Discovery and remodeling ofVibrio natriegensas a microbial platform for efficient formic acid biorefinery

Abstract Formic acid (FA), an organic one-carbon source that is easily produced from CO 2 , has emerged as a promising CO 2 -equivalent feedstock for one-carbon biorefinery. However, developing efficient formatotrophs for an economically competitive FA utilization system remains a grand challenge. Here, we discovered that the gram-negative bacterium Vibrio natriegens has exceptional FA tolerance and metabolic capacity natively. This strain was then remodeled by rewiring the serine cycle and the TCA cycle which resulted in a non-native closed loop (S-TCA) for enhancing FA assimilation. The rational design generated a powerful metabolic sink that enabled rapid emergence of the evolved strains with further significantly improved performance in using FA as the major or sole carbon source. Furthermore, the introduction of a foreign indigoidine-forming pathway into the best-performing V. natriegens strain (S-TCA-2.0) led to production of 29.0 g·L -1 indigoidine and consumption of 233.7 g·L -1 formate within 72 h, achieving an order of magnitude higher formate consumption rate (3.2 g·L -1 ·h -1 ) than the reported highest level in microorganisms. This work represents a significant step towards the development of industrially viable microorganisms for FA biorefinery.