Elimination of Acetate Production to Improve Ethanol Yield During Continuous Synthesis Gas Fermentation by Engineered Biocatalyst Clostridium sp. MTEtOH550

Acetogen strain Clostridum sp. MT653 produced acetate 273?mM (p?<?0.005) and ethanol 250?mM (p?<?0.005) from synthesis gas blend mixture of 64?% CO and 36?%?H₂. Clostridum sp. MT653 was metabolically engineered to the biocatalyst strain Clostridium sp. MTEtOH550. The biocatalyst increased ethanol yield to 590?mM with no acetate production during single-stage continuous syngas fermentation due to expression of synthetic adh cloned in a multi-copy number expression vector. The acetate production was eliminated by inactivation of the pta gene in Clostridium sp. MTEtOH550. Gene introduction and gene elimination were achieved only using Syngas Biofuels Energy, Inc. electroporation generator. The electrotransformation efficiencies were 8.0?±?0.2?×?10⁶ per microgram of transforming DNA of the expression vector at cell viability ~15?%. The frequency of suicidal vector integration to inactivate pta was ~10^?5 per the number of recipient cells. This is the first report on elimination of acetate production and overexpression of synthetic adh gene to engineer acetogen biocatalyst for selective biofuel ethanol production during continuous syngas fermentation.     

Cre-lox66/lox71-Based Elimination of Phosphotransacetylase or Acetaldehyde Dehydrogenase Shifted Carbon Flux in Acetogen Rendering Selective Overproduction of Ethanol or Acetate

Acetogen strain Clostridium sp. MT1121 produced 300?mM acetate (p?<?0.005) and 321?mM ethanol (p?<?0.005) from synthesis gas (syngas) blend 60?% CO and 40?%?H₂. Clostridium sp. MT1121 was metabolically engineered to eliminate production of either acetate or acetaldehyde during syngas fermentation. We used Cre-lox66/lox71-based gene removal system to eliminate either phosphotransacetylase (pta), or acetaldehyde dehydrogenase (aldh). The resulted biocatalyst with eliminated pta increased ethanol yield to 610?mM (p?<?0.005). Inactivation of pta rendered only 502?mM of ethanol (p?<?0.005). The acetogen biocatalyst with eliminated aldh produced 450?mM acetate (p?<?0.005). The role of cell energy pool preservation for re-directed carbon flux is discussed. This is the first report on time- and cost-efficient gene elimination in acetogens using lox66/lox71 gene elimination system.     

Selective n-Butanol Production by Clostridium sp. MTButOH1365 During Continuous Synthesis Gas Fermentation Due to Expression of Synthetic Thiolase, 3-Hydroxy Butyryl-CoA Dehydrogenase, Crotonase, Butyryl-CoA Dehydrogenase, Butyraldehyde Dehydrogenase, and NAD-Dependent Butanol Dehydrogenase

Acetogen Clostridum sp. MT1962 produced 287 mM acetate (p?<?0.005) and 293 mM ethanol (p?<?0.005) fermenting synthesis gas blend 60 % CO and 40 %?H₂ in single-stage continuous fermentation. This strain was metabolically engineered to the biocatalyst Clostridium sp. MTButOH1365. The engineered biocatalyst lost production of ethanol and acetate while initiated the production of 297 mM of n-butanol (p?<?0.005). The metabolic engineering comprised Cre-lox66/lox71-based elimination of phosphotransacetylase and acetaldehyde dehydrogenase along with integration to chromosome synthetic thiolase, 3-hydroxy butyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase, and NAD-dependent butanol dehydrogenase. This is the first report on elimination of acetate and ethanol production genes and expression of synthetic gene cluster encoding n-butanol biosynthesis pathway in acetogen biocatalyst for selective fuel n-butanol production with no antibiotic support for the introduced genes.     

Synthetic 2,3-Butanediol Pathway Integrated Using Tn7-tool and Powered Via Elimination of Sporulation and Acetate Production in Acetogen Biocatalyst

Acetogen Clostridium sp. MT1802 originally producing 336-mM acetate from inorganic carbon of CO₂/CO was engineered to eliminate acetate production and sporulation using Cre-lox66/lox71-approach. The recombinant started producing 105-mM formate expressing synthetic formate dehydrogenase integrated in two copies. Formate-producing recombinant was further engineered to express synthetic formate acetyltransferase, acetolactate synthase, acetolactate decarboxylase, and alcohol dehydrogenase integrated in two copies each using Tn7 tool. The resulted recombinant started producing 102-mM 2,3-butanediol (23BD). 23BD production was confirmed in five independent single step fermentation runs 25 days long each in five repeats using syngas blend 60 % CO and 40 % H₂ (v/v) (p <0.005). 23BD production was 78 % if only CO₂/H₂ blend was fed instead of syngas (p <0.005). 23BD from CO₂/H₂ blend might serve as a commercial route to mitigate global warming in proportion to CO₂ fermentation scale worldwide.