Expression of amplified synthetic ethanol pathway integrated usingTn7-tool and powered at the expense of eliminatedpta,ack,spo0A and spo0J during continuous syngas or CO2/H2blend fermentation

AIMS: To engineer acetogen biocatalyst selectively overproducing ethanol from synthesis gas or CO2 /H2 as the only liquid carbonaceous product. METHODS AND RESULTS: Ethanol-resistant mutant originally capable of producing only acetate from CO2 /CO was engineered to eliminate acetate production and spore formation using our proprietary Cre-lox66/lox71-system. Bi-functional aldehyde/alcohol dehydrogenase was inserted into the chromosome of the engineered mutant using Tn7-based approach. Recombinants with three or six copies of the inserted gene produced 525 mmol l(-1) and 1018 mmol l(-1) of ethanol, respectively, in five independent single-step fermentation runs 25 days each (P < 0.005) in five independent repeats using syngas blend 60% CO and 40% H2 . Ethanol production was 64% if only CO2 + H2 blend was used compared with syngas blend (P < 0.005). CONCLUSIONS: Elimination of genes unnecessary for syngas fermentation can boost artificial integrated pathway performance. SIGNIFICANCE AND IMPACT OF THE STUDY: Cell energy released via elimination of phosphotransacetylase, acetate kinase and early-stage sporulation genes boosted ethanol production. Deletion of sporulation genes added theft-proof feature to the engineered biocatalyst. Production of ethanol from CO2 /H2 blend might be utilized as a tool to mitigate global warming proportional to CO2 fermentation scale.