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Cytosolic isobutanol pathway localization for the production of isobutanol

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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. ...


Browse recent Gevo, Inc. patents - Englewood, CO, US
USPTO Applicaton #: #20110287500 - Class: 435160 (USPTO) - 11/24/11 - Class 435 
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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The Patent Description & Claims data below is from USPTO Patent Application 20110287500, Cytosolic isobutanol pathway localization for the production of isobutanol.

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CROSS-REFERENCE TO RELATED APPLICATIONS

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

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

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

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.

In various embodiments described herein, at least one of the isobutanol pathway enzymes exhibiting cytosolic activity is ALS. In one embodiment, the ALS is derived from a prokaryotic organism, including, but not limited to Bacillus subtilis or L. lactis. In another embodiment, the ALS is derived from a eukaryotic organism, including, but not limited to Magnaporthe grisea, Phaeosphaeria nodorum, Talaromyces stipitatus, and Trichoderma atroviride.

In additional embodiments, at least one of the isobutanol pathway enzymes exhibiting cytosolic activity is KARI. In one embodiment, the KARI is derived from a prokaryotic organism, including, but not limited to Escherichia coli, B. subtilis or L. lactis. In another embodiment, the KARI is derived from a eukaryotic organism, including, but not limited to Piromyces sp. E2, S. cerevisiae, and Arabidopsis. In certain specific embodiments, the KARI comprises an amino acid sequence selected from an organism selected from the group consisting of E. coli, S. cerevisiae, B. subtilis Piromyces sp. E2, Buchnera aphidicola, Spinacia oleracea, Oryza sativa, Chlamydomonas reinhardtii, N. crassa, Schizosaccharomyces pombe, Laccaria bicolor, Ignicoccus hospitalis, Picrophilus torridus, Acidiphilium cryptum, Cyanobacteria/Synechococcus sp., Zymomonas mobilis, Bacteroides thetaiotaomicron, Methanococcus maripaludis, Vibrio fischeri, Shewanella sp, G. forsetii, Psychromonas ingrhamaii, and Cytophaga hutchinsonii. In additional embodiments, the KARI may be an NADH-dependent KARI.

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.

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.

In additional embodiments, the invention provides recombinant microorganisms comprising an isobutanol pathway enzyme fused to a peptide tag, whereby said isobutanol pathway enzyme exhibits increased cytosolic localization and/or cytosolic activity as compared to the parental enzyme. As a result, the recombinant microorganism comprising the tagged isobutanol pathway enzyme will generally exhibit an increased ability to perform a step involved in the conversion of pyruvate to isobutanol in the cytosol. In one embodiment, the peptide tag is non-cleavable. In another embodiment, the peptide tag is fused at the N-terminus of the isobutanol pathway enzyme. In another embodiment, the peptide tag is fused at the C-terminus of the isobutanol pathway 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 various embodiments described herein, the recombinant microorganisms may further comprise a nucleic acid encoding a chaperone protein, wherein said chaperone protein assists the folding of a protein exhibiting cytosolic activity. In a preferred embodiment, the protein exhibiting cytosolic activity is an isobutanol pathway enzyme. In one embodiment, the chaperone may be a native protein. In another embodiment, the chaperone protein may be an exogenous protein. In some embodiments, the chaperone protein may be selected from the group consisting of: endoplasmic reticulum oxidoreductin 1 (Ero1)) including variants of Ero1 that have been suitably altered to reduce or prevent its normal localization to the endoplasmic reticulum; thioredoxins (including, but not limited to, Trx1 and Trx2), thioredoxin reductase (Trr1), glutaredoxins (including, but not limited to, Grx1, Grx2, Grx3, Grx4, Grx5, Grx6, Grx7, and Grx8), glutathione reductase (Glr1), and Jac1, including variants of Jac1 that have been suitably altered to reduce or prevent its normal mitochondrial localization; and homologs or variants thereof.

In some embodiments, the recombinant microorganisms may further comprise one or more genes encoding an iron-sulfur cluster assembly protein. In one embodiment, the iron-sulfur cluster assembly protein encoding genes may be derived from prokaryotic organisms. In one embodiment, the iron-sulfur cluster assembly protein encoding genes are derived from a bacterial organism, including, but not limited to E. coli, L. lactis, Helicobacter pylori, and Entamoeba histolytica. In specific embodiments, the bacterially derived iron-sulfur cluster assembly protein encoding genes are selected from the group consisting of cyaY, iscS, iscU, iscA, hscB, hscA, fdx, isuX, sufA, sufB, sufC, sufD, sufS, sufE, apbC, and homologs or variants thereof.

In another embodiment, the iron-sulfur cluster assembly protein encoding genes may be derived from eukaryotic organisms, including, but not limited to yeasts and plants. In one embodiment, the iron-sulfur cluster protein encoding genes are derived from a yeast organism, including, but not limited to S. cerevisiae. In specific embodiments, the yeast derived genes encoding iron-sulfur cluster assembly proteins are selected from the group consisting of Cfd1, Nbp35, Nar1, Cia1, and homologs or variants thereof. In a further embodiment, the iron-sulfur cluster assembly protein encoding genes may be derived from plant nuclear genes which encode proteins translocated to chloroplast or plant genes found in the chloroplast genome itself.

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.

In certain embodiments described herein, it may be desirable to reduce or eliminate the activity and/or proteins levels of one or more iron-sulfur cluster containing cytosolic proteins. In a specific embodiment, the iron-sulfur cluster containing cytosolic protein is 3-isopropylmalate dehydratase (Leu1p). In one embodiment, the recombinant microorganism comprises a mutation in the LEU1 gene resulting in the reduction of Leu1p protein levels. In another embodiment, the recombinant microorganism comprises a partial deletion in the LEU1 gene resulting in the reduction of Leu1p protein levels. In another embodiment, the recombinant microorganism comprises a complete deletion in the LEU1 gene resulting in the reduction of Leu1p protein levels. In another embodiment, the recombinant microorganism comprises a modification of the regulatory region associated with the LEU1 gene resulting in the reduction of Leu1p protein levels. In yet another embodiment, the recombinant microorganism comprises a modification of a transcriptional regulator for the LEU1 gene resulting in the reduction of Leu1p protein levels.

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.

In various embodiments described herein, the recombinant microorganisms may be engineered to express native genes that catalyze a step in the conversion of pyruvate to isobutanol. In one embodiment, the recombinant microorganism is engineered to increase the activity of a native metabolic pathway gene for conversion of pyruvate to isobutanol. In another embodiment, the recombinant microorganism is further engineered to include at least one enzyme encoded by an exogenous gene and at least one enzyme encoded by a native gene. In yet another embodiment, the recombinant microorganism comprises a reduction in the activity of a native metabolic pathway as compared to a parental microorganism.

In another embodiment, the present invention provides recombinant microorganisms comprising a scaffold system tethered to one or more isobutanol pathway enzymes. In a specific embodiment, the scaffold system is the MAP kinase scaffold (Ste5) system. In a further embodiment, one or more of the isobutanol pathway enzymes may be modified or mutated to comprise a protein domain allowing for binding to the scaffold system.

In various embodiments described herein, the recombinant microorganisms may be microorganisms of the Saccharomyces clade, Saccharomyces sensu stricto microorganisms, 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.

In some embodiments, the recombinant microorganisms may be yeast recombinant microorganisms of the Saccharomyces clade.

In some embodiments, the recombinant microorganisms may be Saccharomyces sensu stricto microorganisms. In one embodiment, the Saccharomyces sensu stricto is selected from the group consisting of S. cerevisiae, S. kudriavzevii, S. mikatae, S. bayanus, S. uvarum. S. carocanis and hybrids thereof.

In some embodiments, the recombinant microorganisms may be Crabtree-negative recombinant yeast microorganisms. In one embodiment, the Crabtree-negative yeast microorganism is classified into a genera selected from the group consisting of Kluyveromyces, Pichia, Hansenula, Issatchenkia, or Candida. In additional embodiments, the Crabtree-negative yeast microorganism is selected from Kluyveromyces lactis, Kluyveromyces marxianus, Pichia anomala, Pichia stipitis, Hansenula anomala, Issatchenkia orientalis, Candida utilis and Kluyveromyces waltii.

In some embodiments, the recombinant microorganisms may be Crabtree-positive recombinant yeast microorganisms. In one embodiment, the Crabtree-positive yeast microorganism is classified into a genera selected from the group consisting of Saccharomyces, Kluyveromyces, Zygosaccharomyces, Debaryomyces, Candida, Pichia and Schizosaccharomyces. In additional embodiments, the Crabtree-positive yeast microorganism is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelli, Saccharomyces kluyveri, Kluyveromyces thermotolerans, Candida glabrata, Z. baiffi, Z. rouxii, Debaryomyces hansenii, Pichia pastorius, Schizosaccharomyces pombe, and Saccharomyces uvarum.

In some embodiments, the recombinant microorganisms may be post-WGD (whole genome duplication) yeast recombinant microorganisms. In one embodiment, the post-WGD yeast recombinant microorganism is classified into a genera selected from the group consisting of Saccharomyces or Candida. In additional embodiments, the post-WGD yeast is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelli, and Candida glabrata.

In some embodiments, the recombinant microorganisms may be pre-WGD (whole genome duplication) yeast recombinant microorganisms. In one embodiment, the pre-WGD yeast recombinant microorganism is classified into a genera selected from the group consisting of Saccharomyces, Kluyveromyces, Candida, Pichia, Debaryomyces, Hansenula, Pachysolen, Yarrowia, Issatchenkia, and Schizosaccharomyces. In additional embodiments, the pre-WGD yeast is selected from the group consisting of Saccharomyces kluyveri, Kluyveromyces thermotolerans, Kluyveromyces marxianus, Kluyveromyces waltii, Kluyveromyces lactis, Candida tropicalis, Pichia pastoris, Pichia anomala, Pichia stipitis, Debaryomyces hansenii, Hansenula anomala, Pachysolen tannophilis, Yarrowia lipolytica, Issatchenkia orientalis, and Schizosaccharomyces pombe.

In some embodiments, the recombinant microorganisms may be microorganisms that are non-fermenting yeast microorganisms, including, but not limited to those, classified into a genera selected from the group consisting of Tricosporon, Rhodotorula, or Myxozyma.

In another aspect, the present invention provides methods of producing isobutanol using one or more recombinant microorganisms of the invention. In one embodiment, the method includes cultivating one or more recombinant microorganisms in a culture medium containing a feedstock providing the carbon source until a recoverable quantity of the isobutanol is produced and optionally, recovering the isobutanol. In one embodiment, the microorganism is selected to produce isobutanol from a carbon source at a yield of at least about 5 percent theoretical. In another embodiment, the microorganism is selected to produce isobutanol at a yield of at least about 10 percent, at least about 15 percent, about least about 20 percent, at least about 25 percent, at least about 30 percent, at least about 35 percent, at least about 40 percent, at least about 45 percent, at least about 50 percent, at least about 55 percent, at least about 60 percent, at least about 65 percent, at least about 70 percent, at least about 75 percent, at least about 80 percent theoretical, at least about 85 percent theoretical, or at least about 90 percent theoretical.

In one embodiment, the microorganism produces isobutanol from a carbon source at a specific productivity of at least about 0.7 mg/L/hr per OD. In another embodiment, the microorganism produces isobutanol from a carbon source at a specific productivity of at least about 1 mg/L/hr per OD, at least about 10 mg/L/hr per OD, at least about 50 mg/L/hr per OD, at least about 100 mg/L/hr per OD, at least about 250 mg/L/hr per OD, or at least about 500 g/L/hr per OD.

BRIEF DESCRIPTION OF DRAWINGS

Illustrative embodiments of the invention are illustrated in the drawings, in which:

FIG. 1 illustrates an exemplary embodiment of an isobutanol pathway.

FIG. 2 illustrates acetoin produced from GEVO 1187 (no ALS), 2280 (B. subtilis AlsS not codon optimized), GEVO 2618 (B. subtilis AlsS), GEVO 2621 (T. atroviride ALS) and GEVO 2622 (T. stipitatus ALS). All acetoin values are normalized to OD600 and reported as mM/OD.

FIG. 3 illustrates the specific activity at pH 7.5 of KARI enzyme in whole cell lysates for GEVO1803 containing empty vector (pGV1102), ilv5ΔN47(pGV1831), ilv5ΔN46(pGV1901), Full length ILV5 (pGV1833) and E. coli ilvC codon optimized for S. cerevisiae (pGV1824).

FIG. 4 illustrates the results from fermentations of GEVO2107 transformed with plasmids for expression of KARI and different DHAD homologs (shown in legend).

FIG. 5 illustrates a phylogenetic tree of 53 representative DHAD homologs following pairwise global alignments and progressive assembly of alignments using Neighbor-Joining phylogeny.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a polynucleotide” includes a plurality of such polynucleotides and reference to “the microorganism” includes reference to one or more microorganisms, and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein.

Any publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.

The term “microorganism” includes prokaryotic and eukaryotic microbial species from the Domains Archaea, Bacteria and Eucarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista. The terms “microbial cells” and “microbes” are used interchangeably with the term microorganism.

The term “genus” is defined as a taxonomic group of related species according to the Taxonomic Outline of Bacteria and Archaea (Garrity et al., 2007, TOBA Release 7.7, Michigan State University Board of Trustees).

The term “species” is defined as a collection of closely related organisms with greater than 97% 16S ribosomal RNA sequence homology and greater than 70% genomic hybridization and sufficiently different from all other organisms so as to be recognized as a distinct unit.

The terms “recombinant microorganism,” “modified microorganism” and “recombinant host cell” are used interchangeably herein and refer to microorganisms that have been genetically modified to express or over-express endogenous polynucleotides, or to express heterologous polynucleotides, such as those included in a vector, or which have an alteration in expression of an endogenous gene. By “alteration” it is meant that the expression of the gene, or level of a RNA molecule or equivalent RNA molecules encoding one or more polypeptides or polypeptide subunits, or activity of one or more polypeptides or polypeptide subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the alteration. For example, the term “alter” can mean “inhibit,” but the use of the word “alter” is not limited to this definition.

The term “expression” with respect to a gene sequence refers to transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein. Thus, as will be clear from the context, expression of a protein results from transcription and translation of the open reading frame sequence. The level of expression of a desired product in a host cell may be determined on the basis of either the amount of corresponding mRNA that is present in the cell, or the amount of the desired product encoded by the selected sequence. For example, mRNA transcribed from a selected sequence can be quantitated by qRT-PCR or by Northern hybridization (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)). Protein encoded by a selected sequence can be quantitated by various methods, e.g., by ELISA, by assaying for the biological activity of the protein, or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay, using antibodies that recognize and bind the protein. The polynucleotide generally encodes a target enzyme involved in a metabolic pathway for producing a desired metabolite. It is understood that the terms “recombinant microorganism” and “recombinant host cell” refer not only to the particular recombinant microorganism but to the progeny or potential progeny of such a microorganism. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

The term “wild-type microorganism” describes a cell that occurs in nature, i.e. a cell that has not been genetically modified. A wild-type microorganism can be genetically modified to express or overexpress a first target enzyme. This microorganism can act as a parental microorganism in the generation of a microorganism modified to express or overexpress a second target enzyme. In turn, the microorganism modified to express or overexpress a first and a second target enzyme can be modified to express or overexpress a third target enzyme.

Accordingly, a “parental microorganism” functions as a reference cell for successive genetic modification events. Each modification event can be accomplished by introducing a nucleic acid molecule in to the reference cell. The introduction facilitates the expression or overexpression of a target enzyme. It is understood that the term “facilitates” encompasses the activation of endogenous polynucleotides encoding a target enzyme through genetic modification of e.g., a promoter sequence in a parental microorganism. It is further understood that the term “facilitates” encompasses the introduction of heterologous polynucleotides encoding a target enzyme in to a parental microorganism.

The term “engineer” refers to any manipulation of a microorganism that results in a detectable change in the microorganism, wherein the manipulation includes but is not limited to inserting a polynucleotide and/or polypeptide heterologous to the microorganism and mutating a polynucleotide and/or polypeptide native to the microorganism.

The term “mutation” as used herein indicates any modification of a nucleic acid and/or polypeptide which results in an altered nucleic acid or polypeptide. Mutations include, for example, point mutations, deletions, or insertions of single or multiple residues in a polynucleotide, which includes alterations arising within a protein-encoding region of a gene as well as alterations in regions outside of a protein-encoding sequence, such as, but not limited to, regulatory or promoter sequences. A genetic alteration may be a mutation of any type. For instance, the mutation may constitute a point mutation, a frame-shift mutation, an insertion, or a deletion of part or all of a gene. In addition, in some embodiments of the modified microorganism, a portion of the microorganism genome has been replaced with a heterologous polynucleotide. In some embodiments, the mutations are naturally-occurring. In other embodiments, the mutations are the results of artificial selection pressure. In still other embodiments, the mutations in the microorganism genome are the result of genetic engineering.

The term “biosynthetic pathway”, also referred to as “metabolic pathway”, refers to a set of anabolic or catabolic biochemical reactions for converting one chemical species into another. Gene products belong to the same “metabolic pathway” if they, in parallel or in series, act on the same substrate, produce the same product, or act on or produce a metabolic intermediate (i.e., metabolite) between the same substrate and metabolite end product.

The term “heterologous” as used herein with reference to molecules and in particular enzymes and polynucleotides, indicates molecules that are expressed in an organism other than the organism from which they originated or are found in nature, independently of the level of expression that can be lower, equal or higher than the level of expression of the molecule in the native microorganism.

On the other hand, the term “native” or “endogenous” as used herein with reference to molecules, and in particular enzymes and polynucleotides, indicates molecules that are expressed in the organism in which they originated or are found in nature, independently of the level of expression that can be lower equal or higher than the level of expression of the molecule in the native microorganism. It is understood that expression of native enzymes or polynucleotides may be modified in recombinant microorganisms.

The term “feedstock” is defined as a raw material or mixture of raw materials supplied to a microorganism or fermentation process from which other products can be made. For example, a carbon source, such as biomass or the carbon compounds derived from biomass are a feedstock for a microorganism that produces a biofuel in a fermentation process. However, a feedstock may contain nutrients other than a carbon source.

The term “substrate” or “suitable substrate” refers to any substance or compound that is converted or meant to be converted into another compound by the action of an enzyme. The term includes not only a single compound, but also combinations of compounds, such as solutions, mixtures and other materials which contain at least one substrate, or derivatives thereof. Further, the term “substrate” encompasses not only compounds that provide a carbon source suitable for use as a starting material, such as any biomass derived sugar, but also intermediate and end product metabolites used in a pathway associated with a recombinant microorganism as described herein.

The term “C2-compound” as used as a carbon source for engineered yeast microorganisms with mutations in all pyruvate decarboxylase (PDC) genes resulting in a reduction of pyruvate decarboxylase activity of said genes refers to organic compounds comprised of two carbon atoms, including but not limited to ethanol and acetate

The term “fermentation” or “fermentation process” is defined as a process in which a microorganism is cultivated in a culture medium containing raw materials, such as feedstock and nutrients, wherein the microorganism converts raw materials, such as a feedstock, into products.

The term “volumetric productivity” or “production rate” is defined as the amount of product formed per volume of medium per unit of time. Volumetric productivity is reported in gram per liter per hour (g/L/h).

The term “specific productivity” or “specific production rate” is defined as the amount of product formed per volume of medium per unit of time per amount of cells. Volumetric productivity is reported in gram or milligram per liter per hour per OD (g/L/h/OD).

The term “yield” is defined as the amount of product obtained per unit weight of raw material and may be expressed as g product per g substrate (g/g). Yield may be expressed as a percentage of the theoretical yield. “Theoretical yield” is defined as the maximum amount of product that can be generated per a given amount of substrate as dictated by the stoichiometry of the metabolic pathway used to make the product. For example, the theoretical yield for one typical conversion of glucose to isobutanol is 0.41 g/g. As such, a yield of isobutanol from glucose of 0.39 g/g would be expressed as 95% of theoretical or 95% theoretical yield.

The term “titer” is defined as the strength of a solution or the concentration of a substance in solution. For example, the titer of a biofuel in a fermentation broth is described as g of biofuel in solution per liter of fermentation broth (g/L).



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stats Patent Info
Application #
US 20110287500 A1
Publish Date
11/24/2011
Document #
File Date
12/21/2014
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Other USPTO Classes
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Genome
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