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The cell energy fraction that powered maintenance and expression of genes encoding pro-phage elements, pta- ack cluster, early sporulation, sugar ABC transporter periplasmic proteins, 6-phosphofructokinase, pyruvate kinase, and fructose-1,6-disphosphatase in acetogen Clostridium sp. MT871 was re-directed to power synthetic operon encoding isobutanol biosynthesis at the expense of these genes achieved via their elimination. Genome tailoring decreased cell duplication time by 7.0 ± 0.1 min ( p < 0.05) compared to the parental strain, with intact genome and cell duplication time of 68 ± 1 min ( p < 0.05). Clostridium sp. MT871 with tailored genome was UVC-mutated to withstand 6.1 % isobutanol in fermentation broth to prevent product inhibition in an engineered commercial biocatalyst producing 5 % (674.5 mM) isobutanol during two-step continuous fermentation of CO/H gas blend. Biocatalyst Clostridium sp. MT871RGIB6 was engineered to express six copies of synthetic operon comprising optimized synthetic format dehydrogenase, pyruvate formate lyase, acetolactate synthase, acetohydroxyacid reductoisomerase, 2,3-dihydroxy-isovalerate dehydratase, branched-chain alpha-ketoacid decarboxylase gene, aldehyde dehydrogenase, and alcohol dehydrogenase, regaining cell duplication time of 68 ± 1 min ( p < 0.05) for the parental strain. This is the first report on isobutanol production by an engineered acetogen biocatalyst suitable for commercial manufacturing of this chemical/fuel using continuous fermentation of CO/H blend thus contributing to the reversal of global warming. [ABSTRACT FROM AUTHOR]

A time- and cost-efficient two-step gene elimination procedure was used for acetogen Clostridium sp. MT1834 capable of fermenting CO/H blend to 245 mM acetate ( p < 0.005). The first step rendered the targeted gene replacement without affecting the total genome size. We replaced the acetate pta- ack cluster with synthetic bi-functional acetaldehyde-alcohol dehydrogenase ( al- adh). Replacement of pta- ack with al- adh rendered initiation of 243 mM ethanol accumulation at the expense of acetate production during CO/H blend continuous fermentation ( p < 0.005). At the second step, al- adh was eliminated to reduce the genome size. Resulting recombinants accumulated 25 mM mevalonate in fermentation broth ( p < 0.005). Cell duplication time for recombinants with reduced genome size decreased by 9.5 % compared to Clostridium sp. MT1834 strain under the same fermentation conditions suggesting better cell energy pool management in the absence of the ack- pta gene cluster in the engineered biocatalyst. If the first gene elimination step was used alone for spo0A gene replacement with two copies of synthetic formate dehydrogenase in recombinants with a shortened genome, mevalonate production was replaced with 76.5 mM formate production in a single step continuous CO/H blend fermentation ( p < 0.005) with cell duplication time almost nearing that of the wild strain. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aims: To engineer acetogen biocatalyst capable of fermenting synthesis gas blend to acetone as the only liquid carbonaceous product. Methods and Results: The metabolic engineering comprised inactivation of phosphotransacetylase via integration of a cassette comprising synthetic genes erm(B), thiolase and HMG-CoA synthase. Acetaldehyde dehydrogenase was inactivated via integration of a cassette consisting of synthetic genes cat, HMG-CoA lyase and acetoacetate decarboxylase. The engineered biocatalyst Clostridum sp. MAceT113 lost production of 253 mmol l−1 ethanol and 296 mmol l−1 acetate and started producing 1·8 mol l−1 acetone in single-stage continuous syngas fermentation. Conclusions: The acetone concentration in culture broth is economical for bulk manufacture because it is about twenty times of that achieved with known acetone-butanol-ethanol fermentation of sugars. Significance and Impact of the Study: The process shows the opportunity to produce acetone from synthesis gas at concentrations comparable with production of acetone from products of petroleum cracking. This is the first report on elimination of acetate and acetaldehyde production and directing carbon flux from Acetyl-CoA to acetone via a non-naturally occurring in acetogen acetone biosynthesis pathway identified in eukaryotic organisms. [ABSTRACT FROM AUTHOR]