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Cytosolic isobutanol pathway localization for the production of isobutanol




Title: 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. ...


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USPTO Applicaton #: #20110287500
Inventors: Jun Urano, Catherine Asleson Dundon, Peter Meinhold, Reid M. Renny Feldman, Aristos Aristidou, Andrew Hawkins, Thomas Buelter, Matthew Peters, Doug Lies, Stephanie Porter-scheinman, Ruth Berry, Ishmeet Kalra


The Patent Description & Claims data below is from USPTO Patent Application 20110287500, Cytosolic isobutanol pathway localization for the production of isobutanol.

CROSS-REFERENCE TO RELATED APPLICATIONS

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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

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

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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 THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: GEVO—041—11US_SeqList_ST25.txt, date recorded: Jul. 2, 2011, file size: 337 kilobytes).

BACKGROUND

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Biofuels have a long history ranging back to the beginning of the 20th century. As early as 1900, Rudolf Diesel demonstrated at the World Exhibition in Paris, France, an engine running on peanut oil. Soon thereafter, Henry Ford demonstrated his Model T running on ethanol derived from corn. Petroleum-derived fuels displaced biofuels in the 1930s and 1940s due to increased supply, and efficiency at a lower cost.

Market fluctuations in the 1970s coupled to the decrease in US oil production led to an increase in crude oil prices and a renewed interest in biofuels. Today, many interest groups, including policy makers, industry planners, aware citizens, and the financial community, are interested in substituting petroleum-derived fuels with biomass-derived biofuels. The leading motivations for developing biofuels are of economical, political, and environmental nature.

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. Also, environmental concerns relating to the possibility of carbon dioxide related climate change is an important social and ethical driving force which is starting to result in government regulations and policies such as caps on carbon dioxide emissions from automobiles, taxes on carbon dioxide emissions, and tax incentives for the use of biofuels.

Ethanol is the most abundant fermentatively produced fuel today but has several drawbacks when compared to gasoline. Butanol, in comparison, has several advantages over ethanol as a fuel: it can be made from the same feedstocks as ethanol but, unlike ethanol, it is compatible with gasoline at any ratio and can also be used as a pure fuel in existing combustion engines without modifications. Unlike ethanol, butanol does not absorb water and can thus be stored and distributed in the existing petrochemical infrastructure. Due to its higher energy content which is close to that of gasoline, the fuel economy (miles per gallon) is better than that of ethanol. Also, butanol-gasoline blends have lower vapor pressure than ethanol-gasoline blends, which is important in reducing evaporative hydrocarbon emissions.

Isobutanol has the same advantages as butanol with the additional advantage of having a higher octane number due to its branched carbon chain. Isobutanol is also useful as a commodity chemical and is also a precursor to MTBE.

Isobutanol has been produced in recombinant microorganisms expressing a heterologous, five-step metabolic pathway (See, e.g., WO/2007/050671 to Donaldson et al., WO/2008/098227 to Liao et al., and WO/2009/103533 to Festel et al.). 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. Thus, recombinant microorganisms exhibiting increased isobutanol productivity, titer, and/or yield are desirable.

SUMMARY

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OF THE INVENTION

The present invention provides cytosolically active dihydroxyacid dehydratase (DHAD) enzymes and recombinant microorganisms comprising said cytosolically active DHAD enzymes. In some embodiments, said recombinant microorganisms may further comprise one or more additional enzymes catalyzing a reaction in an isobutanol producing metabolic pathway. As described herein, the recombinant microorganisms of the present invention are useful for the production of several beneficial metabolites, including, but not limited to isobutanol.

In a first aspect, the invention provides cytosolically active dihydroxyacid dehydratase (DHAD) enzymes. These cytosolically active DHAD enzymes generally exhibit the ability to convert 2,3-dihydroxyisovalerate to ketoisovalerate in the cytosol. The cytosolically active DHAD enzymes of the present invention, as described herein, can include native (i.e. parental) DHAD enzymes that exhibit cytosolic activity, as well DHAD enzymes that have been modified or mutated to increase their cytosolic localization and/or activity as compared to native (i.e. parental) DHAD enzymes.

In various embodiments described herein, the DHAD enzymes may be derived from a prokaryotic organism. In one embodiment, the prokaryotic organism is a bacterial organism. In another embodiment, the bacterial organism is Lactococcus lactis. In a specific embodiment, the DHAD enzyme from L. lactis comprises the amino acid sequence of SEQ ID NO: 18. In another embodiment, the bacterial organism is Francisella tularensis. In a specific embodiment, the DHAD enzyme from F. tularensis comprises the amino acid sequence of SEQ ID NO: 14. In another embodiment, the bacterial organism is Gramella forsetii. In a specific embodiment, the DHAD enzyme from G. forsetii comprises the amino acid sequence of SEQ ID NO: 17.

In alternative embodiments described herein, the DHAD enzyme may be derived from a eukaryotic organism. In one embodiment, the eukaryotic organism is a fungal organism. In an exemplary embodiment, the fungal organism is Neurospora crassa. In a specific embodiment, the DHAD enzyme from N. crassa comprises the amino acid sequence of SEQ ID NO: 165.

In some embodiments, the invention provides modified or mutated DHAD enzymes, wherein said DHAD enzymes exhibit increased cytosolic activity as compared to their parental DHAD enzymes. In another embodiment, the invention provides modified or mutated DHAD enzymes, wherein said DHAD enzymes exhibit increased cytosolic activity as compared to the DHAD enzyme comprised by the amino acid sequence of SEQ ID NO: 11.

In further embodiments, the invention provides DHAD enzymes comprising the amino acid sequence P(I/L)XXXGX(I/L)XIL (SEQ ID NO: 27), wherein X is any natural or non-natural amino acid, and wherein said DHAD enzymes exhibit the ability to convert 2,3-dihydroxyisovalerate to ketoisovalerate in the cytosol.

In some embodiments, the DHAD enzymes of the present invention exhibit a properly folded iron-sulfur cluster domain and/or redox active domain in the cytosol. In one embodiment, the DHAD enzymes comprise a mutated or modified iron-sulfur cluster domain and/or redox active domain.

In another aspect, the present invention provides recombinant microorganisms comprising a cytosolically active DHAD enzyme. In one embodiment, the invention provides recombinant microorganisms comprising a DHAD enzyme derived from a prokaryotic organism, wherein said DHAD enzyme exhibits activity in the cytosol. In one embodiment, the DHAD enzyme is derived from a bacterial organism. In a specific embodiment, the DHAD enzyme is derived from L. lactis and comprises the amino acid sequence of SEQ ID NO: 18. In another embodiment, the invention provides recombinant microorganisms comprising a DHAD enzyme derived from a eukaryotic organism, wherein said DHAD enzyme exhibits activity in the cytosol. In one embodiment, the DHAD enzyme is derived from a fungal organism. In an alternative embodiment, the DHAD enzyme is derived from a yeast organism.

In one embodiment, the invention provides recombinant microorganisms comprising a modified or mutated DHAD enzyme, wherein said DHAD enzyme exhibits increased cytosolic activity as compared to the parental DHAD enzyme. In another embodiment, the invention provides recombinant microorganisms comprising a modified or mutated DHAD enzyme, wherein said DHAD enzyme exhibits increased cytosolic activity as compared to the DHAD enzyme comprised by the amino acid sequence of SEQ ID NO: 11.

In another embodiment, the invention provides recombinant microorganisms comprising a DHAD enzyme comprising the amino acid sequence P(I/L)XXXGX(I/L)XIL (SEQ ID NO: 27), wherein X is any natural or non-natural amino acid, and wherein said DHAD enzymes exhibit the ability to convert 2,3-dihydroxyisovalerate to ketoisovalerate in the cytosol.

In some embodiments, the invention provides recombinant microorganisms comprising a DHAD enzyme fused to a peptide tag, whereby said DHAD enzyme exhibits increased cytosolic localization and/or cytosolic DHAD activity as compared to the parental microorganism. In one embodiment, the peptide tag is non-cleavable. In another embodiment, the peptide tag is fused at the N-terminus of the DHAD enzyme. In another embodiment, the peptide tag is fused at the C-terminus of the DHAD enzyme. In certain embodiments, the peptide tag may be selected from the group consisting of ubiquitin, ubiquitin-like (UBL) proteins, myc, HA-tag, green fluorescent protein (GFP), and the maltose binding protein (MBP).

In certain embodiments described herein, it may be desirable to further overexpress an additional enzyme that converts 2,3-dihydroxyisovalerate (DHIV) to ketoisovalerate (KIV) in the cytosol. In a specific embodiment, the enzyme may be selected from the group consisting of 3-isopropylmalate isomerase (Leu1p) and imidazoleglycerol-phosphate dehydrogenase (His3p).

In various embodiments described herein, the recombinant microorganisms may be further engineered to express an isobutanol producing metabolic pathway comprising at least one exogenous gene that catalyzes a step in the conversion of pyruvate to isobutanol. In one embodiment, the recombinant microorganism may be engineered to express an isobutanol producing metabolic pathway comprising at least two exogenous genes. In another embodiment, the recombinant microorganism may be engineered to express an isobutanol producing metabolic pathway comprising at least three exogenous genes. In another embodiment, the recombinant microorganism may be engineered to express an isobutanol producing metabolic pathway comprising at least four exogenous genes. In another embodiment, the recombinant microorganism may be engineered to express an isobutanol producing metabolic pathway comprising five exogenous genes. Thus, the present invention further provides recombinant microorganisms that comprise an isobutanol producing metabolic pathway and methods of using said recombinant microorganisms to produce isobutanol.

In one embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least one isobutanol pathway enzyme localized in the cytosol. In another embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least two isobutanol pathway enzymes localized in the cytosol. In another embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least three isobutanol pathway enzymes localized in the cytosol. In another embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least four isobutanol pathway enzymes localized in the cytosol. In an exemplary embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with five isobutanol pathway enzymes localized in the cytosol. In a further exemplary embodiment, at least one of the pathway enzymes localized to the cytosol is a cytosolically active DHAD enzyme as disclosed herein.

In various embodiments described herein, the isobutanol pathway enzyme(s) is/are selected from the group consisting of acetolactate synthase (ALS), ketol-acid reductoisomerase (KARI), dihydroxyacid dehydratase (DHAD), 2-keto-acid decarboxylase (KIVD), and alcohol dehydrogenase (ADH).

As described herein, the cytosolically active isobutanol pathway enzymes of the present invention can include native (i.e. parental) enzymes that exhibit cytosolic activity, as well isobutanol pathway enzymes that have been modified or mutated to increase their cytosolic localization and/or activity as compared to native (i.e. parental) pathway enzymes.

In various embodiments described herein, the isobutanol pathway enzymes may be derived from a prokaryotic organism. In alternative embodiments described herein, the isobutanol pathway enzymes may be derived from a eukaryotic organism.

In some embodiments, the invention provides modified or mutated isobutanol pathway enzymes, wherein said isobutanol pathway enzymes exhibit increased cytosolic activity as compared to their parental isobutanol pathway enzymes. In another embodiment, the invention provides modified or mutated isobutanol pathway enzymes, wherein said isobutanol pathway enzymes exhibit increased cytosolic activity as compared to the homologous isobutanol pathway enzyme from S. cerevisiae.




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stats Patent Info
Application #
US 20110287500 A1
Publish Date
11/24/2011
Document #
File Date
12/31/1969
USPTO Class
Other USPTO Classes
International Class
/
Drawings
0


Genome Localization Metabolic Metabolic Pathway Recombinant

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Gevo, Inc.


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Chemistry: Molecular Biology And Microbiology   Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition   Preparing Oxygen-containing Organic Compound   Containing Hydroxy Group   Acyclic   Butanol  

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20111124|20110287500|cytosolic isobutanol pathway localization for the production of isobutanol|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 |Gevo-Inc
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