# Patent

## 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.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to 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, both 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—08US_SeqList_ST25.txt, date recorded: Aug. 10, 2010, 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. bailli, 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 well, 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).
“Aerobic conditions” are defined as conditions under which the oxygen concentration in the fermentation medium is sufficiently high for an aerobic or facultative anaerobic microorganism to use as a terminal electron acceptor.
In contrast, “anaerobic conditions” are defined as conditions under which the oxygen concentration in the fermentation medium is too low for the microorganism to use as a terminal electron acceptor. Anaerobic conditions may be achieved by sparging a fermentation medium with an inert gas such as nitrogen until oxygen is no longer available to the microorganism as a terminal electron acceptor. Alternatively, anaerobic conditions may be achieved by the microorganism consuming the available oxygen of the fermentation until oxygen is unavailable to the microorganism as a terminal electron acceptor.
“Aerobic metabolism” refers to a biochemical process in which oxygen is used as a terminal electron acceptor to make energy, typically in the form of ATP, from carbohydrates. Aerobic metabolism occurs e.g. via glycolysis and the TCA cycle, wherein a single glucose molecule is metabolized completely into carbon dioxide in the presence of oxygen.
In contrast, “anaerobic metabolism” refers to a biochemical process in which oxygen is not the final acceptor of electrons contained in NADH. Anaerobic metabolism can be divided into anaerobic respiration, in which compounds other than oxygen serve as the terminal electron acceptor, and substrate level phosphorylation, in which the electrons from NADH are utilized to generate a reduced product via a “fermentative pathway.”
In “fermentative pathways”, NAD(P)H donates its electrons to a molecule produced by the same metabolic pathway that produced the electrons carried in NAD(P)H. For example, in one of the fermentative pathways of certain yeast strains, NAD(P)H generated through glycolysis transfers its electrons to pyruvate, yielding ethanol. Fermentative pathways are usually active under anaerobic conditions but may also occur under aerobic conditions, under conditions where NADH is not fully oxidized via the respiratory chain. For example, above certain glucose concentrations, Crabtree positive yeasts produce large amounts of ethanol under aerobic conditions.
The term “byproduct” means an undesired product related to the production of a biofuel or biofuel precursor. Byproducts are generally disposed as waste, adding cost to a production process.
The term “non-fermenting yeast” is a yeast species that fails to demonstrate an anaerobic metabolism in which the electrons from NADH are utilized to generate a reduced product via a fermentative pathway such as the production of ethanol and CO2 from glucose. Non-fermentative yeast can be identified by the “Durham Tube Test” (J. A. Barnett, R. W. Payne, and D. Yarrow. 2000. Yeasts Characteristics and Identification. 3rd edition. p. 28-29. Cambridge University Press, Cambridge, UK) or by monitoring the production of fermentation productions such as ethanol and CO2.
The term “polynucleotide” is used herein interchangeably with the term “nucleic acid” and refers to an organic polymer composed of two or more monomers including nucleotides, nucleosides or analogs thereof, including but not limited to single stranded or double stranded, sense or antisense deoxyribonucleic acid (DNA) of any length and, where appropriate, single stranded or double stranded, sense or antisense ribonucleic acid (RNA) of any length, including siRNA. The term “nucleotide” refers to any of several compounds that consist of a ribose or deoxyribose sugar joined to a purine or a pyrimidine base and to a phosphate group, and that are the basic structural units of nucleic acids. The term “nucleoside” refers to a compound (as guanosine or adenosine) that consists of a purine or pyrimidine base combined with deoxyribose or ribose and is found especially in nucleic acids. The term “nucleotide analog” or “nucleoside analog” refers, respectively, to a nucleotide or nucleoside in which one or more individual atoms have been replaced with a different atom or with a different functional group. Accordingly, the term polynucleotide includes nucleic acids of any length, DNA, RNA, analogs and fragments thereof. A polynucleotide of three or more nucleotides is also called nucleotidic oligomer or oligonucleotide.
It is understood that the polynucleotides described herein include “genes” and that the nucleic acid molecules described herein include “vectors” or “plasmids.” Accordingly, the term “gene”, also called a “structural gene” refers to a polynucleotide that codes for a particular sequence of amino acids, which comprise all or part of one or more proteins or enzymes, and may include regulatory (non-transcribed) DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. The transcribed region of the gene may include untranslated regions, including introns, 5′-untranslated region (UTR), and 3′-UTR, as well as the coding sequence.
The term “operon” refers to two or more genes which are transcribed as a single transcriptional unit from a common promoter. In some embodiments, the genes comprising the operon are contiguous genes. It is understood that transcription of an entire operon can be modified (i.e., increased, decreased, or eliminated) by modifying the common promoter. Alternatively, any gene or combination of genes in an operon can be modified to alter the function or activity of the encoded polypeptide. The modification can result in an increase in the activity of the encoded polypeptide. Further, the modification can impart new activities on the encoded polypeptide. Exemplary new activities include the use of alternative substrates and/or the ability to function in alternative environmental conditions.
A “vector” is any means by which a nucleic acid can be propagated and/or transferred between organisms, cells, or cellular components. Vectors include viruses, bacteriophage, pro-viruses, plasmids, phagemids, transposons, and artificial chromosomes such as YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), and PLACs (plant artificial chromosomes), and the like, that are “episomes,” that is, that replicate autonomously or can integrate into a chromosome of a host cell. A vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that are not episomal in nature, or it can be an organism which comprises one or more of the above polynucleotide constructs such as an agrobacterium or a bacterium.
“Transformation” refers to the process by which a vector is introduced into a host cell. Transformation (or transduction, or transfection), can be achieved by any one of a number of means including chemical transformation (e.g. lithium acetate transformation), electroporation, microinjection, biolistics (or particle bombardment-mediated delivery), or agrobacterium mediated transformation.
The term “enzyme” as used herein refers to any substance that catalyzes or promotes one or more chemical or biochemical reactions, which usually includes enzymes totally or partially composed of a polypeptide, but can include enzymes composed of a different, molecule including polynucleotides.
The term “protein”, “peptide” or “polypeptide” as used herein indicates an organic polymer composed of two or more amino acidic monomers and/or analogs thereof. As used herein, the term “amino acid” or “amino acidic monomer” refers to any natural and/or synthetic amino acids including glycine and both D or L optical isomers. The term “amino acid analog” refers to an amino acid in which one or more individual atoms have been replaced, either with a different atom, or with a different functional group. Accordingly, the term polypeptide includes amino acidic polymer of any length including full length proteins, and peptides as well as analogs and fragments thereof. A polypeptide of three or more amino acids is also called a protein oligomer or oligopeptide
The term “homolog”, used with respect to an original enzyme or gene of a first family or species, refers to distinct enzymes or genes of a second family or species which are determined by functional, structural or genomic analyses to be an enzyme or gene of the second family or species which corresponds to the original enzyme or gene of the first family or species. Most often, homologs will have functional, structural or genomic similarities. Techniques are known by which homologs of an enzyme or gene can readily be cloned using genetic probes and PCR. Identity of cloned sequences as homolog can be confirmed using functional assays and/or by genomic mapping of the genes.
A protein has “homology” or is “homologous” to a second protein if the amino acid sequence encoded by a gene has a similar amino acid sequence to that of the second gene. Alternatively, a protein has homology to a second protein if the two proteins have “similar” amino acid sequences. (Thus, the term “homologous proteins” is defined to mean that the two proteins have similar amino acid sequences).
The term “analog” or “analogous” refers to nucleic acid or protein sequences or protein structures that are related to one another in function only and are not from common descent or do not share a common ancestral sequence. Analogs may differ in sequence but may share a similar structure, due to convergent evolution. For example, two enzymes are analogs or analogous if the enzymes catalyze the same reaction of conversion of a substrate to a product, are unrelated in sequence, and irrespective of whether the two enzymes are related in structure.
Cytosolically Localized Isobutanol Pathway Enzymes and Recombinant Microorganisms Comprising the Same
Biosynthetic pathways for the production of isobutanol and 2-methyl-1-butanol by recombinant microorganisms are described by Atsumi et al. (Atsumi et al., 2008, Nature 451: 86-89). One strategy described herein for improving isobutanol production by recombinant microorganisms is the localization of the enzymes catalyzing the biosynthetic isobutanol pathway to the yeast cytosol. Cytosolic localization of the isobutanol pathway enzymes activity is desirable, especially for the production of isobutanol since the ideal biocatalyst (e.g. recombinant microorganism) will have the entire isobutanol pathway functionally expressed in the same compartment (e.g. preferably in the cytosol). In addition, this localization allows the pathway to utilize pyruvate and NAD(P)H that is generated in the cytosol by glycolysis and/or the pentose phosphate pathway without the need for transfer of these metabolites to an alternative compartment (i.e. the mitochondria). However, such a strategy of compartmental localization in yeast is not feasible unless the pathway enzymes exhibit cytosolic activity in that compartment. Thus, if one or more of the cytosolically localized pathway enzymes lacks catalytic activity in the cytosol, high level isobutanol production will not occur. As the present application shows in the Examples below, inefficient cytosolic activity of one or more isobutanol pathway enzymes (e.g. DHAD or ALS) can limit isobutanol production.
The present inventors describe herein cytosolically active isobutanol pathway enzymes and their use in the production of various beneficial metabolites, such as isobutanol and 2-methyl-1-butanol. Using a combination of genetic selection and biochemical analyses, the present inventors have identified a number of isobutanol pathway enzymes, including DHAD enzymes, that have activity in the cytosol. Accordingly, in one aspect, the present application describes the discovery of DHADs with enhanced cytosolic activity and shows that these newly identified, cytosolically active DHADs facilitate improved isobutanol production when co-expressed in the cytosol with the remaining four isobutanol pathway enzymes.
As shown in Example 3 below, the native DHAD of yeast is localized to the mitochondria. Therefore, for economically viable production of isobutanol to occur in the yeast cytosol, the identification of heterologous DHAD enzymes that are “cytosolically active” in yeast (i.e. “active in the cytosol” of the yeast) is important. In addition, the present application shows that in the absence of ALS, KARI, KIVD, and ADH which are “cytosolically active” or “active in the cytosol” in the cytosol of yeast, economically viable isobutanol production will not occur, thus making identification of native and/or heterologous ALS, KARI, KIVD, and ADH enzymes additionally and/or independently important to cytosolic isobutanol production.
As used herein, the term “cytosolically active” or “active in the cytosol” means the enzyme exhibits enzymatic activity in the cytosol of a eukaryotic organism. Cytosolically active enzymes may further be additionally and/or independently characterized as enzymes that generally exhibit a specific cytosolic activity which is greater than the specific mitochondrial activity. In certain respects, a “cytosolically active” enzymes of the present invention exhibit a ratio of the specific activity of the mitochondrial fraction over the specific activity of the whole cell fraction of less than 1, as determined by the method disclosed in Example 3 herein. Cytosolically active enzymes may further be additionally and/or independently characterized as enzymes that, when overexpressed, result in increased activity in the whole cell fraction and do not result in increased activity in the mitochondrial fraction, as determined by the method disclosed in Example 20. Cytosolically active enzymes may further be additionally and/or independently characterized as enzymes that, when overexpressed as one of the five enzymes that together comprise the fivestep biosynthetic pathway for the conversion of pyruvate isobutanol, result in increased isobutanol production compared to enzymes that are not cytosolically active or that are less cytosolically active.
As used herein, the term “cytosolically localized” or “cytosolic localization” means the enzyme is localized in the cytosol of a eukaryotic organism. Cytosolically localized enzymes may further be additionally and/or independently characterized as enzymes that exhibit a cytosolic protein level which is greater than the mitochondrial protein level.
Identification of Cytosolically Active Isobutanol Pathway Enzymes
In one aspect, the present invention encompasses a number of strategies for identifying cytosolically active and/or localized isobutanol pathway enzymes that exhibit cytosolic activity and/or cytosolic localization, as well as methods for modifying said isobutanol pathway enzymes to increase their ability to exhibit cytosolic activity and/or cytosolic localization.
In various embodiments described herein, the isobutanol pathway enzymes may be derived from a prokaryotic organism. In alternative embodiments described herein, the isobutanol pathway enzyme may be derived from a eukaryotic organism. In one embodiment, the eukaryotic organism is a fungal organism. As described herein, the present inventors have found that in general, an enzyme from a fungal source is more likely to show activity in yeast than a bacterial enzyme expressed in yeast. In addition, homologs that are normally expressed in the cytosol are desired, as a normally cytoplasmic enzyme is likely to show higher activity in the cytosol as compared to an enzyme that is relocalized to the cytosol from other organelles, such as the mitochondria. Fungal homologs of various isobutanol pathway enzymes are often localized to the mitochondria. The present inventors have found that fungal homologs of isobutanol pathway enzymes that are cytosolically localized will generally be expected to exhibit higher activity in the cytosol of yeast than those of wild-type yeast strains. Thus, in one embodiment, the present invention provides fungal isobutanol pathway enzyme homologs that are cytosolically active and/or cytosolically localized.
Dihydroxyacid Dehydratase (DHAD)
In additional embodiments, at least one of the isobutanol pathway enzymes exhibiting cytosolic activity is a dihydroxyacid dehydratase (DHAD). In accordance with this embodiment, the present invention provides cytosolically active dihydroxyacid dehydratases (DHADs) and further describes methods for their use in the production of various beneficial metabolites, such as isobutanol and 2-methyl-1-butanol. As noted above, biosynthetic pathways for the production of isobutanol and 2-methyl-1-butanol have been described (Atsumi et al., 2008, Nature 451: 86-89). In these biosynthetic pathways, DHAD catalyzes the conversion of 2,3-dihydroxyisovalerate to 2-ketoisovalerate, and 2,3-dihydroxy-3-methylvalerate to 2-keto-3-methylvarate, respectively. Using a combination of genetic selection and biochemical analyses, the present inventors have identified a number of DHAD homologs that have activity in the cytosol.
Among the many strategies for identifying cytosolically active DHADs, the present inventors performed multiway-protein alignments between several DHAD homologs. Using this analysis, the present inventors identified a protein motif that was surprisingly unique to a subset of DHAD homologs exhibiting cytosolical activity. This protein motif, P(I/L)XXXGX(I/L)XIL (SEQ ID NO: 27) was found in DHAD homologs demonstrating cytosolic activity in yeast. Therefore, in one embodiment, the present 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 enzyme exhibits the ability to convert 2,3-dihydroxyisovalerate to ketoisovalerate in the cytosol. DHAD enzymes harboring this sequence include those derived from L. lactis (SEQ ID NO: 18), G. forsetii (SEQ ID NO: 17), Acidobacteria bacterium Ellin345 (SEQ ID NO: 16), Saccharopolyspora erythraea (SEQ ID NO: 19), Yarrowia lipolytica (SEQ ID NO: 13), Francisella tularensis (SEQ ID NO: 14), Arabidopsis thaliana (SEQ ID NO: 15), Thermotoga petrophila (SEQ ID NO: 10), and Victivallis vadensis (SEQ ID NO: 11). Also encompassed herein are DHAD enzymes that comprise a motif that is at least about 70% similar, at least about 80% similar, or at least about 90% similar to the motif shown in SEQ ID NO: 27.
As described herein, an even more specific version of this motif has been identified by the present inventors. Thus, in a further embodiment, the present invention provides DHAD enzymes comprising the amino acid sequence PIKXXGX(I/L)XIL (SEQ ID NO: 28), wherein X is any natural or non-natural amino acid, and wherein said DHAD enzyme exhibits the ability to convert 2,3-dihydroxyisovalerate to ketoisovalerate in the cytosol. DHAD enzymes harboring this sequence include those derived from L. lactis (SEQ ID NO: 18), G. forsetii (SEQ ID NO: 17), Acidobacteria bacterium Ellin345 (SEQ ID NO: 16), Y. lipolytica (SEQ ID NO: 13), F. tularensis (SEQ ID NO: 14), A. thaliana (SEQ ID NO: 15), T. petrophila (SEQ ID NO: 10), and V. vadensis (SEQ ID NO: 11). Also encompassed herein are DHAD enzymes that comprise a motif that is at least about 70% similar, at least about 80% similar, or at least about 90% similar to the motif shown in SEQ ID NO: 28.
As noted above, one such cytosolically active DHAD identified herein is exemplified by the L. lactis DHAD amino acid sequence of SEQ ID NO: 18, which is encoded by the L. lactis ilvD gene. As described herein, the present inventors have discovered that yeast strains expressing the cytosolically active L. lactis ilvD (DHAD) exhibit higher isobutanol production than yeast strains expressing the S. cerevisiae ILV3 (DHAD), even when the ILV3 from S. cerevisiae is truncated at its N-terminus to remove a putative mitochondrial targeting sequence. In addition to the use and identification of the cytosolically active DHAD homolog from L. lactis, the present invention encompasses a number of different strategies for identifying DHAD enzymes that exhibit cytosolic activity and/or cytosolic localization, as well as methods for modifying DHADs to increase their ability to exhibit cytosolic activity and/or cytosolic localization.
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 L. lactis. In a specific embodiment, the DHAD enzyme from L. lactis comprises the amino acid sequence of SEQ ID NO: 18. In other embodiments, the bacterial organisms are of the genus Lactococcus, Gramella, Acidobacteria, Francisella, Thermotoga and Victivallis. 
In alternative embodiments, 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.
As described herein, the present inventors have found that in general, an enzyme from a fungal source is more likely to show activity in yeast than a bacterial enzyme expressed in yeast. In addition, homologs that are normally expressed in the cytosol are desired, as a normally cytoplasmic enzyme is likely to show higher activity in the cytosol as compared to an enzyme that is relocalized to the cytosol from other organelles, such as the mitochondria. Fungal homologs of various isobutanol pathway enzymes, including DHAD, are often localized to the mitochondria. The present inventors have found that fungal homologs of DHAD that are cytosolically localized will generally be expected to exhibit higher activity in the cytosol of yeast than those of wild-type yeast strains. Thus, in one embodiment, the present invention provides fungal DHAD homologs that are cytosolically active and/or cytosolically localized.
In another embodiment, the eukaryotic organism is a yeast organism. In another embodiment, the eukaryotic organism is selected from the group consisting of the genera Enamoeba and Giardia. 
In various embodiments described herein, the recombinant microorganism may exhibit at least about 5 percent greater dihydroxyacid dehydratase (DHAD) activity in the cytosol as compared to the parental microorganism. In another embodiment, the recombinant microorganism may exhibit 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, at least about 100 percent, at least about 200 percent, or at least about 500 percent greater dihydroxyacid dehydratase (DHAD) activity in the cytosol as compared to the parental microorganism.
In another embodiment, the present invention provides DHAD enzymes that, when overexpressed in yeast, result in increased activity in the whole cell fraction and do not result in increased activity in the mitochondrial fraction. In one embodiment, the DHAD activity in the whole cell fraction is increased by at least about 2-fold. In another embodiment, DHAD activity in the whole cell fraction is increased by at least about 5-fold. In yet another embodiment, DHAD activity in the whole cell fraction is increased by at least about 7-fold. In yet another embodiment, DHAD activity in the whole cell fraction is increased by at least about 10-fold. In yet another embodiment, DHAD activity in the whole cell fraction is increased by at least about 50-fold. In yet another embodiment, DHAD activity in the whole cell fraction is increased by at least about 100-fold.
Acetolactate Synthase (ALS)
As described herein, the isobutanol pathway enzymes in addition to DHAD should preferably be active in the cytosol. These cytosolically active isobutanol pathway enzymes will generally exhibit enzymatic activity in the cytosol. For instance, a cytosolically active ALS should generally exhibit the ability to convert 2 pyruvate to acetolactate in the cytosol. Thus, in various embodiments described herein, at least one of the isobutanol pathway enzymes exhibiting cytosolic activity is acetolactate synthase (ALS). In yeasts such as S. cerevisiae, the native acetolactate synthase, encoded in S. cerevisiae by the ILV2 gene, is naturally expressed in the yeast mitochondria. Unlike the endogenous acetolactate synthase of yeast, expression of heterologous, acetolactate synthases such as the B. subtilis alsS and the L. lactis alsS in yeast occurs in the yeast cytosol (i.e. cytosolically-localized). Thus, cytosolic expression of acetolactate synthase is achieved by transforming a yeast with a gene encoding an acetolactate synthase protein (EC 2.2.1.6).
ALS homologs that could be cytosolically expressed and localized in yeast are predicted to lack a mitochondrial targeting sequence as analyzed using mitoprot (Claros at, 1996, Eur. J. Biochem 241: 779-86). Such cytosolically localized ALS proteins can be used as the first step in the isobutanol pathway. ALS homologs include, but are not limited to, the following: the Serratia marcescens ALS (GenBank Accession No. ADH43113.1) (probability of mitochondrial localization 0.07), the Enterococcus faecalis ALS (GenBank Accession No. NP—814940) (probability of mitochondrial localization 0.21), the Leuconostoc mesenteroides (GenBank Accession No. YP—818010.1) (probability of mitochondrial localization 0.21), the Staphylococcus aureus ALS (GenBank Accession No. YP 417545) (probability of mitochondrial localization 0.13), the Burkholderia cenocepacia ALS (GenBank Accession No. YP—624435) (probability of mitochondrial localization 0.15), the T. atroviride ALS (SEQ ID NO: 71) (probability of mitochondrial localization 0.19), the T. stipitatus ALS (SEQ ID NO: 72) (probability of mitochondrial localization 0.19), and the Magnaporthe grisea ALS (GenBank Accession No. EDJ99221) (probability of mitochondrial localization 0.02), a homolog or variant of any of the foregoing, and a polypeptide having at least 60% identity to anyone of the foregoing and exhibiting cytosolic ALS activity.
In one embodiment, the cytosolically active ALS is derived from a prokaryotic organism, including, but not limited to B. subtilis or L. lactis, which exhibit cytosolic activity. In another embodiment, the ALS may be derived from an eukaryotic organism, including, but not limited to M. grisea, P. nodorum, T. stipitatus, and T. atroviride. 
In some embodiments, an ALS enzyme that is predicted to be mitochondrially localized may be mutated or modified to remove or modify an N-terminal mitochondrial targeting sequence (MTS) to remove or eliminate its ability to target the ALS enzyme to the mitochondria. Removal of the MTS can increase cytosolic localization of the ALS and/or increase the cytosolic activity of the ALS as compared to the parental ALS.
The conversion of two pyruvate molecules to acetolactate can be carried out by either an acetohydroxyacid synthase (AHAS) or an acetolactate synthase (ALS). AHASs are involved in biosynthesis of branched chain amino acids in the mitochondria of yeasts. They are FAD-dependent and are feedback inhibited by branched chain amino acids. ALSs are catabolic and are involved in the conversion of pyruvate to acetoin. ALS are FAD-independent and not feedback inhibited by branched chain amino acids. In addition, ALSs are specific for the conversion of two pyruvates to acetolactate. Therefore, ALSs are favored over AHASs. In addition, in the case of yeast, AHASs are normally mitochondrial, therefore a fungal ALS that is cytoplasmic is favored. Sequence analysis has shown that there is a conserved sequence ‘RFDDR’ found in AHASs that is not conserved among ALSs (Le of al., 2005, Bull. Korean Chem Soc 26: 916-20). This sequence is likely involved in FAD-binding by AHASs and thus could be used to distinguish between the FAD-dependent AHASs and the FAD-independent ALSs. Using this region to distinguish between AHASs and ALSs BLAST searches of fungal sequence databases were performed and resulted in the identification of ALS homologs from several fungal species (M. grisea, P. nodorum, T. atroviride, T. stipitatus, P. marneffei, and Glomerella graminicola). Of these sequences, the ALS homologs from M. grisea, P. nodorum, T. stipitatus, and T. atroviride will generally be expected to be cytosolically localized.
In one embodiment, the recombinant microorganism may exhibit at least about 5 percent greater acetolactate synthase (ALS) activity in the cytosol as compared to the parental microorganism. In another embodiment, the recombinant microorganism may exhibit 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, at least about 100 percent, at least about 200 percent, or at least about 500 percent greater acetolactate synthase (ALS) activity in the cytosol as compared to the parental microorganism.
Ketol-Acid Reductoisomerase (KARI)
In additional embodiments, at least one of the isobutanol pathway enzymes exhibiting cytosolic activity is a ketol-acid reductoisomerase (KARI). A cytosolically active KARI should generally exhibit the ability to convert acetolactate to 2,3-dihydroxyisovalerate in the cytosol.
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. Fungal homologs of KARI are generally mitochondrially localized. The present inventors have identified a fungal homolog from the anaerobic rumenal fungi, Piromyces sp. E2, that is cytosolically localized.
In certain specific embodiments, the KARI comprises an amino acid sequence selected from the group consisting of E. coli (GenBank No: NP—418222, SEQ ID NO: 1), S. cerevisiae (GenBank No: NP—013459, SEQ ID NO: 2), and B. subtilis (GenBank No: CAB14789) and the KARI enzymes from Piromyces sp E2 (GenBank No: CAA76356), B. aphidicola (GenBank No: AAF13807), S. oleracea (GenBank No: CAA40356), O. sativa (GenBank No: NP—001056384, SEQ ID NO: 3), C. reinhardtii (GenBank No: XP—001702649, SEQ ID NO: 6), N. crassa (GenBank No: XP—961335), S. pombe (GenBank No: NP—001018845), L. bicolor (GenBank No: XP—001880867), I. hospitalis (GenBank No: YP—001435197), P. torridus (GenBank No: YP—023851, SEQ ID NO: 7), A. cryptum (GenBank No: YP—001235669, SEQ ID NO: 5), Cyanobacteria/Synechococcus sp. (GenBank No: YP—473733), Z. mobilis (GenBank No: YP—162876: SEQ ID NO. 8), B. thetaiotaomicron (GenBank No: NP 810987), M. maripaludis (GenBank No: YP—001097443, SEQ ID NO: 4), V. fischeri (GenBank No: YP—205911), Shewanella sp (GenBank No: YP—732498.1), G. forsetti (GenBank No: YP 862142), P. ingrhamaii (GenBank No: YP—942294), and C. hutchinsonii (GenBank No: YP—677763), a homolog or variant of any of the foregoing, and a polypeptide having at least 60% identity to anyone of the foregoing and exhibiting cytosolic KARI activity.
In additional embodiments, the KARI may be an NADH-dependent KARI. Thus, in one embodiment, the present invention provides recombinant microorganisms in which the NADPH-dependent enzymes KARI is replaced with an enzyme that preferentially depends on NADH (i.e. a KARI that is NADH-dependent). In one embodiment, such enzymes may be identified in nature. In an alternative embodiment, such enzymes may be generated by protein engineering techniques including but not limited to directed evolution or site-directed mutagenesis. NADH-dependent KARIs useful in various methods of the present invention are described in commonly owned and co-pending applications U.S. Ser. No. 12/610,784 and PCT/US09/62952 (published as WO/2010/051527), which are herein incorporated by reference in their entireties for all purposes.
In one embodiment, a microorganism is, provided in which cofactor usage is balanced during the production of a fermentation product and the microorganism produces the fermentation product at a higher yield compared to a modified microorganism in which the cofactor usage in not balanced. In another embodiment of the present invention, a microorganism is provided in which the cofactor usage is balanced during the production of isobutanol and the microorganism produces isobutanol at a higher yield compared to a modified microorganism in which the cofactor usage in not balanced. Methods for achieving co-factor balance are described in commonly owned and co-pending applications U.S. Ser. No. 12/610,784 and PCT/US09/62952 (published as WO/2010/051527), which are herein incorporated by reference in their entireties for all purposes.
In one embodiment, the recombinant microorganism may exhibit at least about 5 percent greater ketol-acid reductoisomerase (KARI) activity in the cytosol as compared to the parental microorganism. In another embodiment, the recombinant microorganism may exhibit 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, at least about 100 percent, at least about 200 percent, or at least about 500 percent greater ketol-acid reductoisomerase (KARI) activity in the cytosol as compared to the parental microorganism.
Keto-Acid Decarboxylase (KIVD)
A cytosolically active KIVD should generally exhibit the ability to convert ketoisovalerate to isobutyraldehyde in the cytosol. In one embodiment, the cytosolically active KIVD is derived from a prokaryotic organism, including, but not limited to L. lactis, which exhibits cytosolic activity. In a specific embodiment, the KIVD enzyme from L. lactis comprises the amino acid sequence of SEQ ID NO: 173. In additional embodiments, the cytosolically active KIVD is derived from, for example, Enterobacter cloacae (Accession No. P23234.1), Mycobacterium smegmatis (Accession No. A0R480.1), Mycobacterium tuberculosis (Accession No. O53865.1), Mycobacterium avium (Accession No. Q742Q2.1), Azospirillum brasilense (Accession No. P51852.1), B. subtilis (see Oku et al., 1988, J. Biol. Chem. 263: 18386-96), a homolog or variant of any of the foregoing, and a polypeptide having at least 60% identity to anyone of the foregoing and exhibiting cytosolic KIVD activity.
In an alternative embodiment, the KIVD may be derived from an eukaryotic organism.
In one embodiment, the recombinant microorganism may exhibit at least about 5 percent greater 2-keto-acid decarboxylase (KIVD) activity in the cytosol as compared to the parental microorganism. In another embodiment, the recombinant microorganism may exhibit 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, at least about 100 percent, at least about 200 percent, or at least about 500 percent greater 2-keto-acid decarboxylase (KIVD) activity in the cytosol as compared to the parental microorganism.
Alcohol Dehydrogenase (ADH)
A cytosolically active ADH (used interchangeably herein with isobutanol dehydrogenase, “IDH”) should generally exhibit the ability to convert isobutyraldehyde to isobutanol in the cytosol. In one embodiment, the cytosolically active ADH is derived from a prokaryotic organism, including, but not limited to L. lactis. In a specific embodiment, the ADH enzyme from L. lactis comprises the amino acid sequence of SEQ ID NO: 175. In additional embodiments, the ADH is derived from, for example, Lactobacillus brevis (Accession No. YP—794451.1), Pediococcus acidilactici (Accession No. ZP—06197454.1), Bacillus cereus (Accession No. YP—001374103.1), Bacillus thuringiensis (Accession No. ZP—04101989.1), Leptotrichia goodfellowii (Accession No. ZP—06011170.1), Actinobacillus pleuropneumoniae (Accession No. ZP—00134308.2), Streptococcus sanguinis (Accession No. YP—001035842.1), Eikenella corrodens (Accession No. ZP—03713785.1), Exiguobacterium sp. (Accession No. YP—002886170.1), Neisseria elongate (Accession No. ZP—06736067.1), E. coli (Accession No. ZP—06937530.1), Neisseria meningitidis (Accession No. CBA03965.1), Erwinia pyrifoliae (Accession No. CAY75147.1), and Colwellia psychrerythraea (Accession No. YP—270515.1), a homolog or variant of any of the foregoing, and a polypeptide having at least 60% identity to anyone of the foregoing and having cytosolic ADH activity.
In an alternative embodiment, the ADH may be derived from an eukaryotic organism, including, but not limited to S. cerevisiae and D. melanogaster. In a specific embodiment, the ADH enzyme from S. cerevisiae is Adh7. In another specific embodiment, the ADH enzyme from D. melanogaster comprises the amino acid sequence of SEQ ID NO: 176.
In one embodiment, the recombinant microorganism may exhibit at least about 5 percent greater alcohol dehydrogenase (ADH) activity in the cytosol as compared to the parental microorganism. In another embodiment, the recombinant microorganism may exhibit 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, at least about 100 percent, at least about 200 percent, or at least about 500 percent greater alcohol dehydrogenase (ADH) activity in the cytosol as compared to the parental microorganism.
Chimeric Isobutanol Pathway Enzymes
In another aspect, 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. By creating a chimeric protein that incorporates the activities of both ALS and KARI, this will generally be expected to reduce the effect of diffusion and decreasing the time for spontaneous decomposition to occur. By using a flexible linker and/or structural and sequence information to create a protein with the biocatalytic properties of both ALS and KARI, this will generally increase the concentration of 2-acetolactate at the active site of KARI, causing 2-acetolactate to be converted to 2,3-dihydroxyisovalerate near its theoretical maximum (very little effect of diffusion), and thus, the total concentration of 2-acetolactate should remain low correspondingly decreasing its spontaneous decomposition. This will generally have the effect of increasing the rate of conversion of 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.
Isobutanol Pathway Enzymes Attached to a Protein Scaffold
In another aspect, 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.
The present inventors have found that via the use of a protein scaffold, the isobutanol pathway enzymes that act in concert as part of a single pathway can be co-localized. In some embodiments, the scaffold systems are adapted for binding to the isobutanol pathway enzymes. By tethering the enzymes that work together in the pathway to a scaffold protein, they are brought into close physical proximity with each other, thus increasing the efficiency of the isobutanol production.
There are several advantages to keeping pathway enzymes together on a scaffold system. One is that proteins that normally would localize to an intracellular compartment, like the mitochondria, are partitioned onto the scaffold, thus keeping a sizeable portion of the protein population in the cytosol. Another is that the chemical products of each enzyme is physically close to the next enzyme in the pathway, which speeds reaction time and decreases the possibility that the product would be used in a competing pathway. Finally, unstable products of the enzymes would be used more quickly, since the next enzyme in the pathway would be adjacent to use it as a substrate, thus decreasing nonproductive degradation of the product.
In a preferred embodiment, the isobutanol pathway enzymes are arranged in the sequence in which they are needed to function (i.e. ALS followed by KARI followed by DHAD followed by KIVD followed by ADH). In another embodiment, the scaffolded protein complex is targeted to the cytosol by adding localization signals to the scaffold. In yet another embodiment, the scaffolded protein complex is targeted to the cell wall by adding localization signals to the scaffold. As would be understood by one of skill in the art, the scaffold system allows for co-localization of proteins or enzymes in addition to the isobutanol pathway enzymes. Such proteins may include chaperone proteins, proteins for the conversion of xylose to xylulose-5P, cellulases, etc.
Removal and/or Modification of N-Terminal Mitochondrial Targeting Sequences
The localization of the enzymes involved in production of isobutanol is desired to be cytosolic. Cytosolic localization allows for the pathway to utilize pyruvate and NAD(P)H that is generated in the cytosol by glycolysis and/or the pentose phosphate pathway without the need for the transfer of these metabolites to an alternative compartment (i.e. mitochondria). However, the yeast enzymes acetohydroxyacid synthase (AHAS; Ilv2+Ilv6), ketol-acid reductoisomerase (KARI; Ilv5), and dihydroxyacid dehydratase (DHAD; Ilv3) that carry out the first three steps of isobutanol production are physiologically localized to the mitochondria. Mitochondrial matrix proteins are typically targeted to the mitochondria by a N-terminal mitochondrial targeting sequence (MTS), which is then cleaved off in the mitochondria resulting in the ‘mature’ form of the enzyme (Paschen et al., 2001, IUBMB Life 52: 101-112). Indeed, the N-terminal targeting sequences for Ilv6 has been defined (Pang et al., 1999 Biochemistry 38: 5222-31). N-terminal deletions of Ilv5 has also been shown to re-localize this enzyme to the cytosol (Omura, 2008, Appl. Microbiol. Biotechnol. 78: 503-513; See also Omura, WO/2009/078108 A1, hereby incorporated by reference in its entirety).
One mechanism identified by the present inventors for the cytosolic localization of isobutanol pathway enzymes involves the removal and/or modification of N-terminal mitochondrial targeting sequences (MTS). Nuclear genome-encoded proteins destined to reside in the mitochondria often contain an N-terminal Mitochondrial Targeting Sequence (MTS) that is recognized by a set of proteins collectively known as mitochondrial import machinery. Following recognition and import, the MTS is then physically cleaved off of the imported protein. In eukaryotes, homologs of two of the isobutanol pathway enzymes, ketol-acid reductoisomerase (KARI, e.g. S. cerevisiae Ilv5) and dihydroxy acid dehydratase (DHAD, e.g. S. cerevisiae Ilv3), are predicted to be mitochondrial, based upon the presence of an N-terminal MTS as well as several in vivo functional and mutational studies (See e.g., Omura, F., 2008, Appl Gen & Mol Biot 78: 503-513). As described herein, the present inventors have designed isobutanol pathway enzymes, whereby the predicted MTS is removed or modified. In some instances, there exists experimental evidence for the length of the MTS. Specifically, the MTS of Ilv6 has been experimentally defined to be the N-terminal 61 amino acids (Pang et al., 1999, Biochemistry 38: 5222-31). The MTS of Ilv5 has been reported to be the N-terminal 47 residues (Kassow. A., 1992, “Metabolic effects of deleting the region encoding the transit peptide in Saccharomyces cerevisiae ILV5” PhD thesis, University of Copenhagen). In addition, the deletion of the N-terminal 46 amino acids of Ilv5 has been shown to result in an active enzyme that is localized in the cytosol (Omura, F., 2008, Appl Gen & Mol Biot 78: 503-513).
As described herein, the present inventors utilize deletions and/or modifications of the N-terminal MTS to localize the enzymes of the isobutanol pathway to the cytosol. In various embodiments, the MTS can be entirely or partly deleted or its sequence can be modified to eliminate its ability to target the protein to the mitochondria. A benefit of removing the entire MTS is that the resulting protein would essentially be the ‘mature’ form of the enzyme. The use of deletion of the N-terminal MTS can also be expanded to all enzymes/homologs to be used for isobutanol production. This is especially true for homologs from eukaryotic organisms other than S. cerevisiae where the enzymes are localized to the mitochondria. In addition, some bacterial homologs may have a putative MTS. As bacterial enzymes do not undergo an N-terminal cleavage, N-terminal deletions may be deleterious to these enzymes. In such cases, modifications of the sequence to block the MTS function of the N-terminal sequence may be preferable as such alterations would likely be less deleterious to the enzyme's activity. N-terminal MTS can be predicted by MitoProt II (See, e.g., Claros et al., 1996, Eur. J. Biochem. 241: 779-786). Using this program, the lengths of the MTS for Ilv2 and Ilv3 were predicted to be the N-terminal 55 and 20 amino acids, respectively. Modification of the MTS as contemplated herein includes the introduction of one or multiple mutations to inhibit MTS function. It is thought that the mitochondrial import machinery recognizes the aliphatic alpha helix that is formed by the MTS. Thus modifications that may inhibit MTS functions would be amino acid changes that would alter the aliphatic amino acids such as mutating the charged residues. Such modification(s) prevent its recognition by the mitochondrial import machinery and subsequent cleavage of the MTS and import into the mitochondria.
Peptide Tags to Augment Cytosolic Localization of Isobutanol Pathway Enzymes
In additional embodiments, the mitochondrially imported isobutanol pathway enzymes can be expressed as a chimeric fusion protein to augment cytosolic localization. In one embodiment, the isobutanol pathway enzyme is fused to a peptide tag, whereby said isobutanol pathway enzyme exhibits increased cytosolic localization and/or cytosolic activity in yeast as compared to the parental isobutanol pathway enzyme. In one embodiment, the Isobutanol pathway enzyme is fused to, a peptide tag following removal of the N-terminal Mitochondrial Targeting Sequence (MTS). In one embodiment, the peptide tag is non-cleavable. In a preferred embodiment, the peptide tag is fused at the N-terminus of the isobutanol pathway enzyme. Peptide tags useful in the present invention preferably have the following properties: (1) they do not significantly hinder the normal enzymatic function of the isobutanol pathway enzyme; (2) it folds in such as a way as to block recognition of an N-terminal MTS by the normal mitochondrial import machinery; (3) it promotes the stable expression and/or folding of the isobutanol pathway enzyme it precedes; (4) it can be detected, for example, by Western blotting or SDS-PAGE plus Coomassie staining to facilitate analysis of the overexpressed chimeric protein.
Suitable peptide tags for use in the present invention include, but are not limited to, ubiquitin, ubiquitin-like (UBL) proteins, myc, HA-tag, green fluorescent protein (GFP), and the maltose binding protein (MBP). Ubiquitin, and the Ubiquitin-like protein (Ubl's) offer several advantages. For instance, the use of Ubiquitin or similar Ubl's (e.g., SUMO) as a solubility- and expression-enhancing fusion partner has been well documented (Ecker et al., 1989, J Biol Chem 264: 7715-9; Marblestone et al., 2006, Protein Science 15: 182-9). In fact, in S. cerevisiae, several ribosomal proteins are expressed as C-terminal fusions to ubiquitin. Following translation and protein folding, ubiquitin is cleaved from its co-expressed partner by a highly specific ubiquitin hydrolase, which recognizes and requires the extreme C-terminal Gly-Gly motif present in ubiquitin and cleaves immediately following this sequence; a similar pathway removes Ubl proteins from their fusion partners.
The invention described here describes a method to re-localize a normally mitochondrial protein or enzyme by expressing it as fusion with an N-terminal, non-cleavable ubiquitin or ubiquitin-like molecule. In doing so, the re-targeted enzyme enjoys enhanced expression, solubility, and function in the cytosol. In another embodiment, the sequence encoding the MTS can be replaced with a sequence encoding one or more copies of the c-myc epitope tag (amino acids EQKLISEEDL, SEQ ID NO: 9), which will generally not target a protein into the mitochondria and can easily be detected by commercially available antibodies.
Altering the Iron-Sulfur Cluster Domain and/or Redox Active Domain
In general, the yeast cytosol demonstrates a different redox potential than a bacterial cell, as well as the yeast mitochondria. As a result, isobutanol pathway enzymes which exhibit an iron sulfur (FeS) domain and/or redox active domain, may require the redox potential of the native environments to be folded or expressed in a functional form. Expressing some isobutanol pathway enzymes in the yeast cytosol, which can harbor unfavorable redox potential, has the propensity to result in inactive proteins, even if the proteins are expressed. The present inventors have identified a number of different strategies to overcome this problem, which can arise when an isobutanol pathway enzyme which is suited to a particular environment with a specific redox potential is expressed in the yeast cytosol.
In one embodiment, the present invention provides isobutanol pathway enzymes that exhibit a properly folded iron-sulfur cluster domain and/or redox active domain in the cytosol. Such isobutanol pathway enzymes will generally comprise a mutated or modified iron-sulfur cluster domain and/or redox active domain, allowing for a non-native isobutanol pathway enzyme to be expressed in the yeast cytosol in a functional form.
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 DHAD. 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, Accession No. NP—013576.1), including variants of Ero1 that have been suitably altered to reduce or prevent its normal localization to the endoplasmic reticulum; thioredoxins (which includes Trx1, Accession No. NP—013144.1; and Trx2, Accession No. NP—011725.1), thioredoxin reductase (Trr1, Accession No. NP—010640.1); glutaredoxins (which includes Grx1, Accession No. NP—009895.1; Grx2, Accession No. NP—010801.1; Grx3, Accession No. NP—010383.1; Grx4, Accession No. NP—01101.1; Grx5, Accession No. NP—015266.1; Grx6, Accession No. NP—010274.1; Grx7, Accession No. NP—009570.1; Grx8, Accession No. NP—013468.1); glutathione reductase Girl (Accession No. NP—015234.1); and Jac1 (Accession No. NP—011497.1), including variants of Jac1 that have been suitably altered to reduce or prevent its normal mitochondrial localization; and homologs or variants thereof.
As described herein, iron-sulfur cluster assembly for insertion into yeast apo-iron-sulfur proteins begins in yeast mitochondria. To assemble in yeast the active iron-sulfur proteins containing the cluster, either the apo-iron-sulfur protein is imported into the mitochondria from the cytosol and the iron-sulfur cluster is inserted into the protein and the active protein remains localized in the mitochondria; or the iron-sulfur clusters or precursors thereof are exported from the mitochondria to the cytosol and the active protein is assembled in the cytosol or other cellular compartments.
Targeting of yeast mitochondrial iron-sulfur proteins or non-yeast iron-sulfur proteins to the yeast cytosol can result in such proteins not being properly assembled with their iron-sulfur clusters. This present invention overcomes this problem by co-expression and cytosolic targeting in yeast of proteins for iron-sulfur cluster assembly and cluster insertion into apo-iron-sulfur proteins, including iron-sulfur cluster assembly and insertion proteins from organisms other than yeast, together with the apo-iron-sulfur protein to provide assembly of active iron-sulfur proteins in the yeast cytosol.
Therefore, in one embodiment of this invention, the apo-iron-sulfur protein DHAD enzyme encoded by the E. coli ilvD gene is expressed in yeast together with E. coli iron-sulfur cluster assembly and insertion genes comprising either the cyaY, iscS, iscU, iscA, hscB, hscA, fdx and isuX genes or the sufA, sufB, sufC, sufD, sufS and sufE genes. This strategy allows for both the apo-iron-sulfur protein (DHAD) and the iron-sulfur cluster assembly and insertion components (the products of the isc or suf genes) to come from the same organism, causing assembly of the active DHAD iron-sulfur protein in the yeast cytosol. As a modification of this embodiment, for those E. coli iron-sulfur cluster assembly and insertion components that localize to or are predicted to localize to the yeast mitochondria upon expression in yeast, the genes for these components are engineered to eliminate such targeting signals to ensure localization of the components in the yeast cytoplasm. Thus, 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 is prevented. In certain embodiments, it may be preferable to overexpress one or more genes encoding an iron-sulfur cluster assembly protein.
In additional embodiments, iron-sulfur cluster assembly and insertion components from other than E. coli can be co-expressed with the E. coli DHAD protein to provide assembly of the active DHAD iron-sulfur cluster protein. Such iron-sulfur cluster assembly and insertion components from other organisms can consist of the products of the Helicobacter pylori nifS and nifU genes or the Entamoeba histolytica nifS and nifU genes. As a modification of this embodiment, for those non-E. coli iron-sulfur cluster assembly and insertion components that localize to or are predicted to localize to the yeast mitochondria upon expression in yeast, the genes for these components can be engineered to eliminate such targeting signals to ensure localization of the components in the yeast cytoplasm.
As a further modification of this embodiment, in addition to co-expression of these proteins in aerobically-grown yeast, these proteins may be co-expressed in anaerobically-grown yeast to lower the redox state of the yeast cytoplasm to improve assembly of the active iron-sulfur protein.
In another embodiment, the above iron-sulfur cluster assembly and insertion components can be co-expressed with DHAD apo-iron-sulfur enzymes other than the E. coli IlvD gene product to generate active DHAD enzymes in the yeast cytoplasm. As a modification of this embodiment, for those DHAD enzymes that localize to or are predicted to localize to the yeast mitochondria upon expression in yeast, then the genes for these enzymes can be engineered to eliminate such targeting signals to ensure localization of the enzymes in the yeast cytoplasm.
In additional embodiments, the above methods used to generate active DHAD enzymes localized to yeast cytoplasm may be combined with methods to generate active acetolactate synthase, KARI, KIVD and ADH enzymes in the same yeast for the production of isobutanol by yeast.
In another embodiment, production of active iron-sulfur proteins other than DHAD enzymes in yeast cytoplasm can be accomplished by co-expression with iron-sulfur cluster assembly and insertion proteins from organisms other than yeast, with proper targeting of the proteins to the yeast cytoplasm if necessary and expression in anaerobically growing yeast if needed to improve assembly of the active proteins.
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 (Accession No. NP—012263.1), Nbp35 (Accession No. NP—011424.1), Nar1 (Accession No. NP—014159.1), Cia1 (Accession No. NP—010553.1), 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.
As noted above, the iron-sulfur cluster assembly genes may be derived from eukaryotic organisms, including, but not limited to yeasts and plants. In one embodiment, the iron-sulfur cluster genes are derived from a yeast organism, including, but not limited to S. cerevisiae. In specific embodiments, the yeast derived iron-sulfur cluster assembly genes 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 genes may be derived from a plant chloroplast.
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. This modification increases the capacity of a yeast to incorporate [Fe—S] clusters into cytosolically expressed proteins wherein said proteins can be native proteins that are expressed in a non-native compartment or heterologous proteins. This is achieved by deletion of a highly expressed native cytoplasmic [Fe—S]-dependent protein. More specifically, the gene LEU1 is deleted coding for the 3-isopropylmalate dehydratase which catalyses the conversion of 3-isopropylmalate into 2-isopropylmalate as part of the leucine biosynthetic pathway in yeast. Leu1p contains an 4Fe-4S cluster which takes part in the catalysis of the dehydratase. DHAD also contains a 4Fe-4S cluster involved in its dehydratase activity. Therefore, although the two enzymes have different substrate preferences the process of incorporation of the Fe—S cluster is generally similar for the two proteins. Given that Leu1p is present in yeast at 10000 molecules per cell (Ghaemmaghami et al., 2003, Nature 425: 737), deletion of LEU1 therefore ensures that the cell has enough spare capacity to incorporate [Fe—S] clusters into at least 10000 molecules of cytosolically expressed DHAD. Taking into account the specific activity of DHAD (E. coli DHAD is reported to have a specific activity of 63 U/mg) (Flint et al., 1993, J Biological Chem 268: 14732), the LEU1 deletion yeast strain would generally exhibit an increased capacity for DHAD activity in the cytosol as measured in cell lysate.
In alternative embodiments, it may be desirable to further overexpress an additional enzyme that converts 2,3-dihydroxyisovalerate to ketoisovalerate in the cytosol. In a specific embodiment, the enzyme may be selected from the group consisting of 3-isopropylmalate dehydratase (Leu1p) and imidazoleglycerol phosphate dehydrogenase (His3p). Because DHAD activity is limited in the cytosol, alternative dehydratases that convert dihydroxyisovalerate (DHIV) to 2-ketoisovalerate (KIV) and are physiologically localized to the yeast cytosol may be utilized. Leu1p and His3p are dehydratases that potentially may exhibit affinity for DHIV. Leu1p is an Fe—S binding protein that is involved in leucine biosynthesis and is also normally localized to the cytosol. His3p is involved in histidine biosynthesis and is similar to Leu1p, it is generally localized to the cytosol or predicted to be localized to the cytosol. This modification overcomes the problem of a DHAD that is limiting isobutanol production in the cytosol of yeast. The use of an alternative dehydratase that has activity in the cytosol with a low activity towards DHIV may thus be used in place of the DHAD in the isobutanol pathway. As described herein, such enzyme may be further engineered to increase activity with DHIV.
The Microorganism in General
Native producers of 1-butanol, such as Clostridium acetobutylicum, are known, but these organisms also generate byproducts such as acetone, ethanol, and butyrate during fermentations. Furthermore, these microorganisms are relatively difficult to manipulate, with significantly fewer tools available than in more commonly used production hosts such as S. cerevisiae or E. coli. Additionally, the physiology and metabolic regulation of these native producers are much less well understood, impeding rapid progress towards high-efficiency production. Furthermore, no native microorganisms have been identified that can metabolize glucose into isobutanol in industrially relevant quantities.
The production of isobutanol and other fusel alcohols by various yeast species, including Saccharomyces cerevisiae is of special interest to the distillers of alcoholic beverages, for whom fusel alcohols constitute often undesirable off-notes. Production of isobutanol in wild-type yeasts has been documented on various growth media, ranging from grape must from winemaking (Romano et al., 2003, World J. of Microbiol Blot. 19: 311-5), in which 12-219 mg/L isobutanol were produced, to supplemented minimal media (Oliviera et al., 2005, World J. of Microbiol Blot. 21: 1569-76), producing 16-34 mg/L isobutanol. Work from Dickinson et al. (J Biol Chem. 272: 26871-8, 1997) has identified the enzymatic steps utilized in an endogenous S. cerevisiae pathway converting branch-chain amino acids (e.g., valine or leucine) to isobutanol.
Recombinant microorganisms provided herein can express a plurality of heterologous and/or native target enzymes involved in pathways for the production of isobutanol from a suitable carbon source.
Accordingly, “engineered” or “modified” microorganisms are produced via the introduction of genetic material into a host or parental microorganism of choice and/or by modification of the expression of native genes, thereby modifying or altering the cellular physiology and biochemistry of the microorganism. Through the introduction of genetic material and/or the modification of the expression of native genes the parental microorganism acquires new properties, e.g. the ability to produce a new, or greater quantities of, an intracellular metabolite. As described herein, the introduction of genetic material into and/or the modification of the expression of native genes in a parental microorganism results in a new or modified ability to produce isobutanol. The genetic material introduced into and/or the genes modified for expression in the parental microorganism contains gene(s), or parts of genes, coding for one or more of the enzymes involved in a biosynthetic pathway for the production of isobutanol and may also include additional elements for the expression and/or regulation of expression of these genes, e.g. promoter sequences.
In addition to the introduction of a genetic material into a host or parental microorganism, an engineered or modified microorganism can also include alteration, disruption, deletion or knocking-out of a gene or polynucleotide to alter the cellular physiology and biochemistry of the microorganism. Through the alteration, disruption, deletion or knocking-out of a gene or polynucleotide the microorganism acquires new or improved properties (e.g., the ability to produce a new metabolite or greater quantities of an intracellular metabolite, improve the flux of a metabolite down a desired pathway, and/or reduce the production of byproducts).
Recombinant microorganisms provided herein may also produce metabolites in quantities not available in the parental microorganism. A “metabolite” refers to any substance produced by metabolism or a substance necessary for or taking part in a particular metabolic process. A metabolite can be an organic compound that is a starting material (e.g., glucose or pyruvate), an intermediate (e.g., 2-ketoisovalerate), or an end product (e.g., isobutanol) of metabolism. Metabolites can be used to construct more complex molecules, or they can be broken down into simpler ones. Intermediate metabolites may be synthesized from other metabolites, perhaps used to make more complex substances, or broken down into simpler compounds, often with the release of chemical energy.
Exemplary metabolites include glucose, pyruvate, and isobutanol. The metabolite isobutanol can be produced by a recombinant microorganism which expresses or over-expresses a metabolic pathway that converts pyruvate to isobutanol. An exemplary metabolic pathway that converts pyruvate to isobutanol may be comprised of an acetohydroxy acid synthase (ALS), a ketolacid reductoisomerase (KARI), a dihydroxy-acid dehydratase (DHAD), a 2-keto-acid decarboxylase (KIVD), and an alcohol dehydrogenase (ADH).
Accordingly, provided herein are recombinant microorganisms that produce isobutanol and in some aspects may include the elevated expression of target enzymes such as ALS, KARI, DHAD, KIVD, and ADH
The disclosure identifies specific genes useful in the methods, compositions and organisms of the disclosure; however it will be recognized that absolute identity to such genes is not necessary. For example, changes in a particular gene or polynucleotide comprising a sequence encoding a polypeptide or enzyme can be performed and screened for activity. Typically such changes comprise conservative mutation and silent mutations. Such modified or mutated polynucleotides and polypeptides can be screened for expression of a functional enzyme using methods known in the art.
Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantially the same or functionally equivalent polypeptides can also be used to clone and express the polynucleotides encoding such enzymes.
As will be understood by those of skill in the art, it can be advantageous to modify a coding sequence to enhance its expression in a particular host. The genetic code is redundant with 64 possible codons, but most organisms typically use a subset of these codons. The codons that are utilized most often in a species are called optimal codons, and those not utilized very often are classified as rare or low-usage codons. Codons can be substituted to reflect the preferred codon usage of the host, a process sometimes called “codon optimization” or “controlling for species codon bias.”
Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host (Murray et al., 1989, Nucl Acids Res. 17: 477-508) can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. The typical stop codon for monocotyledonous plants is UGA, whereas insects and E. coli commonly use UAA as the stop codon (Dalphin et al., 1996, Nucl Acids Res. 24: 216-8). Methodology for optimizing a nucleotide sequence for expression in a plant is provided, for example, in U.S. Pat. No. 6,015,891, and the references cited therein.
Those of skill in the art will recognize that, due to the degenerate nature of the genetic code, a variety of DNA compounds differing in their nucleotide sequences can be used to encode a given enzyme of the disclosure. The native DNA sequence encoding the biosynthetic enzymes described above are referenced herein merely to illustrate an embodiment of the disclosure, and the disclosure includes DNA compounds of any sequence that encode the amino acid sequences of the polypeptides and proteins of the enzymes utilized in the methods of the disclosure. In similar fashion, a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity. The disclosure includes such polypeptides with different amino acid sequences than the specific proteins described herein so long as they modified or variant polypeptides have the enzymatic anabolic or catabolic activity of the reference polypeptide. Furthermore, the amino acid sequences encoded by the DNA sequences shown herein merely illustrate embodiments of the disclosure.
In addition, homologs of enzymes useful for generating metabolites are encompassed by the microorganisms and methods provided herein.
As used herein, two proteins (or a region of the proteins) are substantially homologous when the amino acid sequences have at least about 30%, 40%, 50% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In one embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, typically at least 40%, more typically at least 50%, even more typically at least 60%, and even more typically at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
When “homologous” is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art (See, e.g., Pearson W. R., 1994, Methods in Mol Biol 25: 365-89.
The following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
Sequence homology for polypeptides, which is also referred to as percent sequence identity, is typically measured using sequence analysis software. See commonly owned and co-pending application US 2009/0226991. A typical algorithm used comparing a molecule sequence to a database containing a large number of sequences from different organisms is the computer program BLAST. When searching a database containing sequences from a large number of different organisms, it is typical to compare amino acid sequences. Database searching using amino acid sequences can be measured by algorithms described in commonly owned and co-pending application US 2009/0226991.
The disclosure provides recombinant microorganisms comprising a biochemical pathway for the production of isobutanol from a suitable substrate at a high yield. A recombinant microorganism of the disclosure comprises one or more recombinant polynucleotides within the genome of the organism or external to the genome within the organism. The microorganism can comprise a reduction in expression, disruption or knockout of a gene found in the wild-type organism and/or introduction of a heterologous polynucleotide and/or expression or overexpression of an endogenous polynucleotide.
In one aspect, the disclosure provides a recombinant microorganism comprising elevated expression of at least one target enzyme as compared to a parental microorganism or encodes an enzyme not found in the parental organism. In another or further aspect, the microorganism comprises a reduction in expression, disruption or knockout of at least one gene encoding an enzyme that competes with a metabolite necessary for the production of isobutanol. The recombinant microorganism produces at least one metabolite involved in a biosynthetic pathway for the production of isobutanol. In general, the recombinant microorganisms comprises at least one recombinant metabolic pathway that comprises a target enzyme and may further include a reduction in activity or expression of an enzyme in a competitive biosynthetic pathway. The pathway acts to modify a substrate or metabolic intermediate in the production of isobutanol. The target enzyme is encoded by, and expressed from, a polynucleotide derived from a suitable biological source. In some embodiments, the polynucleotide comprises a gene derived from a prokaryotic or eukaryotic source and recombinantly engineered into the microorganism of the disclosure. In other embodiments, the polynucleotide comprises a gene that is native to the host organism.
It is understood that a range of microorganisms can be modified to include a recombinant metabolic pathway suitable for the production of isobutanol. In various embodiments, microorganisms may be selected from yeast microorganisms. Yeast microorganisms for the production of isobutanol may be selected based on certain characteristics:
One characteristic may include the property that the microorganism is selected to convert various carbon sources into isobutanol. The term “carbon source” generally refers to a substance suitable to be used as a source of carbon for prokaryotic or eukaryotic cell growth. Examples of suitable carbon sources are described in commonly owned and co-pending application US 2009/0226991. Accordingly, in one embodiment, the recombinant microorganism herein disclosed can convert a variety of carbon sources to products, including but not limited to glucose, galactose, mannose, xylose, arabinose, lactose, sucrose, and mixtures thereof.
The recombinant microorganism may thus further include a pathway for the fermentation of isobutanol from five-carbon (pentose) sugars including xylose. Most yeast species metabolize xylose via a complex route, in which xylose is first reduced to xylitol via a xylose reductase (XR) enzyme. The xylitol is then oxidized to xylulose via a xylitol dehydrogenase (XDH) enzyme. The xylulose is then phosphorylated via a xylulokinase (XK) enzyme. This pathway operates inefficiently in yeast species because it introduces a redox imbalance in the cell. The xylose-to-xylitol step uses NADH as a cofactor, whereas the xylitol-to-xylulose step uses NADPH as a cofactor. Other processes must operate to restore the redox imbalance within the cell. This often means that the organism cannot grow anaerobically on xylose or other pentose sugar. Accordingly, a yeast species that can efficiently ferment xylose and other pentose sugars into a desired fermentation product is therefore very desirable.
Thus, in one aspect, the recombinant is engineered to express a functional exogenous xylose isomerase. Exogenous xylose isomerases functional in yeast are known in the art. See, e.g., Rajgarhia et al, US20060234364, which is herein incorporated by reference in its entirety. In an embodiment according to this aspect, the exogenous xylose isomerase gene is operatively linked to promoter and terminator sequences that are functional in the yeast cell. In a preferred embodiment, the recombinant microorganism further has a deletion or disruption of a native gene that encodes for an enzyme (e.g. XR and/or XDH) that catalyzes the conversion of xylose to xylitol. In a further preferred embodiment, the recombinant microorganism also contains a functional, exogenous xylulokinase (XK) gene operatively linked to promoter and terminator sequences that are functional in the yeast cell. In one embodiment, the xylulokinase (XK) gene is overexpressed.
In one embodiment, the microorganism has reduced or no pyruvate decarboxylase (PDC) activity. PDC catalyzes the decarboxylation of pyruvate to acetaldehyde, which is then reduced to ethanol by ADH via an oxidation of NADH to NADH+. Ethanol production is the main pathway to oxidize the NADH from glycolysis. Deletion of this pathway increases the pyruvate and the reducing equivalents (NADH) available for the isobutanol pathway. Accordingly, deletion of PDC genes can further increase the yield of isobutanol.
In another embodiment, the microorganism has reduced or no glycerol-3-phosphate dehydrogenase (GPD) activity. GPD catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P) via the oxidation of NADH to NAD+. Glycerol is then produced from G3P by Glycerol-3-phosphatase (GPP). Glycerol production is a secondary pathway to oxidize excess NADH from glycolysis. Reduction or elimination of this pathway would increase the pyruvate and reducing equivalents (NADH) available for the isobutanol pathway. Thus, deletion of GPD genes can further increase the yield of isobutanol.
In yet another embodiment, the microorganism has reduced or no PDC activity and reduced or no GPD activity.
In one embodiment, the yeast microorganisms may be selected from the “Saccharomyces Yeast Glade”, as described in commonly owned and co-pending application US 2009/0226991.
The term “Saccharomyces sensu stricto” taxonomy group is a cluster of yeast species that are highly related to S. cerevisiae (Rainieri et al., 2003, J. Biosci Bioengin 96: 1-9). Saccharomyces sensu stricto yeast species include but are not limited to S. cerevisiae, S. cerevisiae, S. kudriavzevii, S. mikatae, S. bayanus, S. uvarum, S. carocanis and hybrids derived from these species (Masneuf et al., 1998, Yeast 7: 61-72).
An ancient whole genome duplication (WGD) event occurred during the evolution of the hemiascomycete yeast and was discovered using comparative genomic tools (Kellis et al., 2004, Nature 428: 617-24; Dujon et al., 2004, Nature 430:35-44; Langkjaer et al., 2003, Nature 428: 848-52; Wolfe et al., 1997, Nature 387: 708-13). Using this major evolutionary event, yeast can be divided into species that diverged from a common ancestor following the WGD event (termed “post-WGD yeast” herein) and species that diverged from the yeast lineage prior to the WGD event (termed “pre-WGD yeast” herein).
Accordingly, in one embodiment, the yeast microorganism may be selected from a post-WGD yeast genus, including but not limited to Saccharomyces and Candida. The favored post-WGD yeast species include: S. cerevisiae, S. uvarum, S. bayanus, S. paradoxus, S. castelli, and C. glabrata. 
In another embodiment, the yeast microorganism may be selected from a pre-whole genome duplication (pre-WGD) yeast genus including but not limited to Saccharomyces, Kluyveromyces, Candida, Pichia, Issatchenkia, Debaryomyces, Hansenula, Yarrowia and, Schizosaccharomyces. Representative pre-WGD yeast species include: S. kluyveri, K. thermotolerans, K. marxianus, K. waltii, K. lactis, C. tropicalis, P. pastoris, P. anomala, P. stipitis, I. orientalis, I. occidentalis, I. scutulata, D. hansenii, H. anomala, Y. lipolytica, and S. pombe. 
A yeast microorganism may be either Crabtree-negative or Crabtree-positive as described in described in commonly owned and co-pending application US 2009/0226991. In one embodiment the yeast microorganism may be selected from yeast with a Crabtree-negative phenotype including but not limited to the following genera: Kluyveromyces, Pichia, Issatchenkia, Hansenula, and Candida. Crabtree-negative species include but are not limited to: K. lactis, K. marxianus, P. anomala, P. stipitis, I. orientalis, I. occidentalis, I. scutulata, H. anomala, and C. utilis. In another embodiment, the yeast microorganism may be selected from a yeast with a Crabtree-positive phenotype, including but not limited to Saccharomyces, Kluyveromyces, Zygosaccharomyces, Debaryomyces, Pichia and Schizosaccharomyces. Crabtree-positive yeast species include but are not limited to: S. cerevisiae, S. uvarum, S. bayanus, S. paradoxus, S. castelli, S. kluyveri, K. thermotolerans, C. glabrata, Z. bailli, Z. rouxii, D. hansenii, P. pastorius, and S. pombe. 
Another characteristic may include the property that the microorganism is that it is non-fermenting. In other words, it cannot metabolize a carbon source anaerobically while the yeast is able to metabolize a carbon source in the presence of oxygen. Nonfermenting yeast refers to both naturally occurring yeasts as well as genetically modified yeast. During anaerobic fermentation with fermentative yeast, the main pathway to oxidize the NADH from glycolysis is through the production of ethanol. Ethanol is produced by alcohol dehydrogenase (ADH) via the reduction of acetaldehyde, which is generated from pyruvate by pyruvate decarboxylase (PDC). In one embodiment, a fermentative yeast can be engineered to be non-fermentative by the reduction or elimination of the native PDC activity. Thus, most of the pyruvate produced by glycolysis is not consumed by PDC and is available for the isobutanol pathway. Deletion of this pathway increases the pyruvate and the reducing equivalents available for the isobutanol pathway. Fermentative pathways contribute to low yield and low productivity of isobutanol. Accordingly, deletion of PDC may increase yield and productivity of isobutanol.
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 one embodiment, a yeast microorganism is engineered to convert a carbon source, such as glucose, to pyruvate by glycolysis and the pyruvate is converted to isobutanol via an engineered isobutanol pathway (See, e.g., WO/2007/050671, WO/2008/098227, and Atsumi et al., 2008, Nature 45: 86-9). Alternative pathways for the production of isobutanol have been described in WO/2007/050671 and in Dickinson et al., 1998, J Biol Chem 273:25751-6.
Accordingly, the engineered isobutanol pathway to convert pyruvate to isobutanol can be comprised of the following reactions:
1. 2 pyruvate→acetolactate+CO2 
2. acetolactate+NAD(P)H→2,3-dihydroxyisovalerate+NAD(P)+
3. 2,3-dihydroxyisovalerate→alpha-ketoisovalerate
4. alpha-ketoisovalerate→4 isobutyraldehyde+CO2 
5. isobutyraldehyde+NAD(P)H isobutanol+NAD(P)
These reactions are carried out by the enzymes 1) Acetolactate Synthase (ALS), 2) Keto-acid Reducto-Isomerase (KARI), 3) Dihydroxy-acid dehydratase (DHAD), 4) Keto-isovalerate decarboxylase (KIVD), and 5) an Alcohol dehydrogenase (ADH) (FIG. 1). In another embodiment, the yeast microorganism is engineered to overexpress these enzymes. For example, these enzymes can be encoded by native genes. Alternatively, these enzymes can be encoded by heterologous genes. For example, ALS can be encoded by the alsS gene of B. subtilis, alsS of L. lactis, or the ilvK gene of K. pneumonia. For example, KARI can be encoded by the ilvC genes of E. coli, C. glutamicum, M. maripaludis, or Piromyces sp E2. For example, DHAD can be encoded by the ilvD genes of E. coli, C. glutamicum, or L. lactis. For example, KIVD can be encoded by the kivD gene of L. lactis. ADH can be encoded by ADH2, ADH6, or ADH7 of S. cerevisiae. 
In one embodiment, pathway steps 2 and 5 may be carried out by KARI and ADH enzymes that utilize NADH (rather than NADPH) as a co-factor. Such enzymes are described in commonly owned and co-pending applications U.S. Ser. No. 12/610,784 and PCT/US09/62952 (published as WO/2010/051527), which are herein incorporated by reference in their entireties for all purposes. The present inventors have found that utilization of NADH-dependent KARI and ADH enzymes to catalyze pathway steps 2 and 5, respectively, surprisingly enables production of isobutanol under anaerobic conditions. Thus, in one embodiment, the recombinant microorganisms of the present invention may use an NADH-dependent KARI to catalyze the conversion of acetolactate (+NADH) to produce 2,3-dihydroxyisovalerate. In another embodiment, the recombinant microorganisms of the present invention may use an NADH dependent ADH to catalyze the conversion of isobutyraldehyde (+NADH) to produce isobutanol. In yet another embodiment, the recombinant microorganisms of the present invention may use both an NADH-dependent KARI to catalyze the conversion of acetolactate (+NADH) to produce 2,3-dihydroxyisovalerate, and an NADH-dependent ADH to catalyze the conversion of isobutyraldehyde (+NADH) to produce isobutanol.
The yeast microorganism of the invention may be engineered to have increased ability to convert pyruvate to isobutanol. In one embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to isobutyraldehyde. In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to keto-isovalerate. In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to 2,3-dihydroxyisovalerate. In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to acetolactate.
Furthermore, any of the genes encoding the foregoing enzymes (or any others mentioned herein (or any of the regulatory elements that control or modulate expression thereof)) may be optimized by genetic/protein engineering techniques, such as directed evolution or rational mutagenesis, which are known to those of ordinary skill in the art. Such action allows those of ordinary skill in the art to optimize the enzymes for expression and activity in yeast.
In addition, genes encoding these enzymes can be identified from other fungal and bacterial species and can be expressed for the modulation of this pathway. A variety of organisms could serve as sources for these enzymes, including, but not limited to, Saccharomyces spp., including S. cerevisiae and S. uvarum, Kluyveromyces spp., including K. thermotolerans, K. lactis, and K. marxianus, Pichia spp., Hansenula spp., including H. polymorpha, Candida spp., Trichosporon spp., Yamadazyma spp., including Y. spp. stipitis, Torulaspora pretoriensis, Schizosaccharomyces spp., including S. pombe, Cryptococcus spp., Aspergillus spp., Neurospora spp., or Ustilago spp. Sources of genes from anaerobic fungi include, but not limited to, Piromyces spp., Orpinomyces spp., or Neocallimastix spp. Sources of prokaryotic enzymes that are useful include, but not limited to, Escherichia. coli, Zymomonas mobilis, Staphylococcus aureus, Bacillus spp., Clostridium spp., Corynebacterium spp., Pseudomonas spp., Lactococcus spp., Enterobacter spp., and Salmonella spp.
Methods in General
Identification of PDC and GPD in a Yeast Microorganism
Any method can be used to identify genes that encode for enzymes with, pyruvate decarboxylase (PDC) activity or glycerol-3-phosphate dehydrogenase (GPD) activity. Suitable methods for the identification of PDC and GPD are described in co-pending applications U.S. Ser. No. 12/343,375 (published as US 2009/0226991), U.S. Ser. No. 12/696,645, and U.S. Ser. No. 12/820,505, which claim priority to U.S. Provisional Application 61/016,483, all of which are herein incorporated by reference in their entireties for all purposes.
Genetic Insertions and Deletions
Any method can be used to introduce a nucleic acid molecule into yeast and many such methods are well known. For example, transformation and electroporation are common methods for introducing nucleic acid into yeast cells. See, e.g., Gietz et al., 1992, Nuc Acids Res. 27: 69-74; Ito et al., 1983, J. Bacteriol. 153: 163-8; and Becker et al., 1991, Methods in Enzymology 194: 182-7.
In an embodiment, the integration of a gene of interest into a DNA fragment or target gene of a yeast microorganism occurs according to the principle of homologous recombination. According to this embodiment, an integration cassette containing a module comprising at least one yeast marker gene and/or the gene to be integrated (internal module) is flanked on either side by DNA fragments homologous to those of the ends of the targeted integration site (recombinogenic sequences). After transforming the yeast with the cassette by appropriate methods, a homologous recombination between the recombinogenic sequences may result in the internal module replacing the chromosomal region in between the two sites of the genome corresponding to the recombinogenic sequences of the integration cassette. (Orr-Weaver et al., 1981, PNAS USA 78: 6354-58).
In an embodiment, the integration cassette for integration of a gene of interest into a yeast microorganism includes the heterologous gene under the control of an appropriate promoter and terminator together with the selectable marker flanked by recombinogenic sequences for integration of a heterologous gene into the yeast chromosome. In an embodiment, the heterologous gene includes an appropriate native gene desired to increase the copy number of a native gene(s). The selectable marker gene can be any marker gene used in yeast, including but not limited to, HIS3, TRP1, LEU2, URA3, bar, ble, hph, and kan. The recombinogenic sequences can be chosen at will, depending on the desired integration site suitable for the desired application.
In another embodiment, integration of a gene into the chromosome of the yeast microorganism may occur via random integration (Kooistra et al., 2004, Yeast 21: 781-792).
Additionally, in an embodiment, certain introduced marker genes are removed from the genome using techniques well known to those skilled in the art. For example, URA3 marker loss can be obtained by plating URA3 containing cells in FOA (5-fluoro-orotic acid) containing medium and selecting for FOA resistant colonies (Boeke et al., 1984, Mol. Gen. Genet 197: 345-47).
The exogenous nucleic acid molecule contained within a yeast cell of the disclosure can be maintained within that cell in any form. For example, exogenous nucleic acid molecules can be integrated into the genome of the cell or maintained in an episomal state that can stably be passed on (“inherited”) to daughter cells. Such extra-chromosomal genetic elements (such as plasmids, etc.) can additionally contain selection markers that ensure the presence of such genetic elements in daughter cells. Moreover, the yeast cells can be stably or transiently transformed. In addition, the yeast cells described herein can contain a single copy, or multiple copies of a particular exogenous nucleic acid molecule as described above.
Reduction of Enzymatic Activity
Yeast microorganisms within the scope of the invention may have reduced enzymatic activity such as reduced glycerol-3-phosphate dehydrogenase activity. The term “reduced” as used herein with respect to a particular enzymatic activity refers to a lower level of enzymatic activity than that measured in a comparable yeast cell of the same species. The term reduced also refers to the elimination of enzymatic activity than that measured in a comparable yeast cell of the same species. Thus, yeast cells lacking glycerol-3-phosphate dehydrogenase activity are considered to have reduced glycerol-3-phosphate dehydrogenase activity since most, if not all, comparable yeast strains have at least some glycerol-3-phosphate dehydrogenase activity. Such reduced enzymatic activities can be the result of lower enzyme concentration, lower specific activity of an enzyme, or a combination thereof. Many different methods can be used to make yeast having reduced enzymatic activity. For example, a yeast cell can be engineered to have a disrupted enzyme-encoding locus using common mutagenesis or knock-out technology. In addition, certain point-mutation(s) can be introduced which results in an enzyme with reduced activity.
Alternatively, antisense technology can be used to reduce enzymatic activity. For example, yeast can be engineered to contain a cDNA that encodes an antisense molecule that prevents an enzyme from being made. The term “antisense molecule” as used herein encompasses any nucleic acid molecule that contains sequences that correspond to the coding strand of an endogenous polypeptide. An antisense molecule also can have flanking sequences (e.g., regulatory sequences). Thus antisense molecules can be ribozymes or antisense oligonucleotides. A ribozyme can have any general structure including, without limitation, hairpin, hammerhead, or axhead structures, provided the molecule cleaves RNA.
Yeast having a reduced enzymatic activity can be identified using many methods. For example, yeast having reduced glycerol-3-phosphate dehydrogenase activity can be easily identified using common methods, which may include, for example, measuring glycerol formation via liquid chromatography.
Overexpression of Heterologous Genes
Methods for overexpressing a polypeptide from a native or heterologous nucleic acid molecule are well known. Such methods include, without limitation, constructing a nucleic acid sequence such that a regulatory element promotes the expression of a nucleic acid sequence that encodes the desired polypeptide. Typically, regulatory elements are DNA sequences that regulate the expression of other DNA sequences at the level of transcription. Thus, regulatory elements include, without limitation, promoters, enhancers, and the like. For example, the exogenous genes can be under the control of an inducible promoter or a constitutive promoter. Moreover, methods for expressing a polypeptide from an exogenous nucleic acid molecule in yeast are well known. For example, nucleic acid constructs that are used for the expression of exogenous polypeptides within Kluyveromyces and Saccharomyces are well known (see, e.g., U.S. Pat. Nos. 4,859,596 and 4,943,529, for Kluyveromyces and, e.g., Gellissen et al., Gene 190(1):87-97 (1997) for Saccharomyces). Yeast plasmids have a selectable marker and an origin of replication. In addition certain plasmids may also contain a centromeric sequence. These centromeric plasmids are generally a single or low copy plasmid. Plasmids without a centromeric sequence and utilizing either a 2 micron (S. cerevisiae) or 1.6 micron (K. lactis) replication origin are high copy plasmids. The selectable marker can be either prototrophic, such as HIS3, TRP1, LEU2, URA3 or ADE2, or antibiotic resistance, such as, bar, ble, hph, or kan.
In another embodiment, heterologous control elements can be used to activate or repress expression of endogenous genes. Additionally, when expression is to be repressed or eliminated, the gene for the relevant enzyme, protein or RNA can be eliminated by known deletion techniques.
As described herein, any yeast within the scope of the disclosure can be identified by selection techniques specific to the particular enzyme being expressed, over-expressed or repressed. Methods of identifying the strains with the desired phenotype are well known to those skilled in the art. Such methods include, without limitation, PCR, RT-PCR, and nucleic acid hybridization techniques such as Northern and Southern analysis, altered growth capabilities on a particular substrate or in the presence of a particular substrate, a chemical compound, a selection agent and the like. In some cases, immunohistochemistry and biochemical, techniques can be used to determine if a cell contains a particular nucleic acid by detecting the expression of the encoded polypeptide. For example, an antibody having specificity for an encoded enzyme can be used to determine whether or not a particular yeast cell contains that encoded enzyme. Further, biochemical techniques can be used to determine if a cell contains a particular nucleic acid molecule encoding an enzymatic polypeptide by detecting a product produced as a result of the expression of the enzymatic polypeptide. For example, transforming a cell with a vector encoding acetolactate synthase and detecting increased acetolactate concentrations compared to a cell without the vector indicates that the vector is both present and that the gene product is active. Methods for detecting specific enzymatic activities or the presence of particular products are well known to those skilled in the art. For example, the presence of acetolactate can be determined as described by Hugenholtz and Starrenburg, 1992, Appl. Micro. Biot. 38:17-22.
Increase of Enzymatic Activity
Yeast microorganisms of the invention may be further engineered to have increased activity of enzymes. The term “increased” as used herein with respect to a particular enzymatic activity refers to a higher level of enzymatic activity than that measured in a comparable yeast cell of the same species. For example, overexpression of a specific enzyme can lead to an increased level of activity in the cells for that enzyme. Increased activities for enzymes involved in glycolysis or the isobutanol pathway would result in increased productivity and yield of isobutanol.
Methods to increase enzymatic activity are known to those skilled in the art. Such techniques may include increasing the expression of the enzyme by increased copy number and/or use of a strong promoter, introduction of mutations to relieve negative regulation of the enzyme, introduction of specific mutations to increase specific activity and/or decrease the Km for the substrate, or by directed evolution. See, e.g., Methods in Molecular Biology (vol. 231), ed. Arnold and Georgiou, Humana Press (2003).
Microorganism Characterized by Producing Isobutanol at High Yield
For a biocatalyst to produce isobutanol most economically, it is desired to produce a high yield. Preferably, the only product produced is isobutanol. Extra products lead to a reduction in product yield and an increase in capital and operating costs, particularly if the extra products have little or no value. Extra products also require additional capital and operating costs to separate these products from isobutanol.
The microorganism may convert one or more carbon sources derived from biomass into isobutanol with a yield of greater than 5% of theoretical. In one embodiment, the yield is greater than 10%. In one embodiment, the yield is greater than 50% of theoretical. In one embodiment, the yield is greater than 60% of theoretical. In another embodiment, the yield is greater than 70% of theoretical. In yet another embodiment, the yield is greater than 80% of theoretical. In yet another embodiment, the yield is greater than 85% of theoretical. In yet another embodiment, the yield is greater than 90% of theoretical. In yet another embodiment, the yield is greater than 95% of theoretical. In still another embodiment, the yield is greater than 97.5% of theoretical.
More specifically, the microorganism converts glucose, which can be derived from biomass into isobutanol with a yield of greater than 5% of theoretical. In one embodiment, the yield is greater than 10% of theoretical. In one embodiment, the yield is greater than 50% of theoretical. In one embodiment the yield is greater than 60% of theoretical. In another embodiment, the yield is greater than 70% of theoretical. In yet another embodiment, the yield is greater than 80% of theoretical. In yet another embodiment, the yield is greater than 85% of theoretical. In yet another embodiment the yield is greater than 90% of theoretical. In yet another embodiment, the yield is greater than 95% of theoretical. In still another embodiment, the yield is greater than 97.5% of theoretical
Microorganism Characterized by Production of Isobutanol from Pyruvate Via an Overexpressed Isobutanol Pathway and a Pdc-Minus and Gpd-Minus Phenotype
In yeast, the conversion of pyruvate to acetaldehyde is a major drain on the pyruvate pool, and, hence, a major source of competition with the isobutanol pathway. This reaction is catalyzed by the pyruvate decarboxylase (PDC) enzyme. Reduction of this enzymatic activity in the yeast microorganism results in an increased availability of pyruvate and reducing equivalents to the isobutanol pathway and may improve isobutanol production and yield in a yeast microorganism that expresses a pyruvate-dependent isobutanol pathway.
Reduction of PDC activity can be accomplished by 1) mutation or deletion of a positive transcriptional regulator for the structural genes encoding for PDC or 2) mutation or deletion of all PDC genes in a given organism. The term “transcriptional regulator” can specify a protein or nucleic acid that works in trans to increase or to decrease the transcription of a different locus in the genome. For example, in S. cerevisiae, the PDC2 gene, which encodes for a positive transcriptional regulator of PDC1,5,6 genes can be deleted; a S. cerevisiae in which the PDC2 gene is deleted is reported to have only  ̃10% of wildtype PDC activity (Hohmann, 1993, Mol Gen Genet 241:657-66). Alternatively, for example, all structural genes for PDC (e.g. in S. cerevisiae, PDC1, PDC5, and PDC6, or in K. lactis, PDC1) are deleted.
Crabtree-positive yeast strains such as S. cerevisiae strain that contains disruptions in all three of the PDC alleles no longer produce ethanol by fermentation. However, a downstream product of the reaction catalyzed by PDC, acetyl-CoA, is needed for anabolic production of necessary molecules. Therefore, the Pdc− mutant is unable to grow solely on glucose, and requires a two-carbon carbon source, either ethanol or acetate, to synthesize acetyl-CoA (Flikweert et al., 1999, FEMS Microbiol Lett. 174: 73-9; and van Maris et al., 2004, Appl. Environ Microbiol. 70: 159-66).
Thus, in an embodiment, such a Crabtree-positive yeast strain may be evolved to generate variants of the PDC mutant yeast that do not have the requirement for a two-carbon molecule and has a growth rate similar to wild type on glucose. Any method, including chemostat evolution or serial dilution may be utilized to generate variants of strains with deletion of three PDC alleles that can grow on glucose as the sole carbon source at a rate similar to wild type (van Maris et al., 2004, Appl Envir Micro 70: 159-66).
Another byproduct that would decrease yield of isobutanol is glycerol. Glycerol is produced by 1) the reduction of the glycolysis intermediate, dihydroxyacetone phosphate (DHAP), to glycerol-3-phosphate (G3P) via the oxidation of NADH to NAD+ by Glycerol-3-phosphate dehydrogenase (GPD) followed by 2) the dephosphorylation of glycerol-3-phosphate to glycerol by glycerol-3-phosphatase (GPP). Production of glycerol results in loss of carbons as well as reducing equivalents. Reduction of GPD activity would increase yield of isobutanol. Reduction of GPD activity in addition to PDC activity would further increase yield of isobutanol. Reduction of glycerol production has been reported to increase yield of ethanol production (Nissen et al., 2000, Yeast 16, 463-74; Nevoigt et al., Method of modifying a yeast cell for the production of ethanol, WO/2009/056984). Disruption of this pathway has also been reported to increase yield of lactate in a yeast engineered to produce lactate instead of ethanol (Dundon et al., Yeast cells having disrupted pathway from dihydroxyacetone phosphate to glycerol, US 2009/0053782).
In one embodiment, the microorganism is a Crabtree-positive yeast with reduced or no GPD activity. In another embodiment, the microorganism is a crabtree positive yeast with reduced or no GPD activity, and expresses an isobutanol biosynthetic pathway and produces isobutanol. In yet another embodiment, the microorganism is a Crabtree-positive yeast with reduced or no GPD activity and with reduced or no PDC activity. In another embodiment, the microorganism is a crabtree positive yeast with reduced or no GPD activity, with reduced or no PDC activity, and expresses an isobutanol biosynthetic pathway and produces isobutanol.
In another embodiment, the microorganism is a Crabtree-negative yeast with reduced or no GPD activity. In another embodiment, the microorganism is a Crabtree-negative yeast with reduced or no GPD activity, expresses the isobutanol biosynthetic pathway and produces isobutanol. In yet another embodiment, the microorganism is a Crabtree-negative yeast with reduced or no GPD activity and with reduced or no PDC activity. In another embodiment, the microorganism is a Crabtree-negative yeast with reduced or no GPD activity, with reduced or no PDC activity, expresses an isobutanol biosynthetic pathway and produces isobutanol.
PDC-minus/GPD-minus yeast production strains are described in co-pending applications U.S. Ser. No. 12/343,375 (published as US 2009/0226991), U.S. Ser. No. 12/696,645, and U.S. Ser. No. 12/820,505, which claim priority to U.S. Provisional Application 61/016,483, all of which are herein incorporated by reference in their entireties for all purposes.
Method of Using Microorganism for High-Yield Isobutanol Fermentation
In a method to produce isobutanol from a carbon source at high yield, the yeast microorganism is cultured in an appropriate culture medium containing a carbon source.
Another exemplary embodiment provides a method for producing isobutanol comprising a recombinant yeast microorganism of the invention in a suitable culture medium containing a carbon source that can be converted to isobutanol by the yeast microorganism of the invention.
In certain embodiments, the method further includes isolating isobutanol from the culture medium. For example, isobutanol may be isolated from the culture medium by any method known to those skilled in the art, such as distillation, pervaporation, or liquid-liquid extraction, including methods disclosed in co-pending applications U.S. Ser. No. 12/342,992 (published as US 2009/0171129) and PCT/US08/88187 (published as WO/2009/086391), which are herein incorporated by reference in their entireties for all purposes.
This invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figure and the Sequence Listing, are incorporated herein by reference for all purposes.
EXAMPLES
General Methods







TABLE 1







Amino acid sequences disclosed herein.








SEQ ID NO
Protein, Accession No.











1
E. coli IlvC, NP_418222


2
S. cerevisiae Ilv5, NP_013459


3
Oryza sativa KARI, NP_001056384


4
Methanococcus maripaludis KARI, YP_001097443


5
Acidiphilium cryptum KARI, YP_001235669


6
Chlamydomonas reinhardtii KARI, XP_001702649


7
Picrophilus torridus KARI, YP_023851


8
Zymomonas mobilis KARI, YP_162876


9
c-myc epitope tag


10
Thermotoga petrophila RKU-1 dihydroxyacid dehydratase (DHAD),



YP_001243973.1


11
Victivailis vadensis ATCC BAA-548 dihydroxyacid dehydratase (DHAD),



ZP_01924101.1


12
Termite group 1 bacterium phylotype Rs-D17 dihydroxyacid dehydratase (DHAD),



YP_001956631.1


13
Yarrowia lipolytica dihydroxyacid dehydratase (DHAD), XP_502180.2


14
Francisella tularensis subsp. tularensis



WY96-3418 dihydroxyacid dehydratase (DHAD), YP_001122023.1


15
Arabidopsis thaliana dihydroxyacid dehydratase (DHAD), AAK64025.1


16
Candidatus Koribacter versatilis Ellin345 dihydroxyacid dehydratase (DHAD),



YP_592184.1 (Acidobacter)


17
Gramella forsetii KT0803 dihydroxyacid dehydratase (DHAD), YP_862145.1


18
Lactococcus lactis subsp. lactis Il1403 dihydroxyacid dehydratase (DHAD),



NP_267379.1


19
Saccharopolyspora erythraea NRRL 2338 dihydroxyacid dehydratase (DHAD),



YP_001103528.2


20
Saccharomyces cerevisiae Ilv3, NP_012550.1


21
Piromyces sp E2 ilvD


22
Ralstonia eutropha JMP134 ilvD, YP_298150.1


23
Chromohalobacter salexigens ilvD, YP_573197.1


24
Picrophilus torridus DSM9790 ilvD, YP_024215.1


25
Sulfolobus tokodaii str. 7 dihydroxyacid dehydratase (DHAD), NP_378168.1


26
Saccharomyces cerevisiae Ilv3ΔN


27
P(I/L))XXXGX(I/L)XIL (conserved motif described in Example 17)


28
PIKXXGX(I/L)XIL (conserved motif described in Example 17)
















TABLE 2







Nucleic acid sequences disclosed herein.








SEQ ID NO
Gene, Accession No.











87
Lactococcus lactis subsp. lactis Il1403 (LI_ilvD)


88
Saccharomyces cerevisiae ILV3 (ScILV3(FL))


89
Saccharomyces cerevisiae ILV3ΔN (ScILV3ΔN)


90
GramsIla forsetii KT0803 (Gf_ilvD)


91
Saccharopolyspora erythraea NRRL 2338 (Se_ilvD)


92
Candidatus Koribacter versatilis Ellin345 ilvD (Acidobacter)


93
Piromcyes sp E2 ilvD (Piromyces ilvD)


94
Ralstonia eutropha JMP134 ilvD, (Re_ilvD)


95
Chromohalobacter salexigens ilvD, (Cs_ilvD)


96
Picrophilus torridus DSM9790 ilvD, (Pt_ilvD)


97
Sulfolobus tokodaii str. 7 ilvD, (St_ilvD)


98
E. coli ilvCQ110V, ( Ec_itvC(Q110V))


99
Lactococcus lactic kivD, (LI_kivD)


100
S. cerevisiae ILV5, (ScILV5)









Determination of optical density. The optical density of the yeast cultures is determined at 600 nm using a DU 800 spectrophotometer (Beckman-Coulter, Fullerton, Calif., USA). Samples are diluted as necessary to yield an optical density of between 0.1 and 0.8.
Gas Chromatography. Analysis of volatile organic compounds, including ethanol and isobutanol was performed on a HP 5890 gas chromatograph fitted with an HP 7673 Autosampler, a DB-FFAP column (J&W; 30 m length, 0.32 mm ID, 0.25_μM film thickness) or equivalent connected to a flame ionization detector (FID). The temperature program was as follows: 200° C. for the injector, 300° C. for the detector, 50° C. oven for 1 minute, 31° C./minute gradient to 140° C., and then hold for 2.5 min. Analysis was performed using authentic standards (>99%, obtained from Sigma-Aldrich), and a 5-point calibration curve with 1-pentanol as the internal standard.
High Performance Liquid Chromatography for quantitative analysis of glucose and organic acids. Analysis of glucose and organic acids was performed on a HP-1100 High Performance Liquid Chromatography system equipped with an Aminex HPX-87H Ion Exclusion column (Bio-Rad, 300×7.8 mm) or equivalent and an H+ cation guard column (Bio-Rad) or equivalent. Organic acids were detected using an HP-1100 UV detector (210 nm, 8 nm 360 nm reference) while glucose was detected using an HP-1100 refractive index detector. The column temperature was 60° C. This method was Isocratic with 0.008 N sulfuric acid in water as the mobile phase. Flow was set at 1 mL/min. Injection volume was 20 μL and the run time was 30 minutes.
High Performance Liquid Chromatography for quantitative analysis of ketoisovalerate and isobutyraldehyde. Analysis of the DNPH derivatives of ketoisovalerate and isobutyraldehyde was performed on a HP-1100 High Performance Liquid Chromatography system equipped with a Hewlett Packard 1200 HPLC stack column (Agilent Eclipse XDB-18, 150×4.0 mm; 5 μm particles [P/N #993967-902] and C18 Guard cartridge). The analytes were detected using an HP-1100 UV detector at 360 nm The column temperature was 50° C. This method was isocratic with 0.1% H3PO4 and 70% acetonitrile in water as mobile phase. Flow was set at 3 mL/min. Injection size was 10 μL and the run time was 2 minutes.
Molecular biology and bacterial cell culture. Standard molecular biology methods for cloning and plasmid construction are generally used, unless otherwise noted (Sambrook, J., Russel, D. W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press).
Standard recombinant DNA and molecular biology techniques used in the Examples are well known in the art and are described by Sambrook, J., Russel, D. W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press; and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987).
General materials and methods suitable for the routine maintenance and growth of bacterial cultures are well known in the art. Techniques suitable for use in the following examples may be found as set out in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds.), American Society for Microbiology, Washington, D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, Mass. (1989).
Yeast transformations—S. cerevisiae. S. cerevisiae strains were transformed by the Lithium Acetate method (Gietz et al., Nucleic Acids Res. 27:69-74 (1992): Cells from 50 mL YPD cultures (YPGal for valine auxotrophs) were collected by centrifugation (2700 rcf, 2 minutes, 25° C.) once the cultures reached an OD600 of 1.0. The cells were washed cells with 50 mL sterile water and collected by centrifugation at 2700 rcf for 2 minutes at 25° C. The cells were washed again with 25 mL sterile water and collected cells by centrifugation at 2700 rcf for 2 minutes at 25° C. The cells were resuspended in 1 mL of 100 mM lithium acetate and transferred to a 1.5 mL eppendorf tube. The cells were collected cells by centrifugation for 20 sec at 18,000 rcf, 25° C. The cells were resuspended cells in a volume of 100 mM lithium acetate that was approximately 4× the volume of the cell pellet. A mixture of DNA (final volume of 15 μl with sterile water), 72 μl 50% PEG, 10 μl 1 M lithium acetate, and 3 μl denatured salmon sperm DNA was prepared for each transformation. In a 1.5 mL tube, 15 μl of the cell suspension was added to the DNA mixture (85 μl), and the transformation suspension was vortexed with 5 short pulses. The transformation was incubated at 30 minutes at 30° C., followed by incubation for 22 minutes at 42° C. The cells were collected by centrifugation for 20 sec at 18,000 rcf, 25° C. The cells were resuspended in 100 μl SOS (1 M sorbitol, 34% (v/v) YP (1% yeast extract, 2% peptone), 6.5 mM CaCl2) or 100 μl YP (1% yeast extract, 2% peptone) and spread over an appropriate selective plate.
Yeast transformations—K. lactis. K. lactis cells were transformed according to a slightly modified version of the protocol as described by Kooistra et al., Yeast 21: 781-792 (2004). Saturated overnight-grown cultures of K. lactis cells were diluted 1:50 into 100 mL YPD and were placed in 30° C. shaker (250 rpm) and grown for 4-5 hours until the culture reached an OD600 of 0.3-0.5. Cells were collected by centrifugation (2 min, 3000×g) and washed with 50 ml cold sterile EB (electroporation buffer; 10 mM Tris-HCl, pH 7.5, 270 mM sucrose, 1 mM MgCl2) at 4° C. Cells were resuspended in 50 mL YPD that contained 25 mM DTT and 20 mM HEPES, pH 8.0 Cells were transferred back into flasks used to grow cells and incubated in 30° C. incubator (without shaking) for 30 minutes. Cells were then collected by centrifugation (2 minutes, 3000×g) and washed with 10 mL ice-cold sterile EB, as above. Cells were then resuspended using one cell pellet volume of ice-cold sterile EB. Sixty microliters of cells were mixed with plasmid DNA and incubated on ice for 15 minutes. For targeted integrations, or transformation of linear DNA, approximately 200-400 ng of non-specific, short (50-500 bp) linear DNA fragments were added to 300-400 ng of the linearized integrating DNA construct. This DNA was either provided by gel-purified AluI-digested salmon sperm DNA, or a mixture of annealed primers 35+36 (yielding a  ̃85 bp linear duplex fragment). Cells were transferred cells to a chilled electroporation (2 mm) cuvette and pulsed using a BioRad Gene Pulser at 1 kV, 400Ω, and 25 uF. The cell suspension was immediately transferred to a 14 mL round-bottom Falcon tube with 1 mL room temperature YPD and allowed to incubate vertically at 30° C., 225 RPM for at 6-18 h. Cells were collected in an 1.7 mL by centrifugation for 10 seconds at maximum speed, and resuspended with 150 μL YPD before being spread onto appropriate selection plates.
Yeast colony PCR with FailSafe® PCR System (EPICENTRE® Biotechnologies, Madison, Wis.; Catalog #FS99250): Cells from each colony were added to 20 μl of colony PCR mix (per reaction mix contains 6.8 μl water, 1.5 μl of each primer, 0.2 μl of FailSafe PCR Enzyme Mix and 10 μl 2× FailSafe Master Mix). Unless otherwise noted, 2× FailSafe Master Mix E was used. The PCR reactions were incubated in a thermocycler using the following touchdown PCR conditions: 1 cycle of 94° C.×2 min, 10 cycles of 94° C.×20 s, 63°-54° C.×20 s (decrease 1° C. per cycle), 72° C.×60 s, 40 cycles of 94° C.×20 s, 53° C.×20 s, 72° C.×60 s and 1 cycle of 72° C.×5 min.
Zymoclean Gel DNA Recovery Kit (Zymo Research, Orange, Calif.; Catalog #D4002) Protocol: DNA fragments were recovered from agarose gels according to manufacturer's protocol.
Zymo Research DNA Clean and Concentrator Kit (Zymo Research, Orange, Calif.; Catalog #D4004) Protocol: DNA fragments were purified according to manufacturer's protocol.
Preparation of cell lysates for in vitro enzyme assays. To grow cultures for cell lysates, triplicate independent cultures of the desired strain were grown overnight in 3 mL of the appropriate medium at 30° C., 250 rpm. The following day, the overnight cultures were diluted into 50 mL fresh medium in 250 mL baffle-bottomed Erlenmeyer flasks and incubated at 30° C. at 250 rpm. Cells were grown for at least 4 generations and the cultures were harvested in mid log phase (OD600 of 1-3) The cells of each culture were collected by centrifugation (2700×g, 5 min, 4° C.). The cell pellets were washed by resuspending in 20 mL of ice cold water. The cells were centrifuged at 2700×g, 4° C. for 5 min, All supernatant was removed from each tube and the tubes were frozen at −80° C. until use.
Lysates were prepared by thawing each cell pellet on ice and preparing a 20% (w/v) cell suspension in lysis buffer. The lysis buffer was varied for each enzyme assay and consisted of: 0.1 M Tris-HCl pH 8.0, 5 mM MgSO4, for DHAD assays, 50 mM potassium phosphate buffer pH 6.0, 1 mM MgSO4 for ALS assays, 250 mM KPO4 pH 7.5, 10 mM MgCl2 for KARI assays, 50 mM NaHPO4, 5 mM MgCl2, for KIVD assays. 10 μL of Yeast/Fungal. Protease Arrest solution (G Biosciences, catalog #788-333) per 1 mL of lysis buffer was used. 800 microliters of cell suspension were added to 1 mL of 0.5 mm glass beads that had been placed in a chilled 1.5 mL tube. Cells were lysed by bead beating (6 rounds, 1 minute per round, 30 beats per second) with 2 minutes chilling on ice in between rounds. The tubes were then centrifuged (20,000×g, 15 min) to pellet debris and the supernatants (cell lysates) were retained in fresh tubes on ice. The protein concentration of each lysate was measured using the BioRad Bradford protein assay reagent (BioRad, Hercules, Calif.) according to manufacturer's instructions.
Preparation of fractionated lysates from S. cerevisiae strains for in vitro enzyme assays. To grow cultures for cell fractionated cell lysates, triplicate independent cultures of the desired strain were grown overnight in 3 mL of the appropriate medium at 30° C., 250 rpm. The following day, the overnight cultures were used to inoculate 1 L cultures of each strain which were grown in the appropriate medium at 30° C. at 250 rpm until they reached an OD600 of approximately 2. The cells were collected by centrifugation (1600×g, 2 min) and the culture medium was decanted. The cell pellets were resuspended in 50 mL sterile deionized water, collected by centrifugation (1600×g, 2 min), and the supernatant was discarded.
To obtain spheroplasts, the cell pellets were resuspended in 0.1M Tris-SO4, pH 9.3, to a final concentration of 0.1 g/mL, and DTT was added to a final concentration of 10 mM. Cells were incubated with gentle (60 rev/min) agitation on an orbital shaker for 20 min at 30° C., and the cells were then collect by centrifugation (1600×g, 2 min) and the supernatant discarded. Each cell pellet was resuspended in spheroplasting buffer, which consists of (final concentrations): 1.2M sorbitol (Amresco, catalog #0691), 20 mM potassium phosphate pH 7.4) and then collected by centrifugation (1600×g, 10 min). Each cell pellet was resuspended in spheroplasting buffer to a final concentration of 01 g cells/mL in a 500 mL centrifuge bottle, and 50 mg of Zymolyase 20T (Seikagaku Biobusiness, Code#120491) was added to each cell suspension. The suspensions were incubated overnight (approximately 16 hrs) at 30° C. with gentle agitation (60 rev/min) on an orbital shaker. The efficacy of spheroplasting was ascertained by diluting an aliquot of each cell suspension 1:10 in either sterile water or in spheroplasting buffer, and comparing the aliquots microscopically (under 40× magnification). In all cases, >90% of the water-diluted cells lysed, indicating efficient spheroplasting. The spheroplasts were centrifuged (3000×g, 10 min, 20° C.), and the supernatant was discarded. Each cell pellet was resuspended in 50 mL spheroplast buffer without Zymolyase, and cells were collected by centrifugation (3000×g, 10 min, 20° C.).
To fractionate spheroplasts, the cells were resuspended to a final concentration of 0.5 g/mL in ice cold mitochondrial isolation buffer (MIB), consisting of (final concentration): 0.6M D-mannitol (BD Difco Cat#217020), 20 mM HEPES-KOH, pH 7.4. For each 1 mL of resulting cell suspension, 0.01 mL of Yeast/Fungal Protease Arrest solution (G Biosciences, catalog #788-333) was added. The cell suspension was subjected to 35 strokes of a Dounce homogenizer with the B (tight) pestle, and the resulting cell suspension was centrifuged (2500×g, 10 min, 4° C.) to collect cell debris and unbroken cells and spheroplasts. Following centrifugation, 2 mL of each sample (1 mL of the pGV1900 transformed cells) were saved in a 2 mL centrifuge tube on ice and designated the “W” (for Whole cell extract) fraction, while the remaining supernatant was transferred to a clean, ice-cold 35 mL Oakridge screw-cap tube and centrifuged (12,000×g, 20 min, 4° C.) to pellet mitochondria and other organellar structures. Following centrifugation, 5 mL of each resulting supernatant was transferred to a clean tube on ice, being careful to avoid the small, loose pellet, and labelled the “S” (soluble cytosol) fraction. The resulting pellets were resuspended in MIB containing Protease Arrest solution, and were labelled the “P” (“pellet”) fractions. The BioRad Protein Assay reagent (BioRad, Hercules, Calif.) was used according to manufacturer's instructions to determine the protein concentration of each fraction.
Preparation of fractionated lysates from K. lactis strains for in vitro enzyme assays Cultures (20 mL YPD) were inoculated with yeast cells (GEVO1742 and GEVO1829) and incubated at 30° C. while shaking at 250 RPM until they reached late-log to stationary phase (OD600 of approximately 10). Cells from the 20 mL cultures were used to inoculate a 250 mL YPD culture at an OD800 of approximately 0.2. The cultures were incubated at 30° C. while shaking at 250 RPM until they reached mid-log(OD600 ̃2).
To prepare spheroplasts, the cells were collected in 500 mL bottles at 5000×g for 5 minutes at room temperature. The pellets were resuspended with 8 mL Spheroplasting Buffer A (25 mM potassium phosphate (pH 7.5), 1 mM MgCl2, 1 mM EDTA, 1.25 mM TPP, 1 mM DTT) without sorbitol and transferred to pre-weighed 50 mL tubes. The cells were collected at 1600×g for 5 minutes at room temperature. The cells were resuspended with 8 mL of Spheroplasting Buffer A with 2.5 M Sorbitol (Amresco Code#0691) and protease inhibitor (G Biosciences Yeast/Fungal ProteaseArrestTM (Catalog #788-333)). Approximately 5 mg of Zymolyase 20T Zymolyase 20T (Seikagaku Biobusiness Code#120491) was added to each cell suspension. The suspensions were incubated at 30° C. with gentle agitation (e.g. 50 RPM), with the tube on its side for good mixing, for 1-2 hours. The efficiency of formation of spheroplasts was verified by dilution of the spheroplast suspension 1:10 into Spheroplasting Buffer A with 2.5 M sorbitol and 1:10 in water. Spheroplasts should remain intact when diluted into the buffer but appear fuzzy or completely disappear when diluted into water. The spheroplasts were collected at 1600×g for 7 minutes at 4° C. The spheroplasts were gently washed with 2 mL of Spheroplasting Buffer A with 2.5 M sorbitol and protease inhibitor, and collected at 1600×g for 7 minutes at 4° C. The spheroplasts were resuspended in 2 mL of Spheroplasting Buffer A with 2.5 M sorbitol and protease inhibitor.
To fractionate the spheroplasts, 8 mL of Spheroplasting Buffer A with 0.2 M sorbitol and protease inhibitor was slowly added to the cell suspension, bringing the final concentration of Sorbitol to 0.66 M. The spheroplasts were broken with 10 strokes using a B (tight fitting) pestle in a 15 mL Dounce homogenizer (Bellco Glass, Inc. Cat#1984-10015) on ice. The homogenate was transferred to a 50 mL tube, and the cell debris was collected by centrifugation at 4° C. for 10 minutes at 1600×g. The supernatant was transferred to a 15 mL tube with a pipette. This supernatant is the “W” fraction. 5 mL of this “W” fraction was transferred to a 35 mL Oakridge tube and centrifuged at 48,000×g for 20 minutes at 4° C. The resulting supernatant was transferred to a 15 mL tube and labeled “S.” The pellet was resuspended in 5 mL of Spheroplasting Buffer A with 0.66 M Sorbitol and protease inhibitor and labeled “P.” All fractions were stored on ice at 4° C. while in use. The BioRad Protein Assay reagant (BioRad, Hercules, Calif.) was used according to manufacturer's instructions to determine the protein concentration of each fraction.
ALS Assay. Cell lysates were prepared and protein concentrations were determined as described above. The colorimetric ALS Assay (FAD-independent) performed here was based on the assay described in Hugenholtz, J. and Starrenburg, J. C., Appl. Microbiol. Biotechnol. (1992) 38:17-22. Reaction buffer was prepared by mixing 900 μl 1M potassium phosphate buffer pH 6.0, 180 μl 100 mM MgSO4, 180 μl 100 mM TPP, 3.96 ml 500 mM pyruvate and 12.78 ml water. For the no substrate control, the volume of pyruvate was replaced with water. Lysates were prepared at a final protein concentration of 2 μg/μl in Spheroplasting Buffer A with 0.66 M sorbitol. To 900 μL ALS Buffer, 100 μL of lysate was added and incubated at 30° C. for 30 min. Acetoin standards were also prepared at concentrations of 2 mM, 1 mM, 0.5 mM, and 0 mM. From each sample and standard, 175 μL was transferred to a fresh 1.5 mL tube. To each sample and standard was added 25 μL 35% (v/v) H2SO4, and all were incubated at 37° C. for 30 mins. After the incubation, the following were added in order, to each standard and sample, with the solutions being mixed by vortexing in between each addition: 50 μL 50% (w/v) NaOH, 50 μL 0.5% creatine, and 50 μL 5% 1-naphthol (in 2.5N NaOH). The samples and standards were incubated at room temperature for 1 hour, being mixed by vortexing every 15 minutes. To a 96 well, half-area, UV-Star, transparent, flat-bottom plate (Catalog #675801, Greiner Bio One, Frickenhausen, Germany), 100 uL of each sample or standard was transferred, and the samples were analyzed by a plate reader by measuring absorbance at 530 nm.
KARI Assay. Cell lysates were prepared and protein concentrations were determined as described above. Acetolactate substrate was made by mixing 50 μl of ethyl-2 acetoxy-2-methyl-acetoacetate with 990 μl of water. Then 10 μl of 2 N NaOH was sequentially added, with vortex mixing between additions, until 260 μl of NaOH was added. The acetolactate was agitated at room temperature for 20 min and then held on ice. NADPH was prepared in 0.01N NaOH (to improve stability) to a concentration of 50 mM. The concentration was determined by reading the OD of a diluted sample at 340 nm in a spectrophotometer and using the molar extinction coefficient of 6.22 M−1 cm−1 to calculate the actual concentration (the OD340 of a 100 μM solution of NAD(P)H should be 0.622). Three buffers were prepared and held on ice. Reaction buffer contained 250 mM KPO4 pH 7.5, 10 mM MgCl2, 1 mM DTT, 10 mM acetolactate, and 0.2 mM NADPH. No substrate buffer contained everything except the acetolactate. No NAD(P)H buffer contained everything except the NADPH. Reactions were performed in triplicate using 10 μl of cell extract with 90 μl of reaction buffer in a 96-well plate in a SpectraMax 340PC multi-plate reader (Molecular Devices, Sunnyvale, Calif.). The reaction was followed at 340 nm by measuring a kinetic curve for 5 minutes, with OD readings taken every 10 seconds. The reactions were performed at 30° C. The reactions were performed in complete, no substrate, and no NAD(P)H buffers. The Vmax for each extract was determined after subtracting the background reading of the no substrate control from the reading in complete buffer.
DHAD Assay. Cell lysates were prepared and protein concentrations were determined as described above. The DHAD activity of each lysate was ascertained as follows. In a fresh 1.5 mL centrifuge tube, 50 μL of each lysate was mixed with 50 μL of 0.1M 2,3-dihydroxyisovalerate (DHIV), 25 μL of 0.1M MgSO4, and 375 μL of 0.05M Tris-HCl pH 8.0, and the mixture was incubated for 30 min at 35° C. Each tube was then heated to 95° C. for 5 min to inactivate any enzymatic activity, and the solution was centrifuged (16,000×g for 5 min) to pellet insoluble debris. To prepare samples for analysis, 100 μL of each reaction were mixed with 100 μL of a solution consisting of 4 parts 15 mM dinitrophenyl hydrazine (DNPH) in acetonitrile with 1 part 50 mM citric acid, pH 3.0, and the mixture was heated to 70° C. for 30 min in a thermocycler. The solution was then analyzed by HPLC as described above in General Methods to quantitate the concentration of ketoisovalerate (KIV) present in the sample.
KIVD Assay. Cell lysates were prepared and protein concentrations were determined as described above. KIVD Assay buffer, containing 1 Roche Protease Inhibitor tablet per 5 mL buffer, was added to each cell pellet to create a 20% (w/v) cell suspension. The KIVD assay buffer was prepared at a final concentration of 0.05 M NaHPO4*H2O, 5 mM MgCl2*8H2O, and 1.5 mM Thiamin pyrophosphate chloride. The reaction substrate, α-keto-isovalerate (3-methyl-2-oxobutanoic acid, Acros Organics), was added where appropriate at 30 mM. Lysates were diluted in reaction buffer at a final protein concentration of 0.1 μg/μL. To 1.5 mL tubes, 50 μL of lysate (5 μg of protein) was mixed with: 200 μL of reaction buffer with or without substrate. The reactions were incubated at 37° C. for 20 minutes, and the reactions were immediately filtered through a 2 μm filter plate. The filtered samples were diluted 1:10 in water, and 100 μL of the 1:10 dilution was mixed with 100 μL of derivatization reagent in a 0.2 ml thin-wall PCR tubes. Derivatization reagent was prepared by mixing 4 ml of 2,4-Dinitrophenyl Hydrazine (DNPH) in 15 mM in HPLC-grade Acetonitrile with 1 ml 50 mM Citric Acid Buffer, pH 3. The samples were incubated at 70° C. for 30 minutes. The samples were analyzed by HPLC.
ADH Assay. Cell lysates were prepared and protein concentrations were determined as described above. Assays (set up in triplicate for each lysate) contained 10 μL of each lysate (or an appropriate dilution of each lysate) plus 90 μl of reaction buffer, which consisted of (final concentrations present in 1× reaction buffer): 0.1M Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.2 mM NADH (or NADPH, where indicated; each diluted from a 4.4 mM spectrophotometrically-confirmed stock), and 11 mM isobutyraldehyde. Where indicated, as controls a parallel set of assay reactions were set up using reaction buffer lacking isobutyraldehyde and/or NAD(P)H, as indicated. For experiments measuring the acetaldehyde-dependent oxidation of NAD(P)H, reaction buffer was prepared in which acetaldehyde was substituted for isobutyraldehyde. In these cases, the reaction buffer contained at least 11 mM acetaldehyde, although the exact amount present is an estimate due to the inherent difficulties of pipetting acetaldehyde solution. Finally, in some cases a parallel set of reactions lacking yeast cell lysate was included as a negative control. After being added (using a multi-channel pipet) to the wells of a 96-well plate, the reactions were immediately placed into a plate reader that had been pre-warmed to 30° C., and the absorbance at 340 nm was measured every 12 seconds over a period of 300 seconds. Kinetic parameters were computed from assays with linear slopes (where necessary, assays were repeated with appropriate dilutions to obtain linear NAD(P)H consumption curves).
Composition of Culture Media
Drugs: When indicated, G418 (Calbiochem, Gibbstown, N.J.) was added at 0.2 g/L, Phleomycin (InvivoGen, San Diego, Calif.) was added at 7.5 mg/L, Hygromycin (InvivoGen, San Diego, Calif.) was added at 0.2 g/L, and 5-fluoro-orotic acid (FOA; Toronto Research Chemicals, North York, Ontario, Canada) was added at 1 g/L.
YP: 1% (w/v) yeast extract, 2% (w/v) peptone.
YPD: YP containing 2% (w/v) glucose unless otherwise noted,
YPGal: YP containing 2% (w/v) galactose
YPE: YP containing 2% (w/v) Ethanol.
SC media: 6.7 g/L DifcoTM Yeast Nitrogen Base, 14 g/L SigmaTM Synthetic Dropout Media supplement (includes amino acids and nutrients excluding histidine, tryptophan, uracil, and leucine; Sigma-Aldrich, St. Louis, Mo.), 0.076 g/L histidine, 0.076 g/L tryptophan, 0.380 g/L leucine, and 0.076 g/L uracil. Drop-out versions of SC media is made by omitting one or more of histidine (H), tryptophan (W), leucine (L), or uracil (U or Ura). When indicated, SC media are supplemented with additional isoleucine (9xI; 0.684 g/L), valine (9xV; 0.684 g/L) or both isoleucine and valine (9xIV). SCD is SC containing 2% (w/v) glucose unless otherwise noted, SCGal is SC containing 2% (w/v) galactose and SCE is SC containing 2% (w/v) ethanol. For example, SCD-Ura+9xIV would be composed of 6.7 g/L DifcoTM Yeast Nitrogen Base, 14 g/L SigmaTM Synthetic Dropout Media supplement (includes amino acids and nutrients excluding histidine, tryptophan, uracil, and leucine), 0.076 g/L histidine, 0.076 g/L tryptophan, 0.380 g/L leucine, 0.684 g/L isoleucine, 0.684 g/L valine, and 20 g/L glucose.
SCD-V+9xI: 6.7 g/L DifcoTM Yeast Nitrogen Base, 0.076 g/L Adenine hemisulfate, 0.076 g/L Alanine 0.076 g/L, Arginine hydrochloride, 0.076 g/L Asparagine monohydrate, 0.076 g/L Aspartic acid, 0.076 g/L Cysteine hydrochloride monohydrate, 0.076 g/L Glutamic acid monosodium salt, 0.076 g/L Glutamine, 0.076 g/L Glycine, 0.076 g/L myo-Inositol, 0.76 g/L Isoleucine, 0.076 g/L Lysine monohydrochloride, 0.076 g/L Methionine, 0.008 g/L p-Aminobenzoic acid potassium salt, 0.076 g/L Phenylalanine, 0.076 g/L Proline, 0.076 g/L Serine, 0.076 g/L Threonine, 0.076 g/L Tyrosine disodium salt, and 20 g/L glucose.
YNB: 6.7 g/L DifcoTM Yeast Nitrogen Base supplemented with indicated nutrients as follows: histidine (H; 0.076 g/L), tryptophan (W; 0.076 g/L), leucine (L; 0.380 g/L), uracil (U or Ura; 0.076 g/L), isoleucine (I; 0.076 g/L), valine (V; 0.076 g/L), and casamino acids (CM; 10 g/L). When indicated, YNB media are supplemented with higher amounts of isoleucine (10xI=0.76 g/L), valine (10xV=0.76 g/L) or both isoleucine and valine (10xIV). YNBD is YNB containing 2% (w/v) glucose unless otherwise noted, YNBGal is YNB containing 2% (w/v) galactose and YNBE is YNB containing 2% (w/v) ethanol. For example, YNBGal+HWLU+10xI+G418 would be composed of 6.7 g/L DifcoTM Yeast Nitrogen Base, 0.076 g/L histidine, 0.076 g/L tryptophan, 0.380 g/L leucine, 0.076 g/L uracil, 0.76 g/L isoleucine, 0.2 g/L G418, and 20 g/L galactose.
Plates: Solid versions of the above described media contain 2% (w/v) agar.
Example 1
Isobutanol Pathway is Partially Cytosolic when Expressed in Yeast
The purpose of this example is to illustrate that three enzymes in the isobutanol biosynthetic pathway (acetolactate synthase, ketoisovalerate decarboxylase, and isobutanol dehydrogenase) are localized to the cytosol when expressed in yeast.







TABLE 3







Genotype of strains disclosed in Example 1.








GEVO No.
Genotype/Source





1287
K. lactis ATCC 200826 MAT α uraA1 trp1 leu2 lysA1



ade1 lac4-8 [pKD1]


1742
K. lactis ATCC 200826 MAT α uraA1 trp1 leu2 lysA1



ade1 lac4-8 [pKD1] pdc1::KanR


1829
K. lactis ATCC 200826 MAT α uraA1 trp1 leu2 lysA1



ade1 lac4-8 [pKD1] pdc1::kanR {PTDH3:Ec_ ilvC-ΔN;



PTEF1:Ec_ilvD-ΔN(codon optimized for K. lactis):



ScLEU2 integrated} {PTEF1:Ll_kivD; PTDH3ScADH7:



KmURA3 integrated} {PCUP1-1:Bs_alsS, TRP1 random



integrated}
















TABLE 4







Plasmids disclosed in Example 1.








pGV No.
Genotype





pGV1503
ScTEF1promoter-kanR bla, pUC ori (GEVO)


pGV1537
KlPDC1 promoter region + Klpdc1 3′UTR sequence,



ScTEF1promoter-kanR bla, pUC ori (GEVO)


pGV1590
TEF1 promoter:Ll-kivd (codon optimized for E. coli):



TDH3 promoter:ADH7:CYC1 terminator, Km-URA3,



1.6 micron ori, bla, pUC ori (GEVO)


pGV1726
CUP1 promoter:Bs-alsS:CYC1 terminator, TRP1, bla,



pUC-ori


pGV1727
TEF1 promoter:Ec-ilvDΔN (codon optimized for



K. lactis):TDH3 promoter:Ec-ilvCΔN:CYC1 terminator,



LEU2, bla, pUC ori (GEVO)









Plasmids

pGV1503 contains an S. cerevisiae TEF1 promoter region driving a G418-resistance gene (kanR).
pGV1537 was constructed by inserting an (AatII plus MfeI)-digested PCR product containing approximately 500 bp each of KIPDC1 5′ and 3′ untranslated regions, into (AatII plus EcoRI)-digested pGV1503. The insert was generated by SOE-PCR. First, the KIPDC1 5′ and 3′ untranslated regions were amplified from K. lactis genomic DNA by primer pairs 1006+1016 and 1017+1009, respectively. Primers 1016 and 1017 were designed to have overlapping sequences. The two fragments were then joined by PCR using primers 1006+1009.
pGV1590 is a K. lactis plasmid for expression of the L. lactis kivD and the S. cerevisiae ADH7. Expression of the L. lactis kivD is driven by the S. cerevisiae TEF1 promoter and expression of the S. cerevisiae ADH7 is driven by the S. cerevisiae TDH3 promoter. pGV1590 was generated by cloning a SalI-NotI fragment carrying the S. cerevisiae ADH7 gene into the XhoI-NotI sites of pGV1585. The S. cerevisiae ADH7 gene fragment originated as a PCR product from S. cerevisiae genomic DNA using primers 410 and 411.
pGV1726 is a yeast integration plasmid (utilizing the S. cerevisiae TRP1 gene as selection marker) for random integration (i.e. for K. lactis). This plasmid does not carry a yeast replication origin, thus is unable to replicate episomally. This plasmid also carries the B. subtilis alsS gene, whose expression is under the control of the S. cerevisiae CUP1 promoter. pGV1726 was generated by cloning a SacI-NgoMIV fragment carrying the S. cerevisiae CUP1 promoter, Bs-alsS ORF and the CYC1 terminator into the same sites of pGV1645. The vector, pGV1645, is a K. lactis expression plasmid that was used for expression of the B. subtilis alsS under the control of the K. lactis PDC1 promoter. This plasmid also carries the S. cerevisiae TRP1 gene as a selection marker and the 1.6 micron replication origin. Digestion of pGV1645 with SacI and NgoMIV removes the K. lactis PDC1 promoter, B. subtilis alsS, CYC1 terminator and the 1.6 micron origin of replication. The insert fragment carrying the S. cerevisiae CUP1 promoter, B. subtilis alsS ORF and the CYC1 terminator was obtained from pGV1649 via digestion with SacI and NgoMIV. The CUP1 promoter originated as a PCR product from S. cerevisiae genomic DNA using primers 637 and 638. The B. subtilis alsS originated as a PCR product from B. subtilis genomic DNA using primers 767 and 697.
pGV1727 is a yeast integration plasmid (utilizing the S. cerevisiae LEU2 gene as selection marker) for random integration (i.e. for K. lactis). This plasmid does not carry a yeast replication origin, thus is unable to replicate episomally. This plasmid carries the E. coli ilvCΔN and ilvCΔN genes, whose expressions are under the control of the S. cerevisiae TEF1 and TDH3 promoters respectively. The E. coli ilvDΔN is a shortened version of E. coli ilvD where the sequence coding for the first 24 amino acids, which encodes for a putative mitochondrial targeting sequence, was removed. Likewise, the E. coli ilvCΔN is a shortened version of E. coli ilvC where the sequence coding for the first 22 amino acids, which is predicted to function as a mitochondrial targeting sequence was removed. pGV1727 was generated by cloning a XhoI-NgoMIV fragment carrying the E. coli ilvCΔN gene and the CYC1 terminator into the same sites of pGV1635. The vector, pGV1635, is a K. lactis expression plasmid that was used for expression of the E. coli ilvDΔN gene under the control of the S. cerevisiae TEF1 promoter. The ilvDΔN gene is followed by the TDH3 promoter, a short MCS (includes an XhoI site), the CYC1 terminator and the 1.6 micron replication origin. This plasmid carries the S. cerevisiae LEU2 gene as a selection marker. Digestion of pGV1635 with XhoI and NgoMIV removes the CYC1 terminator and the 1.6 micron replication origin. This sequence was replaced by the insert fragment carrying the E. coli ilvCΔN and the CYC1 terminator which was obtained from pGV1677 digested with XhoI and NgoMIV. The E. coli ilvDΔN originated as a PCR product from pGV1578 (plasmid carrying E. coli ilvD codon optimized for K. lactis from DNA2.0, Menlo Park, Calif.) using primers 1151 and 1152. The E. coli ilvCΔN originated as a PCR product from pGV1160 (plasmid carrying the full length E. coli ilvC gene) using primers 1149 and 1150. The E. coli ilvC in pGV1160 originated as a PCR product from E. coli genomic DNA using primers 387 and 388.
GEVO1287 was transformed with PmlI-digested pGV1537, yielding GEVO1742. GEVO1829 was constructed by sequentially transforming GEVO1742 with gene fragments from pGV1590, pGV1727, and pGV1726 following the standard lithium acetate protocol. First, a 7.8 kb fragment of pGV1590 generated by digestion with NgoMIV and MfeI was transformed into GEVO1742. Next, this transformant strain was transformed with pGV1727 (FIG. 4) that had been linearized by digestion with BcgI. Finally, this transformant strains was transformed with pGV1726 that had been linearized by digestion with AhdI. The final transformant was GEVO1829.
Cellular fractions were prepared from GEVO1742 and GEVO1829 as described above. The protein concentration used to calculate specific activities from all three fractions (“W,” “S,” and “P”) was measured for the “W” fraction. Below are the results for the assays measuring isobutanol dehydrogenase, acetolactate synthase, and ketoisovalerate decarboxylase activities.
Alcohol Dehydrogenase (ADH) Assay
The results from the assay are summarized in Table 5. The “W” fraction and the “S” fraction of the pathway carrying strain (GEVO1829) contained at least three times the NADPH dependent alcohol dehydrogenase activity found in the same fractions of GEVO1742. The “W” and “S” fractions of GEVO1829 contained more than four times the activity present in the “P” fraction. These data indicated that S. cerevisiae Adh7 activity was predominantly localized to the cytosol.







TABLE 5







Alcohol Dehydrogenase Activity.











Specific Alcohol Dehydrogenase



Sample
Activity (U/mg protein)







1742 W
0.08 ± 0.00



1742 S
0.07 ± 0.02



1742 P
 0.03 ± 0.012



1829 W
0.26 ± 0.00



1829 S
0.25 ± 0.02



1829 P
0.04 ± 0.02










Acetolactate Synthase (ALS) Assay

The results from the assay are summarized in Table 6. The “W” and “S” fractions of the isobutanol pathway carrying strain (GEVO1829) contained ALS activity, while no activity was detected in the same fractions of GEVO1742. The “W” and “S” fractions contained three times higher ALS activity than the “P” fraction. These data indicated that B. subtilis ALS activity was predominantly localized to the cytosol.







TABLE 6







Acetolactate Synthase Activity.











Specific Acetolactate Synthase



Sample
Activity (U/mg protein)







1742 W
0.00 ± 0.00



1742 S
0.00 ± 0.00



1742 P
0.00 ± 0.00



1829 W
0.10 ± 0.01



1829 S
0.10 ± 0.00



1829 P
0.03 ± 0.00










Ketoisovalerate Decarboxylase (KIVD) Assay

The results from the assay are summarized in Table 7. The “W” and “S” fractions of the isobutanol pathway carrying strain (GEVO1829) contained 8-10 times greater activity than in the same fractions of GEVO1742. Furthermore, the activity in “S” fraction was 45× higher than what was detected in “P” fraction. These data indicated that L. lactis KIVD activity was predominantly localized in the cytosol.







TABLE 7







Ketoisovalerate decarboxylase (KIVD) Assay.











Specific Ketoisovalerate Decarboxylase



Sample
Activity (U/mg protein)







1742 W
0.05 ± 0.00



1742 S
0.05 ± 0.04



1742 P
0.03 ± 0.00



1829 W
0.38 ± 0.02



1829 S
0.45 ± 0.04



1829 P
0.01 ± 0.00










Example 2
Construction of an ILV3 Deletion Mutant
The purpose of this example is to describe the construction of an ILV3 deletion mutant of S. cerevisiae, GEVO2244.







TABLE 8







Genotype of strains disclosed in Example 2.








GEVO No.
Genotype/Source





GEVO1147
K. lactis, NRRL Y-1140, (obtained from USDA)


GEVO1188
S. cerevisiae, CEN.PK, (obtained from Euroscarf);



MATα ura3 leu2 his3 trp1


GEVO2145
S. cerevisiae, CEN.PK; MATα ura3 leu2 his3 trp1



ilv3::Kl_URA3


GEVO2244
S. cerevisiae, CEN.PK; MATα ura3 leu2 his3 trp1 ilv3Δ
















TABLE 9







Plasmids disclosed in Example 2.










Plasmid name
Genotype







pUC19
bla, pUC-ori (obtained from Invitrogen)



pGV1299
K. lactis URA3, bla, pUC-ori (GEVO)










Plasmid pGV1299 was constructed by cloning the K. lactis URA3 gene into pUC19. The K. lactis URA3 was obtained by PCR using primers 575 and 576 from K. lactis genomic DNA. The PCR product was digested with EcoRI and BamHI and cloned into pUC19 which was similarly digested. The K. lactis URA3 insert was sequenced (Laragen Inc) to confirm correct sequence.
The ilv3::KI_URA3 integration cassette contained, from 5′ to 3′, the following: 1) a 80 bp homology to ILV3 (position +158 to 237) that functions as the 5′ targeting sequence for the integration, 2) the K. lactis URA3 marker gene, 3) a 60 bp homology to a region ILV3 (position −21 to +39) that is further upstream of the 5′ targeting sequence to facilitate loop-out of the K. lactis URA3 marker, and 4) a 221 bp homology to the 3′ region of ILV3 (position +1759 to 1979) that functions as the 3′ targeting sequence for the integration. This cassette was generated by SOE-PCR. The K. lactis URA3 gene was amplified from pGV1299 using primers 1887 and 1888. Only the 3′ region of ILV3 was initially amplified using primers 1623 and 1892 from genomic DNA and this product was used as template to amplify the 3′ region of ILV3 using primers 1889 and 1890. The K. lactis URA3 and the 3′ region of ILV3 were combined by SOE-PCR using primers 1886 and 1890.
GEVO1188 was transformed with the ilv3::KI_URA3 cassette described above and plated onto YNBD+W+CAA (−Ura) plates. Initially, eight colonies (#1-8) were patched onto YNBD+HUWLIV plates and then replica plated onto YNBD+HUWLI (−V) plates to test for valine auxotrophy. As none of these exhibited valine auxotrophy, an additional eight colonies (#9-16) were streaked out for single colonies and 3 or 4 isolates (A through C or D) from each streak were tested for valine auxotrophy. Isolates A-C from clone #12 exhibited valine auxotrophy.
These isolates were tested for the correct integrations by colony PCR using primer pairs 1916+1920 and 1917+1921 for the 5′ and 3′ junctions, respectively. Correct sized bands were observed with clones #12A through C with primer pair 1916+1920. Correct sized bands were observed with clone 12A when FailSafe Master Mix A or C was used with primer pair 1917+1921. Clone #12A was designated as GEVO2145. The valine auxotrophies of GEVO2145 were reconfirmed by streaking them onto SCD+9xIV and SCD-V+9xI plates. GEVO2145 exhibited no growth on the medium lacking valine (SCD-V+9xI) while it grew on medium supplemented with valine (SCD+9xIV). The parent strain, GEVO1188, grew on both media.
GEVO2145 was streaked onto YNBE+W+CAA+FOA to isolate strains in which the K. lactis URA3 had been excised through homologous recombination, i.e. “looped out”. Five FOA resistant clones (A-E) were tested for auxotrophies for valine and uracil. All five clones exhibited auxotrophies to both nutrients. Clone A was designated GEVO2244. Colony PCR using primers 1891 and 1892 with FailSafe Buffer C was performed and the loss of the KI_URA3 cassette was confirmed.
Example 3
DHAD Activity is Localized to Mitochondria
The purpose of this Example is to demonstrate that the DHAD activity encoded by ScILV3 is localized to the mitochondria.







TABLE 9







Genotype of strains disclosed in Example 3.








GEVO No.
Genotype/Source





Gevo2244
S. cerevisiae, CEN.PK; MATα ura3 leu2 his3 trp1 ilv3Δ
















TABLE 10







Plasmids disclosed in Example 3.










pGV No.
Genotype







pGV1106
pUC ori, bla (AmpR), 2 micron ori, URA3, TDH3




promoter-Myc tag-polylinker-CYC1 terminator



pGV1900
pUC ori, bla (AmpR), 2 micron ori, URA3, TEF1




promoter-ScILV3(FL)










Plasmid pGV1106 is a variant of p426GPD (described, in Mumberg et al, 1995, Gene 119-122). To obtain pGV1106, annealed oligos 271 and 272 were ligated into p426GPD that had been digested with SpeI and XhoI, and the inserted DNA was confirmed by sequencing.
Plasmid pGV1900 was generated by amplifying the full-length, native ScILV3 nucleotide sequence from S. cerevisiae strain CEN.PK genomic DNA using primers 1617 and 1618. The resulting 1.76 kb fragment, which contained the complete ScILV3 coding sequence (SEQ ID NO: 88) flanked by 5′ SalI and 3′ BamHI restriction site sequences was digested with SalI and BamHI and ligated into pGV1662 (described in Example 6) which had been digested with SalI and BamHI.
To measure DHAD activities present in fractionated cell extracts, GEVO2244 was transformed singly with either pGV1106, which served as an empty vector control, or with pGV1900, which is an expression plasmid for ScILV3.
An independent clonal transformant of each plasmid was isolated, and a 1 L culture of each strain was grown in SCGal-Ura+9xIV at 30° C. at 250 rpm. The OD600 was noted, the cells were collected by centrifugation (1600×g, 2 min) and the culture medium was decanted. The cell pellets were resuspended in 50 mL sterile deionized water, collected by centrifugation (1600×g, 2 min), and the supernatant was discarded. The OD600 and total wet cell pellet weight of each culture are listed in Table 11, below:







TABLE 11







OD600 and pellet mass (g) of strain GEVO2244


transformed with the indicated plasmids.













Pellet mass



Plasmid
OD600
(g)







pGV1106
2.2
7.6



pGV1900
1.3
3.8










To obtain spheroplasts, the cell pellets were resuspended in 0.1M Tris-SO4, pH 9.3, to a final concentration of 0.1 g/mL, and DTT was added to a final concentration of 10 mM. Cells were incubated with gentle (60 rev/min) agitation on an orbital shaker for 20 min at 30° C., and the cells were then collect by centrifugation (1600×g, 2 min) and the supernatant discarded. Each cell pellet was resuspended in spheroplasting buffer, which consists of (final concentrations): 1.2M sorbitol (Amresco, catalog #0691), 20 mM potassium phosphate pH 7.4) and then collected by centrifugation (1600×g, 10 min). Each cell pellet was resuspended in spheroplasting buffer to a final concentration of 0.1 g cells/mL in a 500 mL centrifuge bottle, and 50 mg of Zymolyase 20T (Seikagaku Biobusiness, Code#120491) was added to each cell suspension. The suspensions were incubated overnight ( ̃16 hrs) at 30° C. with gentle agitation (60 rev/min) on an orbital shaker. The efficacy of spheroplasting was ascertained by diluting an aliquot of each cell suspension 1:10 in either sterile water or in spheroplasting buffer, and comparing the aliquots microscopically (under 40× magnification). In all cases, >90% of the water-diluted cells lysed, indicating efficient spheroplasting. The spheroplasts were centrifuged (3000×g, 10 min, 20° C.), and the supernatant was discarded. Each cell pellet was resuspended in 50 mL spheroplast buffer without Zymolyase, and cells were collected by centrifugation (3000×g, 10 min, 20° C.).
To fractionate spheroplasts, the cells were resuspended to a final concentration of 0.5 g/mL in ice cold mitochondrial isolation buffer (MIB), consisting of (final concentration): 0.6M D-mannitol (BD Difco Cat#217020), 20 mM HEPES-KOH, pH 7.4. For each 1 mL of resulting cell suspension, 0.01 mL of Yeast/Fungal Protease Arrest solution (G Biosciences, catalog #788-333) was added. The cell suspension was subjected to 35 strokes of a Dounce homogenizer with the B (tight) pestle, and the resulting cell suspension was centrifuged (2500×g, 10 min, 4° C.) to collect cell debris and unbroken cells and spheroplasts. Following centrifugation, 2 mL of each sample (1 mL of the pGV1900 transformed cells) were saved in a 2 mL centrifuge tube on ice and designated the “W” (for Whole cell extract) fraction, while the remaining supernatant was transferred to a clean, ice-cold 35 mL Oakridge screw-cap tube and centrifuged (12,000×g, 20 min, 4° C.) to pellet mitochondria and other organellar structures. Following centrifugation, 5 mL of each resulting supernatant was transferred to a clean tube on ice, being careful to avoid the small, loose pellet, and labelled the “S” (soluble cytosol) fraction. The resulting pellets were resuspended in MIB containing Protease Arrest solution, and were labelled the “P” (“pellet”) fractions. Protein from the “P” fraction was released after dilution 1:5 in DHAD assay buffer (see above) by rapid mixing in a 1.5 mL tube with a Retsch Ball Mill MM301 in the presence of 0.1 mM glass beads. The mixing was performed 4 times for 1 minute.
The BioRad Protein Assay reagant (BioRad, Hercules, Calif.) was used according to manufacturer's instructions to determine the protein concentration of each fraction.
The DHAD activity of each fraction was ascertained as described in the methods above.







TABLE 12







Specific activities (KIV generation) and ratios of specific activities


from fractionated lysates of S. cerevisiae strain GEVO2244 carrying


plasmids to overexpress the indicated DHAD homolog.














Sp. Activity




Lysate (pGV#

[U/mg protein



and fraction*)
DHAD
in fraction]
Std. Dev.







1106 W
—
n.d.




1106 S
—
n.d.



1106 P
—
n.d.



1900 W
ScILV3(FL)
0.0096
0.0018



1900 S
ScILV3(FL)
0.0052
0.0004



1900 P
ScILV3(FL)
0.0340
0.0029







Each data point is the result of triplicate samples.






Cells overexpressing the full-length, native S. cerevisiae Ilv3 contained in a greater proportion of the specific DHAD activity in the mitochondrial fraction (P) versus the cytosolic fraction (S).
Example 4
Replacing Current Mitochondrially Targeted Isobutanol Pathway Enzymes with Fungal Homologs or Functional Analogs that are Targeted to the Cytosol
The purpose of this example is to illustrate that fungal homologs of isobutanol a pathway enzymes exhibit cytosolic activity.







TABLE 13







Genotype of strains disclosed in Example 4.








GEVO No.
Genotype/Source





1187
MATa ura3-52 leu2-3_112 his3Δ1 trp1-289 ADE2



CEN.PK2-1C


2280
MATa ura3-52 leu2-3_112 his3Δ1 trp1-289 ADE2



CEN.PK2-1C integrated pGV1730 at PDC1 locus


2618
MATa ura3-52 leu2-3_112 his3Δ1 trp1-289 ADE2



CEN.PK2-1C integrated pGV2114 at PDC1 locus


2621
MATa ura3-52 leu2-3_112 his3Δ1 trp1-289 ADE2



CEN.PK2-1C integrated pGV2117 at PDC1 locus


2622
MATa ura3-52 leu2-3_112 his3Δ1 trp1-289 ADE2



CEN.PK2-1C integrated pGV2118 at PDC1 locus
















TABLE 14







Plasmids disclosed in Example 4.








pGV No.
Genotype





1730
PCup1-11:Bs_alsS, pUC ORI, AmpR, TRP1, PDC1



3′-fragment-NruI-PDC1 5′-fragment.


2114
PCup1-11:Bs_alsScoSc, pUC ORI, AmpR, TRP1, PDC1



3′-fragment-NruI-PDC1 5′-fragment.


2117
PCup1-11:Ta_alsS, pUC ORI, AmpR, TRP1, PDC1



3′-fragment-NruI-PDC1 5′-fragment.


2118
PCup1-11:Ts_alsS, pUC ORI, AmpR, TRP1, PDC1



3′-fragment-NruI-PDC1 5′-fragment.









Yeast AHASs are normally mitochondrial, thus favoring fungal ALS enzymes for as cytosolically functional isobutanol pathway enzymes. Sequence analysis by Le and Choi (Bull. Korean Chem. Soc. (2005) 26:916-920) showed that there is a conserved sequence ‘RFDDR’ found in AHASs that is not conserved among ALSs. This sequence is likely involved in FAD-binding by AHASs and thus could be used to distinguish between the FAD-dependent AHASs and the FAD-independent ALSs. Using this region to distinguish between AHASs and ALSs BLAST searches of fungal sequence databases were performed and resulted in the identification of ALS homologs from several fungal species (Magnaporthe grisea, Phaeosphaeria nodorum, Trichoderma atroviride (SEQ ID NO: 71), Talaromyces stipitatus (SEQ ID NO: 72), Penicillium marneffei, and Glomerella graminicola). Of these sequences, the ALS homologs from M. grisea, P. nodorum, T. atroviride and T. stipitatus are predicted to be cytoplasmic by Mitoprot II v.1.101 as described in the paper M. G. Claros, P. Vincens. Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur. J. Biochem. 241, 779-786 (1996).
Fungal ALS genes were synthesized by DNA 2.0 with codon optimization biased for S. cerevisiae. The following ALS constructs were made and tested for ALS activity by assaying acetoin in the media during a growth timecourse. All ALS genes were cloned into the integration vector pGV1730 (SEQ ID NO: 69) as described herein.
Plasmid pGV1730 is a yeast integration plasmid used to replace the PDC1 gene in S. cerevisiae with the B. subtilis alsS gene (SEQ ID NO: 70) (not codon optimized for S. cerevisiae) expressed using the S. cerevisiae CUP1 promoter. This plasmid carries the S. cerevisiae TRP1 gene as a selection marker.
Construction of pGV2114: pGV1730 was treated with BamHI and SalI and the 4932 bp vector fragment was purified by gel electrophoresis as described. The B. subtilis AlsS (codon-optimized for expression in S. cerevisiae) gene was ligated to the pGV1730 vector fragment as a BamHI and SalI 1722 bp fragment using standard methods with an approximately 5:1 insert:vector molar ratio and transformed into TOP10 chemically competent E. coli cells. Plasmid DNA was isolated and correct clones were confirmed using restriction enzyme analysis.
Construction of pGV2117. pGV1730 was treated with BamHI and SalI and the 4932 bp vector fragment was purified by gel electrophoresis as described. The T. atroviride ALS gene was ligated to the pGV1730 vector fragment as a BamHI and SalI 1686 bp fragment using standard methods with an approximately 5:1 insert:vector molar ratio and transformed into TOP10 chemically competent E. coli cells. Plasmid DNA was isolated and correct clones were confirmed using restriction enzyme analysis.
Construction of pGV2118. pGV1730 was treated with BamHI and SalI and the 4932 bp vector fragment was purified by gel electrophoresis as described. The T. stipitatus ALS gene was ligated to the pGV1730 vector fragment as a BamHI and SalI 1707 bp fragment using standard methods with an approximately 5:1 insert:vector molar ratio and transformed into TOP10 chemically competent E. coli cells. Plasmid DNA was isolated and correct clones were confirmed using restriction enzyme analysis.
All yeast strains were constructed by treating the plasmid to be integrated with NruI and then transforming the plasmid according to the standard yeast transformation protocol as described herein. Transformants were selected by plating transformed cells onto SCD-W media and growing at 30° C. for 2 days. Primary transformants were single colony purified and then tested for correct integration using colony PCR. Colony PCR was performed using the Yeast colony PCR to check for proper integration of the integrative plasmids used the FailSafeTM PCR System (EPICENTRE® Biotechnologies, Madison, Wis.; Catalog #FS99250) according to the manufacturer protocol The PCR reactions were incubated in a thermocycler using the following conditions: 1 cycle of 94° C. for 2 min, 40 cycles of 94° C. for 30 s, 53° C. for 30 s, 72° C. for 60 s and 1 cycle of 72° C. for 10 min. Presence of the positive PCR product was assessed using agarose gel electrophoresis. Primer pairs for the 5′-end and 3′-end integration sites contained one primer on the plasmid and one primer in the genome.
Yeast strains GEVO1187, 2280, 2618, 2621 and 2622 were grown in YPD overnight at 30° C. A 100 mL culture was inoculated to 1 OD/mL and split into 2.50 mL cultures. This was the time zero. One of the 50 mL cultures received 500 μM CuSO4 at time 2 hours and the other did not. Timepoints consisted of removing 1 mL at times 0, 2, 2.5, 3, 4, 7.5, and 23 hours. At each timepoint the OD600 was determined and acetoin concentrations were determined using GC as described in the General Methods. Before GC samples were treated with H2SO4 to convert intermediates to acetoin. The graph shows the acetoin concentrations in the media of the strains in which transcription of the ALS genes was induced by CuSO4. The acetoin values were normalized to cell OD. Both the T. stipitatus ALS and the T. atroviride ALS showed increased levels of acetoin as compared to the no ALS control (FIG. 2).
ALS activity in whole cell lysates is determined as described in General Methods. Activity in mitochondrial/organellar (P) and cytosolic (S) fractions and whole cell (W) lysates is assayed as described in General Methods
Example 5
Replacing Current Mitochondrially Targeted Isobutanol Pathway Enzymes with Homologs or Functional Analogs from Anaerobic Fungi
The purpose of this example is to illustrate that homologues of isobutanol a pathway enzymes from anaerobic fungi exhibit cytosolic activity.







TABLE 15







Genotype of strains disclosed in Example 5.








GEVO No.
Genotype





GEVO2244
S. cerevisiae, CEN.PK; MATα ura3 leu2 his3 trp1 ilv3Δ
















TABLE 16







Plasmids disclosed in Example 5.










Plasmid name
Genotype







pGV1106
pUC ori, bla (AmpR), 2 μm ori, URA3, TDH3




promoter-Myc tag-polylinker-CYC1 terminator



pGV1662
pUC ori, bla (AmpR), 2 μm ori, URA3, TEF1




promoter-(kivD)



pGV1855
pUC ori, bla (AmpR), 2 μm ori, URA3, TEF1




promoter-Ll_ilvD










Plasmid pGV1106 is described in Example 3, above.
Plasmid pGV1662 (SEQ ID NO: 81) served as the parental plasmid of pGV1855, pGV1900, and pGV2019. The salient features of pGV1662 include the yeast 2 micron origin of replication, the URA3 selectable marker, and the ScTEF1 promoter sequence followed by restriction sites into which an ORF can be cloned to permit its expression under the regulation of the TEF1 promoter.
Plasmid pGV1855 contains the L. lactis ilvD. The L. lactis ilvD sequence was synthesized (DNA2.0, Menlo Park, Calif.) and included a unique SalI and a NotI site at the 5′ and 3′ end of the coding sequence, respectively. The synthesized DNA was digested with SalI and NotI and ligated into vector pGV1662 that had been digested with SalI plus NotI, yielding pGV1855.
The DHAD homolog (ilvD) from the anaerobic fungi Piromyces sp. E2 has a predicted MTS of 49 amino acids at the N-terminus. Thus, a nucleotide sequence encoding the Piromyces ilvD lacking the N-terminal 49 amino acids and with a start codon placed at the N-terminus was synthesized (SEQ ID NO: 73). In addition, a SalI site and a BamHI site were introduced at the 5′ and 3′ ends of this ORF. This fragment was cloned into the SalI and BamHI sites of pGV1662. The resulting plasmid was transformed in to GEVO2242. An empty vector, pGV1106, is used as a negative control. Plasmid, pGV1855, expressing L. lactis ilvD is used as a positive control.
An independent clonal transformant of each plasmid is isolated, and a 1 L culture of each strain is grown in SCGal-Ura+9xIV at 30° C. at 250 rpm. The OD600 is noted, the cells are collected by centrifugation (1600×g, 2 min) and the culture medium is decanted. The cell pellets are resuspended in 50 mL sterile deionized water, collected by centrifugation (1600×g, 2 min), and the supernatant is discarded.
To obtain spheroplasts, the cell pellets are resuspended in 0.1M Tris-SO4, pH 9.3, to a final concentration of 0.1 g/mL, and DTT is added to a final concentration of 10 mM. Cells are incubated with gentle (60 rev/min) agitation on an orbital shaker for 20 min at 30° C., and the cells are then collected by centrifugation (1600×g, 2 min) and the supernatant discarded. Each cell pellet is resuspended in spheroplasting buffer, which consists of (final concentrations): 1.2M sorbitol (Amresco, catalog #0691), 20 mM potassium phosphate pH 7.4) and then collected by centrifugation (1600×g, 10 min). Each cell pellet is resuspended in spheroplasting buffer to a final concentration of 0.1 g cells/mL in a 500 mL centrifuge bottle and 50 mg of Zymolyase 20T (Seikagaku Biobusiness, Code#120491) is added to each cell suspension. The suspensions are incubated overnight (approximately 16 hrs) at 30° C. with gentle agitation (60 rev/min) on an orbital shaker. The efficacy of spheroplasting is ascertained by diluting an aliquot of each cell suspension 1:10 in either sterile water or in spheroplasting buffer, and comparing the aliquots microscopically (under 40× magnification). The spheroplasts are centrifuged (3000×g, 10 min, 20° C.), and the supernatant is discarded. Each cell pellet is resuspended in 50 mL spheroplast buffer without Zymolyase and cells are collected by centrifugation (3000×g, 10 min, 20° C.).
To fractionate spheroplasts, the cells are resuspended to a final concentration of 0.5 g/mL in ice cold mitochondrial isolation buffer (MIB), consisting of (final concentration): 0.6M D-mannitol (BD Difco Cat#217020), 20 mM HEPES-KOH, pH 7.4. For each 1 mL of resulting cell suspension, 0.01 mL of Yeast/Fungal Protease Arrest solution (G Biosciences, catalog #788-333) is added. The cell suspension is subjected to 35 strokes of a Dounce homogenizer with the B (tight) pestle, and the resulting cell suspension is centrifuged (2500×g, 10 min, 4° C.) to collect cell debris and unbroken cells and spheroplasts. Following centrifugation, 2 mL of each sample (1 mL of the pGV1900 transformed cells) are saved in a 2 mL centrifuge tube on ice and designated the “W” (for Whole cell extract) fraction, while the remaining supernatant is transferred to a clean, ice-cold 35 mL Oakridge screw-cap tube and centrifuged (12,000×g, 20 min, 4° C.) to pellet mitochondria and other organellar structures. Following centrifugation, 5 mL of each resulting supernatant is transferred to a clean tube on ice, being careful to avoid the small, loose pellet, and labelled the “S” (soluble cytosol) fraction. The resulting pellets are resuspended in MIB containing Protease Arrest solution, and are labelled the “P” (“pellet”) fractions. The protein concentration of each fraction is determined using the BioRad Protein Assay reagant (BioRad, Hercules, Calif.) according to manufacturer's instructions.
The DHAD activity of each fraction is ascertained using the DHAD assays as described above in the General Methods.
Example 6
Modification of the N-Terminal Mitochondrial Targeting Sequence of an Isobutanol Pathway Enzyme
The purpose of this example is to illustrate that removal or modification of N-terminal mitochondrial targeting sequences allows for cytosolic activity of isobutanol pathway enzymes.







TABLE 17







Genotype of strains disclosed in Example 6.








GEVO No.
Genotype





1803
MATa/alpha ura3/ura3 leu2/leu2 his3/his3 trp1/trp1



pdc1::Bs-alsS, TRP1/PDC1
















TABLE 18







Plasmids disclosed in Example 6.









Plasmid




name
Relevant Genes/Usage
Genotype





pGV1354
Plasmid that contains the
PTDH3:ILV5ΔN47:



ILv5ΔN47 gene.
CYC1 term, bla, ColE1




ORI, URA3, 2μ ori.


pGV1662
Parent vector that has
pTEF1::L. lactis



Ampicillin resistance,
kivD::CYC1 term, bla,



the 2μ origin, a URA3 gene,
ColE1 ORI, URA3, 2μ



the TEF1 promoter, CYC1
ori.



terminator region and an




E. coli origin. It also has




the L. lactis KivD gene that




is removed by cutting the




plasmid with SalI and NotI,




and then gel purifying the




vector portion. SalI and NotI




were used for cloning genes




to be expressed from the




TEF1 promoter.



pGV1810
Plasmid that contains the
pTEF1::ILV5::CYC1 term,



full length ILV5 gene. This
bla, ColE1 ORI, URA3, 2μ



was used as a PCR template
ori.



to generate the




Δ46-ilv5 mutant.



pGV1831
Plasmid that contains the
pTEF1::Sc Ilv5□N47::CYC1



Ilv5ΔN47 gene under control
term, bla, ColE1 ORI,



of the TEF1 promoter.
URA3, 2μ ori.


pGV1833
Plasmid that contains the
pTEF1::Sc ILV5:CYC1



full length ILV5 gene under
term, bla, ColE1 ORI,



control of the TEF1 promoter.
URA3, 2μ ori


pGV1901
The S. cerevisiae KARI with
pTEF1::Δ46ilv5



the N-terminal 46 amino acid
KARI::CYC1 term, bla,



deleted (Δ46) cloned into
ColE1 ORI, URA3, 2μ ori



pGV1662 at the SalI-NotI




sites of the vector. The




S. cerevisiae Δ46 KARI was a




SalI-NotI fragment that was




PCR amplified from




pGV1810 using primers




1809 and 1615.



pGV1824
The E. coli coSc KARI cloned
pTEF1::E. coli coSc



into pGV1662 at SalI-BamHI
KARI:CYC1 term, bla,



sites of the vector.
ColE1 ORI, URA3, 2μ ori









The yeast enzymes acetohydroxyacid synthase (AHAS; ILV2+ILV6), ketol-acid reductoisomerase (KARI; ILV5), and dihydroxyacid dehydratase (DHAD; ILV3) that carry out the first three steps of isobutanol production are physiologically localized to the mitochondria. Mitochondrial matrix proteins are typically targeted to the mitochondria by an N-terminal mitochondrial targeting sequence (MTS), which is then cleaved off in the mitochondria resulting in the ‘mature’ form of the enzyme. N-terminal deletions of ILV5 have been shown to re-localize this enzyme to the cytosol (Omura, 2008, Appl. Microbiol. Biotechnol. 78: 503-513; Omura, WO12009/078108 A1, hereby incorporated by reference in its entirety).
N-terminal mitochondria targeting sequences (MTS) are predicted by MitoProt II software; Claros et al., 1996, Eur. J. Biochem. 241: 779-786. Two N-terminal deletions of the ILV5 gene was constructed, one missing the first 46 amino acids and one missing the first 47 amino acids.
pGV1831 was constructed as follows. pGV1662 was digested with SalI and NotI and the large fragment (6.3 Kb vector backbone) was gel purified by agarose gel electrophoresis. The Ilv5ΔN47 gene was excised from plasmid pGV1354 (SEQ ID NO: 80) using SalI and NotI. The ilv5ΔN47 gene fragment (1.06 Kb) was purified away from the larger vector fragment by agarose gel electrophoresis. The pGV1662 vector and ilv5ΔN47 insert were ligated using standard methods in an approximately 5:1 insert:vector molar ratio and transformed into TOP10 chemically competent E. coli cells. Plasmid DNA was isolated and correct clones were confirmed using restriction enzyme analysis, namely generation of the correct insert size by digesting clones with SalI and NotI enzymes. The clones were verified by sequencing with the primers 351, 1625, and 1626. Purified plasmid DNA was transformed into S. cerevisiae strain GEVO1803 using a standard yeast transformation protocol.
pGV1833 was constructed as follows. pGV1662 was digested with SalI and NotI and the large fragment (6.3 Kb vector backbone) was gel purified by agarose gel electrophoresis. Primers 1615 and 1616 were used to amplify the S. cerevisiae ILV5 gene from the plasmid template pGV1810 by PCR. The correct fragment size was verified with DNA gel electrophoresis. (1.2 Kb). The PCR product was purified after PCR using the Qiagen QIAquick PCR Purification Kit. The PCR product was then digested with XhoI and NotI to generate ends compatible with the pGV1662 backbone (the XhoI end of the PCR product is compatible with the SalI end of the vector, although the ligated DNA fragment can't be recut with either enzyme). After digestion, the PCR product was purified with a Qiagen QIAquick PCR Purification Kit. The two fragments were ligated using standard methods in an approximately 5:1 insert:vector molar ratio and transformed into TOP10 chemically competent. E. coli cells. Plasmid DNA was isolated and correct clones were confirmed using restriction enzyme analysis. In this case, SacI plus NotI digestion yielded a fragment of the predicted size (1.6 Kb). The clones were verified by sequencing with the primers 351, 1625, and 1626. Purified plasmid DNA was transformed into S. cerevisiae strain GEVO1803.
pGV1901 was constructed as follows. pGV1662 was digested with SalI and NotI and the large fragment (6.3 Kb vector backbone) was gel purified by agarose gel electrophoresis. The ILV5 gene was amplified from pGV1810 (SEQ ID NO: 82) using primers 1809 (which removes the first 46 amino acids from the N-terminus while adding a methionine codon) and 1615. The PCR product was digested with SalI and NotI. After digestion, the PCR product was purified on an agarose gel and the proper fragment (1.07 Kb) was recovered using the Zymoclean Gel DNA Recovery Kit. The pGV1662 vector and Ilv5-Δ46 PCR products were ligated using standard methods in an approximately 5:1 insert:vector molar ratio and transformed into TOP10 chemically competent E. coli cells. Plasmid DNA was isolated and correct clones were confirmed with PCR screening of colonies using primers 351 and 1577. The predicted correct PCR product was 580 bp. The clones were sequenced using primers 351, 1625, and 1626. Purified plasmid DNA was transformed into S. cerevisiae strain GEVO1803 using the standard yeast transformation protocol.
pGV1824 contains the E. coli ilvC gene that is codon optimized for S. cerevisiae cloned into the SalI and BamHI of pGV1662 as described above. The sequence of the codon optimized E. coli ilvC is found as SEQ ID NO: 83.
Plasmids were transformed into the yeast strain GEVO1803 and an individual colony was purified from each transformation. KARI assays of whole cell lysates were performed at pH 7.5 as described in General Methods. Results are shown in FIG. 3.
KARI activity in mitochondrial/organellar (P) and cytosolic (S) fractions and whole cell (W) lysates is assayed as described in General Methods
Example 7
Scaffolding Two or More Isobutanol Pathway Enzymes
The purpose of this example is to illustrate how isobutanol pathway enzymes can be scaffolded in order to localize them to the cytosol.
Cellulolytic microorganisms utilize a scaffolded enzyme complex called a cellulosome. In such a complex, numerous enzymes are docked to a single scaffold protein, called a scaffoldin, which contain multiple binding domains called cohesin domains. Each cohesin domain interacts with a dockerin domain. In a cellulosome complex, each cellulytic enzyme also has a dockerin domain that allows it to bind to the scaffoldin.
The cohesin domains of a scaffoldin protein, for example, CipA from Clostridium thermocellum, can be expressed in yeast. The dockerin domains from the cellulolytic enzymes from the same organism, for example Xyn10B, can be fused to the isobutanol enzymes and the fusion proteins expressed in yeast.
The activity of each pathway enzyme in whole cell lysates is determined as described in General Methods. Activity in mitochondrial/organellar (P) and cytosolic (S) fractions and whole cell (W) lysates is assayed as described in General Methods.
Example 8
Adding of Tags, e.g. Ubiquitin Tags, to the N-Terminus of an Isobutanol Pathway Enzyme
The purpose of this is example is to demonstrate that isobutanol pathway enzymes can be targeted to the yeast cytosol. For instance, this example illustrates how a DHAD enzyme can be targeted to the yeast cytosol.







TABLE 18







Genotype of strains disclosed in Example 8.








GEVO No.
Genotype/Source





Gevo2242
S. cerevisiae, CEN.PK; MAT-alpha ura3 leu2 his3 trp1



ilv5O255E pdc1::Bs-alsS, TRP1


Gevo2244
S. cerevisiae, CEN.PK; MATα ura3 leu2 his3 trp1 ilv3Δ
















TABLE 19







Plasmids disclosed in Example 8.










pGV No.
Genotype






pGV1106
pMB1 ori, bla (AmpR), 2 μm ori, URA3, TDH3




promoter-Myc tag-polylinker-CYC1 terminator



pGV1662
pMB1 ori, bla (AmpR), 2 μm ori, URA3,




TEF1 promoter-(kivD)



pGV1784
pUC ori, kanR, Mm_ubiquitin coding sequence



pGV1855
pMB1 ori, bla (AmpR), 2 μm ori, URA3,




TEF1 promoter-Ll_ilvD



pGV1897
pMB1 ori, bla (AmpR), 2 μm ori, URA3,




TEF1 promoter-Mm_ubiquitin(Gly-X)



pGV1900
pMB1 ori, bla (AmpR), 2 μm ori, URA3,




TEF1 promoter-ScILV3(FL)



pGV2019
pUC ori, bla (AmpR), 2 μm ori, URA3,




TEF1 promoter-ScILV3ΔN



pGV2052
pMB1 ori, bla (AmpR), 2 μm ori, URA3,




TEF1 promoter-Mm_ubiquitin(Gly-X)-ScIlv3(FL)



pGV2053
pMB1 ori, bla (AmpR), 2 μm ori, URA3,




TEF1 promoter-Mm_ubiquitin(Gly-X)-ScIlv3ΔN



pGV2054
pMB1 ori, bla (AmpR), 2 μm ori, URA3,




TEF1 promoter-Mm_ubiquitin(Gly-X)-Ll_ilvD



pGV2055
pMB1 ori, bla (AmpR), 2 μm ori, URA3,




TEF1 promoter-Mm_ubiquitin(Gly-X)-Gf_ilvD



pGV2056
pMB1 ori, bla (AmpR), 2 μm ori, URA3,




TEF1 promoter-Mm_ubiquitin(Gly-X)-Se_ilvD









To develop the constructs required to express DHAD as a fusion with an N-terminal ubiquitin, plasmid pGV1784 was synthesized by DNA2.0. This plasmid contained the synthesized sequence for the Mus musculus ubiquitin gene, codon-optimized for expression in S. cerevisiae (SEQ ID NO: 86). Using this plasmid as the template, the M. musculus ubiquitin gene was amplified via PCR using primers 1792 and 1794 to generate a PCR product containing the M. musculus ubiquitin gene codon sequence flanked by restriction sites XhoI and NotI at its 5′ and 3′ ends, respectively, and altered so as to lack the codon for its endogenous C-terminal most glycine residue (denoted as Gly-X). This PCR product was cloned into pGV1662 (described in Example 6), yielding pGV1897.
Plasmid pGV1897 was then used as a recipient cloning vector for sequences encoding S. cerevisiae ILV3 (ScIlv3(FL), SEQ ID NO: 88), S. cerevisiae Ilv3ΔN (ScIlv3ΔN, SEQ ID NO: 89), L. lactis (Ll_ilvD SEQ ID NO: 87), G. forsetti ilvD (Gf_ilvD, SEQ ID NO: 90), and S. erythraea ilvD (Se_ilvD, SEQ ID NO: 91), yielding plasmids pGV2052-2056, respectively.
The DHAD activity exhibited by cells transformed with each of the resulting constructs is ascertained by in vitro assay. GEVO2244 is transformed (singly) with pGV2052-2056, pGV1106 (empty control vector), pGV1855 (expressing native, unfused Ll_ilvD) or pGV1900 (expressing native, full-length Sc_ILV3(FL)). Lysates of transformants are prepared and DHAD activity in mitochondrial/organellar (P) and cytosolic (S) fractions and whole cell (W) lysates is assayed as described in Example 3.
In an analogous manner, a desired ALS (e.g., the B. subtilis alsS) or KARI gene whose product is known or predicted to be mitochondrial can be re-targeted to the cytosol by means of the methods detailed in this example. The nucleotide sequence encoding for a full-length, or variant, ALS or KARI is amplified by PCR using primers that introduce restriction sites convenient for cloning the final product as an in-frame fusion of the M. musculus ubiquitin gene. The resulting construct is transformed into a host S. cerevisiae cell suitable for assaying the in vitro activity of the expressed M. musculus ubiquitin-gene chimeric fusion protein, using methods described in Example 3.
Example 9
Dihydroxy Acid Dehydratase Limits Isobutanol Production in Yeast
This example illustrates the specific activity of various DHAD homologs in yeast. The example also illustrates that high specific activity of the Lactococcus lactis IlvD enzyme (SEQ ID NO: 18) correlates with an increase in isobutanol production.
Plasmid pGV1106 was used as a control and is described in Example 3. Plasmid pGV1662 (described in Example 6) served as the parental plasmid of pGV1855, pGV1900, and pGV2019 (see Example 5). Plasmids pGV1851-1855 and pGV1904-1907 are all variants of pGV1662 (See Table 20), in which the kivD ORF sequence present in pGV1662 was excised and replaced with a sequence encoding a DHAD homolog, as indicated below.







TABLE 20







Plasmids disclosed in Example 9.








pGV No.
Genotype





pGV1851
pUC ori, bla (AmpR), 2 μm ori, URA3, TEF1 promoter-



Gramella forsetti ilvD


pGV1852
pUC ori, bla (AmpR), 2 μm ori, URA3, TEF1 promoter-



Chromohalobacter salexigens ilvD


pGV1853
pUC ori, bla (AmpR), 2 μm ori, URA3, TEF1 promoter-



Ralstonia eutropha ilvD


pGV1854
pUC ori, bla (AmpR), 2 μm ori, URA3, TEF1 promoter-



Saccharopolyspora erythraea ilvD


pGV1855
pUC ori, bla (AmpR), 2 μm ori, URA3, TEF1 promoter-



Ll_ilvD


pGV1900
pUC ori, bla (AmpR), 2 μm ori, URA3, TEF1 promoter-



ScILV3(FL)


pGV1904
pUC ori, bla (AmpR), 2 μm ori, URA3, TEF1 promoter-



Acidobacteria bacterium Ellin345 ilvD


pGV1905
pUC ori, bla (AmpR), 2 μm ori, URA3, TEF1 promoter-



Picrophilus torridus DSM 9790 ilvD


pGV1906
pUC ori, bla (AmpR), 2 μm ori, URA3, TEF1 promoter-



Piromyces species E2 ilvD


pGV1907
pUC ori, bla (AmpR), 2 μm ori, URA3, TEF1 promoter-



Sulfolobus tokodaii strain 7 ilvD









Plasmid pGV1851 contains the G. forsetti ilvD gene (SEQ ID NO: 90). Plasmid pGV1852 contains the C. salexigens gene (SEQ ID NO: 95). Plasmid pGV1853 contains the R. eutropha gene (SEQ ID NO: 94). Plasmid pGV1854 contains the S. erythraea ilvD (SEQ ID NO: 91). Plasmid pGV1855 contains the L. lactis ilvD (SEQ ID NO: 87). Plasmid pGV1900 contains the S. cerevisiae ILV3 (SEQ ID NO: 88). Plasmid pGV1904 contains the A. bacterium Ellin345 ilvD (SEQ ID NO: 92). Plasmid pGV1905 contains the P. torridus DSM 9790 ilvD (SEQ ID NO: 96). Plasmid pGV1906 contains the Piromyces sp. E2 ilvD (SEQ ID NO: 93). Plasmid pGV1907 contains the S. tokodaii ilvD (SEQ ID NO: 97). All sequences (except that of the S. cerevisiae ILV3 (full length) were synthesized with 5′ SalI and 3′ NotI sites by DNA2.0 (Menlo Park, Calif.), digested with SalI and NotI, and ligated into pGV1662 which had also been digested with SalI and NotI. For plasmid pGV1900, the sequence containing the open reading frame of the S. cerevisiae ILV3 (full length) was amplified from S. cerevisiae genomic DNA using primers 1617 and 1618, and the resulting 1.8 kb fragment was digested with SalI plus BamHI and cloned into pGV1662. Various DHADs were tested for in vitro activity using whole cell lysates. In this case, the DHADs were expressed in a yeast deficient for DHAD activity (GEVO2244; ilv3Δ) (see Example 2) to minimize endogenous background activity.
To grow cultures for cell lysates, triplicate independent cultures of each desired strain were grown overnight in 3 mL SCD-Ura+9xIV at 30° C., 250 rpm. The following day, the overnight cultures were diluted 1:50 into 50 mL fresh. SCD-Ura in a 250 mL baffle-bottomed Erlenmeyer flask and incubated at 30° C. at 250 rpm. After approximately 10 hours, the OD600 of all cultures were measured, and the cells of each culture were collected by centrifugation (2700×g, 5 min). The cell pellets were washed by resuspending in 1 mL of water, and the suspension was placed in a 1.5 mL tube and the cells were collected by centrifugation (16,000×g, 30 seconds). All supernatant was removed from each tube and the tubes were frozen at −80° C. until use.
Lysates were prepared by resuspending each cell pellet in 0.7 mL of lysis buffer. Lysate lysis buffer consisted of: 0.1M Tris-HCl pH 8.0, 5 mM MgSO4, with 10 μL of Yeast/Fungal Protease Arrest solution (G Biosciences, catalog #788-333) per 1 mL of lysis buffer. Eight hundred microliters of cell suspension were added to 1 mL of 0.5 mm glass beads that had been placed in a chilled 1.5 mL tube. Cells were lysed by bead beating (6 rounds, 1 minute per round, 30 beats per second) with 2 minutes chilling on ice in between rounds. The tubes were then centrifuged (20,000×g, 15 min) to pellet debris and the supernatant (cell lysates) were retained in fresh tubes on ice. The protein concentration of each lysate was measured using the BioRad Bradford protein assay reagent (BioRad, Hercules, Calif.) according to manufacturer's instructions.
The DHAD activity of each lysate was ascertained as follows. In a fresh 1.5 mL centrifuge tube, 50 μL of each lysate was mixed with 50 μL of 0.1M 2,3-dihydroxyisovalerate (DHIV), 25 μL of 0.1M MgSO4, and 375 μL of 0.05M Tris-HCl pH 8.0, and the mixture was incubated for 30 min at 35° C. Each tube was then heated to 95° C. for 5 min to inactivate any enzymatic activity, and the solution was centrifuged (16,000×g for 5 min) to pellet insoluble debris. To prepare samples for analysis, 100 μL of each reaction were mixed with 100 μL of a solution consisting of 4 parts 15 mM dinitrophenyl hydrazine (DNPH) in acetonitrile with 1 part 50 mM citric acid, pH 3.0, and the mixture was heated to 70° C. for 30 min in a thermocycler. The solution was then analyzed by HPLC as described above in General Methods to quantitate the concentration of ketoisovalerate (KIV) present in the sample. The results are shown in Table 21.







TABLE 21







Specific activities (KIV generation) from lysates


of S. cerevisiae strain GEVO2244 carrying plasmids


to overexpress the indicated DHAD homolog.











Specific activity




(U/mg total


Plasmid
Gene
protein)





pGV1106
Control (i.e. no DHAD)
n.d.


pGV1851
Gramella forsetti ilvD
0.012


pGV1852
Chromohalobacter salexigens
n.d.



(SEQ ID NO: 95)



pGV1853
Ralstonia eutropha
n.d.



(SEQ ID NO: 94)



pGV1854
Saccharopolyspora erythraea ilvD
0.002


pGV1855
Lactococcus lactis ilvD
0.027


pGV1900
Saccharomyces cerevisiae ILV3(FL)
0.148


pGV1904
Acidobacteria bacterium
0.004



Ellin345 DHAD



pGV1905
Picrophilus torridus
n.d.



DSM 9790 DHAD



pGV1906
Piromyces Sp E2 DHAD
0.016


pGV1907
Sulfolobus tokodaii
0.001



str. 7 DHAD





Each data point is the result of triplicate samples.


* n.d., not detectable






Example 10
Dihydroxy Acid Dehydratase Limits Isobutanol Production in Yeast
This example illustrates that high specific DHAD activity, and in particular the high specific activity of the L. lactis IlvD enzyme (SEQ ID NO: 18) correlates with an increase in isobutanol production.







TABLE 22







Genotype of strains disclosed in Example 10.








GEVO No.
Genotype/Source





GEVO1186
S.cerevisiae, CEN.PK; MATa/α ura3/ura3 leu2/leu2



his3/his3 trp1/trp1


GEVO1188
S.cerevisiae, CEN.PK; MATα ura3 leu2 his3 trp1


GEVO1803
MATa/α ura3/ura3 leu2/leu2 his3/his3 trp1/trp1



pdc1::Bs-alsS, TRP1/PDC1


GEVO2107
MATa/α ura3/ura3 leu2/leu2 his3/his3 trp1/trp1



pdc1::Bs-alsS, TRP1/PDC1 pdc6::{ScTEF1p-Ll_kivd



ScTDH3p-Dm_ADH URA3}/PDC6
















TABLE 23







Plasmids disclosed in Example 10.








pGV No.
Genotype





p423GPD
PTDH3:MCS:TCYC1, HIS3, 2-micron, bla, pUC ori



(Mumberg, D. et al. (1995) Gene 156:119-122;



obtained from ATCC)


pGV1103
PTDH3:myc-tag:MCS:TCYC1, HIS3, 2 micron, bla,



pUC ori


pGV1730
PCUP1:Bs-alsS:TPDC1IPDC1-3′ region:PDC1-5′ region,



TRP1, bla, pUC ori


pGV1914
PTEF1:Ll_kivD PTDH3:Dm_ADH PDC6 5′, 3′ targeting



homology URA3 pUC ori bla(ampR)


pGV1974
PTEF1:Sc_ILV3ΔN:PTDH3:Ec_ilvCQ110V-coSc:TCYC1,



HIS3, 2 micron, bla, pUC ori bla(ampR)


pGV1981
PTEF1:Lactococcus lactis ilvD-coSc:PTDH3:



Ec_ilvCQ110V-coSc:TCYC1, HIS3, 2 micron, bla,



pUC ori


pGV2001
PTEF1:PTDH3:Ec_ilvCQ110V-coSc:TCYC1, HIS3, 2 micron,



bla, pUC ori









Plasmid pGV1103 was generated by inserting a linker (primers 271 annealed to primer 272) containing a myc-tag and a new MCS (SalI-EcoRI-SmaI-BamHI-NotI) into the SpeI and XhoI sites of p423GPD. The construction of plasmid pGV1730 is described in Example 4.
pGV1914 (SEQ ID NO: 117) is a yeast integrating vector that includes the S. cerevisiae URA3 gene as a selection marker and contains homologous sequence for targeting the HpaI-digested, linearized plasmid for integration at the PDC6 locus of S. cerevisiae. pGV1914 carries the D. melanogaster adh (Dm_ADH) (SEQ ID NO: 116) and L. lactis kivd (Ll_kivD) genes, expressed under the control of the S. cerevisiae TDH3 and TEF1 promoters, respectively. The open reading frame sequence of DmADH was originally amplified by PCR from clone RH54514 (available from the Drosophila Genome Resource Center).
Plasmid pGV1974 is a yeast high copy plasmid with HIS3 as a marker for the expression of E. coli ilvC110V (SEQ ID NO: 98) and S. cerevisiae ILV3ΔN (SEQ ID NO: 89). pGV1974 was generated by cloning a SacI-NotI fragment (4.9 kb, SEQ ID NO: 118) carrying the S. cerevisiae TEF1 promoter: S. cerevisiae ilv3ΔN: S. cerevisiae TDH3 promoter: E. coli ilvCQ110V into the SacI-NotI sites of pGV1103 (5.4 kb), a yeast expression plasmid carrying the HIS3 marker.
Plasmid pGV1981 is a yeast high copy plasmid with HIS3 as a marker for the expression of E. coli ilvCQ110V and L. lactis ilvD. pGV1981 was generated by cloning a SalI-BamHI fragment (1.7 kb) carrying the L. lactis ilvD ORF (SEQ ID NO: 87 with a SalI and BamHI sites introduces at the 5′ and 3′ ends, respectively) into the SalI-BamHI of pGV1974 (8.5 kb), replacing the S. cerevisiae Ilv34N ORF.
Plasmid pGV2001 is a yeast high copy plasmid with HIS3 as a marker for the expression of E. coli ilvVQ110V. pGV2001 was generated by digesting pGV1974 with SalI-BamHI to remove the S. cerevisiae Ilv3ΔN ORF. The digest was treated with Klenow to fill-in the 5′ overhangs, the larger 8.5 kb fragment was isolated and self-ligated.
GEVO1803 was made by transforming GEVO1186 with the 6.7 kb pGV1730 (contains S. cerevisiae TRP1 marker and the CUP1 promoter-driven B. subtilis alsS) that had been linearized by digestion with NruI. Completion of the digest was confirmed by running a small sample on a gel. The digested DNA was then purified using Zymo Research DNA Clean and Concentrator and used in the transformation. Trp+ clones were confirmed for the correct integration into the PDC1 locus by colony PCR using primer pairs 1440+1441 and 1442+1443 for the 5′ and 3′ junctions, respectively. Expression of B. subtilis alsS was confirmed by qRT-PCR using, primer pairs 1323+1324.
GEVO2107 was made by transforming GEVO1803 with linearized, HpaI-digested pGV1914. Correct integration of pGV1914 at the PDC6 locus was confirmed by analyzing candidate Ura+ colonies by colony PCR using primers 1440 plus 1441, or 1443 plus 1633, to detect the 5′ and 3′ junctions of the integrated construct, respectively. Expression of all transgenes were confirmed by qRT-PCR using primer pairs 1321 plus 1322, 1587 plus 1588, and 1633 plus 1634 to examine Bs_alsS, Ll_kivD, and Dm_ADH transcript levels, respectively.
GEVO 2107 was transformed with plasmids that contained either a KARI alone (pGV2001 with E. coli ilvCQ110V) or the same KARI with a DHAD (pGV1974 with the S. cerevisiae Ilv3ΔN or pGV1981 with the L. lactis ilvD). Fermentations were carried out with three independent transformants for each DHAD homolog being tested, as well as the no DHAD control plasmid. Seed cultures were grown in SCD-H medium to mid-log phase. The fermentations were initiated by collecting cells and resuspending in 25 mL of SCD-H (5% glucose) medium to an OD600 of 1. Fermentations were performed aerobically in 125 mL unbaffled flasks shaken at 250 rpm at 30° C. At 0, 24, 48 and 72 hours, the OD600 was checked and 2 mL samples were taken. These samples were centrifuged at 18,000×g in a microcentrifuge and 1.5 mL of the clarified media was transferred to a 1.5 mL Eppendorf tube. The clarified media was stored at 4° C. until analyzed by GC and HPLC as described in General Methods. At 24 and 48 hours, 2.5 mL of glucose from a 400 g/L stock solution was added to the cultures. FIG. 4 shows the production of isobutanol in these fermentations. All values were adjusted for the dilution caused by the volume change from adding glucose. An increased amount of isobutanol was produced from the cells expressing the L. lactis ilvD.
Example 11
Assaying DHAD Activity in Fractionated Cell Extracts
The purpose of this Example is to describe how DHAD activity can be measured in fractionated cellular extracts that are enriched for either mitochondrial or soluble cytosolic components.
Plasmids pGV1106, pGV1662, pGV1855, pGV1900 are described in Example 9 above. To measure the DHAD activities present in fractionated cell extracts, the strain GEVO2244 was transformed singly with either pGV1106, which served as an empty vector control, or with one of: pGV1855, pGV1900, or pGV2019, which are expression plasmids for L. lactis IlvD, S. cerevisiae ILV3 (full length), and S. cerevisiae ILV3ΔN, respectively.
An independent clonal transformant of each plasmid was isolated, and a 1 L culture of each strain was grown in SCGal-Ura+9xIV at 30° C. at 250 rpm. The OD600 was noted, the cells were collected by centrifugation (1600×g, 2 min) and the culture medium was decanted. The cell pellets were resuspended in 50 mL sterile deionized water, collected by centrifugation (1600×g, 2 min), and the supernatant was discarded. The OD600 and total wet cell pellet weight of each culture are listed in Table 24, below:







TABLE 24







OD600 and pellet mass (g) of strain GEVO2244


transformed with the indicated plasmids.













Pellet mass



Plasmid
OD600
(g)






pGV1106
2.2
7.6



pGV1855
2.3
7.7



pGV1900
1.3
3.8



pGV2019
2.6
8.4









To obtain spheroplasts, the cell pellets were resuspended in 0.1M Tris-SO4, pH 9.3 to a final concentration of 0.1 g/mL, and DTT was added to a final concentration of 10 mM. Cells were incubated with gentle (60 rev/min) agitation on an orbital shaker for 20 min at 30° C., and the cells were then collect by centrifugation (1600×g, 2 min) and the supernatant discarded. Each cell pellet was resuspended in spheroplasting buffer, which consists of (final concentrations): 1.2M sorbitol (Amresco, catalog #0691), 20 mM potassium phosphate pH 7A) and then collected by centrifugation (1600×g, 10 min). Each cell pellet was resuspended in spheroplasting buffer to a final concentration of 0.1 g cells/mL in a 500 mL centrifuge bottle, and 50 mg of Zymolyase 20T (Seikagaku Biobusiness, Code#120491) was added to each cell suspension. The suspensions were incubated overnight (approximately 16 hrs) at 30° C. with gentle agitation (60 rev/min) on an orbital shaker. The efficacy of spheroplasting was ascertained by diluting an aliquot of each cell suspension 1:10 in either sterile water or in spheroplasting buffer, and comparing the aliquots microscopically (under 40× magnification). In all cases, >90% of the water-diluted cells lysed, indicating efficient spheroplasting. The spheroplasts were centrifuged (3000×g, 10 min, 20° C.), and the supernatant was discarded. Each cell pellet was resuspended in 50 mL spheroplast buffer without Zymolyase, and cells were collected by centrifugation (3000×g, 10 min, 20° C.).
To fractionate spheroplasts, the cells were resuspended to a final concentration of 0.5 g/mL in ice cold mitochondrial isolation buffer (MIB), consisting of (final concentration): 0.6M D-mannitol (BD Difco Cat#217020), 20 mM HEPES-KOH, pH 7.4. For each 1 mL of resulting cell suspension, 0.01 mL of Yeast/Fungal Protease Arrest solution (G Biosciences, catalog #788-333) was added. The cell suspension was subjected to 35 strokes of a Dounce homogenizer with the B (tight) pestle, and the resulting cell suspension was centrifuged (2500×g, 10 min, 4° C.) to collect cell debris and unbroken cells and spheroplasts. Following centrifugation, 2 mL of each sample (1 mL of the pGV1900 transformed cells) were saved in a 2 mL centrifuge tube on ice and designated the “W” (for Whole cell extract) fraction, while the remaining supernatant was transferred to a clean, ice-cold 35 mL Oakridge screw-cap tube and centrifuged (12,000×g, 20 min, 4° C.) to pellet mitochondria and other organellar structures. Following centrifugation, 5 mL of each resulting supernatant was transferred to a clean tube on ice, being careful to avoid the small, loose pellet, and labelled the “S” (soluble cytosol) fraction. The resulting pellets were resuspended in MIB containing Protease Arrest solution, and were labelled the “P” (“pellet”) fractions. Protein from the “P” fraction was released after dilution 1:5 in DHAD assay buffer (see above) by rapid mixing in a 1.5 mL tube with a Retsch Ball Mill MM301 in the presence of 0.1 mM glass beads. The bead-beating was performed 4 times for 1 minute, 30 beats per second, after which insoluble debris was removed by centrifugation (20,000×g, 10 min, 4° C.) and the soluble portion retained for use.
The BioRad Protein Assay reagant (BioRad, Hercules, Calif.) was used according to manufacturer's instructions to determine the protein concentration of each fraction; the data are summarized in Table 25, below:







TABLE 25







Protein concentrations of mitochondrial/organellar


(P) and cytosolic (S) fractions and whole cell


(W) lysates, prepared as described in the text.










plasmid/fraction
protein [μg/μL]













1106 P
20.3



1855 P
17.7



1900 P
9.2



2019 P
19.7



1106 S
12.3



1855 S
12.9



1900 S
7.9



2019 S
12.4



1106 W
14.0



1855 W
15.0



1900 W
7.9



2019 W
14.7









The DHAD activity of each fraction was ascertained as follows. In a fresh 1.5 mL centrifuge tube, 50 μL of each fraction was mixed with 50 μL of 0.1M 2,3-dihydroxyisovalerate (DHIV), 25 μL of 0.1M MgSO4, and 375 μL of 0.05M Tris-HCl pH 8.0, and the mixture was incubated for 30 min at 35° C. Each reaction was carried out in triplicate. Each tube was then heated to 95° C. for 5 min to inactivate any enzymatic activity, and the solution was centrifuged (16,000×g for 5 min) to pellet insoluble debris. To prepare samples for analysis, 100 μL of each reaction were mixed with 100 μL of a solution consisting of 4 parts 15 mM dinitrophenyl hydrazine (DNPH) in acetonitrile with 1 part 50 mM citric acid, pH 3.0, and the mixture was heated to 70° C. for 30 min in a thermocycler. Analysis of ketoisovalerate via HPLC was carried out as described in General Methods. Data from the experiment are summarized below in Table 26.







TABLE 26







Specific activities (KIV generation) and ratios of specific activities


from fractionated lysates of S. cerevisiae strain GEVO2244 carrying


plasmids to overexpress the indicated DHAD homolog.













Sp. Activity

Ratio of Sp.


Lysate

[U/mg

Activities


(pGV# and

protein in
Std.
(Cyto or Mito


fraction*)
DHAD
fraction]
Dev.
to Whole-Cell)





1106 WCL
—
n.d.




1106 cyto
—
n.d.




1106 mito
—
n.d.




1855 WCL
Ll_ilvD
0.0006
4.7E−05



1855 cyto
Ll_ilvD
0.0011
0.0001
1.76


1855 mito
Ll_ilvD
2E−05
3.5E−05
0.03


1900 WCL
ScILV3(FL)
0.0096
0.0018



1900 cyto
ScILV3(FL)
0.0052
0.0004
0.54


1900 mito
ScILV3(FL)
0.0340
0.0029
3.53





Each data point is the result of triplicate samples.


*WCL, whole cell lysate; cyto, cytosolic-enriched fraction; mito, mitochondrial (organellar)-enriched fraction






Cells overexpressing the L. lactis ilvD generated a significantly greater proportion of DHAD activity in the cytosolic fraction versus the mitochondrial fraction, whereas cells overexpressing the full-length, native (mitochondrial) S. cerevisiae ILV3 resulted in a greater proportion of the specific activity residing in the mitochondrial fraction.
Example 12
Alternative, Native Dehydratases with DHAD Activity
This example describes how the overexpression of native dehydratases in S. cerevisiae for the conversion of 2,3-dihydroxyisovalerate to ketoisovalerate is measured.







TABLE 27







Plasmids disclosed in Example 12.








pGV No.
Genotype





p426TEF
PTEF1:MCS:TCYC1, URA3, 2-micron, bla, pUC-ori



(Mumberg, D. et al. (1995) Gene 156:119-122;



obtained from ATCC)


1102
PTEF1:HA-tag:MCS:TCYC1, URA3, 2-micron, bla,



pUC-ori


1106
PTDH3:myc-tag:MCS:TCYC1, URA3, 2-micron, bla,



pUC-ori


1662
PTEF1:Ll_kivd:TCYC1, URA3, 2-micron, bla,



pUC-ori


1894
PTEF1:Ec_ilvCQ110V-coSc:TCYC1, URA3, 2-micron,



bla, pUC-ori


2000
PTEF1:Sc_ILV3ΔN:PTDH3:Ec_ilvCQ110V-coSc:TCYC1,



URA3, 2-micron, bla, pUC-ori


2111
PTEF1:Ll_ilvD:PTDH3:Ec_ilvCQ110V-coSc:TCYC1,



URA3, 2-micron, bla, pUC-ori


2112
PTEF1:Sc_LEU1:PTDH3:Ec_ilvCQ110V-coSc:TCYC1,



URA3, 2-micron, bla, pUC-ori


2113
PTEF1:Sc_HIS3:PTDH3:Ec_ilvCQ110V-coSc:TCYC1,



URA3, 2-micron, bla, pUC-ori









Plasmid pGV1102 was generated by inserting a linker (primers 269 annealed to primer 270) containing a HA-tag and a new MCS (SalI-EcoRI-SmaI-BamHI-NotI) into the SpeI and XhoI sites of p426TEF. Plasmids pGV1106 and pGV1662 are described in Examples 3 and 5, respectively. Plasmid pGV1894 is a yeast high copy plasmid with URA3 as a marker for the expression of E. coli ilvCQ110V and was generated by cloning a XhoI-NotI fragment (1.5 kb) carrying the E. coli ilvCQ110V ORF (SEQ ID NO: 98) into the SalI-NotI of pGV1:662 (6.3 kb), replacing the L. lactis kivD ORF. Plasmids pGV2000, pGV2111, pGV2112, and pGV2113 are yeast high copy plasmids with URA3 as a marker for the expression of E. coli ilvCQ110V and a DHAD. pGV2000 is generated by cloning a SacI-NotI fragment (4.9 kb) from pGV1974 (described in Example 10) carrying the S. cerevisiae TEF1 promoter: S. cerevisiae Ilv3ΔN: S. cerevisiae TDH3 promoter: E. coli ilvCQ110V into the SacI-NotI sites of pGV1106 (6.6 kb), a yeast expression plasmid carrying the URA3 marker. pGV2111 is generated by cloning a SalI-BamHI fragment (1.7 kb) carrying the L. lactis ilvD ORF (SEQ. ID NO: 97 with SalI and BamHI sites introduced at the 5′ and 3′ ends, respectively) into the SalI-BamHI of pGV2000 (8.4 kb), replacing the S. cerevisiae Ilv3ΔN ORF. pGV2112 is generated by cloning the S. cerevisiae LEU1 gene as a SalI-BamHI fragment (2.3 kb), generated by PCR using primers 2163 and 1842 using genomic DNA as template, into the SalI-BamHI of pGV2000 (8.4 kb), replacing the S. cerevisiae Ilv3ΔN ORF. pGV2113 is generated by cloning the S. cerevisiae HIS3 gene as a SalI-BamHI fragment (0.7 kb), generated by PCR using primers 2183 and 2184 using genomic DNA as template, into the SalI-BamHI of pGV2000 (8.4 kb), replacing the S. cerevisiae Ilv3ΔN ORF.
DHADs are tested for in vitro activity using whole cell lysates. The DHADs as well as LEU1 and HIS3 are expressed from pGV2000, pGV2112, and pGV2113 GEVO2244 to minimize endogenous DHAD background activity. A plasmid that does not express DHAD, pGV1894, and a plasmid that expresses the L. lactis ilvD, pGV2111, are used as negative and positive controls, respectively
To grow cultures for cell lysates, triplicate independent cultures of each desired strain are grown overnight in 3 mL YNBD+HLW+10xIV at 30° C., 250 rpm. The following day, the overnight cultures are diluted 1:50 into 50 mL fresh YNBD+HLW+10xIV in a 250 mL baffle-bottomed Erlenmeyer flask and incubated at 30° C. at 250 rpm. After approximately 10 hours, the OD600 of all cultures are measured, and the cells of each culture are collected by centrifugation (2700×g, 5 min). The cell pellets are washed by resuspending in 1 mL of water, and the suspension is placed in a 1.5 mL tube and the cells are collected by centrifugation (16,000×g, 30 seconds). All supernatant is removed from each tube and the tubes are frozen at −80° C. until use.
Lysates are prepared by resuspending each cell pellet in 0.7 ml of lysis buffer. Lysate lysis buffer consisted of: 0.1M Tris-HCl pH 8.0, 5 mM MgSO4, with 10 μL of Yeast/Fungal Protease Arrest solution (G Biosciences, catalog #788-333) per 1 mL of lysis buffer. Eight hundred microliters of cell suspension are added to 1 mL of 0.5 mm glass beads that had been placed in a chilled 1.5 mL tube. Cells are lysed by bead beating (6 rounds, 1 minute per round, 30 beats per second) with 2 minutes chilling on ice in between rounds. The tubes are then centrifuged (20,000×g, 15 min) to pellet debris and the supernatant (cell lysates) are retained in fresh tubes on ice. The protein concentration of each lysate is measured using the BioRad Bradford protein assay reagent (BioRad, Hercules, Calif.) according to manufacturer's instructions.
The DHAD activity of each lysate is ascertained as follows. In a fresh 1.5 mL centrifuge tube, 50 μL of each lysate is mixed with 50 μL of 0.1M 2,3-dihydroxyisovalerate (DHIV), 25 μL of 0.1M MgSO4, and 375 μL of 0.05M Tris-HCl pH 8.0, and the mixture is incubated for 30 min at 35° C. Each tube is then heated to 95° C. for 5 min to inactivate any enzymatic activity, and the solution is centrifuged (16,000×g for 5 min) to pellet insoluble debris. To prepare samples for analysis, 100 μL of each reaction are mixed with 100 μL of a solution consisting of 4 parts 15 mM dinitrophenyl hydrazine (DNPH) in acetonitrile with 1 part 50 mM citric acid, pH 3.0, and the mixture is heated to 70° C. for 30 min in a thermocycler. The solution is then analyzed by HPLC as described above in General Methods to quantitate the concentration of ketoisovalerate (KIV) present in the sample.
DHADs are tested for in vitro activity using whole cell lysates. The DHADs are expressed in a yeast deficient for DHAD activity (GEVO2244; ilv3Δ) to minimize endogenous background activity.
Example 13
Cloning of Low-Abundance, Endogenous Cytosolic Iron-Sulfur Cluster Assembly Machinery for Overexpression in S. Cerevisiae 
The purpose of this example is to describe how three known components of the S. cerevisiae cytosolic iron-sulfur assembly machinery were cloned to permit their overexpression in S. cerevisiae, to increase cytosolic DHAD activity.
In the yeast S. cerevisiae, at four least genes—CIA1, CFD1, NAR1, and NBP35—encode activities that contribute to the proper assembly and/or transfer of iron-sulfur [Fe—S] clusters of cytosolic proteins. Of these four genes, three—CFD1, NAR1, and NBP35—have been shown to be expressed at very low levels during aerobic growth on glucose (Ghaemmaghami et al., 2003, Nature, 425: 737-741). These three genes thus represent attractive candidates for overexpression to increase the cellular capacity for proper cytosolic [Fe—S] cluster protein assembly.







TABLE 27







Plasmids disclosed in Example 13.








pGV No.
Genotype





pGV2074
pUC ori, bla (AmpR), 2 μm ori, TPI1 promoter-hph



(HygroR), PGK1 promoter, TEF1 promoter, TDH3 promoter


pGV2127
pUC ori, bla (AmpR), 2 μm ori, TPI1 promoter-hph



(HygroR), PGK1 promoter, TEF1 promoter, TDH3



promoter-CFD1


pGV2138
pUC ori, bla (AmpR), 2 μm ori, TPI1 promoter-hph



(HygroR), PGK1 promoter, TEF1 promoter-NAR1, TDH3



promoter-CFD1


pGV2144
pUC ori, bla (AmpR), 2 μm ori, TPI1 promoter-hph



(HygroR), PGK1 promoter-NBP35, TEF1 promoter,



TDH3 promoter


pGV2147
pUC ori, bla (AmpR), 2 μm ori, TPI1 promoter-hph



(HygroR), PGK1 promoter-NBP35, TEF1 promoter-NAR1,



TDH3 promoter-CFD1









To clone the sequences for CFD1, NAR1, and NBP35 into an appropriate S. cerevisiae expression vector, the following steps were carried out: Vector pGV2074 (SEQ ID NO: 133) was used as a parental plasmid for subsequent cloning steps described below. The salient features of pGV2074 include a bacterial origin of replication (pUC) and selectable marker (bla), an S. cerevisiae 2 μm origin of replication and selectable marker (the hph gene, conferring resistance to hygromycin, operably linked to the TPI1 promoter region), and sequences containing the S. cerevisiae promoters for the PGK1, TDH3 and TEF1 genes, each followed by one or more unique restriction sites to facilitate the introduction of coding sequences.
First, the CFD1 coding sequence was amplified from S. cerevisiae genomic DNA by PCR, using primers 2195 and 2196, which also added 5′ XhoI and 3′ NotI sites, respectively. The resulting  ̃890 bp product was digested with XhoI plus NotI and ligated into pGV2074 that had been digested with XhoI plus NotI, yielding the plasmid pGV2127. All sequences amplified by PCR were confirmed by DNA sequencing. Next, the NAR1 coding sequence was amplified from S. cerevisiae genomic DNA by PCR, using primers 2197 and 2198, which added 5′ SalI and 3′ BamHI sites, respectively. The resulting  ̃1485 bp product was digested with SalI plus BamHI and cloned into pGV2127 which had also been digested with SalI plus BamHI, thereby yielding pGV2138. Next, the NBP35 coding sequence was amplified S. cerevisiae genomic DNA by PCR, using primers 2259 and 2260, which added 5′ BglII and 3′ KpnI and XhoI (from 5′ to 3′) sites, respectively. The resulting  ̃995 bp product was digested with BglII plus XhoI and ligated into pGV2074 that had been digested with BglII plus SalI, yielding pGV2144. Finally, pGV2144 was digested with AvrII plus BamHI, and the resulting 1.78 kb fragment (which contained the PGK1 promoter and the NBP35 ORF sequence) was gel purified and ligated into the vector pGV2138 that had been digested with AvrII plus BglII, yielding pGV2147.
Example 14
Cloning of Heterologous Cytosolic Iron-Sulfur Cluster Assembly Machinery for Overexpression in S. Cerevisiae 
The purpose of this example is to describe how one or more cytosolic iron-sulfur assembly machinery components, from various species, can be cloned to permit their overexpression in S. cerevisiae, thereby increasing cytosolic DHAD activity.
In addition to the endogenous cytosolic iron-sulfur assembly machinery found in S. cerevisiae, homologous sequences and activities have been identified in other microbial and eukaryotic species. In one example, the ApbC protein of Salmonella enterica serovar Typhimurium has been shown, in vitro, to bind and effectively transfer iron-sulfur clusters to a known cytosolic [Fe—S] cluster-containing S. cerevisiae substrate, Leu1 (Boyd et al., 2008, Biochemistry, 47: 8195-202). Thus, a number of other useful homologs of the known S. cerevisiae cytosolic iron-sulfur assembly machinery components exist and present attractive candidates for overexpression in S. cerevisiae. Table 28 lists several exemplary homologs and their GenBank accession numbers, as identified by previous homology searches (Boyd et al., 2009, J. Biol Chem 284: 110-118). Also included in the table are two closely related S. cerevisiae homologs, Nbp35 and Cfd1. Of note, Ind1 is reported to be localized to and functional in the mitochondria (Bych et al., 2008, EMBO J. 27: 1736-46), whereas Hcf101 is reported to participate in iron-sulfur cluster assembly in Arabidopsis chloroplasts (Lezhneva et al., 2004, Plant J. Cell Mol Biol 37: 174-185).







TABLE 28







Functionally homologous proteins involved


in iron-sulfur cluster formation.








Gene
Source, Accession Number





ApbC
Salmonella enterica serovar Typhimurium LT2, NP_461098


Ind1
Yarrowia lypolytica, YALI0B18590g


Hcf101
Arabidopsis thaliana, AAR97892.1


Nubp1
Homo sapiens, NP_002475.2


Nbp35
S. cerevisiae, CAA96797.1


Cfd1
S. cerevisiae, AAS56623









The cloning of one or more of these genes is carried out using techniques well known to one skilled in the art. Oligonucleotide primers are designed that are homologous to the 5′ and 3′ ends of each desired reading, and which furthermore incorporate a restriction site sequence convenient for the cloning of each reading frame into vector pGV2074. A standard PCR reaction is used to amplify each gene, either from the genome of each host organism, or from an in vitro synthesized DNA fragment, and the resulting PCR product is cloned into an expression vector (pGV2074). In the case of a protein known to be targeted to the mitochondria, such as Yarrowia lypolytica Ind1, PCR primers are designed to amplify the majority of the coding sequence while excluding the known N-terminal mitochondrial targeting sequence (Bych et al., 2008, EMBO J. 27: 1736-46).
Example 15
Overexpression of S. Cerevisiae Cytosolic Iron-Sulfur Assembly Machinery to Increase Cytosolic DHAD Activity
The purpose of this example is to describe how a plasmid expressing one or more iron-sulfur assembly machinery components is co-expressed with a DHAD, thereby increasing the cytosolic activity of the DHAD.
Strain GEVO2244 is simultaneously co-transformed with one of: pGV1851, pGV1852, pGV1853, pGV1854, pGV1855, pGV1904, pGV1905, pGV1906, or pGV1907 (pGV1851-55 and pGV1904-07 are described in Table 20); plus, one of either: pGV2074 (Table 27) (which serves as an empty-vector control) or pGV2147 (Table 27) (which serves as the cytosolic Fe—S cluster machinery overexpression plasmid), and doubly-transformed cells are selected by plating onto SCD-Ura+9xIV containing 0.1 g/L Hygromycin B.
Three independent isolates from each transformation are cultured in SCD-Ura+9xIV containing 0.1 g/L Hygromycin B to obtain a cell mass suitable for preparation of a lysate, as described in Example 3. Lysates are prepared from each culture, and the resulting lysates are assayed for DHAD activity as described in Example 3. To further confirm that the increased DHAD activity is due specifically to increased cytosolic activity, cultures of GEVO2244 containing pGV1855 plus either pGV2074 or pGV2147 are grown in SCD-Ura+9xIV containing 0.1 g/L Hygromycin B as otherwise described in Example 11. Fractionated lysates are prepared and in vitro assays to measure DHAD activity are further carried out as described in Example 11.
Example 16
Deletion of LEU1
The purpose of this example is to describe the deletion of LEU1 to increase the iron-sulfur cluster availability in the yeast cytosol.







TABLE 29







Plasmids disclosed in Example 16.










pGV No.
Genotype






pGV1299
K. lactis URA3, bla, pUC-ori (GEVO)



PGV1981
PTEF1:Lactococcus lactis ilvD-coSc:PTDH3:




Ec_ilvCQ110V-coSc:TCYC1, HIS3, 2-micron, bla,




pUC-ori



pGV2001
PTEF1:PTDH3:Ec_ilvCQ110V-coSc:TCYC1, HIS3,




2-micron, bla, pUC-ori









The LEU1 gene was deleted by transforming cells with a leu1::K. lactis URA3 deletion cassette that was generated by two rounds of PCR. Initially, the K. lactis URA3 gene was amplified with primers 2171 and 2172 from pGV1299 (described in Example 2). These primers add 40 bp of the LEU1 promoter and terminator sequences to the 5′ and 3′ ends of the K. lactis URA3 gene. This PCR product was then used as a template for a PCR using primers 2170 and 2173. Primer 2170 adds an additional 36 bp of the LEU1 promoter sequence at the 5′ end and primer 2173 adds an additional 38 bp of the LEU1 terminator sequence at the 3′ end. This PCR product was transformed into GEVO2244 (described in Example 2) to generate GEVO2570. The 5′ junction of the integrations were confirmed by colony PCR using primers 2226 and 587. The 3′ junction of the integrations were confirmed by colony PCR using primers 588 and 2175. The loss of the LEU1 gene was confirmed by a lack of PCR product using primers 2167 and 2227.
GEVO2570 has a deletion in ILV3. GEVO2570 is used to measure DHAD activity in the presence of L. lactis ilvD overexpressed as described in Examples 2 and 4. A plasmid (pGV2001) with no DHAD is used as a negative control.
Example 17
Conserved Motif Amongst Cytosolically Active DHAD Enzymes
This example illustrates that a DHAD enzymes with a specific amino acid sequence motif are more likely to be functional when expressed in the yeast cytosol.
Based on the data from biochemical assays (see Example 10), several DHAD homologs were identified that exhibit at least some cytosolic activity. A total of ten different homologs were tested using biochemical assays. The DHADs were expressed from 2 micron yeast vectors and transformed into GEVO2244. The homologs were then ranked based on their measured specific activity in both whole cell lysates and in cytosolic fractions.
Based on these data, four DHAD homologs: L. lactis (SEQ ID NO: 18), G. forsetii (SEQ ID NO: 17), Acidobacteria (SEQ ID NO: 16), and S. erythraea (SEQ ID NO: 19) exhibit cytosolic DHAD activity. Four DHAD homologs exhibit no cytosolic DHAD activity: R. eutropha (SEQ ID NO: 22), C. salexigens (SEQ ID NO: 23), P. torridus (SEQ ID NO: 24), and S. tokodaii (SEQ. ID NO: 25). One motif-containing homolog was inconclusive: Piromyces sp. E2 (SEQ ID NO: 21), which did not complement the GEVO2242 valine auxotrophy and had detectable biochemical DHAD activity. Since, this homolog has a putative organellar targeting sequence, the protein is likely to be mitochondrially located explaining its inability to complement the GEVO2242 auxotrophy, despite containing the motif.
A multiple sequence alignment (MSA) was created using the Align Multiple Sequences tool of Clone Manager 9 Professional Addition Software using the “Multi-Way” function. This function performs exhaustive pairwise global alignments of all sequences and progressive assembly of alignments using Neighbor-Joining phylogeny. A total of 53 representative DHAD homologs (FIG. 5) were aligned using the following using the BLOSUM62 scoring matrix setting. This alignment generated the tree in FIG. 5.
Many of the DHAD homologs exhibiting cytosolic activity are related by overall homology (>40%) homology when compared to the S. cerevisiae DHAD encoded by S. cerevisiae ILV3 (e.g. L. lactis, G. forsetii, Acidobacteria, and S. erythraea). However, the 40% homology cut-off still includes several DHAD homologs that do not exhibit cytosolic DHAD activity (e.g. R. eutropha, C. salexigens, P. torridus, and S. tokodaii). The Piromyces sp. E2 DHAD failed to complement in the genetic/biochemistry assay but this result is still consistent with our motif hypothesis since the protein still retained its mitochondrial localization signal. Therefore, a common sequence motif, unique to DHAD homologs that are cytosolically active, was identified: P(I/L)XXXGX(I/L)XIL (SEQ ID NO: 27), where (I/L) indicates an isoleucine or leucine at that position, and X indicates any natural or non-natural amino acid. This motif can be found in all DHAD homologs exhibiting cytosolically activity. Furthermore, an even more specific version of this motif was identified that is conserved in all of DHAD homologs exhibiting cytosolic activity except for the S. erythraea DHAD: PIKXXGX(I/L)XIL (SEQ ID NO: 28). This motif is conserved amongst the majority if not all eukaryotic homologs of DHAD.
Six additional DHAD homologs were identified: SEQ ID NOs: 10-15 as specified in Table 1. These DHAD homologs (SEQ ID NOs: 10-15) contain the motifs PYHKEGGLGIL (SEQ ID NO: 145), PYSEKGGLAIL (SEQ ID NO: 146), PYKPEGGIAIL (SEQ ID NO: 147), PLKPSGHLQIL (SEQ ID NO: 148), PIKKTGHLQIL (SEQ. ID NO: 149), and PIKETGHIQIL (SEQ ID NO: 150), respectively.
Example 18
Use of Cytosolically Localized DHADs for the Production of Isobutanol
The following example illustrates the use of DHADs that have cytosolic activity in yeast and when expressed in the context of an isobutanol biosynthetic pathway lead to isobutanol production.
A yeast strain that contains one integrated copy of the B. subtilis alsS gene codon-optimized for expression in S. cerevisiae (SEQ ID NO: 144), two integrated copies of the L. lactis kivD gene (SEQ ID NOs: 99 and 151), one integrated copy of L. lactis adhARE1 gene (SEQ ID NO: 152), and one integrated copy of the S. cerevisiae AFT1 gene (SEQ ID NO: 153) was transformed with high copy three-component isobutanol pathway plasmids containing a KARI (Ec_ilvC_coScP2D1-A1-his6, SEQ ID NO: 154), an ADH (L. lactis adhARE1, SEQ ID NO: 152) and a DHAD which was expressed from the S. cerevisiae PDC1-286 promoter. The DHAD varied according to Table 31. Isobutanol titer and DHAD activity of each strain was compared to that of a control strain that did not express a DHAD in the plasmid. Strains, plasmids, and DHADs are listed in Tables 30, 31, and 32, respectively.







TABLE 30







Genotype of strains disclosed in Example 18.








GEVO No.
Genotype





GEVO3868
S. cerevisiae, CEN.PK2, MATa ura3 leu2 his3 trp1



gpd1::TKl_URA3 gpd2::TKl_URA3 tma29::TKl_URA3 pdc1::PPDC1-



Ll_kivD2_coSc5-PFBA1-LEU2-TLEU2-PADH1-Bs_alsS1_coSc-



TCYC1-PPGK1-Ll_kivD2_coEc-PENO2-Sp_HIS5 pdc5::TKl_URA3



pdc6::PTDH3-Sc_AFT1-PENO2-Ll_adhARE1-T-Kl_URA3_short-PFBA1-



Kl_URA3-TKl_URA3 {evolved for C2 supplement-



independence, glucose tolerance and faster growth}
















TABLE 31







Plasmids disclosed in Example 18.









Plasmid




Name
DHAD
Genotype





pGV2663
none
PTDH3-Ec_ilvC_coScP2D1-A1-his6,




PENO2-Ll_adhARE1, 2μ-ori,




pUC ori, bla, G418r


pGV2635
L. lactis
PPDC1-286-Ll_ilvD_coSc,




PTDH3-Ec_ilvC_coScP2D1-A1-his6,




PENO2-Ll_adhARE1, 2μ-ori,




pUC ori, bla, G418r


pGV2671
S. cerevisiae
PPDC1-286-Sc_ilv3_ΔN20,




PTDH3-Ec_ilvC_coScP2D1-A1-his6,




PENO2-Ll_adhARE1, 2μ-ori,




pUC ori, bla, G418r


pGV2672
G. forsetii
PPDC1-286-Gf_ilvD_coSc,




PTDH3-Ec_ilvC_coScP2D1-A1-his6,




PENO2-Ll_adhARE1, 2μ-ori,




pUC ori, bla, G418r


pGV2673
S. erythraea
PPDC1-286-Se_ilvD_coSc,




PTDH3-Ec_ilvC_coScP2D1-A1-his6,




PENO2-Ll_adhARE1, 2μ-ori,




pUC ori, bla, G418r


pGV2674
F. tularensis
PPDC1-286-Ft_ilvD_coSc,




PTDH3-Ec_ilvC_coScP2D1-A1-his6,




PENO2-Ll_adhARE1 2μ-ori,




pUC ori, bla, G418r


pGV2675
S. cerevisiae
PPDC1-286-Sc_ilv3_ΔN19,



ilv3ΔN19
PTDH3-Ec_ilvC_coScP2D1-A1-his6,




PENO2-Ll_adhARE1 2μ-ori,




pUC ori, bla, G418r


pGV2676
S. cerevisiae
PPDC1-286-Sc_ilv3_ΔN23,



ilv3ΔN23
PTDH3-Ec_ilvC_coScP2D1-A1-his6,




PENO2-Ll_adhARE1 2μ-ori,




pUC ori, bla, G418r


pGV2677
N. crassa
PPDC1-286-Nc_ilvD2_coSc,



ilvD2
PTDH3-Ec_ilvC_coScP2D1-A1-his6,




PENO2-Ll_adhARE1 2μ-ori,




pUC ori, bla, G418r


pGV2678
Acidobacteria
PPDC1-286-Ab_ilvD_coSc,



bacterium
PTDH3-Ec_ilvC_coScP2D1-A1-his6,




PENO2-Ll_adhARE1 2μ-ori,




pUC ori, bla, G418r


pGV2679
Acaryochloris
PPDC1-286-Am_ilvD_coSc,



marina
PTDH3-Ec_ilvC_coScP2D1-A1-his6,




PENO2-Ll_adhARE1 2μ-ori,




pUC ori, bla, G418r


pGV2680
Lyngbya spp.
PPDC1-286-Lsp_ilvD_coSc,




PTDH3-Ec_ilvC_coScP2D1-A1-his6,




PENO2-Ll_adhARE1 2μ-ori,




pUC ori, bla, G418r


pGV2681
E. coli
PPDC1-286-Ec_ilvD_coKl,




PTDH3-Ec_ilvC_coScP2D1-A1-his6,




PENO2-Ll_adhARE1 2μ-ori,




pUC ori, bla, G418r
















TABLE 32







DHAD sequences disclosed in Example 18.












SEQ ID NO
SEQ ID NO


DHAD
Abbreviation
(DNA)
(protein)













L. lactis
Ll_ilvD_coSc
155
18


S. cerevisiae
Sc_ilv3_ΔN20
89
26


ilv3ΔN20





G. forsetii
Gf_ilvD_coSc
90
17


S. erythraea
Se_ilvD_coSc
91
19


F. tularensis
Ft_ilvD_coSc
156
14


S. cerevisiae
Sc_ilv3_ΔN19
157
163


ilv3ΔN19





S. cerevisiae
Sc_ilv3_ΔN23
158
164


ilv3ΔN23





N. crassa
Nc_ilvD2_coSc
159
165


ilvD2





A. bacterium
Ab_ilvD_coSc
92
16


A. marina
Am_ilvD_coSc
160
166


Lyngbya spp.
Lsp_ilvD_coSc
161
167


E. coli
Ec_ilvD_coKl
162
168









Cloning techniques included digestion with restriction enzymes, gel purification of DNA fragments (using the Zymoclean Gel DNA Recovery Kit, Cat# D4002, Zymo Research Corp, Orange, Calif.), ligation of two DNA fragments using the DNA Ligation Kit (Mighty Mix Cat# TAK 6023, Clontech Laboratories, Madison, Wis.), and bacterial transformations into competent E. coli cells (Xtreme Efficiency DH5α Competent Cells, Cat# ABP-CE-CC02096P, Allele Biotechnology, San Diego, Calif.). Plasmid DNA was purified from E. coli cells using the Qiagen QIAprep Spin Miniprep Kit (Cat#27106, Qiagen, Valencia, Calif.).
Yeast media used for this example include YP medium (1% (w/v) yeast extract, 2% (w/v) peptone), YPD medium (YP medium containing 2% (w/v) glucose), YPD supplemented with glycerol and ethanol (YPD medium containing 1% (v/v) 80% glycerol and 1% (v/v) ethanol. The antibiotic G418 was added to agar plates to a final concentration of 0.2 g/L. Precultures were grown in YP medium supplemented with 5% glucose, 1% ethanol, and 0.2 g/L G418. Fermentations were carried out in YP medium containing 8% glucose, 1% v/v of ergosterol and Tween-80 in 100% ethanol, 200 mM MES (pH 6.5), and 0.2 μg/mL G418.
A large patch of S. cerevisiae strain. GEVO3868 was grown on an YPD plate. Cells from the patch were scraped from the plate, resuspended in 2 mL YPD containing 1% v/v ethanol containing 1% v/v 80% glycerol and placed in the 30° C. orbital shaker overnight. The following morning, 1 mL of the overnight culture was used to inoculate 50 mL YPD containing 1% ethanol containing 1% v/v 80% glycerol and returned to the 30° C. orbital shaker. After 6 hours, the cells were at an OD600 of 0.55. They were diluted to an OD600 of 0.1 in the same media and grown overnight at 30° C. In the morning the cells were diluted to an OD600 of 0.6, grown for 3 hours at 30° C. until the OD600 was 1.1, and the cells were collected by centrifugation at 2700 rcf for 2 min at room temperature. The medium was removed, 50 mL sterile milliQ water was used to wash the cells, and the cells were centrifuged for 2 min at 2700 rcf at room temperature. After removing the supernatant, the cells were washed with 25 mL sterile milliQ water and centrifuged at 2700 rcf for 2 min at room temperature. The supernatant was removed and the cells were resuspended in 1 mL 100 mM lithium acetate. The cells were centrifuged for 10 sec, the supernatant removed, and the cells resuspended in 400 μL 100 mM lithium acetate. The cells were transformed as follows. First, a mixture of plasmid DNA (final volume of 15 μl with sterile water), 72 μl 50% PEG, 10 μl 1M lithium acetate, and 3 μl of denatured salmon sperm DNA (10 mg/mL) was prepared for each transformation. In a sterile 1.5 mL tube, 15 μl of the cell suspension was added to the DNA mixture (100 μl), and the transformation suspension was vortexed for 5 short pulses. The transformation was incubated for 30 min at 30° C., followed by incubation for 22 min at 42° C. The cells were collected by centrifugation (18,000×g, 10 seconds, 25° C.). After removing the supernatant, the cells were resuspended in 400 μl YPD. After an overnight recovery shaking at 30° C. and 250 RPM, the cells were spread over selective plates, YPD containing 0.2 g/L G418. Transformants were then single colony purified onto selective plates.
For fermentations, 3 mL cultures of GEVO3868 transformed with each 2p plasmid were started in YPD containing 1% ethanol containing 0.2 g/L G418 and incubated overnight at 30° C. and 250 RPM. There were three biological replicates of each strain for 39 cultures total. After the OD600 of these cultures were taken the next day, the appropriate amount of culture was used to inoculate 50 mL of YP with 5% glucose containing 1% ethanol containing 0.2 g/L G418 (baffled flask) to an OD600 of approximately 0.1. These cultures were incubated at 30° C. and 250 RPM overnight. The next day, the cultures containing the S. cerevisiae ilv3ΔN20, the S. cerevisiae ilv3ΔN19, and the S. cerevisiae ilv3ΔN23 did not reach an OD600 of 5 (0.6-2.4) so incubation continued for another 24 h at 30° C. and 250 RPM. The remaining 30 cultures had reached an OD600 of approximately 5 and were centrifuged in 50 mL Falcon tubes at 2700 rcf for 5 min at 25° C. The cells from the 30 cultures were resuspended in 50 mL YP with 8% glucose, 1% (v/v) ethanol, ergosterol, Tween-80, 200 mM MES (pH 6.5), and 0.2 g/L G418. The cultures were transferred to 250 mL unbaffled flasks with closed screw caps and incubated at 30° C. and 75 RPM. The next day, the remaining 9 cultures were at a higher OD600 (3-5) and prepared for the fermentation as described above. At 24 and 48 h after transfer to 250 mL unbaffled flasks with closed screw caps, samples of each of the 39 flasks were taken to determine OD600 and prepared for gas chromatography as follows. 2 mL of sample (per flask) was removed and OD600 was determined. The remaining sample was centrifuged for 10 min at maximum speed. 1 mL of the supernatant was analyzed by gas chromatography as described. For the final 72 h timepoint, the same procedures were used for measuring OD600 and analysis by gas chromatography. In addition samples were analyzed by high performance liquid chromatography. Cells were also prepared for enzyme assays. After 3×15 mL Falcon tubes per flask were weighed (total of 117), 14 mL of the appropriate sample was transferred into the Falcon tubes. After centrifugation at 3000×g for 5 min at 4° C., the supernatant was removed and the cells washed in 3 mL cold, sterile water. The tubes were centrifuged as per above for 2 min, the supernatant removed, and the tubes reweighed to determine total cell weight. The Falcon tubes were stored at −80° C.
Analysis of organic acid metabolites was performed on an HP-1200 HPLC system equipped with two Restek RFQ 150×7.8 mm columns in series. Organic acid metabolites were detected using an HP-1100 UV detector (210 nm) and refractive index. The column temperature was 60° C. This method was isocratic with 0.0180 NH2SO4 (in Milli-Q water) as mobile phase. Flow was set to 1.1 mL/min. Injection volume was 20 μL and run time was 16 min. Analysis was performed using authentic standards (>99%, obtained from Sigma-Aldrich, with the exception of 2,3-dihydroxyisovalerate (DHIV), which was custom synthesized according to Cioffi et al., 1980, Anal Biochem 104: 485 and a 5-point calibration curve.
Analysis of volatile organic compounds, including ethanol and isobutanol was performed on a HP 5890, 6890 or 7890 gas chromatograph fitted with an HP 7673 Autosampler, a DB-FFAP column (J&W; 30 m length, 0.32 mm ID, 0.25 μM film thickness) or equivalent connected to a flame ionization detector (FID). The temperature program was as follows: 230° C. for the injector, 300° C. for the detector, 100° C. oven for 1 minute, 70° C./minute gradient to 230° C., and then hold for 2.5 min. Analysis was performed using authentic standards (>99%, obtained from Sigma-Aldrich, and a 5-point calibration curve with 1-pentanol as the internal standard.
For DHAD activity assays cells were thawed on ice and resuspended in lysis buffer (50 mM Tris pH 8.0 and 5 mM MgSO4) for a 20% cell suspension by mass. 1000 μl of glass beads (0.5 mm diameter) were added to a 1.5 ml Eppendorf tube and 875 μl of cell suspension was added. Yeast cells were lysed using a Retsch MM301 mixer mill (Retsch Inc. Newtown, Pa.), mixing 6×1 min each at full speed with 1 min incubations on ice between each bead-beating step. The tubes were centrifuged for 10 min at 23,500×g at 4° C. and the supernatant was removed for use. These lysates were held on ice until assayed. Yeast lysate protein concentration was determined using the BioRad Bradford Protein Assay Reagent Kit (Cat#500-0006, BioRad Laboratories, Hercules, Calif.) and using BSA for the standard curve. Briefly 10 μL standard or lysate were added into a microcentrifuge tube. The samples were diluted to fit in the linear range of the standard curve (1:40). 500 μL of diluted and filtered Bio-Rad protein assay dye was added to the blank and samples and then vortexed. Samples were incubated at room temperature for 6 min, transferred into cuvettes and the OD595 was determined in a spectrophotometer. The linear regression of the standards was then used to calculate the protein concentration in each sample. For DHAD assays technical triplicates were performed for each sample. In addition, a no lysate control with lysis buffer was performed. To assay each sample, 10 μL of an appropriate dilution of lysate in assay buffer was mixed with 90 μL of assay buffer (5 μL of 0.1 M MgSO4, 10 μL of 0.1 M DHIV, and 75 μL 50 mM Tris pH 8.0), and incubated in a thermocycler for 30 minutes at 35° C., then at 95° C. for 5 minutes. Cell debris and precipitant were removed from the samples by centrifugation at 3000×g for 5 min.
Finally, 75 μL of supernatant was transferred to new PCR tubes and analyzed by Liquid Chromatography for the 2-keto-isovalerate (KIV) product. DNPH reagent (12 mM 2,4-Dinitrophenyl Hydrazine 20 mM Citric Acid pH 3.0 80% Acetonitrile 20% MilliQ H2O) was added to each sample in a 1:1 ratio. Samples were incubated for 30 min at 70° C. in a thermo-cycler (Eppendorf, Mastercycler). Analysis of KIV was performed on an HP-1200 High Performance Liquid Chromatography system equipped with an Eclipse XDB C-18 reverse phase column (Agilent) and a C-18 reverse phase column guard (Phenomenex). Ketoisovalerate was detected using an HP-1100 UV detector (360 nm). The column temperature was 50° C. This method was isocratic with 70% acetonitrile 2.5% phosphoric acid (4%), 27.5% water as mobile phase. Flow was set to 3 mL/min. Injection size was 10 μL and run time was 2 min.
The data at 72 hours are summarized in Table 33. The data demonstrates that the DHADs contained in plasmids pGV2635, 2677, 2674, 2672, 2673 and 2676 led to production of isobutanol titers of at least 2.5 g/L and are considered to be significantly active in the cytosolic isobutanol pathway. The DHADs contained in plasmids pGV2675, 2681, 2680, 2678, 2679, 2671, and 2676 led to production of isobutanol titers below 2.5 g/L and are considered to be inactive or poorly active in the cytosolic isobutanol pathway.







TABLE 33







Isobutanol production with selected DHADs.










Plasmid

Isobutanol produced
DHAD activity


(DHAD Gene)
OD600
[g/L]
(U/mg)





pGV2635
8.6 ± 0.6
9.02 ± 0.28
0.62 ± 0.01


(L. lactis)





pGV2677
9.4 ± 0.6
6.30 ± 0.85
0.42 ± 0.02


(N. crassa)





pGV2674
7.5 ± 0.7
6.22 ± 0.31
0.30 ± 0.00


(F. tularensis)





pGV2672
8.1 ± 0.6
6.10 ± 0.26
0.20 ± 0.00


(G. forsetii)





pGV2673
8.0 ± 1.1
3.23 ± 0.12
0.03 ± 0.00


(S. erythraea)





pGV2676
5.2 ± 0.2
2.67 ± 0.06
0.02 ± 0.00


(S. cerevisiae





ilv3ΔN23)





pGV2675
5.0 ± 0.2
2.27 ± 0.16
0.09 ± 0.00


(S. cerevisiae





ilv3ΔN19)





pGV2681
6.9 ± 0.6
2.21 ± 0.09
0.03 ± 0.00


(E. coli)





pGV2680
6.9 ± 1.3
2.13 ± 0.09
0.02 ± 0.00


(Lyngbya spp.)





pGV2678
7.5 ± 0.2
2.06 ± 0.17
0.03 ± 0.00


(Acidobacteria)





pGV2679
7.5 ± 0.6
2.05 ± 0.06
0.03 ± 0.00


(A. marina)





pGV2671
5.5 ± 0.0
1.92 ± 0.03
0.44 ± 0.01


(S. cerevisiae)





pGV2663
6.7 ± 0.2
1.53 ± 0.18
0.01 ± 0.01


(none)









Example 19
Overexpression of the L. Lactis ilvD in K. Lactis and K. Marxianus 
The purpose of this example is to demonstrate activity of L. lactis DHAD in K. lactis and in K. marxianus. 
Strains, plasmids, and sequences disclosed herein are listed in Tables 34, 35, and 36, respectively.







TABLE 34







Genotype of strains disclosed in Example 19.








GEVO Number
Genotype





K. marxianus
K. marxianus NRRL-Y-7571 ura3-delta2


strain
pdc1Δ::Ll.kivd2 coSc. PTDH3:Dm_ADH:PFBA1:


GEVO2504
URA3:PSc_FBA1:31COX4_MTS:Bs_alsS1_coSc


K. marxianus
ura3-delta2 pdc1Δ::Δ::{Ll_kivd2 co:PSc_TDH3:


strain
Ec_ilvCQ110V coSC:PSc_TPI1:G418R:PSc_CUP1:


GEVO2543
Bs_alsS1_coSc}


K. marxianus
ura3-delta2 pdc1Δ::{Ll_kivd2 co:PSc_TDH3:


strain
Ec_ilvCQ110V coSC:PSc_TPI1:G418R:PSc_CUP1:


GEVO2598
Bs_alsS1_coSc} + random integration of



{PSc_TEF1:Ll_ilvD_coSc URA3}


K. lactis
MATalpha uraA1 trp1 leu2 lysA1 ade1


strain
lac4-8 [pKD1] ATCC 200826


GEVO1287
















TABLE 35







Piasmids disclosed in Example 19.









Plasmid




Name
Relevant Genes/Usage
Genotype





pGV2271
Empty 1.6 micron
1.6μ ori, bla, hygroR



vector that can




be maintained in




K. lactis. Encodes




hygromycin resistance.



pGV2273
1.6 micron vector
PTDH3:Ec_ilvC_P2D1-A1, PTEF1:



for expression of
Ll_ilvD_coSc, PPGK1:



KARI, KIVD, DHAD
Ll_kivD2_coEc, PENO2:Ll_adhA



and ADH in
1.6μ ori, bla, HygroR



K. lactis



pGV2069
2 micron plasmid
PTDH3:Ec_ilvC_coScQ110V, PTEF1:



for expression of
Ll_ilvD_coSc, PPGK1:



KIVD, DHAD, KARI,
Ll_kivD2_coEc, PCUP1:



and ALS in
Bs_alsS1_coSc, PENO2:



K. marxianus
Dm_adhA, 2μ ori, bla, G418


pGV1855
2 micron plasmid
PTEF1:Ll_ilvD, 2μ ori,



for expression of
bla, URA



DHAD in




K. marxianus
















TABLE 36







Amino acid and nucleotide sequences of enzymes


and genes disclosed in Example 19.













Corresponding





Protein


Enz.
Source
Gene (SEQ ID NO)
(SEQ ID NO)





ALS
B. subtilis
Bs_alsS1_coSc
Bs_AlsS1_coSc




(SEQ ID NO: 144)
(SEQ ID NO: 169)


KARI
E. coli
Ec_ilvC_coScQ110V
Ec_ilvC_coScQ110V




(SEQ ID NO: 98)
(SEQ ID NO: 170)



E. coli
Ec_ilvC_coScP2D1-A1
Ec_ilvC_coScP2D1-A1




(SEQ ID NO: 171)
(SEQ ID NO: 172)


KIVD
L. lactis
Ll_kivd2_coEc
Ll_Kivd2_coEc




(SEQ ID NO: 99)
(SEQ ID NO: 173)


DHAD
L. lactis
Ll_ilvD_coSc
Ll_ilvD_coSc




(SEQ ID NO: 155)
(SEQ ID NO: 18)


ADH
L. lactis
Ll_adhA
Ll_adhA




(SEQ ID NO: 174)
(SEQ ID NO: 175)



D.
Dm_adh
Dm_adh



melanogaster
(SEQ ID NO: 116)
(SEQ ID NO: 176)









To generate GEVO2543, GEVO2504 was transformed with pGV2069 to integrate into the genome three genes: Bs_alsS1_coSc (SEQ ID NO: 144), Ec_ilvC_coScQ110V (SEQ ID NO: 98), and Ll_kivd2_coEc (SEQ ID NO: 99). To generate GEVO2598, GEVO2543 was transformed pGV1855 to integrate the L. lactis ilvD gene which was codon optimized for S. cerevisiae (gene sequence SEQ ID NO: 155, also referred to as Ll_ilvD_coSc; protein sequence SEQ ID NO: 18) into the chromosome. GEVO1287 was transformed with either pGV2271 (control plasmid) or pGV2273, which contains Ll_ilvD_coSc.
GEVO2543, GEVO2598 and GEVO1287 transformed with pGV2271 or pGV2273 were inoculated into 3 mL of YPD (for GEVO2543 and GEVO2598) or YPD supplemented with 0.1 g/L hygromycin (for GEVO1287) for an overnight culture. After approximately 18 hours, a 50 ml YPD culture in a baffled 250 ml shake flask was inoculated to 0.15 OD600 and shaken at 250 rpms for approximately 9 hours. Next, DHAD activity and protein concentrations were measured.
Over-expression of the L. lactis ilvD gene resulted in an increase in DHAD activity (U/mg total cell lysate protein). Table 37 shows the DHAD activity (U/mg total cell lysate protein) averages from technical triplicates comparing strains expressing the L. lactis DHAD to strains not expressing the L. lactis DHAD gene.







TABLE 37







DHAD activity in whole cell yeast lysates.










Strain
Activity [mU/mg]






K. marxianus strain GEVO2543
0.010 ± 0.002



(no DHAD)




K. marxianus strain GEVO2598
0.016 ± 0.001



(DHAD)




K. lactis strain GEVO1287 +
0.052 ± 0.003



pGV2271 (No DHAD)




K. lactis strain GEVO1287 +
0.122 ± 0.011



pGV2273 (DHAD)









Example 20
L. Lactis ilvD Activity is Localized to the Yeast Cytosol
The purpose of this example is to demonstrate that the Lactococcus lactis ilvD protein localizes to the cytosol when expressed in a yeast strain.
The S. cerevisiae strain GEVO1187 (S. cerevisiae CEN.PK2, MATa ura3 leu2 his3 trp1 ADE2) was transformed with plasmid pGV2484, a 2 micron plasmid expressing the L. lactis ilvD gene which was codon optimized for S. cerevisiae (gene sequence SEQ ID NO: 155, also referred to as Ll_ilvD_coSc; protein sequence SEQ ID NO: 18) under the S. cerevisiae TEF1 promoter (PTEF1:LLl_ilvD_coSc, 2μ ori, bla, G418R). Briefly, the strain was grown in YPD to an OD600 of 0.6-0.8. Cells were washed in H2O, and then resuspended in 100 mM Lithium acetate. In a 1.5 mL tube, 15 μL of the cell suspension was added to a mixture of DNA (final volume of 15 μl with sterile water), 72 μl 50% PEG, 10 μl 1M lithium acetate, and 3 μl of denatured salmon sperm DNA (10 mg/mL). The transformation suspension was vortexed for 5 short pulses. The mixture was incubated at 30° C. for 30 minutes, followed by incubation for 22 minutes at 42° C. The cells were collected by centrifugation (18,000×g, 10 seconds, 25° C.). The cells were resuspended in 1 ml YPD medium (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose, pH 5) and after an overnight recovery shaking at 30° C. and 250 rpms, the cells were spread over YPD agar plates supplemented with 0.2 g/L G418. Transformants were then single colony purified onto G418 selective plates.
All isolations of crude mitochondrial fractions were performed in duplicate. GEVO1187 and GEVO1187 transformed with pGV2484 were each grown in 100 mL of YPG medium (1% (w/v) yeast extract, 2% (w/v) peptone, 3% (v/v) glycerol, pH5) overnight at 30° C. and 250 rpm. This overnight culture was used to inoculate 840 mL of YPG in a 2800 mL baffled flask at an OD600 of 0.03, and cells were grown at 30° C. and 250 rpm for 20-28 h. At an OD600 of about 2.0, cells were harvested by centrifugation at 3000×g for 5 minutes, resuspended in 100 mL H2O followed by centrifugation at 3000×g for 5 minutes. Cells were incubated in 2 mL/g CWW (cell wet weight) of DTT buffer (100 mM Tris-H2SO4 pH 9.4, 10 mM DTT) for 20 minutes at 30° C. Cells were resuspended in 7 mL/g CWW Zymolyase buffer (1.2 M sorbitol, 20 mM Potassium phosphate pH 7.4) and then centrifuged at 3000×g for 5 minutes. Cells were spheroplasted by incubating in Zymolyase buffer with Zymolyase (Seikagaku Biobusiness Corporation #120491-1; 3 mg/g CWW) for 45 minutes at 30° C. on a rocking platform. 100 OD of spheroplasts were set aside for whole cell lysate preparation (see below). Spheroplasts were resuspended in Zymolyase buffer and centrifuged at 3000×g for 5 minutes before resuspension in 6.5 mL/g CWW homogenization buffer (chilled to 4° C.; 6.5 mL/g 0.6 M sorbitol, 10 mM Tris-HCl pH 7.4, 1 mM EDTA, 1 mM PMSF, 0.2% (w/v) BSA). Spheroplasts were homogenized on ice with 15 strokes of a pre-chilled glass-Teflon homogenizer (40 mL capacity), and the sample was diluted 2-fold with homogenization buffer. Cell debris and nuclei were pelleted by serial supernatant centrifugations of 1500×g for 5 minutes, and 4000×g for 5 minutes. The mitochondrial fraction was isolated by centrifugation at 12,000×g for 15 minutes. The crude mitochondrial pellet was resuspended in 10 mL SEM buffer (250 mM sucrose, 1 mM EDTA, 10 mM MOPS-KOH pH 7.2), centrifuged at 4000×g for 5 minutes to further remove cellular debris and nuclei before recovering the mitochondrial fraction by centrifugation at 12,000×g for 15 minutes. The mitochondrial fraction may contain markers of the plasma membrane, the endoplasmic reticulum, and vacuoles in addition to markers of the mitochondria. Mitochondrial pellet was resuspended in 750 μL SEM Buffer+Protease Arrest (GBiosciences #786-108).
Preparation of whole cell yeast lysates was performed using the 100 ODs of yeast cells set aside after spheroplasting (see above) by resuspending cells in 20% (w/v) SEM Buffer+1× Protease Arrest (GBiosciences #786-108). 1000 μl of glass beads (0.5 mm diameter) were added to a 1.5 ml eppendorf tube, and 875 μl of cell suspension was added. Yeast cells were lysed using a Retsch MM301 mixer mill (Retsch Inc. Newtown, Pa.), mixing 6×1 min each at full speed with 1 min incubations on ice between each bead-beating step. The tubes were centrifuged for 10 min at 23,500×g at 4° C., the supernatant was removed, aliquoted, flash frozen in liquid nitrogen, and stored at −80° C.
The resuspended mitochondrial fraction (see above) was added to 1000 μl of glass beads (0.1 mm diameter) in a 1.5 ml Eppendorf tube. Additional buffer was added if necessary to fill the tube completely. The mitochondrial fraction was lysed using a Retsch MM301 mixer mill (Retsch Inc. Newtown, Pa.), mixing 3×1 minute each at full speed with 1 minute incubations on ice between each bead-beating step. The tubes were centrifuged for 10 min at 23,500×g at 4° C., the supernatant was removed, aliquoted, flash frozen in liquid nitrogen, and stored at −80° C.
Whole cell yeast lysate and mitochondrial fraction lysate protein concentration was determined using the BioRad Bradford Protein Assay Reagent Kit (Cat#500-0006, BioRad Laboratories, Hercules, Calif.) and using BSA for the standard curve. Briefly, 10 μL standard or lysate were added into a microcentrifuge tube. The samples were diluted to fit in the linear range of the standard curve (1:10-1:40). 500 μL of diluted and filtered Bio-Rad protein assay dye was added to the blank and samples and then vortexed. Samples were incubated at room temperature for 6 mins, transferred into cuvettes and the OD595 was determined in a spectrophotometer. The linear regression of the standards was then used to calculate the protein concentration in each sample.
Three samples of each of the mitochondrial and whole cell yeast lysates were assayed for DHAD activity, along with no lysate controls. Table 38 shows the DHAD activity (U/mg protein) averages from duplicate cultures comparing strains GEVO1187 (no DHAD expression) to GEVO1187 transformed with pGV2484 (L. lactis DHAD expressed from pGV2484). DHAD activity was measured in the whole cell yeast lysate and the mitochondrial fraction lysate. Expression of DHAD from pGV2484 resulted in about a 7-fold increase in DHAD activity in the whole cell yeast lysate. Expression of DHAD from pGV2484 did not affect DHAD activity localized to the mitochondrial fraction. Subtracting the background activity in the GEVO1187 whole cell yeast lysate of 0.27 mU/mg from the activity in the whole cell yeast lysate of GEVO1187 transformed with pGV2484 of 1.87 mU/mg shows an increase in 1.60 mU/mg. These data suggest that L. lactis DHAD activity does not localize to the organellar structures harvested in the mitochondrial fraction, and is therefore cytosolic when expressed in a yeast strain.







TABLE 38







DHAD activity in whole cell yeast lysates


and mitochondrial fraction lysates.













Activity



Strain
Lysate
[mU/mg]






GEVO1187
Whole cell
0.27 ± 0.07



GEVO1187 transformed
Whole cell
1.87 ± 0.14



with pGV2484





GEVO1187
Mitochondrial
3.76 ± 0.01



GEVO1187 transformed
Mitochondrial
3.85 ± 0.13



with pGV2484









Example 21
Overexpression of the L. Lactis ilvD in Issatchenkia Orientalis 
The purpose of this example is to demonstrate cytosolic activity of L. lactis DHAD in I. orientalis. 
An engineered strain derived from the wild-type I. orientalis strain ATCC PTA-6658 was further modified to contain copies of all five isobutanol pathway genes integrated into the chromosome. First, both alleles of the PDC1 locus were deleted in series (See e.g. WO/2007/106524, which is herein incorporated by reference in its entirety). The deletion event also simultaneously integrated a copy of B. subtilis alsS gene and a copy of the L. lactis kivD gene which encode SEQ ID NOs: 169 and 173, respectively. This resulted in a Pdc− strain with two integrated copies of the B. subtilis alsS gene and two integrated copies of the L. lactis kivD gene (pdc1Δ::Ll_kivD: Bs_alsS pdc1Δ::Ll_kivD: Bs_alsS). This strain was further engineered to delete a single allele of the GPD1 locus (See e.g. WO/2007/106524). The deletion event also simultaneously integrated a single copy of the L. lactis adhARE1, the E. coli ilvCP2D1-A1, and L. lactis ilvD which encode the proteins shown in SEQ ID NOs: 177, 172, and 18, respectively. This results in a Pdc− Gpd+ strain with one integrated copy of the Ll_adhARE1, Ec_ilvCP2D1-A1, and Ll_ilvD genes (GPD1/gpd1Δ::[Ll_adhARE1: Ec_ilvCP2D1-A1: URA3:Ll_ilvD]). This strain is GEVO4306 (Table 39).
To generate a control strain which does not express the pathway genes, both alleles of the PDC1 locus were deleted in series but with no simultaneous integration of heterologous genes. Next one of the two GPD1 alleles was deleted with no simultaneous integration of heterologous genes. The resulting control strain is GEVO4308 (pdc1Δ::loxP/pdc1Δ::loxP GPD1/gpd1Δ::loxP:URA3:loxP) (Table 39).







TABLE 39







Genotype of strains disclosed in Example 21.








GEVO Number
Genotype





4306
pdc1Δ::[Ll_kivD:Bs_alsSl pdc1Δ::Ll_kivD:



Bs_alsS] GPD1/gpd1Δ::[Ll_adhARE1:



Ec_ilvCP2D1-A1:URA3:Ll_ilvD]


4308
pdc1Δ::loxP/pdc1Δ::loxP GPD1/



gpd1Δ::loxP:URA3:loxP









Over-expression of the L. lactis ilvD gene resulted in an increase in DHAD activity (U/mg total cell lysate protein). Table 40 shows the DHAD activity (U/mg total cell lysate protein) averages from technical triplicates comparing the strain expressing the L. lactis DHAD gene to the strain not expressing the L. lactis DHAD gene. Expression of the L. lactis ilvD gene, when expressed with the remainder of the isobutanol pathway, resulted in isobutanol production as seen in Table 40.







TABLE 40







DHAD activity in whole cell yeast lysates and


isobutanol titer after 72 hr fermentation.












Activity
Isobutanol



Strain
[mU/mg]
titer g/L






GEVO4306
0.041 ± 0.009
0.56 ± 0.01



GEVO4308
0.012 ± 0.002
0.00 ± 0.00









Example 22
Cytosolic ALS Homologs that Support Isobutanol Production
This example demonstrates isobutanol production using expression of cytosolically localized ALS genes in the presence of the rest of the isobutanol pathway. The ALS genes were integrated into the PDC1 locus of S. cerevisiae strain GEVO1187 and isobutanol production was achieved by expression from plasmid of the other genes in the isobutanol pathway. Isobutanol production in strains carrying the ALS genes from T. atroviride (Ta_ALS) and T. stipitatus (Ts_ALS) was compared to isobutanol production in strains carrying the ALS gene from B. subtilis. Plasmids described in this example are listed in Table 41.







TABLE 41







Plasmids disclosed in Example 22.









Plasmid




name
Relevant Genes/Usage
Genotype





pGV1730
Integration plasmid that will
See Table 14.



integrate PCUP1-1:Bs_alsS2 into




PDC1 using digestion with NruI




for targeting. This was the




parent vector for cloning the




ALS homologs.



pGV1773
Vector with Bacillus subtilis
PPDC1:Bs_AlsS1_coSc,



AlsS codon optimized for
PTDH3:Ll_kivD,



S. cerevisiae.
PADH1:Sc_ADH7_coSc,




URA3 5′-end,




pUC ORI, kanR.


pGV1802
DNA2.0 plasmid carrying the
Ta_ALS_coSc in DNA



Trichoderma atroviride
2.0 vector



ALS.



pGV1803
DNA2.0 plasmid carrying the
Ts_ALS_coSc in DNA



Talaromyces stipitatus
2.0 vector



ALS.



pGV2082
High copy 2μ plasmid with 4
Ec_ilvC_coScQ110V,



isobutanol pathway genes
Ll_ilvD_coSc,



without an ALS gene.
Ll_kivD2_coEc, and




Dm_ADH, 2μ ori, bla,




G418R.


pGV2114
Integration plasmid that will
See Table 14.



integrate into PDC1 using




digestion with NruI for




targeting. It carries the




Bacillus subtilis AlsS gene




codon optimized for




S. cerevisiae.



pGV2117
Integration plasmid that will
See Table 14.



integrate into PDC1 using




digestion with NruI for




targeting. It carries the




Trichoderma atroviride




ALS gene codon optimized for




S. cerevisiae.



pGV2118
Integration plasmid that will
See Table 14.



integrate into PDC1 using




digestion with NruI for




targeting. It carries the




Talaromyces stipitatus




ALS gene codon optimized for




S. cerevisiae.









Strains with integrated ALS genes expressed from the CUP1 promoter were transformed with pGV2082 (which carries the other 4 isobutanol pathway genes Ec_ilvC_coScQ110V (SEQ ID NO: 98), Ll_ilvD (SEQ ID NO: 155), Ll_kivd2_coEc (SEQ ID NO: 99), and Dm_ADH (SEQ ID NO: 116).
GEVO2618, GEVO2621, and GEVO2622 (see Table 13) were each transformed with pGV2082. Control strains GEVO2280 (B. subtilis alsS2) (Table 13) and GEVO1187 (no ALS) (Table 13) were also transformed with pGV2082.
Fermentations of the transformed strains GEVO1187, GEVO2280, GEVO2618, GEVO2621, GEVO2622 were performed. Strains encoding the ALS from T. atroviride (SEQ ID NO: 71) and T. stipitatus (SEQ ID NO: 72) produced more isobutanol than the strain containing the B. subtilis als2. The strain containing Bs_Als1_coSc produced the most isobutanol. Table 42 shows the final OD, glucose consumption, and isobutanol titer for each of the strains. The integration of the cytosolic genes Ta_ALS_coSc and Ts_ALS_coSc led to production of isobutanol that was in each case 6-fold above that of a strain without an integrated ALS gene, demonstrating that these strains are producing isobutanol using a cytosolic pathway.







TABLE 42







Results of fermentations with cytosolic ALS homologs at 72 hrs.














Glucose
Isobutanol



Strain
OD600
consumed g/L
produced g/L






GEVO1187
10.9 ± 0.3 
233 ± 36
0.3 ± 0.0



GEVO2280
9.9 ± 0.3
274 ± 26
 1.3 ± 0.11



GEVO2618
9.4 ± 0.2
138 ± 9 
2.6 ± .09



GEVO2621
9.9 ± 0.3
161 ± 52
1.9 ± .18



GEVO2622
10.8 ± 0.6 
182 ± 47
1.8 ± .15









The foregoing detailed description has been given for clearness of understanding only and no unnecessary limitations should be understood there from as modifications will be obvious to those skilled in the art.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.
The disclosures, including the claims, figures and/or drawings, of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entireties.

## Claims

1. A recombinant yeast microorganism for producing isobutanol, the recombinant yeast microorganism comprising an isobutanol producing metabolic pathway, wherein said isobutanol producing metabolic pathway comprises the following substrate to product conversions:
(i) pyruvate to acetolactate;
(ii) acetolactate to 2,3-dihydroxyisovalerate;
(iii) 2,3-dihydroxyisovalerate to α-ketoisovalerate;
(iv) α-ketoisovalerate to isobutyraldehyde; and
(v) isobutyraldehyde to isobutanol;

wherein said recombinant yeast microorganism expresses:
an exogenous nucleic acid encoding dihydroxy acid dehydratase to catalyze the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate, said exogenous nucleic acid encoding dihydroxy acid dehydratase comprising an amino acid motif which is at least 90% identical to the motif of SEQ ID NO: 27, wherein said dihydroxy acid dehydratase is active in the cytosol of said recombinant yeast microorganism, and wherein the recombinant yeast microorganism has been engineered to inactivate one or more endogenous pyruvate decarboxylase (PDC) genes.

2. The recombinant yeast microorganism of claim 1, wherein said dihydroxy acid dehydratase is obtained from a prokaryotic organism.

3. The recombinant yeast microorganism of claim 2, wherein said prokaryotic organism is selected from the group consisting of Lactococcus lactis, Francisella tularensis, Acidobacteria bacterium, Saccharopolyspora erythraea, Thermotoga petrophila, Gramella forsetii, and Victivallis vadensis.

4. The recombinant yeast microorganism of claim 1, wherein said dihydroxy acid dehydratase is obtained from a eukaryotic organism.

5. The recombinant yeast microorganism of claim 4, wherein said eukaryotic organism is selected from the group consisting of Yarrowia lipolytica, Neurospora crassa, Arabidopsis thaliana, and Piromyces sp. E2.

6. The recombinant yeast microorganism of claim 1, wherein said one or more endogenous PDC genes is selected from the group consisting of PDC1, PDC5, and PDC6.

7. The recombinant yeast microorganism of claim 1, wherein said recombinant yeast microorganism has been engineered to reduce the activity of one or more endogenous glycerol-3-phosphate dehydrogenase (GPD) genes.

8. The recombinant yeast microorganism of claim 7, wherein said one or more endogenous GPD genes is selected from the group consisting of GPD1 and GPD2.

9. The recombinant yeast microorganism of claim 1, wherein said recombinant yeast microorganism is a yeast microorganism of the Saccharomyces clade.

10. A method of producing isobutanol, comprising:
(a) providing a recombinant yeast microorganism according to claim 1; and
(b) cultivating said recombinant yeast microorganism in a culture medium containing a feedstock providing a carbon source, until the isobutanol is produced.

11. The recombinant yeast microorganism of claim 1, wherein the substrate to product conversion of (i) is catalyzed by an exogenous acetolactate synthase encoded by alsS of Bacillus subtilis.

12. The recombinant yeast microorganism of claim 1, wherein the substrate to product conversion of (i) is catalyzed by an exogenous acetolactate synthase encoded by alsS of Lactococcus lactis.

13. The recombinant yeast microorganism of claim 1, wherein the substrate to product conversion of (i) is catalyzed by an endogenous acetolactate synthase encoded by ILV2.

14. The recombinant yeast microorganism of claim 1, wherein the substrate to product conversion of (ii) is catalyzed by an exogenous ketol-acid reductoisomerase obtained from a microorganism selected from the group consisting of Escherichia coli, Bacillus subtilis, Lactococcus lactis, Methanococcus maripaludis, and Shewanella sp.

15. The recombinant yeast microorganism of claim 1, wherein the substrate to product conversion of (ii) is catalyzed by an endogenous ketol-acid reductoisomerase encoded by ILV5.

16. The recombinant yeast microorganism of claim 1, wherein the substrate to product conversion of (iv) is catalyzed by an exogenous 2-keto-acid decarboxylase encoded by kivD of Lactococcus lactis.

17. The recombinant yeast microorganism of claim 1, wherein the substrate to product conversion of (iv) is catalyzed by an exogenous 2-keto-acid decarboxylase obtained from a microorganism selected from the group consisting of Enterobacter cloacae, Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium avium, Azospirillum brasilense, and Bacillus subtilis.

18. The recombinant yeast microorganism of claim 1, wherein the substrate to product conversion of (v) is catalyzed by an exogenous alcohol dehydrogenase obtained from a microorganism selected from the group consisting of Lactococcus lactis, Lactococcus brevis, Pediococcus acidilactici, Bacillus cereus, Bacillus thuringiensis, Leptotrichia goodfellowii, Actinobacillus pleuropneumonias, Streptococcus sanguinis, Eikenella corrodens, Neisseria elongate, Escherichia coli, Neisseria meningitidis, Erwinia pyrifoliae, and Colwellia psychrerythraea.

19. The recombinant yeast microorganism of claim 1, wherein the substrate to product conversion of (v) is catalyzed by an engineered alcohol dehydrogenase obtained from Lactococcus lactis, wherein said engineered alcohol dehydrogenase comprises SEQ ID NO: 177.

20. The recombinant yeast microorganism of claim 1, wherein the substrate to product conversion of (v) is catalyzed by an endogenous alcohol dehydrogenase encoded by a gene selected from the group consisting of ADH2, ADH6, and ADH7.