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Tyurin M.,Syngas Biofuels Energy Inc.
Journal of Industrial Microbiology and Biotechnology | Year: 2013

A time- and cost-efficient two-step gene elimination procedure was used for acetogen Clostridium sp. MT1834 capable of fermenting CO2/H 2 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 CO2/H2 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 CO2/H2 blend fermentation (p < 0.005) with cell duplication time almost nearing that of the wild strain. © 2013 Society for Industrial Microbiology and Biotechnology.

Acetogen Clostridum sp. MT683 produced 272 mM acetate (p<0.005) and 263 mM ethanol (p<0.005) fermenting syngas (60% CO + 40% H2) in a single stage continuous fermentation with zero CO2 emission. Inactivation of phosphotransacetylase (pta) gene in Clostridium sp. MT683 eliminated acetate production and increased ethanol yield to 363 mM (p<0.005). Ethanol production in Clostridum sp. MT683 was further increased when the synthetic acetaldehyde dehydrogenase (aldh) from Clostridium ljungdahlii was cloned in the multi-copy expression vector in this strain. The resulted biocatalyst increased ethanol yield to 576 mM (p<0.005). Electrotransformation efficiencies were (9.30 + 0.15) × 106 transformants/μg of the expression vector DNA at cell viability ~15%. The integration frequency of pta inactivation was (2.12 + 0.02) × 10-5. Recorded in real time pulse current oscillations reflected the cell membranes electropermeabilization events. This is the first report on inactivation of pta and expression of synthetic aldh in the acetogen biocatalyst for selective biofuel ethanol production during continuous single step syngas fermentation.

Tyurin M.,Syngas Biofuels Energy Inc. | Kiriukhin M.,Ajinomoto Co.
Applied Biochemistry and Biotechnology | Year: 2013

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. © 2013 Springer Science+Business Media New York.

Tyurin M.,Syngas Biofuels Energy Inc. | Kiriukhin M.,Ajinomoto Co.
World Journal of Microbiology and Biotechnology | Year: 2013

Methanol-resistant mutant acetogen Clostridium sp. MT1424 originally producing only 365 mM acetate from CO2/CO was engineered to eliminate acetate production and spore formation using Cre-lox66/lox71-system to power subsequent methanol production via expressing synthetic methanol dehydrogenase, formaldehyde dehydrogenase and formate dehydrogenase, three copies of each, assembled in cluster and integrated to chromosome using Tn7-based approach. Production of 2.2 M methanol was steady (p < 0.005) in single step fermentations of 20 % CO2 + 80 % H2 blend (v/v) 25 day runs each in five independent repeats. If the integrated cluster comprised only three copies of formate dehydrogenase the respective recombinants produced 95 mM formate (p < 0.005) under the same conditions. For commercialization, the suggested source of inorganic carbon would be CO2 waste of IGCC power plant. Hydrogen may be produced in situ via powered by solar panels electrolysis. © 2013 Springer Science+Business Media Dordrecht.

Berzin V.,Syngas Biofuels Energy Inc. | Kiriukhin M.,Ajinomoto Co. | Tyurin M.,Syngas Biofuels Energy Inc.
Applied Biochemistry and Biotechnology | Year: 2012

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 %H 2. 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±0.2×10 6 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. © 2012 Springer Science+Business Media, LLC.

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