Discovery and remodeling of *Vibrio natriegens* as a microbial platform for efficient formic acid biorefinery

The study addresses the challenge of efficiently assimilating and converting formic acid (FA), a CO2-derived C1 feedstock, into value-added products using microbial hosts. While FA is attractive due to ease of storage and high-selectivity chemical production from CO2 or syngas, most native FA-utilizers grow slowly and existing engineered hosts (e.g., Escherichia coli) suffer from low FA tolerance and limited metabolic capacity, leading to low FA utilization efficiency compared to traditional substrates like sugars. The research aims to discover and develop a superior microbial chassis with innate FA tolerance and metabolic versatility, and to engineer it to achieve high-rate FA consumption and product formation. Vibrio natriegens, a bacterium with an exceptionally short doubling time and robust physiology, is investigated for its native FA tolerance and subsequently remodeled to enhance FA assimilation and conversion.

FA assimilation in microbes typically proceeds via the Wood–Ljungdahl pathway, serine cycle, or reductive glycine pathway, all relying on the tetrahydrofolate (THF) cycle initiated by formate–tetrahydrofolate ligase (FTL). Pyruvate formate-lyase (PFL) can also assimilate FA to pyruvate. Native FA-utilizing microbes generally show slow growth and low efficiency. Engineering efforts in E. coli reconstructed the THF cycle and reverse glycine cleavage system to enable growth on FA/CO2 or FA/methanol, but FA utilization rates remain low due to weak tolerance and metabolic capacity. Energy conservation systems are critical in growth on C1 substrates. The authors position V. natriegens as a potentially superior chassis due to rapid growth and broad metabolic capabilities, and propose integrating the serine cycle with the TCA cycle to create a strong metabolic pull that can increase FA assimilation flux.

- Characterization of native FA tolerance and metabolism: V. natriegens was grown in LBv2 and modified M9 media with graded sodium formate (HCOONa·2H2O) concentrations. Growth (OD600) and formate consumption (HPLC, Aminex HPX-87H) were quantified. - Genomic and transcriptomic analysis: Annotated FA-related genes (fdh family, ftl, folD, pfl) and downstream pathways (reductive glycine, serine cycle, TCA). RNA-seq compared gene expression with/without 40 g·L−1 sodium formate; qRT-PCR validated selected genes. - Genetic perturbations to validate pathways: Constructed deletion mutants via MuGENT, targeting six fdh genes (PN96_05840, PN96_05845, PN96_05850, PN96_05880, PN96_21155, PN96_22795), ftl (PN96_20840), and pfl (PN96_08455), singly and in combination. Phenotyped growth and formate consumption. - Design and construction of an S-TCA metabolic sink: Engineered integration of serine cycle with TCA to pull FA-assimilation flux by deleting madh (PN96_06470, PN96_19465, PN96_11695), maeAB (PN96_07295, PN96_14755), aceB (PN96_10585), and serA (PN96_00930), yielding strain S-TCA-1.0. Assessed transcription of S-TCA genes and intracellular metabolites associated with the cycle. - Adaptive laboratory evolution (ALE): Evolved S-TCA-1.0 under increasing sodium formate in LBv2, with periodic UV mutagenesis, to obtain S-TCA-2.0 tolerant to high FA. Parallel ALE on wild type served as comparison. Evaluated growth and consumption in high FA. - Whole-genome resequencing of evolved isolates: Identified recurrent mutations across three S-TCA-2.0 isolates; analyzed potential relevance to flux redistribution (e.g., fumA, sdhC). - 13C tracing and assimilation quantification: Grew S-TCA-2.0 in modified M9 with 13C-formate to estimate specific formate assimilation rate and fraction in biomass. For product-pathway linkage, used 13C-formate to determine labeling in amino acids of S-TCA-2.0-IE. - Product pathway integration and fed-batch fermentation: Introduced indigoidine biosynthesis genes (idgs from Streptomyces lividans/lavendulae CGMCC 4.1386 and sfp from Bacillus subtilis 168) on a plasmid into S-TCA-2.0 to create S-TCA-2.0-IE. Conducted fed-batch in LBv2 starting at ~60 g·L−1 formate with sequential additions every 12 h. Quantified indigoidine by extraction and spectrophotometric assay calibrated with purified standard; monitored formate by HPLC and growth by OD600. - Metabolomics and targeted assays: LC–MS/MS profiled S-TCA metabolites; measured specific intermediates (e.g., glycine) and amino-acid labeling patterns by GC–MS/UHPLC–MS.

- Native FA tolerance and consumption: In LBv2, V. natriegens grew with 20–40 g·L−1 sodium formate (192–385 mM), consuming all formate within 24 h; at 60 g·L−1 (577 mM), growth was minimal but 69 mM formate was still consumed in 24 h. In M9 + 4 g·L−1 glucose, 288 mM formate fully inhibited growth; at 192 mM, cells consumed 95 mM in 24 h (~3.96 mM·h−1). Overall, native consumption exceeded many reported microbes. - Pathway genetics: Genome encodes multiple FDHs (two fdhFs, fdhA, fdol, fdoH, fdhD), THF-cycle genes (ftl, folD) and pfl. RNA-seq under FA stress showed up-regulation of FDHs (up to 9.7-fold, PN96_05850), pfl (8.9-fold), serine-cycle, reductive glycine, and TCA-cycle genes, indicating activation of dissimilation and assimilation coupled to enhanced energy metabolism. - Functional validation: Deleting fdh or ftl significantly impaired growth and formate consumption; deleting pfl had no effect, indicating low PFL activity toward formate. Triple deletion (fdh, ftl, pfl) reduced but did not abolish formate consumption, suggesting additional/alternative routes. - S-TCA design outcome: Engineered S-TCA-1.0 showed reduced total consumption and growth versus wild type at 40 g·L−1 FA, but substantially higher formate consumption per unit biomass, with correct expression of S-TCA genes and elevated levels of several S-TCA metabolites, confirming enhanced per-cell flux. - ALE to S-TCA-2.0: Evolution yielded a strain growing with up to 140 g·L−1 sodium formate (1,346 mM). In media initially containing 85 g·L−1 (817 mM), S-TCA-2.0 reached OD600 ~1.6 and consumed 78.9 g·L−1 (759 mM) within 24 h, at 1.42 g·L−1·h−1 (31.6 mM·h−1) – several-fold above prior reports. Parallel ALE of wild type adapted more slowly and stalled near 70 g·L−1. - Mutations in evolved isolates: Nine recurrent gene mutations identified (rpoS, sprT, ZapC, actP, glpR, fumA[A44E], hutH, sdhC[indel], PN96_19530). fumA and sdhC alterations likely modulate TCA/S-TCA flux partitioning. - Assimilation fraction: Specific formate assimilation in S-TCA-2.0 was 43.3 mg·gDCW−1·h−1, ~12.1% of total consumed formate (lower bound; secreted metabolites not accounted for). - Product synthesis from formate: S-TCA-2.0-IE produced visible indigoidine in 24 h at 85 g·L−1 FA. 13C-formate labeling showed incorporation (mostly m+1) across 11 amino acids, with serine (45.7%) and methionine (39.4%) most labeled, consistent with proximity to THF-cycle flux. - Fed-batch performance: With initial 60 g·L−1 FA and five 60 g·L−1 pulses (total ~360 g·L−1 added; consumed ~165.3 g·L−1), S-TCA-2.0-IE reached OD600 ~3.1 at 60 h and produced 29.0 g·L−1 indigoidine in 72 h with a formate consumption rate of 2.3 g·L−1·h−1. Without FA addition, indigoidine reached only 4.7 g·L−1.

The findings demonstrate that Vibrio natriegens possesses innate tolerance and metabolic capacity for formate superior to many microbes, and that coupling a designed serine–TCA (S-TCA) loop to FA assimilation effectively increases flux by leveraging the TCA cycle as a metabolic sink. Despite an initial fitness cost from pathway rewiring, adaptive laboratory evolution rapidly restored growth and dramatically enhanced formate utilization, indicating high metabolic plasticity of V. natriegens. Transcriptomics under FA stress revealed activation of FDH-mediated oxidation (generating reducing power) and up-regulation of downstream assimilation pathways (reductive glycine, serine cycle, TCA), which likely pull THF-cycle flux. Genetic knockouts confirmed essential roles for fdh and ftl in FA metabolism, while residual consumption after triple deletions hints at alternative or incomplete pathways. Evolved mutations in fumA and sdhC suggest that tuning branch points in the S-TCA/TCA cycles can reduce flux diversion and optimize throughput. The engineered strain efficiently co-utilized formate to boost indigoidine synthesis, with isotopic data confirming incorporation of formate-derived carbon into amino acids and product precursors, though a large fraction of formate served for energy and cofactor generation. Collectively, integrating rational pathway design with ALE presents a generalizable route to build high-performance FA-utilizing platforms.

This work identifies Vibrio natriegens as a powerful chassis for formic acid biorefinery and establishes a successful strategy that combines a designed serine–TCA metabolic sink with adaptive laboratory evolution to achieve high formate tolerance and consumption. The evolved strain S-TCA-2.0 consumes formate at record rates and, when equipped with an indigoidine pathway, produces up to 29.0 g·L−1 indigoidine while consuming ~165 g·L−1 formate in 72 h. The genetic insights (e.g., fumA, sdhC mutations) and demonstrated pathway integration provide design rules for further optimization and application to other products and hosts. Future work should improve assimilation yield (greater incorporation into biomass and products), elucidate potential alternative assimilation routes, fine-tune S-TCA flux control (e.g., GCS/serine cycle expression balance), and enable efficient growth on FA as the sole carbon source.

- Growth and performance were primarily demonstrated in nutrient-rich LBv2 medium; like other systems, robust growth on formate as the sole organic carbon source remains challenging. - A substantial portion of consumed formate was oxidized to CO2 to supply energy/reducing power, with only a modest fraction directly assimilated into biomass and product carbon, limiting carbon yield. - The engineered S-TCA pathway likely contains bottlenecks; overexpression of some GCS and serine-cycle genes negatively impacted consumption, indicating the need for finer control. - Despite deletion of fdh, ftl, and pfl, residual formate consumption persisted, implying uncharacterized routes whose identities and contributions are unclear. - Reported production and rates were under specific culture conditions; scalability and performance in minimal media or industrial settings require further validation.