Cytosolic isobutanol pathway localization for the production of isobutanol

Abstract

The present invention provides recombinant microorganisms comprising isobutanol producing metabolic pathway with at least one isobutanol pathway enzyme localized in the cytosol, wherein said recombinant microorganism is selected to produce isobutanol from a carbon source. Methods of using said recombinant microorganisms to produce isobutanol are also provided. In various aspects of the invention, the recombinant microorganisms may comprise a cytosolically active isobutanol pathway enzymes. In some embodiments, the invention provides mutated, modified, and / or chimeric isobutanol pathway enzymes with cytosolic 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 of U.S. application Ser. No. 12 / 855,276, filed Aug. 12, 2010, which claims the benefit of U.S. Provisional Application Ser. No. 61 / 272,058, filed Aug. 12, 2009, and U.S. Provisional Application Ser. No. 61 / 272,059, filed Aug. 12, 2009, each of which are herein incorporated by reference in their entireties for all purposes.ACKNOWLEDGMENT OF GOVERNMENTAL SUPPORT[0002]This invention was made with government support under Contract No. IIP-0823122, awarded by the National Science Foundation, and under Contract No. EP-D-09-023, awarded by the Environmental Protection Agency. The government has certain rights in the invention.TECHNICAL FIELD[0003]Recombinant 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 recombinant microorganisms and enzymatic preparations therefrom.DESCRIPTION OF TH...

AI Technical Summary

Benefits of technology

[0031]In various embodiments described herein, the isobutanol pathway enzyme may be mutated or modified to remove an N-terminal mitochondrial targeting sequence (MTS). Removal of the MTS can increase cytosolic localization of the isobutanol pathway enzyme and / or increase the cytosolic activity of the isobutanol pathway enzyme as compared to the parental isobutanol pathway enzyme.

[0032]In some embodiments, the MTS may be modified or mutated to reduce or eliminate its ability to target the isobutanol pathway enzyme to the mitochondria. Selected modification of the MTS can increase cytosolic localization of the isobutanol pathway enzyme and / or increase the cytosolic activity of the isobutanol pathway enzyme as compared to the parental isobutanol pathway enzyme.

[0037]In some embodiments, one or more genes encoding an iron-sulfur cluster assembly protein may be mutated or modified to remove a signal peptide, whereby localization of the product of said one or more genes to the mitochondria or other subcellular compartment is prevented. In certain embodiments, it may be preferable to overexpress one or more genes encoding an iron-sulfur cluster assembly protein.

[0039]In additional embodiments, the present invention provides recombinant microorganisms comprising chimeric proteins consisting of isobutanol pathway enzymes. In one embodiment, the chimeric proteins consist of ALS and at least one additional protein. In a specific embodiment, the additional protein is KARI. In a preferred embodiment, the chimeric protein exhibits the biocatalytic properties of both ALS and KARI. Such a chimeric protein allows for an increase in the concentration of 2-acetolactate at the active site of KARI as compared to the parental microorganism, giving the recombinant microorganism an enhanced ability to convert 2-acetolactate to 2,3-dihydroxyisovalerate. In another embodiment, the chimeric proteins consist of KARI and at least one additional protein. In a specific embodiment, the additional protein is DHAD. In a preferred embodiment, the chimeric protein exhibits the biocatalytic properties of both KARI and DHAD. In each of the various embodiments described herein, the proteins may be connected via a flexible linker.

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, the microorganisms produced have fallen short of commercial relevance due to their low performance characteristics, including, for example low productivity, low titer, low yield, and the requirement for oxygen during the fermentation process.

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