Syngas Biofuels Energy Inc.

Monte Alto, TX, United States

Syngas Biofuels Energy Inc.

Monte Alto, TX, United States
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Kiriukhin M.,Syngas Biofuels Energy Inc. | Kiriukhin M.,Ajinomoto Co. | Tyurin M.,Syngas Biofuels Energy Inc. | Tyurin M.,Ajinomoto Co.
Bioprocess and Biosystems Engineering | Year: 2014

Naturally mevalonate-resistant acetogen Clostridium sp. MT1243 produced only 425 mM acetate during syngas fermentation. Using Clostridium sp. MT1243 we engineered biocatalyst selectively producing mevalonate from synthesis gas or CO2/H2 blend. Acetate production and spore formation were eliminated from Clostridium sp. MT1243 using Cre-lox66/lox71-system. Cell energy released via elimination of phosphotransacetylase, acetate kinase and early stage sporulation genes powered mevalonate accumulation in fermentation broth due to expression of synthetic thiolase, HMG-synthase, and HMG-reductase, three copies of each, integrated using Tn7-approach. Recombinants produced 145 mM mevalonate in five independent single-step fermentation runs 25 days each in five repeats using syngas blend 60 % CO and 40 % H2 (v/v) (p < 0.005). Mevalonate production was 97 mM if only CO2/H2 blend was fed instead of syngas (p < 0.005). Mevalonate from CO 2/H2 blend might serve as a commercial route to mitigate global warming in proportion to CO2 fermentation scale worldwide. © 2013 Springer-Verlag Berlin Heidelberg.


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.


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

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. © Springer Science+Business Media, LLC 2012.


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. © 2013 The Society for Applied Microbiology.


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.


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.


Berzin V.,Syngas Biofuels Energy Inc. | Kiriukhin M.,Ajinomoto Co. | Tyurin M.,Syngas Biofuels Energy Inc.
Archives of Microbiology | Year: 2013

Plasmid-free acetogen Clostridium sp. MT962 electrotransformed with a small cryptic plasmid pMT351 was used to develop time- and cost-effective methods for plasmid elimination. Elimination of pMT351 restored production of acetate and ethanol to the levels of the plasmid-free strain with no dry cell weight changes. Destabilizing cell membrane via microwave at 2.45 GHz, or exposure to a single 12 ms square electric pulse at 35 kV cm-1, eliminated pMT351 in 42-47 % of cells. Plasmid elimination with a single square electric pulse required 10 versus 0.1 J needed to introduce the same 3,202-bp plasmid into the cells as calculated per cell sample of Clostridium sp. MT962. Microwave caused visible changes in repPCR pattern and increased ethanol production at the expense of acetate. This is the first report on microwave of microwave ovens, wireless routers, and mobile devices causing chromosomal DNA aberrations in microbes along with carbon flux change. © 2012 Springer-Verlag Berlin Heidelberg.


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.


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. © Springer Science+Business Media New York 2013.

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