assayed codon-optimized C-terminally tagged SimR | Trypsin assay: 1 nmol tagged SimR with 1 pmol bovine trypsin in 100 uL, 50 mM Tris pH 8, 20 mM CaCl2, 150 mM NaCl at 4 C. DNA preincubation is equimolar for 5 min; SDS-PAGE samples at 0/5/10/15 min. Band persistence is not a measured protease-rate constant or an in vivo half-life. Supplement S8 compares the binding 17mer with the 23mer. Its caption says the 17mer does not protect as well, while the main text uses stronger wording. Retain the observed comparison, not an absolute claim of zero protection. | DOI:10.1093/nar/gkr640
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| Assayed codon-optimized C-terminally tagged SimR | shifts_in_emsa | Supplement S5B left-repeat 17-bp duplex | Supplement S5 uses radiolabelled duplex and 8% native PAGE, not the 5% gel for the 138-bp probe. Displayed SimR series is 0/1.5/3.1/6.2/12.5/25/50 nM. S5B prints TTCGTACGGTGTACGAA, changing native position 14 T to C. Binding is shown for this sequence; its label is not proof that it is identical to the crystallized DNA. | DOI:10.1093/nar/gkr640
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| Assayed codon-optimized C-terminally tagged SimR | no_shift_detected_in_emsa | Supplement S5B right-repeat 17-bp duplex | Supplement S5 uses radiolabelled duplex and 8% native PAGE, not the 5% gel for the 138-bp probe. Displayed SimR series is 0/1.5/3.1/6.2/12.5/25/50 nM. S5B prints TTCATACGGTGTATGAA (native position 4 G to A); no evident shifted band is displayed through 50 nM. Do not generalize this to all symmetric repeats. | DOI:10.1093/nar/gkr640
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| Supplement S5B left-repeat 17-bp duplex | differs_in_sequence_from | Deposited 3ZQL engineered 17-bp duplex | Deposited 3ZQL/main-text TTCGTACGGCGTACGAA has native positions 10 and 14 changed T to C. S5B left-repeat TTCGTACGGTGTACGAA has only position 14 changed; the sequences differ at position 10. The main text cites S5B for the crystal oligo's affinity, but the literal panel does not establish that comparison for the exact deposited sequence. This is an evidence-identity comparison, not a biochemical causal edge or a source correction. | DOI:10.1093/nar/gkr640 |
| Wild-type OX-region 23-bp duplex | slows_observed_tryptic_digestion_of | Assayed codon-optimized C-terminally tagged SimR | Trypsin assay: 1 nmol tagged SimR with 1 pmol bovine trypsin in 100 uL, 50 mM Tris pH 8, 20 mM CaCl2, 150 mM NaCl at 4 C. DNA preincubation is equimolar for 5 min; SDS-PAGE samples at 0/5/10/15 min. Band persistence is not a measured protease-rate constant or an in vivo half-life. The 23mer retains more full-length band than its relevant shorter/no-DNA comparison. This supports protection in the reconstituted assay, not absolute protease resistance or independent measurement of a disorder-to-order transition. | DOI:10.1093/nar/gkr640
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| Wild-type OX-region 25-bp duplex | slows_observed_tryptic_digestion_of | Assayed codon-optimized C-terminally tagged SimR | Trypsin assay: 1 nmol tagged SimR with 1 pmol bovine trypsin in 100 uL, 50 mM Tris pH 8, 20 mM CaCl2, 150 mM NaCl at 4 C. DNA preincubation is equimolar for 5 min; SDS-PAGE samples at 0/5/10/15 min. Band persistence is not a measured protease-rate constant or an in vivo half-life. The 25mer retains more full-length band than its relevant shorter/no-DNA comparison. This supports protection in the reconstituted assay, not absolute protease resistance or independent measurement of a disorder-to-order transition. | DOI:10.1093/nar/gkr640
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| Wild-type OX-region 31-bp duplex | slows_observed_tryptic_digestion_of | Assayed codon-optimized C-terminally tagged SimR | Trypsin assay: 1 nmol tagged SimR with 1 pmol bovine trypsin in 100 uL, 50 mM Tris pH 8, 20 mM CaCl2, 150 mM NaCl at 4 C. DNA preincubation is equimolar for 5 min; SDS-PAGE samples at 0/5/10/15 min. Band persistence is not a measured protease-rate constant or an in vivo half-life. The 31mer retains more full-length band than its relevant shorter/no-DNA comparison. This supports protection in the reconstituted assay, not absolute protease resistance or independent measurement of a disorder-to-order transition. | DOI:10.1093/nar/gkr640
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 10 nodes, 13 edges
SimR operator contacts and static DNA/ligand/apo structure comparisons in producer-cluster regulation. Crystal packing, paper-to-native residue offsets and unresolved owner stereochemistry constrain interpretation (review #536). This is not a drug-target graph or an observed kinetic transition pathway.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Assayed codon-optimized C-terminally tagged SimR | has_native_sequence_parent | Native SimR / SimReg2 protein | The paper calls its parent 259 aa. Supplement S2 exactly matches native Q9AMH9 residues 3..261, omitting the native MW start; deposited protein is those 259 residues plus LEHHHHHH (267 aa). Paper positions map to UniProt with +2. Synthetic codon-optimized simR, pIJ10499, was expressed in E. coli and purified by nickel affinity. This edge does not assert native N-terminal processing, a corrected native annotation, or equivalence of tagged and native proteins. | DOI:10.1093/nar/gkr640 |
| 3ZQL tagged SimR dimer-DNA structural complex | contains_protein_construct | Assayed codon-optimized C-terminally tagged SimR | 3ZQL is a 2.99-A crystal structure. Its asymmetric unit contains two dimer-DNA complexes, not an established physiological tetramer. Modeled paper-numbered ranges are A7..241, C7..242, and B/D7..15 plus 26..242; missing residues must not be treated as resolved atoms merely because the entity sequence includes them. | DOI:10.1093/nar/gkr640 |
| 3ZQL tagged SimR dimer-DNA structural complex | contains_engineered_duplex | 3ZQL engineered OX 17-bp duplex | Deposited strands TTCGTACGGCGTACGAA and TTCGTACGCCGTACGAA are reverse complements. The former differs from native OX at positions 10/14 and from S5B left-repeat at position 10. This structure demonstrates the deposited complex, not an affinity measurement for the S5B oligo or a native unmodified operator structure. | DOI:10.1093/nar/gkr640 |
| Assayed codon-optimized C-terminally tagged SimR | contains_n_terminal_region | TFR arm of the assayed SimR construct | The paper defines a 28-aa TFR arm (native Q9AMH9 residues 3..30), including paper R18/R19/R22/R25 (native R20/R21/R24/R27). Paper residues 8..10 are a proposed anchor; the Figure 4 caption instead labels 8..11. Do not silently turn either annotation into an independently isolated protein. | DOI:10.1093/nar/gkr640
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| Assayed codon-optimized C-terminally tagged SimR | contains_dna_recognition_region | HTH region of the assayed SimR construct | Paper alpha2 49..58 and alpha3 61..67 form the HTH within core DBD29..67; add 2 for native Q9AMH9 numbering. Domains are regions of the engineered protein, not distinct UniProt proteins or separate gene products. | DOI:10.1093/nar/gkr640
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| HTH region of the assayed SimR construct | makes_structural_major_groove_contacts_with | 3ZQL engineered OX 17-bp duplex | Paper M62 and Y66 contribute base contacts; R51 recognizes G11. Reported backbone contacts include S49/M50/G60/S63/Y65/Y67 and K71 just outside the core HTH. Native positions are all +2. Figure 5 distinguishes base versus phosphate contacts; not every contact is independently tested by a mutant or quantified energetically. Alpha3 is described as 3_10-helical in this DNA complex rather than the alpha-helical ligand/apo models. | DOI:10.1093/nar/gkr640
|
| TFR arm of the assayed SimR construct | contacts_minor_groove_of_crystal_neighbor | Symmetry-related neighboring crystal DNA duplex | The ordered arm contacts the adjacent symmetry-related duplex, not the same 17mer bound by its own HTH. Paper R18 (native R20) contacts C3/T2 and R22 (native R24) the C3/G4 phosphates. Paper R19 (native R21) makes no DNA contact in this model; that is not universal dispensability. The other arm is disordered in this dimer. No cooperative adjacent-native-operator binding is inferred from the crystal neighbor. | DOI:10.1093/nar/gkr640
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| 3ZQL engineered OX 17-bp duplex | stacks_with_in_crystal | Symmetry-related neighboring crystal DNA duplex | Duplex ends form a pseudo-continuous filament through the crystal. Unequal end stacking gives different minor-groove geometries. Curves+ reports about 15-degree bending for the modeled DNA; this is not native DNA bending measured in solution or proof that the crystallographic neighbor is a second genomic operator. | DOI:10.1093/nar/gkr640
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| 2Y30 tagged SimR-SD8 structural complex | contains_protein_construct | Assayed codon-optimized C-terminally tagged SimR | 2Y30 is a 2.30-A SD8-bound deposition with the same native3..261-plus-LEHHHHHH protein sequence as 3ZQL. This public coordinate/sequence record and the NAR comparison were inspected; full text of the separate JMB ligand paper was not available here, so no uninspected ligand-assay detail is imported. | https://www.rcsb.org/structure/2Y30 |
| 2Y30 tagged SimR-SD8 structural complex | contains_deposited_ligand | Source-assigned SD8 ligand, CCD SM8 | 2Y30 names simocyclinone D8 as CCD SM8. SM8 has fully specified InChIKey PLEGMCYXNQPJNV-VHSVXIAXSA-N, whereas this owner retains incomplete PLEGMCYXNQPJNV-TYXWGSLYSA-N. Connectivity and already assigned centers agree, but six owner stereocentres remain unspecified. The ligand node is not grounded as exact identity to the owner and does not replace its structure or resolve the shared-MIBiG discussion. It is not C4 in 2Y31. | https://www.rcsb.org/structure/2Y30 |
| 2Y2Z tagged apo SimR structural state | contains_protein_construct | Assayed codon-optimized C-terminally tagged SimR | 2Y2Z is the 1.95-A apo deposition with native3..261-plus-LEHHHHHH sequence. An ordered TFR arm in this crystal is stabilized by symmetry-related arms (Supplement S3); this does not prove that the arm is rigid in solution. | DOI:10.1093/nar/gkr640 |
| 2Y30 tagged SimR-SD8 structural complex | has_static_conformational_difference_from | 3ZQL tagged SimR dimer-DNA structural complex | NAR Figure 9/S11/S12 compare static models: about 16-degree relative subunit rotation; recognition-helix separation 41.7 A for SD8 versus 36.8 A for DNA, assessed at paper Tyr65 (native Tyr67) C-alpha atoms and averaged for the DNA complexes. This is not a measured binding trajectory, rotation rate or direct kinetic proof of ligand-induced derepression. Cores largely superpose; the paper distinguishes wrapping-arm/alpha8 movement from whole DBD pendulum motion. | DOI:10.1093/nar/gkr640
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| 2Y2Z tagged apo SimR structural state | has_static_conformational_difference_from | 3ZQL tagged SimR dimer-DNA structural complex | The reported recognition-helix spacing is 42.3 A for the apo crystal versus 36.8 A for DNA. The discussion explicitly allows flexible apo protein to sample DNA-compatible states. Do not infer that apo SimR cannot bind DNA under any conditions or that the crystallographic conformation is the only solution state. | DOI:10.1093/nar/gkr640
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 22 nodes, 28 edges
Producer-cluster SimX/SimEx2 resistance genetics and the proposed SD8 export role. The two deposited SimX variants remain distinct, and the tested residue-126 allele is unresolved. Native simX::apr genetics, heterologous expression, MIC and disk readouts are separate; no direct transport flux or SimEx2 functional-null claim is inferred (review #537). Existing owner stereochemistry and mechanism are unchanged.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Assayed SimX protein, residue-126 allele unresolved | has_candidate_native_sequence | Deposited native SimX / SimEx with Ala126 | This 534-aa deposited SimX has Ala at residue 126. Both deposited candidates fit the expression primers, but their internal CDS nucleotide 377 and protein residue 126 differ. The thesis identifies native Tu6040 and the cloning primers without depositing the assayed expression allele. Do not assign either accession as uniquely identical to the assayed protein, treat them as two demonstrated paralogs, or assume one is a sequencing error. Canonical nodes denote the individual deposited sequences. | https://ueaeprints.uea.ac.uk/id/eprint/35207/ |
| Assayed SimX protein, residue-126 allele unresolved | has_candidate_native_sequence | Deposited native SimX / Sim17 with Gly126 | This 534-aa deposited SimX has Gly at residue 126. Both deposited candidates fit the expression primers, but their internal CDS nucleotide 377 and protein residue 126 differ. The thesis identifies native Tu6040 and the cloning primers without depositing the assayed expression allele. Do not assign either accession as uniquely identical to the assayed protein, treat them as two demonstrated paralogs, or assume one is a sequencing error. Canonical nodes denote the individual deposited sequences. | https://ueaeprints.uea.ac.uk/id/eprint/35207/ |
| Deposited native SimX / SimEx with Ala126 | differs_at_one_residue_from | Deposited native SimX / Sim17 with Gly126 | Direct sequence comparison: Ala126 versus Gly126; coding nucleotide 377 C versus G. All other aligned coding nucleotides and protein residues agree. This is a deposited-sequence comparison, not a mutation experiment, an activity comparison or a demonstrated evolutionary event. | https://www.ncbi.nlm.nih.gov/nuccore/AF324838.2 |
| pIJ10480 ermEp*-simX expression construct | is_reported_to_express | Assayed SimX protein, residue-126 allele unresolved | simEX1-F-NdeI/simEX1-R-HindIII PCR product was cloned into NdeI/HindIII-cut integrative pIJ10257 under ermEp*, yielding pIJ10480. Literal reconstruction against either native sequence predicts the full 1605-bp CDS (534 aa plus stop), 1620 bp including primer tails, without an encoded terminal tag. Matching terminal primers does not resolve Ala/Gly126 or prove the full plasmid sequence, expression level or membrane localization. | https://ueaeprints.uea.ac.uk/id/eprint/35207/ |
| pIJ10481 ermEp*-simEX2 expression construct | is_reported_to_express | Intended SimEx2 product of pIJ10481 | simEX2-F-NdeI/simEX2-R-HindIII PCR product was inserted into NdeI/HindIII-cut pIJ10257 under ermEp* as pIJ10481. This denotes the intended expression construct; the resistance experiment does not separately quantify its protein abundance, folding or localization, so a negative phenotype cannot establish a native transport-function null. | https://ueaeprints.uea.ac.uk/id/eprint/35207/
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| Intended SimEx2 product of pIJ10481 | has_predicted_native_protein_sequence | Deposited native SimEx2 protein | Literal primer reconstruction changes native GTG to ATG but preserves the 1242-bp coding span including its stop, predicting the same 413-aa CDS-aware translated protein. The 1298-bp tailed amplicon includes sequence beyond the stop; its maximal matching native suffix is 12946..14227, not evidence for a one-base gene truncation. This is a primer-derived intended product, not an independently sequenced whole plasmid or an experimentally verified mature protein. The native Na/H-antiporter annotation is a prediction, not a measured ion-transport mechanism. | https://ueaeprints.uea.ac.uk/id/eprint/35207/ |
| Reported native-promoter simX construct without simR | is_reported_to_express | Assayed SimX protein, residue-126 allele unresolved | The source reports integrative simX under its native promoter with simR absent. No construct identifier or complete insert sequence is established for this paragraph; do not substitute pIJ10480 (ermEp*) or a simXp-luxAB reporter. The result is a heterologous expression phenotype, not native-promoter activity measured in Tu6040. | https://ueaeprints.uea.ac.uk/id/eprint/35207/
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| S. lividans 1326 carrying pIJ10480 | carries | pIJ10480 ermEp*-simX expression construct | S. lividans 1326 is the heterologous host, not the native SD8 producer. Transfer of this expression construct does not establish heterologous SD8 biosynthesis. | https://ueaeprints.uea.ac.uk/id/eprint/35207/
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| S. lividans 1326 carrying pIJ10481 | carries | pIJ10481 ermEp*-simEX2 expression construct | S. lividans 1326 is the heterologous host, not the native SD8 producer. Transfer of this expression construct does not establish heterologous SD8 biosynthesis. | https://ueaeprints.uea.ac.uk/id/eprint/35207/
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| S. lividans 1326 carrying native-promoter simX | carries | Reported native-promoter simX construct without simR | S. lividans 1326 is the heterologous host, not the native SD8 producer. Transfer of this expression construct does not establish heterologous SD8 biosynthesis. | https://ueaeprints.uea.ac.uk/id/eprint/35207/
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| S. lividans 1326 carrying pIJ10480 | has_assay_phenotype | Reported SD8 MIC of 65 ug/mL | MIC assay: approximately 5e4 spores per well in a 96-well plate, two-fold SD8 dilutions in SMMS, scored daily for five days; endpoint is the lowest concentration without observed growth. DMSO is at most 1% v/v. Methods name medium and subsequently agar, so this is not relabelled broth microdilution. No replicate count or uncertainty is given for these MIC values; they are not ug-per-disk measurements. The ermEp*-simX strain is reported at 65 ug/mL, compared with 2 ug/mL controls. | https://ueaeprints.uea.ac.uk/id/eprint/35207/
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| S. lividans 1326 carrying pIJ10481 | has_assay_phenotype | Reported SD8 MIC of 2 ug/mL | MIC assay: approximately 5e4 spores per well in a 96-well plate, two-fold SD8 dilutions in SMMS, scored daily for five days; endpoint is the lowest concentration without observed growth. DMSO is at most 1% v/v. Methods name medium and subsequently agar, so this is not relabelled broth microdilution. No replicate count or uncertainty is given for these MIC values; they are not ug-per-disk measurements. The ermEp*-simEX2 strain is reported at 2 ug/mL, matching host-only and empty-vector controls. This is not proof that SimEx2 cannot transport any substrate. | https://ueaeprints.uea.ac.uk/id/eprint/35207/
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| S. lividans 1326 host-only and pIJ10257 controls | has_assay_phenotype | Reported SD8 MIC of 2 ug/mL | MIC assay: approximately 5e4 spores per well in a 96-well plate, two-fold SD8 dilutions in SMMS, scored daily for five days; endpoint is the lowest concentration without observed growth. DMSO is at most 1% v/v. Methods name medium and subsequently agar, so this is not relabelled broth microdilution. No replicate count or uncertainty is given for these MIC values; they are not ug-per-disk measurements. Both the host-only strain and the pIJ10257 empty-vector strain are reported at 2 ug/mL. | https://ueaeprints.uea.ac.uk/id/eprint/35207/
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| S. lividans 1326 carrying native-promoter simX | has_assay_phenotype | Reported SD8 MIC of 4 ug/mL | MIC assay: approximately 5e4 spores per well in a 96-well plate, two-fold SD8 dilutions in SMMS, scored daily for five days; endpoint is the lowest concentration without observed growth. DMSO is at most 1% v/v. Methods name medium and subsequently agar, so this is not relabelled broth microdilution. No replicate count or uncertainty is given for these MIC values; they are not ug-per-disk measurements. The source reports 4 ug/mL for native-promoter simX without simR, a modest change rather than the ermEp* result. | https://ueaeprints.uea.ac.uk/id/eprint/35207/
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| Reported SD8 MIC of 65 ug/mL | is_measured_against | Source-assigned simocyclinone D8 used in sensitivity assays | The assay compound is source-assigned SD8, distinct from SC4 in Figure 4.1. No complete stereochemical identity is reconstructed from the drawing or MIC. The owner remains PLEGMCYXNQPJNV-TYXWGSLYSA-N with unresolved stereocentres; this assay node does not resolve the shared-MIBiG discussion or merge it with a fully specified structural deposition. | https://ueaeprints.uea.ac.uk/id/eprint/35207/
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| Reported SD8 MIC of 2 ug/mL | is_measured_against | Source-assigned simocyclinone D8 used in sensitivity assays | The assay compound is source-assigned SD8, distinct from SC4 in Figure 4.1. No complete stereochemical identity is reconstructed from the drawing or MIC. The owner remains PLEGMCYXNQPJNV-TYXWGSLYSA-N with unresolved stereocentres; this assay node does not resolve the shared-MIBiG discussion or merge it with a fully specified structural deposition. | https://ueaeprints.uea.ac.uk/id/eprint/35207/
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| Reported SD8 MIC of 4 ug/mL | is_measured_against | Source-assigned simocyclinone D8 used in sensitivity assays | The assay compound is source-assigned SD8, distinct from SC4 in Figure 4.1. No complete stereochemical identity is reconstructed from the drawing or MIC. The owner remains PLEGMCYXNQPJNV-TYXWGSLYSA-N with unresolved stereocentres; this assay node does not resolve the shared-MIBiG discussion or merge it with a fully specified structural deposition. | https://ueaeprints.uea.ac.uk/id/eprint/35207/
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| S. lividans 1326 host-only and pIJ10257 controls | provides_controls_for | Reported SD8 MIC of 65 ug/mL | Host-only and empty-vector MIC values provide the comparison for the ermEp*-simX result. Figure 4.2 separately displays empty vector, ermEp*-simX and ermEp*-simEX2 disk assays: approximately 5e4 spores on R2 agar, 0/0.2/0.5/1/10 ug SD8 per disk, 30 C for four days. The plate images support the qualitative resistance contrast, not the numerical MIC or a transport rate; no zone diameters are extracted. | https://ueaeprints.uea.ac.uk/id/eprint/35207/
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| S. lividans 1326 host-only and pIJ10257 controls | provides_controls_for | Reported SD8 MIC of 4 ug/mL | The native-promoter simX MIC is compared with the text-reported host-only/parent-vector baseline. No separately identified matched native-promoter empty construct is supplied in this result; do not infer equal copy number, expression or vector context solely from the word integrative. | https://ueaeprints.uea.ac.uk/id/eprint/35207/
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| S. lividans 1326 carrying pIJ10480 | has_text_reported_assay_phenotype | No reported extra resistance to selected other antibiotics | The source states that ermEp*-simX did not add resistance to novobiocin, erythromycin, lincomycin, rifampicin, bacitracin or nisin. The inspected paragraph does not provide their doses, individual MICs or a displayed panel. Keep this as a limited source-reported negative comparison, not demonstrated exclusive substrate specificity under all conditions. | https://ueaeprints.uea.ac.uk/id/eprint/35207/
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| simX locus of the assayed native stock | encodes | Assayed SimX protein, residue-126 allele unresolved | The study names the native producer-cluster gene simEX1/simX. Its allele in the assayed stock is not resolved to either deposited residue-126 variant. The gene and canonical protein sequences are distinct identity levels. | https://ueaeprints.uea.ac.uk/id/eprint/35207/
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| S. antibioticus Tu6040 simX::apr mutant | disrupts | simX locus of the assayed native stock | The strain table specifies simX::apr. PCR targeting used the pIJ773 apr/oriT cassette with 39-nt homologous arms and lambda RED/pIJ790 in the Galm et al. VII-8g cosmid; transfer was into native Tu6040. Literal arms map to AF324838.2 58144..58182(+) and 59782..59820(-), retaining ATG and TAG while replacing 58183..59781 (1599 bp) in the model. This is not a demonstrated marker-free allele, observed mutant sequence, or proof of no polar effects. Cosmid provenance alone does not resolve the assayed wild-type or expression-plasmid Ala/Gly126 identity. | https://ueaeprints.uea.ac.uk/id/eprint/35207/ |
| S. antibioticus Tu6040 wild-type comparator | retains_native_locus | simX locus of the assayed native stock | The native comparator is S. antibioticus Tu6040, distinct from the heterologous S. lividans strains. The wild-type label does not independently identify its deposited SimX sequence variant. | https://ueaeprints.uea.ac.uk/id/eprint/35207/
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| S. antibioticus Tu6040 simX::apr mutant | has_assay_phenotype | Increased native-mutant SD8 disk sensitivity | Relative to wild type, simX::apr is more sensitive to exogenous SD8 in the displayed disk assay: approximately 5e4 spores on SMMS agar, 0/1/10/100 ug SD8 per disk, 30 C for four days. This is not the heterologous R2 plate or the five-day MIC assay. No native-mutant MIC, quantified zone diameter, statistical effect size or displayed complementation experiment is supplied by these inspected results. | https://ueaeprints.uea.ac.uk/id/eprint/35207/
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| S. antibioticus Tu6040 wild-type comparator | provides_native_comparator_for | Increased native-mutant SD8 disk sensitivity | Figure 4.3 displays wild-type and simX::apr plates at the same listed disk doses. The images are a qualitative comparison; differences in lawn growth and photography are not normalized into a quantitative resistance ratio. No rescue or independent replicate panel is inferred. | https://ueaeprints.uea.ac.uk/id/eprint/35207/
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| Increased native-mutant SD8 disk sensitivity | is_measured_against | Source-assigned simocyclinone D8 used in sensitivity assays | Exogenous SD8 is the applied compound. The source does not measure native SD8 production yield or intracellular/extracellular concentration in this disk experiment. | https://ueaeprints.uea.ac.uk/id/eprint/35207/
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| Assayed SimX protein, residue-126 allele unresolved | is_proposed_to_mediate | Proposed SimX-mediated SD8 export | The author interprets pump-like sequence context, heterologous resistance and native disruption sensitivity as support for SD8 efflux. This graph retains that mechanism as an inference: these experiments do not directly measure transport, membrane localization, proton coupling, kinetics or intracellular drug depletion. There is no independent transport comparison between the two deposited SimX variants. | https://ueaeprints.uea.ac.uk/id/eprint/35207/
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| Proposed SimX-mediated SD8 export | has_proposed_substrate | Source-assigned simocyclinone D8 used in sensitivity assays | SD8 is the proposed exported product. This is not an experimentally demonstrated exclusive substrate list, direct SimX-SD8 binding measurement or demonstrated SC4 transport. Other regulatory experiments do not by themselves prove either transport claim. | https://ueaeprints.uea.ac.uk/id/eprint/35207/
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Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 18 nodes, 25 edges
SimC7 deletion and complementation in the heterologous sim cluster, including the engineered SimX-expressing host background and chemical assignment of 7-oxo-SD8. Native sequence identity, intended alleles, source-reported rescue and conflicting construct/yield descriptions remain distinct (review #538). The 2015 main article and relevant primary thesis sections were inspected; the journal supplementary file was not obtained. Neither native-producer deletion nor complete owner stereochemistry is established by these experiments.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Native simC7 locus | encodes | Deposited native SimC7 ketoreductase | This node denotes the deposited native protein. The scar mutant, tagged enzyme and complementation plasmids are separate entities; none is assigned the native accession as an exact engineered-sequence identifier. | https://www.ncbi.nlm.nih.gov/protein/AEU17892.1 |
| PAC-12I containing the sim biosynthetic cluster | contains | Native simC7 locus | The paper reports PAC-12I with a roughly 95-kb insert containing the approximately 72-kb, 49-gene cluster and about 19-kb/4-kb flanks. Thesis Table 2.4 instead describes an 85-kb BamHI genomic fragment; the whole insert and restriction fragment are not assumed identical measurements. PAC sequencing was reported, but a complete public experimental insert was not independently compared base-for-base here. | DOI:10.1016/j.jmb.2015.03.019 |
| PAC-12I delta-simC7 scar construct | is_derived_from | PAC-12I containing the sim biosynthetic cluster | PCR targeting in E. coli used lambda RED and an apramycin cassette amplified from pIJ773_del_oriT; FLP excision removed the marker and reportedly left an 81-bp in-frame scar. PCR and EcoRI digestion verified the construct. This is not a scarless deletion, the earlier native simX::apr allele, or a sequenced final junction. | DOI:10.1016/j.jmb.2015.03.019 |
| PAC-12I delta-simC7 scar construct | disrupts | Native simC7 locus | Literal 39-nt deletion arms map to AF324838.2:9308..9346(+) and 10196..10234(-), retaining native ATG/TAG and replacing the intervening 849 bp. An 81-bp scar would give an 87-bp start-to-stop allele. Check primers span 9192..10350, predicting 1159 bp natively versus 391 bp for that scar model. These are curator reconstructions, not measured mutant sequence or observed gel sizes; possible local regulatory effects are not excluded by calling the scar in-frame. | https://ueaeprints.uea.ac.uk/id/eprint/61276/ |
| S. coelicolor M1152ex1 SimX-expressing host | carries | pIJ10480 ermEp*-simEx1 expression construct | M1152ex1 was created by introducing the integrative pIJ10480 construct into S. coelicolor M1152. All subsequent SimC7 cluster experiments use this engineered background; the native producer S. antibioticus Tu6040 was not deleted in this study. | DOI:10.1016/j.jmb.2015.03.019
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| pIJ10480 ermEp*-simEx1 expression construct | is_reported_to_express | Assayed SimX / SimEx1 protein, allele unresolved | The construct drives simEx1/SimX from ermEp*. This experiment does not resolve the native Ala/Gly126 SimX ambiguity documented in the existing resistance graph (review #537), nor does enhanced resistance directly measure transport flux. | DOI:10.1016/j.jmb.2015.03.019
|
| Assayed SimX / SimEx1 protein, allele unresolved | has_candidate_native_sequence | Deposited native SimX / SimEx with Ala126 | This 534-aa deposited SimX (AF324838.2:58180..59784(+), AAK06799.1, UniProtKB:Q9AMH8) has Ala at residue 126. Both deposited Streptomyces antibioticus Tu 6040 SimX sequences fit the expression primers, but neither DOI:10.1016/j.jmb.2015.03.019 nor the thesis deposits the integrated pIJ10480 allele sequence in M1152ex1. Canonical nodes denote the individual deposited candidate sequences without collapsing the assayed SimX node onto one accession. | DOI:10.1016/j.jmb.2015.03.019 |
| Assayed SimX / SimEx1 protein, allele unresolved | has_candidate_native_sequence | Deposited native SimX / Sim17 with Gly126 | This 534-aa deposited SimX (AF322256.1:13386..14990(+), AAL15595.1, UniProtKB:Q93FA2) has Gly at residue 126. Both deposited Streptomyces antibioticus Tu 6040 SimX sequences fit the expression primers, but neither DOI:10.1016/j.jmb.2015.03.019 nor the thesis deposits the integrated pIJ10480 allele sequence in M1152ex1. Canonical nodes denote the individual deposited candidate sequences without collapsing the assayed SimX node onto one accession. | DOI:10.1016/j.jmb.2015.03.019 |
| S. coelicolor M1152ex1 SimX-expressing host | has_reported_resistance_relative_to | S. coelicolor M1152 heterologous host | Reported SD8 MIC changes from 1 to 100 ug/mL after introduction of pIJ10480. This is the M1152 comparison, not the earlier S. lividans 2-to-65 ug/mL result. The thesis specifies 200-uL 96-well SMMS assays, 1e5 spores per well, 30 C for six days and a growth-inhibition endpoint; no flux, energy coupling or quantitative MIC uncertainty is measured here. | DOI:10.1016/j.jmb.2015.03.019 |
| M1152ex1 carrying intact PAC-12I | has_engineered_host_background | S. coelicolor M1152ex1 SimX-expressing host | This experimental strain retains the SimX-expressing M1152ex1 background. The genomic source of the PAC is native Tu6040, but its assay host is S. coelicolor. | DOI:10.1016/j.jmb.2015.03.019
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| M1152ex1 carrying intact PAC-12I | carries | PAC-12I containing the sim biosynthetic cluster | The indicated PAC was mobilized into the heterologous host; the intact-cluster and scar-mutant strains are compared without conflating their construct identities. | DOI:10.1016/j.jmb.2015.03.019
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| M1152ex1 carrying PAC-12I delta-simC7 | has_engineered_host_background | S. coelicolor M1152ex1 SimX-expressing host | This experimental strain retains the SimX-expressing M1152ex1 background. The genomic source of the PAC is native Tu6040, but its assay host is S. coelicolor. | DOI:10.1016/j.jmb.2015.03.019
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| M1152ex1 carrying PAC-12I delta-simC7 | carries | PAC-12I delta-simC7 scar construct | The indicated PAC was mobilized into the heterologous host; the intact-cluster and scar-mutant strains are compared without conflating their construct identities. | DOI:10.1016/j.jmb.2015.03.019
|
| M1152ex1 carrying intact PAC-12I | produces | Source-assigned simocyclinone D8 | Culture extracts match source-assigned SD8 by LC-MS-UV, against an isolated standard. Production conditions were six days at 30 C and 250 rpm in defined medium with glutamine 5.84 g/L and glycerol 20 mL/L. The article reports 24-56 mg/L versus 9-14 mg/L for native Tu6040. Thesis p.115 instead prints 24-56 mg/mL, while Figure 3.3 uses an mg/L axis; the conflict is preserved, not silently corrected. That thesis growth curve also used 1e8 spores for the cluster strain versus 1e7 for host-only controls. It is not an equal-inoculum growth-rate experiment. Figure 3.2 qualitatively shows partial alleviation of cluster-associated growth/sporulation impairment by SimX on SFM agar after nine days at 30 C. | DOI:10.1016/j.jmb.2015.03.019 |
| S. coelicolor M1152ex1 SimX-expressing host | has_no_detected_production_of | Source-assigned simocyclinone D8 | The M1152ex1 control without the sim cluster produced no detected simocyclinones under the reported production conditions. This is a negative assay control, not proof of absence under every culture condition or a numerical zero yield. | DOI:10.1016/j.jmb.2015.03.019
|
| M1152ex1 carrying PAC-12I delta-simC7 | produces | Source-assigned 7-oxo-simocyclinone D8 | The principal mutant product is assigned as 7-oxo-SD8, not failure of all simocyclinone synthesis. Figure 2 compares mutant, intact-cluster and SD8-standard extracts after six days; their displayed retention times are 20.65, 19.53 and 19.48 min. Approximately 63 mg was purified from 4 L with reported >98% purity and 27% recovery. Retention time alone is not the structural proof: HRMS, fragmentation and NMR supply the assignment. | DOI:10.1016/j.jmb.2015.03.019 |
| Source-assigned 7-oxo-simocyclinone D8 | has_source_assigned_structural_difference_from | Source-assigned simocyclinone D8 | The reported difference is a C-7 carbonyl instead of the C-7 hydroxyl on the angucyclinone, with the tetraene linker retained. Two-Da lower parent mass, polyketide-localized MS/MS differences, missing H-7, shifted H-6, and carbonyl/HMBC NMR evidence support this assignment. These source-assigned assay compounds are distinct from MGD8N2A and other congeners. No uninspected complete chemical identifier is invented; the owner retains six unspecified stereocentres and its open shared-MIBiG discussion remain unresolved. | DOI:10.1016/j.jmb.2015.03.019
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| Delta-simC7 host carrying pGM1190-NB-C7 | is_complemented_derivative_of | M1152ex1 carrying PAC-12I delta-simC7 | Complementation was performed in the M1152ex1/PAC-12I delta-simC7 background, not in native Tu6040. The genotype is not silently replaced by the intact-PAC control. | DOI:10.1016/j.jmb.2015.03.019 |
| Delta-simC7 host carrying pGM1190-NB-C7 | carries | pGM1190-NB-C7, promoter description disputed | The named pGM1190 derivative provides an in-trans simC7 copy. The source does not establish equal expression, copy number or promoter activity between the rescue constructs. | https://ueaeprints.uea.ac.uk/id/eprint/61276/
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| Delta-simC7 host carrying pGM1190-pB7-NB-C7 | is_complemented_derivative_of | M1152ex1 carrying PAC-12I delta-simC7 | Complementation was performed in the M1152ex1/PAC-12I delta-simC7 background, not in native Tu6040. The genotype is not silently replaced by the intact-PAC control. | DOI:10.1016/j.jmb.2015.03.019 |
| Delta-simC7 host carrying pGM1190-pB7-NB-C7 | carries | pGM1190-pB7-NB-C7 complementation construct | The named pGM1190 derivative provides an in-trans simC7 copy. The source does not establish equal expression, copy number or promoter activity between the rescue constructs. | https://ueaeprints.uea.ac.uk/id/eprint/61276/
|
| pGM1190-NB-C7, promoter description disputed | provides_reported_intact_copy_of | Native simC7 locus | Methods p.85, Table 2.4 and Figure 4.16 describe pGM1190-NB-C7 as ptipA::simC7. Results p.146 instead explain its low yield by a missing promoter. This unresolved source conflict is not repaired or used to assign a demonstrated promoter mechanism. The reverse-primer name/site discrepancy in Table 2.5 also prevents literal reconstruction of the final cloning junction from names alone. | https://ueaeprints.uea.ac.uk/id/eprint/61276/
|
| pGM1190-pB7-NB-C7 complementation construct | provides_reported_intact_copy_of | Native simC7 locus | The second rescue fuses simC7 to the approximately 400-bp region upstream of simB7 in pGM1190. Literal 20-nt promoter-primer arms map to 3812..3831(+) and 4189..4208(-), predicting a 397-bp native segment and 415-bp tailed PCR product. This reconstruction does not establish an experimental plasmid junction or directly map a promoter. The proposed simB7-to-simC7 seven-gene operon remains putative. | DOI:10.1016/j.jmb.2015.03.019 |
| Delta-simC7 host carrying pGM1190-NB-C7 | has_reported_low_production_of | Source-assigned simocyclinone D8 | The thesis reports low SD8 yield from the first rescue and shows a source-assigned SD8 peak in Figure 4.16. The promoter explanation is disputed within the source. Panel-normalized intensity is not an absolute yield or a quantified fraction of wild-type rescue; a matched empty-rescue-vector comparison is not shown in this panel. | https://ueaeprints.uea.ac.uk/id/eprint/61276/
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| Delta-simC7 host carrying pGM1190-pB7-NB-C7 | restores_detectable_production_of | Source-assigned simocyclinone D8 | The article reports complementation with the simB7-upstream fusion as data not shown. The thesis Figure 4.16 displays LC-MS-UV chromatograms at 200-700 nm after six days at 30 C and reports HRMS confirmation without displaying those spectra. The normalized traces and SD8/7-oxo standards support product rescue, not an absolute wild-type-equivalent yield or exclusion of all polar effects. | DOI:10.1016/j.jmb.2015.03.019 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 15 nodes, 16 edges
Purified N-terminally His-tagged SimC7 converts source-assigned 7-oxo-SD8 to SD8 with NADH or NADPH and supports the slower reverse reaction with NAD+. Controls, construct discrepancies and concentration conflicts remain explicit (review #538). The earlier physiological substrate is a hypothesis, so no terminal in-vivo pathway order or kinetic constants are invented. This graph does not duplicate gyrase-target bioactivity mechanisms.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Reported pET15b-NB-C7 expression construct | is_reported_to_express | Assayed N-terminally His-tagged SimC7 | The article describes PCR from PAC-12I, pJET1.2/blunt cloning, NdeI/BamHI subcloning into pET15b and sequencing for errors. Protein was expressed in E. coli Rosetta (DE3)pLysS at 25 C, induced near OD600 0.4 with 0.5 mM IPTG for 16 h, purified on Ni-NTA and quantified by Bradford assay. The thesis also describes later altered temperature/salt protocols; those are not substituted for the 2015 method. Table 2.4 p.53 labels the pET15b insert ptipA::simC7 despite its parent-vector T7 description; retain the discrepancy. | DOI:10.1016/j.jmb.2015.03.019 |
| Assayed N-terminally His-tagged SimC7 | has_native_parent_sequence | Deposited native SimC7 ketoreductase | The intended recombinant product adds MGSSHHHHHHSSGLVPRGSH at the N terminus (304 aa total), without claiming tag removal. Native CDS translation agrees with G9VYV4. Both printed reverse primers share the native stop-region suffix and predict the full 855-nt CDS and 870-bp tailed amplicon with the NdeI forward primer. However, the primer named simC7-HindIII-R contains GGATCC, while simC7-BamHI-R contains AGGCTT and neither a BamHI nor HindIII site. The paper reports sequence checking, but its complete plasmid sequence was not inspected here; literal primer names do not prove the final junction or expressed sequence. | https://ueaeprints.uea.ac.uk/id/eprint/61276/ |
| Assayed N-terminally His-tagged SimC7 | catalyzes | Assayed C-7 ketoreduction | Purified tagged SimC7 converts 7-oxo-SD8 to SD8 in the Figure 6b assay. Main methods report 25-200 uM substrate, 0.3 mM NAD(P)H, 50 mM HEPES pH 7.2 and 500 nM enzyme; Figure 6 incubates for one hour at room temperature. The thesis Figure 4.17 specifies 100 uM substrate and 250 uL reactions. Thesis p.99 also equates 500 nM with 24.18 ug/mL, inconsistent with its reported 32,235-Da tagged mass (500 nM implies about 16.12 ug/mL). Neither concentration is silently corrected or used to calculate catalytic efficiency. This direct enzyme experiment, not the knockout alone, establishes ketoreduction. | DOI:10.1016/j.jmb.2015.03.019 |
| Assayed C-7 ketoreduction | uses_substrate | Source-assigned 7-oxo-simocyclinone D8 | Purified, source-assigned 7-oxo-SD8 is the tested substrate. Its use does not establish that complete 7-oxo-SD8 is the physiological substrate or that SimC7 acts after attachment of every other SD8 moiety. | DOI:10.1016/j.jmb.2015.03.019
|
| Assayed C-7 ketoreduction | forms_product | Source-assigned simocyclinone D8 | Product is assigned as SD8 by HPLC against a standard, with tandem-MS confirmation reported. Equal-volume methanol quenching, 100 C for ten minutes and centrifugation stop the assay. Main quantification uses reversed-phase HPLC at 360 nm, 25-95% methanol over 20 min then five minutes at 95%, against 0.1% formic acid in water; standards span 7.5-2100 pmol. The journal supplemental standard curve was not inspected. Thesis methods report duplicate sample analyses and triplicate standards, not a reported number of independent biological enzyme preparations. | DOI:10.1016/j.jmb.2015.03.019 |
| NADH in the reduction assay | supports | Assayed C-7 ketoreduction | Figure 6b shows SD8 product with SimC7 and this reduced cofactor; the main method reports 0.3 mM NAD(P)H. This establishes activity under the assayed conditions, not an in-vivo cofactor preference or measured Km. | DOI:10.1016/j.jmb.2015.03.019
|
| NADPH in the reduction assay | supports | Assayed C-7 ketoreduction | Figure 6b shows SD8 product with SimC7 and this reduced cofactor; the main method reports 0.3 mM NAD(P)H. This establishes activity under the assayed conditions, not an in-vivo cofactor preference or measured Km. | DOI:10.1016/j.jmb.2015.03.019
|
| NADH/NADPH reduction controls without SimC7 | provides_omission_controls_for | Assayed C-7 ketoreduction | Separate minus-SimC7 traces containing NADH or NADPH retain the 7-oxo-SD8 peak instead of the SD8 product peak. These are assay controls for the displayed conditions, not numerical upper bounds on spontaneous conversion. | DOI:10.1016/j.jmb.2015.03.019
|
| SimC7 reduction control without reduced cofactor | provides_omission_control_for | Assayed C-7 ketoreduction | The plus-SimC7/minus-NAD(P)H trace retains 7-oxo-SD8. It constrains the cofactor requirement in this assay without establishing all possible electron donors or excluding every trace side reaction. | DOI:10.1016/j.jmb.2015.03.019
|
| Assayed N-terminally His-tagged SimC7 | catalyzes | Assayed reverse C-7 oxidation | The reverse experiment converts some SD8 to 7-oxo-SD8 and is reported as much slower. Residual SD8 is visible in Figure 6c. This is not an equilibrium constant, a quantitative rate comparison or evidence that reverse oxidation is the physiological direction. | DOI:10.1016/j.jmb.2015.03.019
|
| Assayed reverse C-7 oxidation | uses_substrate | Source-assigned simocyclinone D8 | In the reverse experiment the input is SD8, despite the general method and thesis Figure 4.17 caption describing 7-oxo-SD8 as substrate. The panel and results define the reversed substrate/product direction. | DOI:10.1016/j.jmb.2015.03.019 |
| Assayed reverse C-7 oxidation | forms_product | Source-assigned 7-oxo-simocyclinone D8 | The new reverse-reaction peak is assigned as 7-oxo-SD8; tandem-MS confirmation is reported. This does not revise the owner structure or merge 7-oxo-SD8 into the SD8 record as an equivalent compound. | DOI:10.1016/j.jmb.2015.03.019
|
| NAD+ in the reverse assay | supports | Assayed reverse C-7 oxidation | The displayed and described reverse assay uses 300 mM NAD+. The scheme and caption say NAD(P)+, but direct reverse activity with NADP+ is not demonstrated in the inspected panel. The very high oxidized-cofactor concentration must not be relabelled 0.3 mM or physiological. | DOI:10.1016/j.jmb.2015.03.019
|
| NAD+ oxidation control without SimC7 | provides_omission_control_for | Assayed reverse C-7 oxidation | The minus-SimC7/plus-NAD+ trace retains the SD8 peak without the displayed 7-oxo-SD8 product peak. This is the relevant reverse-assay control, distinct from the NADH/NADPH reduction controls. | DOI:10.1016/j.jmb.2015.03.019
|
| Assayed N-terminally His-tagged SimC7 | has_text_reported_assay_phenotype | Text-reported NADPH preference without kinetic constants | Both sources report NADPH preference but mark the comparison data not shown. The thesis says conversion was nearly complete by 30 seconds and attempts at lower enzyme concentration did not yield sufficient kinetic data; below 10 nM the reaction was too slow. Do not derive kcat, Km, cofactor specificity ratios or a quantitative initial rate from endpoint chromatograms or the unshown comparison. Absorbance overlap prevented a simple NAD(P)H spectrophotometric assay. | DOI:10.1016/j.jmb.2015.03.019 |
| Deposited native SimC7 ketoreductase | has_proposed_physiological_substrate | Proposed earlier 7-oxo A-group angucyclinone substrate | The authors infer that SimC7 normally reduces an earlier angucyclinone intermediate, before glycosylation and attachment of the tetraene and aminocoumarin. Appropriate-mass A-group species in the mutant are supporting observations, not a purified assay of a fully resolved earlier substrate. The exact physiological substrate and pathway position are therefore not encoded as a demonstrated ordered step. | DOI:10.1016/j.jmb.2015.03.019
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 14 nodes, 24 edges
Six engineered SimC7 variants constrain the proposed ketoreduction mechanism through HPLC activity measurements and a displayed subset of far-UV CD controls. Native G9VYV4 is distinct from every tagged assay protein. The paper/thesis uncertainty disagreement, residual activity and limited structural control remain explicit (review #539). The 2016 main paper was inspected in the public author-thesis appendix, not as an independently fetched final PDF; the journal supplement was not obtained. This graph does not turn mutant phenotypes into direct observation of proton transfer or a phenolate intermediate.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Reported pET15b-NB-C7 mutagenesis template | is_reported_to_express | N-terminally His-tagged wild-type SimC7 comparator | The pET15b-NB-C7 construct supplies the tagged wild-type comparator and the template for PCR-based Q5 mutagenesis. The paper reports N-terminally His-tagged E. coli expression, Ni-affinity purification and sequence verification. Thesis pp.87-88 distinguishes a later low-salt protocol for crystal and mutant proteins (18 C induction with 0.5 mM IPTG for 16 h) from earlier production. The historical template promoter/primer discrepancies remain documented in the existing SimC7 graphs; no uninspected complete plasmid junction is asserted here. | DOI:10.1016/j.chembiol.2016.07.018 |
| N-terminally His-tagged wild-type SimC7 comparator | has_native_parent_sequence | Deposited native SimC7 ketoreductase | The intended 304-aa recombinant protein comprises the 20-aa N-terminal extension MGSSHHHHHHSSGLVPRGSH plus native 284-aa SimC7. There is no claim of tag removal. The native accession identifies the parent, not an exact tagged or mutant experimental molecule. Residue labels S95/I108/H112 use native numbering; their positions would be 115/128/132 when the extension is counted. | https://www.ncbi.nlm.nih.gov/nuccore/AF324838.2 |
| N-terminally His-tagged SimC7 S95A | is_site_directed_variant_of | N-terminally His-tagged wild-type SimC7 comparator | S95A denotes the engineered tagged protein, using native residue numbering. The printed replacement codon is TCG to GCC. Q5 amplification used pET15b-NB-C7, DpnI template digestion, kinase/ligase treatment and E. coli DH5alpha transformation; sequencing was reported. The independent primer audit reconstructs the intended local substitution, not the full experimental plasmid or protein sample. No exact UniProt accession is assigned to this variant. | DOI:10.1016/j.chembiol.2016.07.018 |
| N-terminally His-tagged SimC7 I108A | is_site_directed_variant_of | N-terminally His-tagged wild-type SimC7 comparator | I108A denotes the engineered tagged protein, using native residue numbering. The printed replacement codon is ATC to GCG. Q5 amplification used pET15b-NB-C7, DpnI template digestion, kinase/ligase treatment and E. coli DH5alpha transformation; sequencing was reported. The independent primer audit reconstructs the intended local substitution, not the full experimental plasmid or protein sample. No exact UniProt accession is assigned to this variant. | DOI:10.1016/j.chembiol.2016.07.018 |
| N-terminally His-tagged SimC7 I108D | is_site_directed_variant_of | N-terminally His-tagged wild-type SimC7 comparator | I108D denotes the engineered tagged protein, using native residue numbering. The printed replacement codon is ATC to GAT. Q5 amplification used pET15b-NB-C7, DpnI template digestion, kinase/ligase treatment and E. coli DH5alpha transformation; sequencing was reported. The independent primer audit reconstructs the intended local substitution, not the full experimental plasmid or protein sample. No exact UniProt accession is assigned to this variant. | DOI:10.1016/j.chembiol.2016.07.018 |
| N-terminally His-tagged SimC7 H112A | is_site_directed_variant_of | N-terminally His-tagged wild-type SimC7 comparator | H112A denotes the engineered tagged protein, using native residue numbering. The printed replacement codon is CAC to GCC. Q5 amplification used pET15b-NB-C7, DpnI template digestion, kinase/ligase treatment and E. coli DH5alpha transformation; sequencing was reported. The independent primer audit reconstructs the intended local substitution, not the full experimental plasmid or protein sample. No exact UniProt accession is assigned to this variant. | DOI:10.1016/j.chembiol.2016.07.018 |
| N-terminally His-tagged SimC7 H112N | is_site_directed_variant_of | N-terminally His-tagged wild-type SimC7 comparator | H112N denotes the engineered tagged protein, using native residue numbering. The printed replacement codon is CAC to AAC. Q5 amplification used pET15b-NB-C7, DpnI template digestion, kinase/ligase treatment and E. coli DH5alpha transformation; sequencing was reported. The independent primer audit reconstructs the intended local substitution, not the full experimental plasmid or protein sample. No exact UniProt accession is assigned to this variant. | DOI:10.1016/j.chembiol.2016.07.018 |
| N-terminally His-tagged SimC7 H112Q | is_site_directed_variant_of | N-terminally His-tagged wild-type SimC7 comparator | H112Q denotes the engineered tagged protein, using native residue numbering. The printed replacement codon is CAC to CAG. Q5 amplification used pET15b-NB-C7, DpnI template digestion, kinase/ligase treatment and E. coli DH5alpha transformation; sequencing was reported. The independent primer audit reconstructs the intended local substitution, not the full experimental plasmid or protein sample. No exact UniProt accession is assigned to this variant. | DOI:10.1016/j.chembiol.2016.07.018 |
| N-terminally His-tagged wild-type SimC7 comparator | has_measured_activity_in | HPLC quantification of SimC7-dependent SD8 formation | Tagged wild type: reported conversion 100.0 +/- 7.3% relative to tagged wild type; specific activity 13.9 +/- 1.0 nmol/min/mg in Figure 4C. The paper calls the errors standard errors for three independent experiments. Thesis Table 5.6 calls the errors standard deviations; the same means are not pooled or treated as independent replication. Normalization to 100% does not establish absolute substrate depletion. These are HPLC-derived specific activities, not kcat/Km or independently reconstructed initial rates. | DOI:10.1016/j.chembiol.2016.07.018 |
| N-terminally His-tagged SimC7 S95A | has_measured_activity_in | HPLC quantification of SimC7-dependent SD8 formation | S95A: reported conversion 86.1 +/- 7.1% relative to tagged wild type; specific activity 11.9 +/- 1.0 nmol/min/mg in Figure 4C. The paper calls the errors standard errors for three independent experiments. Thesis Table 5.6 calls the errors standard deviations; the same means are not pooled or treated as independent replication. Normalization to 100% does not establish absolute substrate depletion. These are HPLC-derived specific activities, not kcat/Km or independently reconstructed initial rates. Retained activity under this condition is not proof of complete mechanistic dispensability or unchanged substrate/cofactor binding. | DOI:10.1016/j.chembiol.2016.07.018 |
| N-terminally His-tagged SimC7 I108A | has_measured_activity_in | HPLC quantification of SimC7-dependent SD8 formation | I108A: reported conversion 91.5 +/- 13.8% relative to tagged wild type; specific activity 12.7 +/- 1.9 nmol/min/mg in Figure 4C. The paper calls the errors standard errors for three independent experiments. Thesis Table 5.6 calls the errors standard deviations; the same means are not pooled or treated as independent replication. Normalization to 100% does not establish absolute substrate depletion. These are HPLC-derived specific activities, not kcat/Km or independently reconstructed initial rates. Retained activity under this condition is not proof of complete mechanistic dispensability or unchanged substrate/cofactor binding. | DOI:10.1016/j.chembiol.2016.07.018 |
| N-terminally His-tagged SimC7 I108D | has_measured_activity_in | HPLC quantification of SimC7-dependent SD8 formation | I108D: reported conversion 1.2 +/- 0.8% relative to tagged wild type; specific activity 0.2 +/- 0.1 nmol/min/mg in Figure 4C. The paper calls the errors standard errors for three independent experiments. Thesis Table 5.6 calls the errors standard deviations; the same means are not pooled or treated as independent replication. Normalization to 100% does not establish absolute substrate depletion. These are HPLC-derived specific activities, not kcat/Km or independently reconstructed initial rates. The nonzero reported mean is retained despite prose describing activity as abolished; no detection limit or exact zero is inferred. | DOI:10.1016/j.chembiol.2016.07.018 |
| N-terminally His-tagged SimC7 H112A | has_measured_activity_in | HPLC quantification of SimC7-dependent SD8 formation | H112A: reported conversion 12.5 +/- 3.6% relative to tagged wild type; specific activity 1.7 +/- 0.5 nmol/min/mg in Figure 4C. The paper calls the errors standard errors for three independent experiments. Thesis Table 5.6 calls the errors standard deviations; the same means are not pooled or treated as independent replication. Normalization to 100% does not establish absolute substrate depletion. These are HPLC-derived specific activities, not kcat/Km or independently reconstructed initial rates. | DOI:10.1016/j.chembiol.2016.07.018 |
| N-terminally His-tagged SimC7 H112N | has_measured_activity_in | HPLC quantification of SimC7-dependent SD8 formation | H112N: reported conversion 5.8 +/- 2.5% relative to tagged wild type; specific activity 0.8 +/- 0.4 nmol/min/mg in Figure 4C. The paper calls the errors standard errors for three independent experiments. Thesis Table 5.6 calls the errors standard deviations; the same means are not pooled or treated as independent replication. Normalization to 100% does not establish absolute substrate depletion. These are HPLC-derived specific activities, not kcat/Km or independently reconstructed initial rates. H112N has an additional numeric conflict: Table 5.6 gives 0.8 +/- 0.3 nmol/min/mg, not the paper's 0.8 +/- 0.4. | DOI:10.1016/j.chembiol.2016.07.018 |
| N-terminally His-tagged SimC7 H112Q | has_measured_activity_in | HPLC quantification of SimC7-dependent SD8 formation | H112Q: reported conversion 1.0 +/- 1.4% relative to tagged wild type; specific activity 0.1 +/- 0.2 nmol/min/mg in Figure 4C. The paper calls the errors standard errors for three independent experiments. Thesis Table 5.6 calls the errors standard deviations; the same means are not pooled or treated as independent replication. Normalization to 100% does not establish absolute substrate depletion. These are HPLC-derived specific activities, not kcat/Km or independently reconstructed initial rates. The nonzero reported mean is retained despite prose describing activity as abolished; no detection limit or exact zero is inferred. | DOI:10.1016/j.chembiol.2016.07.018 |
| HPLC quantification of SimC7-dependent SD8 formation | uses_substrate | Source-assigned 7-oxo-simocyclinone D8 assay substrate | Thesis mutant methods specify 0.2 mM source-assigned 7-oxo-SD8, 500 nM enzyme (reported as 16.12 ug/mL) and 50 mM HEPES pH 7.2. This mutant paragraph is distinct from the conflicting 24.18 ug/mL in the generic assay paragraph. Reaction duration and temperature were not independently established for this mutant table in the inspected methods; the earlier one-hour assay and CD temperature are not substituted. The final physiological substrate and pathway order remain unresolved, as in the prior SimC7 component. | DOI:10.1016/j.chembiol.2016.07.018 |
| HPLC quantification of SimC7-dependent SD8 formation | quantifies_product | Source-assigned simocyclinone D8 assay product | The variant method quantifies the SD8 HPLC peak against purified standards spanning 7.5-9000 pmol, using a 70-95% methanol gradient over 10 min. Three independent experiments and two technical replicates are reported. Methanol quenching, heating for ten minutes at 100 C and centrifugation precede analysis; the heating step is not the enzyme reaction temperature. Chemical assignments and the owner's incomplete stereochemistry are not upgraded by this relative-conversion assay. | DOI:10.1016/j.chembiol.2016.07.018 |
| NADH used in the SimC7 mutant assay | is_reduced_cofactor_in | HPLC quantification of SimC7-dependent SD8 formation | The mutant methods specify 0.3 mM NADH. They do not report an NADPH mutant comparison or measure cofactor-binding affinity; the prior wild-type NADH/NADPH activity experiments are a separate claim. | https://ueaeprints.uea.ac.uk/id/eprint/61276/
|
| Displayed far-UV CD comparison of wild type and four SimC7 mutants | provides_limited_structural_control_for | HPLC quantification of SimC7-dependent SD8 formation | The displayed CD experiment compares tagged wild type with I108D, H112A, H112N and H112Q. The main paper and thesis methods more broadly report CD integrity checks, but the inspected thesis panel does not display S95A or I108A. Similar far-UV spectra constrain gross secondary-structure changes; they do not exclude local active-site rearrangements or prove the proposed proton-transfer mechanism. This comparison is an experimental control, not a biochemical interaction between CD and ketoreduction. | DOI:10.1016/j.chembiol.2016.07.018 |
| N-terminally His-tagged wild-type SimC7 comparator | has_reference_spectrum_in | Displayed far-UV CD comparison of wild type and four SimC7 mutants | The tagged wild-type spectrum is the comparator. Thesis methods use 0.2 mg/mL protein in 20 mM potassium phosphate pH 7.2 and 5% glycerol, measured at 20 C with a 0.5-mm cuvette. Four scans over 190-260 nm are averaged (2-nm bandwidth, 0.5-nm step). These are CD conditions, not the kinetic assay. The plot does not establish identical tertiary structure, local binding geometry or statistical equivalence. | https://ueaeprints.uea.ac.uk/id/eprint/61276/
|
| N-terminally His-tagged SimC7 I108D | has_reported_similar_spectrum_in | Displayed far-UV CD comparison of wild type and four SimC7 mutants | The displayed I108D spectrum is reported as similar to tagged wild type. Thesis methods use 0.2 mg/mL protein in 20 mM potassium phosphate pH 7.2 and 5% glycerol, measured at 20 C with a 0.5-mm cuvette. Four scans over 190-260 nm are averaged (2-nm bandwidth, 0.5-nm step). These are CD conditions, not the kinetic assay. The plot does not establish identical tertiary structure, local binding geometry or statistical equivalence. | https://ueaeprints.uea.ac.uk/id/eprint/61276/
|
| N-terminally His-tagged SimC7 H112A | has_reported_similar_spectrum_in | Displayed far-UV CD comparison of wild type and four SimC7 mutants | The displayed H112A spectrum is reported as similar to tagged wild type. Thesis methods use 0.2 mg/mL protein in 20 mM potassium phosphate pH 7.2 and 5% glycerol, measured at 20 C with a 0.5-mm cuvette. Four scans over 190-260 nm are averaged (2-nm bandwidth, 0.5-nm step). These are CD conditions, not the kinetic assay. The plot does not establish identical tertiary structure, local binding geometry or statistical equivalence. | https://ueaeprints.uea.ac.uk/id/eprint/61276/
|
| N-terminally His-tagged SimC7 H112N | has_reported_similar_spectrum_in | Displayed far-UV CD comparison of wild type and four SimC7 mutants | The displayed H112N spectrum is reported as similar to tagged wild type. Thesis methods use 0.2 mg/mL protein in 20 mM potassium phosphate pH 7.2 and 5% glycerol, measured at 20 C with a 0.5-mm cuvette. Four scans over 190-260 nm are averaged (2-nm bandwidth, 0.5-nm step). These are CD conditions, not the kinetic assay. The plot does not establish identical tertiary structure, local binding geometry or statistical equivalence. | https://ueaeprints.uea.ac.uk/id/eprint/61276/
|
| N-terminally His-tagged SimC7 H112Q | has_reported_similar_spectrum_in | Displayed far-UV CD comparison of wild type and four SimC7 mutants | The displayed H112Q spectrum is reported as similar to tagged wild type. Thesis methods use 0.2 mg/mL protein in 20 mM potassium phosphate pH 7.2 and 5% glycerol, measured at 20 C with a 0.5-mm cuvette. Four scans over 190-260 nm are averaged (2-nm bandwidth, 0.5-nm step). These are CD conditions, not the kinetic assay. The plot does not establish identical tertiary structure, local binding geometry or statistical equivalence. | https://ueaeprints.uea.ac.uk/id/eprint/61276/
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 12 nodes, 13 edges
SimL/SimD5 is assigned the aminocoumarin amide-ligation role in simocyclinone biosynthesis. Exact native Q9F5J5 is distinct from the two recombinant preparations. The inspected 2005 evidence is abstract-level only: natural C4 acceptance, broad acid-substrate activity and engineered novobiocin-analogue tailoring are separate claims. No uninspected kinetic conditions, exact construct sequence, complete native D8 reconstitution or final in-vivo step order is asserted. Review #540 preserves these evidence and identity boundaries.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Native SimL / SimD5 amide synthetase | is_source_assigned_to | Source-assigned simocyclinone aminocoumarin amide-ligation step | Luft et al. assign SimL to formation of the amide joining the aminocoumarin and complex acyl moieties of simocyclinone. Native protein identity is independently sequence-grounded; exact experimental constructs remain ungrounded. | DOI:10.1007/s00203-005-0770-0 |
| SimL purified from Streptomyces lividans T7 by Luft et al. | is_reported_recombinant_preparation_of | Native SimL / SimD5 amide synthetase | The abstract describes cloned SimL, S. lividans T7 expression and a near-homogeneous 60-kDa preparation. No complete engineered sequence or tag identity was inspected. | DOI:10.1007/s00203-005-0770-0
|
| SimL purified from Streptomyces lividans T7 by Luft et al. | catalyzes | Luft et al. acid activation and aminocoumarin amide formation | The abstract reports acid activation and transfer to the aminocoumarin amino group without a 4'-phosphopantetheinyl cofactor requirement. | DOI:10.1007/s00203-005-0770-0
|
| Source-assigned simocyclinone C4 acid substrate | is_reported_accepted_acid_substrate_in | Luft et al. acid activation and aminocoumarin amide formation | C4 is explicitly named as an accepted natural substrate. Its exact assayed stereochemical form and reaction product were not independently reconstructed from the abstract. | DOI:10.1007/s00203-005-0770-0
|
| Reported aminocoumarin cosubstrate in the Luft study | is_reported_amine_partner_in | Luft et al. acid activation and aminocoumarin amide formation | The inspected abstract identifies the aminocoumarin amino group, not a fully specified assay reagent; no exact chemical accession is assigned. | DOI:10.1007/s00203-005-0770-0
|
| ATP used in the reported SimL reactions | is_required_in_reported_reaction | Luft et al. acid activation and aminocoumarin amide formation | Acid activation is reported as ATP-dependent. This is not a measured ATP-binding affinity or reconstructed stoichiometric assay. | DOI:10.1007/s00203-005-0770-0
|
| Luft et al. acid activation and aminocoumarin amide formation | supports_source_assignment_of | Source-assigned simocyclinone aminocoumarin amide-ligation step | The biochemical report supports the assigned assembly role; this graph does not assert a native genetic knockout, complete D8 reconstitution, or its final in-vivo reaction order. | DOI:10.1007/s00203-005-0770-0
|
| SimL purified from Escherichia coli by Pacholec et al. | is_reported_recombinant_preparation_of | Native SimL / SimD5 amide synthetase | Pacholec et al. report expression of S. antibioticus SimL and purification from E. coli. The exact construct is unresolved here; it is not equated with Luft or Metzger preparations. | DOI:10.1021/bi047303g
|
| SimL purified from Escherichia coli by Pacholec et al. | catalyzes | Pacholec et al. polyenoic-acid amide-formation assay | The reported activity is amide formation with several polyenoic acids. No numerical rate, substrate preference, or native-D8 product identity is inferred from this abstract. | DOI:10.1021/bi047303g
|
| ATP used in the reported SimL reactions | is_required_in_reported_reaction | Pacholec et al. polyenoic-acid amide-formation assay | The reaction is reported as ATP-dependent; experimental concentrations and kinetic parameters were not inspected. | DOI:10.1021/bi047303g
|
| Reported retinoic-acid substrate | is_reported_acid_substrate_in | Pacholec et al. polyenoic-acid amide-formation assay | Retinoic acid is named in the abstract. Its exact assayed isomer and product structure are not assigned here. | DOI:10.1021/bi047303g
|
| Reported fumagillin substrate | is_reported_acid_substrate_in | Pacholec et al. polyenoic-acid amide-formation assay | Fumagillin is named in the abstract. This surrogate reaction does not show its involvement in native simocyclinone biosynthesis. | DOI:10.1021/bi047303g
|
| Pacholec et al. polyenoic-acid amide-formation assay | supplies_a_reported_product_for | Engineered novobiocin-analogue tailoring of a SimL product | NovM, NovP and NovN from the novobiocin pathway were used to tailor a SimL product into an analogue with a tetraenoate moiety replacing the usual prenylhydroxybenzoate. The inspected abstract does not uniquely map this product to either named acid above. These three enzymes are not assigned native D8 roles. | DOI:10.1021/bi047303g
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 9 nodes, 9 edges
Primary Metzger thesis experiments characterize a distinct pUM06-derived SimL preparation. N-terminal His8-tag removal leaves a reported cloning scar; neither recombinant state is exact native Q9F5J5. HPLC specific activity on prenylhydroxybenzoate and a methylated aminocoumarin is a surrogate-substrate result, not native C4 turnover or D8 reconstitution. Literal primers, fragment sizes, purification data and the SimL-specific gel were checked; review #540 limitations remain.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Reported SimL expression construct pUM06 | is_reported_to_express | pUM06-derived N-terminal His8-tagged SimL | Metzger cloned simL from pSH2 by PCR into pGEM-T as pUM01, then subcloned its reported 1560-bp NcoI-BglII fragment into pHis8 as pUM06. The reported PCR length is 1569 bp. Construct sequencing was reported. Expression used E. coli BL21(DE3)pLysS, 0.5 mM IPTG and 6 h at 20 C after growth to OD600 0.6. This is a later, distinct preparation, not the full construct from either 2005 study. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
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| pUM06-derived N-terminal His8-tagged SimL | has_native_sequence_core_from | Native SimL / SimD5 amide synthetase | Native SimL has 519 residues. The literal forward primer GACCATGGAGGCAACGAGCACTACGTCCGG and reverse primer GAGATCTCATTCGCCATGCGTGGCCTCCCG reconstruct a 1569-bp product with NcoI and BglII top-strand cuts at 4 and 1564, a 1560-bp cut-to-cut span. The reverse primer changes native CDS nt1548 C to G (ACC to ACG at Thr516), without changing the amino acid. The BglII site overlaps the final base of the retained native stop. The in-frame insert core preserves native residues3-519. This local reconstruction is not an independent complete-plasmid sequence or verification of every tag/vector junction. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf |
| pUM06-derived N-terminal His8-tagged SimL | is_processed_to | Thrombin-cleaved SimL retaining the reported N-terminal scar | His-affinity purification is followed by thrombin cleavage (0.1 U/mg, 15 h at 4 C), removal of thrombin and tag, and Superdex200 purification. The source reports post-cleavage GSHGGN... for SimL; native protein instead begins MEGNEH.... Tag removal therefore does not restore exact native sequence. No complete experimental protein sequence is assigned to either state. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Thrombin-cleaved SimL retaining the reported N-terminal scar | has_measured_specific_activity_in | Metzger SimL surrogate-substrate HPLC assay | Table3.5 reports 1.0 nkat/mg for the purified SimL preparation with 3-dimethylallyl-4-hydroxybenzoic acid and the methylated aminocoumarin. Methods specify a 100-uL reaction, 50 mM Tris-HCl pH8.0, 5 mM MgCl2 and 15 min at 30 C. The cited assay paragraph does not specify the SimL enzyme loading, and this table supplies no replicate count or uncertainty. This is a reported specific activity, not kcat/Km or an independently reconstructed initial rate. The acid is a surrogate, not simocyclinone C4. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Thrombin-cleaved SimL retaining the reported N-terminal scar | is_characterized_as_the_preparation_used_in | Metzger SimL surrogate-substrate HPLC assay | Table3.5 reports 20 mg protein per 1 L culture and final concentration 19.5 mg/mL, not assay enzyme loading. Mass-specific extinction coefficients are 0.91 with tag and 0.94 without tag L/g/cm; the methods use sequence-derived coefficients and A280. Figure3.23 shows the SimL final preparation, 15 ug loaded, and gives an expected mass of 55.3 kDa, not a precise measured mass. Figure3.22, SeMet yields/activity and the subsequently obtained crystals concern NovL, not SimL. The thesis reports no SimL crystals in its own screening; no global structural-data absence or equivalence with the separately reported 60-kDa preparation is claimed. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| 3-Dimethylallyl-4-hydroxybenzoic acid assay substrate | is_acid_substrate_in | Metzger SimL surrogate-substrate HPLC assay | The method uses 1 mM acid; results specifically identify 3-dimethylallyl-4-hydroxybenzoic acid for SimL. This experimental substrate is not relabelled as the native simocyclinone acyl moiety. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| 3-Amino-4,7-dihydroxy-8-methylcoumarin assay substrate | is_amine_substrate_in | Metzger SimL surrogate-substrate HPLC assay | The assay uses 1 mM 3-amino-4,7-dihydroxy-8-methylcoumarin. This specified surrogate is not silently equated with the aminocoumarin reagent in the abstract-only Luft experiment. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| ATP used in the Metzger amide-synthetase assay | is_assay_reagent_in | Metzger SimL surrogate-substrate HPLC assay | The method uses 1 mM ATP with 5 mM MgCl2. Including ATP in this assay is not itself a no-ATP control or metal-selectivity test. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
| Novobiocic acid HPLC standard | is_quantification_standard_for | Metzger SimL surrogate-substrate HPLC assay | The method specifies 1 mM novobiocic acid as standard. Reactions are stopped with 5 uL 1.5 M trichloroacetic acid, ethyl-acetate extracted and analyzed by Eclipse XDB-C18 HPLC at 330 nm, 1 mL/min, with the Table2.7 methanol/water gradient containing 1% formic acid. The use of this standard does not assign an exact chemical identifier to every assay product or to native D8. | https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/49469/1/pdf/Diss_Metzger_gesamt.pdf
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 10 nodes, 10 edges
Native-producer SimB3 insertion genetics reported by Trefzer et al. support involvement in simocyclinone biosynthesis, but not a purified 2,3-dehydratase reaction or exclusion of polar effects. The two deposited 482-aa proteins differ at residue 134 and remain distinct; neither accession denotes the engineered Sim-C2 allele. Reported internal-fragment size, the uncorrected degenerate-primer discrepancy, HPLC detection limits and source-assigned A1 identity are retained (review #541). This is separate from the paper's multigene Sim-PKSII insertion and does not adopt its tetraene-pathway hypotheses.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Native simB3 locus targeted in the Trefzer study | encodes_deposited_native_protein | Deposited SimB3, putative sugar 2,3-dehydratase (Leu134) | The 1449-nt CDS including stop at AF324838.2:71408..72856(+) encodes AAK06810.1. CDS-aware table-11 translation and deposited translation exactly match the 482-aa UniProt Q9AMH2 sequence. This is the source-linked native sequence, not a sequence of the disrupted allele or a purified experimental protein. | https://www.ncbi.nlm.nih.gov/nuccore/AF324838.2 |
| Alternate deposited Sim20 / SimB3 protein (Pro134) | differs_in_deposited_sequence_from | Deposited SimB3, putative sugar 2,3-dehydratase (Leu134) | AF322256.1:26613..28061(+) encodes 482-aa AAL15606.1 / Q93F99 (gene sim20). Exact CDS-aware translation agrees with Q93F99, but comparison to Q9AMH2 gives Leu134 versus Pro134. CDS positions 401/402 change CTG to CCT; position 243 changes CGG to CGC synonymously at Arg81. Both deposits name Tu6040 and taxon 1890. The discrepancy is not resolved as natural polymorphism, sequencing error or a measured functional difference, and the two proteins are not merged as identical examples. | https://www.ncbi.nlm.nih.gov/nuccore/AF322256.1 |
| pSP1-C2 internal-fragment insertion construct | contains_reported_internal_fragment_of | Native simB3 locus targeted in the Trefzer study | Methods report a 0.8-kb internal simB3 PCR fragment (Figure 2 fragment A) ligated into the singular XbaI site of erythromycin resistance vector pSP1. The exact insert and whole plasmid were not independently sequenced here. The gene-discovery primers as printed are PCR-D1a CAGGCSACSWSSAACTACAC and PCR-D1b SWRGAASCGSCCSCCCTCCT (R=A/G, W=A/T, S=C/G). The former has no exact IUPAC-compatible match on either strand of AF324838.2; the latter maps uniquely to 72666..72685(-). No primer is silently repaired and no exact amplicon length or failed experimental amplification is inferred. | DOI:10.1128/AAC.46.5.1174-1182.2002 |
| S. antibioticus Tu6040 insertion mutant Sim-C2 | has_reported_chromosomal_integration_of | pSP1-C2 internal-fragment insertion construct | Unmethylated plasmid was prepared in E. coli ET12567 and introduced into native-producer protoplasts; erythromycin selection and PCR supported integration. Sim-C2 is an insertional disruption, not a clean whole-gene deletion. The inspected article does not report SimB3 complementation, a complete sequenced mutant allele or exclusion of polar effects. | DOI:10.1128/AAC.46.5.1174-1182.2002
|
| S. antibioticus Tu6040 wild-type comparator | is_reported_production_comparator_for | S. antibioticus Tu6040 insertion mutant Sim-C2 | Figure 3 compares culture extracts from wild type and Sim-C2. The methods describe 2% mannitol / 2% soybean meal, pH 7.5, 28 C, with 20 mL liquid culture in a single-baffled 100-mL Erlenmeyer flask; mutant maintenance uses 50 ug/mL erythromycin. Production, extraction and HPLC details are referred to earlier publications. The shown comparison is not a matched empty-vector strain or a purified substrate-turnover experiment. | DOI:10.1128/AAC.46.5.1174-1182.2002
|
| S. antibioticus Tu6040 wild-type comparator | has_assay_phenotype | Wild-type D8 peak detected by HPLC-UV/Vis | The wild-type culture extract has a source-assigned D8 peak, retention time 10.8 min, with the displayed UV/visible spectrum. This is a qualitative HPLC-UV/Vis comparison, not a reported absolute titer, mass-spectrometric identification or independent resolution of the owner's six unspecified stereocentres. | DOI:10.1128/AAC.46.5.1174-1182.2002
|
| S. antibioticus Tu6040 insertion mutant Sim-C2 | has_assay_phenotype | D8 not detected in Sim-C2 by HPLC-UV/Vis | D8 was not detected in the Sim-C2 culture extract under the reported HPLC-UV/Vis conditions. No numerical detection limit, replicate count, uncertainty, fold change or absolute zero is assigned from this figure. Loss of the product signal does not isolate a single enzyme reaction or distinguish the disrupted gene's effect from possible effects on neighboring expression. | DOI:10.1128/AAC.46.5.1174-1182.2002
|
| S. antibioticus Tu6040 insertion mutant Sim-C2 | has_assay_phenotype | Source-assigned simocyclinone A1 accumulation in Sim-C2 | The source assigns the accumulated 5.8-min HPLC-UV/Vis peak to simocyclinone A1. This graph preserves that historical assignment as a phenotype; it does not mint an exact chemical accession, infer all A1 stereochemistry, or call the extract a pure compound. Figure 3 does not provide an absolute A1 titer. | DOI:10.1128/AAC.46.5.1174-1182.2002
|
| Deposited SimB3, putative sugar 2,3-dehydratase (Leu134) | is_proposed_to_participate_in | Inferred SimB3 contribution to dNDP-D-olivose biosynthesis | The source proposes a dNDP-4-keto-6-deoxyglucose 2,3-dehydratase role from sequence similarity and the deoxysugar-pathway model. Table 1 compares SpnO (AAG23276, 54% identity), which is a homolog, not the native SimB3 accession. Genetic production effects do not establish purified catalysis, substrate specificity or kinetics. | DOI:10.1128/AAC.46.5.1174-1182.2002
|
| Source-assigned simocyclinone A1 accumulation in Sim-C2 | is_consistent_with_source_proposal_of | Inferred SimB3 contribution to dNDP-D-olivose biosynthesis | The authors interpret A1 accumulation together with loss of the D8 signal as disruption of deoxysugar assembly while angucycline production persists. This is an inference from metabolite profiles, not a direct measurement proving absence of every dTDP-D-olivose molecule or a SimB3-specific catalytic block independent of insertion effects. | DOI:10.1128/AAC.46.5.1174-1182.2002
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 8 nodes, 8 edges
Native S. antibioticus Tu6040 SimC1B KS-region replacement and retained source-assigned D8 production, from the Bilyk et al. primary supporting information. G9VYV9 denotes the native protein, not the engineered allele. The primer-site/prose conflict and qualitative genotype and metabolite readouts are explicit (review #542). This is not a clean whole-gene deletion, purified catalysis or proof that every SimC1B domain is dispensable under all conditions. The historical type-I-PKS tetraene-assembly assignment is not adopted as an established function. The full main article was not obtained.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Native simC1B locus | encodes_native_protein | Native SimC1B modular polyketide synthase | The 6597-nt CDS including stop at AF324838.2:15910..22506(+) encodes AEU17897.1. Its CDS-aware table-11 translation and deposited translation exactly match the 2198-aa G9VYV9 sequence. The native producer is S. antibioticus Tu6040 (Tue6040 in the deposit), taxon 1890. This grounding does not identify the sequence or expression state of any engineered protein. | https://www.ncbi.nlm.nih.gov/nuccore/AF324838.2 |
| pKGKSdelta KS-region replacement construct | was_designed_to_replace_KS_encoding_region_of | Native simC1B locus | Methods p.33 describe two 2.5-kb homology arms fused by overlap PCR in pJET1.2, an hph cassette flanked by loxP, and transfer to pKG1139. They call the central site SspI and external sites StuI. The literal primer table on p.41 instead labels and contains central EcoRV (GATATC) and external SspI (AATATT), with no StuI (AGGCCT) site in these four primers. The table is headed S5 but listed as S6 in the contents. Neither the source text nor primers are silently corrected. No complete plasmid or inserted hph/loxP sequence was independently audited. | DOI:10.1021/acschembio.5b00669.s001
|
| S. antibioticus Tu6040 deltaKS mutant | was_generated_using | pKGKSdelta KS-region replacement construct | The supplement reports screening for loss of vector resistance after double crossover and confirmation of intragenic deletions by PCR. Figure S8a specifically maps the Tu6040 deltaKS replacement. The hph/loxP cassette remains shown; no Cre-excised scar-only allele or complete experimental mutant sequence is asserted. The inspected supplement does not show SimC1B complementation or exclude all secondary genomic effects. The nearby smcKSII complementation experiments are in a different producer and are not SimC1B rescue tests. | DOI:10.1021/acschembio.5b00669.s001
|
| S. antibioticus Tu6040 deltaKS mutant | has_reported_KS_region_replacement_in | Native simC1B locus | Figure S8a replaces the KS-encoding segment with hph/loxP while retaining the region labelled AT, DH, ER and KR; this is not deletion of the full simC1B gene. Literal primers are KS_R_fus-f TTCGGCAGTATCTGAAGAAGGATATCGCGCAGCCCGACGCACCGGG; KS_F_fus-r CCCGGTGCGTCGGGCTGCGCGATATCCTTCTTCAGATACTGCCGAA; KS_R-SspI-r ACTAATATTCCGCCCCATGGGTCAGGACGA; and KS_F-SspI-f ACTAATATTGGTCTGGCCGGGACGACGCTC. Separating their artificial sites gives native arms 13410..15909 and 17410..19909 of AF324838.2, each 2500 nt, surrounding 15910..17409 (the first 1500 CDS nt / 500 aa). This is a primer-derived model, not a sequenced allele or a measured domain boundary. The reverse inner primer has an incidental 21-nt native suffix crossing its artificial-site boundary; that match does not change the 20-nt designed arm. No exact mutant protein, folding state or residual catalytic activity is inferred from the replacement diagram. | DOI:10.1021/acschembio.5b00669.s001 |
| S. antibioticus Tu6040 wild-type Southern comparator | is_hybridization_comparator_for | S. antibioticus Tu6040 deltaKS mutant | Figure S8aB compares wild-type and deltaKS chromosomal DNA with an hph probe, alongside a DNA ladder and the pKGKSdelta cassette control. No corresponding hph band is visible in the wild-type lane. This comparator concerns hybridization, not matched fermentation yield or protein abundance; it does not establish absence of every unintended mutation. | DOI:10.1021/acschembio.5b00669.s001
|
| S. antibioticus Tu6040 deltaKS mutant | has_genotyping_readout | Mutant hph hybridization near the predicted 5.9-kb position | The source marks the mutant hph-hybridizing band near a predicted 5.9-kb fragment position. The value is reported from the figure/caption, not independently reconstructed from a specified restriction digest or fully sequenced junctions. Band appearance is not an absolute copy-number measurement or proof of whole-genome specificity. | DOI:10.1021/acschembio.5b00669.s001
|
| S. antibioticus Tu6040 deltaKS mutant | retains_source_assigned_product_signal | Source-assigned SD8 signal in deltaKS crude extract | Figure S8b shows the mutant crude-extract HPLC-ESI/MS chromatogram, with SD8 labelled m/z 930.65 [M-H]-. The displayed trace has an mAU intensity axis; the source ion assignment is retained without silently converting the number to an accurate-mass measurement. D8 detection after this intervention argues against a strict requirement for the targeted intact KS region under the assayed conditions. It does not establish unchanged wild-type yield, absence of all SimC1B functions, or the alternative enzymatic route. No absolute titer, replicate count, uncertainty or exact retention time is supplied. Main-article fermentation conditions were not obtained; Kitasatospora culture conditions and the supplement's printed mg/mL conjugation selection values are not turned into a validated Tu6040 production protocol. The owner's six unspecified stereocentres remain unresolved. | DOI:10.1021/acschembio.5b00669.s001 |
| S. antibioticus Tu6040 deltaKS mutant | has_source_assigned_product_signal | Source-assigned SD6 signal in deltaKS crude extract | The same Figure S8b labels a second signal as SD6, m/z 890.37 [M+H]+. Its positive-ion annotation is kept distinct from the negative-ion SD8 annotation. This is a source assignment in a crude extract, not a new exact chemical record, independently confirmed stereochemistry, absolute yield or measured SD6-to-SD8 conversion. Relative peak heights do not establish molar product ratios. | DOI:10.1021/acschembio.5b00669.s001
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 11 nodes, 12 edges
SimH/SimY tyrosine-adenylation evidence for the simocyclinone D8 aminocoumarin branch, from Boll et al. 2011. Native Q9F5J4 and Q9F5J2, alternate Q9AMH5, recombinant preparations and assay readouts remain distinct (review #543). The source length and tag-state limits are preserved. ATP-PPi exchange is not direct PCP loading, complete D8 production or proof of physiological flux. The inspected primary text is the author-reproduced manuscript and supplement in the public thesis, not independently retrieved final journal full text.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Purified His6-SimH from the ybdZ-deletion expression host | was_prepared_from_native_sequence_named_as | Native SimH tyrosine-adenylating enzyme | The paper explicitly identifies AAL15600 (997 aa). AAL15600.1 from AF322256.1:19223..22216(+) and AAG34184.1 from AF321122.1:2435..5428(+) both translate exactly to Q9F5J4 using CDS-aware table 11. The producer is S. antibioticus Tu6040, taxon 1890. pBB25 expresses an N-terminal His6 fusion in E. coli BL21(DE3) with a ybdZ deletion and pSU20_sfp (B. subtilis Sfp). Sfp is preparation context, not a measured PCP-loading fraction. The N-terminal tag was retained. No native accession is assigned to the full recombinant protein, whose vector junctions were not independently sequenced. Literal primers GGGAATTCCATATGGCCATGCCATCCGGCA and GCCCTCGAGTCACTTCACGGCCGTTGTGG predict a 3014-nt amplicon containing the 2994-nt CDS with its stop; this is a reconstruction, not a measured complete plasmid. The source reports checking entire gene sequences. Induction was 0.4 mM IPTG for 14 h at 20 C after OD600 0.6. Ni-affinity and gel filtration gave 9.4 mg SimH per liter culture from the deletion host; this is not assay loading or a reported ybdZ+ yield. The source assigns no established biosynthetic function to the additional N-terminal condensation-like region. | DOI:10.1074/jbc.M111.288092 |
| Alternate deposited SimD6 sequence | differs_in_deposited_sequence_from | Native SimH tyrosine-adenylating enzyme | Q9AMH5 exactly matches the 1010-aa AAK06804.1 translation of AF324838.2:63978..67010(+), gene simD6. Compared with paper-linked Q9F5J4, it contains the 13-aa N-terminal extension MANESPVMTLENN and two aligned differences: His274 versus Tyr287 and Asp396 versus His409 (short/long numbering). The cause of these deposited differences is unresolved. The SimH forward primer also matches internally at 64017 of AF324838.2; that predicted 997-aa product retains both residue differences. Primers alone do not choose the assay allele. The paper explicitly names AAL15600; its measured kinetics are not transferred to alternate Q9AMH5. | https://www.ncbi.nlm.nih.gov/nuccore/AF324838.2 |
| Purified recombinant SimY with reported tag removal | was_prepared_from_native_sequence_named_as | Native SimY MbtH-like protein | The paper names AAG34186 but reports 69 aa. AAG34186.1 at AF321122.1:6727..6939(+), AAL15602.1 at AF322256.1:23515..23727(+) and AAK06806.1 at AF324838.2:68309..68521(+) all have 213-nt CDSs including stop and translate exactly to the 70-aa Q9F5J2 sequence. The 69/70-aa discrepancy is unresolved; initiator removal or an error is not assumed. pBB28 in E. coli Rosetta2(DE3) initially produced N-His6-SimY. General methods and results report MbtH-tag removal with 0.4 units thrombin/mg protein for 8 h at 4 C before gel filtration, yet Figure S2 labels N-His6-SimY and a calculated 9.4-kDa mass. The gel may depict a different purification stage; it does not identify the functional-assay tag state. No exact cleavage scar or complete assay sequence is independently verified. Primers GGGAATTCCATATGGCCAACCCGTTTGACGA and GCCCTCGAGTCAGCTGGGGTCCGTCG predict a 233-nt amplicon retaining the native coding start and stop; this is not a sequenced plasmid. The reported yield is 27 mg/liter culture, not an assay concentration. The native accession is not assigned to the recombinant preparation. | DOI:10.1074/jbc.M111.288092 |
| Reconstituted His6-SimH plus prepared SimY reaction | contains_prepared_enzyme | Purified His6-SimH from the ybdZ-deletion expression host | Standard 100-uL reactions contained 1 uM tyrosine-activating enzyme, 95 mM Tris-HCl pH 8, 5 mM MgCl2, 5 mM TCEP, 2 mM ATP, 1.5 mM L-tyrosine and 1 mM [32P]PPi. These are assay conditions, not protein-expression concentrations. | DOI:10.1074/jbc.M111.288092 |
| Reconstituted His6-SimH plus prepared SimY reaction | contains_prepared_helper | Purified recombinant SimY with reported tag removal | SimY was added at 1.2 uM in the standard assay, giving SimH:SimY 1:1.2. Figure 4 instead varies the helper amount; the standard ratio is not imposed on its titration or on the analytical gel-filtration load. | DOI:10.1074/jbc.M111.288092 |
| Purified His6-SimH from the ybdZ-deletion expression host | has_low_unassisted_assay_readout | SimH-alone activity at most 0.03 nkat/mg protein | Figure 3 explicitly labels SimH-alone <=0.03 nkat/mg protein. This bound is neither zero nor an exact 0.03 measurement. The caption reports means of two independent reactions for SimH; the six-reaction exception concerns CloH. No precise SimH-plus-SimY specific activity is digitized from the bar height. The pooled 99.0-99.8% discussion range is not assigned specifically to SimH. | DOI:10.1074/jbc.M111.288092 |
| Purified recombinant SimY with reported tag removal | has_helper_only_control_readout | No activity reported for MbtH-only controls | The results report no activity in the controls containing MbtH-like proteins alone. No numerical detection limit or SimY-alone kinetic fit is supplied; this is an assay control, not proof that SimY has no function. | DOI:10.1074/jbc.M111.288092 |
| Reconstituted His6-SimH plus prepared SimY reaction | supports_measured_adenylation_assay | L-tyrosine-dependent ATP-PPi exchange | Adding SimY made the SimH adenylation signal readily detectable. Reactions ran for 5 min at 30 C, initiated by enzyme addition; charcoal isolation and scintillation measured radiolabel exchange. The assay supports the adenylation step, not direct covalent tyrosyl-PCP loading or synthesis of a complete aminocoumarin or D8 molecule. | DOI:10.1074/jbc.M111.288092 |
| Reconstituted His6-SimH plus prepared SimY reaction | has_reported_kinetic_readout | SimH plus SimY L-tyrosine kinetic parameters | At SimH:SimY 1:1.2, varying L-tyrosine and nonlinear regression in Prism 5 gave Km 164 uM in Table 1 (rounded), kcat 1.01 s^-1 and kcat/Km 6180 M^-1 s^-1. Figure S3 prints Km 163.5 +/- 2.6 uM; the inspected text/caption does not define that uncertainty as SD or SE. These are reported parameters for the prepared mixture under the exchange-assay conditions, not intrinsic constants of free native SimH or a D8 pathway flux. No kcat uncertainty is invented. Very low unassisted activity prevented a SimH-alone kinetic investigation. | DOI:10.1074/jbc.M111.288092 |
| Purified His6-SimH from the ybdZ+ expression host | was_prepared_from_native_sequence_named_as | Native SimH tyrosine-adenylating enzyme | The same pBB25 construct and pSU20_sfp were used in BL21(DE3) with intact ybdZ for a separate preparation. It is not the deletion-host preparation and is not assigned the native accession. No exact sequence of a copurifying helper or quantitative SimH ybdZ+ purification yield is provided by the inspected results. | DOI:10.1074/jbc.M111.288092 |
| Purified His6-SimH from the ybdZ+ expression host | did_not_show_more_unassisted_activity_than | Purified His6-SimH from the ybdZ-deletion expression host | The reported host comparison found no increase in SimH activity from intact-ybdZ hosts. It does not prove that YbdZ cannot bind SimH or quantify its occupancy. The 20-30% unassisted activity and suggested copurification for CloH/Pcza361.18 are not SimH measurements. | DOI:10.1074/jbc.M111.288092 |
| Purified recombinant SimY with reported tag removal | stimulates_in_vitro_adenylation_by | Purified His6-SimH from the ybdZ-deletion expression host | The paired absence/presence comparison supports stimulation by prepared SimY. This functional comparison alone does not prove a physical interface, absolute in-vivo requirement or unique specificity for SimY. The separate cross-activation experiment reports other MbtH-like proteins can stimulate SimH; those heterologous helpers are not asserted here as native D8 biosynthetic partners. | DOI:10.1074/jbc.M111.288092 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 10 nodes, 11 edges
Association of separately prepared recombinant SimH and SimY, with apparent gel-filtration masses and an activity titration supporting a proposed heterotetramer (review #543). The observed co-eluting complex is not assigned a subunit accession or a verified native in-vivo stoichiometry. Source-reported tag removal and the different Figure S2 label remain explicit. The public primary author manuscript and supplement were inspected; final journal full text was not independently retrieved.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Purified His6-SimH used in association experiments | was_prepared_from_native_sequence_named_as | Native SimH tyrosine-adenylating enzyme | The source links SimH to AAL15600, whose 997-aa native sequence matches Q9F5J4 and the independent AAG34184.1 deposit. The protein used in association experiments is a recombinant N-His6 preparation, not the accession-defined native sequence. General methods describe pBB25 in BL21(DE3) with or without ybdZ deletion and with pSU20_sfp; the analytical SEC section does not separately identify which host preparation supplied that experiment. It is not silently assigned the deletion-host provenance of Figure 3. Entire gene sequences were reported checked by the authors, but complete experimental vector and tag junctions were not independently obtained. The literal primers cannot distinguish paper-linked SimH from the two-residue-different shortened product of alternate simD6/AAK06804.1. The explicit paper accession supports the selected native grounding, not a claim to have sequenced the complete assayed protein. | DOI:10.1074/jbc.M111.288092 |
| Purified recombinant SimY with reported tag removal | was_prepared_from_native_sequence_named_as | Native SimY MbtH-like protein | The paper names AAG34186 but reports 69 aa. AAG34186.1 at AF321122.1:6727..6939(+), AAL15602.1 at AF322256.1:23515..23727(+) and AAK06806.1 at AF324838.2:68309..68521(+) all have 213-nt CDSs including stop and translate exactly to the 70-aa Q9F5J2 sequence. The 69/70-aa discrepancy is unresolved; initiator removal or an error is not assumed. pBB28 in E. coli Rosetta2(DE3) initially produced N-His6-SimY. General methods and results report MbtH-tag removal with 0.4 units thrombin/mg protein for 8 h at 4 C before gel filtration, yet Figure S2 labels N-His6-SimY and a calculated 9.4-kDa mass. The gel may depict a different purification stage; it does not identify the functional-assay tag state. No exact cleavage scar or complete assay sequence is independently verified. Primers GGGAATTCCATATGGCCAACCCGTTTGACGA and GCCCTCGAGTCAGCTGGGGTCCGTCG predict a 233-nt amplicon retaining the native coding start and stop; this is not a sequenced plasmid. The reported yield is 27 mg/liter culture, not an assay concentration. The native accession is not assigned to the recombinant preparation. | DOI:10.1074/jbc.M111.288092 |
| Purified His6-SimH used in association experiments | is_a_component_of_observed_fraction | Observed co-eluting recombinant SimH/SimY complex | Analytical gel filtration of a mixture and SDS-PAGE of the isolated approximately 208-kDa fraction support association of the recombinant proteins. This is not a structure-based interface determination or native-cell oligomer measurement. The SimH native accession is not an identifier for the whole complex. | DOI:10.1074/jbc.M111.288092 |
| Purified recombinant SimY with reported tag removal | is_a_component_of_observed_fraction | Observed co-eluting recombinant SimH/SimY complex | A SimY band accompanies SimH in the isolated fraction. General methods report tag cleavage; the N-His6-SimY label in Figure S2 does not independently establish the exact SimY sequence used in this fraction. The protein complex is left ungrounded rather than assigned a subunit accession or an unverified ComplexPortal record. | DOI:10.1074/jbc.M111.288092 |
| Purified His6-SimH used in association experiments | has_analytical_gel_filtration_readout | Dominant apparent 650-kDa SimH-alone aggregate peak | SimH alone eluted predominantly near an apparent mass of 650 kDa, interpreted by the source as aggregation. The source gives 107.3 kDa as the calculated tagged monomer mass; neither value is a measured native monomer mass or an exact number of aggregate subunits. Analytical SEC used a Superdex 200 10/300 GL, 24-mL bed, 20 mM Tris-HCl pH 8 and 150 mM NaCl with log-mass/Kav calibration standards. | DOI:10.1074/jbc.M111.288092 |
| Observed co-eluting recombinant SimH/SimY complex | has_mixture_gel_filtration_readout | Dominant apparent 208-kDa SimH/SimY mixture peak | The SimH/SimY mixture has a dominant approximately 208-kDa peak by calibrated SEC. Figure 5A also shows residual approximately 650-kDa and <=15-kDa signals; not all material became one homogeneous complex. These are hydrodynamic apparent masses. The inspected SEC method does not specify the sample loading concentration, injection volume or mixture ratio; the separate 1:1.2 activity-assay ratio is not substituted for a missing SEC loading ratio. | DOI:10.1074/jbc.M111.288092 |
| Observed co-eluting recombinant SimH/SimY complex | has_isolated_fraction_SDS_PAGE_readout | SimH and SimY bands in the isolated 208-kDa fraction | SDS-PAGE of the isolated 208-kDa peak shows bands assigned to SimH and SimY. Band presence supports composition and co-elution, not an exact molar ratio from staining intensity, a unique oligomer size or a mapped binding interface. | DOI:10.1074/jbc.M111.288092 |
| Purified His6-SimH used in association experiments | has_helper_titration_response | Activity plateau at a 1:1 SimY:SimH molar ratio | The L-tyrosine adenylation activity increased as SimY was added and plateaued at approximately equimolar SimY:SimH. Figure 4 measures activity, not directly bound subunits. No dissociation constant, Hill coefficient or binding occupancy is inferred from this curve. | DOI:10.1074/jbc.M111.288092 |
| Purified recombinant SimY with reported tag removal | was_varied_in_activity_titration | Activity plateau at a 1:1 SimY:SimH molar ratio | Different SimY amounts were assayed with SimH. Further helper beyond the approximately 1:1 ratio did not increase activity in the displayed experiment. This does not prove a heterodimer, and it is not the distinct CloH/CloY pETDUET coexpression experiment. | DOI:10.1074/jbc.M111.288092 |
| Dominant apparent 208-kDa SimH/SimY mixture peak | is_consistent_with_source_proposed_model | Source-proposed (SimH)2(SimY)2 heterotetramer | The authors compare the apparent 208-kDa complex with a calculated 229-kDa (SimH)2(SimY)2 assembly. The 229-kDa value is the source calculation, not independently verified from complete assayed tag/cleavage sequences. SEC alone does not directly count subunits or exclude alternative shapes or oligomers. This remains the source-supported model, not an experimentally solved native structure. | DOI:10.1074/jbc.M111.288092 |
| Activity plateau at a 1:1 SimY:SimH molar ratio | supports_relative_composition_in_proposed_model | Source-proposed (SimH)2(SimY)2 heterotetramer | The activity plateau is compatible with equal relative amounts of helper and adenylating enzyme. Combined with SEC it motivated the 2:2 proposal; it does not uniquely determine that stoichiometry or establish which oligomer exists in the native producer. | DOI:10.1074/jbc.M111.288092 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 20 nodes, 26 edges
SimJ1/SimD2 native genetics, methyl-aminocoumarin feeding and the confounded double-crossover follow-up from Ute Galm's 2003 primary thesis (review #544). Native Q9F5J1 is distinct from engineered alleles; single crossovers retain differently arranged intact and deleted copies. D-met feeding rescue is not restored chlorinated D8. Qualitative product assignments, the restriction-size and culture-fraction discrepancies, and source-inferred catalysis remain explicit. The related 2002 journal paper was not independently obtained in full; thesis-only results are cited to the thesis. PDF page numbers are four greater than printed page numbers.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Native simJ1 locus, also named simD2 | encodes_native_protein | Native SimJ1 (SimD2) protein | AAL15603.1 at AF322256.1:23738..24559(+), AAG34187.1 at AF321122.1:6950..7771(+) and AAK06807.1 at AF324838.2:68532..69353(+) each have an 822-nt CDS including stop and translate exactly to the 273-aa Q9F5J1 sequence using CDS-aware table 11. The last deposit calls the gene simD2; all name S. antibioticus Tu6040/Tue6040, taxon 1890. This is not the distinct 246-aa SimJ2/SimC6 protein, and the native accession does not identify an engineered allele. Earlier SimReg1 graphs mention partial simD2 flanks in constructs, not an independently assayed SimJ1 protein. | https://www.ncbi.nlm.nih.gov/nuccore/AF322256.1 |
| pUG013 internal simJ1-deletion construct | was_designed_for_internal_deletion_of | Native simJ1 locus, also named simD2 | Cosmid VII-8g arms were cloned XbaI/BamHI then BamHI/EcoRI into pBSKT to form pUG012; its approximately 2.8-kb XbaI/EcoRI insert was transferred to pKC1132, yielding pUG013 with apramycin selection. The source reports an internal in-frame 756-bp deletion, not whole-gene removal. Literal primers are P01f GTACGGCGGTCTAGAAGGAC, P01r CATTCGGGATCCTTCCCTGC, P02f CGACGGCGGATCCCAGTGAA and P02r GGCCACCGAATTCGGCCACA. Their declared native coordinates and introduced sites reproduce 1392- and 1434-nt PCR products. A BamHI fusion model loses 756 nt net and leaves a 66-nt CDS including stop, predicting a 21-aa remnant (MNQPWPATRADRPNICREGSQ). This model is not a sequenced experimental allele, verified expression product or full plasmid. The single-crossover strains retain both copies in different arrangements. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1 |
| S. antibioticus UG J1-15 single-crossover strain | was_generated_with | pUG013 internal simJ1-deletion construct | PEG-mediated protoplast transformation and apramycin selection yielded UG J1-15 as a single-crossover isolate. The source reports pUG013 integration by homologous recombination; pUG012/thiostrepton selection instead gave many spontaneous-resistant colonies and is not equivalent evidence of integration. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| S. antibioticus UG J1-21 single-crossover strain | was_generated_with | pUG013 internal simJ1-deletion construct | UG J1-21 is another pUG013 single-crossover arrangement, not a clean simJ1 deletion. The source map retains an intact copy separated from the putative native transcriptional unit by vector and resistance-gene DNA. Its inferred transcriptional silencing is not a measured RNA or protein abundance. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| S. antibioticus UG J1-15 single-crossover strain | has_reported_genotyping_readout | Southern discrimination of single-crossover arrangements | Figure 5 places the intact copy at the end of the proposed simKLHIYJ1 unit in UG J1-15. BglII/NotI and PvuII/EcoRI Southern patterns support the source-assigned arrangement. Native AF322256.1 fragment lengths 3788 and 3893 bp agree with the displayed WT 3.79/3.89-kb rounding. The source reports a 1242-bp downstream probe (1.2 kb in methods), consistent with the primer-derived BamHI/EcoRV interval. No complete single-crossover vector junction was sequenced in this independent audit. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1 |
| S. antibioticus UG J1-21 single-crossover strain | has_reported_genotyping_readout | Southern discrimination of single-crossover arrangements | Figure 5 instead places the deleted copy adjacent to the putative native transcriptional unit in UG J1-21. The results report BglII/EcoRV bands at 2.77 and 1.97 kb (not pictured there). The deposited native interval is 2729 bp, not 2770 bp; the intended 756-nt deletion model gives 1973 bp. This source-versus-native discrepancy is unresolved. Figure 7 later uses coarse 2.8/2.0-kb labels. Reported bands, reconstructed lengths and exact experimental allele sequences are not interchangeable. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1 |
| Original S. antibioticus Tu6040 wild type | has_source_assigned_product_profile | Source-assigned D4/D8 chromatographic profile | Figure 6A shows D4 and D8 signals in the original WT; Figure 6B labels [M-H]- assignments of 896.2 and 930.4, respectively. These are source assignments, not independently verified accurate masses or complete stereochemical determinations. Section 9.1.2 specifies acidifying 50 mL whole culture to pH 4, extracting twice with 50 mL ethyl acetate and reconstituting in 5 mL methanol. The results call the analyzed material culture filtrate; this fraction discrepancy remains unresolved. The specific simocyclinone HPLC used a 0.1% phosphoric acid/acetonitrile gradient at 2 mL/min, not the preceding generic 1 mL/min RP-18 method. No absolute yield, detection threshold or replicate count is invented. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| S. antibioticus UG J1-15 single-crossover strain | retains_source_assigned_product_profile | Source-assigned D4/D8 chromatographic profile | UG J1-15 produces the same source-assigned D-series identities as WT, D4 and D8. Figure 6 uses different intensity scales across panels; this is not proof of identical molar productivity, unchanged protein expression or a purified reaction rate. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| S. antibioticus UG J1-21 single-crossover strain | lacks_detected_D_series_profile | Source-assigned D4/D8 chromatographic profile | The source reports complete loss of D4/D8 production in UG J1-21, with no corresponding assigned peaks in Figure 6C. The graph records a qualitative nondetection phenotype, not a numerical zero or a universal detection limit. It supports a role for the manipulated region in aminocoumarin-containing product formation but does not isolate a biochemical catalytic step or exclude all polar and insertion effects. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| S. antibioticus UG J1-21 single-crossover strain | accumulates_source_assigned_signal | Source-assigned C4 accumulation | The mutant accumulates assigned C4, described as retaining the two polyketide units and sugar but lacking the aminocoumarin moiety. Figure 6 gives [M-H]- 721.0. HPLC and HPLC-MS support the source assignment; no new exact chemical record, absolute C4 titer or independently verified stereochemical structure is asserted here. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| S. antibioticus UG J1-21 single-crossover strain | has_unresolved_product_signal | Unresolved same-mass C4a product signal | A second same-mass signal is called C4a by the source. Its structure was not established in the inspected experiment. It is not merged with C4 or assigned a particular isomer identity from mass alone. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| UG J1-21 methyl-aminocoumarin feeding condition | uses_mutant_background | S. antibioticus UG J1-21 single-crossover strain | The feeding experiment used UG J1-21 in 50 mL complex production medium 1 at 28 C and 120 rpm. Section 4.1.3 defines that medium as glycerol 20 g/L and full-fat soy flour 20 g/L in tap water, pH 7.5. These feeding-specific conditions are not replaced by the broader temperature and shaking ranges in the general culture section, and are not assumed for every unrelated experiment. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| UG J1-21 methyl-aminocoumarin feeding condition | was_supplied_with | Novobiocin-derived 3-amino-4,7-dihydroxy-8-methylcoumarin | At 42 h after inoculation and again 5 h later, 1 mg novobiocin-derived 8-methyl-aminocoumarin in 10 uL methanol was added. Cultures continued for another 43 h before extraction/HPLC. This substrate differs from the native 8-chloro aminocoumarin of D8; no exact compound accession is chosen from a similar name. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| UG J1-21 methyl-aminocoumarin feeding condition | has_source_assigned_analogue_readout | Source-assigned methylated D-met product signal | Figure 6D reports D-met, [M-H]- 910.2, with a methyl substituent in place of the D8 chlorine. Feeding restores formation of an aminocoumarin-containing D-series analogue, not native chlorinated D8. Residual C4/C4a signals are visible; complete conversion or full WT yield is not claimed. This is metabolic bypass feeding, not simJ1 gene complementation, direct SimJ1 catalysis or a separately measured purified SimL reaction. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| Parallel methanol-only control conditions | were_reported_as_parallel_controls_for | UG J1-21 methyl-aminocoumarin feeding condition | Methods specify parallel original WT and mutant cultures without the aminocoumarin feed but with 10-uL methanol additions, extracted and analyzed likewise. This records the control design; it does not invent a numerical solvent effect or silently identify every displayed WT trace as that particular solvent-control culture. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| Source-assigned UG J1-293 double-crossover strain | was_recovered_after_passage_of_integrants_generated_with | pUG013 internal simJ1-deletion construct | After repeated antibiotic-free YMG passages, apramycin-sensitive isolates were examined for a second crossover. The source calls UG J1-293 a double-crossover genotype. Its specific single-crossover ancestor and whole experimental genome are not independently identified here. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| Source-assigned UG J1-692 double-crossover strain | was_recovered_after_passage_of_integrants_generated_with | pUG013 internal simJ1-deletion construct | UG J1-692 is the second source-assigned double-crossover isolate from that selection. Selection phenotype alone does not establish its allele or loss of every vector sequence; the source used Southern analysis to classify it. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| WT-genotype revertant recovered after passage | was_recovered_after_passage_of_integrants_generated_with | pUG013 internal simJ1-deletion construct | A separate apramycin-sensitive isolate was classified as reverted to the WT simJ1 genotype. It is distinct from the original unpassaged WT comparator and is not assumed to be wild type across its entire genome or physiology. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| Source-assigned UG J1-293 double-crossover strain | has_source_assigned_genotyping_result | Source-reported double-crossover and revertant Southern genotypes | The source assigns UG J1-293 to one of the two isolates with the approximately 2.0-kb BglII/EcoRV deletion-band classification. Figure 7 and its caption are the genotype evidence, not complete allele sequencing or proof of an otherwise isogenic genome. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| Source-assigned UG J1-692 double-crossover strain | has_source_assigned_genotyping_result | Source-reported double-crossover and revertant Southern genotypes | The second source-classified deletion isolate is UG J1-692. The caption assigns lanes 1 and 4 to the two double crossovers. Coarse 2.0-kb labelling is kept distinct from the independently reconstructed 1973-bp intended allele fragment and from an actual sequenced genome. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1 |
| WT-genotype revertant recovered after passage | has_source_assigned_genotyping_result | Source-reported double-crossover and revertant Southern genotypes | The source calls the approximately 2.8-kb-only isolate a WT-genotype revertant. Another apramycin-sensitive clone retained both bands and was still classified as a single crossover; resistance loss alone therefore did not establish a second crossover. The 2729-bp native reconstruction and source 2.77/2.8-kb descriptions remain separately attributed. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1 |
| Source-assigned UG J1-293 double-crossover strain | has_shared_followup_production_loss | Shared loss of all detected simocyclinones after passage | Repeated cultivation of UG J1-293 yielded no simocyclinones according to the source, rather than the expected C-series-only phenotype. No exact replicate count, detection limit or titer is given for this follow-up. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| Source-assigned UG J1-692 double-crossover strain | has_shared_followup_production_loss | Shared loss of all detected simocyclinones after passage | UG J1-692 likewise lost all reported simocyclinone production. This broader loss is not interpreted as specific proof of the proposed SimJ1 oxidation reaction. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| WT-genotype revertant recovered after passage | also_has_followup_production_loss | Shared loss of all detected simocyclinones after passage | The WT-genotype revertant also produced no simocyclinones in the repeated cultures. This shared loss is a confound for interpreting the double crossovers: it cannot be attributed specifically to absence of simJ1. The cause was not resolved and the source stopped this follow-up; no particular secondary mutation is invented. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| Original S. antibioticus Tu6040 wild type | is_parallel_positive_production_comparator_for | Shared loss of all detected simocyclinones after passage | The original WT cultured in parallel still produced D8 while both deletion-classified isolates and the revertant lost all reported simocyclinones. This is a positive production comparator, not a purified-enzyme control or proof that the passaged strains differ only at simJ1. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| Native SimJ1 (SimD2) protein | is_proposed_to_contribute_to | Proposed beta-hydroxytyrosyl oxidation step | The thesis proposes oxidation of a beta-hydroxytyrosyl intermediate to a beta-ketotyrosyl intermediate, based on sequence similarity and pathway reasoning. These experiments show genetics and feeding, not purified SimJ1 turnover or a directly determined substrate. The source argues against polarity from 170/322-bp intergenic distances but could not unambiguously identify the downstream -10/-35 promoter regions. The simKLHIYJ1 transcriptional unit is also proposed from spacing, not mapped transcripts. Lack of rescue by endogenous SimJ2 in this context is not a universal inactivity claim; the separate SimJ2 mutant production profiles were explicitly not examined in the thesis. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 10 nodes, 11 edges
Native SimJ2/SimD4 Q9F5I9 and genetically targeted constructs from the Galm 2003 and Schafer 2016 primary theses (review #546). Historical SimC6 maps this protein by literal primers, not by the current GenBank gene name alone. The Galm single-crossover clones were genotyped, but their metabolite production was explicitly not examined. Reported deletion/frame and fragment sizes remain separate from native and literal-primer reconstructions. The later PAC construct is a source-listed resource, not a measured catalytic or production outcome. No purified SimJ2 reaction, functional-null allele or established modular-PKS tetraene role is asserted. Existing simD4-region DNA assays and promoter reporters are preserved without promotion to protein activity. Galm PDF page numbers exceed printed pages by four; Schafer numbers coincide.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Native simJ2 locus, also named simD4 | encodes_native_protein | Native SimJ2 (SimD4; historical SimC6) protein | Three 741-nt CDSs including stop translate exactly to the 246-aa native Q9F5I9 sequence using CDS-aware table 11: AAL15605.1 at AF322256.1:25806..26546(+), AAG34189.1 at AF321122.1:9018..9758(+) and AAK06809.1 at AF324838.2:70601..71341(+). The first two deposits call the gene simJ2; the third calls it simD4. All identify S. antibioticus Tu6040/Tue6040, taxon 1890. This native protein is distinct from 273-aa SimJ1 and the 487-aa AEU17894.1 putative halogenase currently named simC6 in AF324838.2. It does not identify either engineered allele. Database oxidoreductase annotations do not constitute purified native activity measurements. | https://www.ncbi.nlm.nih.gov/nuccore/AF322256.1 |
| pUG014 source-reported internal simJ2-deletion construct | was_designed_for_internal_deletion_of | Native simJ2 locus, also named simD4 | The source describes pUG010 then pUG014 assembly in pBSKT from cosmid VII-8g upstream XbaI/EcoRI and downstream EcoRI/HindIII PCR fragments, and calls the resulting deletion 639 bp and in frame. Literal primers are P01f CAGACGATCTAGATCGACGG, P01r CCACATCGAATTCCTCGCGA, P02f TCGACGGCGGAATTCCGATCTGAC and P02r CTCCTCGCCAAGCTTGATGATCTG. Their printed coordinates reproduce the reported 1379/1409-nt amplicons, but EcoRI fusion gives a 640-nt net deletion and a 101-nt residual coding span, which is not divisible by three. This is an unresolved source-versus-primer-model discrepancy, not proof of the actual allele or its expression. No primer is silently repaired and no residual protein is assigned. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1 |
| pUG015 apramycin-selectable simJ2 targeting construct | derives_targeting_insert_from | pUG014 source-reported internal simJ2-deletion construct | The reported approximately 2.8-kb XbaI/HindIII insert was transferred from pUG014 to the apramycin-selectable suicide vector pKC1132. Table 14 lists pUG014 at 7.2 kb and pUG015 at 6.3 kb. These are source-reported sizes, not independently sequenced full plasmids or evidence that the actual deletion resolves the 639/640-nt conflict. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| Source-listed S. antibioticus simJ2 single-crossover clone set | were_generated_with | pUG015 apramycin-selectable simJ2 targeting construct | PEG-mediated protoplast transformation of native S. antibioticus Tu6040 and apramycin selection yielded the three examined clones. Table 16 lists UG J2-7, UG J2-8 and UG J2-10 as pUG015-containing simJ2 single-crossover mutants. The results describe three clones collectively, without pictured individual Southern lanes or clone-specific production measurements. The pUG014/thiostrepton route instead gave many spontaneous-resistant colonies, so resistance alone is not equated with targeted integration. These are not three production-assay replicates. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| Source-listed S. antibioticus simJ2 single-crossover clone set | have_reported_genotyping_readout | Reported EcoRV/PvuII Southern genotyping profiles | After EcoRV/PvuII digestion and S-simJ2 hybridization, all three examined clones reportedly had both the 1.9-kb WT-associated and 1.2-kb construct-associated bands. The Southern is explicitly not pictured. These source-assigned single crossovers are not clean double-crossover replacements or proof of protein abundance, intact-copy expression or complete junction sequences. Their secondary-metabolite production was explicitly not examined. No D8 loss, retained production, dispensability or catalytic rate is inferred. The thesis stopped this follow-up based on its separate SimJ1 observations; those do not supply a SimJ2-mutant outcome. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| S. antibioticus Tu6040 wild-type Southern comparator | provides_genomic_DNA_control_for | Reported EcoRV/PvuII Southern genotyping profiles | The source reports only a 1.9-kb hybridizing band for WT genomic DNA. Native AF322256.1 has a probe-containing EcoRV/PvuII interval of 1826 bp, between cut positions 25791 and 27617. The source band label and native reconstruction remain separately attributed; no unshown gel is remeasured or assigned greater precision. This control concerns genotyping, not a simultaneous product titer. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1 |
| pUG015 apramycin-selectable simJ2 targeting construct | provides_plasmid_DNA_control_for | Reported EcoRV/PvuII Southern genotyping profiles | The source reports only a 1.2-kb hybridizing band for pUG015 DNA. The literal-primer deletion model yields a 1186-bp EcoRV/PvuII interval, consistent with that coarse label. This does not verify the complete experimental plasmid or make the source-reported in-frame allele consistent with the printed primers. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1 |
| S-simJ2 DIG-labelled downstream DNA probe | was_generated_from | pUG014 source-reported internal simJ2-deletion construct | Methods describe a 1.1-kb EcoRI/PvuII fragment from pUG014, gel isolation and DIG labelling. The introduced EcoRI cut maps to 26534 in the primer-derived model; the downstream native PvuII cut is 27617, giving 1083 bp. This supports the reported approximate probe size, not a deposited sequence of the experimental probe. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1 |
| S-simJ2 DIG-labelled downstream DNA probe | was_used_in | Reported EcoRV/PvuII Southern genotyping profiles | S-simJ2 hybridization is the locus-probing readout for the three selected clones and the WT/plasmid controls. Band presence is not a measurement of SimJ2 RNA, protein or biosynthetic catalytic function. | https://bibliographie.uni-tuebingen.de/xmlui/bitstream/handle/10900/48524/pdf/Dissertation_UG1.pdf?isAllowed=y&sequence=1
|
| Source-listed PAC-12I-deltaC6 construct | is_listed_as_derivative_of | PAC-12I sim-cluster parent construct | The Schafer vector table lists PAC-12I-deltaC6 as an in-frame simC6 deletion and attributes it to T. Le and O. Shorinola, unpublished. This is a later cluster construct, not one of the Galm native single-crossover clones. The inspected listing and primer tables do not supply a SimC6-specific production or activity outcome. Separate SimC7 experiments and historical pathway drawings are not substituted for such an outcome. | https://ueaeprints.uea.ac.uk/id/eprint/61276/
|
| Source-listed PAC-12I-deltaC6 construct | targets_same_native_locus | Native simJ2 locus, also named simD4 | The later del_simC6-F and del_simC6-R native arms are ggatcgaccgggaatgacgatatccggagttgcgtgatg and tgaacaggtcggcccgcggcaggccgggtcagaccgtca. Both 39-nt arms match AF324838.2 exactly and uniquely: 70565..70603(+) and 71339..71377(-), retaining native start/stop boundaries around AAK06809.1/simD4. The appended cassette tails are ATTCCGGGGATCCGTCGACC and TGTAGGCTGGAGCTGCTTC. The 24-nt check primers caggcctgtccactggaaccttgc and gagggacgccgacctcgtgaagcg map 70446..70469(+) and 71471..71494(-). Thus this historical SimC6 denotes SimJ2/SimD4, not AEU17894.1. These mappings identify the intended target; the complete experimental cassette, scar and expressed allele are not independently sequenced. | https://ueaeprints.uea.ac.uk/id/eprint/61276/ |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Explore across X-Mech
AntibioticMech (simocyclinone D8) ↗
MediaIngredientMech (naturalproductmech:mibig-e03313fa44) ↗
TaxonMech: Streptomyces antibioticus (NCBITaxon:1890) ↗
TaxonMech: Streptomyces antibioticus (NCBITaxon:1890) ↗
PathwayMech (mibig:BGC0001072) ↗
PathwayMech (mibig:BGC0000270) ↗
Open questions 1
shared-structure — This structure is reported by more than one MIBiG entry: BGC0000270, BGC0001072. Confirm they describe the same compound rather than an upstream cross-reference error.
Provenance
| Source concept | Source | Version |
|---|---|---|
| BGC0001072 | MIBIG | 5 |
| BGC0000270 | MIBIG | 5 |
This page is generated from data/natural_products/polyketides/simocyclinone-d8.yaml, which is generated from the committed inventories. Neither is edited by hand.