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Engineered microorganisms capable of producing target compounds under anaerobic conditions

a technology of anaerobic conditions and microorganisms, applied in microorganisms, biofuels, enzymes, etc., can solve the problems of increasing the demand for domestically produced biofuels, increasing the cost of fuel, increasing the demand for alternative fuels, etc., and achieves the effect of increasing the rate, titer and productivity

Inactive Publication Date: 2010-06-10
GEVO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides recombinant microorganisms that produce C3-C5 alcohols under anaerobic and aerobic conditions. These microorganisms have an engineered metabolic pathway that increases the production of C3-C5 alcohols compared to a parental microorganism with a native metabolic pathway. The recombinant microorganisms have an overexpressed transhydrogenase that converts NADH to NADPH, or an NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase, or enzymes that have increased activity using NADH as a cofactor. The KARI enzyme is modified or mutated to enhance the cell's ability to produce beneficial metabolites such as isobutanol. The invention also provides modified or mutated KARI enzymes that preferentially utilize NADH.

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 biofuel today but has several drawbacks when compared to gasoline.
However, the production of isobutanol using these microorganisms is feasible only under aerobic conditions and the maximum yield that can be achieved is limited.

Method used

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  • Engineered microorganisms capable of producing target compounds under anaerobic conditions
  • Engineered microorganisms capable of producing target compounds under anaerobic conditions
  • Engineered microorganisms capable of producing target compounds under anaerobic conditions

Examples

Experimental program
Comparison scheme
Effect test

example 1

EXAMPLE 1

Low-Level Anaerobic Production of Isobutanol

[0471]This example illustrates that a modified microorganism which is engineered to overexpress an isobutanol producing pathway produces a low amount of isobutanol under anaerobic conditions.

[0472]Overnight cultures of GEVO1859 were started from glycerol stocks stored at −80° C. of previously transformed strains. These cultures were started in 3 mL M9 minimal medium (Miller, J. H. A Short Course in Bacterial Genetics: A laboratory manual and handbook for Escherichia coli and related bacteria. 1992. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), supplemented with 10 g / L yeast extract, 10 μM ferric citrate and trace metals, containing 8.5% glucose and the appropriate antibiotics in snap cap tubes about 14 h prior to the start of the fermentation. Isobutanol fermentations were then carried out in screw cap flasks containing 20 mL of the same medium that was inoculated with 0.2 mL of the overnight culture. The cells ...

example 2

EXAMPLE 2

Determination of Transhydrogenase Activity

[0477]This example illustrates that an isobutanol producing microorganism which carries a plasmid for the expression of the E. coli PntAB transhydrogenase (SEQ ID NO: 2 and SEQ ID NO: 4) contains increased transhydrogenase activity.

[0478]A fermentation was performed with a strain expressing the tet repressor (GEVO1385) and carrying the plasmids pGV1655 (SEQ ID NO: 109) and pGV1698 (SEQ ID NO: 112) for expression of the isobutanol pathway. The E. coli transhydrogenase PntAB was expressed from a third plasmid pGV1685 (SEQ ID NO: 111), which contained the E. coli pntAB genes under control of the PLtet promoter. The appropriate empty vector control carries the plasmid pGV1490 (SEQ ID NO: 104).

[0479]GEVO1385 was transformed with pGV1698, pGV1655, and either pGV1685 or pGV1490. Transformed cells were plated on LB-plates containing the appropriate antibiotics and the plates were incubated overnight at 37° C. Overnight cultures were starte...

example 3

EXAMPLE 3

Overexpression of pntAB Improves Isobutanol Fermentation Performance

[0481]This example illustrates that overexpression of a transhydrogenase, exemplified by the E. coli pntAB operon (SEQ ID NO: 1 and SEQ ID NO: 3) on a low copy plasmid improves isobutanol production under micro-aerobic conditions.

[0482]GEVO1748 was transformed with plasmids pGV1698 (SEQ ID NO: 112) and one of either pGV1720 (SEQ ID NO: 115) (control) or pGV1745 (SEQ ID NO: 117) (E. coli pntAB).

[0483]The aforementioned strains were plated on LB-plates containing the appropriate antibiotics and incubated overnight at 37° C. Overnight cultures were started in 3 ml. EZ-Rich medium (Neidhardt, F. C., P. L. Bloch, and D. F. Smith. 1974. Culture medium for enterobacteria. J Bacteriol. 119:736-47) containing 5% glucose and the appropriate antibiotics in snap cap tubes about 14 h prior to the start of the fermentation. Isobutanol fermentations were then carried out in EZ-Rich Medium containing 5% glucose and the ap...

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Abstract

The present invention is generally provides recombinant microorganisms comprising engineered metabolic pathways capable of producing C3-C5 alcohols under aerobic and anaerobic conditions. The invention further provides ketol-acid reductoisomerase enzymes which have been mutated or modified to increase their NADH-dependent activity or to switch the cofactor preference from NADPH to NADH and are expressed in the modified microorganisms. In addition, the invention provides isobutyraldehyde dehydrogenase enzymes expressed in modified microorganisms. Also provided are methods of producing beneficial metabolites under aerobic and anaerobic conditions by contacting a suitable substrate with the modified microorganisms of the present invention.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to U.S. Provisional Application Ser. No. 61 / 110,543, filed Oct. 31, 2008; U.S. Provisional Application Ser. No. 61 / 121,830, filed Dec. 11, 2008; U.S. Provisional Application Ser. No. 61 / 184,580, filed Jun. 5, 2009; U.S. Provisional Application Ser. No. 61 / 184,605, filed Jun. 5, 2009; and U.S. Provisional Application Ser. No. 61 / 239,618, filed Sep. 3, 2009. This application is related to U.S. patent application Ser. No. 12 / 263,442, entitled “Methods for the Economical Production of Biofuel Precursors that is also a Biofuel from Biomass,” filed Oct. 31, 2008. This application is also related to the U.S. patent application Ser. No. 12 / 263,436, entitled “Methods for the Economical Production of Biofuel from Biomass,” filed Oct. 31, 2008. Accordingly, this application incorporates by reference in its entirety all subject matter of the above-referenced applications to the extent such subject matter is not incons...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P7/16C12N9/02C12N1/21C12N1/15C12N1/19
CPCC12N9/0006C12N9/0008Y02E50/10C12P7/16Y02E50/17C12N9/0036
Inventor BUELTER, THOMASMEINHOLD, PETERRENNY FELDMAN, REID M.ECKL, EVAHAWKINS, ANDREWARISTIDOU, ARISTOSASLESON DUNDON, CATHERINELIES, DOUGBASTIAN, SABINEARNOLD, FRANCESURANO, JUN
Owner GEVO INC
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