Metabolic reprogramming of the acetogen Clostridium sp. AWRP via adaptive laboratory evolution for improved formate-based growth
Formate is a promising, sustainable feedstock for microbial conversion into value-added products via the Wood–Ljungdahl pathway. Based on the identification of three distinct formate dehydrogenases in Clostridium sp. AWRP, this study investigated the potential of this acetogen for formate-based growth. Although the wild-type strain exhibited an extended lag phase due to formate toxicity, adaptive laboratory evolution yielded the adapted strain F30T, which demonstrated a significantly enhanced growth rate and tolerance to formate concentrations up to 300 mM. Whole-genome and transcriptomic analyses of the F30T strain revealed a sophisticated, multi-layered strategy for mitigating organic acid stress and optimizing energy conservation. Adaptive laboratory evolution resulted in six key non-synonymous mutations, including those in ackA and adhE1 , and triggered the differential expression of 948 genes out of 4199 genes under the formate-supplemented condition. Specifically, F30T achieved intracellular pH buffering and supplemental ATP generation by upregulating the arginine deiminase pathway, histidine biosynthesis, and ethanolamine utilization. To maintain growth near the thermodynamic limit, the strain implemented a stringent energy-saving program by downregulating the F₀F₁-type ATP synthase and the methyl-branch of the Wood–Ljungdahl pathway, while simultaneously enhancing stress resilience through molecular chaperone upregulation. In a pH-stat fed-batch system using formic acid, the adapted F30T strain showed a higher biomass yield while achieving a similar acetate yield from formate as the wild-type strain. This study demonstrated that adaptive laboratory evolution is a highly effective strategy for enhancing the formate utilization and stress tolerance of Clostridium sp. AWRP. Through integrated genomic and transcriptomic analyses, the molecular basis of the F30T strain’s improved performance was elucidated, identifying key mutations and coordinated metabolic and regulatory mechanisms that maintain cellular homeostasis and maximize energy efficiency. These findings provide valuable molecular insights to aid the targeted design and optimization of formate-utilizing acetogens for use in microbial cell factories aimed at sustainable C1-based bioproduction.