Optimization of n -butanol synthesis in Lactobacillus brevis via the functional expression of thl , hbd , crt and ter

N -butanol is an important chemical and can be naturally synthesized by Clostridium species; however, the poor n-butanol tolerance of Clostridium impedes the further improvement in titer. In this study, Lactobacillus brevis , which possesses a higher butanol tolerance, was selected as host for heterologous butanol production. The Clostridium acetobutylicum genes thl , hbd , and crt which encode thiolase, β-hydroxybutyryl-CoA dehydrogenase, and crotonase, and the Treponema denticola gene ter , which encodes trans -enoyl-CoA reductase were cloned into a single plasmid to express the butanol synthesis pathway in L. brevis . A titer of 40 mg/L n -butanol was initially achieved with plasmid pLY15-opt, in which all pathway genes are codon-optimized. A titer of 450 mg/L of n-butanol was then synthesized when ter was further overexpressed in this pathway . The role of metabolic flux was reinforced with pLY15, in which only the ter gene was codon-optimized, which greatly increased the n-butanol titer to 817 mg/L. Our strategy significantly improved n-butanol synthesis in L. brevis and the final titer is the highest achieved amongst butanol-tolerant lactic acid bacteria. Graphic abstract