(176z) Extending Carbon Chain Length in Genome-Engineered Clostridium Tyrobutyricum for Butanol and Hexanol Production with Enhanced Carbon Flux and NADH Availability

Clostridium tyrobutyricum overexpressing the heterologous aldehyde/alcohol dehydrogenase (AAD) encoded by the adhE2 from Clostridium acetobutylicum is a promising n-butanol producer. Further extending the carbon chain length from butanol to hexanol is of high interest because of the higher cetane index and energy density of hexanol, which can also be chemically converted into 1-hexene. The feasibility of n-hexanol biosynthesis in C. tyrobutyricum was investigated by extending the coenzyme A (CoA) dependent n-butanol synthesis pathway. First, the C4-acyl-CoA intermediates acetyl-CoA acetyltransferase (atoB), 3-hydroxybutyryl-coA dehydrogenase (hbd), crotonase (crt), and trans-enoyl-CoA reductase (ter) from different organisms were overexpressed to further increase the carbon flux from acetyl-CoA to butyryl-CoA in C. tyrobutyricum ΔMΔIΔuppΔcat1::adhE2. Then, by overexpressing b-ketothiolase (BktB), butyryl-CoA was further extended to hexanoyl-CoA, which can be reduced to yield hexanol by AAD. To further increase butanol and hexanol production in engineered C. tyrobutyricum, the endogenous hydrogenase (hyd) and redox-sensing protein Rex were knocked out via iterative genome-editing using CRISPR-Cas system. These mutants were evaluated for their batch fermentation kinetics in serum bottles and bioreactors, and the results will be reported.