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Yeast organism producing isobutanol at a high yield

a technology of isobutanol and yeast, which is applied in the direction of lyase, transferase, carbon-carbon lyase, etc., can solve the problems of increasing the demand for domestically produced biofuels, increasing the cost of fuel, and increasing the demand for alternative fuels, so as to reduce the activity of pyruvate decarboxylase (pdc), reduce the activity of glycerol-3-phosphate dehydrogena

Inactive Publication Date: 2012-02-02
GEVO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0010]The present inventors have observed that by combining the expression of a cytosolically localized acetolactate synthase enzyme with reduced pyruvate decarboxylase (PDC) activity and / or reduced glycerol-3-phosphate dehydrogenase (GPD) activity, an unexpectedly high flux from pyruvate to acetolactate can be achieved. Thus, the invention provides yeast cells that are engineered to exhibit an efficient conversion of pyruvate to acetolactate in the cytoplasm due to suppression of competing metabolic pathways. Therefore, as would be understood in the art, the present invention has utility for the production of any acetolactate-derived product, including, but not limited to, isobutanol, 2-butanol, 1-butanol, 2-butanone, 2,3-butanediol, valine, leucine, and 3-methyl-1-butanol.

Problems solved by technology

One is the threat of ‘peak oil’, the point at which the consumption rate of crude oil exceeds the supply rate, thus leading to significantly increased fuel cost results in an increased demand for alternative fuels.
In addition, instability in the Middle East and other oil-rich regions has increased the demand for domestically produced biofuels.
Ethanol is the most abundant fermentatively produced fuel today but has several drawbacks when compared to gasoline.
However, these yeast microorganisms fall short of commercial relevance due to their low performance characteristics, including low productivity, low titer, low yield, and the requirement for oxygen during the fermentation process.
One of the primary reasons for the sub-optimal performance observed in existing isobutanol-producing microorganisms is the undesirable conversion of pathway intermediates to unwanted by-products.

Method used

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  • Yeast organism producing isobutanol at a high yield
  • Yeast organism producing isobutanol at a high yield
  • Yeast organism producing isobutanol at a high yield

Examples

Experimental program
Comparison scheme
Effect test

example 1

Construction and Confirmation of PDC Deletion in K. lactis

[0346]The purpose of this Example is to describe how a PDC-deletion variant of a member of the Saccharomyces clade, Crabtree-negative yeast, pre-WGD yeast K. lactis was constructed and confirmed.

[0347]Construction of plasmid pGV1537: Plasmid pGV1537 (SEQ ID NO: 1) was constructed by the following series of steps. All PCR reactions carried out to generate pGV1537 used KOD polymerase (Novagen, Inc., Gibbstown, N.J.) and standard reaction conditions according to the manufacturer. A first round of two PCR reactions was carried out, wherein one PCR reaction contained primers 1006 and 1016 and used approximately 100 ng of genomic DNA from K. lactis strain GEV01287 as a template. The other first-round PCR reaction contained primers 1017 and 1009 and approximately 10Ong of genomic DNA from K. lactis strain GEVO1287 as a template. The two resulting PCR products (approximately 530 bp and 630 bp in size, respectively) were gel purified...

example 2

Construction and Confirmation of PDC Deletion in S. cerevisiae

[0351]The purpose of this Example is to describe how a PDC deletion variant of a member of the Saccharomyces sensu stricto yeast group, the Saccharomyces yeast clade, a Crabtree-positive yeast, and a post-WGD yeast, S. cerevisiae was constructed and confirmed.

[0352]Strains GEVO1537 and GEVO1538 were incubated in 1% potassium acetate for 3-4 days which induces sporulation. The resulting haploid spores were recovered by random spore analysis. Briefly, a culture of sporulating cells was examined microscopically to ensure that a sufficient fraction of cells had sporulated (>10%). Five (5) mL of a culture of sporulated cells were collected by centrifugation (5 minutes at 3000×g) and washed once in 1 mL of water. The cells were resuspended in 5 mL water to which was added 0.5 mL of a 1 mg / mL solution (freshly made) of Zymolyase-T (in water) as well as 10 μL of β-mercaptoethanol. The cell suspension was incubated overnight at 3...

example 3

Other Pdc-Minus S. cerevisiae Strains.

[0359]S. cerevisiae engineered to be deficient in PDC activity have been previously described: (Flikweert, M. T., van der Zanden, L., Janssen, W. M. T. M, Steensma, H. Y., van Dijken J. P., Pronk J. T. (1996) Yeast 12(3):247-57). Such strains may be obtained from these sources.

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Abstract

The present invention provides recombinant microorganisms comprising an isobutanol producing metabolic pathway and methods of using said recombinant microorganisms to produce isobutanol. In various aspects of the invention, the recombinant microorganisms may comprise a modification resulting in the reduction of pyruvate decarboxylase and / or glycerol-3-phosphate dehydrogenase activity. In various embodiments described herein, the recombinant microorganisms may be microorganisms of the Saccharomyces clade, Crabtree-negative yeast microorganisms, Crabtree-positive yeast microorganisms, post-WGD (whole genome duplication) yeast microorganisms, pre-WGD (whole genome duplication) yeast microorganisms, and non-fermenting yeast microorganisms.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a divisional application of U.S. application Ser. No. 12 / 820,505, filed Jun. 22, 2010, which claims priority to U.S. Provisional Application Ser. No. 61 / 219,173, filed Jun. 22, 2009. U.S. application Ser. No. 12 / 820,505 is also a continuation-in-part of U.S. application Ser. No. 12 / 696,645, filed Jan. 29, 2010, which is a divisional application of U.S. application Ser. No. 12 / 343,375, filed Dec. 23, 2008, now U.S. Pat. No. 8,017,375, which claims, as does the present application, the benefit of U.S. Provisional Application Ser. No. 61 / 016,483, filed Dec. 23, 2007, all of which are herein incorporated by reference in their entireties for all purposes.TECHNICAL FIELD[0002]Metabolically engineered microorganisms and methods of producing such organisms are provided. Also provided are methods of producing metabolites that are biofuels by contacting a suitable substrate with metabolically engineered microorganisms and enzyma...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P7/16C12N1/19
CPCC12N9/0006C12N9/0008C12N9/88C12N9/90C12P7/16C12N15/81C12Y202/01006C12Y401/01001C12Y402/01009Y02E50/10C12Y101/05003C12N9/1022C12Y101/01001C12Y101/01008C12Y101/01086C12Y401/01074
Inventor FELDMAN, REID M. RENNYGUNAWARDENA, UVINIURANO, JUNMEINHOLD, PETERARISTIDOU, ARISTOSDUNDON, CATHERINE ASLESONSMITH, CHRISTOPHER
Owner GEVO INC
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