Stepwise engineering of Clostridium ljungdahlii for efficient ethanol production from single-carbon gases

Abstract

Ethanol, a cornerstone biofuel, has been successfully produced on an industrial scale via fermentation of industrial off-gases (CO/CO₂) using gas-fermenting Clostridium strains. To further enhance clostridial ethanol synthesis during gas fermentation, efforts have focused on the rational design and genetic modification of Clostridium strains. However, progress remains limited, indicating substantial potential for further optimization and improvement. In this study, we systematically and genetically engineered Clostridium ljungdahlii DSM 13528, a well-characterized gas-fermenting Clostridium strain, to achieve highly efficient ethanol synthesis via gas fermentation. By implementing a three-pronged metabolic strategy—strengthening ethanol synthesis pathways, improving acetate assimilation, and reducing 2,3-butanediol byproduct formation—we maximized carbon flux toward ethanol biosynthesis. The optimized strain exhibited a remarkable ethanol synthesis capacity for fermenting a CO–CO₂–H₂ mixture, producing 30.1 g/L of ethanol within 102 h. This study established fundamental metabolic engineering principles for developing high-performance gas-fermenting Clostridium strains for sustainable ethanol synthesis from CO/CO₂ substrates. This strategy can be readily adapted to industrial gas-fermenting Clostridium strains to enable more economically viable ethanol production.