Synthetic 2,3-Butanediol Pathway Integrated Using Tn7-tool and Powered Via Elimination of Sporulation and Acetate Production in Acetogen Biocatalyst
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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.
KeywordsAcetogens Formate 2,3-butanediol CO2 Fermentation Tn7-based Gene Integration
The research was supported by the funds of angel friends of MT family. Syngas Biofuels Energy, Inc. and its successors are the sole distributors of the electroporation and electrofusion equipment: www.syngasbiofuelsenergy.com.
Conflict of Interest
The authors declare that they have no conflict of interest.
- 3.Tyurin, M., & Kiriukhin, M. (2013). Ethanol overproduction during continuous syngas fermentation due to expression of amplified ethanol biosynthesis pathway integrated using Tn7-tool and powered at the expense of eliminated acetic acid production and spore formation. Journal of Applied Microbiology. doi: 10.1111/jam.12123.Google Scholar
- 6.Köpke, M., Michalcea, C., Liew, F.-M., Tizard, J. H., Ali, M. S., Conolly, J. J., et al. (2011). 2, 3-butanediol production by acetogenic bacteria, an alternative route to chemical synthesis, using industrial waste gas. Applied and Environmental Microbiology, 77(15), 5467–5475.CrossRefGoogle Scholar
- 9.Gaddy, J. L., Arora, D. D., Ko, C.-W., Phillips, J. R., Basu, R., Wikstrom, C. V., Clausen, E. C. (2001). Methods for increasing the production of ethanol from microbial fermentation. US Patent 7285402.Google Scholar
- 15.Drake, H. L. (1994). Acetogenesis. NY, London: Chapman & Hall.Google Scholar
- 16.Berzin, V., Kiriukhin, M., & Tyurin, M. (2012). Cre-lox66/lox71-based elimination of phosphotransacetylase or acetaldehyde dehydrogenase shifted carbon flux in acetogen rendering selective overproduction of ethanol or acetate. Applied Biochemistry and Biotechnology. doi: 10.1007/s12010-012-9864-8.Google Scholar
- 19.Tyurin, M., Lynd, L. R., & Wiegel, J. (2006). Genetic delivery systems for strictly anaerobic thermophiles. In F. A. Rainey & A. Oren (Eds.), Methods in Microbiology (1st ed., Vol. 35, pp. 307–328). Amsterdam: Elsevier, Academic Press.Google Scholar
- 20.Berzin, V., Kiriukhin, M., & Tyurin, M. (2013). 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. Applied Biochemistry and Biotechnology. doi: 10.1007/s12010-012-0060-7.Google Scholar
- 21.Tyurin, MV. (1992). The method and apparatus for electric cell treatment using partial power capacitor discharge via the sample and the power tetrode. Russian Patent 2005776.Google Scholar
- 24.Tyurin, M., Kiriukhin, M., & Berzin, V. (2012). Electrofusion of cells of acetogen Clostridium sp. MT351 with erm(B) or cat in the chromosome. Journal of Biotech Research, 4, 1–12.Google Scholar
- 25.Berzin, V., & Tyurin, M. (2012). Acetogen biocatalyst Clostridium sp. MTEtOH871 engineered with our proprietary electrotransformation technology and equipment: continuous synthesis gas fermentation for selective ethanol production. Journal of Biotech Research, 4, 54–64.Google Scholar
- 28.Ross, S. M. (2000). Introduction to probability and statistics for engineers and scientists (2nd ed.). San Diego: Academic.Google Scholar