Microbial utilization of one-carbon (C1) compounds for producing value-added products is a significant area of research, offering a sustainable route using abundant carbon sources. Formic acid (FA), efficiently derived from CO2 or syngas, is a promising C1 feedstock. Its liquid state simplifies storage compared to gaseous alternatives, making it a convenient CO2-equivalent carbon source. Native FA-utilizing microorganisms employ pathways like the Wood-Ljungdahl pathway, the serine cycle, and the reductive glycine pathway, all relying on a core FA-fixing module: the tetrahydrofolate (THF) cycle initiated by formate-tetrahydrofolate ligase (FTL). Pyruvate formate-lyase (PFL) also plays a role. However, these microorganisms often exhibit slow growth and inefficient FA utilization. Previous efforts have focused on engineering microorganisms like *Escherichia coli*, but their efficiency remains limited by weak FA tolerance and metabolic capacity. Therefore, identifying and utilizing a more suitable microbial chassis is crucial for establishing efficient FA bioconversion platforms. *Vibrio natriegens*, known for its rapid growth (shortest doubling time among known bacteria), is explored as a potential candidate. Its FA tolerance and metabolic capacity, however, were previously unknown.

The assimilation of formate by microorganisms has been studied extensively. The Wood-Ljungdahl pathway, serine cycle, and reductive glycine pathway are well-established mechanisms for incorporating formate into metabolism. These pathways typically utilize formate-tetrahydrofolate ligase (FTL) to initiate the process. However, existing microorganisms that utilize formate often exhibit limitations in growth rate and efficiency. Engineered *Escherichia coli* strains have shown some improvement in formate assimilation through the reconstruction of the THF cycle and the reverse glycine cleavage pathway, but their efficiency remains far below that of traditional carbon sources like sugars. This underscores the need for a more suitable microbial chassis with inherent formate tolerance and metabolic capacity.

The study began by evaluating the growth and formate consumption of *V. natriegens* at various formate concentrations in LBv2 and M9 minimal media. The genomic analysis of *V. natriegens* identified genes related to formate assimilation (FTL, THF cycle genes, PFL) and dissimilation (multiple *fdh* genes encoding formate dehydrogenases). Comparative transcriptomic analysis (RNA-seq and qRT-PCR) examined transcriptional changes following formate addition. Gene deletion experiments were performed to validate the roles of predicted pathways in formate metabolism. To enhance formate utilization, the researchers designed a metabolic sink by integrating the serine and TCA cycles. This involved manipulating genes involved in malate dehydrogenase (*madh*), malate synthase (*maeAB*), and serine synthesis (*serA*) to redirect metabolic flux through the TCA cycle. The resulting strain, S-TCA-1.0, was then subjected to adaptive laboratory evolution (ALE) with progressively increasing formate concentrations, leading to the evolved strain S-TCA-2.0. Genome sequencing compared S-TCA-2.0 to S-TCA-1.0 to identify mutations potentially responsible for enhanced formate utilization. ¹³C isotopomer analysis quantified formate assimilation. Finally, the indigoidine synthesis pathway was introduced into S-TCA-2.0 to evaluate its potential for producing valuable compounds from formate, and fed-batch cultivation was performed to assess the strain's capability for indigoidine production.

*V. natriegens* exhibited superior formate consumption compared to other reported microorganisms. Genomic and transcriptomic analyses revealed the involvement of multiple formate dehydrogenases, the THF cycle, the reductive glycine pathway, the serine cycle, and the TCA cycle in formate metabolism. Deletion experiments confirmed the importance of formate dissimilation and assimilation pathways. The engineered S-TCA cycle, combining the serine and TCA cycles, significantly enhanced formate metabolic flux per unit biomass, although it initially impaired overall growth. Adaptive laboratory evolution (ALE) of S-TCA-1.0 yielded S-TCA-2.0, a strain capable of extraordinarily high formate consumption (1.42 g L⁻¹.h⁻¹, several times higher than previously reported). Genome sequencing of S-TCA-2.0 isolates identified several mutations, including ones in *fumA* (fumarate hydratase) and *sdhC* (succinate dehydrogenase), that may contribute to its enhanced formate utilization. ¹³C isotopomer analysis showed approximately 12.1% of consumed formate was assimilated. Finally, strain S-TCA-2.0-IE, engineered to produce indigoidine, yielded 29.0 g L⁻¹ indigoidine and consumed 165.3 g L⁻¹ formate within 72 h in a fed-batch culture, a high level compared to previously reported microbial indigoidine production.

This study demonstrates the remarkable potential of *V. natriegens* as a microbial platform for formic acid biorefinery. The combination of rational metabolic engineering and adaptive laboratory evolution proved highly effective in improving formate utilization. The creation of the artificial S-TCA cycle was pivotal in driving the metabolic flux toward enhanced formate assimilation and conversion. Although formate assimilation through the THF cycle is energy-consuming, the co-utilization of other carbon sources or formate oxidation via FDH likely contributes to the energy balance. The high indigoidine production by S-TCA-2.0-IE in the LBv2 medium suggests that formate oxidation generates reducing power and supports the efficient utilization of other carbon sources for indigoidine synthesis. The findings highlight the metabolic flexibility of *V. natriegens* and provide valuable insights for engineering other microbial chassis for formate utilization. Further optimization of the S-TCA cycle, such as through the manipulation of glycine cleavage system and serine cycle genes, shows potential for further enhancement.

This research successfully established *Vibrio natriegens* as a high-performance microbial chassis for formate biorefinery. The creation of the S-TCA metabolic loop and the use of adaptive laboratory evolution proved to be a powerful strategy for enhancing formate utilization efficiency. The high yield of indigoidine produced demonstrates the industrial potential of this engineered strain. Future research could focus on further optimization of the S-TCA pathway, exploring other valuable products synthesized from formate, and adapting this metabolic engineering strategy to other microbial hosts.

While the study demonstrates impressive formate utilization and indigoidine production, the use of LBv2 medium, containing yeast extract and peptone, means that the precise contribution of formate as the sole carbon source is not fully elucidated. The identification of specific mutations responsible for the enhanced phenotype in S-TCA-2.0 requires further investigation beyond the nine identified mutations. The relatively high concentration of formate used may not be economically viable at an industrial scale. Further research is needed to optimize the process for lower formate concentrations.