Student or educator? Sign up with your school email and get 6 months of Pro free.
CoChat
Sign Up
PreprintCited by 1Microbial Natural Products and BiosynthesisMicrobial Metabolic Engineering and BioproductionLipid metabolism and biosynthesis

The DOI matches its Crossref record; 1 of the 1 references it cites were found in a registry or an index.

Repurposing a Fully-Reducing Polyketide Synthase for 2-Methyl Guerbet-like Lipid Production

Open access
Herrera, Michael Andrés, McColm, Stephen, Craigie, Louise-Marie, Simpson, Joanna, Brown, Fraser, Clarke, David, Carr, Reuben, Campopiano, Dominic·2024·ACS Catalysis
DOI 10.26434/chemrxiv-2024-0lrh8PMC PMC11574752

Preprint — published as 10.1021/acscatal.4c04714 · Paper page

Open this paper in CoChat — free account

Abstract

In Nature, thousands of diverse and bioactive polyketides are assembled by a family of multifunctional, “assembly line” enzyme complexes called Polyketide Synthases (PKS). Since the late 20th century, there have been several attempts to decode, re-arrange and “re-programme” the PKS assembly line to generate valuable materials such as biofuels and platform chemicals. By mastering the biosynthetic logic underpinning PKSs, there exists the potential to develop these enzymes into scalable biocatalysts for sustainable chemical production. Here, the first module from Mycobacterium tuberculosis (Mt) PKS12 – an unorthodox, “modularly-iterative” PKS – was modified and repurposed towards the production of 2-methyl Guerbet lipids, which have wide applications in industry. We established a robust method for the recombinant expression and purification of this modified module (named [M1*]), and we demonstrated its synthetic utility in generating several 2-methyl Guerbet-like lipids (C13-C21). Furthermore, we studied and applied the promiscuous thioesterase (TE) activity of a neighbouring β-ketoacyl synthase (KS) to release [M1*]-bound condensation products in a one-pot biosynthetic cascade. Finally, by coupling the E. coli fatty acyl-CoA synthetase FadD to [M1*], we could generate our primary target compound – 2-methyltetradecanoic acid – from lauric acid, highlighting the potential to derive valuable lipids from inexpensive fatty acids. This work demonstrates the biosynthetic potential of [M1*] in generating industrially-useful Guerbet-like chemicals, whilst also contributing to the broader exploration and application of PKS modules for sustainable chemical manufacturing.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.26434/chemrxiv-2024-0lrh8

Cites

1 of 1 references verified0 citation errors · 0 not checkable

Statistics

Figures

Scheme 1 Proposed Utilization of MtPKS12-derived [M1*] to Produce 2-Methyl Guerbet-like Lipids The domain color coding shown here is used throughout this work.
Scheme 1 Proposed Utilization of MtPKS12-derived [M1*] to Produce 2-Methyl Guerbet-like Lipids The domain color coding shown here is used throughout this work.CC BY · PubMed Central
Figure 1 (A) Composite, color-coded structural model of MtPKS12-derived [M1*]. All domain structures were predicted using ColabFold. The crystal structure deposited by MuCullough et al. (PDB 8G7W)24 was used as a template to approximate the relative organization of the predicted reducing region ([DH1][KR1][ER1]) using structural alignment tools. (B) SEC purification of [M1*] monitored by UV–vis spectroscopy (280 nm) and SDS-PAGE. A HiMark prestained protein standard was used as the protein ladder.
Figure 1 (A) Composite, color-coded structural model of MtPKS12-derived [M1*]. All domain structures were predicted using ColabFold. The crystal structure deposited by MuCullough et al. (PDB 8G7W)24 was used as a template to approximate the relative organization of the predicted reducing region ([DH1][KR1][ER1]) using structural alignment tools. (B) SEC purification of [M1*] monitored by UV–vis spectroscopy (280 nm) and SDS-PAGE. A HiMark prestained protein standard was used as the protein ladder.CC BY · PubMed Central
Figure 2 (A) NADPH-dependent reduction of ketone 6 to alcohol 7 by MtPKS12 [KR1]. [KR1] is composed of structural [KRS] and catalytic [KRC] subdomains, separated in sequence by [ER1]. The AF2 predicted structure of the MtPKS12 [KRS1][ER1][KRC1] didomain is shown ([ER1] is grayed out). (B) Monitoring MtPKS12 [KR1]-catalyzed reduction of 6 by NADPH depletion (340 nm). All reactions were performed in technical triplicate. Standard deviations are shown as colored error bands. (C) GC/EI-MS chromatograms showing the MtPKS12 [KR1]-catalyzed reduction of 6 to 7. The reaction reaches equilibrium after 120 min (see also Figure S11). Negative controls (sampled after 600 min) are also shown. (D) Michaelis–Menten curve for MtPKS12 [KRS1][ER1][KRC1] using ketone 6. Error bars represent standard error of three technical replicates. (E) Michaelis–Menten curve for [M1*] using ketone 6. Error bars represent the standard error of three technical replicates. (F) kcat/KM comparison between MtPKS12 [KRS1][ER1][KRC1] and [M1*] against ketone 6. Error bars represent the standard error of three technical replicates.
Figure 2 (A) NADPH-dependent reduction of ketone 6 to alcohol 7 by MtPKS12 [KR1]. [KR1] is composed of structural [KRS] and catalytic [KRC] subdomains, separated in sequence by [ER1]. The AF2 predicted structure of the MtPKS12 [KRS1][ER1][KRC1] didomain is shown ([ER1] is grayed out). (B) Monitoring MtPKS12 [KR1]-catalyzed reduction of 6 by NADPH depletion (340 nm). All reactions were performed in technical triplicate. Standard deviations are shown as colored error bands. (C) GC/EI-MS chromatograms showing the MtPKS12 [KR1]-catalyzed reduction of 6 to 7. The reaction reaches equilibrium after 120 min (see also Figure S11). Negative controls (sampled after 600 min) are also shown. (D) Michaelis–Menten curve for MtPKS12 [KRS1][ER1][KRC1] using ketone 6. Error bars represent standard error of three technical replicates. (E) Michaelis–Menten curve for [M1*] using ketone 6. Error bars represent the standard error of three technical replicates. (F) kcat/KM comparison between MtPKS12 [KRS1][ER1][KRC1] and [M1*] against ketone 6. Error bars represent the standard error of three technical replicates.CC BY · PubMed Central
Figure 3 GC/EI-MS analysis of [M1*]-derived 5a–e, detected using SIM. The chromatograms show the simultaneous detection of the molecular ion (bottom chromatograms, black) and the common McLafferty product ion (top inverted chromatograms, colored) for each analyte. (A) SIM detection of 5a. (B) SIM detection of 5b. (C) SIM detection of 5c. (D) SIM detection of 5d. (E) SIM detection of 5e. (F) Relative abundance of [M1*]-derived products, calculated from the intensity of the common McLafferty product ion.
Figure 3 GC/EI-MS analysis of [M1*]-derived 5a–e, detected using SIM. The chromatograms show the simultaneous detection of the molecular ion (bottom chromatograms, black) and the common McLafferty product ion (top inverted chromatograms, colored) for each analyte. (A) SIM detection of 5a. (B) SIM detection of 5b. (C) SIM detection of 5c. (D) SIM detection of 5d. (E) SIM detection of 5e. (F) Relative abundance of [M1*]-derived products, calculated from the intensity of the common McLafferty product ion.CC BY · PubMed Central
Figure 4 (A) GC/EI-MS chromatograms demonstrating the promiscuous TE activity of MtPKS12 KS domains, alongside negative controls. Chromatograms were recorded after 19.5 h of incubation with 2b. The proposed hydrolysis of the acyl-[KS1] Michaelis complex is also shown. (B) [KS1] TE activity against a range of acyl-CoAs (2a, 2b, 2d, and 2e), detected using the DTNB assay. Spectrophotometric detection of the TNB2– anion (412 nm) was recorded after 18 h. Background absorbance from nonenzymatic acyl-CoA hydrolysis, as well as the reaction of DTNB with surface-exposed [KS1][AT1] cysteines, was subtracted from all experimental values. Error bars represent the standard deviation of three technical replicates. (C) Representative GC/EI-MS chromatograms showing the TE activity of wild-type (WT) [KS2] and mutant H2361D [KS2] against 2b. (D) Quantified 1b titers using WT and H2361D [KS2]. Error bars represent the standard deviation of three technical replicates. (E) Proposed [KS2]-mediated acyl transfer and hydrolysis of 4b giving 5b. (F) GC/EI-MS chromatograms showing the successful [KS2]-mediated release of 5b. The instrument was configured for SIM (m/z = 74).
Figure 4 (A) GC/EI-MS chromatograms demonstrating the promiscuous TE activity of MtPKS12 KS domains, alongside negative controls. Chromatograms were recorded after 19.5 h of incubation with 2b. The proposed hydrolysis of the acyl-[KS1] Michaelis complex is also shown. (B) [KS1] TE activity against a range of acyl-CoAs (2a, 2b, 2d, and 2e), detected using the DTNB assay. Spectrophotometric detection of the TNB2– anion (412 nm) was recorded after 18 h. Background absorbance from nonenzymatic acyl-CoA hydrolysis, as well as the reaction of DTNB with surface-exposed [KS1][AT1] cysteines, was subtracted from all experimental values. Error bars represent the standard deviation of three technical replicates. (C) Representative GC/EI-MS chromatograms showing the TE activity of wild-type (WT) [KS2] and mutant H2361D [KS2] against 2b. (D) Quantified 1b titers using WT and H2361D [KS2]. Error bars represent the standard deviation of three technical replicates. (E) Proposed [KS2]-mediated acyl transfer and hydrolysis of 4b giving 5b. (F) GC/EI-MS chromatograms showing the successful [KS2]-mediated release of 5b. The instrument was configured for SIM (m/z = 74).CC BY · PubMed Central
Figure 5 (A) Proposed coupling of EcFadD to [M1*] to produce 5b from 1b via 2b. (B) Spectrophotometric detection of the EcFadD-catalyzed conversion of 1b to 2b using the ammonium molybdate assay. The released PPi forms a blue phosphomolybdate complex in the presence of a reducing agent (λmax = 580 nm). (C) GC/EI-MS chromatograms showing the successful EcFadD-[M1*] coupled production of 5b from 1b. The instrument was configured for SIM (m/z = 74).
Figure 5 (A) Proposed coupling of EcFadD to [M1*] to produce 5b from 1b via 2b. (B) Spectrophotometric detection of the EcFadD-catalyzed conversion of 1b to 2b using the ammonium molybdate assay. The released PPi forms a blue phosphomolybdate complex in the presence of a reducing agent (λmax = 580 nm). (C) GC/EI-MS chromatograms showing the successful EcFadD-[M1*] coupled production of 5b from 1b. The instrument was configured for SIM (m/z = 74).CC BY · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Show all 1 references

Metadata

Authors
Herrera, M. A., McColm, S., Craigie, L.-M., Simpson, J., Brown, F., Clarke, D., Carr, R., & Campopiano, D.
Affiliations
  • University of Edinburgh (GB)
  • Ingenza (United Kingdom) (GB)
Corresponding author
Campopiano, Dominic
ORCID
Venue
ACS Catalysis
Publisher
American Chemical Society (ACS)
Published
2024-06-25
Volume
14
Issue
22
Pages
16834-16842
ISSN
2155-5435 (ISSN-L)
DOI
10.26434/chemrxiv-2024-0lrh8
PMC
PMC11574752
OpenAlex
W4403942416
Cited by
1
References
1
Citations by year
2025: 1
FWCI
0.28
Type
journal article
Language
en
Open access
Licence
Locations
  • ACS Catalysis (publishedVersion, cc-by)
  • ChemRxiv (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Edinburgh Research Explorer (University of Edinburgh) (submittedVersion, cc-by)
Funders
  • Industrial Biotechnology Innovation Centre (BB/S506953/1)
  • Engineering and Physical Sciences Research Council (EP/K039717/1)
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
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Lipid metabolism and biosynthesis
Keywords
polyketide synthase, beta-ketoacyl synthase, lauric acid, polyketide biosynthesis, recombinant protein expression

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review

How to cite

Cite CoChat for the checks on this page, and the paper for its own findings.

This page
CoChat. (2026). Citation check: Repurposing a Fully-Reducing Polyketide Synthase for 2-Methyl Guerbet-like Lipid Production. https://app.cochat.ai/papers/10.26434/chemrxiv-2024-0lrh8
The paper
Herrera, M. A., McColm, S., Craigie, L.-M., Simpson, J., Brown, F., Clarke, D., Carr, R., & Campopiano, D. (2024). Repurposing a Fully-Reducing Polyketide Synthase for 2-Methyl Guerbet-like Lipid Production. ACS Catalysis. https://doi.org/10.26434/chemrxiv-2024-0lrh8

Data sources

Bibliographic data: Semantic Scholar, OpenAlex, Crossref.

Contains information from the Semantic Scholar Academic Graph Datasets, made available under the ODC Attribution License (ODC-BY 1.0).

Semantic Scholar’s data is described in: Kinney, R., et al. (2023). The Semantic Scholar Open Data Platform. arXiv:2301.10140.