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DOE OSTI2025

Automated Strain Construction for Biosynthetic Pathway Screening in Yeast

Automation accelerates the Design-Build-Test-Learn (DBTL) cycle for synthetic biology; however, most strain construction pipelines lack robotic integration. Here, in this study, we present the workflow design and source code for a modular, integrated protocol that automates the Build step in Saccharomyces cerevisiae. We programmed the Hamilton Microlab VANTAGE to integrate off-deck hardware via its central robotic arm, enabling automated steps that increased throughput to 2,000 transformations per week. We developed a user interface with the Hamilton VENUS software to support on-demand parameter customization. As a proof of concept, we screened a gene library in an engineered yeast strain producing verazine, a key intermediate in the biosynthesis of steroidal alkaloids. Our pipeline rapidly identified pathway bottlenecks and genes that enhanced verazine production by 2.0- to 5-fold. This technical note provides resources for synthetic biologists designing yeast workflows for biofoundries to screen libraries for pathway discovery/optimization, combinatorial biosynthesis, and protein engineering.

automation↗

DOE OSTI2026

Hydrazinoacetic acid is a biosynthetic precursor of the bacterially produced nitramine, N -nitroglycine

Nitramines [R(R′)N–NO 2 ; R,R′=H or alkyl] are valuable synthetic products, but knowledge of the biosynthetic processes that generate these compounds is limited. This work sought to elucidate the biosynthesis of a nitramine natural product, N-nitroglycine (NNG) by Streptomyces noursei . Stable isotope studies showed that S. noursei cells supplemented with L-(ε- 15 N)lysine, ( 15 N)glycine, or ( 13 C)hydrazinoacetic acid (HAA) incorporated 67%, 88%, and 67% of the isotope label into NNG, respectively, indicating that these compounds are biosynthetic precursors of NNG. Liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) of 15 N-Lys-labeled NNG confirmed that the nitro nitrogen of NNG originates from Lys. Bioinformatics analysis of the S. noursei genome showed evidence for a biosynthetic gene cluster (BGC) that contained machinery for HAA biosynthesis ( nngKLM ), consistent with the results of the isotope labeling. In vitro reconstitution of the gene products produced HAA. The borders of this BGC were defined by cross-referencing the predicted BGC with previously published differential proteomics data. Furthermore, we show that azaserine is produced alongside NNG in S. noursei cultures, linking the two biosynthetic pathways via a proposed nitrosamine biosynthetic intermediate. Finally, the oxygen balance for NNG is −20.2% for the formation of carbon dioxide (CO 2 ), which is comparable to that of hexahydro-1,3,5- trinitro-1,3,5-triazene (common name: RDX; −21.6%). Crystal structure data of NNG indicate that the unit crystalizes as a pure material, not a hydrate, suggesting a favorable energetic crystallization phase. The combined results suggest a route that, with further development, could lead to sustainable production of energetic nitramines via synthetic biology or biocatalytic approaches.

biosynthesis↗

DOE OSTI2024

An efficient cre‐based workflow for genomic integration and expression of large biosynthetic pathways in Eubacterium limosum

Abstract Acetogenic Clostridia are obligate anaerobes that have emerged as promising microbes for the renewable production of biochemicals owing to their ability to efficiently metabolize sustainable single‐carbon feedstocks. Additionally, Clostridia are increasingly recognized for their biosynthetic potential, with recent discoveries of diverse secondary metabolites ranging from antibiotics to pigments to modulators of the human gut microbiota. Lack of efficient methods for genomic integration and expression of large heterologous DNA constructs remains a major challenge in studying biosynthesis in Clostridia and using them for metabolic engineering applications. To overcome this problem, we harnessed chassis‐independent recombinase‐assisted genome engineering (CRAGE) to develop a workflow for facile integration of large gene clusters (>10 kb) into the human gut acetogen Eubacterium limosum . We then integrated a non‐ribosomal peptide synthetase gene cluster from the gut anaerobe Clostridium leptum , which previously produced no detectable product in traditional heterologous hosts. Chromosomal expression in E. limosum without further optimization led to production of phevalin at 2.4 mg/L. These results further expand the molecular toolkit for a highly tractable member of the Clostridia, paving the way for sophisticated pathway engineering efforts, and highlighting the potential of E. limosum as a Clostridial chassis for exploration of anaerobic natural product biosynthesis.

Sanford, Patrick A.↗

DOE OSTI2024

Oleaginous Yeast Biology Elucidated With Comparative Transcriptomics

ABSTRACT Extremophilic yeasts have favorable metabolic and tolerance traits for biomanufacturing‐ like lipid biosynthesis, flavinogenesis, and halotolerance – yet the connection between these favorable phenotypes and strain genotype is not well understood. To this end, this study compares the phenotypes and gene expression patterns of biotechnologically relevant yeasts Yarrowia lipolytica , Debaryomyces hansenii , and Debaryomyces subglobosus grown under nitrogen starvation, iron starvation, and salt stress. To analyze the large data set across species and conditions, two approaches were used: a “network‐first” approach where a generalized metabolic network serves as a scaffold for mapping genes and a “cluster‐first” approach where unsupervised machine learning co‐expression analysis clusters genes. Both approaches provide insight into strain behavior. The network‐first approach corroborates that Yarrowia upregulates lipid biosynthesis during nitrogen starvation and provides new evidence that riboflavin overproduction in Debaryomyces yeasts is overflow metabolism that is routed to flavin cofactor production under salt stress. The cluster‐first approach does not rely on annotation; therefore, the coexpression analysis can identify known and novel genes involved in stress responses, mainly transcription factors and transporters. Therefore, this work links the genotype to the phenotype of biotechnologically relevant yeasts and demonstrates the utility of complementary computational approaches to gain insight from transcriptomics data across species and conditions.

Weintraub, Sarah J. [Department of Bioinformatics ↗

DOE OSTI2024

Omics-driven onboarding of the carotenoid producing red yeast Xanthophyllomyces dendrorhous CBS 6938

Transcriptomics is a powerful approach for functional genomics and systems biology, yet it can also be used for genetic part discovery. Here, we derive constitutive and light-regulated promoters directly from transcriptomics data of the basidiomycete red yeast Xanthophyllomyces dendrorhous CBS 6938 (anamorph Phaffia rhodozyma) and use these promoters with other genetic elements to create a modular synthetic biology parts collection for this organism. X. dendrorhous is currently the sole biotechnologically relevant yeast in the Tremellomycete class-it produces large amounts of astaxanthin, especially under oxidative stress and exposure to light. Thus, we performed transcriptomics on X. dendrorhous under different wavelengths of light (red, green, blue, and ultraviolet) and oxidative stress. Differential gene expression analysis (DGE) revealed that terpenoid biosynthesis was primarily upregulated by light through crtI, while oxidative stress upregulated several genes in the pathway. Further gene ontology (GO) analysis revealed a complex survival response to ultraviolet (UV) where X. dendrorhous upregulates aromatic amino acid and tetraterpenoid biosynthesis and downregulates central carbon metabolism and respiration. The DGE data was also used to identify 26 constitutive and regulated genes, and then, putative promoters for each of the 26 genes were derived from the genome. Simultaneously, a modular cloning system for X. dendrorhous was developed, including integration sites, terminators, selection markers, and reporters. Each of the 26 putative promoters were integrated into the genome and characterized by luciferase assay in the dark and under UV light. The putative constitutive promoters were constitutive in the synthetic genetic context, but so were many of the putative regulated promoters. Notably, one putative promoter, derived from a hypothetical gene, showed ninefold activation upon UV exposure. Thus, this study reveals metabolic pathway regulation and develops a genetic parts collection for X. dendrorhous from transcriptomic data. Therefore, this study demonstrates that combining systems biology and synthetic biology into an omics-to-parts workflow can simultaneously provide useful biological insight and genetic tools for nonconventional microbes, particularly those without a related model organism. This approach can enhance current efforts to engineer diverse microbes.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2025

Bleach Rescues Nannochloropsis from an Obligate Parasite and Alters Microbial and Metabolite Signatures of Outdoor Cultures

Chemical agents are commonly used to protect algal crops. Yet, few studies have characterized the effects of these agents on associated microbial communities to understand effects on microbial functions relevant to algal crop production and protection. Here, we used shotgun metagenomic sequencing and untargeted exometabolite profiling to link the application of bleach, a -cidal agent used to protect algae from pests, to changes in community composition, metabolic pathways, and exometabolies - at a whole community level. Bleach protected the algal crop from crashing but altered bacterial diversity. Analysis of metagenome-assembled genomes (MAGs) revealed a classic predator-prey cycle between Oligoflexus and our target alga Nannochloropsis. Olifoflexus genomes from our study were notably similar to a previously identified BALO (Bdellovibrio and like organism), FD111, known to kill Nannochloropsis cultures, providing strong evidence that an FD111-like organism was responsible for the crash. Metabolic pathway composition differed between bleached and unbleached ponds, with abundance of twelve pathways related to stress tolerance, including the superpathway of methylglyoxal degradation, lipid IVA biosynthesis, and ectoine biosynthesis, greater in bleached ponds compared to unbleached ponds. Virulence factors related to adherence, biofilm formation, motility, and pathogenicity increased dramatically in bleached ponds with time, although this increase was not coupled with an increase in pathogens - algal or otherwise - or a decline in algal health. Our study highlights the importance of coupling 16S rRNA gene sequencing with whole genome data and other -omics tools to sketch a larger picture of community structure and function in crop systems. Moreover, our results highlight that continued long-term bleaching may lead to negative effects to crop health or downstream adverse health effects to humans or animals, depending on the algal product (i.e. human supplements or animal feedstocks). Future work on alternative treatment methods that would reduce resistance is necessary in the field.

09 BIOMASS FUELS↗

DOE OSTI2025

Alpha ketoacid decarboxylases: Diversity, structures, reaction mechanisms, and applications for biomanufacturing of platform chemicals and fuels

In living cells, alpha-ketoacid decarboxylases (KDCs, EC 4.1.1.-) are a class of enzymes that convert alpha-ketoacids into aldehydes through decarboxylation. These aldehydes serve as either drop-in chemicals or precursors for the biosynthesis of alcohols, carboxylic acids, esters, and alkanes. These compounds play crucial roles in cellular metabolism and fitness and the bioeconomy, facilitating the sustainable and renewable biomanufacturing of platform chemicals and fuels. This review explores the diversity and classification of KDCs, detailing their structures, mechanisms, and functions. We highlight recent advancements in repurposing KDCs to enhance their efficiency and robustness for biomanufacturing. Additionally, we present modular KDC-dependent metabolic pathways for the microbial biosynthesis of aldehydes, alcohols, carboxylic acids, esters, and alkanes. Lastly, we discuss recent developments in the modular cell engineering technology that can potentially be applied to harness the diversity of KDC-dependent pathways for biomanufacturing platform chemicals and fuels.

09 BIOMASS FUELS↗

DOE OSTI2025

Persistent fatty acid catabolism during plant oil synthesis

Plant lipids are an essential energy source for diets and are a sustainable alternative to petroleum-based fuels and feedstocks. Fatty acid breakdown during seed germination is crucial for seedling establishment but unexpected during seed filling. Here, we demonstrate that the simultaneous biosynthesis and degradation of fatty acids begins early and continues across all phases of oil filling and throughout the photoperiod. Tests in camelina, rapeseed, and an engineered high-oil tobacco line confirmed that concomitant synthesis and breakdown in oil-producing tissues over development is the rule rather than the exception. Furthermore, we show that transgenics, designed to elevate fatty acid biosynthesis, failed to achieve anticipated increases in storage lipid levels due to increased degradation, potentially explaining the underperformance of engineered lines compared to expectations more generally.

coenzyme A↗

DOE OSTI2026

Bioelectrocatalytic conversion of CO₂ to PHA bioplastics using engineered methylotrophs

The sustainable generation of biodegradable plastics represents an opportunity to capture atmospheric CO 2 while reducing plastic waste accumulation in the environment. This study implements an integrated platform for bioelectrocatalytic CO 2 conversion to medium-chain-length polyhydroxyalkanoates (mcl-PHAs). Immobilizing cobalt phthalocyanine electrocatalysts on a covalent-organic framework in a gas recirculation electrolyzer enabled CO 2 -to-methanol conversion with a carbon conversion efficiency of 98%. Integration of polymer biosynthesis pathways enabled Methylotuvimicrobium alcaliphilum 20Z R to produce ~20% mcl-PHA of the dry cell weight with a CO 2 -to-bioproducts carbon conversion efficiency of 50%. This cell line was adapted to high sodium bicarbonate media, eliminating costly intermediate separation steps while improving economic potential. Transcriptomic analysis revealed sulfate transporters and peptidoglycan biosynthesis as key pathways involved in sodium bicarbonate halotolerance. Altogether, this research presents a foundation for integrating divergent chemical and biological processes into a transformative electrobiomanufacturing platform, addressing the need for alternative pipelines for generating valuable plastics and chemicals.

CO2 utilization↗

DOE OSTI2025

A variant of the MenB strain of Synechocystis sp. PCC 6803 disrupts a stress response pathway allowing DMPBQ to occupy the A 1 sites of photosystem I

Phylloquinone (PhQ) plays a unique role in photosynthesis as the A1A and A1B intermediates in light-driven electron transfer in Photosystem I (PSI). When PhQ biosynthesis is inhibited by deletion of the menB gene in the cyanobacterium Synechocystis sp. PCC 6803, previous studies have shown that plastoquinone-9 (PQ-9) occupies the A 1 sites. However, a recent cryo-electron microscopy structure of a strain of ΔmenB from the year 2023 revealed an unusual quinone in the A 1 sites with a benzoquinone headgroup similar to PQ-9 and a phytyl tail similar to PhQ. Here, we investigate its biosynthesis and binding properties. Mass spectrometry confirms that PSI from the 2023 ΔmenB strain contains 2,3-dimethyl-5-phytyl-1,4-benzoquinone (DMPBQ), while PSI from the original strain contains PQ-9. Whole genome sequencing reveals that a mutation in slr1737 (tocopherol cyclase) leads to the accumulation of DMPBQ, an intermediate in the tocopherol biosynthetic pathway. Transient optical and electron paramagnetic resonance spectroscopy studies show that when DMPBQ occupies the A 1 sites, it does not exchange with exogenously supplied PhQ in contrast to PQ-9, which exchanges readily. We propose that the slr1737 mutation in the 2023 ΔmenB strain is a likely candidate for the source of the phenotype. This new strain has sacrificed tocopherol synthesis, the absence of which is known to have no effect on growth under low stress conditions, resulting in incorporation of DMPBQ in the A 1 sites. The tighter binding and function of DMPBQ likely allow this ΔmenB strain to outcompete its peers under optimal growth conditions and thus dominate the population.

2,3-dimethyl-5-phytyl-1,4-benzoquinone↗

DOE OSTI2025

Integrated lipidomic and proteomic profiling reveals metabolic network disruption by SARS-CoV-2 variants

The rapid evolution of SARS-CoV-2 has produced myriad viral strains with increasing transmissibility and capacity for immune evasion. While effective vaccination campaigns have reduced the fatalities associated with SARS-CoV-2, infections continue, and a detailed understanding of how this virus manipulates host biochemical pathways remains elusive. We asked both whether the patterns of host lipid rewiring remained consistent across variants and whether the changes in the abundance of lipid classes are related to changes in the expression of the enzymes involved in their biosynthesis. We compared global nontargeted lipidomics on A549-ACE2 cells infected with the delta variant (B.1.617.2), or the omicron (B.1.1.529) variant to our previous results of global nontargeted lipidomics on A549-ACE2 cells infected with the original WA1 strain and further performed quantitative proteomics to assess changes in the host proteome. We found that metabolic rewiring, both on the lipid and the enzymatic level, is remarkably consistent across all three variants. We further mapped changes in the expression of host metabolic enzymes, linking enzyme expression to alterations in the abundance of specific lipids during infection. This analysis identified key proteins related to virus-mediated changes in lipid abundance, including fatty acid synthase (FASN), lysosomal acid lipase (LIPA), and ORMDL, a regulator of sphingolipid biosynthesis. These integrated lipidomic and proteomic experiments shed light on the importance of the complex network of host metabolism networks that support SARS-CoV-2 infection and suggest that lipid metabolism may be a promising avenue for uncovering conserved therapeutic targets.

SARS-CoV-2↗

DOE OSTI2025

Engineering Pseudomonas putida for production of 3-hydroxyacids using hybrid type I polyketide synthases

Engineered type I polyketide synthases (T1PKSs) are a potentially transformative platform for the biosynthesis of small molecules. Due to their modular nature, T1PKSs can be rationally designed to produce a wide range of bulk or specialty chemicals. While heterologous PKS expression is best studied in microbes of the genus Streptomyces, recent studies have focused on the exploration of non-native PKS hosts. The biotechnological production of chemicals in fast growing and industrial relevant hosts has numerous economic and logistic advantages. With its native ability to utilize alternative feedstocks, Pseudomonas putida has emerged as a promising workhorse for the sustainable production of small molecules. Here, we outline the assessment of P. putida as a host for the expression of engineered T1PKSs and production of 3-hydroxyacids. After establishing the functional expression of an engineered T1PKS, we successfully expanded and increased the pool of available acyl-CoAs needed for the synthesis of polyketides using transposon sequencing and protein degradation tagging. This work demonstrates the potential of T1PKSs in P. putida as a production platform for the sustainable biosynthesis of unnatural polyketides.

Schmidt, Matthias↗

DOE OSTI2026

Lignin’s Indispensable role in orchestrating seed stone formation: Insights from jujuba, peach and pear with future prospective on pitless fruits

A hard endocarp (i.e., stone) inside fruit is a characteristic of drupe fruits such as jujube, peach, mango, etc. Hard stone significantly affects the quality and downstream processing of fruits. The complex aromatic polymer lignin deposition in the secondary cell wall determines stone hardness. Lignin comprises phenylpropanoid units formed by hydroxycinnamoyl alcohol, which includes coniferyl, sinapyl, and p-coumaroyl alcohols. Lignin biosynthesis pathway involves a series of complex enzymatic reactions initiated from phenylalanine ammonia lyase and ends up polymerizing lignin monomers by laccase and peroxidase enzymes. Phytohormones, particularly auxin, gibberellins, and Ca²⁺ signaling, further modulate endocarp lignification by regulating transcriptional networks and lignin biosynthetic genes, thereby fine-tuning secondary cell wall thickening and stone hardness in drupe fruits. Lignin biosynthesis is controlled by both structural genes and transcriptional regulators. The structural genes encoding lignin biosynthetic enzymes include LAC12–1, PAL2, C4H, C3H, CSE, CCoAOMT, F5H, CAD, and PRX1. In addition, several transcription factors regulating secondary cell wall and lignin deposition, such as MYB24, bZIP48, and bZIP33 play key regulatory roles. Conversely, delignification or suppression of stone formation is associated with transcription factors (Pistillata, MYB32, FUL, and REPLUMLESS) and post-transcriptional regulators, including miR397a, miR31-3p, and miR8-5p. Accurate alteration in the expression of these genes will result in the attainment of stoneless fruits for cheap and hazel-free downstream processing.

Fruit endocarp↗

DOE OSTI2026

Lipid accumulation in nitrogen and phosphorus-limited yeast is caused by less growth-related dilution

Oleaginous yeasts are used commercially to produce oleochemicals and hold potential also for biodiesel production. In response to nitrogen or phosphorous limitation, oleaginous yeasts accumulate lipids in the form of triacylglycerols. Previous work has investigated potential mechanisms by which nutrient limitation induces lipid biosynthesis without verifying whether lipid biosynthesis flux is actually enhanced. Here, in this work, we show, using 13C-glucose tracing, that in nitrogen or phosphorous limitation, lipid accumulation occurs without consistent increases in biosynthetic flux. Instead, the main driver of increased lipid pools is decreased growth-related dilution. This conclusion holds across two divergent oleaginous yeasts: Rhodotorula toruloides and Yarrowia lipolytica . Quantitative proteomics shows a substantial proteome reallocation in response to nitrogen and phosphorous limitation, with ribosomal proteins strongly downregulated, while lipid enzymes are preserved but not consistently upregulated in absolute quantity. Thus, nutrient limitation, rather than triggering greatly enhanced lipid synthesis, results in roughly sustained lipid enzyme levels and biosynthetic flux. Due to slower lipid dilution by cell division, this suffices to drive marked lipid accumulation.

09 BIOMASS FUELS↗

DOE OSTI2026

Precursor-Dependent Routing of Aromatic Amino Acids Determines Lignin Structure in Grasses by Sensitivity-Enhanced Solid-State NMR

Lignin biosynthesis in grasses exhibits unique metabolic flexibility, yet the precursor-specific routing of carbon into lignin polymers remains poorly resolved in planta. Here, we combine 13 C-isotope labeling with solid-state NMR under sensitivity-enhancement by dynamic nuclear polarization (DNP), to directly track phenylalanine- and tyrosine-derived carbon incorporation into the lignin polymer in Brachypodium distachyon. Precursor-specific 13 C labeling reveals that phenylalanine is the dominant contributor to canonical guaiacyl and syringyl lignins, whereas tyrosine preferentially enriches hydroxyphenyl lignin and hydroxycinnamates, including ferulates characteristic of grass cell walls. Two-dimensional 13 C− 13 C correlation NMR resolves distinct lignin moieties arising from each precursor. Disruption of pcoumarate 3-hydroxylase (C3H) selectively impairs phenylalanine-derived lignification, while tyrosine-derived lignin remains comparatively unchanged, maintaining polymer assembly through alternative metabolic routes. These findings show precursordependent control of lignin composition and reveal tyrosine-mediated lignification as a compensatory pathway in grasses. This work also establishes precursor-resolved solid-state NMR and DNP as a powerful framework for dissecting lignin biosynthesis and metabolic plasticity in plant cell walls.

Biopolymers↗

DOE OSTI2024

Rubisco supplies pyruvate for the 2- C -methyl-D-erythritol-4-phosphate pathway

RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE (Rubisco) produces pyruvate in the chloroplast through β-elimination of the aci-carbanion intermediate. Here we show that this side reaction supplies pyruvate for isoprenoid, fatty acid and branched-chain amino acid biosynthesis in photosynthetically active tissue. 13 C labelling studies of intact Arabidopsis plants demonstrate that the total carbon commitment to pyruvate is too large for phosphoenolpyruvate to serve as a precursor. Low oxygen stimulates Rubisco carboxylase activity and increases pyruvate production and flux through the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, which supplies the precursors for plastidic isoprenoid biosynthesis. Metabolome analysis of mutants defective in phosphoenolpyruvate or pyruvate import and biochemical characterization of isolated chloroplasts further support Rubisco as the main source of pyruvate in chloroplasts. Seedlings incorporated exogenous, 13 C-labelled pyruvate into MEP pathway intermediates, while adult plants did not, underscoring the developmental transition in pyruvate sourcing. Rubisco β-elimination leading to pyruvate constituted 0.7% of the product profile in in vitro assays, which translates to 2% of the total carbon leaving the Calvin–Benson–Bassham cycle. These insights solve the “pyruvate paradox”, improve the fit of metabolic models for central metabolism and connect the MEP pathway directly to carbon assimilation.

Plant physiology↗

DOE OSTI2024

A single diiron enzyme catalyses the oxidative rearrangement of tryptophan to indole nitrile

Abstract Nitriles are uncommon in nature and are typically constructed from oximes through the oxidative decarboxylation of amino acid substrates or from the derivatization of carboxylic acids. Here we report a third nitrile biosynthesis strategy featuring the cyanobacterial nitrile synthase AetD. During the biosynthesis of the eagle-killing neurotoxin, aetokthonotoxin, AetD transforms the 2-aminopropionate portion of 5,7-dibromo-l-tryptophan to a nitrile. Employing a combination of structural, biochemical and biophysical techniques, we characterized AetD as a non-haem diiron enzyme that belongs to the emerging haem-oxygenase-like dimetal oxidase superfamily. High-resolution crystal structures of AetD together with the identification of catalytically relevant products provide mechanistic insights into how AetD affords this unique transformation, which we propose proceeds via an aziridine intermediate. Our work presents a unique template for nitrile biogenesis and portrays a substrate binding and metallocofactor assembly mechanism that may be shared among other haem-oxygenase-like dimetal oxidase enzymes.

Chemistry↗

DOE OSTI2025

Engineering controllable alteration of malonyl-CoA levels to enhance polyketide production

Heterologous expression of polyketide synthase (PKS) genes in Escherichia coli has enabled the production of various valuable natural and synthetic products. However, the limited availability of malonyl-CoA (M-CoA) in E. coli remains a substantial impediment to high-titer polyketide production. Here we address this limitation by disrupting the native M-CoA biosynthetic pathway and introducing an orthogonal pathway comprising a malonate transporter and M-CoA ligase, enabling efficient M-CoA biosynthesis under malonate supplementation. This approach substantially increases M-CoA levels, enhancing fatty acid and polyketide titers while reducing the promiscuous activity of PKSs toward undesired acyl-CoA substrates. Subsequent adaptive laboratory evolution of these strains provides insights into M-CoA regulation and identifies mutations that further boost M-CoA and polyketide production. This strategy improves E. coli as a host for polyketide biosynthesis and advances understanding of M-CoA metabolism in microbial systems.

Klass, Sarah H↗