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

Abstract

Aims: To engineer acetogen biocatalyst selectively overproducing ethanol from synthesis gas or CO 2 /H 2 as the only liquid carbonaceous product. Methods and Results: Ethanol-resistant mutant originally capable of producing only acetate from CO 2 /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 À1 and 1018 mmol l À1 of ethanol, respectively, in five independent single-step fermentation runs 25 days each (P < 0Á005) in five independent repeats using syngas blend 60% CO and 40% H 2 . Ethanol production was 64% if only CO 2 + H 2 blend was used compared with syngas blend (P < 0Á005). 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 CO 2 /H 2 blend might be utilized as a tool to mitigate global warming proportional to CO 2 fermentation scale.

Ethanol and Acetic Acid Production from Carbon Monoxide in a Clostridium Strain in Batch and Continuous Gas-Fed Bioreactors

“…However, the exact mechanisms involved in energy conservation remain still unclear. Very recently, metabolically engineered acetogens have been used to selectively produce metabolites from CO [ 6 , 7 ], although it is also possible to produce specific metabolites of interest from CO, in wild type bacteria, through manipulation of the medium composition and/or operating conditions in bioreactors [ 8 , 9 ]. Several acetogens are known to produce acetic acid, as major end metabolite, from CO, including Moorella thermoacetica , Acetobacterium woodii , Eubacterium limosum KIST 612, Peptostreptococcus productus U-1 and Clostridium aceticum [ 3 ]; whereas Clostridium ljungdahlii , Clostridium autoethanogenum , Clostridium ragsdalei and Alkalibaculum bacchi are ethanologenic acetogens, able to produce ethanol besides acetic acid [ 2 , 3 ].…”

New challenges for syngas fermentation: towards production of biopolymers

“…In another study, Kiriukhin and Tyurin eliminated genes unnecessary for syngas fermentation to boost an artificial integrated pathway performance. They engineered an acetogenic biocatalyst to selectively overproduce ethanol from syngas or CO 2 /H 2 blends as the only liquid carbonaceous end product.…”