(599cg) How to Increase Butanol Production By Metabolic Cell-Process Engineering of Clostridium Tyrobutyricum?

Biobutanol is a sustainable and environmentally friendly biofuel that is a potential substitute of imported petroleum in future. The overall goal of this study is to increase butanol production by acidogenic Clostridium tyrobutyricum using Metabolic Cell-Process Engineering. We first performed comparative proteomics analysis to identify the proteins with significant expression change using three strains, including wild type C. tyrobutyricum producing 20 g/L of butyrate, ACKKO producing 38 g/L of butyrate, and ACKKO-adhE2 producing 16 g/L of butanol. The integrated proteomics and fermentation production analysis demonstrated that both the carbon redistribution enzymes related to primary metabolism and the enzymes rebalancing redox regulated butanol production. Therefore, we engineered the wild type C. tyrobutyricum by redistributing carbon via aldehyde/alcohol dehydrogenase and rebalancing redox via NAD⁺-dependent formate dehydrogenase using double vector system. It is found that the butanol titer by the carbon and redox balanced mutant (2.7 g/L) was 1.7 fold higher than that produced by the carbon rebalanced mutant (1.0 g/L). It is clear that redox balance was important to butanol formation, which can be targeted to further engineer high butanol producing strain. In addition, the metabolic process engineering was performed to screen the medium supplement, showing that butanol selectivity was increased by more than 30% with redox supplement. The metabolite analysis also identified the amino acids correlated with high butanol formation. We are confident that the butanol can be further improved by the metabolic cell-process engineering by integrating rational cell engineering with fermentation process engineering.