phenalinolactone A
naturalproductmech:mibig-e29f83c77b 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REVIEWED records curator sign-off on identity, structure, filing class and producer claims; DEPRECATED retains a superseded or out-of-scope record.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 | LULQRLKBANNSOX-AFCHNXSNSA-N |
|---|---|
| SMILES | [H][C@]12CC[C@@]3(C)[C@H](C4=C(O)C(=O)OC4O)C(C)=CC[C@@]3([H])[C@]1(C)[C@@H](O)C[C@@H](OC(C)=O)C2(CO[C@H]1CC[C@@H](OC)[C@H](C)O1)COC(=O)C1=CC=C(C)N1 |
| Stereochemistry | undefined stereocentres — the InChIKey does not distinguish this compound from its stereoisomers |
| Cross-references | npatlas:NPA016342 |
Filing pathway
Unclassified — 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 terpene, saccharide, from MIBiG.
Producer organisms 1
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 sp. Tu6071 NCBITaxon:355249 |
BGC_CHARACTERIZED | mibig:BGC0000654 | PMID:16632249 |
Occurrences 2
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 NCBITaxon:1883 |
LOTUS | DOI:10.1016/J.CHEMBIOL.2006.01.011 |
| Streptomyces NCBITaxon:1883 |
LOTUS | DOI:10.1038/JA.2010.165 |
Biosynthetic gene clusters 1
| Accession | Host | Locus evidence | Genome |
|---|---|---|---|
| mibig:BGC0000654 | Streptomyces sp. Tu6071 NCBITaxon:355249 |
CLUSTER_DEMONSTRATED | genbank:DQ230532.1 |
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 6 nodes, 6 edges
PlaM1 evidence in Streptomyces sp. Tu6071 from the Duerr 2005 thesis. Native protein identity is separate from the mutant and rescue constructs; culture phenotypes are distinct from inferred chemistry. Cultures were grown in NL111V at 28 C; analytical harvests were after 6-7 days.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Internal plaM1 deletion in Tu6071-derived K1/1 | inactivates_encoding_gene_of | Native PlaM1 parent | K1/1 is a double crossover with a 414-bp internal PstI deletion, not whole-gene removal, supported by colony PCR and apramycin-resistance loss. Native PstI sites are 414 bp apart, consistent with an in-frame lesion. Possible SCplaM1 polar effects are not attributed to PlaM1 alone. Curator comparison: thesis appendix page 126 has C at 13742 versus deposited T (terminal Arg CGG versus CGA): synonymous, not identical DNA. GenPept/GenBank/MIBiG and translated thesis proteins agree. UniParc UPI000068F395 links active EMBL ABB69739.1 and inactive UniProtKB/TrEMBL Q2I754 (plaM1, taxon 355249) with zero active UniProtKB entries; no homolog substituted. | https://freidok.uni-freiburg.de/data/2280 |
| Internal plaM1 deletion in Tu6071-derived K1/1 | disrupts_normal | Detectable phenalinolactone A and D production | Mutant-culture HPLC/ESI-MS showed PL CD1 dominating instead of parental A/D, not loss of all natural products or a quantified detection limit. | https://freidok.uni-freiburg.de/data/2280
|
| Internal plaM1 deletion in Tu6071-derived K1/1 | causes_accumulation_of | PL CD1 and related mutant-congener accumulation | PL CD1 (m/z 684 [M-H]-) is 4''-O-demethylphenalinolactone D, not A, by comparative NMR: missing sugar methoxy and shifted C-4/H-4 signals. Other congeners include PL CD2 and PL X3/X4; minor proposed demethylated A analog PL X5 is MS-assigned, not fully NMR-elucidated. | https://freidok.uni-freiburg.de/data/2280
|
| pSET-plaM1 genomic-fragment complementation | supplies_wild_type_encoding_gene_for | Native PlaM1 parent | ermE*-driven pSET152-derived pSET-plaM1 carries a genomic fragment. Curator mapping places 22c_F/22c_R changes outside PlaM1 but includes partial plaA4/plaP6: neither isolated CDS nor purified-protein assay. Methods name both cosmids 1O12 and 4D08; exact clone provenance is unresolved. | https://freidok.uni-freiburg.de/data/2280 |
| pSET-plaM1 genomic-fragment complementation | restores_detectable | Detectable phenalinolactone A and D production | All six complemented-conjugant cultures recovered the wild-type HPLC/ESI-MS profile: genetic support, not measured PlaM1 turnover or isolated-protein sufficiency. | https://freidok.uni-freiburg.de/data/2280
|
| Native PlaM1 parent | is_proposed_to_catalyze | Proposed late 4''-O-methylation of attached L-amicetose | Demethylated glycoside, rescue and whole-cell feeding support late sugar methylation, including proposed PL CD1-to-D conversion. The thesis shows neither purified PlaM1 conversion, an experimentally established methyl donor, nor direct PlaM1 CD1-to-A turnover. | https://freidok.uni-freiburg.de/data/2280
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 6 nodes, 6 edges
PlaO4 evidence in Streptomyces sp. Tu6071 from the Duerr 2005 thesis. Native protein identity is separate from the mutant and rescue constructs; culture phenotypes are distinct from inferred chemistry. Cultures were grown in NL111V at 28 C; analytical harvests were after 6-7 days.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Internal plaO4 disruption in Tu6071-derived 42.A.35 | inactivates_encoding_gene_of | Native PlaO4 parent | Double-crossover 42.A.35 was selected by apramycin-resistance loss and colony PCR. The reported internal RsrII frameshifting deletion is 880 bp; native sites are 884 bp apart. Exact mutant junction is unverified. The preceding single crossover's short-only PCR does not exclude its wild-type allele. Curator comparison: appendix pages 138-139 match PlaO4 CDS; thesis G versus deposited A at 38851 lies in plaA5, not a PlaO4 substitution. GenPept/GenBank/MIBiG and translated thesis proteins agree. UniParc UPI000068F3A9 links active EMBL ABB69759.1 and inactive UniProtKB/TrEMBL Q2I770 (plaO4, taxon 355249) with zero active UniProtKB entries; no homolog substituted. | https://freidok.uni-freiburg.de/data/2280 |
| Internal plaO4 disruption in Tu6071-derived 42.A.35 | disrupts_normal | Detectable phenalinolactone A and D production | Mutant extracts showed PL CD8-CD11; complementation reverted this culture-production phenotype, not a direct activity assay or loss of all metabolites. | https://freidok.uni-freiburg.de/data/2280
|
| Internal plaO4 disruption in Tu6071-derived 42.A.35 | causes_accumulation_of | PL CD8-CD11 mutant-congener accumulation | Preparative cultures yielded PL CD8; CD9 was the largest analytical peak (Table 5). CD8 NMR lacks sugar/O-3 acetyl signals; C-21 methylene becomes a methyl linked to C-4 by HMBC. Its gamma-butyrolactone remains. Other congeners have HPLC/ESI-MS/UV evidence, not all complete NMR. | https://freidok.uni-freiburg.de/data/2280
|
| pSET-plaO4 genomic-fragment complementation | supplies_wild_type_encoding_gene_for | Native PlaO4 parent | ermE*-driven pSET-plaO4 carries a genomic fragment. Curator mapping places 42c_F/42c_R changes outside PlaO4 but includes partial plaV/plaA5: neither isolated CDS nor reconstituted monooxygenase system. | https://freidok.uni-freiburg.de/data/2280 |
| pSET-plaO4 genomic-fragment complementation | restores_detectable | Detectable phenalinolactone A and D production | All three complemented-conjugant cultures recovered the wild-type HPLC/ESI-MS profile: genetic rescue, not purified PlaO4 substrate conversion. | https://freidok.uni-freiburg.de/data/2280
|
| Native PlaO4 parent | is_proposed_to_catalyze | Proposed C-21 hydroxylation of the axial C-4 methyl group | Deletion, rescue and CD8 structure support C-21 hydroxylation, replacing the homology-based C-19 hypothesis. CD8 also lacks C-1 hydroxylation; the thesis argues against dual PlaO4 specificity without a direct exclusion assay. Substrate, redox partners and order remain unresolved. | https://freidok.uni-freiburg.de/data/2280
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 8 nodes, 8 edges
PlaO1 evidence in Streptomyces sp. Tu6071 from the Duerr 2005 thesis. Native protein identity is separate from the mutant and rescue constructs; culture phenotypes are distinct from inferred chemistry. Cultures were grown in NL111V at 28 C; analytical harvests were after 6-7 days.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Internal plaO1 deletion in Tu6071-derived 24.4.3 | inactivates_encoding_gene_of | Native PlaO1 parent | Double-crossover 24.4.3 has an internal 264-bp PstI deletion, distinguished from its single-crossover parent by colony PCR. Results 3.4.3 says in-frame; methods 5.9.3 says frameshift. Native sites 264 bp apart support in-frame; exact mutant junction is unverified. Curator comparison: appendix page 127 has C at 16420 versus deposited T (GTG versus ATG initiator). Code 11 initiator-Met gives identical complete protein, not DNA. GenPept/GenBank/MIBiG and translated thesis proteins agree. UniProt mapping failed; no homolog substituted. | https://freidok.uni-freiburg.de/data/2280 |
| Internal plaO1 deletion in Tu6071-derived 24.4.3 | abolishes_detectable | Detectable phenalinolactone A and D production | Mutant HPLC/ESI-MS extracts lacked wild-type phenalinolactones. Different metabolites appeared at 235 nm but poorly at 280 nm: not abolition of all secondary metabolism. | https://freidok.uni-freiburg.de/data/2280
|
| Internal plaO1 deletion in Tu6071-derived 24.4.3 | causes_accumulation_of | PL CD6 and PL CD7 accumulation | HR-ESI-MS/NMR identify C23 terpene CD6 (m/z 373 [M-H]-) with a C-14 beta,gamma-unsaturated alpha-ketoacid side chain, no gamma-butyrolactone; CD7 (m/z 371 [M-H]-) has a C-3 ketone instead of alcohol. Table 7 formulas are deprotonated ions, not neutral molecules. Neither is A. | https://freidok.uni-freiburg.de/data/2280
|
| pSET-plaO1 genomic-fragment complementation | supplies_wild_type_encoding_gene_for | Native PlaO1 parent | ermE*-driven pSET-plaO1 carries a genomic fragment. Curator mapping places 24c_F/24c_R changes outside PlaO1 but includes partial plaP6/plaT1. The native accession does not denote the deleted mutant as full-length active enzyme. | https://freidok.uni-freiburg.de/data/2280 |
| pSET-plaO1 genomic-fragment complementation | restores_detectable | Detectable phenalinolactone A and D production | All three complemented-conjugant cultures recovered A/D by HPLC/MS: genetic support, not a purified-enzyme reaction. | https://freidok.uni-freiburg.de/data/2280
|
| Native PlaO1 parent | is_proposed_to_catalyze | Proposed oxidative contribution to gamma-butyrolactone formation | Mutant and precursor structures support lactone biosynthesis. Genetic/isotope evidence suggests C-16 hydroxylation/rearrangement, not purified PlaO1 turnover. No demonstrated cyclopropanone intermediate, enzyme-specific closure, measured cofactor requirement or proven sequence is asserted. | https://freidok.uni-freiburg.de/data/2280
|
| PL CD1 feeding to delta-plaO1 cultures | restores_detectable | Detectable phenalinolactone A and D production | Whole-cell feeding: 1 mg CD1/20 ml mutant culture after 28 h growth yielded A/D, methylated A-2/D-2 and CD1-2 after 63 h. This bypass supports CD1 biosynthetic competence, not PlaO1 turnover or isolated PlaM1 activity. Methods specify medium-only control; its outcome is not reported here as a separate negative. | https://freidok.uni-freiburg.de/data/2280
|
| Native PlaO1 parent | has a sequence-identical UniProtKB example | PlaO1 (Streptomyces sp. Tu6071 UniProtKB example) | UniProtKB:Q2I752 is the active Streptomyces sp. Tu6071 plaO1 entry sharing 100% amino-acid sequence identity (UPI000068F397) with DQ230532.1 ABB69741.1 (PlaO1). | https://www.uniprot.org/uniprotkb/Q2I752 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 10 nodes, 12 edges
Native Tu6071 disruption and rescue evidence for PlaT1-PlaT3 from Schnell 2007. Analytical cultures used NL111 medium at 28 C, with standard harvest near 168 h and HPLC/ESI-MS. These experiments establish production phenotypes, not isolated enzyme reactions or a reaction order.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Tu6071-derived XT1 internal PlaT1 gene lesion | inactivates_encoding_gene_of | Native PlaT1 parent | BamHI fill-in frameshift, not deletion; plasmid sequence-checked. Apramycin sensitivity and PCR/BamHI identified double crossovers 7/8, not complete mutant-genome sequencing. | https://freidok.uni-freiburg.de/data/3799 |
| Tu6071-derived XT1 internal PlaT1 gene lesion | abolishes_detectable | Detectable phenalinolactone A and D production | Both double crossovers lacked detectable A/D or recognizable intermediates; parallel wild type produced A/D. Sampling at 96, 120 and 144 h and chloroform extraction also found no intermediates. | https://freidok.uni-freiburg.de/data/3799
|
| pS-25co genomic-fragment complementation | supplies_wild_type_encoding_gene_for | Native PlaT1 parent | ermE*-driven/pSET152-derived pS-25co rescue. Curator Co25-F/R alignment: DQ230532.1:16385..17855; primer changes outside PlaT1, partial plaO1/plaT2 included. Not isolated CDS. | https://freidok.uni-freiburg.de/data/3799 |
| pS-25co genomic-fragment complementation | restores_detectable | Detectable phenalinolactone A and D production | All three complemented cultures recovered A/D; PCR/restriction supported the construct. Neither all off-target changes nor purified-enzyme sufficiency is resolved. | https://freidok.uni-freiburg.de/data/3799
|
| Tu6071-derived XT2 internal PlaT2 gene lesion | inactivates_encoding_gene_of | Native PlaT2 parent | EcoNI fill-in frameshift, not whole-gene removal; plasmid sequence-checked. Apramycin sensitivity and PCR/EcoNI identified double crossovers 3/4/5. The preceding single crossover's mutant-only amplicon does not exclude its wild-type allele. | https://freidok.uni-freiburg.de/data/3799 |
| Tu6071-derived XT2 internal PlaT2 gene lesion | abolishes_detectable | Detectable phenalinolactone A and D production | Double crossovers 3/5 lacked detectable A/D or recognizable intermediates unlike parallel wild type. Single-crossover CD6/CD7 may reflect plaO1 polarity, not PlaT2 chemistry. | https://freidok.uni-freiburg.de/data/3799
|
| pS-26co genomic-fragment complementation | supplies_wild_type_encoding_gene_for | Native PlaT2 parent | ermE*-driven/pSET152-derived pS-26co rescue. Curator 26co-F/R alignment: DQ230532.1:17732..19513; primer changes outside PlaT2, partial plaT1/plaT3 included. Not isolated CDS. | https://freidok.uni-freiburg.de/data/3799 |
| pS-26co genomic-fragment complementation | restores_detectable | Detectable phenalinolactone A and D production | All four complemented cultures recovered detectable A/D: genetic rescue, not purified cyclase activity. | https://freidok.uni-freiburg.de/data/3799
|
| Tu6071-derived XT3 internal PlaT3 gene lesion | inactivates_encoding_gene_of | Native PlaT3 parent | Internal 223-bp BstEII deletion causes downstream frameshift, not whole-gene removal; native cuts are 223 bp apart. Apramycin sensitivity and shorter PCR versus cosmid identified the double crossover. Earlier single-crossover control PCR failed. | https://freidok.uni-freiburg.de/data/3799 |
| Tu6071-derived XT3 internal PlaT3 gene lesion | abolishes_detectable | Detectable phenalinolactone A and D production | Double-crossover ethyl-acetate/chloroform extracts lacked detectable A/D or recognizable intermediates; the single crossover retained low A/D. These are detection-limited culture observations. | https://freidok.uni-freiburg.de/data/3799
|
| pS-27co genomic-fragment complementation | supplies_wild_type_encoding_gene_for | Native PlaT3 parent | ermE*-driven/pSET152-derived pS-27co rescue. Curator Co27-F/R alignment: DQ230532.1:19411..20594; primer changes outside PlaT3, partial plaT2/plaH included. Not isolated CDS. | https://freidok.uni-freiburg.de/data/3799 |
| pS-27co genomic-fragment complementation | restores_detectable | Detectable phenalinolactone A and D production | All three complemented clones recovered A/D, reported slightly above wild type without numerical fold change. PCR supported intact introduced gene; not direct prenyltransferase activity. | https://freidok.uni-freiburg.de/data/3799
|
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Causal graph — biosynthesis 14 nodes, 19 edges
Alsup et al. 2024 heterologous backbone reconstitution with native Tu6071 genes. Hosts were S. albus J1074 and S. venezuelae ATCC 10712; HA cultures were screened after 7 days at 28 C by LC-MS against empty-vector controls. The four-gene cassette makes scaffold analogs, not phenalinolactone A. Enzyme roles and order below are inferences from gene combinations, not purified-protein turnover. All four full-cassette genes were amplified from genomic DNA and TEDA-assembled under sp44 promoter/sr40 RBS. Authors report sequence-confirming plasmids; curator primer mapping supports native CDS boundaries, not independent full-plasmid sequencing. UniParc archives UPI000068F398 (PlaT1, ABB69742.1), UPI000068F399 (PlaT2, ABB69743.1), UPI000068F39A (PlaT3, ABB69744.1) and UPI000068F3A4 (PlaT4, ABB69754.1) link only inactive UniProtKB/TrEMBL entries Q2I763, Q2I762, Q2I761 and Q2I775 with zero active UniProtKB accessions; exact native GenPept accessions are used.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| pJR3008 plaT4321 heterologous cultures | supplies_wild_type_encoding_gene_for | Native PlaT1 parent | PlaT1 in pJR3008; shared construction/identity qualifications in graph description. | DOI:10.1039/D4CB00138A |
| pJR3008 plaT4321 heterologous cultures | supplies_wild_type_encoding_gene_for | Native PlaT2 parent | PlaT2 in pJR3008; shared construction/identity qualifications in graph description. | DOI:10.1039/D4CB00138A |
| pJR3008 plaT4321 heterologous cultures | supplies_wild_type_encoding_gene_for | Native PlaT3 parent | PlaT3 in pJR3008; shared construction/identity qualifications in graph description. | DOI:10.1039/D4CB00138A |
| pJR3008 plaT4321 heterologous cultures | supplies_wild_type_encoding_gene_for | Native PlaT4 parent | PlaT4 in pJR3008; shared construction/identity qualifications in graph description. | DOI:10.1039/D4CB00138A |
| pJR3008 plaT4321 heterologous cultures | produces_detectable | Cyclized amino-acid-tail products 1 and 2 | S. albus produced 1/2; S. venezuelae only 1. Isolated S. albus products have HRMS/NMR support: C-3 ketone in 1, hydroxyl in 2. Both bear amino-acid tails without the CD6/CD7 C-15/C-16 olefin; neither is CD6/CD7 or mature A. C-17 absolute configuration, host amination and 2-to-1 oxidation remain unresolved. | DOI:10.1039/D4CB00138A
|
| pJR3007 plaT431 cultures lacking plaT2 | omits_encoding_gene_of | Native PlaT2 parent | pJR3007 encodes plaT4/plaT3/plaT1, omitting plaT2 in heterologous hosts; not a native deletion. | DOI:10.1039/D4CB00138A
|
| pJR3007 plaT431 cultures lacking plaT2 | abolishes_detectable | Cyclized amino-acid-tail products 1 and 2 | Removing plaT2 abolished detectable 1/2 and other tricyclic products in both hosts under the reported screening conditions. | DOI:10.1039/D4CB00138A
|
| pJR3007 plaT431 cultures lacking plaT2 | produces_detectable | Metabolites 3-10 lacking the tricyclic diterpene core | S. venezuelae::plaT431 produced 3-10; S. albus::plaT431 8-10. Isolated 3-7/10 were structurally characterized; 8/9 are MS proposals. All lack the tricyclic diterpene core, but 6/7 retain tetrahydrofuran rings: not all acyclic, native intermediates or direct PlaT1 products. | DOI:10.1039/D4CB00138A
|
| plaT4, plaT41 and plaT421 host cultures lacking plaT3 | omits_encoding_gene_of | Native PlaT3 parent | pJR3001, pJR3002 and pJR3005 encode plaT4, plaT41 and plaT421 respectively. | DOI:10.1039/D4CB00138A
|
| plaT4, plaT41 and plaT421 host cultures lacking plaT3 | fails_to_produce_detectable | Metabolites 3-10 lacking the tricyclic diterpene core | No-plaT3 constructs lacked monitored diterpenoids. PlaT3-dependent 11-13 are proposed unoxidized-prenyl-tail structures, MS-only after failed NMR isolation. Compound 11 negative-ion masses conflict between Figure 3D and HRESIMS text/Figure S54; no corrected exact identity/mass is chosen. | DOI:10.1039/D4CB00138A
|
| pJR3004 plaT43(D67A) heterologous cultures | tests_substitution_in | Native PlaT3 parent | Accession denotes native PlaT3, not D67A. Curator SI-primer alignment changes residue-67 GAC to GCC; F1 maps upstream to plaT4 in the coupled construct. No independent mutant-plasmid deposit inspected. | DOI:10.1039/D4CB00138A |
| pJR3004 plaT43(D67A) heterologous cultures | fails_to_produce_detectable | Metabolites 3-10 lacking the tricyclic diterpene core | plaT43(D67A) cultures lacked 3-10 in both hosts; S. albus traces also lacked 11-13. Supports a PlaT3-dependent step, not isolated-protein activity or abundance/stability. | DOI:10.1039/D4CB00138A
|
| Native PlaT4 parent | is_proposed_to_catalyze | Proposed geranylgeranyl diphosphate supply | Model assigns GGPP synthesis to PlaT4. All combinations include plaT4: no independent omission test or purified turnover. | DOI:10.1039/D4CB00138A
|
| Native PlaT3 parent | is_proposed_to_catalyze | Proposed prenylation of a three-carbon acceptor | Gene combinations/D67A support prenylation before oxidation/cyclization. Proposed substrates: GGPP and a three-carbon alpha-keto acid of unresolved identity/origin; amino-acid acceptor not excluded. | DOI:10.1039/D4CB00138A
|
| Native PlaT1 parent | is_proposed_to_catalyze | Proposed terminal epoxidation of the prenylated intermediate | Revised model: prenylation then terminal epoxidation, without purified PlaT1 turnover or isolated epoxy intermediate. Oxidized 3-10 also occur without plaT1, host-dependently; not all oxidation is PlaT1's. | DOI:10.1039/D4CB00138A
|
| Native PlaT2 parent | is_proposed_to_catalyze | Proposed epoxide-initiated tricyclic-core formation | Omission/product structures support epoxide-initiated anti/anti/syn-core cyclization with observed C-3 hydroxyl orientation, not isolated-enzyme kinetics or complete 1/2 absolute configuration. | DOI:10.1039/D4CB00138A
|
| Proposed geranylgeranyl diphosphate supply | is_proposed_to_supply_substrate_for | Proposed prenylation of a three-carbon acceptor | GGPP supply precedes inferred prenylation; not physical gene order or observed purified-enzyme cascade. | DOI:10.1039/D4CB00138A
|
| Proposed prenylation of a three-carbon acceptor | is_inferred_to_precede | Proposed terminal epoxidation of the prenylated intermediate | 2024 reconstitution revises epoxidation-first proposal; older native knockouts did not resolve order. | DOI:10.1039/D4CB00138A
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| Proposed terminal epoxidation of the prenylated intermediate | is_inferred_to_precede | Proposed epoxide-initiated tricyclic-core formation | Order concerns productive backbone formation, not every extract host-oxidation/degradation reaction. | DOI:10.1039/D4CB00138A
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 11 nodes, 12 edges
PlaO5 C-1 hydroxylation evidence from Schnell 2007. Native Tu6071 production phenotypes are separate from complementation with untagged or His6-tagged constructs and the purified-protein assay. Spinach electron-transfer proteins are assay substitutes, not identified native pathway partners.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Tu6071-XO5 internal plaO5 deletion | inactivates_encoding_gene_of | Native PlaO5 parent | XO5 double-crossover clone 83: apramycin-sensitive with shorter colony PCR. Internal 363-bp in-frame deletion, not whole-gene removal. Curator PCR/PstI-junction reconstruction predicts the same net lesion; no independently sequenced mutant genome/junction is asserted. | https://freidok.uni-freiburg.de/data/3799 |
| Tu6071-XO5 internal plaO5 deletion | abolishes_detectable | Detectable native phenalinolactone A production | XO5 lacked the parallel wild-type A signal; preceding single crossover retained it. NL111, 28 C, harvest near 168 h, HPLC/UV/MS: detection-limited A loss, not all phenalinolactones. | https://freidok.uni-freiburg.de/data/3799
|
| Tu6071-XO5 internal plaO5 deletion | changes_production_profile_to | PL D, PL X1 and mutant-congener profile | D/X1 and other congeners remained. NMR identifies HS2 as D-related pyrrole-tail methoxy derivative; other HS proposals do not all have complete NMR. No quantified fold increase or direct PlaO5 synthesis of these congeners inferred. | https://freidok.uni-freiburg.de/data/3799
|
| pS-O5co genomic-fragment complementation | supplies_wild_type_encoding_gene_for | Native PlaO5 parent | ermE*-driven/pSET152-derived pS-O5co rescue. Curator O5co2-F/O5co-R mapping: 1312 bp, 44241..45552; changes outside plaO5, noncoding flanks but no neighboring CDS. Not a separate rescue-plasmid deposit. | https://freidok.uni-freiburg.de/data/3799 |
| pS-O5co genomic-fragment complementation | restores_detectable | Detectable native phenalinolactone A production | All three complemented cultures recovered detectable A/D: supports genetics, not exclusion of every off-target effect or measured turnover. | https://freidok.uni-freiburg.de/data/3799
|
| Recombinant N-terminal His6-PlaO5 preparation | is_engineered_derivative_of | Native PlaO5 parent | pRSET-O5 in E. coli BL21(DE3)pLysS; Ni-NTA purification. Thesis reports 446-aa N-terminal His6 fusion, sequence-checked insert, 43% MALDI peptide-mass fingerprint coverage, not complete protein sequencing. Native accession is not tagged construct. Curator comparison: printed 409-aa parent matches GenPept/GenBank/MIBiG; expression-primer changes outside CDS. UniParc UPI000068F3AE links active EMBL ABB69764.1 and inactive UniProtKB/TrEMBL Q2I777 (plaO5, taxon 355249) with zero active UniProtKB entries. | https://freidok.uni-freiburg.de/data/3799 |
| pSET-O5NTag complementation of Tu6071-XO5 | expresses | Recombinant N-terminal His6-PlaO5 preparation | Tagged pRSET-O5 insert transferred to ermE*-driven integrating vector; authors report sequence checking. Co-XO5his primers target engineered template, not just native DNA. | https://freidok.uni-freiburg.de/data/3799
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| pSET-O5NTag complementation of Tu6071-XO5 | restores_detectable | Detectable native phenalinolactone A production | Tagged rescue restored A/D profile: retained function, not identical kinetics or complete tag neutrality. | https://freidok.uni-freiburg.de/data/3799
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| Recombinant N-terminal His6-PlaO5 preparation | catalyzes | Assayed C-1 hydroxylation of phenalinolactone D to A | Purified tagged PlaO5 supports D-to-A conversion at pH 7.5, 37 C, 120 min with recombinant His-tagged spinach ferredoxin, commercial spinach ferredoxin reductase, NADPH/NADP+ and glucose-6-phosphate/dehydrogenase regeneration. No native redox partner, separate cofactor-necessity test, kinetic constant or conversion yield established. The 2010 abstract is not counted as independent replication. | https://freidok.uni-freiburg.de/data/3799 |
| Assayed C-1 hydroxylation of phenalinolactone D to A | has_substrate | Phenalinolactone D supplied in the assay preparation | D supplied, but Figure 3.51 also shows D2/reported A2: not claimed homogeneous or proof of broad substrate scope. | https://freidok.uni-freiburg.de/data/3799
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| Assayed C-1 hydroxylation of phenalinolactone D to A | produces | Phenalinolactone A assigned in the assay | Product assigned A: retention 21.4 min under reported gradient, UV maxima near 276/244 nm, m/z 714 [M-H]-. Not fresh NMR, co-injection or complete absolute stereochemistry. | https://freidok.uni-freiburg.de/data/3799
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| Matched assay without His6-PlaO5 | fails_to_form_detectable | Phenalinolactone A assigned in the assay | Matched no-His6-PlaO5 control lacked new A peak: supports PlaO5-dependent conversion, not separate omission tests for every redox component. | https://freidok.uni-freiburg.de/data/3799
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Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 6 nodes, 6 edges
Daum 2009 native Tu6071 PlaV genetics. NL111 cultures harvested after 7-8 days; ethyl-acetate supernatant extracts analyzed by HPLC/ESI-MS (plaESI). Production phenotypes and genomic-fragment rescue support inferred 3-O-acetylation, not purified-protein catalysis.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Tu6071 delta-plaV internal deletion | inactivates_encoding_gene_of | Native PlaV parent | pKC1132-Vdel gives a reported internal 378-bp in-frame deletion, not whole-gene removal. Apramycin-sensitive double crossover has 1.4-kb PCR versus 1.8-kb wild type; single-crossover/plasmid controls are shown. Native GenPept/GenBank/MIBiG translations agree; curator BstEII reconstruction predicts 378 bp but does not sequence the mutant. Native S65/K68 and Figure 3-16 disagree with thesis prose S66/K69. No catalytic-residue or beta-lactam-resistance function is established. UniParc UPI000068F3A8 links active EMBL ABB69758.1 and inactive UniProtKB/TrEMBL Q2I771 (plaV, taxon 355249) with zero active UniProtKB entries; no homolog mapping substituted. | https://freidok.uni-freiburg.de/data/6815 |
| Tu6071 delta-plaV internal deletion | abolishes_detectable | Detectable phenalinolactone A and D production | Mutant extract lacked A/D seen in wild type, while other phenalinolactones remained. This is assay-limited production loss, not loss of all metabolites or a quantified detection limit. | https://freidok.uni-freiburg.de/data/6815
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| Tu6071 delta-plaV internal deletion | changes_production_profile_to | PL-X1 and PL-MD1/MD2 mutant profile | Major PL-X1/PL-MD1 and minor PL-MD2. PL-X1 NMR/HRMS supports 3-O-deacetyl-D, but C-3 hydroxyl relative configuration is unresolved. PL-MD1 is a proposed X1 aglycone: HRMS formula C29H39NO8, 26/29 13C signals, three possibly solvent-hidden; less than 1 mg measured, no informative HSQC/HMBC, and ethyl-acetate contamination noted. PL-MD2 is only an MS/UV-based hydroxylated-X1 proposal, without confirmed hydroxylation site. No exact structure grounding for these congeners. | https://freidok.uni-freiburg.de/data/6815
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| pSET-plaV genomic-fragment complementation | supplies_wild_type_encoding_gene_for | Native PlaV parent | pSET152-derived pSET-plaV uses ermE-up. Curator plaV-F/R mapping gives DQ230532.1:35941..37726 (1786 bp): primer changes outside PlaV CDS, but partial plaU/plaO4 included. Not isolated CDS or a separately deposited rescue-plasmid sequence. | https://freidok.uni-freiburg.de/data/6815 |
| pSET-plaV genomic-fragment complementation | restores_detectable | Detectable phenalinolactone A and D production | Complementation restored A/D; amounts described as wild-type-like, without a quantified fold change here. Methods compare three complemented clones with wild type and mutant+pSET-1cerm vector control (not claimed empty: vector carries urdGT1c). Genetic support does not prove isolated PlaV sufficiency or exclude every off-target effect. | https://freidok.uni-freiburg.de/data/6815
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| Native PlaV parent | is_proposed_to_catalyze | Proposed phenalinolactone 3-O-acetylation | Deletion/rescue and deacetylated products support 3-O-acetylation, including proposed X1-to-D conversion. Donor, reversibility, exact pathway order and SxxK catalytic mechanism are hypotheses, not purified-PlaV results. Motif mutagenesis was ongoing, not reported as completed; lack of marked mutant growth inhibition does not establish self-resistance. No bioactivity target is inferred. | https://freidok.uni-freiburg.de/data/6815
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Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 5 nodes, 5 edges
Partial native Tu6071 genetics from Daum 2009: disruption of the putative SDR genes plaU and plaZ did not abolish detectable PL-A/PL-D under the tested conditions. Native protein parents are distinct from mutant alleles. The PlaU deletion-junction discrepancy is unresolved; the combined mutant has a PlaZ single-crossover insertion, not two clean deletions. No purified PlaU/PlaZ catalysis, measured cofactor preference, or universal biosynthetic dispensability is demonstrated. PlaU C-3 oxidation, minor/shunt product roles, extracluster compensation and silence remain hypotheses.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Tu6071 reported delta-plaU internal deletion | disrupts_encoding_gene_of | Native PlaU parent (putative SDR) | The thesis reports a 156-bp in-frame deletion made by NheI/SfiI restriction and blunting of pBSK-12-P, transferred as pKC1132-Udel. DC4/DC15 colony PCR gives about 0.8 kb versus 0.95 kb in wild type; revertants DC17/DC27 and plasmid controls are shown. DC4 PstI/NheI Southern has about 8.3 kb versus wild-type 5.4/3.0 kb; the single crossover retains both patterns. These support disruption, not an independently sequenced junction. Native DQ230532.1 has intragenic SfiI/NheI top-strand cuts after 35490/35630; standard blunting predicts 143 bp, not 156 bp or an in-frame lesion. This curator reconstruction does not establish the actual mutant sequence and does not silently correct the thesis. Native GenPept/MIBiG and the printed 244-aa PlaU alignment agree. UniParc UPI000068F3A7 links active EMBL ABB69757.1 and inactive UniProtKB/TrEMBL Q2I772 (plaU, taxon 355249) with zero active UniProtKB entries; no homolog substituted. | https://freidok.uni-freiburg.de/data/6815 |
| Tu6071 reported delta-plaU internal deletion | retains_detectable_profile_of | Detectable PL-A/PL-D under tested conditions | HPLC/ESI-MS (plaESI) of the mutant crude extract was reported unchanged from wild type; discussion identifies retained PL-A/PL-D. The standard production method uses NL111 for 7-8 days and ethyl-acetate extraction of supernatant. This is an assay-bounded negative phenotype, not quantified equivalence, a detection-limit measurement, or exclusion of minor/shunt products and redundancy. | https://freidok.uni-freiburg.de/data/6815
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| Tu6071 reported delta-plaU internal deletion | provides_genetic_background_for | Tu6071 delta-plaU with plaZ insertion (SCZ) | pKC1132-SCZ was introduced into the delta-plaU strain to test possible PlaU/PlaZ compensation. The internal plaZ PCR fragment is about 0.52 kb (SCZ-F/SCZ-R, HindIII/XbaI), not the full CDS. A single-crossover insertion was used after attempts at a second recombination yielded revertants. No two-clean-deletion genotype or complete mutant-genome sequence is asserted. | https://freidok.uni-freiburg.de/data/6815
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| Tu6071 delta-plaU with plaZ insertion (SCZ) | disrupts_encoding_gene_of | Native PlaZ parent (putative SDR) | Vector/genomic junction PCR (aac-F/C-SCZ-R) yields about 2.2 kb in three single-crossover clones. The delta-plaU parent is the negative control; plasmid positive control uses SCZ-R instead of C-SCZ-R. Native 237-aa ABB69767.1 matches GenBank/MIBiG translation, not an assayed purified protein or a mutant protein sequence. UniParc UPI000068F3B1 links active EMBL ABB69767.1 and inactive UniProtKB/TrEMBL Q2I782 (plaZ, taxon 355249) with zero active UniProtKB entries; no active UniProtKB mapping claimed. | https://freidok.uni-freiburg.de/data/6815 |
| Tu6071 delta-plaU with plaZ insertion (SCZ) | retains_detectable | Detectable PL-A/PL-D under tested conditions | PL-A and PL-D were detected in combined-mutant crude extract by HPLC/ESI-MS (plaESI). Exconjugants were cultured with 35 ug/mL apramycin because single-crossover integration can revert; the methods compare with wild type and the single mutant. The result does not prove identical yields, exclude residual gene activity or extracluster compensation, or establish no function in any condition. Proposed C-3 oxidation and minor-product roles remain untested here; no necessity/catalysis edge is inferred. | https://freidok.uni-freiburg.de/data/6815
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Causal graph — biosynthesis 4 nodes, 4 edges
Partial native Tu6071 PlaO2 genetics from Schnell 2007. The internal deletion retained the observed wild-type phenalinolactone profile under the tested culture/analytical conditions. No reaction, universal dispensability, complete absence of protein activity, or purified catalysis is established. A described complementation construct was not a completed rescue experiment. Native parent identity is separate from the single-crossover and deletion alleles.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Tu6071-scO2 single-crossover clone 1 | contains_recombined_alleles_of | Native PlaO2 parent (putative cytochrome P450) | pKC-O2del was made from a cosmid 3-1O12 genomic PCR fragment, with an internal NcoI deletion. All 12 tested exconjugants showed both approximately 1.23-kb intact and 0.93-kb deleted O2co-F/R amplicons; clone 1 became scO2. Its PstI Southern had both wild-type and deleted bands. This is not a clean knockout: an intact copy is retained. | https://freidok.uni-freiburg.de/data/3799
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| Tu6071-XO2 internal plaO2 deletion (clone 49) | disrupts_encoding_gene_of | Native PlaO2 parent (putative cytochrome P450) | After four passages, 5 of 128 colonies were apramycin-sensitive: 49/119 had the shorter PCR product; 60/63/75 were wild-type revertants. Clone 49 became XO2. The reported 300-bp in-frame NcoI deletion agrees with native coding-oriented cuts 363/663. PstI Southern gives about 0.9 kb in XO2 versus 1.2 kb in wild type (expected 891/1191 bp). O2blot probe is reported as 565 bp; curator native alignment instead spans 566 bp and finds a printed O2blot-F C versus native T at genomic 21398. These qualifications do not negate the PCR/Southern result or establish an independently sequenced mutant junction. Native 397-aa GenPept, GenBank and MIBiG translations agree; no mutant protein sequence is assigned. UniParc UPI000068F39C links active EMBL ABB69746.1 and inactive UniProtKB/TrEMBL Q2I759 (plaO2, taxon 355249) with zero active UniProtKB entries; no homolog substituted. | https://freidok.uni-freiburg.de/data/3799 |
| Tu6071-scO2 single-crossover clone 1 | retains_detectable_profile_of | Detectable PL-A/PL-D profile under tested conditions | HPLC/ESI-MS of scO2 culture extract detected the wild-type major metabolites PL A and D without new derivatives reported. An intact gene copy remains in this strain; this observation cannot show loss-of-function dispensability or exclude all polar effects. | https://freidok.uni-freiburg.de/data/3799
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| Tu6071-XO2 internal plaO2 deletion (clone 49) | retains_detectable_profile_of | Detectable PL-A/PL-D profile under tested conditions | XO2 culture-extract HPLC/ESI-MS was reported indistinguishable from wild type. Standard analytical cultures used 25 mL NL111, 28 C and 180 rpm, with roughly 168-h sampling and ethyl-acetate extraction. No detection limit, equivalence test, or exclusion of minor products, residual activity or compensation is given. Results 3.2.4.4 explicitly state that complementation was omitted because the phenotype was unchanged; methods describing pS-O2co preparation are not evidence of performed rescue. | https://freidok.uni-freiburg.de/data/3799
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Causal graph — biosynthesis 11 nodes, 14 edges
Native Tu6071 PlaO3 internal-deletion, untagged and His6-tagged complementation evidence from Schnell 2007. XO3 accumulated the NMR-characterized PL HS6/HS7 pair in place of the wild-type major product profile. C-20 hydroxylation of HS6 is inferred from genetics and metabolite structures; the proposed immediate product was not isolated here. Recombinant expression, partial peptide identification and whole-cell tagged rescue do not establish purified turnover, unchanged kinetics, or native electron-transfer partners. HS6/HS7 are distinct mutant products, not phenalinolactone A identifiers.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Tu6071-XO3 internal plaO3 deletion (clone 92) | disrupts_encoding_gene_of | Native PlaO3 parent | XO3 is apramycin-sensitive double-crossover clone 92 with the shorter O3co-F/R PCR product. The source reports a 246-bp in-frame SphI/PflMI deletion, not whole-gene removal; expected amplicons are 1277/1031 bp. Native sequence has two intragenic PflMI sites (top cuts after 24382/24424) and SphI after 24625. Complete digestion and standard blunting predict deletion 24380..24625 (246 bp), consistent with the report. PflMI has a 3-base 3-prime overhang, not the 4 bases stated for both enzymes on p. 153. Figure 3.18(b) calls its plasmid control pKC-O2del, while the accompanying text says pKC-O3del. These source discrepancies remain explicit; native reconstruction is not an independently sequenced XO3 allele. The native 401-aa GenPept/GenBank/MIBiG and complete printed parent sequence match. UniParc UPI000068F39E links active EMBL ABB69748.1 and inactive UniProtKB/TrEMBL Q2I769 (PlaO3, taxon 355249) with zero active UniProtKB entries; no homolog used. | https://freidok.uni-freiburg.de/data/3799 |
| Tu6071-scO3 single-crossover clone 7 | contains_recombined_alleles_of | Native PlaO3 parent | All ten tested exconjugants showed intact and deleted PCR products; clone 7 became scO3. Four passages followed by screening 128 colonies yielded 38 apparently sensitive clones, many still showing both bands; clone 92 had the desired shorter product. Do not interpret scO3 itself as a clean knockout. | https://freidok.uni-freiburg.de/data/3799
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| Tu6071-scO3 single-crossover clone 7 | retains_detectable_profile_of | Native PL-A/PL-D major-product profile | At 72, 96, 121, 138, 168 and 192 h, ethyl-acetate extracts of scO3 were compared by HPLC/ESI-MS with parallel wild-type cultures. PL A was clearly detected, with no conspicuous new peaks or changed profile reported. This bounds the source claim of no polar effect to that assay; an intact copy remains. | https://freidok.uni-freiburg.de/data/3799
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| Tu6071-XO3 internal plaO3 deletion (clone 92) | replaces_major_product_profile_of | Native PL-A/PL-D major-product profile | XO3 instead showed HS6/HS7-dominated culture-extract chromatograms, compared with PL A/D in wild type. Figures 3.19 and 3.44 support loss of the wild-type major-product pattern, not an absolute zero concentration or quantified detection limit for every mature congener. | https://freidok.uni-freiburg.de/data/3799
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| Tu6071-XO3 internal plaO3 deletion (clone 92) | accumulates | PL HS6 (C-3 alcohol; C-20 methyl) | PL HS6 was isolated from a 3-L NL111 XO3 culture. 1D/2D NMR in methanol-d4 assigns the cyclized diterpene core, gamma-butyrolactone and C-3 alcohol (H-3 3.13 ppm; C-3 79.6 ppm); both C-20/C-21 remain methyl groups. UV maximum 244 nm and retention 17.5 min were reported. The text calls 390 an m/z value while Table 3.2 calls it mass in Da; the ion/adduct is not assigned here. The missing pyrrole chromophore distinguishes it from mature PL A. | https://freidok.uni-freiburg.de/data/3799
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| Tu6071-XO3 internal plaO3 deletion (clone 92) | accumulates | PL HS7 (C-3 ketone analog of HS6) | PL HS7 was isolated alongside HS6 and NMR-characterized as its C-3 ketone analog: C-3 220.8 ppm with no attached H-3/HSQC signal. It retains the gamma-butyrolactone and C-20/C-21 methyl groups. Retention 19.9 min, UV maximum 244 nm, and source m/z/mass value 388 are reported without an ion/adduct assignment here. The redox-pair structure does not identify the enzyme responsible for C-3 oxidation or prove HS7 is a productive pathway intermediate. | https://freidok.uni-freiburg.de/data/3799
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| pS-O3co genomic-fragment complementation of XO3 | reintroduces_encoding_sequence_for | Native PlaO3 parent | pS-O3co carries a cosmid-derived O3co-F/R fragment in pSET-1cermE (EcoRI/XbaI insert, MunI/XbaI-opened vector). Native primer alignment spans 23700..24976 (1277 bp), including the final 30 bp of neighboring plaM2, complete plaO3 and downstream noncoding sequence. It is not an isolated-ORF-only experiment or expression of full PlaM2. | https://freidok.uni-freiburg.de/data/3799 |
| pS-O3co genomic-fragment complementation of XO3 | restores_detectable | Native PL-A/PL-D major-product profile | After conjugation, six clones were selected and three were cultured analytically; all three produced clearly detectable PL A and D. Figure 3.44 shows untagged rescue alongside wild type, XO3 and tagged rescue. This supports pathway function, not wild-type titer equivalence or a purified substrate-to-product assay. | https://freidok.uni-freiburg.de/data/3799
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| Recombinant N-terminal His6-PlaO3 construct | engineered_version_of | Native PlaO3 parent | The reported 421-aa N-terminal His6 fusion was expressed from pET15-O3 in E. coli BL21(DE3)pLysS and purified by Ni-NTA. Insert sequence checking is reported; the entire printed 401-aa parent matches native ABB69748.1, not the full fusion. MALDI peptide fingerprinting assigned the approximately 45-kDa band to PlaO3 with reported 35% coverage; the nearby 46-kDa band was an E. coli protein. No claim of homogeneous preparation or complete experimental sequence coverage is made. Figure 3.43(b) says PlaT3, but the adjacent PlaO3 text and MALDI analysis identify the preparation reviewed here. | https://freidok.uni-freiburg.de/data/3799 |
| pSET-O3NTag complementation of XO3 | expresses | Recombinant N-terminal His6-PlaO3 construct | The tagged coding sequence was amplified from pET-O3 using Co-XO3his-F/R and transferred into pSET-1cermE as pSET-O3NTag. Sequencing is reported to show no mutations or frameshifts in the insert. This is expression in the producer mutant, not extracellular purified-enzyme feeding. | https://freidok.uni-freiburg.de/data/3799
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| pSET-O3NTag complementation of XO3 | restores_detectable | Native PL-A/PL-D major-product profile | pSET-O3NTag restored detectable PL A/D production in XO3. The differently scaled HPLC traces in Figure 3.44 do not establish unchanged yields, kinetics, or total product profiles. The rescue shows that the fusion retains pathway function in this host context; it does not prove the tag has no effect in all conditions or demonstrate purified turnover. | https://freidok.uni-freiburg.de/data/3799
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| Native PlaO3 parent | is_inferred_to_catalyze | Inferred C-20 hydroxylation of PL HS6 | The thesis assigns PlaO3 to C-20 hydroxylation from the deletion, complementation and accumulated-metabolite structures. Its discussion explicitly proposes future in-vitro verification of PlaO3/PlaO4 selectivity. This is not the purified PlaO5 assay, does not assign C-3 oxidation to PlaO3, and does not identify native electron-transfer partners. | https://freidok.uni-freiburg.de/data/3799 |
| PL HS6 (C-3 alcohol; C-20 methyl) | is_proposed_substrate_for | Inferred C-20 hydroxylation of PL HS6 | HS6 is the proposed acceptor in Figure 4.5; the assignment is inferred, not a direct substrate-scope assay. Its C-20 methyl is distinguished from the C-21 methyl assigned to PlaO4 in the downstream proposal. | https://freidok.uni-freiburg.de/data/3799 |
| Inferred C-20 hydroxylation of PL HS6 | is_proposed_to_form | Proposed C-20-hydroxylated HS6 intermediate | The immediate C-20-hydroxylated intermediate is explicitly not detected in this study. Pyrrole attachment and later tailoring separate this proposal from mature PL A; neither the native PlaO3 protein nor its tagged rescue is claimed to make mature PL A in one catalytic step. | https://freidok.uni-freiburg.de/data/3799 |
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Causal graph — biosynthesis 12 nodes, 11 edges
Schnell 2007 joint plaT5/plaT6 construct experiments in native Tu6071. Native PlaT5/PlaT6 identities are distinct from engineered plasmids. Some integrative-vector clones increased phenalinolactone output, but the source-described empty vector also increased it and integration sites were not mapped. The episomal construct changed the A/D profile without a reported total increase. Neither an individual protein effect nor rate-limiting pathway control is demonstrated. HMBPP/DXP synthase roles are homology-based proposals, not purified activity or measured protein overexpression. The pSET-OE map-label discrepancy is retained.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Joint plaT5/plaT6 gene-introduction design | introduces_encoding_sequence_for | Native PlaT5 parent (putative HMBPP synthase) | The two genes were jointly amplified from cosmid 3-1O12, not manipulated separately. Native PlaT5 is 386 aa, DQ230532.1:32273..33433 on the positive strand. GenPept, native GenBank and MIBiG translations agree. The accession identifies the native parent, not a complete sequenced plasmid or assayed purified preparation. UniParc UPI000068F3A5 links active EMBL ABB69755.1 and inactive UniProtKB/TrEMBL Q2I774 (plaT5, taxon 355249) with zero active UniProtKB entries; no homolog was substituted. | https://freidok.uni-freiburg.de/data/3799 |
| Joint plaT5/plaT6 gene-introduction design | introduces_encoding_sequence_for | Native PlaT6 parent (putative DXP synthase) | The same joint fragment includes native PlaT6, 593 aa, DQ230532.1:33430..35211 on the positive strand, with a four-base native CDS overlap with plaT5. GenPept, GenBank and MIBiG translations agree. The native accession is not a measured construct-specific protein sequence. UniParc UPI000068F3A6 links active EMBL ABB69756.1 and inactive UniProtKB/TrEMBL Q2I773 (plaT6, taxon 355249) with zero active UniProtKB entries; an ortholog is not used as its identity. | https://freidok.uni-freiburg.de/data/3799 |
| Tu6071 pSET-OE integrative-construct clones | introduces_joint_cassette | Joint plaT5/plaT6 gene-introduction design | pSET-OE was made with the approximately 3.0-kb ORF38/39-F/R PCR product, cloned EcoRI/XbaI into MunI/XbaI-opened pSET-1cermE. Native primer alignment spans 32221..35251 (3031 bp), including the final 12 bp of plaT4, complete plaT5/plaT6 and flanking sequence, not a full third enzyme. Primer substitutions lie outside both target CDSs. The pSET-OE map on p. 188 labels the promoter-proximal short CDS T6 and next long CDS T5, opposite the native primer-supported plaT5-plaT6 order. This map-label discrepancy is not silently corrected or treated as a sequenced construct. Apramycin-resistant exconjugants were selected; the integration sites were not mapped. | https://freidok.uni-freiburg.de/data/3799 |
| Tu6071 pUo-OE episomal-construct clones | introduces_joint_cassette | Joint plaT5/plaT6 gene-introduction design | Table 5.14 describes transfer of the same approximately 3.0-kb fragment from pSET-OE into pUWLoriT through EcoRI/XbaI. pUo-OE is described as a nonintegrating high-copy plasmid; actual copy number was not quantified in the reported experiment. Thiostrepton-resistant exconjugants were selected. The pUo-OE map labels ORF38 and ORF39; p. 82 also calls the intended construct pUWL-OE. Construct introduction is not a measurement of increased transcript or protein abundance. | https://freidok.uni-freiburg.de/data/3799
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| Tu6071 pSET-OE integrative-construct clones | has_observed_profile | Variable PL-A/PL-D production across pSET-OE clones | Ten XHM clones were compared with ten wild-type cultures by HPLC/DAD peak area, then cultures were repeated. Clones 2/5/6/8/10 initially showed higher reported output; clone 9 joined that group in the repeat, while other clones were lower. PL D predominated in many clones; XHM6 instead favored PL A. XHM2/8 lagged wild type for A, and XHM5/10 showed early D followed by later A. Figures 3.38-3.39 plot D time courses for selected clones, not all-clone A gains. XHM5 was used for subsequent feeding experiments, not as proof of enzyme-specific rate control. These AUC observations are not calibrated titers, fluxes, or statistically established yield effects. | https://freidok.uni-freiburg.de/data/3799
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| Tu6071 source-described empty pSET-1cermE control | has_observed_profile | Production increase with pSET-1cermE vector control | The source-described empty pSET-1cermE control also increased phenalinolactone production. No control-specific numerical titers or mapped integration site are supplied in this account. Table 5.13 describes pSET-1cermE as containing urdGT1c, while the results call it an empty control; its exact control-backbone sequence is not independently established here. Do not silently rename this comparator as bare pSET152 or infer an extra active glycosyltransferase from vector nomenclature. | https://freidok.uni-freiburg.de/data/3799
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| Tu6071 source-described empty pSET-1cermE control | limits_causal_attribution_of | Variable PL-A/PL-D production across pSET-OE clones | Because the empty-vector comparator also increased production and integration sites were untested, the phenotype does not isolate a PlaT5/PlaT6 effect. Activation of cluster or regulator expression by the ermE promoter at a favorable integration site is the source hypothesis, not an observed locus-specific mechanism. The source explicitly says rate-limiting IPP-synthesis control by these enzymes was not shown. This is lack of such proof, not evidence that either protein lacks pathway function. | https://freidok.uni-freiburg.de/data/3799
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| Tu6071 pUo-OE episomal-construct clones | has_observed_profile | pUo-OE D-dominant A/D profile without reported total increase | Three pUo-OE clones, three empty-pUWLoriT controls and three wild-type cultures were compared by HPLC/DAD AUC and cultured a second time. The source reports roughly similar total A+D output in pUo-OE and wild type, but D was dominant in pUo-OE and A in wild type. Figures 3.40-3.41 show separate time-dependent A/D signals; they do not prove quantitative equivalence, fixed ratios at all times or improved A yield. The shared cassette does not distinguish the individual contributions of PlaT5 and PlaT6. | https://freidok.uni-freiburg.de/data/3799
|
| Tu6071 empty pUWLoriT control | shows_lower_reported_output_than | pUo-OE D-dominant A/D profile without reported total increase | The empty-pUWLoriT control cultures were reported to produce somewhat less phenalinolactone than both wild type and pUo-OE. The comparator is a separate control group in each of the two culture runs, not the same strain as the integrative-vector control. No calibrated concentration, exact effect size, variance model or individual-enzyme causal effect is assigned. | https://freidok.uni-freiburg.de/data/3799
|
| Native PlaT5 parent (putative HMBPP synthase) | has_proposed_activity | Proposed HMBPP synthesis in the MEP pathway | Sequence comparisons and the MEP-pathway scheme motivate a putative HMBPP synthase role at a late precursor-supply step. This is not a purified PlaT5 reaction assay, measured cofactor requirement, native electron-partner assignment or direct conversion to mature phenalinolactone A. The tested co-introduction does not demonstrate that PlaT5 is rate-limiting. | https://freidok.uni-freiburg.de/data/3799 |
| Native PlaT6 parent (putative DXP synthase) | has_proposed_activity | Proposed DXP synthesis in the MEP pathway | Sequence comparisons motivate a putative DXP synthase assignment. The source pathway scheme describes the pyruvate plus glyceraldehyde-3-phosphate condensation at the entry to the MEP pathway; it does not assay purified PlaT6, establish its kinetic parameters or show direct synthesis of mature phenalinolactone A. The joint construct does not demonstrate independent PlaT6 activity or rate limitation. | https://freidok.uni-freiburg.de/data/3799 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 14 nodes, 21 edges
Myronovskyi et al. 2011 regulator genetics in the phenalinolactone-producing Tu6071 locus. Marker-free internal disruptions and integrated promoter-GusA reporters are separate from exact native PlaR1/PlaR2/PlaR3 proteins. Reporter changes are not direct DNA binding, native RNA measurements or native protein abundance. Production results are reported as phenalinolactone without a congener-resolved titer here; do not generalize them to every congener. Results report trace production for deltaR2, despite cessation wording in Discussion. The plaM1/plaM2 text discrepancy and construct limitations remain explicit. Quantities below come from paper text; Figure 6 bars/error bars were not independently digitized or visually verified.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Tu6071 deltaR1 marker-free internal disruption | targets_gene_encoding | Native PlaR1 parent (SARP-family annotation) | Native parent ABB69733.1: 265 aa, DQ230532.1:complement(6556..7353), Tu6071 taxon 355249. Native GenBank, MIBiG and GenPept translations match. The study used pKGLP2-based replacement with a loxP-flanked apramycin marker, followed by Cre-mediated marker excision; marker-free is not a claim of scarless editing. PCR verified R1/R3 replacements and Southern hybridization R2. Curator alignment of Table 2 inner homology arms places an internal native interval between them at 6615..7201 (587 bp); residual coding sequence remains. These are native-coordinate reconstructions, not sequenced mutant alleles or measured residual protein products. Q2I751 is deleted as redundant; UniParc UPI000068F38F retains the exact sequence but no active UniProtKB cross-reference in the inspected entry. The exact 265-aa native parent is grounded, not an inferred mutant protein. | DOI:10.1128/AEM.00434-11 |
| Tu6071 deltaR2 marker-free internal disruption | targets_gene_encoding | Native PlaR2 parent (TetR-family annotation) | Native parent ABB69734.1: 200 aa, DQ230532.1:complement(7806..8408), Tu6071 taxon 355249. Native GenBank, MIBiG and GenPept translations match. The study used pKGLP2-based replacement with a loxP-flanked apramycin marker, followed by Cre-mediated marker excision; marker-free is not a claim of scarless editing. PCR verified R1/R3 replacements and Southern hybridization R2. Curator alignment of Table 2 inner homology arms places an internal native interval between them at 7829..8386 (558 bp); residual coding sequence remains. These are native-coordinate reconstructions, not sequenced mutant alleles or measured residual protein products. Q2I750 is deleted as redundant; UniParc UPI000068F390 retains the exact sequence but no active UniProtKB cross-reference in the inspected entry. A TetR-family annotation does not by itself establish direct repression of these promoters. | DOI:10.1128/AEM.00434-11 |
| Tu6071 deltaR3 marker-free internal disruption | targets_gene_encoding | Native PlaR3 parent (HxlR-related interpretation) | Native parent ABB69766.1: 119 aa, DQ230532.1:complement(47334..47693), Tu6071 taxon 355249. Native GenBank, MIBiG and GenPept translations match. The study used pKGLP2-based replacement with a loxP-flanked apramycin marker, followed by Cre-mediated marker excision; marker-free is not a claim of scarless editing. PCR verified R1/R3 replacements and Southern hybridization R2. Curator alignment of Table 2 inner homology arms places an internal native interval between them at 47381..47678 (298 bp); residual coding sequence remains. These are native-coordinate reconstructions, not sequenced mutant alleles or measured residual protein products. The legacy GenPept protein name says putative TetR regulator, whereas the paper and the GenPept domain annotation support an HxlR-related interpretation. Neither demonstrates a formaldehyde response. Methods describe R3.2 cloning with XbaI/BamHI, while Table 2 primers carry BamHI/EcoRV tails; the exact engineered junction is not asserted. A bounded official gene/taxon and EMBL cross-reference query found no active exact UniProtKB entry. | DOI:10.1128/AEM.00434-11 |
| Tu6071 deltaR1 marker-free internal disruption | has_reported_production_phenotype | Phenalinolactone production reported abolished in deltaR1 | LC-MS analysis reports abolished phenalinolactone production in the marker-free deltaR1 strain. Production cultures used NL111. This is the reported detection phenotype, not a numerical zero titer, quantified detection limit, purified PlaR1 reaction or direct promoter binding. The assayed congener breakdown is not given in this account; no complementation-based exclusion of polar effects is asserted. | DOI:10.1128/AEM.00434-11
|
| Tu6071 deltaR2 marker-free internal disruption | has_reported_production_phenotype | Trace phenalinolactone production reported in deltaR2 | Results report trace phenalinolactone production in deltaR2, whereas Discussion calls it cessation. Preserve this disagreement and the trace result; do not encode complete abolition. NL111 production cultures and LC-MS are distinct from TSB-grown promoter-reporter assays. No exact residual titer, congener ratio or universal PlaR2 requirement is assigned. | DOI:10.1128/AEM.00434-11
|
| Tu6071 deltaR3 marker-free internal disruption | has_reported_production_phenotype | Phenalinolactone production reported unchanged in deltaR3 | The LC-MS account describes deltaR3 phenalinolactone production as the same as wild type under the tested conditions. This is bounded negative evidence, not statistical equivalence, all-condition dispensability or absence of regulatory effects. The same mutant changes reporter outputs. | DOI:10.1128/AEM.00434-11
|
| Tu6071 deltaR1 marker-free internal disruption | has_reported_promoter_reporter_effect | Engineered plaR1 promoter-GusA reporter activity | Reported activity is 168% of the corresponding wild-type reporter; wild type is the relative 100% reference. The pGUS promoter-GusA fusion was integrated in Tu6071 and assayed in TSB-grown cultures by p-nitrophenyl-glucuronide hydrolysis (OD415, 20 min at 37 C). The study states means with standard errors from three independent experiments. No significance test or exact reporter-culture harvest time is inferred here. Upstream aadA/T4 terminators were designed to block readthrough. The native homology span of the cloned promoter fragment is 7365..7864 (-) by curator primer alignment, not an exact minimal promoter or regulator-binding site. This assay supports a regulatory effect in an engineered reporter context, not direct binding or native protein abundance. | DOI:10.1128/AEM.00434-11 |
| Tu6071 deltaR1 marker-free internal disruption | has_reported_promoter_reporter_effect | Engineered plaR2 promoter-GusA reporter activity | The text reports no detected change versus the corresponding wild-type reporter; do not replace that statement with an invented exact 100% measurement. The pGUS promoter-GusA fusion was integrated in Tu6071 and assayed in TSB-grown cultures by p-nitrophenyl-glucuronide hydrolysis (OD415, 20 min at 37 C). The study states means with standard errors from three independent experiments. No significance test or exact reporter-culture harvest time is inferred here. Upstream aadA/T4 terminators were designed to block readthrough. The native homology span of the cloned promoter fragment is 8428..8934 (-) by curator primer alignment, not an exact minimal promoter or regulator-binding site. This assay supports a regulatory effect in an engineered reporter context, not direct binding or native protein abundance. | DOI:10.1128/AEM.00434-11 |
| Tu6071 deltaR1 marker-free internal disruption | has_reported_promoter_reporter_effect | Engineered plaR3 promoter-GusA reporter activity | Reported activity is 35.5% of the corresponding wild-type reporter; wild type is the relative 100% reference. The pGUS promoter-GusA fusion was integrated in Tu6071 and assayed in TSB-grown cultures by p-nitrophenyl-glucuronide hydrolysis (OD415, 20 min at 37 C). The study states means with standard errors from three independent experiments. No significance test or exact reporter-culture harvest time is inferred here. Upstream aadA/T4 terminators were designed to block readthrough. The native homology span of the cloned promoter fragment is 47714..48213 (-) by curator primer alignment, not an exact minimal promoter or regulator-binding site. This assay supports a regulatory effect in an engineered reporter context, not direct binding or native protein abundance. | DOI:10.1128/AEM.00434-11 |
| Tu6071 deltaR1 marker-free internal disruption | has_reported_promoter_reporter_effect | Engineered plaM2 promoter-GusA reporter activity | Reported activity is 81.5% of the corresponding wild-type reporter; wild type is the relative 100% reference. The pGUS promoter-GusA fusion was integrated in Tu6071 and assayed in TSB-grown cultures by p-nitrophenyl-glucuronide hydrolysis (OD415, 20 min at 37 C). The study states means with standard errors from three independent experiments. No significance test or exact reporter-culture harvest time is inferred here. Upstream aadA/T4 terminators were designed to block readthrough. The native homology span of the cloned promoter fragment is 22371..22863 (+) by curator primer alignment, not an exact minimal promoter or regulator-binding site. This assay supports a regulatory effect in an engineered reporter context, not direct binding or native protein abundance. One Results sentence names plaM1, but Table 1, Table 2, the methods, reporter labels and detailed results identify plaM2. Primer alignment supports the plaM2 region; no plaM1 regulatory edge is inferred. | DOI:10.1128/AEM.00434-11 |
| Tu6071 deltaR1 marker-free internal disruption | has_reported_promoter_reporter_effect | Engineered plaO2 promoter-GusA reporter activity | Reported activity is 58% of the corresponding wild-type reporter; wild type is the relative 100% reference. The pGUS promoter-GusA fusion was integrated in Tu6071 and assayed in TSB-grown cultures by p-nitrophenyl-glucuronide hydrolysis (OD415, 20 min at 37 C). The study states means with standard errors from three independent experiments. No significance test or exact reporter-culture harvest time is inferred here. Upstream aadA/T4 terminators were designed to block readthrough. The native homology span of the cloned promoter fragment is 22650..23150 (-) by curator primer alignment, not an exact minimal promoter or regulator-binding site. This assay supports a regulatory effect in an engineered reporter context, not direct binding or native protein abundance. | DOI:10.1128/AEM.00434-11 |
| Tu6071 deltaR2 marker-free internal disruption | has_reported_promoter_reporter_effect | Engineered plaR1 promoter-GusA reporter activity | The text reports no detected change versus the corresponding wild-type reporter; do not replace that statement with an invented exact 100% measurement. The pGUS promoter-GusA fusion was integrated in Tu6071 and assayed in TSB-grown cultures by p-nitrophenyl-glucuronide hydrolysis (OD415, 20 min at 37 C). The study states means with standard errors from three independent experiments. No significance test or exact reporter-culture harvest time is inferred here. Upstream aadA/T4 terminators were designed to block readthrough. The native homology span of the cloned promoter fragment is 7365..7864 (-) by curator primer alignment, not an exact minimal promoter or regulator-binding site. This assay supports a regulatory effect in an engineered reporter context, not direct binding or native protein abundance. | DOI:10.1128/AEM.00434-11 |
| Tu6071 deltaR2 marker-free internal disruption | has_reported_promoter_reporter_effect | Engineered plaR2 promoter-GusA reporter activity | The text reports no detected change versus the corresponding wild-type reporter; do not replace that statement with an invented exact 100% measurement. The pGUS promoter-GusA fusion was integrated in Tu6071 and assayed in TSB-grown cultures by p-nitrophenyl-glucuronide hydrolysis (OD415, 20 min at 37 C). The study states means with standard errors from three independent experiments. No significance test or exact reporter-culture harvest time is inferred here. Upstream aadA/T4 terminators were designed to block readthrough. The native homology span of the cloned promoter fragment is 8428..8934 (-) by curator primer alignment, not an exact minimal promoter or regulator-binding site. This assay supports a regulatory effect in an engineered reporter context, not direct binding or native protein abundance. | DOI:10.1128/AEM.00434-11 |
| Tu6071 deltaR2 marker-free internal disruption | has_reported_promoter_reporter_effect | Engineered plaR3 promoter-GusA reporter activity | Reported activity is 19% of the corresponding wild-type reporter; wild type is the relative 100% reference. The pGUS promoter-GusA fusion was integrated in Tu6071 and assayed in TSB-grown cultures by p-nitrophenyl-glucuronide hydrolysis (OD415, 20 min at 37 C). The study states means with standard errors from three independent experiments. No significance test or exact reporter-culture harvest time is inferred here. Upstream aadA/T4 terminators were designed to block readthrough. The native homology span of the cloned promoter fragment is 47714..48213 (-) by curator primer alignment, not an exact minimal promoter or regulator-binding site. This assay supports a regulatory effect in an engineered reporter context, not direct binding or native protein abundance. | DOI:10.1128/AEM.00434-11 |
| Tu6071 deltaR2 marker-free internal disruption | has_reported_promoter_reporter_effect | Engineered plaM2 promoter-GusA reporter activity | Reported activity is 46% of the corresponding wild-type reporter; wild type is the relative 100% reference. The pGUS promoter-GusA fusion was integrated in Tu6071 and assayed in TSB-grown cultures by p-nitrophenyl-glucuronide hydrolysis (OD415, 20 min at 37 C). The study states means with standard errors from three independent experiments. No significance test or exact reporter-culture harvest time is inferred here. Upstream aadA/T4 terminators were designed to block readthrough. The native homology span of the cloned promoter fragment is 22371..22863 (+) by curator primer alignment, not an exact minimal promoter or regulator-binding site. This assay supports a regulatory effect in an engineered reporter context, not direct binding or native protein abundance. One Results sentence names plaM1, but Table 1, Table 2, the methods, reporter labels and detailed results identify plaM2. Primer alignment supports the plaM2 region; no plaM1 regulatory edge is inferred. | DOI:10.1128/AEM.00434-11 |
| Tu6071 deltaR2 marker-free internal disruption | has_reported_promoter_reporter_effect | Engineered plaO2 promoter-GusA reporter activity | Reported activity is 58% of the corresponding wild-type reporter; wild type is the relative 100% reference. The pGUS promoter-GusA fusion was integrated in Tu6071 and assayed in TSB-grown cultures by p-nitrophenyl-glucuronide hydrolysis (OD415, 20 min at 37 C). The study states means with standard errors from three independent experiments. No significance test or exact reporter-culture harvest time is inferred here. Upstream aadA/T4 terminators were designed to block readthrough. The native homology span of the cloned promoter fragment is 22650..23150 (-) by curator primer alignment, not an exact minimal promoter or regulator-binding site. This assay supports a regulatory effect in an engineered reporter context, not direct binding or native protein abundance. | DOI:10.1128/AEM.00434-11 |
| Tu6071 deltaR3 marker-free internal disruption | has_reported_promoter_reporter_effect | Engineered plaR1 promoter-GusA reporter activity | The text reports no detected change versus the corresponding wild-type reporter; do not replace that statement with an invented exact 100% measurement. The pGUS promoter-GusA fusion was integrated in Tu6071 and assayed in TSB-grown cultures by p-nitrophenyl-glucuronide hydrolysis (OD415, 20 min at 37 C). The study states means with standard errors from three independent experiments. No significance test or exact reporter-culture harvest time is inferred here. Upstream aadA/T4 terminators were designed to block readthrough. The native homology span of the cloned promoter fragment is 7365..7864 (-) by curator primer alignment, not an exact minimal promoter or regulator-binding site. This assay supports a regulatory effect in an engineered reporter context, not direct binding or native protein abundance. | DOI:10.1128/AEM.00434-11 |
| Tu6071 deltaR3 marker-free internal disruption | has_reported_promoter_reporter_effect | Engineered plaR2 promoter-GusA reporter activity | Reported activity is 207% of the corresponding wild-type reporter; wild type is the relative 100% reference. The pGUS promoter-GusA fusion was integrated in Tu6071 and assayed in TSB-grown cultures by p-nitrophenyl-glucuronide hydrolysis (OD415, 20 min at 37 C). The study states means with standard errors from three independent experiments. No significance test or exact reporter-culture harvest time is inferred here. Upstream aadA/T4 terminators were designed to block readthrough. The native homology span of the cloned promoter fragment is 8428..8934 (-) by curator primer alignment, not an exact minimal promoter or regulator-binding site. This assay supports a regulatory effect in an engineered reporter context, not direct binding or native protein abundance. | DOI:10.1128/AEM.00434-11 |
| Tu6071 deltaR3 marker-free internal disruption | has_reported_promoter_reporter_effect | Engineered plaR3 promoter-GusA reporter activity | Reported activity is 63% of the corresponding wild-type reporter; wild type is the relative 100% reference. The pGUS promoter-GusA fusion was integrated in Tu6071 and assayed in TSB-grown cultures by p-nitrophenyl-glucuronide hydrolysis (OD415, 20 min at 37 C). The study states means with standard errors from three independent experiments. No significance test or exact reporter-culture harvest time is inferred here. Upstream aadA/T4 terminators were designed to block readthrough. The native homology span of the cloned promoter fragment is 47714..48213 (-) by curator primer alignment, not an exact minimal promoter or regulator-binding site. This assay supports a regulatory effect in an engineered reporter context, not direct binding or native protein abundance. | DOI:10.1128/AEM.00434-11 |
| Tu6071 deltaR3 marker-free internal disruption | has_reported_promoter_reporter_effect | Engineered plaM2 promoter-GusA reporter activity | Reported activity is 209% of the corresponding wild-type reporter; wild type is the relative 100% reference. The pGUS promoter-GusA fusion was integrated in Tu6071 and assayed in TSB-grown cultures by p-nitrophenyl-glucuronide hydrolysis (OD415, 20 min at 37 C). The study states means with standard errors from three independent experiments. No significance test or exact reporter-culture harvest time is inferred here. Upstream aadA/T4 terminators were designed to block readthrough. The native homology span of the cloned promoter fragment is 22371..22863 (+) by curator primer alignment, not an exact minimal promoter or regulator-binding site. This assay supports a regulatory effect in an engineered reporter context, not direct binding or native protein abundance. One Results sentence names plaM1, but Table 1, Table 2, the methods, reporter labels and detailed results identify plaM2. Primer alignment supports the plaM2 region; no plaM1 regulatory edge is inferred. | DOI:10.1128/AEM.00434-11 |
| Tu6071 deltaR3 marker-free internal disruption | has_reported_promoter_reporter_effect | Engineered plaO2 promoter-GusA reporter activity | Reported activity is 139% of the corresponding wild-type reporter; wild type is the relative 100% reference. The pGUS promoter-GusA fusion was integrated in Tu6071 and assayed in TSB-grown cultures by p-nitrophenyl-glucuronide hydrolysis (OD415, 20 min at 37 C). The study states means with standard errors from three independent experiments. No significance test or exact reporter-culture harvest time is inferred here. Upstream aadA/T4 terminators were designed to block readthrough. The native homology span of the cloned promoter fragment is 22650..23150 (-) by curator primer alignment, not an exact minimal promoter or regulator-binding site. This assay supports a regulatory effect in an engineered reporter context, not direct binding or native protein abundance. | DOI:10.1128/AEM.00434-11 |
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Causal graph — biosynthesis 6 nodes, 6 edges
Myronovskyi et al. 2011 PlaM2-HL4-GusA reporter in native Tu6071. The exact native PlaM2 parent is not the engineered fusion. GusA signal tracks a translational-reporter construct, not native methyltransferase turnover or an absolute protein concentration. Reporter and six-day phenalinolactone productivity rank media differently. Congener-specific titers and native methylation chemistry are not assigned. Numerical values are reported text values; Figure 8 was not independently digitized or visually verified.
| Subject | Predicate | Object | Notes | Evidence |
|---|---|---|---|---|
| Engineered PlaM2-HL4-GusA fusion | uses_native_parent_sequence_from | Native PlaM2 parent (putative methyltransferase) | PlaM2 native parent ABB69747.1 is 281 aa, DQ230532.1:22884..23729 (+), Tu6071 taxon 355249; GenPept, GenBank and MIBiG translations match. The forward primer includes the upstream region and the M2gusfus reverse homology ends at 23726, before the native stop codon. The gene was fused through HL4 to GusA lacking its own start codon. The parent accession is not an identifier for this fusion or its complete junction. GenPept retains a putative rRNA-methyltransferase name; this reporter study does not demonstrate an rRNA or natural-product substrate. UniParc UPI000068F39D links active EMBL ABB69747.1 and inactive UniProtKB/TrEMBL Q2I758 (plaM2, taxon 355249) with zero active UniProtKB entries. | DOI:10.1128/AEM.00434-11 |
| Tu6071 pGUSHL4plaM2aadA reporter strain | carries_expression_construct_for | Engineered PlaM2-HL4-GusA fusion | The pTESa-based integrative pGUSHL4aadA vector contains an HL4 linker and start-codon-free gusA. PlaM2 without its stop codon was inserted through XbaI/EcoRV to produce pGUSHL4plaM2aadA, transferred into Tu6071. The upstream aadA cassette with T4 terminators limits readthrough by design. Construct carriage and GusA activity do not establish native PlaM2 catalytic competence or a measured copy number. | DOI:10.1128/AEM.00434-11
|
| Promoterless plaM2 fusion control construct | is_promoterless_comparator_for | Tu6071 pGUSHL4plaM2aadA reporter strain | The source constructed pGUSHL4plaM2aadAcontr with a promoterless plaM2 PCR fragment as control. The m2fuscontrxba forward homology starts at 22852, rather than the experimental upstream 22371 region. This identifies the described control design, not a quantified zero background signal or a complete sequenced plasmid. | DOI:10.1128/AEM.00434-11 |
| Tu6071 pGUSHL4plaM2aadA reporter strain | has_measured_reporter_profile | Medium- and time-dependent PlaM2-GusA reporter signal | Text reports 24-h GusA activities of 0.3, 0.07, 0.0065 and 0.047 mU/mg in SG, TSB, NL111 and HA, respectively. Follow-up in SG/NL111 gave 0.49/0.27 mU/mg on day 3 and 0.44/0.27 mU/mg on day 5. Units are retained as reported; these are not molar PlaM2 abundance or methyl-transfer rates. Lysate p-nitrophenyl-glucuronide hydrolysis was read at OD415 after 20 min at 37 C; the paper specifies three independent experiments with means and standard errors. Mass-normalization details and Figure 8 error bars are not re-established from the inspected text. Lower day-5 SG activity was attributed speculatively to autolysis, not supported here by a separate lysis measurement. | DOI:10.1128/AEM.00434-11
|
| Tu6071 pGUSHL4plaM2aadA reporter strain | has_reported_medium_productivity_profile | Six-day medium-dependent phenalinolactone output | Six-day extracts from SG, HA, TSB and NL111 were compared by LC-MS. The text reports no detected phenalinolactone in TSB, about four times the SG amount in HA and about five times in NL111, recalculated to equal wet biomass. These are relative phenalinolactone outputs, not absolute congener-specific titers or equivalence of culture growth. The production sampling time differs from the reporter time course. | DOI:10.1128/AEM.00434-11
|
| Medium- and time-dependent PlaM2-GusA reporter signal | does_not_rank_media_like | Six-day medium-dependent phenalinolactone output | SG gives the larger PlaM2-GusA signals, while NL111 gives the largest reported six-day product output. Reporter expression therefore does not identify the highest-productivity medium in this experiment. Precursor/cofactor limitation is the authors' proposed explanation, not a direct measurement of limiting metabolites or a PlaM2 reaction. | DOI:10.1128/AEM.00434-11
|
Every edge carries its own citation; an uncited edge is refused by the corpus tests.
Explore across X-Mech
AntibioticMech (phenalinolactone A) ↗
MediaIngredientMech (naturalproductmech:mibig-e29f83c77b) ↗
TaxonMech: Streptomyces sp. Tu6071 (NCBITaxon:355249) ↗
PathwayMech (mibig:BGC0000654) ↗
Provenance
| Source concept | Source | Version |
|---|---|---|
| BGC0000654 | MIBIG | 4 |
This page is generated from data/natural_products/unclassified/phenalinolactone-a.yaml, which is generated from the committed inventories. Neither is edited by hand.