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Cited by 28Microbial Natural Products and BiosynthesisMicrobial Metabolic Engineering and Bioproductionbiodegradable polymer synthesis and properties

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Assessing and harnessing updated polyketide synthase modules through combinatorial engineering

Open access
Ray, Katherine A., Lutgens, Joshua D., Bista, Ramesh, Zhang, Jie, Desai, Ronak R., Hirsch, Melissa, Miyazawa, Takeshi, Cordova, Antonio, Keatinge-Clay, Adrian T.·2024·Nature Communications
DOI 10.1038/s41467-024-50844-6PMC PMC11294587

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Abstract

The modular nature of polyketide assembly lines and the significance of their products make them prime targets for combinatorial engineering. While short synthases constructed using the recently updated module boundary have been shown to outperform those using the traditional boundary, larger synthases constructed using the updated boundary have not been investigated. Here we describe our design and implementation of a BioBricks-like platform to rapidly construct 5 triketide, 25 tetraketide, and 125 pentaketide synthases from the updated modules of the Pikromycin synthase. Every combinatorial possibility of modules 2-6 inserted between the first and last modules of the native synthase was constructed and assayed. Anticipated products were observed from 60% of the triketide synthases, 32% of the tetraketide synthases, and 6.4% of the pentaketide synthases. Ketosynthase gatekeeping and module-skipping were determined to be the principal impediments to obtaining functional synthases. The platform was also used to create functional hybrid synthases through the incorporation of modules from the Erythromycin, Spinosyn, and Rapamycin assembly lines. The relaxed gatekeeping observed from a ketosynthase in the Rapamycin synthase is especially encouraging in the quest to produce designer polyketides.

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Figures

Fig. 1 The pikromycin PKS. The 7 modules (P1–P7, updated definition) of this natural polyketide assembly line collaborate to biosynthesize the heptaketide precursor of pikromycin, narbonolide. AT acyltransferase, KR ketoreductase, DH dehydratase, ER enoylreductase, ACP acyl carrier protein, KS ketosynthase, TE thioesterase, KSQ priming KS, KR0 epimerase, CDD & NDD docking domain motifs.
Fig. 1 The pikromycin PKS. The 7 modules (P1–P7, updated definition) of this natural polyketide assembly line collaborate to biosynthesize the heptaketide precursor of pikromycin, narbonolide. AT acyltransferase, KR ketoreductase, DH dehydratase, ER enoylreductase, ACP acyl carrier protein, KS ketosynthase, TE thioesterase, KSQ priming KS, KR0 epimerase, CDD & NDD docking domain motifs.CC BY-NC-ND · PubMed Central
Fig. 2 Platform for cloning modules and constructing PKS expression plasmids. a Cloning plasmids were made by inserting synthetic DNA between the HindIII and XbaI sites of pUC19. The insertion of amplicons encoding the N- & C-terminal portions of a module, such as the 6th module of the pikromycin synthase (P6), places cognate CDD and NDD docking motifs between its ACP and KS domains. b The P1-P7 expression plasmid was made by inserting DNA encoding P1, HindIII-N12-SpeI, and P7 between the KpnI and AvrII sites of pCDF-1b. Module-encoding DNA cut from the cloning plasmids with HindIII and XbaI can be sequentially inserted to construct synthases like P1-P2-P4-P6-P7 (Supplementary Fig. 1, Supplementary Data 1).
Fig. 2 Platform for cloning modules and constructing PKS expression plasmids. a Cloning plasmids were made by inserting synthetic DNA between the HindIII and XbaI sites of pUC19. The insertion of amplicons encoding the N- & C-terminal portions of a module, such as the 6th module of the pikromycin synthase (P6), places cognate CDD and NDD docking motifs between its ACP and KS domains. b The P1-P7 expression plasmid was made by inserting DNA encoding P1, HindIII-N12-SpeI, and P7 between the KpnI and AvrII sites of pCDF-1b. Module-encoding DNA cut from the cloning plasmids with HindIII and XbaI can be sequentially inserted to construct synthases like P1-P2-P4-P6-P7 (Supplementary Fig. 1, Supplementary Data 1).CC BY-NC-ND · PubMed Central
Fig. 3 Polyketides anticipated from the P1-X-P7, P1-X-Y-P7, and P1-X-Y-Z-P7 synthases. Green background for polyketides 1–19 indicates that the anticipated m/z was detected by high-resolution mass spectrometry (Supplementary Figs. 3–60 and Supplementary Tables 3 and 4). Stereochemistries are based on how modules operate within the pikromycin synthase. Because synthases in which P3 or P5 is the penultimate module generate β-keto acids that undergo spontaneous decarboxylation, their decarboxylated products are shown.
Fig. 3 Polyketides anticipated from the P1-X-P7, P1-X-Y-P7, and P1-X-Y-Z-P7 synthases. Green background for polyketides 1–19 indicates that the anticipated m/z was detected by high-resolution mass spectrometry (Supplementary Figs. 3–60 and Supplementary Tables 3 and 4). Stereochemistries are based on how modules operate within the pikromycin synthase. Because synthases in which P3 or P5 is the penultimate module generate β-keto acids that undergo spontaneous decarboxylation, their decarboxylated products are shown.CC BY-NC-ND · PubMed Central
Fig. 4 Polyketide products characterized by NMR and crystallography. a ¹H NMR spectrum of 4, the anticipated product of P1-P2-P3-P7. b Crystal structure of 17 (absolute stereochemistry), the anticipated product of P1-P2-P3-P6-P7 (CCDC number 2278377). AT acyltransferase, KR ketoreductase, DH dehydratase, ER enoylreductase, ACP acyl carrier protein, KS ketosynthase, TE thioesterase, KSQ priming KS, CDD & NDD docking domain motifs.
Fig. 4 Polyketide products characterized by NMR and crystallography. a ¹H NMR spectrum of 4, the anticipated product of P1-P2-P3-P7. b Crystal structure of 17 (absolute stereochemistry), the anticipated product of P1-P2-P3-P6-P7 (CCDC number 2278377). AT acyltransferase, KR ketoreductase, DH dehydratase, ER enoylreductase, ACP acyl carrier protein, KS ketosynthase, TE thioesterase, KSQ priming KS, CDD & NDD docking domain motifs.CC BY-NC-ND · PubMed Central
Fig. 5 Hybrid synthases. The 6th module of the erythromycin synthase, E6, functions equivalently to P6 in the hybrid synthases shown in the top two rows. The 2nd module of the spinosyn synthase, S2, collaborates with pikromycin modules in P1-S2-P7 to yield the expected triketide lactone, 20. As no pikromycin module was observed to function immediately downstream of P6, the 4th module of the rapamycin synthase, R4, hypothesized to contain a more substrate-permissive KS, was placed downstream of P6 in P1-P6-R4-P7. Results suggest the anticipated tetraketide, 21, is produced (Supplementary Figs. 82 and 83). That P1-P2-R4-P7 apparently also produces its anticipated tetraketide, 5, indicates that RapKS4 can accept stereoisomeric intermediates with opposite stereochemistries at the γ- and δ-carbons and is relatively promiscuous, as hypothesized. AT acyltransferase, KR ketoreductase, DH dehydratase, ER enoylreductase, ACP acyl carrier protein, KS ketosynthase, TE thioesterase, KSQ priming KS, KR0 epimerase in P4 but KR with unknown function in R4, DH0 DH with unknown function; ER0 ER with unknown function, CDD & NDD docking domain motifs.
Fig. 5 Hybrid synthases. The 6th module of the erythromycin synthase, E6, functions equivalently to P6 in the hybrid synthases shown in the top two rows. The 2nd module of the spinosyn synthase, S2, collaborates with pikromycin modules in P1-S2-P7 to yield the expected triketide lactone, 20. As no pikromycin module was observed to function immediately downstream of P6, the 4th module of the rapamycin synthase, R4, hypothesized to contain a more substrate-permissive KS, was placed downstream of P6 in P1-P6-R4-P7. Results suggest the anticipated tetraketide, 21, is produced (Supplementary Figs. 82 and 83). That P1-P2-R4-P7 apparently also produces its anticipated tetraketide, 5, indicates that RapKS4 can accept stereoisomeric intermediates with opposite stereochemistries at the γ- and δ-carbons and is relatively promiscuous, as hypothesized. AT acyltransferase, KR ketoreductase, DH dehydratase, ER enoylreductase, ACP acyl carrier protein, KS ketosynthase, TE thioesterase, KSQ priming KS, KR0 epimerase in P4 but KR with unknown function in R4, DH0 DH with unknown function; ER0 ER with unknown function, CDD & NDD docking domain motifs.CC BY-NC-ND · PubMed Central
Fig. 6 Apparent gatekeeping of intermediates by pikromycin KSs. On the left, stereodiagrams show how native polyketide intermediates may be bound to the reactive cysteines of PikKS2–PikKS6 (KS structures generated by AlphaFold). The α-, β-, γ-, and δ-carbons of the intermediates as well as important gatekeeping residues are labeled15. On the right, intermediates apparently accepted or excluded by KSs within the engineered PKSs are tabulated.
Fig. 6 Apparent gatekeeping of intermediates by pikromycin KSs. On the left, stereodiagrams show how native polyketide intermediates may be bound to the reactive cysteines of PikKS2–PikKS6 (KS structures generated by AlphaFold). The α-, β-, γ-, and δ-carbons of the intermediates as well as important gatekeeping residues are labeled15. On the right, intermediates apparently accepted or excluded by KSs within the engineered PKSs are tabulated.CC BY-NC-ND · PubMed Central

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Authors
Ray, K. A., Lutgens, J. D., Bista, R., Zhang, J., Desai, R. R., Hirsch, M., Miyazawa, T., Cordova, A., & Keatinge-Clay, A. T.
Affiliations
The University of Texas at Austin (US)
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2024-08-01
Volume
15
Issue
1
Pages
6485-6485
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-024-50844-6
PMC
PMC11294587
OpenAlex
W4401220553
Cited by
28
References
54
Citations by year
2026: 14 · 2025: 13 · 2024: 1
FWCI
7.95
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by-nc-nd)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (GM145992)
  • Welch Foundation (F-1712)
  • National Science Foundation (MRSEC Cooperative Agreement No. DMR-1720595)
Topics
  • Life Sciences › Immunology and Microbiology › Applied Microbiology and Biotechnology › Microbial Natural Products and Biosynthesis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
  • Physical Sciences › Materials Science › Biomaterials › biodegradable polymer synthesis and properties
Keywords
polyketide synthase, combinatorial engineering, module boundaries, erythromycin, spinosyns

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