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Keywords:

  • aatTn7;
  • acetogens;
  • ack ;
  • al-adh;
  • gene elimination;
  • pta ;
  • sporulation;
  • syngas fermentation

Abstract

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.