WO2013178699A1

WO2013178699A1 - Isopropanol production by bacterial hosts

Info

Publication number: WO2013178699A1
Application number: PCT/EP2013/061103
Authority: WO
Inventors: Torsten BAK; Steen Joergensen; Brian KOBMANN; Bjarke Christensen
Original assignee: Novozymes A/S
Priority date: 2012-05-31
Filing date: 2013-05-29
Publication date: 2013-12-05

Abstract

Provided herein are Lactobacillusmutants having a disruption to an endogenous gene encoding an acetate kinase, wherein the mutants produce increased amounts of recombinant isopropanol. Also described aremethods forproducing the mutants and methods of using the mutants to produce isopropanol.

Description

ISOPROPANOL PRODUCTION BY BACTERIAL HOSTS

Cross-Reference to Related Applications

This application claims priority from U.S. provisional application Serial No. 61/653,908, filed on May 31 , 2012, the content of which is fully incorporated herein by reference.

Reference to a Sequence Listing

This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

Background

Concerns related to future supply of oil have prompted research in the area of renewable energy and renewable sources of other raw materials. Biofuels, such as ethanol and bioplastics (e.g., particularly polylactic acid) are examples of products that can be made directly from agricultural sources using microorganisms. Additional desired products may then be derived using non-enzymatic chemical conversions, e.g., dehydration of ethanol to ethylene.

Polymerization of ethylene provides polyethylene, a type of plastic with a wide range of useful applications. Ethylene is traditionally produced by refined non-renewable fossil fuels, but dehydration of biologically-derived ethanol to ethylene offers an alternative route to ethylene from renewable carbon sources, i.e., ethanol from fermentation of fermentable sugars. This process has been utilized for the production of "Green Polyethylene" that - save for minute differences in the carbon isotope distribution - is identical to polyethylene produced from oil.

Similarly, isopropanol and n-propanol can be dehydrated to propylene, which in turn can be polymerized to polypropylene. As with polyethylene, using biologically-derived propanol starting material (i.e., isopropanol or n-propanol) would result in "Green Polypropylene." However, unlike polyethylene, the production of the polypropylene starting material from renewable sources has proved challenging. Proposed efforts at propanol production have been reported in WO 2009/049274, WO 2009/103026, WO 2009/131286, WO 2010/071697, WO 201 1/031897, WO 201 1/029166, WO 201 1/022651 , WO 2012/058603. It is clear that the successful development of a process for the biological production of propanol requires careful selection of enzymes in the metabolic pathways as well as an efficient overall metabolic engineering strategy. It would be advantageous in the art to improve isopropanol production, as a result of genetic engineering using recombinant DNA techniques.

Summary Described herein, inter alia, are Lactobacillus host cells having a disruption to an endogenous gene encoding an acetate kinase, resulting in decreased acetate production. In some aspects, the Lactobacillus mutants comprise an active isopropanol pathway and produce increased amounts of isopropanol compared to cells without the gene disruption. In some aspects, the mutants produce a greater amount of isopropanol when cultivated in the presence of fructose. In some aspects, the Lactobacillus mutant is a Lactobacillus reuteri mutant.

Also described are methods for obtaining the Lactobacillus mutants, comprising disrupting an endogenous gene encoding an acetate kinase in a Lactobacillus strain having an active isopropanol pathway.

Also described are methods for obtaining a Lactobacillus mutants described herein, comprising: (a) providing a Lactobacillus strain; (b) disrupting an endogenous gene encoding an acetate kinase in the Lactobacillus strain; (c) transforming the Lactobacillus strain with one or more heterologous isopropanol pathway genes resulting in the strain having an active isopropanol pathway; and (d) isolating the resulting Lactobacillus mutant.

Also described are methods of producing isopropanol, comprising: (a) cultivating a

Lactobacillus mutant described herein having an active isopropanol pathway; and (b) recovering the isopropanol.

Brief Description of the Figures Figure 1 shows an exemplary metabolic isopropanol pathway for the production of isopropanol from glucose.

Figure 2 shows a plasmid map for pSJ 10600.

Figure 3 shows a plasmid map for pSJ10603.

Figure 4 shows a plasmid map for pJP042.

Figure 5 shows the results of a fermentation experiment using a L. reuteri acetate kinase mutant and control strains.

Figure 6 shows the isopropanol production from a L. reuteri acetate kinase mutant with and without supplemental fructose.

Figure 7 shows the isopropanol+acteone production from a L. reuteri acetate kinase mutant with and without supplemental fructose. Figure 8 shows the isopropanol production from a L. reuteri acetate kinase mutant with and without supplemental 1 ,2-propanediol.

Figure 9 shows the isopropanol+acteone production from a L. reuteri acetate kinase mutant with and without supplemental 1 ,2-propanediol.

Definitions

Disruption: The term "disruption" means that a coding region and/or control sequence of a referenced gene is partially or entirely modified (such as by deletion, insertion, and/or substitution of one or more nucleotides) resulting in the absence (inactivation) or decrease in expression, and/or the absence or decrease of enzyme activity of the encoded polypeptide. The effects of disruption can be measured using techniques known in the art such as detecting the absence or decrease of enzyme activity using from cell-free extract measurements referenced herein; or by the absence or decrease of corresponding mRNA (e.g., at least 25% decrease, at least 50% decrease, at least 60% decrease, at least 70% decrease, at least 80% decrease, or at least 90% decrease); the absence or decrease in the amount of corresponding polypeptide having enzyme activity (e.g., at least 25% decrease, at least 50% decrease, at least 60% decrease, at least 70% decrease, at least 80% decrease, or at least 90% decrease); or the absence or decrease of the specific activity of the corresponding polypeptide having enzyme activity (e.g., at least 25% decrease, at least 50% decrease, at least 60% decrease, at least 70% decrease, at least 80% decrease, or at least 90% decrease). Disruptions of a particular gene of interest can be generated by methods known in the art, e.g., by directed homologous recombination (see Methods in Yeast Genetics (1997 edition), Adams, Gottschling, Kaiser, and Stems, Cold Spring Harbor Press (1998)).

Parent: The term "parent" or "parent Lactobacillus strain" means a Lactobacillus strain to which a disruption is made to produce a mutant Lactobacillus strain described herein. The parent may be a naturally occurring (wild-type) or previously modified Lactobacillus strain.

Mutant: As used herein, the term "mutant" means a Lactobacillus host cell comprising a disruption to an endogenous acetate kinase gene.

Acetate Kinase: The term "acetate kinase" is defined herein as a transferase enzyme that catalyzes the chemical reaction of acetyl-phosphate and ADP to acetate and ATP (e.g., EC 2.7.2.1 ). The acetate kinase may be monomeric or in the form of a protein complex comprising two or more subunits under cellular conditions. Acetate kinase activity may be determined from cell-free extracts as described in the art, e.g., as described in S. Mukhopadhyay et al., 2008, Bioorg Chem. 36: 65-69.

Active isopropanol pathway: As used herein, a host cell having an "active isopropanol pathway" produces active enzymes necessary to catalyze each reaction in a metabolic pathway from a fermentable sugar to isopropanol, and therefore is capable of producing isopropanol in measurable yields when cultivated under fermentation conditions in the presence of at least one fermentable sugar. A host cell having an active isopropanol pathway comprises one or more isopropanol pathway genes. An "isopropanol pathway gene" as used herein refers to a gene that encodes an enzyme involved in an active isopropanol pathway.

The active enzymes necessary to catalyze each reaction in an active isopropanol pathway may result from activities of endogenous gene expression, activities of heterologous gene expression, or from a combination of activities of endogenous and heterologous gene expression.

Thiolase: The term "thiolase" is defined herein as an acyltransferase that catalyzes the chemical reaction of two molecules of acetyl-CoA to acetoacetyl-CoA and CoA (e.g., EC 2.3.1 .9). The thiolase may be monomeric or in the form of a protein complex comprising two or more subunits (e.g., two heteromeric subunits) under cellular conditions. Thiolase activity may be determined from cell-free extracts as described in the art, e.g., as described in D. P. Wiesenborn et al., 1988, Appl. Environ. Microbiol. 54:2717-2722. For example, thiolase activity may be measured spectrophotometrically by monitoring the condensation reaction coupled to the oxidation of NADH using 3-hydroxyacyl-CoA dehydrogenase in 100 mM Tris hydrochloride (pH 7.4), 1 .0 mM acetyl-CoA, 0.2 mM NADH, 1 mM dithiothreitol, and 2 U of 3-hydroxyacyl-CoA dehydrogenase. After equilibration of the cuvette contents at 30°C for 2 min, the reaction is initiated by the addition of about 125 ng of thiolase in 10 μΙ_. The absorbance decrease at 340 nm due to oxidation of NADH is measured, and an extinction coefficient of 6.22 mM^"1 cm^"1 used.

CoA-transferase: As used herein, the term "CoA-transferase" is defined as any enzyme that catalyzes the removal of coenzyme A from acetoacetyl-CoA to generate acetoacetate. In some aspects, the CoA-transferase is an acetoacetyl- CoA:acetate/butyrate CoA transferase (e.g., the CoA-transferase is a butyrate- acetoacetate CoA transferase of EC 2.8.3.9) that converts acetoacetyl-CoA and acetate to acetoacetate and acetyl-CoA and/or converts acetoacetyl-CoA and butyrate to acetoacetate and butyryl-CoA. In some aspects, the CoA-transferase is an acetoacetyl-CoA hydrolase of EC 3.1 .2.1 1 .

In some aspects, the CoA-transferase is a succinyl-CoA:acetoacetate transferase. As used herein, "succinyl-CoA:acetoacetate transferase" is an acetotransferase that catalyzes the chemical reaction of acetoacetyl-CoA and succinate to acetoacetate and succinyl-CoA (EC 2.8.3.5). The succinyl-CoA:acetoacetate transferase may be in the form of a protein complex comprising two or more subunits (e.g., two heteromeric subunits) as described herein. Succinyl-CoA:acetoacetate transferase activity may be determined from cell-free extracts as described in the art, e.g., as described in L. Stols et al., 1989, Protein Expression and Purification 53:396-403. For example, succinyl-CoA:acetoacetate transferase activity may be measured spectrophotometrically by monitoring the formation of the enolate anion of acetoacetyl-CoA, wherein absorbance is measured at 310nm/30°C over 4 minutes in an assay buffer of 67 mM lithium acetoacetate, 300 μΜ succinyl-CoA, and 15 mM MgCI₂ in 50 mM Tris, pH 9.1 .

Acetoacetate decarboxylase: The term "acetoacetate decarboxylase" is defined herein as an enzyme that catalyzes the chemical reaction of acetoacetate to carbon dioxide and acetone (e.g., EC 4.1 .1 .4). The acetoacetate decarboxylase may be monomeric or in the form of a protein complex comprising two or more subunits (e.g., two heteromeric subunits) under cellular conditions. Acetoacetate decarboxylase activity may be determined from cell-free extracts as described in the art, e.g., as described in D.J. Petersen, et al., 1990, Appl. Environ. Microbiol. 56, 3491 -3498. For example, acetoacetate decarboxylase activity may be measured spectrophotometrically by monitoring the depletion of acetoacetate at 270 nm in 5 nM acetoacetate, 0.1 M K₂P0₄, pH 5.9 at 26°C.

Isopropanol dehydrogenase: The term "isopropanol dehydrogenase" is defined herein as any suitable oxidoreductase that catalyzes the reduction of acetone to isopropanol (e.g., any suitable enzyme of EC1 .1 .1 .1 or EC 1 .1 .1 .80). The isopropanol dehydrogenase may be monomeric or in the form of a protein complex comprising two or more subunits (e.g., two heteromeric subunits) under cellular conditions. Isopropanol dehydrogenase activity may be determined spectrophotometrically from cell-free extracts as described in the art, e.g., by decrease in absorbance at 340 nm in an assay containing 200 μΜ NADPH and 10 mM acetone in 25 mM potassium phosphate, pH 7.2 at 25°C.

Mature polypeptide sequence: The term "mature polypeptide sequence" means the portion of the referenced polypeptide sequence after any post-translational sequence modifications (such as N-terminal processing and/or C-terminal truncation). The mature polypeptide sequence may be predicted, e.g., based on the SignalP program (Nielsen et al., Protein Engineering 1997, 10, 1 -6) or the InterProScan program (The European Bioinformatics Institute). In some instances, the mature polypeptide sequence may be identical to the entire referenced polypeptide sequence. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptide sequences (i.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide.

Coding sequence: The term "coding sequence" or "coding region" means a polynucleotide sequence, which specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA. The coding sequence may be a sequence of genomic DNA, cDNA, a synthetic polynucleotide, and/or a recombinant polynucleotide.

Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means the portion of the referenced polynucleotide sequence that encodes a mature polypeptide sequence. The mature polypeptide coding sequence may be predicted, e.g., based on the SignalP program (supra) or the InterProScan program (supra). In some instances, the mature polypeptide coding sequence may be identical to the entire referenced polynucleotide sequence.

Sequence Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

For purposes described herein, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, J. Mol. Biol. 1970, 48, 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., Trends Genet 2000, 16, 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment) For purposes described herein, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

Heterologous gene: The term "heterologous gene" is defined herein as a gene that is not native to the host cell; an endogenous gene in which one or more (e.g., two, several) structural modifications have been made to the coding region; an endogenous gene whose expression is quantitatively altered as a result of manipulation of the DNA by recombinant DNA techniques, e.g., a different (foreign) promoter linked to the polynucleotide; or an endogenous gene whose expression is quantitatively altered by the introduction of one or more extra copies of the gene into the host cell.

Endogenous gene: The term "endogenous gene" means a gene that is native to the parent Lactobacillus strain.

Nucleic acid construct: The term "nucleic acid construct" means a polynucleotide comprising one or more (e.g., two, several) control sequences. The polynucleotide may be single-stranded or double-stranded, and may be isolated from a naturally occurring gene, modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature, or synthetic.

Control sequence: The term "control sequence" means a nucleic acid sequence necessary for polypeptide expression. Control sequences may be native or foreign to the polynucleotide encoding the polypeptide, and native or foreign to each other. Such control sequences include, but are not limited to, a leader sequence, polyadenylation sequence, propeptide sequence, promoter sequence, signal peptide sequence, and transcription terminator sequence. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.

Operably linked: The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.

Expression: The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression can be measured— for example, to detect increased expression— by techniques known in the art, such as measuring levels of mRNA and/or translated polypeptide.

Expression vector: The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences, wherein the control sequences provide for expression of the polynucleotide encoding the polypeptide. At a minimum, the expression vector comprises a promoter sequence, and transcriptional and translational stop signal sequences.

Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The term "recombinant host cell" is defined herein as a non-naturally occurring host cell comprising one or more (e.g., several) heterologous polynucleotides.

Allelic variant: The term "allelic variant" means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

Fermentable medium: The term "fermentable medium" or "fermentation medium" refers to a medium comprising one or more (e.g., several) sugars, such as glucose, fructose, sucrose, cellobiose, xylose, xylulose, arabinose, mannose, galactose, and/or soluble oligosaccharides, wherein the medium is capable, in part, of being converted (fermented) by a host cell into a desired product, such as isopropanol. In some instances, the fermentation medium is derived from a natural source, such as sugar cane, starch, or cellulose, and may be the result of pretreating the source by enzymatic hydrolysis (saccharification).

Reference to "about" a value or parameter herein includes aspects that are directed to that value or parameter per se. For example, description referring to "about X" includes the aspect "X". When used in combination with measured values, "about" includes a range that encompasses at least the uncertainty associated with the method of measuring the particular value, and can include a range of plus or minus two standard deviations around the stated value.

As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. It is understood that the aspects described herein include "consisting" and/or "consisting essentially of" aspects.

Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Detailed Description

Disrupted Lactobacillus Host Cells and Methods of Production

Described herein, inter alia, are Lactobacillus host cells comprising a disrupted endogenous acetate kinase gene. In some Lactobacillus host cells comprise an active isopropanol pathway, wherein the cells produces a greater amount of isopropanol compared to the cell without the disruption when cultivated under identical conditions. In some aspects, the endogenous gene is inactivated.

The Lactobacillus host cells may be any suitable Lactobacillus strain, such as a wild-type Lactobacillus strain or a metabolically engineered strain thereof. In some aspects, the Lactobacillus host cell is a Lactobacillus plantarum, Lactobacillus fructivorans, or Lactobacillus reuteri host cell. In one aspect, the Lactobacillus host cell is a Lactobacillus plantarum host cell. In another aspect, the Lactobacillus host cell is a Lactobacillus fructivorans host cell. In another aspect, the Lactobacillus host cell is a Lactobacillus reuteri host cell.

Additional Lactobacillus host cells contemplated include, but are not limited to, L. acetotolerans, L. acidifarinae, L. acidipiscis, L. acidophilus, L. agilis, L. algidus, L. alimentarius, L. amylolyticus, L. amylophilus, L. amylotrophicus, L. amylovorus, L. animalis,

L. antri, L. apodemi, L. aquaticus, L. arizonensis, L. aviarius, L. bavaricus, L. bifermentans, L. bobalius, L. brevis, L. buchneri, L. bulgaricus, L. cacaonum, L. camelliae, L. capillatus, L. carni, L. casei, L. catenaformis, L. cellobiosus, L. ceti, L. coleohominis, L. collinoides, L. composti, L. concavus, L. confusus, L. coryniformis, L. crispatus, L. crustorum, L. curvatus,

L. cypricasei, L. delbrueckii, L. dextrinicus, L. diolivorans, L. divergens, L. durianis, L. equi,

L. equicursoris, L. equigenerosi, L. fabifermentans, L. farciminis, L. farraginis, L. ferintoshensis, L. fermentum, L. fornicalis, L. fructivorans, L. fructosus, L. frumenti, L. fuchuensis, L. gallinarum, L. gasseri, L. gastricus, L. ghanensis, L. graminis, L. halotolerans, L. hammesii, L. hamsteri, L. harbinensis, L. hayakitensis, L. helveticus, L. heterohiochii, L. hilgardii, L. homohiochii, L. hordei, L. iners, L. ingluviei, L. intestinalis, L. jensenii, L. johnsonii, L. kalixensis, L. kandleri, L. kefiranofaciens, L. kefiranofaciens, L. kefirgranum, L. kefiri, L. kimchii, L. kisonensis, L. kitasatonis, L. kunkeei, L. lactis, L. leichmannii, L. lindneri, L. malefermentans, L. mali, L. maltaromicus, L. manihotivorans, L. mindensis, L. minor, L. minutus, L. mucosae, L. murinus, L. nagelii, L. namurensis, L. nantensis, L. nodensis, L. oeni, L. oligofermentans, L. oris, L. otakiensis, L. panis, L. pantheris, L. parabrevis, L. parabuchneri, L. paracasei, L. paracollinoides, L. parafarraginis, L. parakefiri, L. paralimentarius, L. paraplantarum, L. pentosus, L. perolens, L. piscicola, L. plantarum, L. pobuzihii, L. pontis, L. psittaci, L. rapi, L. rennini, L. reuteri, L. rhamnosus, L. rimae, L. rogosae, L. rossiae, L. ruminis, L. saerimneri, L. sakei, L. salivarius, L. sanfranciscensis, L. satsumensis, L. secaliphilus, L. senmaizukei, L. sharpeae, L. siliginis, L. similis, L. sobrius, L. spicheri, L. sucicola, L. suebicus, L. sunkii, L. suntoryeus, L. taiwanensis, L. thailandensis, L. thermotolerans, L. trichodes, L. tucceti, L. uli, L. ultunensis, L. uvarum, L. vaccinostercus, L. vaginalis, L. versmoldensis, L. viridescens, L. vitulinus, L. xylosus, L. yamanashiensis, L. zeae, and L. zymae.

The disrupted gene may be any suitable endogenous gene encoding an acetate kinase. An example of an endogenous gene encoding the acetate kinase includes the Lactobacillus reuteri gene having the coding sequence shown in SEQ ID NO: 59, which encodes the amino acid sequences of SEQ ID NO: 60. Additional target endogenous genes encoding the acetate kinase for disruption can be selected for a particular Lactobacillus host cell based on an appropriate acetate kinase polypeptide encoded for that particular host, e.g., as shown in SEQ ID NOs: 66-248.

In some aspects, the endogenous gene encodes an acetate kinase having at least 60%, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 60 or 66-248. In some aspects, the endogenous gene encodes an acetate kinase having a sequence that differs by no more than ten amino acids, e.g., by no more than five amino acids, by no more than four amino acids, by no more than three amino acids, by no more than two amino acids, or by one amino acid from any one of SEQ ID NOs: 60 or 66-248. In some aspects, the endogenous gene encodes an acetate kinase comprising or consisting of any one of SEQ ID NOs: 60 or 66-248.

In other aspects of the mutants and related methods, the coding sequence of the endogenous gene encoding an acetate kinase has at least 60%, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 59. In some aspects, the coding sequence of the endogenous gene comprises or consists of SEQ ID NO: 59.

In other aspects of the mutants and related methods, the coding sequence of the gene encoding the acetate kinase hybridizes under at least low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with the full-length complementary strand of SEQ ID NO: 59 (see, e.g., J. Sambrook, E.F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York). For polynucleotides of at least 100 nucleotides in length, very low to very high stringency conditions are defined as prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/mL sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and 5 medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures for 12 to 24 hours optimally. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 45°C (very low stringency), at 50°C (low stringency), at 55°C (medium stringency), at 60°C (medium-high stringency), at 65°C (high stringency), and at 70°C (very high stringency).

i o For polynucleotides of about 15 nucleotides to about 70 nucleotides in length, stringency conditions are defined as prehybridization and hybridization at about 5°C to about 10°C below the calculated T_m using the calculation according to Bolton and McCarthy (1962, Proc. Natl. Acad. Sci. USA 48:1390) in 0.9 M NaCI, 0.09 M Tris-HCI pH 7.6, 6 mM EDTA, 0.5% NP-40, 1 X Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium

15 monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mL following standard Southern blotting procedures for 12 to 24 hours optimally. The carrier material is finally washed once in 6X SCC plus 0.1 % SDS for 15 minutes and twice each for 15 minutes using 6X SSC at 5°C to 10°C below the calculated T_m.

The Lactobacillus mutants described herein may be constructed by disrupting the

20 referenced gene encoding the acetate kinase using methods well known in the art, including those methods described herein. A portion of the gene can be disrupted such as the coding region or a control sequence required for expression of the coding region. Such a control sequence of the gene may be a promoter sequence or a functional part thereof, i.e., a part that is sufficient for affecting expression of the gene. For example, a promoter

25 sequence may be inactivated resulting in no expression or a weaker promoter may be substituted for the native promoter sequence to reduce expression of the coding sequence. Other control sequences for possible modification include, but are not limited to, a leader, propeptide sequence, signal sequence, transcription terminator, and transcriptional activator.

30 The Lactobacillus mutants may be constructed by gene deletion techniques to eliminate or reduce expression of the gene. Gene deletion techniques enable the partial or complete removal of the gene thereby eliminating their expression. In such methods, deletion of the gene is accomplished by homologous recombination using a plasmid that has been constructed to contiguously contain the 5' and 3' regions flanking the gene.

35 The Lactobacillus mutants may also be constructed by introducing, substituting, and/or removing one or more (e.g., two, several) nucleotides in the gene or a control sequence thereof required for the transcription or translation thereof. For example, nucleotides may be inserted or removed for the introduction of a stop codon, the removal of the start codon, or a frame-shift of the open reading frame. Such a modification may be accomplished by site-directed mutagenesis or PCR generated mutagenesis in accordance 5 with methods known in the art. See, for example, Botstein and Shortle, Science 1985, 229, 4719; Lo et al., Proc. Natl. Acad. Sci. U.S.A. 1985, 81, 2285; Higuchi et al., Nucleic Acids Res 1988, 16, 7351 ; Shimada, Meth. Mol. Biol. 1996, 57, 157; Ho et al., Gene 1989, 77, 61 ; Horton et al., Gene 1989, 77, 61 ; and Sarkar and Sommer, BioTechniques 1990, 8, 404.

i o The Lactobacillus mutants may also be constructed by gene disruption techniques by inserting into the gene a disruptive nucleic acid construct comprising a nucleic acid fragment homologous to the gene that will create a duplication of the region of homology and incorporate construct DNA between the duplicated regions. Such a gene disruption can eliminate gene expression if the inserted construct separates the promoter of the gene from

15 the coding region or interrupts the coding sequence such that a non-functional gene product results. A disrupting construct may be simply a selectable marker gene accompanied by 5' and 3' regions homologous to the gene. The selectable marker enables identification of transformants containing the disrupted gene.

The Lactobacillus mutants may also be constructed by the process of gene

20 conversion (see, for example, Iglesias and Trautner, Molecular General Genetics 1983,

189, 73-76). For example, in the gene conversion method, a nucleotide sequence corresponding to the gene is mutagenized in vitro to produce a defective nucleotide sequence, which is then transformed into the Lactobacillus strain to produce a defective gene. By homologous recombination, the defective nucleotide sequence replaces the

25 endogenous gene. It may be desirable that the defective nucleotide sequence also comprises a marker for selection of transformants containing the defective gene.

The Lactobacillus mutants may also be constructed by established anti-sense techniques using a nucleotide sequence complementary to the nucleotide sequence of the gene (Parish and Stoker, FEMS Microbiol. Lett. 1997, 154, 151 -157). More specifically,

30 expression of the gene by a Lactobacillus strain may be reduced or inactivated by introducing a nucleotide sequence complementary to the nucleotide sequence of the gene, which may be transcribed in the strain and is capable of hybridizing to the mRNA produced in the strain. Under conditions allowing the complementary anti-sense nucleotide sequence to hybridize to the mRNA, the amount of protein translated is thus reduced or eliminated.

35 The Lactobacillus mutants may be further constructed by random or specific mutagenesis using methods well known in the art, including, but not limited to, chemical mutagenesis (see, for example, Hopwood, The Isolation of Mutants in Methods in Microbiology (J.R. Norris and D.W. Ribbons, eds.) pp. 363-433, Academic Press, New York, 1970). Modification of the gene may be performed by subjecting the parent strain to mutagenesis and screening for mutant strains in which expression of the gene has been reduced or inactivated. The mutagenesis, which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, use of a suitable oligonucleotide, or subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing methods.

Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N- nitrosoguanidine (MNNG), N-methyl-N'-nitrosogaunidine (NTG) O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues. When such agents are used, the mutagenesis is typically performed by incubating the parent strain to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and selecting for mutants exhibiting reduced or no expression of the gene.

A nucleotide sequence homologous or complementary to a gene described herein may be used from other microbial sources to disrupt the corresponding gene in a Lactobacillus strain of choice.

In one aspect, the modification of a gene in the Lactobacillus host cell is unmarked with a selectable marker. Removal of the selectable marker gene may be accomplished by culturing the mutants on a counter-selection medium. Where the selectable marker gene contains repeats flanking its 5' and 3' ends, the repeats will facilitate the looping out of the selectable marker gene by homologous recombination when the mutant strain is submitted to counter-selection. The selectable marker gene may also be removed by homologous recombination by introducing into the mutant strain a nucleic acid fragment comprising 5' and 3' regions of the defective gene, but lacking the selectable marker gene, followed by selecting on the counter-selection medium. By homologous recombination, the defective gene containing the selectable marker gene is replaced with the nucleic acid fragment lacking the selectable marker gene. Other methods known in the art may also be used.

Also described are methods of producing the Lactobacillus mutants described herein. In one aspect is a method for obtaining a Lactobacillus mutant described herein, comprising disrupting an endogenous gene encoding an acetate kinase in a Lactobacillus strain having an active isopropanol pathway (described below in more detail). In another aspect is a method for obtaining a Lactobacillus mutant described herein, comprising: (a) providing a Lactobacillus strain; (b) disrupting an endogenous gene encoding an acetate kinase in the Lactobacillus strain; (c) transforming the Lactobacillus strain with one or more heterologous isopropanol pathway genes resulting in the strain having an active isopropanol pathway; and (d) isolating the resulting Lactobacillus host cell.

As can be appreciated by one of skill in the art, steps (b) disrupting an endogenous gene encoding an acetate kinase in the Lactobacillus strain; and (c) transforming the Lactobacillus strain with one or more heterologous isopropanol pathway genes resulting in the strain having an active isopropanol pathway, can be conducted in any order— that is, the disruption to the endogenous acetate kinase may occur to a strain already having an active isopropanol pathway, or the disruption to the endogenous acetate kinase may occur to a strain lacking an active isopropanol pathway, followed by introduction of the active isopropanol pathway. Isopropanol Pathways and Methods of Producing Isopropanol

The Lactobacillus mutants described herein can be used in metabolic engineering, e.g., in the production of isopropanol when comprising an active isopropanol pathway. Isopropanol pathway genes and corresponding engineered Lactobacillus transformants for fermentation of isopropanol are known in the art (e.g., see PCT/US201 1/58405, the content of which is hereby incorporated in its entirety). One exemplary isopropanol pathway for the production of isopropanol from glucose is depicted in Figure 1 , wherein cellular acetyl-CoA is converted to acetoacetyl-CoA by a thiolase, acetoacetyl-CoA is converted to acetoacetate by a CoA-transferase, acetoacetate is converted to acetone by an acetoacetate decarboxylase, and acetone is converted to isopropanol by an isopropanol dehydrogenase. Any suitable isopropanol pathway gene encoding a thiolase, CoA transferase, acetoacetate decarboxylase, and/or isopropanol dehydrogenase may be used to produce isopropanol. Thus, the mutant comprising an active isopropanol pathway may comprise thiolase activity, CoA transferase activity, acetoacetate decarboxylase activity and/or isopropanol dehydrogenase activity.

The Lactobacillus mutants may comprise any one or combination of a plurality of the heterologous isopropanol pathway genes described. For example, in one aspect, the recombinant host cell comprises a heterologous thiolase gene, a heterologous CoA- transferase gene, a heterologous acetoacetate decarboxylase gene, and/or a heterologous isopropanol dehydrogenase gene described herein. In some aspects, the mutant produces (or is capable of producing) a greater amount of isopropanol compared to the mutant without the heterologous polynucleotides when cultivated under the same conditions. In some of these aspects, the mutant lacks an endogenous thiolase gene, lacks an endogenous CoA-transferase gene, lacks an endogenous acetoacetate decarboxylase gene, and/or lacks an endogenous isopropanol dehydrogenase gene.

In one aspect, the Lactobacillus mutant comprises one or more (e.g., two, several) heterologous polynucleotides encoding a thiolase described herein. In one aspect, the mutant comprises one or more heterologous polynucleotides encoding a CoA-transferase described herein. In one aspect, the mutant comprises one or more heterologous polynucleotides encoding an acetoacetate decarboxylase described herein. In one aspect, the mutant comprises one or more heterologous polynucleotides encoding an isopropanol dehydrogenase described herein.

The thiolase, CoA-transferase, acetoacetate decarboxylase, and isopropanol dehydrogenase, and activities thereof, can be detected using methods known in the art or as described herein. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. See, for example, Sambrook et al ., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001 ); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999); and Hanai et al., Appl. Environ. Microbiol. 73:7814-7818 (2007)).

In some aspects, the mutant comprises one or more (e.g., two, several) heterologous isopropanol pathway genes described herein, wherein the host cell secretes (and/or is capable of secreting) an increased level of isopropanol compared to the mutant without the one or more heterologous isopropanol pathway genes when cultivated under the same conditions. In some aspects, the mutant secretes and/or is capable of secreting an increased level of isopropanol of at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 100%, at least 150%, at least 200%, at least 300%, or at 500% compared to the mutant without the one or more heterologous isopropanol pathway genes, when cultivated under the same conditions. Examples of suitable cultivation conditions are described below and will be readily apparent to one of skill in the art based on the teachings herein.

In any of these aspects, the host cell produces (and/or is capable of producing) isopropanol at a yield of at least than 10%, e.g., at least than 20%, at least than 30%, at least than 40%, at least than 50%, at least than 60%, at least than 70%, at least than 80%, or at least than 90%, of theoretical.

In any of these aspects, the mutant has an isopropanol volumetric productivity greater than about 0.1 g/L per hour, e.g., greater than about 0.2 g/L per hour, 0.5 g/L per hour, 0.6 g/L per hour, 0.7 g/L per hour, 0.8 g/L per hour, 0.9 g/L per hour, 1.0 g/L per hour, 1 .1 g/L per hour, 1 .2 g/L per hour, 1 .3 g/L per hour, 1 .5 g/L per hour, 1 .75 g/L per hour, 2.0 g/L per hour, 2.25 g/L per hour, 2.5 g/L per hour, or 3.0 g/L per hour; or between about 0.1 g/L per hour and about 2.0 g/L per hour, e.g., between about 0.3 g/L per hour and about 1 .7 g/L per hour, about 0.5 g/L per hour and about 1 .5 g/L per hour, about 0.7 g/L per hour and about 1 .3 g/L per hour, about 0.8 g/L per hour and about 1 .2 g/L per hour, or about 0.9 g/L per hour and about 1.1 g/L per hour.

Thiolases

In some aspects, the host cells comprise a heterologous gene that encodes a thiolase. The thiolase can be any thiolase that is suitable for the host cells and their methods of use described herein, such as a naturally occurring thiolase or a variant thereof that retains thiolase activity. In one aspect, the thiolase is present in the cytosol of the host cells.

In some aspects, the host cells comprising a heterologous thiolase gene have an increased level of thiolase activity compared to the host cells without the heterologous thiolase gene, when cultivated under the same conditions. In some aspects, the host cells have an increased level of thiolase activity of at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 100%, at least 150%, at least 200%, at least 300%, or at 500% compared to the host cells without the heterologous thiolase gene, when cultivated under the same conditions.

Exemplary thiolase genes that can be used with the host cells and methods of use described herein include, but are not limited to, the Clostridium acetobutylicum thiolase gene comprising SEQ ID NO: 1 (which encodes the thiolase of SEQ ID NO: 3), the Lactobacillus reuteri thiolase gene encoding the thiolase of SEQ ID NO: 26, the Lactobacillus brevis thiolase gene comprising SEQ ID NO: 41 (which encodes the thiolase of SEQ ID NO: 42), the Propionibacterium freudenreichii thiolase gene comprising SEQ ID NO: 39 (which encodes the thiolase of SEQ ID NO: 40), an E. coli thiolase (NP_416728, Martin et al., Nat. Biotechnology 21 :796-802 (2003)), a S. cere visiae thiolase (NP_015297, Hiser et al., J. Biol. Chem. 269:31383 -31389 (1994)), a C. pasteurianum thiolase (e.g., protein ID ABAI8857.I), a C. beijerinckii thiolase (e.g., protein ID EAP59904.1 or EAP59331 .1 ), a Clostridium perfringens thiolase (e.g., protein ID ABG86544.I, ABG83108.I), a Clostridium diflicile thiolase (e.g., protein ID CAJ67900.1 or ZP _01231975.1 ), a Thermoanaerobacterium thermosaccharolyticum thiolase (e.g., protein ID CAB07500.1 ), a Thermoanaerobacter tengcongensis thiolase (e.g., A.L\.M23825.1 ), a Carboxydothermus hydrogenoformans thiolase (e.g., protein ID ABB13995.1), a Desulfotomaculum reducens Ml-I thiolase (e.g., protein ID EAR45123.1 ), or a Candida tropicalis thiolase (e.g., protein ID BAA02716.1 or BAA02715.1 ).

In one aspect, the thiolase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the thiolase encoded by any thiolase gene described herein (e.g., any thiolase of SEQ ID NO: 3, 26, 40 or 42), or the mature polypeptide sequence thereof. In one aspect, the thiolase sequence differs by no more than ten amino acids, e.g., by no more than five amino acids, by no more than four amino acids, by no more than three amino acids, by no more than two amino acids, or by one amino acid from the thiolase encoded by any thiolase gene described herein (e.g., any thiolase of SEQ ID NO: 3, 26, 40 or 42), or the mature polypeptide sequence thereof. In one aspect, the thiolase comprises or consists of the amino acid sequence of the thiolase encoded by any thiolase gene described herein (e.g., any thiolase of SEQ ID NO: 3, 26, 40 or 42), the mature polypeptide sequence, allelic variant, or a fragment thereof having thiolase activity. In one aspect, the thiolase has an amino acid substitution, deletion, and/or insertion of one or more (e.g., two, several) amino acids. In some aspects, the total number of amino acid substitutions, deletions and/or insertions is not more than 10, e.g., not more than 9, 8, 7, 6, 5, 4, 3, 2, or 1 .

The amino acid changes are generally of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, LeuA al, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that the physico- chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the thiolase, alter the substrate specificity, change the pH optimum, and the like.

Essential amino acids in a thiolase can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081 -1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for thiolase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the thiolase or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identities of essential amino acids can also be inferred from analysis of identities with other thiolases that are related to the referenced thiolase.

Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152- 2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error- prone PCR, phage display (e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active thiolases can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

In one aspect, the coding sequence of the heterologous thiolase gene hybridizes under at least low stringency conditions, e.g., medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with the coding sequence of any thiolase gene described herein (e.g., any thiolase gene of SEQ ID NO: 1 , 2, 25, 39, or 41 ), or the full-length complementary strand thereof (see, e.g., J. Sambrook, et al., supra).

In one aspect, the coding sequence of the heterologous thiolase gene has at least 65%, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the coding sequence of any thiolase gene described herein (e.g., any thiolase gene of SEQ I D NO: 1 , 2, 25, 39, or 41 ), or the mature polypeptide coding sequence thereof.

In one aspect, the coding sequence of the heterologous thiolase gene comprises the coding sequence of any thiolase gene described herein (e.g., any thiolase gene of SEQ ID NO: 1 , 2, 25, 39, or 41 ), or the mature polypeptide coding sequence thereof. In one aspect, the coding sequence of the heterologous thiolase gene comprises a subsequence of any thiolase gene described herein (e.g., any thiolase gene of SEQ I D NO: 1 , 2, 25, 39, or 41 ), or the mature polypeptide coding sequence thereof, wherein the subsequence encodes a polypeptide having thiolase activity. In one aspect, the number of nucleotides residues in the subsequence is at least 75%, e.g., at least 80%, 85%, 90%, or 95% of the number of the referenced sequence.

The thiolase may be a fused polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the thiolase. A fused polypeptide may be produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide encoding the thiolase. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter(s) and terminator. Fusion proteins may also be constructed using intein technology in which fusions are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251 ; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991 , Biotechnology 9: 378-381 ; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48. Techniques used to isolate or clone the thiolase genes used in any of the aspects mentioned herein, are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the polynucleotides from such genomic DNA can be effected, e.g., by using the well-known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shares structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleotide sequence-based amplification (NASBA) may be used.

The thiolase genes described herein, or subsequences thereof; as well as the corresponding amino acid sequences, or fragments thereof; may be used to design nucleic acid probes to identify and clone DNA encoding a thiolase from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, e.g., at least 14 nucleotides, at least 25 nucleotides, at least 35 nucleotides, at least 70 nucleotides in lengths. The probes may be longer, e.g., at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides in lengths. Even longer probes may be used, e.g., at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).

A genomic DNA or cDNA library prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having thiolase activity. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that is homologous with SEQ ID NO: 1 , 3, or a subsequence thereof, the carrier material may be used in a Southern blot.

For purposes of the probes described above, hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to the coding sequence of the thiolase gene, the full-length complementary strand thereof, or a subsequence of the foregoing; under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film.

The thiolase genes described herein may be obtained from a microorganism of any genus. As used herein, the term "obtained from" in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a cell in which the polynucleotide from the source has been inserted.

The thiolase may be a bacterial thiolase. For example, the thiolase may be a Gram- positive bacterial thiolase such as a Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, or Oceanobacillus lactate dehydrogenase, or a Gram-negative bacterial thiolase such as an E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, llyobacter, Neisseria, or Ureaplasma thiolase.

In one aspect, the thiolase is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis thiolase.

In another aspect, the thiolase is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus lactate dehydrogenase. In another aspect, the lactate dehydrogenase is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans thiolase.

The thiolase may be a fungal thiolase. In one aspect, the fungal thiolase is a yeast thiolase such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia thiolase.

In another aspect, the fungal thiolase is a filamentous fungal thiolase such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria thiolase.

In another aspect, the thiolase is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces luyveri, Saccharomyces norbensis, or Saccharomyces oviformis thiolase.

In another aspect, the thiolase is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus flavus, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus sojae, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium grammearum, Fusarium grammum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride thiolase.

It will be understood that for the aforementioned species, both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, are encompassed regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

The thiolase gene may also be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, silage, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, silage, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. The thiolase genes may then be derived by similarly screening a genomic or cDNA library of another microorganism or mixed DNA sample. Once a thiolase gene has been detected with suitable probe(s) as described herein, the sequence may be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., J. Sambrook, E.F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).

CoA-transferases

In some aspects, the host cells comprise a heterologous gene that encodes a CoA- transferase. The CoA-transferase can be any CoA-transferase that is suitable for the host cells and their methods of use described herein, such as a naturally occurring CoA- transferase or a variant thereof that retains CoA-transferase activity. In some aspects, the CoA-transferase is an acetoacetyl-CoA:acetate/butyrate CoA transferase. In some aspects, the CoA-transferase is an acetoacetyl-CoA hydrolase. In some aspects, the CoA- transferase is a succinyl-CoA:acetoacetate transferase. In some aspects, the CoA- transferase is present in the cytosol of the host cells. In some aspects, the CoA-transferase is a protein complex comprising a first CoA-transferase subunit and the second CoA- transferase subunit wherein the subunits comprise different amino acid sequences.

In some aspects, the host cells comprising a heterologous CoA-transferase gene have an increased level of CoA-transferase activity compared to the host cells without the heterologous CoA-transferase gene, when cultivated under the same conditions. In some aspects, the host cells have an increased level of CoA-transferase activity of at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 100%, at least 150%, at least 200%, at least 300%, or at 500% compared to the host cells without the heterologous CoA-transferase gene, when cultivated under the same conditions.

Exemplary succinyl-CoA:acetoacetate transferase genes that can be used with the host cells and methods of use described herein include, but are not limited to, a Bacillus subtilis succinyl-CoA:acetoacetate transferase gene comprising SEQ ID NO: 4 and SEQ ID NO: 7 (which encodes a protein complex comprising subunits of SEQ ID NO: 6 and SEQ ID NO: 9, respectively), a Bacillus mojavensis succinyl-CoA:acetoacetate transferase gene comprising SEQ ID NO: 10 and SEQ ID NO: 13 (which encodes a protein complex comprising subunits of SEQ ID NO: 12 and SEQ ID NO: 15, respectively), a Helicobacter pylori succinyl-CoA:acetoacetate transferase (YP_627417, YP_627418, Corthesy-Theulaz, et al., J Biol Chem 272:25659-25667 (1997)), and a Homo sapiens succinyl- CoA:acetoacetate transferase (NP_000427, NP071403, Fukao, T., et al., Genomics 68:144-151 (2000); Tanaka, H., et al., Mol Hum Reprod 8:16-23 (2002)).

Exemplary acetoacetyl-CoA:acetate/butyrate CoA transferase genes that can be used with the host cells and methods of use described herein include, but are not limited to, an E. coli acetoacetyl-CoA:acetate CoA transferase (comprising SEQ ID NO: 27 and SEQ ID NO: 29, which encodes a protein complex comprising subunits of SEQ ID NO: 28 and SEQ ID NO: 30, respectively; NP 416726.1 , NP_416725.1 ; Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007)), a Clostridium acetobutylicum acetoacetyl-CoA:acetate CoA transferase (comprising SEQ ID NO: 31 and SEQ ID NO: 33, which encodes a protein complex comprising subunits of SEQ ID NO: 32 and SEQ ID NO: 34, respectively NPJ 49326.1 , NPJ 49327.1 ; Jojima et al., Appl Microbiol Biotechnol 77:1219-1224 (2008)), and a Clostridium saccharoperbutylacetonicum acetoacetyl-CoA:acetate CoA transferase (AAP42564.1 , AAP42565.1 ; Kosaka et al., Biosci. Biotechnol Biochem. 71 :58- 68 (2007)).

Exemplary acetoacetyl-CoA hydrolase genes that can be used with the host cells and methods of use described herein include, but are not limited to, acyl-CoA hydrolases, 3-hydroxyisobutyryl-CoA hydrolases, acetyl-CoA hydrolases, and dicarboxylic acid thioesterases, such as a Rattus norvegicus 3-hydroxyisobutyryl-CoA hydrolase (Q5XIE6.2; Shimomura et al., J Biol. Chem. 269 :14248-14253 (1994)), a Homo sapiens 3- hydroxyisobutyryl-CoA hydrolase (Q6NVY1 .2; Shimomura et al., supra), a Rattus norvegicus acetyl-CoA hydrolase (NP 570103.1 ; Robinson et al., Res. Commun. 71 :959- 965 (1976)), a Saccharomyces cerevisiae acetyl-CoA hydrolase (NP_009538; Buu et al., J. Biol. Chem. 278: 17203-17209 (2003)), a Homo sapiens dicarboxylic acid thioesterase (CAA15502; Westin et al., J Biol. Chem. 280:38125-38132 (2005)), and an Escherichia coli dicarboxylic acid thioesterase (Naggert et al., J Biol. Chem. 266: 1 1044-1 1050 (1991 )).

In one aspect, the CoA-transferase is a protein complex wherein one or more subunits have at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the CoA-transferase subunits encoded by any CoA-transferase subunit gene described herein (e.g., any CoA-transferase subunit of the complexes of SEQ ID NOs: 6+9, 12+15, 28+30, or 32+34), or the mature polypeptide sequence thereof. In one aspect, the sequence of the CoA-transferase subunits differ by no more than ten amino acids, e.g., by no more than five amino acids, by no more than four amino acids, by no more than three amino acids, by no more than two amino acids, or by one amino acid from the CoA- transferase subunits encoded by any CoA-transferase subunit gene described herein (e.g., any CoA-transferase subunit of the complexes of SEQ ID NOs: 6+9, 12+15, 28+30, or 32+34), or the mature polypeptide sequence thereof. In one aspect, the CoA-transferase is a protein complex comprising or consisting of the amino acid sequences of any CoA- transferase protein complex described herein (e.g., any CoA-transferase complex of SEQ ID NOs: 6+9, 12+15, 28+30, or 32+34). In one aspect, the sequence of the CoA- transferase subunit has an amino acid substitution, deletion, and/or insertion of one or more (e.g., two, several) amino acids. In some aspects, the total number of amino acid 5 substitutions, deletions and/or insertions is not more than 10, e.g., not more than 9, 8, 7, 6, 5, 4, 3, 2, or 1 .

In one aspect, the coding sequence of the heterologous CoA-transferase subunit gene hybridizes under at least low stringency conditions, e.g., medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high i o stringency conditions with the coding sequence of any CoA-transferase subunit gene described herein (e.g., a subunit gene comprising SEQ ID NO: 4, 5, 7, 8, 10, 1 1 , 13, 14), or the full-length complementary strand thereof (see, e.g., J. Sambrook, et al., supra).

In one aspect, the coding sequence of the heterologous CoA-transferase subunit gene has at least 65%, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least

15 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the coding sequence of any CoA-transferase subunit gene described herein (e.g., a subunit gene comprising SEQ ID NO: 4, 5, 7, 8, 10, 1 1 , 13, 14), or the mature polypeptide coding sequence thereof.

20 In one aspect, the coding sequence of the heterologous CoA-transferase subunit gene comprises the coding sequence of any CoA-transferase subunit gene described herein (e.g., a subunit gene comprising SEQ ID NO: 4, 5, 7, 8, 10, 1 1 , 13, 14), or the mature polypeptide coding sequence thereof. In one aspect, the coding sequence of the heterologous CoA-transferase gene comprises a subsequence of any CoA-transferase

25 gene described herein, or the mature polypeptide coding sequence thereof. In one aspect, the number of nucleotides residues in the subsequence is at least 75%, e.g., at least 80%, 85%, 90%, or 95% of the number of the referenced sequence.

The CoA-transferases (and subunits thereof) can also include fused polypeptides or cleavable fusion polypeptides, as described supra.

30 The CoA-transferase genes can also be used to design nucleic acid probes to identify and clone DNA encoding CoA-transferases from strains of different genera or species, as described supra.

Techniques used to isolate or clone a polynucleotide encoding a CoA-transferase, and polypeptide subunits thereof, are described supra.

35 The CoA-transferase genes may also be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.,) as described supra.

Acetoacetate decarboxylases

In some aspects, the host cells comprise a heterologous gene that encodes an acetoacetate decarboxylase. The acetoacetate decarboxylase can be any acetoacetate decarboxylase that is suitable for the host cells and their methods of use described herein, such as a naturally occurring acetoacetate decarboxylase or a variant thereof that retains acetoacetate decarboxylase activity. In one aspect, the acetoacetate decarboxylase is present in the cytosol of the host cells.

In some aspects, the host cells comprising a heterologous acetoacetate decarboxylase gene have an increased level of acetoacetate decarboxylase activity compared to the host cells without the heterologous acetoacetate decarboxylase gene, when cultivated under the same conditions. In some aspects, the host cells have an increased level of acetoacetate decarboxylase activity of at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 100%, at least 150%, at least 200%, at least 300%, or at 500% compared to the host cells without the heterologous acetoacetate decarboxylase gene, when cultivated under the same conditions.

Exemplary acetoacetate decarboxylase genes that can be used with the host cells and methods of use described herein include, but are not limited to, a Clostridium beijerinckii acetoacetate decarboxylase of SEQ ID NO: 16 (which encodes the acetoacetate decarboxylase of SEQ ID NO: 18), a Lactobacillus salivarius acetoacetate decarboxylase gene comprising SEQ ID NO: 43 (which encodes the acetoacetate decarboxylase of SEQ ID NO: 44), a Lactobacillus plantarum acetoacetate decarboxylase gene comprising SEQ ID NO: 45 (which encodes the acetoacetate decarboxylase of SEQ ID NO: 46), a C. acetobutylicum acetoacetate decarboxylase gene (NP_149328.1 , which encodes the acetoacetate decarboxylase of SEQ ID NO: 36; see Petersen and Bennett, Appl. Environ. Microbiol 56:3491 -3498 (1990)) and a Clostridium saccharoperbutylacetonicum acetoacetate decarboxylase (AAP42566.1 , Kosaka, et al., Biosci. Biotechnol Biochem. 71 :58-68 (2007)).

In one aspect, the acetoacetate decarboxylase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the acetoacetate decarboxylase encoded by any acetoacetate decarboxylase gene described herein (e.g., any acetoacetate decarboxylase of SEQ ID NO: 18, 36, 44, or 46), or the mature polypeptide sequence thereof. In one aspect, the acetoacetate decarboxylase sequence differs by no more than ten amino acids, e.g., by no more than five amino acids, by no more than four amino acids, by no more than three amino acids, by no more than two amino acids, or by one amino acid from the acetoacetate decarboxylase encoded by any acetoacetate decarboxylase gene described 5 herein (e.g., any acetoacetate decarboxylase of SEQ ID NO: 18, 36, 44, or 46), or the mature polypeptide sequence thereof. In one aspect, the acetoacetate decarboxylase comprises or consists of the amino acid sequence of the acetoacetate decarboxylase encoded by any acetoacetate decarboxylase gene described herein (e.g., any acetoacetate decarboxylase of SEQ ID NO: 18, 36, 44, or 46), the mature polypeptide sequence, allelic i o variant, or a fragment thereof having acetoacetate decarboxylase activity. In one aspect, the acetoacetate decarboxylase has an amino acid substitution, deletion, and/or insertion of one or more (e.g., two, several) amino acids. In some aspects, the total number of amino acid substitutions, deletions and/or insertions is not more than 10, e.g., not more than 9, 8, 7, 6, 5, 4, 3, 2, or 1 .

15 In one aspect, the coding sequence of the heterologous acetoacetate decarboxylase gene hybridizes under at least low stringency conditions, e.g., medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with the coding sequence of any acetoacetate decarboxylase gene described herein (e.g., any acetoacetate decarboxylase gene of SEQ

20 ID NO: 16, 17, 35, 43, or 45), or the full-length complementary strand thereof (see, e.g., J.

Sambrook, et al., supra).

In one aspect, the coding sequence of the heterologous acetoacetate decarboxylase gene has at least 65%, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least

25 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the coding sequence of any acetoacetate decarboxylase gene described herein (e.g., any acetoacetate decarboxylase gene of SEQ ID NO: 16, 17, 35, 43, or 45), or the mature polypeptide coding sequence thereof.

In one aspect, the coding sequence of the heterologous acetoacetate

30 decarboxylase gene comprises the coding sequence of any acetoacetate decarboxylase gene described herein (e.g., any acetoacetate decarboxylase gene of SEQ I D NO: 16, 17, 35, 43, or 45), or the mature polypeptide coding sequence thereof. In one aspect, the coding sequence of the heterologous acetoacetate decarboxylase gene comprises a subsequence of any acetoacetate decarboxylase gene described herein, or the mature

35 polypeptide coding sequence thereof. In one aspect, the number of nucleotides residues in the subsequence is at least 75%, e.g., at least 80%, 85%, 90%, or 95% of the number of the referenced sequence.

The acetoacetate decarboxylases can also include fused polypeptides or cleavable fusion polypeptides, as described supra.

The acetoacetate decarboxylase genes can also be used to design nucleic acid probes to identify and clone DNA encoding acetoacetate decarboxylases from strains of different genera or species, as described supra.

Techniques used to isolate or clone a polynucleotide encoding a acetoacetate decarboxylase are described supra.

The acetoacetate decarboxylase genes may also be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.,) as described supra.

Isopropanol dehydrogenases

In some aspects, the host cells comprise a heterologous gene that encodes an isopropanol dehydrogenase. The isopropanol dehydrogenase can be any isopropanol dehydrogenase that is suitable for the host cells and their methods of use described herein, such as a naturally occurring isopropanol dehydrogenase or a variant thereof that retains isopropanol dehydrogenase activity. In one aspect, the isopropanol dehydrogenase is present in the cytosol of the host cells.

In some aspects, the host cells comprising a heterologous isopropanol dehydrogenase gene have an increased level of isopropanol dehydrogenase activity compared to the host cells without the heterologous isopropanol dehydrogenase gene, when cultivated under the same conditions. In some aspects, the host cells have an increased level of isopropanol dehydrogenase activity of at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 100%, at least 150%, at least 200%, at least 300%, or at 500% compared to the host cells without the heterologous isopropanol dehydrogenase gene, when cultivated under the same conditions.

Exemplary isopropanol dehydrogenase genes that can be used with the host cells and methods of use described herein include, but are not limited to, a Clostridium beijerinckii isopropanol dehydrogenase of SEQ ID NO: 19 (which encodes the isopropanol dehydrogenase of SEQ ID NO: 21 ), a Thermoanaerobacter ethanolicus isopropanol dehydrogenase gene comprising SEQ ID NO: 22 (which encodes the isopropanol dehydrogenase of SEQ ID NO: 24), a Lactobacillus fermentum isopropanol dehydrogenase gene comprising SEQ ID NO: 47 (which encodes the isopropanol dehydrogenase of SEQ ID NO: 48), a Lactobacillus antri isopropanol dehydrogenase gene comprising SEQ ID NO: 37 (which encodes the isopropanol dehydrogenase of SEQ ID NO: 38), a Thermoanaerobacter brockii isopropanol dehydrogenase (P14941 .1 , Hanai et al., Appl. Environ. Microbiol. 73:7814-7818 (2007); Peretz et al., Anaerobe 3:259-270 (1997)), a 5 Ralstonia eutropha isopropanol dehydrogenase (formerly Alcaligenes eutrophus) (YP_299391 .1 , Steinbuchel and Schlegel et al., Eur. J. Biochem. 141 :555-564 (1984)), a Burkholderia sp. AIU 652 isopropanol dehydrogenase, and a Phytomonas species isopropanol dehydrogenase (AAP39869.1 , Uttaro and Opperdoes et al., Mol. Biochem. Parasitol. 85:213-219 (1997)).

i o In one aspect, the isopropanol dehydrogenase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the isopropanol dehydrogenase encoded by any isopropanol dehydrogenase gene described herein (e.g., any isopropanol dehydrogenase

15 of SEQ ID NO: 21 , 24, 38, or 48), or the mature polypeptide sequence thereof. In one aspect, the isopropanol dehydrogenase sequence differs by no more than ten amino acids, e.g., by no more than five amino acids, by no more than four amino acids, by no more than three amino acids, by no more than two amino acids, or by one amino acid from the isopropanol dehydrogenase encoded by any isopropanol dehydrogenase gene described

20 herein (e.g., any isopropanol dehydrogenase of SEQ ID NO: 21 , 24, 38, or 48), or the mature polypeptide sequence thereof. In one aspect, the isopropanol dehydrogenase comprises or consists of the amino acid sequence of the isopropanol dehydrogenase encoded by any isopropanol dehydrogenase gene described herein (e.g., any isopropanol dehydrogenase of SEQ I D NO: 21 , 24, 38, or 48), the mature polypeptide sequence, allelic

25 variant, or a fragment thereof having isopropanol dehydrogenase activity. In one aspect, the isopropanol dehydrogenase has an amino acid substitution, deletion, and/or insertion of one or more (e.g., two, several) amino acids. In some aspects, the total number of amino acid substitutions, deletions and/or insertions is not more than 10, e.g., not more than 9, 8, 7, 6, 5, 4, 3, 2, or 1 .

30 In one aspect, the coding sequence of the heterologous isopropanol dehydrogenase gene hybridizes under at least low stringency conditions, e.g., medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with the coding sequence of any isopropanol dehydrogenase gene described herein (e.g., any isopropanol dehydrogenase gene of SEQ

35 ID NO: 19, 20, 22, 23, 37, or 47), or the full-length complementary strand thereof (see, e.g., J. Sambrook, et al., supra). In one aspect, the coding sequence of the heterologous isopropanol dehydrogenase gene has at least 65%, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the coding sequence of any isopropanol dehydrogenase gene described herein (e.g., any isopropanol dehydrogenase gene of SEQ ID NO: 19, 20, 22, 23, 37, or 47), or the mature polypeptide coding sequence thereof.

In one aspect, the coding sequence of the heterologous isopropanol dehydrogenase gene comprises the coding sequence of any isopropanol dehydrogenase gene described herein (e.g., any isopropanol dehydrogenase gene of SEQ ID NO: 19, 20, 22, 23, 37, or 47), or the mature polypeptide coding sequence thereof. In one aspect, the coding sequence of the heterologous isopropanol dehydrogenase gene comprises a subsequence of any isopropanol dehydrogenase gene described herein, or the mature polypeptide coding sequence thereof. In one aspect, the number of nucleotides residues in the subsequence is at least 75%, e.g., at least 80%, 85%, 90%, or 95% of the number of the referenced sequence.

The isopropanol dehydrogenases can also include fused polypeptides or cleavable fusion polypeptides, as described supra.

The isopropanol dehydrogenase genes can also be used to design nucleic acid probes to identify and clone DNA encoding isopropanol dehydrogenases from strains of different genera or species, as described supra.

Techniques used to isolate or clone a polynucleotide encoding an isopropanol dehydrogenase are described supra.

The isopropanol dehydrogenase genes may also be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.,) as described supra.

Expression Vectors and Nucleic Acid Constructs

The recombinant host cells and methods may utilize expression vectors comprising the coding sequence of one or more (e.g., two, several) heterologous isopropanol pathway genes (e.g., the coding sequence of a thiolase, CoA-transferase, acetoacetate decarboxylase, and/or isopropanol dehydrogenase described herein) linked to one or more control sequences that direct expression in a suitable host cell under conditions compatible with the control sequence(s). Such expression vectors may be used in any of the host cells and methods describe herein. The polynucleotides described herein may be manipulated in a variety of ways to provide for expression of a desired polypeptide. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more (e.g., two, several) convenient restriction sites to allow for insertion or substitution of the polynucleotide at such sites. Alternatively, the polynucleotide(s) may be expressed by inserting the polynucleotide(s) or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

In one aspect, each gene coding sequence is contained on an independent vector. In one aspect, at least two of the gene coding sequences are contained on a single vector. In one aspect, at least three of the gene coding sequences are contained on a single vector. In one aspect, at least four of the gene coding sequences are contained on a single vector. In one aspect, all the gene coding sequences are contained on a single vector. Polynucleotides encoding heteromeric subunits of a protein complex (e.g., a CoA- transferase) may be contained in a single heterologous polynucleotide on a single vector or alternatively contained in separate heterologous polynucleotides on separate vectors.

The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

The expression vector may contain any suitable promoter sequence that is recognized by a host cell for expression of any isopropanol pathway gene described herein. The promoter sequence contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

Each gene coding sequence described herein may be operably linked to a promoter that is foreign to the gene. For example, in one aspect, the gene coding sequence encoding a thiolase, CoA-transferase, acetoacetate decarboxylase, and/or isopropanol dehydrogenase is operably linked to promoter foreign to the polynucleotide.

As described supra, polynucleotides encoding heteromeric subunits of a protein complex may be contained in a single heterologous polynucleotide (e.g., a single plasmid), or alternatively contained in separate heterologous polynucleotides (e.g., on separate plasmids). For example, in one aspect, the heterologous polynucleotide encoding a first subunit, and the heterologous polynucleotide encoding a second subunit are contained in a single heterologous polynucleotide operably linked to a promoter that is foreign to both the heterologous polynucleotide encoding the first subunit and the heterologous polynucleotide encoding the second subunit. In one aspect, the heterologous polynucleotide encoding a first subunit, and the heterologous polynucleotide encoding a second subunit are each contained in separate unlinked heterologous polynucleotides, wherein the heterologous polynucleotide encoding the first subunit is operably linked to a foreign promoter, and the heterologous polynucleotide encoding the second subunit is operably linked to a foreign promoter. The promoters in the foregoing may be the same or different.

Examples of suitable promoters for directing the transcription of the nucleic acid constructs in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, E. coli lac operon, E. coli trc promoter (Egon et al., 1988, Gene 69: 301 -315), Streptomyces coelicolor agarase gene {dagA), and prokaryotic beta- lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731 ), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21 -25). Further promoters are described in "Useful proteins from recombinant bacteria" in Gilbert et al., 1980, Scientific American, 242: 74-94; and in Sambrook et al., 1989, supra. Additional exemplary synthetic promoters useful for gene expression in Lactobacillus can be found in Rud et al., 2006, Microbiology 152, 101 1 -1019.

The control sequence may also be a suitable transcription terminator sequence, which is recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3'-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell of choice may be used.

The control sequence may also be a suitable leader sequence, when transcribed is a nontranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5'-terminus of the polynucleotide encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used.

The control sequence may also be a polyadenylation sequence; a sequence operably linked to the 3'-terminus of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell of choice may be used.

It may also be desirable to add regulatory sequences that allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the polynucleotide encoding the polypeptide would be operably linked with the regulatory sequence.

The vectors may contain one or more (e.g., two, several) selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or

Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, erythromycin, kanamycin, spectinomycin, or tetracycline resistance.

The vectors may contain one or more (e.g., two, several) elements that permit integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicator" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB1 10, pE194, pTA1060, and ρΑΜβΙ permitting replication in Bacillus.

More than one copy of a polynucleotide described herein may be inserted into a host cell to increase production of a polypeptide. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to construct the recombinant expression vectors described herein are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).

A construct or vector (or multiple constructs or vectors) comprising the one or more (e.g., two, several) heterologous isopropanol pathway genes may be introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self- replicating extra-chromosomal vector as described earlier. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.

The introduction of a construct or vector containing one or more heterologous isopropanol pathway genes into a Lactobacillus cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 1 1 1 - 1 15), by using competent cells (see, e.g., Young and Spizizen, 1961 , J. Bacteriol. 81 : 823- 829, or Dubnau and Davidoff-Abelson, 1971 , J. Mol. Biol. 56: 209-221 ), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751 ), or by conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169: 5271 -5278).

Methods of producing isopropanol

The mutant cells described herein may be used for the production of isopropanol. In one aspect is a method of producing isopropanol, comprising: (a) cultivating any one of the Lactobacillus mutants described herein in a medium under suitable conditions to produce the isopropanol; and (b) recovering the isopropanol.

The mutants comprising an active isopropanol pathway may be cultivated in a nutrient medium suitable for isopropanol production using methods well known in the art. For example, the cell may be cultivated by shake flask cultivation, and small-scale or large- scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable fermentation medium and under conditions allowing isopropanol production.

The mutants may produce isopropanol in a fermentable medium comprising any one or more (e.g., two, several) sugars, such as glucose, fructose, sucrose, cellobiose, xylose, xylulose, arabinose, mannose, galactose, and/or soluble oligosaccharides. In some instances, the fermentable medium is derived from a natural source, such as sugar cane, starch, or cellulose, and may be the result of pretreating the source by enzymatic hydrolysis (saccharification). In some aspects, the fermentable medium comprises sugar cane juice. Suitable media are available from commercial suppliers, may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection), or may be prepared from commercially available ingredients.

The applicants have surprisingly found that the mutants produce greater amounts of isopropanol when fermented in the presence of fructose. Accordingly, in one aspect, the Lactobacillus host cell comprising an active isopropanol pathway and a disruption to an endogenous gene encoding an acetate kinase produces a greater amount of isopropanol when cultivated in the presence of fructose, such as at least 0.1 %, 0.25%, 0.5%, 0.75%, 1 %, 2%, 3%, 4%, 5% or 10% fructose.

The applicants have also found that the mutants produce greater amounts of isopropanol when fermented in the presence of 1 ,2-propanediol. Accordingly, in one aspect, the Lactobacillus host cell comprising an active isopropanol pathway and a disruption to an endogenous gene encoding an acetate kinase produces a greater amount of isopropanol when cultivated in the presence of 1 ,2-propanediol, such as at least 0.1 %, 0.25%, 0.5%, 0.75%, 1 %, 2%, 3%, 4%, 5% or 10% 1 ,2-propanediol.

In addition to the appropriate carbon sources from one or more (e.g., two, several) sugar(s), the fermentable medium may contain other nutrients or stimulators known to those skilled in the art, such as macronutrients (e.g., nitrogen sources) and micronutrients (e.g., vitamins, mineral salts, and metallic cofactors). In some aspects, the carbon source can be preferentially supplied with at least one nitrogen source, such as yeast extract, N₂, peptone (e.g., Bacto™ Peptone), or soytone (e.g., Bacto™ Soytone). Nonlimiting examples of vitamins include multivitamins, biotin, pantothenate, nicotinic acid, meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and Vitamins A, B, C, D, and E. Examples of mineral salts and metallic cofactors include, but are not limited to Na, P, K, Mg, S, Ca, Fe, Zn, Mn, Co, and Cu.

Suitable conditions used for the methods of propanol production may be determined by one skilled in the art in light of the teachings herein. In some aspects of the methods, the host cells are cultivated for about 12 hours to about 216 hours, such as about 24 hours to about 144 hours, or about 36 hours to about 96 hours. The temperature is typically between about 26°C to about 60°C, e.g., about 34°C to about 50°C, and at a pH of about 3.0 to about 8.0, such as about 3.0 to about 7.0, about 3.0 to about 6.0, about 3.0 to about 5.0, about 3.5 to about 4.5, about 4.0 to about 8.0, about 4.0 to about 7.0, about 4.0 to about 6.0, about 4.0 to about 5.0, about 5.0 to about 8.0, about 5.0 to about 7.0, or about 5.0 to about 6.0 or less than about 6.0, less than about 5.5, less than about 5.0, less than about 4.5, less than about 4.0, less than about 3.5, less than about 3.0, or less than about 2.5. In some aspects of the methods, the resulting intracellular pH of the host cell is about 2.0 to about 8.0, such as about 2.0 to about 7.0, about 2.0 to about 6.0, about 2.0 to about 5.0, about 1 .5 to about 4.5, about 3.0 to about 8.0, such as about 3.0 to about 7.0, about 3.0 to about 6.0, about 3.0 to about 5.0, about 3.5 to about 4.5, about 4.0 to about 8.0, about 4.0 to about 7.0, about 4.0 to about 6.0, about 4.0 to about 5.0, about 5.0 to about 8.0, about 5.0 to about 7.0, or about 5.0 to about 6.0, or less than about 6.0, less than about 5.5, less than about 5.0, less than about 4.5, less than about 4.0, less than about 3.5, less than about 3.0, or less than about 2.5. Cultivation may be performed under anaerobic, microaerobic, or aerobic conditions, as appropriate. In some aspects, the cultivation is performed under anaerobic conditions.

Cultivation may be performed under anaerobic, substantially anaerobic (microaerobic), or aerobic conditions, as appropriate. Briefly, anaerobic refers to an environment devoid of oxygen, substantially anaerobic (microaerobic) refers to an environment in which the concentration of oxygen is less than air, and aerobic refers to an environment wherein the oxygen concentration is approximately equal to or greater than that of the air. Substantially anaerobic conditions include, for example, a culture, batch fermentation or continuous fermentation such that the dissolved oxygen concentration in the medium remains less than 10% of saturation. Substantially anaerobic conditions also includes growing or resting cells in liquid medium or on solid agar inside a sealed chamber maintained with an atmosphere of less than 1 % oxygen. The percent of oxygen can be maintained by, for example, sparging the culture with an N₂/C0₂ mixture or other suitable non-oxygen gas or gases. In some embodiments, the cultivation is performed under anaerobic conditions or substantially anaerobic conditions.

The methods of described herein can employ any suitable fermentation operation mode. For example, a batch mode fermentation may be used with a close system where culture media and host microorganism, set at the beginning of fermentation, have no additional input except for the reagents certain reagents, e.g., for pH control, foam control or others required for process sustenance. The process described herein can also be employed in Fed-batch or continuous mode.

The methods described herein may be practiced in several bioreactor configurations, such as stirred tank, bubble column, airlift reactor and others known to those skilled in the art. The methods may be performed in free cell culture or in immobilized cell culture as appropriate. Any material support for immobilized cell culture may be used, such as alginates, fibrous bed, or argyle materials such as chrysotile, montmorillonite KSF and montmorillonite K-10.

In one aspect of the methods, the isopropanol is produced at a titer greater than about 10 g/L, e.g., greater than about 25 g/L, 50 g/L, 75 g/L, 100 g/L, 125 g/L, 150 g/L, 160 g/L, 170 g/L, 180 g/L, 190 g/L, 200 g/L, 210 g/L, 225 g/L, 250 g/L, 275 g/L, 300 g/L, 325 g/L, 350 g/L, 400 g/L, or 500g/L; or between about 10 g/L and about 500 g/L, e.g., between about 50 g/L and about 350 g/L, about 100 g/L and about 300 g/L, about 150 g/L and about 250 g/L, about 175 g/L and about 225 g/L, or about 190 g/L and about 210 g/L. In one aspect of the methods, the isopropanol is produced at a titer greater than about 0.01 gram per gram of carbohydrate, e.g., greater than about 0.02, 0.05, 0.75, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 .0 gram per gram of carbohydrate.

In one aspect of the methods, the amount of produced isopropanol, is at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 50%, or at least 100% greater compared to cultivating the host cell without the one or more (e.g., two, several) heterologous isopropanol pathway genes when cultivated under the same conditions.

The isopropanol can be optionally recovered from the fermentation medium using any procedure known in the art including, but not limited to, chromatography (e.g., size exclusion chromatography, adsorption chromatography, ion exchange chromatography), electrophoretic procedures, differential solubility, osmosis, distillation, extraction (e.g., liquid-liquid extraction), pervaporation, extractive filtration, membrane filtration, membrane separation, reverse, or ultrafiltration. In one aspect, the isopropanol is separated from other fermented material and purified by conventional methods of distillation. Accordingly, in one aspect, the method further comprises purifying the recovered isopropanol by distillation.

The recombinant isopropanol may also be purified by the chemical conversion of impurities (contaminants) to products more easily removed from isopropanol by the procedures described above (e.g., chromatography, electrophoretic procedures, differential solubility, distillation, or extraction) and/or by direct chemical conversion of impurities to isopropanol. For example, in one aspect, the method further comprises purifying the recovered isopropanol by converting acetone contaminant to isopropanol. Conversion of acetone to isopropanol may be accomplished using any suitable reducing agent known in the art (e.g., lithium aluminium hydride (LiAIH₄), a sodium species (such as sodium amalgam or sodium borohydride (NaBH₄)), tin species (such as tin(ll) chloride), hydrazine, zinc-mercury amalgam (Zn(Hg)), diisobutylaluminum hydride (DIBAH), oxalic acid (C₂H₂0₄), formic acid (HCOOH), Ascorbic acid, iron species (such as iron(ll) sulfate), and the like).

In some aspects of the methods, the recombinant isopropanol preparation before and/or after being optionally purified is substantially pure. With respect to the methods of producing isopropanol, "substantially pure" intends a recovered preparation that contains no more than 15% impurity, wherein impurity intends compounds other than propanol but does not include the n-propanol isomer. This, a substantially pure preparation of isopropanol may contain n-propanol in excess of 15%. In one variation, a substantially pure preparation is provided wherein the preparation contains no more than 25% impurity, or no more than 20% impurity, or no more than 10% impurity, or no more than 5% impurity, or no more than 3% impurity, or no more than 1 % impurity, or no more than 0.5% impurity.

The Lactobacillus mutants may also be engineered to coproduce isopropanol and n- propanol, as described in PCT/US201 1/58405, the content of which is hereby incorporated in its entirety (particularly the discussions on coproduction of isopropanol and n-propanol).

The isopropanol produced by any of the methods described herein may be converted to propylene. Propylene can be produced by the chemical dehydration of isopropanol using acidic catalysts known in the art, such as acidic alumina, zeolites, and other metallic oxides; acidic organic-sulfonic acid resins; mineral acids such as phosphoric and sulfuric acids; and Lewis acids such as boron trifluoride and aluminum compounds (March, Jerry. Advanced Organic Chemistry. New York: John Wiley and Sons, 1992). Suitable temperatures for dehydration of isopropanol to propylene typically range from about 180°C to about 600°C, e.g., 300°C to about 500°C, or 350°C to about 450°C.

The dehydration reaction of isopropanol is typically conduced in an adiabatic or isothermal reactor, which can also be a fixed or a fluidized bed reactor; and can be optimized using residence time ranging from about 0.1 to about 60 seconds, e.g., from about 1 to about 30 seconds. Non-converted alcohol can be recycled to the dehydration reactor.

In one aspect is a method of producing propylene, comprising: (a) cultivating a mutant host cell described herein (e.g., a recombinant Lactobacillus host cell comprising an active isopropanol pathway and a disruption to an endogenous gene encoding an acetate kinase) in a medium under suitable conditions to produce isopropanol; (b) recovering the isopropanol; (c) dehydrating the isopropanol under suitable conditions to produce propylene; and (d) recovering the propylene.

Contaminants that may be generated during dehydration may be removed through purification using techniques known in the art. For example, propylene can be washed with water or a caustic solution to remove acidic compounds like carbon dioxide and/or fed into beds to absorb polar compounds like water or for the removal of, e.g., carbon monoxide. Alternatively, a distillation column can be used to separate higher hydrocarbons such as propane, butane, butylene and higher compounds. The separation of propylene from contaminants like ethylene may be carried out by methods known in the art, such as cryogenic distillation.

Suitable assays to test for the production of isopropanol and propylene for the methods of production and mutants described herein can be performed using methods known in the art. For example, final isopropanol product and intermediates (e.g., acetone), as well as other organic compounds, can be analyzed by methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography Mass Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine procedures well known in the art. The release of isopropanol in the fermentation broth can also be tested with the culture supernatant. Byproducts and residual sugar in the fermentation medium (e.g., glucose) can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775 -779 (2005)), or using other suitable assay and detection methods well known in the art.

The propylene produced from isopropanol may be further converted to polypropylene or polypropylene copolymers by polymerization processes known in the art. Suitable temperatures typically range from about 105°C to about 300°C for bulk polymerization, or from about 50°C to about 100°C for polymerization in suspension. Alternatively, polypropylene can be produced in a gas phase reactor in the presence of a polymerization catalyst such as Ziegler-Natta or metalocene catalysts with temperatures ranging from about 60°C to about 80°C.

The following examples are provided by way of illustration and are not intended to be limiting of the invention.

Examples

Chemicals used as buffers and substrates were commercial products of at least reagent grade. Host Strains

Lactobacillus reuteri SJ10655 (Q4ZXV)

A strain described as Lactobacillus reuteri DSM20016 was obtained from a public strain collection. This strain was subcultured in MRS medium, and an aliquot frozen as

SJ10468. SJ10468 was inoculated into MRS medium, propagated without shaking for one day at 37°C, and spread on MRS agar plates to obtain single colonies. After two days of growth at 37°C, a single colony was reisolated on a MRS agar plate, the plate incubated at

37°C for three days, and the cell growth on the plate was scraped off and stored in the strain collection as SJ 10655 (alternative name: 04ZXV).

The same cell growth was used to inoculate a 10 ml MRS culture, which was incubated without shaking at 37°C for 3 days, whereafter cells were harvested by centrifugation and genomic DNA was prepared using a QIAamp DNA Blood Kit (Qiagen,

Hilden, Germany) and sent for genome sequencing.

The genome sequence revealed that the isolate SJ 10655 (04ZXV) has a genome essentially identical to that of JCM1 1 12, rather than to that of the closely related strain DSM20016. JCM1 1 12 and DSM20016 are derived from the same original isolate, L. reuteri

F275 (Morita et al. DNA research, 2008, 15, 151 -161 .)

Bacillus subtilis DN 1885 (see Diderichsen et al. J. Bacteriol. 1990, 172, 4315-4321 ).

Escherichia coli SJ2 (see Diderichsen et al. J. Bacteriol. 1990, 172, 4315-4321 ).

Escherichia coli MG1655 (see Blattner et al. Science 1997, 277, 1453-1462).

Escherichia coli TG1

TG1 is a commonly used cloning strain and was obtained from a commercial supplier; it has the following genotype: F'[traD36 laclq A(lacZ) M15 proA+B+] glnV (supE) thi-1 A(mcrB-hsdSM)5 (rK- mK- McrB-) thi A(lac-proAB). Media

LB plates were composed of 37 g LB agar (Sigma cat no. L3027) and double distilled water to 1 L.

LBPGS plates were composed of 37 g LB agar (Sigma cat no. L3027), 0.5% starch (Merck cat. no. 101252), 0.01 M K₂P0₄, 0.4% glucose, and double distilled water to 1 L.

TY bouillon medium was composed of 20 g tryptone (Difco cat no. 21 1699), 5 g yeast extract (Difco cat no. 212750), 7^*10^"3 g ferrochloride, 1 ^*10^"3 g manganese(ll)-chloride, 1 .5^*10^"3 g magnesium sulfate, and double distilled water to 1 L.

Minimal medium (MM) was composed of 20 g glucose, 1 .1 g KH₂P0₄, 8.9 g K₂HP0₄; 1 .0 g (NH₄)₂S0₄; 0.5 g Na-citrate; 5.0 g MgS0₄-7H₂0; 4.8 mg MnS0₄-H₂0; 2 mg thiamine; 0.4 mg/L biotin; 0.135 g FeCI₃-6H₂0; 10 mg ZnCI₂-4H₂0; 10 mg CaCI₂-6H₂0; 10 mg Na₂Mo0₄-2H₂0; 9.5 mg CuS0₄-5H₂0; 2.5 mg H₃B0₃; and double distilled water to 1 L, pH adjusted to 7 with HCI.

MRS medium was obtained from Difco™, as either Difco™ Lactobacilli MRS Agar or Difco™ Lactobacilli MRS Broth, having the following compositions— Difco™ Lactobacilli MRS Agar: Proteose Peptone No. 3 (10.0 g), Beef Extract (10.0 g), Yeast Extract (5.0 g), Dextrose (20.0 g), Polysorbate 80 (1 .0 g), Ammonium Citrate (2.0 g), Sodium Acetate (5.0 g), Magnesium Sulfate (0.1 g), Manganese Sulfate (0.05 g), Dipotassium Phosphate (2.0 g), Agar (15.0 g) and water to 1 L. Difco™ Lactobacilli MRS Broth: Consists of the same ingredients without the agar.

LC (Lactobacillus Carrying) medium (LCM) was composed of Trypticase (10 g), Tryptose (3 g), Yeast extract (5 g), KH₂P0₄ (3 g), Tween 80 (1 ml), sodium-acetate (1 g), ammonium citrate (1 .5 g), Cystein-HCI (0.2 g), MgS0₄.7H₂0 (12 mg), FeS0₄.7H₂0 (0.68 mg), MnS0₄.2H₂0 (25 mg), and double distilled water to 1 L, pH adjusted to 7.0. Stearile glucose is added after autoclaving, to 1 % (5 ml of a 20 % glucose stock solution/100 ml medium).

Example 1 : Transformation protocol for Lactobacillus strains.

Lactobacillus strains

Unless noted otherwise, plasmid DNA constructed in E. coli was purified from 2 ml of an overnight culture grown in TY medium, and supplemented with appropriate antibiotics using a QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) as described by the manufacturer. The plasmid DNA was recovered in a volume of 50 microliters, and one microliter of this plasmid preparation was used for electroporation of Lactobacillus.

Plasmid DNA was transformed into Lactobacillus strains by electroporation. The L. reuteri strains were prepared for electroporation as follows: The strain was inoculated from a frozen stock culture into LCM medium, and incubated without shaking at 37°C overnight. A 5 ml aliquot was transferred into 500 ml LCM medium and incubated at 37°C without shaking until OD₆oo reached approximately 0.8. The cells were harvested by centrifugation as above, resuspended and washed 2 times in 50 ml of ion-exchanged sterile water at room temperature, and harvested by centrifugation. The cells were finally gently resuspended in 2.5 ml of 30% PEG1500, and 50 microliter aliquots were quickly frozen in an alcohol/dry ice bath, and stored at -80°C until use. Variations to the electroporation procedures below are described in the respective examples.

Electroporation procedure A: The frozen cells were thawed on ice, and 2 microliter of a DNA suspension in TE buffer was added. 40 microliters of the mixture was transferred to an ice-cold 2 mm electroporation cuvette, kept on ice for 1 -3 minutes, and electroporation carried out in a BioRad Gene Pulser™ with a setting of 1 .5 kV; 25 microFarad; 400 Ohms. 500 microliter of LCM was added, and the mixture incubated without shaking for 2 hours at 37°C before plating. Cells were plated on either LCM agar plates (LCM medium solidified with % agar) or MRS agar plates, supplemented with the required antibiotics, and incubated in an anaerobic chamber (Oxoid; equipped with Anaerogen sachet).

Electroporation procedure B: The frozen cells were thawed on ice, and 1 microliter of a DNA suspension in TE buffer was added. 40 microliters of the mixture was transferred to an ice-cold 1 mm electroporation cuvette, kept on ice for 1 -3 minutes, and electroporation carried out in a BioRad Gene Pulser™ with a setting of 1 .2 kV; 25 microFarad; 400 Ohms. 500 microliter of LCM was added, and the mixture incubated without shaking for 4 hours at 37 °C before plating on MRS agar plates, supplemented with the required antibiotics, and incubation in an anaerobic chamber.

E. coli strains

Transformation of E. coli was conducted by electroporation using either a BioRad Gene Pulser™ (BioRad, Hercules, CA, USA) as described by the manufacturer, or by using chemically competent cells prepared following ordinary textbook procedures commonly known in the art.

Example 2: Peptide-inducible pSIP expression vectors.

The peptide-inducible expression vectors pSIP409, pSIP410, and pSIP41 1 (S0rvig, et al. Microbiology 2005, 151 , 2439-2449.) were received from Lars Axelsson, Nofima Mat AS, Norway. pSIP409 and pSIP410 were transformed into E. coli SJ2 by electroporation, selecting erythromycin resistance (150 microgram/ml) on LB agar plates at 37°C. Two transformants containing pSIP409 were kept as SJ10517 and SJ10518, and two transformants containing pSIP410 were kept as SJ10519 and SJ10520.

pSIP41 1 was transformed into naturally competent Bacillus subtilis DN 1885 cells, essentially as described (Yasbin et al. J Bacteriol 1975, 121 , 296-304), selecting for erythromycin resistance (5 microgram/ml) on LBPGS plates at 37°C. Two such transformants were kept as SJ 10513 and SJ10514.

pSIP41 1 was in addition transformed into £ coli MG1655 by electroporation, selecting erythromycin resistance (200 microgram/ml) on LB agar plates at 37°C, and two transformants kept as SJ 10542 and SJ 10543.

For use in induction of gene expression from these vectors in Lactobacillus, the inducing peptide, here named M-19-R and having the following amino acid sequence: "Met- Ala-Gly-Asn-Ser-Ser-Asn-Phe-lle-His-Lys-lle-Lys-Gln-lle-Phe-Thr-His-Arg", was obtained from "Polypeptide Laboratories France, 7 rue de Boulogne, 67100 Strasbourg, France".

Example 3: Construction of pVS2 -based vectors pSJ 10600 and pSJ 10603 for constitutive expression.

A set of constitutive expression vectors were constructed based on the plasmid pVS2 (von Wright et al., Appl. Environ. Microbiol. 1987, 53, 1584-1588) and promoters described by Rud et al. (Rud et al. Microbiology 2006, 152, 101 1 -1019). A DNA fragment containing the P1 1 promoter with a selection of flanking restriction sites, and another fragment containing P27 with a selection of flanking restriction sites, was chemically synthesized by Geneart AG (Regenburg, Germany).

The DNA fragment containing P1 1 with flanking restriction sites, and the DNA fragment containing P27 with flanking restriction sites are shown in SEQ ID NOs: 49 and 50, respectively. Both DNA fragments were obtained in the form of DNA preparations, where the fragments had been inserted into the standard Geneart vector, pMA. The vector containing P1 1 was transformed into £ coli SJ2 cells, and a transformant kept as SJ 10560, containing plasmid pSJ10560. The vector containing P27 was transformed into £ coli SJ2 cells, and a transformant kept as SJ 10561 , containing plasmid pSJ10561 .

The promoter-containing fragments, in the form of 176 bp Hindi 11 fragments, were excised from the Geneart vectors and ligated to Hindlll-digested pUC19. The P1 1 - containing fragment was excised from the vector prepared from SJ 10560, ligated to pUC19, and correct transformants of £. coli SJ2 were kept as SJ10585 and SJ10586, containing pSJ10585 and pSJ10586, respectively. The P27 containing fragment was excised from the vector prepared from SJ10561 , ligated to pUC19, and correct transformants of £ coli SJ2 were kept as SJ 10587 and SJ 10588, containing pSJ 10587 and pSJ 10588, respectively.

Plasmid pVS2 was obtained in Lactobacillus plantarum NC8, a strain kept as 5 SJ10491 , extracted from this strain by standard plasmid preparation procedures known in the art, and transformed into £ coli MG1655 selecting erythromycin resistance (200 microgram/ml) on LB agar plates at 37 oC. Two such transformants were kept as SJ 10583 and SJ10584.

To insert P1 1 into pVS2, the P1 1 -containing 176 bp Hindi 11 fragment was excised i o and purified by agarose gel electrophoresis from pSJ 10585, and ligated to Hindi ll-digested pVS2, which had been prepared from SJ 10583. The ligation mixture was transformed by electroporation into £. coli MG1655, selecting erythromycin resistance (200 microgram/ml) on LB agar plates, and two transformants, which both harbor plasmids with the promoter insert in one particular of the two possible orientations, were kept as SJ 10600 and

15 SJ10601 , containing pSJ10600 (Figure 2) and pSJ10601 .

Another transformant, having the promoter insert in the other of the two possible orientations, was kept as SJ10602, containing pSJ10602. The plasmid preparation from SJ 10602 appeared to contain less DNA than the comparable preparations from SJ 10600 and SJ10601 , and, upon further work, pSJ10602 appeared to be rather unstable, with

20 deletion derivatives dominating in the plasmid population.

To insert P27 into pVS2, the P27-containing 176 bp Hindi 11 fragment was excised and purified by agarose gel electrophoresis from pSJ10588, and ligated to Hindi ll-digested pVS2, which had been prepared from SJ 10583. The ligation mixture was transformed by electroporation into £ coli MG1655, selecting erythromycin resistance (200 microgram/ml)

25 on LB agar plates, and two transformants, which both harbor plasmids with the promoter insert in one particular of the two possible orientations, were kept as SJ 10603 and SJ10604, containing pSJ10603 (Figure 3) and pSJ10604.

Another transformant, having the promoter insert in the other of the two possible orientations, was kept as SJ10605, containing pSJ10605. The promoter orientation in this

30 plasmid is the same as in pSJ10602, described above. The plasmid preparation from SJ 10605 appeared to contain less DNA than the comparable preparations from SJ 10603 and SJ10604, and, upon further work, pSJ10605 appeared to be rather unstable, with deletion derivatives dominating in the plasmid population. Example 4: Cloning of isopropanol pathway genes.

Cloning of a Clostridium acetobutylicum thiolase gene and construction of vector pSJ-10705.

The 1 176 bp coding sequence (without stop codon) of a thiolase gene identified in Clostridium acetobutylicum was designed for optimized expression in the three organisms Escherichia coli, Lactobacillus plantarum, and Lactobacillus reuteri and synthetically constructed into pSJ10705. The DNA fragment containing the codon optimized coding sequence was designed with the sequence 5'-AAGCTTTC-3' immediately prior to the start codon (to add a Hindi 11 site and convert the start region to a Ncol-compatible BspHI site), and the sequence 5'- TAGTCTAGACTCGAGGAATTCGGTACC-3' immediately downstream (to add a stop codon, and restriction sites Xbal-Xhol-EcoRI-Kpnl).

The resulting sequence was then submitted to and synthesized by Geneart AG (Regenburg, Germany) and delivered in the pMA backbone vector containing the β- lactamase encoding gene blaTEM-1 . The DNA preparation delivered from Geneart was transformed into E. coli SJ2 by electroporation, selecting ampicillin resistance (200 microgram/ml) and two transformants kept, as SJ 10705 (SJ2/pSJ 10705) and SJ 10706 (SJ2/pSJ 10706).

The wild-type nucleotide sequence (WT), codon-optimized nucleotide sequence (CO), and deduced amino acid sequence of the C. acetobutylicum thiolase gene are SEQ ID NOs: 1 , 2, and 3, respectively. The coding sequence is 1 179 bp including the stop codon and the encoded predicted protein is 392 amino acids. Using the SignalP program (Nielsen et al., 1997, Protein Engineering 10: 1 -6), no signal peptide in the sequence was predicted. Based on this program, the predicted mature protein contains 392 amino acids with a predicted molecular mass of 41 .4 kDa and an isoelectric pH of 7.08.

Cloning of a Lactobacillus reuteri thiolase gene and construction of vector pSJ 10694.

The 1 176 bp thiolase coding sequence (withough stop codon) from Lactobacillus reuteri was amplified from chromosomal DNA of SJ10468 (supra) using primers 671826 and 671827 shown below.

Primer 671826:

5'-AGTCAAGCTTCCATGGAGAAGGTTTACATTGTTGC-3' (SEQ ID NO: 51 )

Primer 671827:

5'-ATGCGGTACCGAATTCCTCGAGTCTAGACTAAATTTTCTTAAGCAGAACCG-3' (SEQ ID NO: 52)

The PCR reaction was programmed for 94°C for 2 minutes; and then 19 cycles each at 95°C for 30 seconds, 59°C for 1 minute, and 72°C for 2 minute; then one cycle at 72°C for 5 minutes. A PCR amplified fragment of approximately 1 .2 kb was digested with Ncol + EcoRI, purified by agarose gel electrophoresis, and then ligated to the agarose gel electrophoresis purified EcoRI-Ncol vector fragment of plasmid pSIP409. The ligation mixture was transformed into E. coli SJ2, selecting ampicillin resistance (200 microgram/ml), and a transformant, deemed correct by restriction digest and DNA sequencing, was kept as SJ10694 (SJ2/pSJ 10694).

The codon-optimized nucleotide sequence (CO), and deduced amino acid sequence of the L. reuteri thiolase gene are SEQ ID NOs: 25 and 26, respectively. The coding sequence is 1 179 bp including the stop codon and the encoded predicted protein is 392 amino acids. Using the SignalP program (Nielsen et al., 1997, Protein Engineering 10: 1 -6), no signal peptide in the sequence was predicted. Based on this program, the predicted mature protein contains 392 amino acids with a predicted molecular mass of 41 .0 kDa and an isoelectric pH of 5.4.

Cloning of a Propionibacterium freudenreichii thiolase gene and construction of vector PSJ10676.

The 1 152 bp coding sequence (without stop codon) of a thiolase gene identified in Propionibacterium freudenreichii was optimized for expression in the three organisms Escherichia coli, Lactobacillus plantarum, and Lactobacillus reuteri and synthetically constructed into pSJ10676. The DNA fragment containing the codon optimized CDS was designed with the sequence 5'-AAGCTTTC-3' immediately prior to the start codon (to add a Hindlll site and convert the start region to a Ncol-compatible BspHI site), and the sequence 5'-TAGTCTAGACTCGAGGAATTCGGTACC-3' (SEQ ID NO: 53) immediately downstream (to add a stop codon, and restriction sites Xbal-Xhol-EcoRI-Kpnl).

The resulting sequence was then submitted to and synthesized by Geneart AG (Regenburg, Germany) and delivered in the pMA backbone vector containing the β- lactamase encoding gene blaTEM-1 . The DNA preparation delivered from Geneart was transformed into E. coli SJ2 by electroporation, selecting ampicillin resistance (200 microgram/ml) and two transformants kept, as SJ 10676 (SJ2/pSJ 10676) and SJ 10677 (SJ2/pSJ 10677).

The codon-optimized nucleotide sequence (CO), and deduced amino acid sequence of the P. freudenreichii thiolase gene are SEQ ID NOs: 39 and 40, respectively. The coding sequence is 1 155 bp including the stop codon and the encoded predicted protein is 384 amino acids. Using the SignalP program (Nielsen et al., 1997, Protein Engineering 10: 1 -6), no signal peptide in the sequence was predicted. Based on this program, the predicted mature protein contains 384 amino acids with a predicted molecular mass of 39.8 kDa and an isoelectric pH of 6.1 .

Cloning of a Lactobacillus brevis thiolase gene and construction of vector pSJ 10699.

The 1 167 bp coding sequence (without stop codon) of a thiolase gene identified in Lactobacillus brevis was optimized for expression in the three organisms Escherichia coli, 5 Lactobacillus plantarum, and Lactobacillus reuteri and synthetically constructed into pSJ 10699. The DNA fragment containing the codon optimized CDS was designed with the sequence 5'- AAGCTTCC-3' immediately prior to the start codon (to add a Hindi 11 site and convert the start region to a Ncol site), and the sequence 5'- TAGTCTAGACTCGAGGAATTCGGTACC-3' (SEQ ID NO: 53) immediately downstream (to i o add a stop codon, and restriction sites Xbal-Xhol-EcoRI-Kpnl).

The resulting sequence was then submitted to and synthesized by Geneart AG (Regenburg, Germany) and delivered in the pMA backbone vector containing the β- lactamase encoding gene blaTEM-1 . The DNA preparation delivered from Geneart was transformed into E. coli SJ2 by electroporation, selecting ampicillin resistance (200

15 microgram/ml) and two transformants kept, as SJ 10699 (SJ2/pSJ 10699) and SJ 10700 (SJ2/pSJ 10700).

The codon-optimized nucleotide sequence (CO), and deduced amino acid sequence of the L. brevis thiolase gene are SEQ ID NOs: 41 and 42, respectively. The coding sequence is 1 170 bp including the stop codon and the encoded predicted protein is 20 389 amino acids. Using the SignalP program (Nielsen et al., 1997, Protein Engineering 10:

1 -6), no signal peptide in the sequence was predicted. Based on this program, the predicted mature protein contains 389 amino acids with a predicted molecular mass of 40.4 kDa and an isoelectric pH of 6.5.

Cloning of B. subtilis succinyl-CoA:acetoacetate transferase genes and construction of

25 vectors pSJ 10695 and pSJ 10697.

The 699 bp coding sequence (without stop codon) of the scoA subunit of the B. subtilis succinyl-CoA:acetoacetate transferase and the 648 bp coding sequence of the scoB subunit of the B. subtilis succinyl-CoA:acetoacetate transferase were designed for optimized expression in the three organisms Escherichia coli, Lactobacillus plantarum, and

30 Lactobacillus reuteri and synthetically constructed into pSJ 10695 and pSJ 10697, respectively.

The DNA fragment containing the codon-optimized scoA coding sequence was designed with the sequence 5'-AAGCT TCTCG AGACT ATTAC AAGGA GATTT TAGCC-3' (SEQ ID NO: 54) immediately prior to the start codon (to add a Hindll l site, a Lactobacillus 35 RBS, and to have the start codon within a Ncol site), and an EcoRI restriction site immediately downstream. The designed construct was obtained from Geneart AG and transformed as described above, resulting in SJ 10695 (SJ2/pSJ 10695) and SJ 10696 (SJ2/pSJ 10696).

The wild-type nucleotide sequence (WT), codon-optimized nucleotide sequence (CO), and deduced amino acid sequence of the B. subtilis scoA subunit of the succinyl- CoA:acetoacetate transferase are SEQ ID NOs: 4, 5, and 6, respectively. The coding sequence is 702 bp including the stop codon and the encoded predicted protein is 233 amino acids. Using the SignalP program (Nielsen et al., supra), no signal peptide in the sequence was predicted. Based on this program, the predicted mature protein contains 233 amino acids with a predicted molecular mass of 25.1 kDa and an isoelectric pH of 6.50.

The DNA fragment containing the codon optimized scoB coding sequence was designed with the sequence 5'-GAATT CACTA TTACA AGGAG ATTTT AGTC-3' (SEQ ID NO: 55) immediately prior to the start codon (to add a EcoRI site, a Lactobacillus RBS, and to have the start codon within a Ncol-compatible BspHI site), and Eagl and Kpnl restriction sites immediately downstream. The designed construct was obtained from Geneart AG and transformed as described above, resulting in SJ 10697 (SJ2/pSJ 10697) and SJ 10698 (SJ2/pSJ 10698).

The wild-type nucleotide sequence (WT), codon-optimized nucleotide sequence (CO), and deduced amino acid sequence of the B. subtilis scoB subunit of the succinyl- CoA:acetoacetate transferase are SEQ ID NOs: 7, 8, and 9, respectively. The coding sequence is 651 bp including the stop codon and the encoded predicted protein is 216 amino acids. Using the SignalP program (Nielsen et al., supra), no signal peptide in the sequence was predicted. Based on this program, the predicted mature protein contains 216 amino acids with a predicted molecular mass of 23.4 kDa and an isoelectric pH of 5.07. Cloning of B. mojavensis succinyl-CoA:acetoacetate transferase genes and construction of vectors pSJ10721 and pSJ10723.

The 71 1 bp coding sequence (without stop codon) of the scoA subunit of the B. mojavensis succinyl-CoA:acetoacetate transferase and the 654 bp coding sequence

(without stop codon) of the scoB subunit of the B. mojavensis succinyl-CoA:acetoacetate transferase were designed for optimized expression in the three organisms Escherichia coli, Lactobacillus plantarum, and Lactobacillus reuteri and synthetically constructed into pSJ10721 and pSJ 10723, respectively.

The DNA fragment containing the codon-optimized scoA coding sequence was designed with the sequence 5'-AAGCT TCTCG AGACT ATTAC AAGGA GATTT TAGCC-3' (SEQ ID NO: 54) immediately prior to the start codon (to add a Hindll l site, a Lactobacillus RBS, and to have the start codon within a Ncol site), and an EcoRI restriction site immediately downstream. The designed construct was obtained from Geneart AG and transformed as described above, resulting in SJ10721 (SJ2/pSJ 10721 ) and SJ10722 (SJ2/pSJ 10722).

The wild-type nucleotide sequence (WT), codon-optimized nucleotide sequence

(CO), and deduced amino acid sequence of the B. mojavensis scoA subunit of the succinyl- CoA:acetoacetate transferase are SEQ ID NOs: 10, 1 1 , and 12, respectively. The coding sequence is 714 bp including the stop codon and the encoded predicted protein is 237 amino acids. Using the SignalP program (Nielsen et al., supra), no signal peptide in the sequence was predicted. Based on this program, the predicted mature protein contains 237 amino acids with a predicted molecular mass of 25.5 kDa and an isoelectric pH of 5.82.

The DNA fragment containing the codon optimized scoB nucleotide coding sequence was designed with the sequence 5'-GAATT CACTA TTACA AGGAG ATTTT AGTC-3' (SEQ ID NO: 55) immediately prior to the start codon (to add a EcoRI site, a Lactobacillus RBS, and to have the start codon within a Ncol-compatible BspHI site), and Eagl and Kpnl restriction sites immediately downstream. The designed construct was obtained from Geneart AG and transformed as described above, resulting in SJ 10723 (SJ2/pSJ 10723) and SJ 10724 (SJ2/pSJ 10724).

The wild-type nucleotide sequence (WT), codon-optimized nucleotide sequence (CO), and deduced amino acid sequence of the B. mojavensis scoB subunit of the succinyl- CoA:acetoacetate transferase are SEQ ID NOs: 13, 14, and 15, respectively. The coding sequence is 657 bp including the stop codon and the encoded predicted protein is 218 amino acids. Using the SignalP program (Nielsen et al., supra), no signal peptide in the sequence was predicted. Based on this program, the predicted mature protein contains 218 amino acids with a predicted molecular mass of 23.7 kDa and an isoelectric pH of 5.40.

Cloning of E. coli acetoacetyl-CoA transferase genes and construction of vectors pSJ 10715 and PSJ10717.

The 648 bp coding sequence (without stop codon) of the atoA subunit (uniprot:P76459) of the E. coli acetyl-CoA transferase and the 660 bp coding sequence (without stop codon) of the atoD subunit (uniprot:P76458) of the E. coli acetyl-CoA transferase were optimized for expression in the three organisms Escherichia coli, Lactobacillus plantarum, and Lactobacillus reuteri and synthetically constructed into pSJ10715 and pSJ 10717, respectively.

The DNA fragment containing the codon-optimized atoA subunit nucleotide coding sequence was designed with the sequence 5'-AAGCT TCTCG AGACT ATTAC AAGGA GATTT TAGCC-3' (SEQ ID NO: 54) immediately prior to the start codon (to add Hindi 11 and Xhol sites, a Lactobacillus RBS, and to have the start codon within a Ncol site), and an EcoRI restriction site immediately downstream. The designed construct was obtained from Geneart AG and transformed as described above, resulting in SJ10715 (SJ2/pSJ10715) and SJ10716 (SJ2/pSJ10716).

The codon-optimized nucleotide sequence (CO) and deduced amino acid sequence of the E. coli atoA subunit of the acetoacetyl-CoA transferase are SEQ ID NOs: 27 and 28, respectively. The coding sequence is 651 bp including the stop codon and the encoded predicted protein is 216 amino acids. Using the SignalP program (Nielsen et al., supra), no signal peptide in the sequence was predicted. Based on this program, the predicted mature protein contains 216 amino acids with a predicted molecular mass of 23.0 kDa and an isoelectric pH of 5.9.

The DNA fragment containing the codon optimized atoD nucleotide coding sequence was designed with the sequence 5'-GAATT CACTA TTACA AGGAG ATTTT AGTC-3' (SEQ ID NO: 55) immediately prior to the start codon (to add a EcoRI site, a Lactobacillus RBS, and to have the start codon within a Ncol-compatible BspHI site), and Eagl and Kpnl restriction sites immediately downstream. The designed construct was obtained from Geneart AG and transformed as described above, resulting in SJ10717 (SJ2/pSJ 10717) and SJ10718 (SJ2/pSJ10718).

The codon-optimized nucleotide sequence (CO) and deduced amino acid sequence of the E. coli atoD subunit of the acetoacetyl-CoA transferase are SEQ ID NOs: 29 and 30, respectively. The coding sequence is 663 bp including the stop codon and the encoded predicted protein is 220 amino acids. Using the SignalP program (Nielsen et al., supra), no signal peptide in the sequence was predicted. Based on this program, the predicted mature protein contains 220 amino acids with a predicted molecular mass of 23.5 kDa and an isoelectric pH of 4.9.

Cloning of Clostridium acetobutylicum acetoacetyl-CoA transferase genes and construction of vectors pSJ 10727 and pSJ 10731 .

The 654 bp coding sequence (without stop codon) of the ctfA subunit (uniprot:P33752) of the C. acetobutylicum acetyl-CoA transferase and the 663 bp coding sequence (without stop codon) of the ctfB subunit (uniprot:P23673) of the C. acetobutylicum acetyl-CoA transferase were optimized for expression in the three organisms Escherichia coli, Lactobacillus plantarum, and Lactobacillus reuteri and synthetically constructed into pSJ10727 and pSJ 10731 , respectively.

The DNA fragment containing the codon optimized ctfA subunit coding sequence was designed with the sequence 5'-AAGCT TCTCG AGACT ATTAC AAGGA GATTT TAGTC-3' (SEQ ID NO: 56) immediately prior to the start codon (to add Hindlll and Xhol sites, a Lactobacillus RBS, and to have the start codon within a Ncol-compatible BspHI site), and an EcoRI restriction site immediately downstream. The designed construct was 5 obtained from Geneart AG and transformed as described above, resulting in SJ 10727 (SJ2/pSJ 10727) and SJ 10728 (SJ2/pSJ 10728).

The codon-optimized nucleotide sequence (CO) and deduced amino acid sequence of the C. acetobutylicum ctfA subunit of the acetoacetyl-CoA transferase are SEQ ID NOs: 31 and 32, respectively. The coding sequence is 657 bp including the stop codon and the i o encoded predicted protein is 218 amino acids. Using the SignalP program (Nielsen et al., supra), no signal peptide in the sequence was predicted. Based on this program, the predicted mature protein contains 218 amino acids with a predicted molecular mass of 23.6 kDa and an isoelectric pH of 9.3.

The DNA fragment containing the codon optimized ctfB subunit coding sequence

15 was designed with the sequence 5'-GAATT CACTA TTACA AGGAG ATTTT AGTC-3' (SEQ ID NO: 55) immediately prior to the start codon (to add a EcoRI site, a Lactobacillus RBS, and to have the start codon within a Ncol-compatible BspHI site), and Eagl and Kpnl restriction sites immediately downstream. The designed construct was obtained from Geneart AG and transformed as described above, resulting in SJ10731 (SJ2/pSJ 10731 )

20 and S J 10732 (S J2/pS J 10732).

The codon-optimized nucleotide sequence (CO) and deduced amino acid sequence of the C. acetobutylicum ctfB subunit of the acetoacetyl-CoA transferase are SEQ ID NOs: 33 and 34, respectively. The coding sequence is 666 bp including the stop codon and the encoded predicted protein is 221 amino acids. Using the SignalP program (Nielsen et al.,

25 supra), no signal peptide in the sequence was predicted. Based on this program, the predicted mature protein contains 221 amino acids with a predicted molecular mass of 23.6 kDa and an isoelectric pH of 8.5.

Cloning of a Clostridium acetobutylicum acetoacetate decarboxylase gene and construction of vector pSJ1071 1 .

30 The 777 bp coding sequence (without stop codon) of the acetoacetate decarboxylase (uniprot:P23670) from C. acetobutylicum was optimized for expression in the three organisms Escherichia coli, Lactobacillus plantarum, and Lactobacillus reuteri and synthetically constructed into pSJ1071 1 .

The DNA fragment containing the codon-optimized acetoacetate decarboxylase

35 coding sequence (adc) was designed with the sequence 5'-AAGCT TCGGC CGACT ATTAC AAGGA GATTT TAGCC-3' (SEQ ID NO: 57) immediately prior to the start codon (to add Hindi 11 and Eagl sites and a Lactobacillus RBS), and a Kpnl restriction site immediately downstream. The designed construct was obtained from Geneart AG and transformed as described above, resulting in SJ1071 1 (SJ2/pSJ1071 1 ) and SJ10712 (SJ2/pSJ10712).

The codon-optimized nucleotide sequence (CO) and deduced amino acid sequence of the C. acetobutylicum acetoacetate decarboxylase gene are SEQ ID NOs: 35 and 36, respectively. The coding sequence is 780 bp including the stop codon and the encoded predicted protein is 259 amino acids. Using the SignalP program (Nielsen et al., supra), no signal peptide in the sequence was predicted. Based on this program, the predicted mature protein contains 259 amino acids with a predicted molecular mass of 27.5 kDa and an isoelectric pH of 6.2.

Cloning of a Clostridium beijerinckii acetoacetate decarboxylase gene and construction of vector PSJ 10713.

The 738 bp coding sequence (without stop codon) of the acetoacetate decarboxylase (uniprot:Q716S5) from C. beijerinckii was optimized for expression in the three organisms Escherichia coli, Lactobacillus plantarum, and Lactobacillus reuteri and synthetically constructed into pSJ10713.

The DNA fragment containing the codon optimized acetoacetate decarboxylase coding sequence (adc Cb) was designed with the sequence 5'-AAGCT TCGGC CGACT ATTAC AAGGA GATTT TAGCC-3' (SEQ ID NO: 57) immediately prior to the start codon (to add Hindi 11 and Eagl sites and a Lactobacillus RBS), and a Kpnl restriction site immediately downstream. The desigined construct was obtained from Geneart AG and transformed as described above, resulting in SJ10713 (SJ2/pSJ10713) and SJ10714 (SJ2/pSJ10714).

The wild-type nucleotide sequence (WT), codon-optimized nucleotide sequence (CO), and deduced amino acid sequence of the C. beijerinckii acetoacetate decarboxylase gene is SEQ ID NO: 16, 17, and 18, respectively. The coding sequence is 741 bp including the stop codon and the encoded predicted protein is 246 amino acids. Using the SignalP program (Nielsen et al., supra), no signal peptide in the sequence was predicted. Based on this program, the predicted mature protein contains 246 amino acids with a predicted molecular mass of 27.5 kDa and an isoelectric pH of 6.18.

Cloning of a Lactobacillus salivarius acetoacetate decarboxylase gene and construction of vector pSJ 10707.

The 831 bp CDS (without stop codon) of the acetoacetate decarboxylase (SWISSPROT:Q1 WVG5) from L. salivarius was optimized for expression in the three organisms Escherichia coli, Lactobacillus plantarum, and Lactobacillus reuteri and synthetically constructed into pSJ 10707.

The DNA fragment containing the codon optimized acetoacetate decarboxylase 5 CDS (adc Ls) was designed with the sequence 5'-AAGCT TCGGC CGACT ATTAC AAGGA GATTT TAGAC-3' (SEQ ID NO: 57) immediately prior to the start codon (to add Hindi 11 and Eagl sites and a Lactobacillus RBS), and a Kpnl restriction site immediately downstream. The constructs were obtained from Geneart AG and transformed as previously described, resulting in SJ10707 (SJ2/pSJ 10707) and SJ10708 (SJ2/pSJ 10708).

i o The codon-optimized nucleotide sequence (CO) and deduced amino acid sequence of the L. salivarius acetoacetate decarboxylase gene is SEQ ID NO: 43 and 44, respectively. The coding sequence is 834 bp including the stop codon and the encoded predicted protein is 277 amino acids. Using the SignalP program (Nielsen et al., supra), no signal peptide in the sequence was predicted. Based on this program, the predicted

15 mature protein contains 277 amino acids with a predicted molecular mass of 30.9 kDa and an isoelectric pH of 4.6.

Cloning of a Lactobacillus plantarum acetoacetate decarboxylase gene and construction of vector pSJ 10701 .

The 843 bp CDS (without stop codon) of the acetoacetate decarboxylase

20 (SWISSPROT:Q890G0) from L. plantarum was optimized for expression in the three organisms Escherichia coli, Lactobacillus plantarum, and Lactobacillus reuteri and synthetically constructed into pSJ10701 .

The DNA fragment containing the codon optimized acetoacetate decarboxylase CDS (adc Lp) was designed with the sequence 5'-AAGCT TCGGC CGACT ATTAC

25 AAGGA GATTT TAGCC-3' (SEQ ID NO: 57) immediately prior to the start codon (to add Hindi 11 and Eagl sites and a Lactobacillus RBS), and a Kpnl restriction site immediately downstream. The constructs were obtained from Geneart AG and transformed as previously described, resulting in SJ10701 (SJ2/pSJ 10701 ) and SJ 10702 (SJ2/pSJ 10702).

The codon-optimized nucleotide sequence (CO) and deduced amino acid sequence

30 of the L. plantarum acetoacetate decarboxylase gene is SEQ ID NO: 45 and 46, respectively. The coding sequence is 846 bp including the stop codon and the encoded predicted protein is 281 amino acids. Using the SignalP program (Nielsen et al., supra), no signal peptide in the sequence was predicted. Based on this program, the predicted mature protein contains 281 amino acids with a predicted molecular mass of 30.8 kDa and

35 an isoelectric pH of 4.7. Cloning of a Thermoanaerobacter ethanolicus isopropanol dehydrogenase gene and construction of vector pSJ 10719.

The 1056 bp coding sequence (without stop codon) of the isopropanol dehydrogenase (uniprot:Q2MJT8) from T. ethanolicus was optimized for expression in the 5 three organisms Escherichia coli, Lactobacillus plantarum, and Lactobacillus reuteri and synthetically constructed into pSJ10719.

The DNA fragment containing the codon optimized isopropanol dehydrogenase coding sequence (adh Te) was designed with the sequence 5'-GGTAC CACTA TTACA

AGGAG ATTTT AGTC-3' (SEQ ID NO: 58) immediately prior to the start codon (to add a i o Kpnl site, a Lactobacillus RBS, and to have the start codon within a Ncol-compatible BspHI site), and Xmal and Hind 111 restriction sites immediately downstream. The desigined construct was obtained from Geneart AG and transformed as described above, resulting in

SJ10719 (SJ2/pSJ10719) and SJ10720 (SJ2/pSJ 10720).

The wild-type nucleotide sequence (WT), codon-optimized nucleotide sequence 15 (CO), and deduced amino acid sequence of the T. ethanolicus isopropanol dehydrogenase gene is SEQ ID NO: 22, 23, and 24, respectively. The coding sequence is 1059 bp including the stop codon and the encoded predicted protein is 352 amino acids. Using the

SignalP program (Nielsen et al., supra), no signal peptide in the sequence was predicted.

Based on this program, the predicted mature protein contains 352 amino acids with a 20 predicted molecular mass of 37.7 kDa and an isoelectric pH of 6.23.

Cloning of a Clostridium beijerinckii isopropanol dehydrogenase gene and construction of vector pSJ 10725.

The 1053 bp coding sequence (without stop codon) of the isopropanol dehydrogenase (uniprot:P25984) from C. beijerinckii was optimized for expression in the 25 three organisms Escherichia coli, Lactobacillus plantarum, and Lactobacillus reuteri and synthetically constructed into pSJ 10725.

The DNA fragment containing the codon optimized isopropanol dehydrogenase coding sequence (adh Cb) was designed with the sequence 5'-GGTAC CACTA TTACA AGGAG ATTTT AGTC-3' (SEQ ID NO: 58) immediately prior to the start codon (to add a 30 Kpnl site, a Lactobacillus RBS, and to have the start codon within a Ncol-compatible BspHI site), and Xmal and Hind 111 restriction sites immediately downstream. The desigined construct was obtained from Geneart AG and transformed as described above, resulting in SJ 10725 (SJ2/pSJ 10725) and SJ 10726 (SJ2/pSJ 10726).

The wild-type nucleotide sequence (WT), codon-optimized nucleotide sequence 35 (CO), and deduced amino acid sequence of the C. beijerinckii isopropanol dehydrogenase gene is SEQ ID NO: 19, 20, and 21 , respectively. The coding sequence is 1056 bp including the stop codon and the encoded predicted protein is 351 amino acids. Using the SignalP program (Nielsen et al., supra), no signal peptide in the sequence was predicted. Based on this program, the predicted mature protein contains 351 amino acids with a 5 predicted molecular mass of 37.8 kDa and an isoelectric pH of 6.64.

Cloning of a Lactobacillus antri isopropanol dehydrogenase gene and construction of vector pSJ 10709.

The 1068 bp coding sequence (without stop codon) of the isopropanol dehydrogenase (SWISSPROT:C8P9V7) from L. antri was optimized for expression in the i o three organisms Escherichia coli, Lactobacillus plantarum, and Lactobacillus reuteri and synthetically constructed into pSJ 10709.

The DNA fragment containing the codon-optimized isopropanol dehydrogenase coding sequence (sadh La) was designed with the sequence 5'-GGTAC CACTA TTACA AGGAG ATTTT AGTC-3' (SEQ ID NO: 58) immediately prior to the start codon (to add a

15 Kpnl site and a Lactobacillus RBS), and Xmal and Hind 111 restriction sites immediately downstream. The desigined construct was obtained from Geneart AG and transformed as described above, resulting in SJ10709 (SJ2/pSJ 10709) and SJ10710 (SJ2/pSJ10710).

The codon-optimized nucleotide sequence (CO) and deduced amino acid sequence of the L. antri isopropanol dehydrogenase gene is SEQ ID NO: 37 and 38, respectively.

20 The coding sequence is 1071 bp including the stop codon and the encoded predicted protein is 356 amino acids. Using the SignalP program (Nielsen et al., supra), no signal peptide in the sequence was predicted. Based on this program, the predicted mature protein contains 356 amino acids with a predicted molecular mass of 38.0 kDa and an isoelectric pH of 4.9.

25 Cloning of a Lactobacillus fermentum isopropanol dehydrogenase gene and construction of vector pSJ 10703.

The 1068 bp CDS (without stop codon) of the isopropanol dehydrogenase (SWISSPROT:B2GDH6) from L. fermentum was optimized for expression in the three organisms Escherichia coli, Lactobacillus plantarum, and Lactobacillus reuteri and 30 synthetically constructed into pSJ 10703.

The DNA fragment containing the codon optimized isopropanol dehydrogenase CDS (sadh Lf) was designed with the sequence 5'-GGTAC CACTA TTACA AGGAG ATTTT AGTC-3' (SEQ ID NO: 58) immediately prior to the start codon (to add a Kpnl site and a Lactobacillus RBS), and Xmal and Hind 111 restriction sites immediately downstream. The 35 constructs were obtained from Geneart AG and transformed as previously described, resulting in SJ 10703 (SJ2/pSJ 10703) and SJ 10704 (SJ2/pSJ 10704).

The codon-optimized nucleotide sequence (CO) and deduced amino acid sequence of the L. fermentum isopropanol dehydrogenase gene is SEQ ID NO: 47 and 48, respectively. The coding sequence is 1071 bp including the stop codon and the encoded predicted protein is 356 amino acids. Using the SignalP program (Nielsen et al., supra), no signal peptide in the sequence was predicted. Based on this program, the predicted mature protein contains 356 amino acids with a predicted molecular mass of 37.9 kDa and an isoelectric pH of 5.2. Example 5: Construction and transformation of pathway constructs for isopropanol production in E. coli.

Construction of pSJ10843 containing a C. beijerinckii acetoacetate decarboxylase gene and a C. beijerinckii alcohol dehydrogenase gene.

Plasmids pSJ10725 and pSJ10713 were digested individually with Kpnl+AlwNI. Plasmid pSJ 10725 was further digested with Pvul to reduce the size of unwanted fragments. The resulting 1689 bp fragment of pSJ 10725 and the 2557 bp fragment of pSJ10713 were each purified using gel electrophoresis and subsequently ligated as outlined herein. An aliquot of the ligation mixture was used for transformation of E. coli SJ2 chemically competent cells, and transformants selected on LB plates with 200 microgram/ml ampicillin. Four colonies, picked among more than 100 transformants, were all deemed to contain the desired recombinant plasmid by restriction analysis using Hindi 11 , and two of these were kept, resulting in SJ 10843 (SJ2/pSJ 10843) and SJ 10844 (SJ2/pSJ 10844).

Construction of pSJ10841 containing a C. acetobutylicum acetoacetate decarboxylase gene and a C. beijerinckii alcohol dehydrogenase gene.

Plasmids pSJ10725 and pSJ1071 1 were digested individually with Kpnl+AlwNI; in addition, pSJ 10725 was digested with Pvul to reduce the size of unwanted fragments. The resulting 1689 bp fragment of pSJ 10725 and the 2596 bp fragment of pSJ1071 1 were each purified using gel electrophoresis and subsequently ligated as outlined herein. An aliquot of the ligation mixture was used for transformation of E. coli SJ2 chemically competent cells, and transformants selected on LB plates with 200 microgram/ml ampicillin. 4 colonies, picked among more than 100 transformants, were all deemed to contain the desired recombinant plasmid by restriction analysis using Bsgl,and two of these were kept, resulting in SJ10841 (SJ2/pSJ10841 ) and SJ10842 (SJ2/pSJ 10842).

Construction of pSJ 10748 containing a B. subtilis succinyl-CoA:acetoacetate transferase genes.

Plasmids pSJ10697 and pSJ10695 were each digested with EcoRI and Kpnl. The resulting 690 bp fragment of pSJ 10697 and the 3106 bp fragment of pSJ 10695 were each purified using gel electrophoresis and subsequently ligated as outlined herein.

5 An aliquot of the ligation mixture was used for transformation of £ coli SJ2 by electroporation, and transformants selected on LB plates with 200 microgram/ml ampicillin. 3 colonies, picked among more than 50 transformants, were all deemed to contain the desired recombinant plasmid by restriction analysis using Pvul, and two of these were kept, resulting in SJ 10748 (SJ2/pSJ 10748) and SJ 10749 (SJ2/pSJ 10749).

i o Construction of pSJ 10777 containing a B. mojavensis succinyl-CoA:acetoacetate transferase genes.

Plasmids pSJ10723 and pSJ10721 were each digested with EcoRI + Kpnl. The resulting 696 bp fragment of pSJ 10723 and the 31 18 bp fragment of pSJ 10721 were each purified using gel electrophoresis and subsequently ligated as outlined herein.

15 An aliquot of the ligation mixture was used for transformation of £ coli SJ2 chemically competent cells, and transformants selected on LB plates with 200 microgram/ml ampicillin. 4 colonies, picked among more than 500 transformants, were analyzed and one, deemed to contain the desired recombinant plasmid by restriction analysis using Pvul, was kept, resulting in SJ10777 (SJ2/pSJ 10777).

20 Construction of pSJ10750 containing a E. coli acetoacetyl-CoA transferase genes.

Plasmids pSJ10717 and pSJ10715 were each digested with EcoRI + Kpnl. The resulting 702 bp fragment of pSJ 10717 and the 3051 bp fragment of pSJ 10715 were each purified using gel electrophoresis and subsequently ligated as outlined herein.

An aliquot of the ligation mixture was used for transformation of £ coli SJ2 by 25 electroporation, and transformants selected on LB plates with 200 microgram/ml ampicillin.

3 colonies, picked among more than 50 transformants, were all deemed to contain the desired recombinant plasmid by restriction analysis using ApaLI, and two of these were kept, resulting in SJ10750 (SJ2/pSJ 10750) and SJ10751 (SJ2/pSJ 10751 ).

Construction of pSJ10752 containing a Clostridium acetobutylicum acetoacetyl-CoA 30 transferase genes.

Plasmids pSJ10731 and pSJ10727 were each digested with EcoRI + Kpnl. The resulting 705 bp fragment of pSJ 10731 and the 3061 bp fragment of pSJ 10727 were each purified using gel electrophoresis and subsequently ligated as outlined herein.

An aliquot of the ligation mixture was used for transformation of £. coli SJ2 by 35 electroporation, and transformants selected on LB plates with 200 microgram/ml ampicillin. 3 colonies, picked among more than 50 transformants, were all deemed to contain the desired recombinant plasmid by restriction analysis using Pvul, and two of these were kept, resulting in SJ 10752 (SJ2/pSJ 10752) and SJ 10753 (SJ2/pSJ 10753).

Construction of expression vector pSJ10798 containing a Clostridium acetobutylicum thiolase gene.

Plasmid pSJ10705 was digested with BspHI and EcoRI, whereas pSJ10600 was digested with Ncol and EcoRI. The resulting 1 193 bp fragment of pSJ10705 and the 5147 bp fragment of pSJ 10600 were each purified using gel electrophoresis and subsequently ligated as outlined herein.

An aliquot of the ligation mixture was used for transformation of E. coli TG1 by electroporation, and transformants selected on LB plates with 200 microgram/ml erythromycin. 3 of 4 colonies analyzed were deemed to contain the desired recombinant plasmid by restriction analysis using Nsil as well as DNA sequencing, and two of these were kept, resulting in SJ 10798 (TG1/pSJ10798) and SJ10799 (TG1/pSJ10799).

Construction of expression vector pSJ10796 containing a L. reuteri thiolase gene.

Plasmid pSJ10694 was digested with Ncol and EcoRI, and the resulting 1 .19 kb fragment purified using gel electrophoresis. Plasmid pSJ10600 was digested with Ncol and EcoRI, and the 5.2 kb fragment purified using gel electrophoresis. The purified fragments were mixed, ligated, and the ligation mixture transformed into TG1 electrocompetent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Four of the resulting colonies were analyzed and deemed to contain the desired recombinant plasmid by restriction analysis using Nsil, and two of these, further verified by DNA sequencing, were kept, resulting in SJ 10796 (TG1/pSJ10796) and SJ10797 (TG1/pSJ10797).

Construction of expression vector pSJ 10795 containing a Propionibacterium freudenreichii thiolase gene.

Plasmid pSJ10676 was digested with BspHI and EcoRI, and the resulting 1 .17 kb fragment purified using gel electrophoresis. Plasmid pSJ10600 was digested with Ncol and EcoRI, and the 5.2 kb fragment purified using gel electrophoresis. The purified fragments were mixed, ligated, and the ligation mixture transformed into TG1 electrocompetent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Four of the resulting colonies were analyzed and deemed to contain the desired recombinant plasmid by restriction analysis using Nsil, and one of these, further verified by DNA sequencing, was kept, resulting in SJ10795 (TG1/pSJ10795).

Construction of expression vector pSJ 10743 containing a Lactobacillus brevis thiolase gene.

Plasmid pSJ10699 was digested with Ncol and EcoRI, and the resulting 1 .18 kb fragment purified using gel electrophoresis. Plasmid pSJ10600 was digested with Ncol and EcoRI, and the 5.2 kb fragment purified using gel electrophoresis. The purified fragments 5 were mixed, ligated, and the ligation mixture transformed into MG1655 electrocompetent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. 16 of the resulting colonies were analyzed and two, deemed to contain the desired recombinant plasmid by restriction analysis using Clal and further verified by DNA sequencing, were kept, resulting in SJ10743 (TG1/pSJ10743) and SJ10757 (TG1/pSJ10757).

i o Construction of expression vector pSJ10886 containing a Bacillus subtilis succinyl- CoA:acetoacetate transferase genes.

Plasmid pSJ10748 was digested with Ncol and Kpnl, and the resulting 1 .4 kb fragment purified using gel electrophoresis. Plasmid pSJ10600 was digested with Ncol and Kpnl, and the resulting 5.1 kb fragment purified using gel electrophoresis. The purified

15 fragments were mixed, ligated, and the ligation mixture transformed into TG1 chemically competent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Four of the resulting colonies were analyzed and deemed to contain the desired recombinant plasmid by restriction analysis using Hind III, and two of these, further verified by DNA sequencing, were kept, resulting in SJ10886 (TG1/pSJ10886) and SJ10887

20 (TG1/pSJ10887).

Construction of expression vector pSJ10888 containing E. coli acetoacetyl-CoA transferase genes.

Plasmid pSJ10750 was digested with Ncol and Kpnl, and the resulting 1 .35 kb fragment purified using gel electrophoresis. Plasmid pSJ10600 was digested with Ncol and

25 Kpnl, and the resulting 5.1 kb fragment purified using gel electrophoresis. The purified fragments were mixed, ligated, and the ligation mixture transformed into TG1 chemically competent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Four of the resulting colonies were analyzed and deemed to contain the desired recombinant plasmid by restriction analysis using Hind III, and two of these, further verified

30 by DNA sequencing, were kept, resulting in SJ10888 (TG1/pSJ10888) and SJ10889 (TG1/pSJ10889).

Construction of expression vector pSJ10756 containing a C. beijerinckii acetoacetate decarboxylase gene.

Plasmid pSJ10713 was digested with Eagl and Kpnl, and the resulting 0.77 kb 35 fragment purified using gel electrophoresis. Plasmid pSJ10600 was digested with Eagl and Kpnl, and the resulting 5.1 kb fragment purified using gel electrophoresis. The purified fragments were mixed, ligated, and the ligation mixture transformed into TG1 chemically competent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Four of the resulting colonies were analyzed and one, deemed to contain the desired 5 recombinant plasmid by restriction analysis using Clal and verified by DNA sequencing, was kept as SJ10756 (TG1/pSJ10756).

Construction of expression vector pSJ 10754 containing a C. acetobutylicum acetoacetate decarboxylase gene.

Plasmid pSJ1071 1 was digested with Eagl and Kpnl, and the resulting 0.81 kb i o fragment purified using gel electrophoresis. Plasmid pSJ10600 was digested with Eagl and Kpnl, and the resulting 5.1 kb fragment purified using gel electrophoresis. The purified fragments were mixed, ligated, and the ligation mixture transformed into MG1655 electrocompetent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Four of the resulting colonies were analyzed, three deemed to contain the desired 15 recombinant plasmid by restriction analysis using Clal and two, verified by DNA sequencing, were kept as SJ10754 (MG1655/pSJ 10754) and SJ10755 (MG1655/pSJ 10755).

Construction of expression vector pSJ 10780 containing a L. salvarius acetoacetate decarboxylase gene.

20 Plasmid pSJ10707 was digested with Pcil and Kpnl, and the resulting 0.84 kb fragment purified using gel electrophoresis. Plasmid pSJ10600 was digested with Ncol and Kpnl, and the resulting 5.1 kb fragment purified using gel electrophoresis. The purified fragments were mixed, ligated, and the ligation mixture transformed into MG1655 chemically competent cells, selecting erythromycin resistance (200 microgram/ml) on LB

25 plates at 37°C. Four of the resulting colonies were analyzed, all deemed to contain the desired recombinant plasmid by restriction analysis using Clal and two, verified by DNA sequencing, were kept as SJ10780 (MG1655/pSJ 10780) and SJ10781 (MG1655/pSJ 10781 ).

Construction of expression vector pSJ 10778 containing a L. plantarum acetoacetate

30 decarboxylase gene.

Plasmid pSJ10701 was digested with Ncol and Kpnl, and the resulting 0.85 kb fragment purified using gel electrophoresis. Plasmid pSJ10600 was digested with Ncol and Kpnl, and the resulting 5.1 kb fragment purified using gel electrophoresis. The purified fragments were mixed, ligated, and the ligation mixture transformed into MG1655

35 chemically competent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Four of the resulting colonies were analyzed, all deemed to contain the desired recombinant plasmid by restriction analysis using Clal and two, verified by DNA sequencing, were kept as SJ10778 (MG1655/pSJ 10778) and SJ10779 (MG1655/pSJ 10779).

5 Construction of expression vector pSJ 10768 containing a Lactobacillus antri isopropanol dehydrogenase gene.

Plasmid pSJ10709 was digested with Kpnl and Xmal, and the resulting 1 .1 kb fragment purified using gel electrophoresis. Plasmid pSJ10600 was digested with Xmal and Kpnl, and the resulting 5.1 kb fragment purified using gel electrophoresis. The purified i o fragments were mixed, ligated, and the ligation mixture transformed into TG1 electrocompetent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Four of the resulting colonies were analyzed and two deemed to contain the desired recombinant plasmid by restriction analysis using Clal and verified by DNA sequencing, were kept as SJ 10768 (TG1/pSJ10768) and SJ 10769 (TG1/pSJ 10769).

15 Construction of expression vectors pSJ 10745, pSJ 10763, pSJ 10764, and pSJ 10767, containing a Thermoanaerobacter ethanolicus isopropanol dehydrogenase gene.

Plasmid pSJ10719 was digested with BspHI and Xmal, and the resulting 1 .06 kb fragment purified using gel electrophoresis. Plasmid pSJ10600 was digested with Ncol and Xmal, and the resulting 5.1 kb fragment purified using gel electrophoresis. The purified

20 fragments were mixed and ligated. The ligation mixture was transformed into MG1655 electrocompetent cells, and one of the resulting colonies, deemed to contain the desired recombinant plasmid by restriction analysis using Clal and verified by DNA sequencing, was kept as SJ10745 (MG1655/pSJ 10745). The ligation mixture was also tranformed into electrocompetent E. coli JM103, where two of four colonies were deemed to contain the

25 desired plasmid by restriction analysis using Clal, and these kept as SJ10763 (JM103/pSJ10763) and SJ10764 (JM103/pSJ10764).

Finally, the ligation mixture was transformed into electrocompetent TG1 , where three of four colonies were deemed to contain the desired plasmid by restriction analysis using Clal, and one, SJ10767 (JM103/pSJ10767), was verified by DNA sequencing.

30 Construction of expression vector pSJ 10782 containing a Clostridium beijerinckii isopropanol dehydrogenase gene.

Plasmid pSJ10725 was digested with BspHI and Xmal, and the resulting 1 .06 kb fragment purified using gel electrophoresis. Plasmid pSJ10600 was digested with Ncol and Xmal, and the resulting 5.1 kb fragment purified using gel electrophoresis. The purified

35 fragments were mixed, ligated, and the ligation mixture transformed into MG1655 electrocompetent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Four of the resulting colonies were analyzed and two, deemed to contain the desired recombinant plasmid by restriction analysis using Clal and verified by DNA sequencing, were kept as SJ 10782 (TG1/pSJ10782) and SJ 10783 (TG1/pSJ 10783).

5 Construction of expression vector pSJ 10762 containing a Lactobacillus fermentum isopropanol dehydrogenase gene.

Plasmid pSJ10703 was digested with BspHI and Xmal, and the resulting 1 .1 kb fragment purified using gel electrophoresis. Plasmid pSJ10600 was digested with Xmal and Ncol, and the resulting 5.1 kb fragment purified using gel electrophoresis. The purified i o fragments were mixed, ligated, and the ligation mixture transformed into JM103 as well as TG1 electrocompetent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Transformants were analyzed and two (one from each host strain), deemed to contain the desired recombinant plasmid by restriction analysis using Clal and verified by DNA sequencing, were kept as SJ10762 (JM103/pSJ10762) and SJ10765

15 (TG1/pSJ10765). Transformant SJ10766 (JM103/pSJ10766) was also verified to contain the Lactobacillus fermentum isopropanol dehydrogenase gene.

Construction of expression vector pSJ 10954 containing a C. acetobutylicum thiolase gene, B. mojavensis succinyl-CoA:acetoacetate transferase genes (both subunits), a C. beijerinckii acetoacetate decarboxylase gene, and a C. beijerinckii alcohol dehydrogenase 20 gene.

Plasmid pSJ10798 was digested with Xhol and Xmal, and the resulting 6.3 kb fragment purified using gel electrophoresis. Plasmid pSJ10777 was digested with Xhol and Eagl, and the resulting 1 .43 kb fragment purified using gel electrophoresis. Plasmid pSJ10843 was digested with Eagl and Xmal, and the resulting 1 .85 kb fragment purified

25 using gel electrophoresis. The three purified fragments were mixed, ligated, and the ligation mixture transformed into TG1 chemically competent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Four of the resulting colonies were analyzed and deemed to contain the desired recombinant plasmid by restriction analysis using Xbal, and two of these were kept, resulting in SJ10954 (TG1/pSJ 10954) and SJ10955

30 (TG1/pSJ10955).

Construction of expression vector pSJ 10956 containing a C. acetobutylicum thiolase gene, B. mojavensis succinyl-CoA:acetoacetate transferase genes (both subunits), a C. acetobutylicum acetoacetate decarboxylase gene, and a C. beijerinckii alcohol dehydrogenase gene.

35 Plasmid pSJ10798 was digested with Xhol and Xmal, and the resulting 6.3 kb fragment purified using gel electrophoresis. Plasmid pSJ10777 was digested with Xhol and Eagl, and the resulting 1 .43 kb fragment purified using gel electrophoresis. Plasmid pSJ10841 was digested with Eagl and Xmal, and the resulting 1 .89 kb fragment purified using gel electrophoresis. The three purified fragments were mixed, ligated, and the ligation 5 mixture transformed into TG1 chemically competent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Four of the resulting colonies were analyzed and deemed to contain the desired recombinant plasmid by restriction analysis using Xbal , and two of these were kept, resulting in SJ10956 (TG1/pSJ 10956) and SJ10957 (TG1/pSJ10957).

i o From an independent construction process (digestion, fragment purification, ligation, transformation by electroporation) one transformant, deemed to contain the desired recombinant plasmid by restriction analysis using Xbal, was kept as SJ 10926 (TG1 PSJ10926).

Construction of expression vector pSJ 10942 containing a C. acetobutylicum thiolase gene, 15 B. subtilis succinyl-CoA:acetoacetate transferase genes (both subunits), a C. beijerinckii acetoacetate decarboxylase gene, and a C. beijerinckii alcohol dehydrogenase gene.

Plasmid pSJ10798 was digested with Xhol and Xmal, and the resulting 6.3 kb fragment purified using gel electrophoresis. Plasmid pSJ10748 was digested with Xhol and Eagl, and the resulting 1 .43 kb fragment purified using gel electrophoresis. Plasmid 20 pSJ10843 was digested with Eagl and Xmal, and the resulting 1 .85 kb fragment purified using gel electrophoresis. The three purified fragments were mixed, ligated, and the ligation mixture transformed into TG1 chemically competent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Four of the resulting colonies were analyzed and deemed to contain the desired recombinant plasmid by restriction analysis using Xbal, and 25 two of these were kept, resulting in SJ10942 (TG1/pSJ 10942) and SJ10943 (TG1/pSJ10943).

Construction of expression vector pSJ 10944 containing a C. acetobutylicum thiolase gene, B. subtilis succinyl-CoA:acetoacetate transferase genes (both subunits), a C. acetobutylicum acetoacetate decarboxylase gene, and a C. beijerinckii alcohol

30 dehydrogenase gene.

Plasmid pSJ10798 was digested with Xhol and Xmal, and the resulting 6.3 kb fragment purified using gel electrophoresis. Plasmid pSJ10748 was digested with Xhol and Eagl, and the resulting 1 .43 kb fragment purified using gel electrophoresis. Plasmid pSJ10841 was digested with Eagl and Xmal, and the resulting 1 .89 kb fragment purified

35 using gel electrophoresis. The three purified fragments were mixed, ligated, and the ligation mixture transformed into TG1 chemically competent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Four of the resulting colonies were analyzed and deemed to contain the desired recombinant plasmid by restriction analysis using Xbal, and two of these were kept, resulting in SJ10944 (TG1/pSJ 10944) and SJ10945 5 (TG1/pSJ10945).

Construction of expression vector pSJ 10946 containing a C. acetobutylicum thiolase gene, an E. coli acetoacetyl-CoA transferase genes (both subunits), a C. beijerinckii acetoacetate decarboxylase gene, and a C. beijerinckii alcohol dehydrogenase gene.

Plasmid pSJ10798 was digested with Xhol and Xmal, and the resulting 6.3 kb i o fragment purified using gel electrophoresis. Plasmid pSJ10750 was digested with Xhol and Eagl, and the resulting 1 .37 kb fragment purified using gel electrophoresis. Plasmid pSJ10843 was digested with Eagl and Xmal, and the resulting 1 .85 kb fragment purified using gel electrophoresis. The three purified fragments were mixed, ligated, and the ligation mixture transformed into TG1 chemically competent cells, selecting erythromycin resistance

15 (200 microgram/ml) on LB plates at 37°C. Four of the resulting colonies were analyzed and deemed to contain the desired recombinant plasmid by restriction analysis using Xbal, and two of these were kept, resulting in SJ10946 (TG1/pSJ 10946) and SJ10947 (TG1/pSJ10947).

Construction of expression vector pSJ 10948 containing a C. acetobutylicum thiolase gene, 20 E. coli acetoacetyl-CoA transferase genes (both subunits), a C. acetobutylicum acetoacetate decarboxylase gene, and a C. beijerinckii alcohol dehydrogenase gene.

Plasmid pSJ10798 was digested with Xhol and Xmal, and the resulting 6.3 kb fragment purified using gel electrophoresis. Plasmid pSJ10750 was digested with Xhol and Eagl, and the resulting 1 .37 kb fragment purified using gel electrophoresis. Plasmid 25 pSJ10841 was digested with Eagl and Xmal, and the resulting 1 .89 kb fragment purified using gel electrophoresis. The three purified fragments were mixed, ligated, and the ligation mixture transformed into TG1 chemically competent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Four of the resulting colonies were analyzed and deemed to contain the desired recombinant plasmid by restriction analysis using Xbal, and 30 two of these were kept, resulting in SJ10948 (TG1/pSJ 10948) and SJ10949 (TG1/pSJ10949).

Construction of expression vector pSJ 10950 containing a C. acetobutylicum thiolase gene, C. acetobutylicum acetoacetyl-CoA transferase genes (both subunits), a C. beijerinckii acetoacetate decarboxylase gene, and a C. beijerinckii alcohol dehydrogenase gene.

35 Plasmid pSJ10798 was digested with Xhol and Xmal, and the resulting 6.3 kb fragment purified using gel electrophoresis. Plasmid pSJ10752 was digested with Xhol and Eagl, and the resulting 1 .38 kb fragment purified using gel electrophoresis. Plasmid pSJ10843 was digested with Eagl and Xmal, and the resulting 1 .85 kb fragment purified using gel electrophoresis. The three purified fragments were mixed, ligated, and the ligation 5 mixture transformed into TG1 chemically competent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Four of the resulting colonies were analyzed and deemed to contain the desired recombinant plasmid by restriction analysis using Xbal, and two of these were kept, resulting in SJ10950 (TG1/pSJ 10950) and SJ10951 (TG1/pSJ10951 ).

i o Construction of expression vector pSJ 10952 containing a C. acetobutylicum thiolase gene, C. acetobutylicum acetoacetyl-CoA transferase genes (both subunits), a C. acetobutylicum acetoacetate decarboxylase gene, and a C. beijerinckii alcohol dehydrogenase gene.

Plasmid pSJ10798 was digested with Xhol and Xmal, and the resulting 6.3 kb fragment purified using gel electrophoresis. Plasmid pSJ10752 was digested with Xhol and

15 Eagl, and the resulting 1 .38 kb fragment purified using gel electrophoresis. Plasmid pSJ10841 was digested with Eagl and Xmal, and the resulting 1 .89 kb fragment purified using gel electrophoresis. The three purified fragments were mixed, ligated, and the ligation mixture transformed into TG1 chemically competent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Four of the resulting colonies were analyzed and

20 deemed to contain the desired recombinant plasmid by restriction analysis using Xbal, and two of these were kept, resulting in SJ10952 (TG1/pSJ 10952) and SJ10953 (TG1/pSJ10953).

Construction of expression vector pSJ 10790 containing a C. acetobutylicum thiolase gene, B. mojavensis succinyl-CoA:acetoacetate transferase genes (both subunits), a C.

25 beijerinckii acetoacetate decarboxylase gene, and a C. beijerinckii alcohol dehydrogenase gene under control of the P1 1 promoter.

Plasmid pTRGU00178 (see US Provisional Patent Application No. 61/408, 138, filed October 29, 2010) was digested with Ncol and BamHI, and the resulting 1 .2 kb fragment purified using gel electrophoresis. pTRGU00178 was also digested with BamHI and Sail,

30 and the resulting 2.1 kb fragment purified using gel electrophoresis. pSIP409 was digested with Ncol and Xhol, and the resulting 5.7 kb fragment purified using gel electrophoresis. The three purified fragments were mixed, ligated, and the ligation mixture transformed into SJ2 electrocompetent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Two transformants, deemed to contain the desired recombinant plasmid by

35 restriction analysis using EcoRI, Bglll, and Hindlll, were kept as SJ10562 (SJ2/pSJ 10562) and SJ 10563 (SJ2/pSJ 10563).

Plasmid pSJ10562 was digested with Xbal and Notl, and the resulting 7.57 kb fragment purified using gel electrophoresis. Plasmid pTRGU00200 (supra) was digested with Xbal and Notl, and the resulting 2.52 kb fragment purified using gel electrophoresis. The purified fragments were mixed, ligated, and the ligation mixture transformed into MG1655 electrocompetent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Two transformants, deemed to contain the desired recombinant plasmid by restriction analysis using Notl + Xbal, were kept as SJ10593 (MG1655/pSJ 10593) and SJ10594 (MG1655/pSJ 10594).

Plasmid pTRGU00200 was digested with EcoRI and BamHI, and the resulting 1 .2 kb fragment purified using gel electrophoresis. pSJ 10600 was digested with EcoRI and BamHI, and the resulting 5.2 kb fragment purified using gel electrophoresis. The purified fragments were mixed, ligated, and the ligation mixture transformed into MG1655 electrocompetent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Two transformants, deemed to contain the desired recombinant plasmid by restriction analysis using EcoRI + BamHI, were kept as SJ10690 (MG1655/pSJ 10690) and SJ10691 (MG1655/pSJ10691 ).

Plasmid pSJ10593 was digested with BamHI and Xbal, and the resulting 3.25 kb fragment purified using gel electrophoresis. pSJ 10690 was digested with BamHI and Xbal , and the resulting 6.3 kb fragment purified using gel electrophoresis. The purified fragments were mixed, ligated, and the ligation mixture transformed into TG1 electrocompetent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Two transformants, deemed to contain the desired recombinant plasmid by restriction analysis using Nsil, were kept as SJ10790 (TG1/pSJ 10790) and SJ 10791 (TG1/pSJ 10791 ).

Construction of pSJ10792 containing a C. acetobutylicum thiolase gene, B. mojavensis succinyl-CoA:acetoacetate transferase genes (both subunits), a C. beijerinckii acetoacetate decarboxylase gene, and a C. beijerinckii alcohol dehydrogenase gene under control of the P27 promoter.

Plasmid pTRGU00200 was digested with EcoRI and BamHI, and the resulting 1 .2 kb fragment purified using gel electrophoresis. pSJ 10603 was digested with EcoRI and BamHI, and the resulting 5.2 kb fragment purified using gel electrophoresis. The purified fragments were mixed, ligated, and the ligation mixture transformed into MG1655 electrocompetent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Two transformants, deemed to contain the desired recombinant plasmid by restriction analysis using EcoRI + BamHI, were kept as SJ10692 (MG1655/pSJ 10692) and SJ10693 (MG1655/pSJ 10693).

Plasmid pSJ10593 was digested with BamHI and Xbal, and the resulting 3.25 kb fragment purified using gel electrophoresis. pSJ 10692 was digested with BamHI and Xbal, and the resulting 6.3 kb fragment purified using gel electrophoresis. The purified fragments 5 were mixed, ligated, and the ligation mixture transformed into TG1 electrocompetent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Two transformants, deemed to contain the desired recombinant plasmid by restriction analysis using Nsil, were kept as SJ10792 (TG1/pSJ 10792) and SJ 10793 (TG1/pSJ10793).

Construction of expression vector pSJ1 1208 containing a L. reuteri thiolase gene, B. i o mojavensis succinyl-CoA:acetoacetate transferase genes (both subunits), a C. beijerinckii acetoacetate decarboxylase gene, and a C. beijerinckii alcohol dehydrogenase gene.

Plasmid pSJ10796 (described below) was digested with Xhol and Xmal, and the resulting 6.3 kb fragment purified using gel electrophoresis. Plasmid pSJ10954 was digested with Xhol and Xmal, and the resulting 3.28 kb fragment purified using gel

15 electrophoresis. The purified fragments were mixed, ligated, and the ligation mixture transformed into TG1 chemically competent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Three of the resulting colonies were analyzed and deemed to contain the desired recombinant plasmid by restriction analysis using Xbal, and two of these were kept, resulting in SJ1 1208 (TG1/pSJ1 1208) and SJ1 1209

20 (TG1/pSJ1 1209).

Construction of expression vector pSJ1 1204 containing a L. reuteri thiolase gene, B. subtilis succinyl-CoA:acetoacetate transferase genes (both subunits), a C. beijerinckii acetoacetate decarboxylase gene, and a C. beijerinckii alcohol dehydrogenase gene.

Plasmid pSJ10796 (described below) was digested with Xhol and Xmal, and the

25 resulting 6.3 kb fragment purified using gel electrophoresis. Plasmid pSJ10942 was digested with Xhol and Xmal, and the resulting 3.26 kb fragment purified using gel electrophoresis. The purified fragments were mixed, ligated, and the ligation mixture transformed into TG1 chemically competent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Four of the resulting colonies were analyzed and

30 deemed to contain the desired recombinant plasmid by restriction analysis using Xbal, and two of these were kept, resulting in SJ1 1204 (TG1/pSJ1 1204) and SJ1 1205 (TG1/pSJ1 1205).

Construction of expression vector pSJ1 1230 containing a L. reuteri thiolase gene, E. coli acetoacetyl-CoA transferase genes (both subunits), a C. beijerinckii acetoacetate 35 decarboxylase gene, and a C. beijerinckii alcohol dehydrogenase gene. Plasmid pSJ10796 (described below) was digested with Xhol and Xmal, and the resulting 6.3 kb fragment purified using gel electrophoresis. Plasmid pSJ10946 was digested with Xhol and Xmal, and the resulting 3.23 kb fragment purified using gel electrophoresis. The purified fragments were mixed, ligated, and the ligation mixture 5 transformed into TG1 chemically competent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Seven of the resulting colonies were analyzed and 5 deemed to contain the desired recombinant plasmid by restriction analysis using Xbal, and two of these were kept, resulting in SJ1 1230 (TG1/pSJ1 1230) and SJ1 1231 (TG1/pSJ 1 1231 ).

i o Construction of expression vector pSJ1 1206 containing a L. reuteri thiolase gene, C. acetobutylicum acetoacetyl-CoA transferase genes (both subunits), a C. beijerinckii acetoacetate decarboxylase gene, and a C. beijerinckii alcohol dehydrogenase gene.

Plasmid pSJ10796 (described below) was digested with Xhol and Xmal, and the resulting 6.3 kb fragment purified using gel electrophoresis. Plasmid pSJ10951 was

15 digested with Xhol and Xmal, and the resulting 3.23 kb fragment purified using gel electrophoresis. The purified fragments were mixed, ligated, and the ligation mixture transformed into TG1 chemically competent cells, selecting erythromycin resistance (200 microgram/ml) on LB plates at 37°C. Four of the resulting colonies were analyzed and two, deemed to contain the desired recombinant plasmid by restriction analysis using Xbal, were

20 kept as SJ1 1206 (TG1/pSJ1 1206) and SJ1 1207 (TG1/pSJ1 1207).

Example 6: Construction of a Lactobacillus reuteri strain with disruption to an acetate kinase gene (ackA1).

Introduction of pJP042 into host strain L. reuteri JCM1 1 12 (04ZXV) and isolation of strain 25 TRGU768

MRS medium containing 5 μg/ml erythromycin was inoculated with a L. reuteri MM4 strain harboring pJP042 (Pijkeren and Britton Nuc. Acids Res. 2012, 1-13; Figure 4) and incubated overnight at 37°C. The strain was subcultured in 10 ml MRS containing 5 μg/ml erythromycin to OD₆oo=0.1 . The culture was incubated at 37°C for approximately 4 hours to

30 OD₆oo=0.8 and centrifuged at 8000 x g for 5 minutes. The supernatant was discarded and the cells were resuspended in 10 ml SET buffer (0.1 M NaCI, 1 mM EDTA, 10 mM Tris-CI). The suspension was centrifuged at 8000 x g for 5 minutes and the supernatant was discarded. The cells were then resuspended in 1 ml lysis buffer (6.7 % saccharose, 50 mM Tris-CI pH 8, 0.1 mM EDTA). Lysozyme was added to 10 mg/ml and the mixture was

35 incubated at 37°C for 1 hour. The lysate was then centrifuged at 8000 x g for 5 minutes. The plasmid pJP042 DNA was isolated from the supernatant using a PureYield MiniPrep kit (Promega, USA) following the directions of the manufacturer.

JCM1 1 12 cells were made competent from an overnight culture in MRS containing 5 μg ml erythromycin by subculturing in 40 ml MRS containing 5 μg ml erythromycin to 5 OD=0.1 and harvested at OD=0.8. The cells were kept on ice and washed carefully twice with 40 ml ice cold Wash Buffer (0.5M sucrose, 10% (V/V) glycerol), and resuspended in 400 μΙ Wash Buffer.

5 μΙ of isolated pJP042 (supra) was added to 100 μΙ freshly prepared competent cells (supra) and electroporated in a BioRad Gene Pulser™ with a setting of 2.5 kV, 25 i o microFarad and 400 Ohms. To this was added 1 ml MRS medium and the cells incubated at 37 °C for 3 hours. The electroporated cells were incubated anaerobically overnight at 37°C on MRS agar plates (MRS medium containing 15 g/l Bacto Agar) containing 5 μg ml erythromycin for selection of pJP042 transformants. Erythromycin resistant colonies were checked for presence of pJP042 with colony PCR using primers flanking the recT1. Out of

15 1 1 transformants, 2 were isolated and confirmed to harbor pJP042. One of these strains was stored as TRGU768 in 10% glycerol at -80 °C.

Disruption of ackA 1 via homologous recombination and isolation of strain TRGU975

To disrupt the ackA 1 gene by recombineering, the four oligonucleotides below were designed using PyRec 3.1 (obtained from Robert Britton, Microbial Genomics Laboratory,

20 Michigan State University, Ml, USA).

o516: 5'- ATCGTACCTT CAGCTACTAC ATCTTCACTT GGCATATCAA ATAAAGCTTA TTTAAGTGTT GAACTACCAG CGTTAACTGC AATTGTTTTT-3 ' (SEQ ID NO: 61 ) o517: 5'-TGTAAAGTTGTACCATCAAGATGCTG-3' (SEQ ID NO: 62)

o518: 5'-ATCTTCACTTGGCATATCAAATAAAGCTT-3' (SEQ ID NO: 63)

25 o519: 5'-TACTTACGAGCACCATACTTCTCATA-3' (SEQ ID NO: 64)

The four oligonucleotides were designed to construct and screen for mutants with an in-frame stop codon and a Hind\\\ restriction site. Sequence o516 was used for the recombineering and incorporation of the nucleotides AGCTT, which in the complementary direction implements a stop codon in the reading frame and thus results in disruption of

30 gene translation. Sequences o517, o518, and o519 were used in a PCR screen of all colonies screened. A 572 bp amplicon indicates that the mutations had been incorporated, whereas a single 1024 bp amplicon indicates that the o518 sequence did not anneal due to the mismatch between the oligo and the wild type sequence.

An overnight culture of TRGU768 was subcultured in 40 ml MRS medium containing

35 5 μg ml erythromycin to OD₆oo 0.1 . After approximately 2 hours incubation at 37°C, OD₆oo reached approximately 0.55 and recT1 expression was induced by addition of induction peptide (8 μΙ; 50 Mg/ml) MAGNSSNFIHKIKQIFTHR (SEQ ID NO: 65). The incubation at 37°C was prolonged for 20 minutes. Competent cells were then prepared by centrifugation and washing of the cells twice in 40 ml ice-cold Wash-Buffer (0.5M sucrose, 10% (v/v) glycerol). Finally the cells were resuspended in 800 ul Wash Buffer. 100 μΙ of the resuspended cells was used for each transformation. The cells were then transformed by electroporation with 5 μΙ o516 (20 μg μl) as described in the procedure above. After two hours incubation in 1 ml MRS medium at 37°C the cells were incubated anaerobically overnight on MRS agar plates.

300 colonies were screened with PCR reactions composed of 0.1 μΙ o517, 0.1 μΙ o518, 0.1 μΙ o519, 25 μΙ sterile deionized water, and 1 illustra PuReTaq Ready-To-Go™ PCR bead (GE Helathcare, Pollards Wood, UK). All PCR reactions resulted in a single 1024 bp amplicon, indicating that the cells were not mutated. After an additional 1 day of anaerobic incubation of the MRS agar plates with colonies, 4 additional colonies had appeared. These colonies were tested with PCR and resulted in one correct ackA 1 mutant, TRGU957, as shown by the presence of a 572 bp amplicon.

As the mutagenic oligo o516 in the recombineering event is incorporated into one of the DNA strings of the chromosomal double helix, the colony identified could be a mixed genotype consisting of mutants and wildtype L. reuteri cells. Hence, 25 μΙ of an overnight culture of TRGU957 was streaked onto MRS agar plates to obtain single colonies. After overnight incubation, 16 colonies were tested by PCR with o517, o518, and o519 and all resulted in 1024 bp and 572 bp amplicons indicating the presence of the correct mutations. The 16 PCR products were further digested with Hind\\\ and resulted in full digest of the 1024 bp amplicons, thereby verifying the absence of wild type cells in the selected colonies, and confirming that the colonies were pure genotypes. To test for the presence of pJP042, all 16 colonies were tested in liquid cultures in MRS with and without 5 μg ml erythromycin. One colony, TRGU975, was shown to be erythromycin sensitive and was stored in 10% glycerol at -80°C.

PCR of TRGU975 with primers o517 and 0519 resulted in a 1024 bp amplicon which was subjected to DNA sequencing. Sequence analysis confirmed the presence of the desired mutation in the genome.

Example 7: Construction of Lactobacillus reuteri strains containing an active isopropanol pathway Lactobacillus reuteri SJ11322 (L. reuteri strain containing an active isopropanol pathway) SJ 10655 (04ZXV) was transformed using protocol A with pSJ 10769 (described above), a pVS2-based plasmid containing an alcohol-dehydrogenase expression construct, resulting in SJ1 1016. SJ1 1016 was propagated in MRS medium with 0.25 microgram/ml novobiocin, to cure the strain for the plasmid, plated on MRS agar plates, and erythromycin sensitive colonies identified by replica plating. One selected strain, SJ 1 1044, was prepared for electroporation along with the original strain SJ 10655 and no difference in electroporation frequency, using pSJ10600 (described below) as a test plasmid, was observed.

L. reuteri SJ1 1044 was transformed with pSJ1 1231 by electroporation using protocol A (supra) and a transformant was kept as SJ1 1278. Several reisolations were performed to provide a pure culture of the L. reuteri transformant, which was subsequently kept as SJ 1 1322.

Lactobacillus reuteri SJ11422/SJ11423 (L. reuteri strain ontaining an active isopropanol pathway and an acetate kinase disruption)

L. reuteri strain TRGu975 (Example 10) was transformed with pSJ1 1231 by electroporation using protocol B (supra) and two transformants were kept as SJ 1 1422 and SJ1 1423.

Example 8: Small-scale isopropanol production for strains with and without an acetate kinase disruption

L reuteri strains SJ1 1322, SJ 1 1422, and SJ1 1423 were inoculated from the frozen stocks into 2 ml MRS medium supplemented with erythromycin (10 microgram/ml), and incubated overnight in stationary, closed Eppendorf tubes at 37°C. Aliquots from these cultures were used to inoculate new 2 ml MRS medium cultures supplemented with erythromycin (10 microgram/ml) and further supplemented as indicated in Table 2 below, and these cultures incubated in stationary, closed Eppendorf tubes for 3 days at 30°C.

Supernatants were subsequently analyzed for 1 -propanol, 2-propanol, acetone, and ethanol content by GC-FID. Samples were diluted 1 +1 with 0.05% tetrahydrofuran in methanol and analyzed using the GC parameters are listed in Table 1 . Results are shown in Table 2.

Table 1 .

Temp, gradient 0 - 4.5 min: 50°C

4.5 - 9.93 min: 50 - 240°C linear gradient

Detection FID

Internal Tetrahydrofuran 2.4

standard

External Acetone (Analytical grade) 2.0

standards 1 -propanol (Analytical grade) 5.4

2-propanol (HPLC grade) 3.3

Ethanol 3.5

Table 2.

Knockout strains showed significant increase in isopropanol yield when fermented prescence of fructose compared to control strains.

Example 9: Isopropanol production for strains with and without an acetate kinase disruption

Strain SJ1 1322 and the ackA1 mutant strain SJ1 1422 both harboring the same plasmid containing the isopropanol operon described above were cultivated under identical conditions for comparison of isopropanol production.

To create a pre-culture for the labscale tank fermentation, the frozen strains (glycerol preserved) were each inoculated into a sugar cane juice based medium (BRIX=5) containing 10 g/L Bacto yeast extract, 1 g/L Tween 80, 50 mg/L MnS0₄ H₂0 and 10 mg/L erythromycin. The cultures were then incubated at 37°C overnight.

For each experiment, a 40 mL sample of the overnight culture was used to inoculate a 3 liter bioreactor containing 2 liter of sugar cane fermentation medium composed of sugar cane juice (BRIX=20), 1 mL/L pluronic/Dowfax 63N, 20 g/L Bacto Yeast Extract, 1 g/L Tween 80, 25 mg/L MnS0₄ H₂0, 650 mg/L phytic acid, 1 g/L serine, 1 g/L threonine, 400 mg/L uridine, 1 g/L monosodium glutamate and 10 mg/L erythromycin. The erythromycin, monosodium glutamate and uridine were added by sterile filtration after autoclavation.

Nitrogen was continuously added to the slowly agitated medium (400 RPM); the temperature was held constant at 30°C; pH was held constant at pH 5.5 by addition of NH₃ (aq.) (approximately 10% (w/w)).

Samples were taken throughout the fermentations which lasted 71 hours. The isopropanol content of the samples was measured using GC-FID as described herein. The isopropanol concentration profiles are shown in the Figure 5. The ackA1 mutant strain SJ1 1422 containing an acetate kinse disruption (GPP133) showed significantly higher isopropanol production compared to the non-mutant control strain SJ1 1322 (GPP134).

Example 10: Effect of fructose on isopropanol production using strain SJ11422

Mutant strain SJ1 1422 (containing an active isopropanol pathway and an actetate kinase disruption) was grown with and without addition of 20 g/L fructose to the fermentation medium to observe the effects of fructose on isopropanol production in strain having an acetate kinase disruption.

To create a pre-culture for the labscale tank fermentation, the frozen strain (glycerol preserved) was inoculated into a sugar cane juice based medium (BRIX=5) containing 10 g/L Bacto yeast extract, 1 g/L Tween 80, 50 mg/L MnS0₄ H₂0 and erythromycin 10 mg/L.

The culture was incubated at 37 °C overnight.

40 mL of this pre-culture was used to inoculated a 3 liter bioreactor containing 2 liters of sugar cane fermentation medium composed of sugar cane juice (BRIX=10), 1 mL/L pluronic/Dowfax 63N, 10 g/L Bacto Yeast Extract, 1 g/L Tween 80, 25 mg/L MnS0₄ H₂0,

650 mg/L phytic acid, 1 g/L serine, 1 g/L threonine, 400 mg/L uridine, 1 g/L monosodium glutamate, 2 g/L potassium dihydrogenphosphate, 2 g/L sodium acetate trihydrate and 10 mg/L erythromycin, with or without 20 g/L fructose. Erythromycin, monosodium glutamate and uridine were added by sterile filtration after autoclavation.

Nitrogen was continuously bobbled through the slowly agitated medium (400 RPM); the temperature was held constant at 37 °C; pH was held constant at pH 5.5 by addition of

NH₃ (aq.) (approximately 10% (w/w)).

Samples were taken throughout the 72 hour fermentations. The isopropanol and acetone contents of the samples were measured using GC-FID as described herein. The isopropanol concentration profiles are shown in the Figure 6. The ackA1 mutant strain

SJ1 1422 containing an acetate kinse disruption showed significantly higher isopropanol production with supplemental fructose (GPP146) compared to the same fermentation without supplemental fructose (GPP144).

Acetone is a known intermediate in the current active isopropanol pathway. The combined isopropanol+acetone concentration profiles from the fermentation above are shown in the Figure 7. The ackA1 mutant strain SJ1 1422 containing an acetate kinse disruption showed significantly higher isopropanol+acetone production with supplemental fructose (GPP146) compared to the same fermentation without supplemental fructose (GPP144). Example 11 : Effect of 1 ,2-propanediol on isopropanol production using strain SJ11422

Mutant strain SJ1 1422 (containing an active isopropanol pathway and an actetate kinase disruption) was grown with and without addition of 5 g/L 1 ,2-propanediol to the fermentation medium.

To create a pre-culture for the labscale tank fermentation, the frozen strain (glycerol preserved) was inoculated into a sugar cane juice based medium (BRIX=5) containing 10 g/L Bacto yeast extract, 1 g/L Tween 80, 50 mg/L MnS0₄ H₂0 and erythromycin 10 mg/L. The culture was incubated at 37 °C overnight.

40 mL of this pre-culture was used to inoculated a 3 liter bioreactor containing 2 liters of sugar cane fermentation medium composed of sugar cane juice (BRIX=10), 1 mL/L pluronic/Dowfax 63N, 10 g/L Bacto Yeast Extract, 1 g/L Tween 80, 25 mg/L MnS0₄ H₂0, 650 mg/L phytic acid, 1 g/L serine, 1 g/L threonine, 400 mg/L uridine, 1 g/L monosodium glutamate, 2 g/L potassium dihydrogenphosphate, 2 g/L sodium acetate trihydrate and 10 mg/L erythromycin, with or without 5 g/L 1 ,2-propanediol. Erythromycin, monosodium glutamate and uridine were added by sterile filtration after autoclavation.

Nitrogen was continuously bobbled through the slowly agitated medium (400 RPM); the temperature was held constant at 37 °C; pH was held constant at pH 5.5 by addition of NH₃ (aq.) (approximately 10% (w/w)).

Samples were taken throughout the 72 hour fermentations. The isopropanol and acetone contents of the samples were measured using GC-FID as described herein. The isopropanol concentration profiles are shown in the Figure 8. The ackA1 mutant strain SJ1 1422 containing an acetate kinse disruption (GPP145) showed increased isopropanol production with supplemental 1 ,2-propanediol after about 50 hours compared to the same fermentation without supplemental 1 ,2-propanediol (GPP144).

Acetone is a known intermediate in the current active isopropanol pathway. The combined isopropanol+acetone concentration profiles from the fermentation above are shown in the Figure 9. The ackA1 mutant strain SJ1 1422 containing an acetate kinse disruption showed significantly higher isopropanol+acetone production with 1 ,2-propanediol (GPP145) compared to the same fermentation without supplemental 1 ,2-propanediol (GPP144).

Example 12: Construction of a Lactobacillus reuteri strain with disruption to an acetate kinase gene (ackA1) by insertion of a chloramphenicol acetyltransferase (cat) gene.

To inactivate the acetate kinase gene, and to be able to replace this gene by other DNA constructs, a construct was designed that carried the acetate kinase gene region, modified so that it comprises an upstream region of approximately 1 .5 kb, retaining the first 9 codons of the acetate kinase coding region, followed by a BamHI restriction site, followed by the stop codon of the acetate kinase coding region and further approximately 1 .5 kb sequence of downstream DNA. Double homologus recombination with the chromosome involving this construct would thus bring about a deletion of a majority of the acetate kinase coding region.

Construction of pSJ1 1093 containing the acetate kinase upstream region.

The acetate kinase upstream region was amplified by PCR, using chromosomal DNA from L. reuteri SJ 10655 as template, and oligonucleotide primers 684225 and 684226 shown below.

684225: 5'-GACTGAATTCCCGCGGTTATTGCTGTACAGTCAGAC-3' (SEQ ID NO: 249) 684226: 5'-GACTGGATCCAGCGTTAACTGCAATTGTTTTTG-3' (SEQ ID NO: 250)

A PCR fragment of the desired size was obtained, digested with EcoRI + BamHI, and purified by agarose gel electrophoresis. Vector pUC19 DNA was digested with EcoRI + BamHI, treated with alkaline phosphatase, and purified by agarose gel electrophoresis. The vector and PCR fragment were mixed, ligated, and the ligation mixture transformed into £. coli SJ2 selecting ampicillin resistance (200 microgram/ml). Two correct transformants, verified by restriction digests and DNA sequencing of the PCR amplified insert, were kept as S J 1 1093 (S J2/pS J 1 1093) and S J 1 1094 (S J2/pSJ 1 1094).

Construction of pSJ1 1095, containing the acetate kinase downstream region.

The acetate kinase downstream region was amplified by PCR, using chromosomal DNA from L. reuteri SJ10655 as template, and oligonucleotide primers 684227 and 684228 shown below.

684227: 5'-GACTGGATCCTAAGCACTAAATAATTCGTCAAAAGA-3' (SEQ ID NO: 251 ) 684228: 5'-GACTAAGCTTCTCGAGGTTTTCAGCGACAAAATCTG-3' (SEQ ID NO: 252)

A PCR fragment of the desired size was obtained, digested with BamHI + Hind III, and purified by agarose gel electrophoresis. Vector pUC19 DNA was digested with BamHI + Hindi 11 , treated with alkaline phosphatase, and purified by agarose gel electrophoresis. 5 The vector and PCR fragment were mixed, ligated, and the ligation mixture transformed into £ coli SJ2 selecting ampicillin resistance (200 microgram/ml). Two correct transformants, verified by restriction digests and DNA sequencing of the PCR amplified insert, were kept as S J 1 1095 (S J2/pS J 1 1095) and S J 1 1096 (S J2/pSJ 1 1096). i o Construction of pSJ 1 1227, containing acetate kinase upstream and downstream regions.

The acetate kinase downstream region was excised from pSJ1 1095 as a 1 .5 kb BamHI-Hindlll fragment, and purified by agarose gel electrophoresis. This region was ligated to the 4.1 kb Hindlll-BamHI fragment of pSJ1 1093, which had been treated with alkaline phosphatase and purified by agarose gel electrophoresis. The ligation mixture was

15 transformed into chemically competent SJ2 cells, selecting ampicillin resistance (200 microgram/ml). Four of the six transformants were deemed correct by restriction analysis, and two kept as SJ1 1227 (SJ2/pSJ1 1227) and SJ1 1228 (SJ2/pSJ1 1228).

Construction of pSJ1 1280, containing acetate kinase upstream and downstream regions

20 flanking an antibiotic resistance cassette.

To insert a chloramphenicol resistance gene flanked by resolvase sites, the appropriate 1 .2 kb fragment was prepared from pSJ3372 by digestion with Bell + BamHI and purified by agarose gel electrophoresis (prepared from a dam- £. coli host; see WO 96/23073, Figure 9 and examples). The fragment was mixed and ligated with a BamHI-

25 digested, alkaline phosphatase treated and agarose gel purified pSJ 1 1227 DNA fragment.

The ligation mixture was transformed into £ coli SJ2 by electroporation as described above, selecting both chloramphenicol resistance (10 microgram/ml) and ampicillin resistance (200 microgram/ml). A resulting transformant, containing a plasmid deemed correct by restriction analysis, was kept as SJ1 1280 (SJ2/pSJ1 1280).

30

Construction of pSJ1 1503, a temperature-sensitive plasmid for replication in Lactobacillus containing acetate kinase upstream and downstream regions flanking an antibiotic resistance cassette.

A plasmid with the above characteristics was constructed by combination of 35 pSJ1 1280 and the vector pTN1 , which has been described as temperature-sensitive for replication in various Lactobacilli (Neu, T., and Henrich, B. (2003). Appl. Environ. Microbiol., 69, 1377-1382).

Specifically, pSJ1 1280 was digested with Hindi 11 , treated with alkaline phosphatase, and the linearized 6.8 kb DNA fragment was purified by agarose gel electrophoresis. pTN1 was digested with Hind III, and the 3.3 kb linearized fragment purified by gel electrophoresis. The two fragments were mixed, ligated, and the ligation mixture transformed into E. coli TG1 by electroporation, selecting erythromycin (100 microgram/ml) and chloramphenicol (10 microgram/ml) resistance at 37°C. By replica plating, all transformants were found to be ampicillin, erythromycin and chloramphenicol resistant. Plasmids were extracted from six transformants, and two plasmids, kept as SJ1 1502 (TG1/pSJ1 1502) and SJ 1 1503 (TG1/pSJ1 1503), had a restriction pattern indicating that the plasmids pSJ1 1280 and pTN1 were combined in an orientation where the pUC19 replication origin present on pSJ1 1280 was situated next to the ermR gene of pTN 1 .

Deletion of the L. reuteri acetate kinase gene.

Plasmids pSJ 1 1502 and pSJ 1 1503 were transformed into electrocompetent cells of

L. reuteri SJ1 1400, using electroporation protocol B previously described, and transformants selected anaerobically on MRS plates with 10 microgram/ml erythromycin at 37°C. After 2 days, plates were moved to 33°C for further 3 days of anaerobic incubation, whereafter plasmids were prepared and four strains, deemed by restriction digests to contain correct plasmids, were kept as SJ1 1521 (a pSJ 1 1502 transformant) and SJ1 1522, SJ1 1523 and SJ1 1524 (all pSJ1 1503 transformants). These strains were verified to grow on MRS with 6 microgram/ml chloramphenicol.

To select for cells in which the plasmid was integrated into the chromosome, the strains above were plated on MRS plates with either 6 microgram/ml chloramphenicol, 10 microgram/ml erythromycin, or with both antibiotics simultaneously and followed by incubation anaerobiaclly at 45°C for 3 days. Colonies appeared on all plates, and several were checked by PCR amplification with primer pairs that were designed to amplify from within the cat gene across the acetate kinase upstream region into flanking chromosomal DNA (primers 309520 and 692431 below), or from within the cat gene across the acetate kinase downstream region into flanking chromosomal DNA (primers 309519 and 692432 below).

309519: 5'-TCAAATGAACTTTAATAAAATTGATTTAG-3' (SEQ ID NO: 253)

309520: 5'-GCCAGTCATTAGGCCTATC-3' (SEQ ID NO: 254)

692431 : 5'-TTTGAATTAATGGAGGCTCGT-3' (SEQ ID NO: 255)

692432: 5'-CGCGGTAACATTAATATCATGA-3' (SEQ ID NO: 256)

PCR amplifications revealed colonies in which integration in either of the homologous regions were detected, as well as some colonies that appeared of mixed phenotype, having integrations in one as well as in the other region.

Colonies revealing integration were inoculated in liquid MRS medium without antibiotics, at 30 oC, and after overnight incubation, these overnight cultures were plated 5 on MRS with 6 microgram/ml chloramphenicol, at 45°C. The overnight cultures were also used to inoculate further overnight cultures without antibiotics, at 30°C. Next day, platings and further inoculations were performed as before.

Colonies arising on the chloramphenicol plates at 45°C were patched on MRS with 6 microgram/ml chloramphenicol, as well as on MRS with 10 microgram/ml erythromycin, and i o chloramphenicol resistant, erythromycin sensitive strains were identified. Such strains were reisolated, the resistance phenotype verified again, and strains were finally characterized by PCR amplifications across the ack locus to ascertain that these strains carried the chloramphenicol resistance gene replacing most of the acetate kinase coding region.

Strains kept were SJ1 1534 (from SJ 1 1522), SJ1 1535 and SJ1 1536 (both from SJ 1 1524), 15 and S J 1 1537 and S J 1 1538 (both from S J 1 1523).

Site-specific deletion of the chromosomally integrated cat gene using the resolvase from pAMbetal .

Plasmid pSJ3008 (AKA pWT) is a derivative of pAMbetal which carries the 20 pAMbetal resolvase gene (described in WO 96/23073, Figure 3 and example 6). This plasmid may be used to express the resolvase protein which can act on the two res sites (recognition sites for the resolvase) flanking the cat gene on the plasmid pSJ3372 (see WO 96/23073, Figure 9 and examples), resulting in the site-specific deletion of the cat gene from the construct. When pSJ3372 was used in the construction of pSJ1 1280, fragments 25 were used so that the cat gene inserted into pSJ1 1280 was flanked on either side with functional res sites. Consequently the cat gene, now present in the chromosome of e.g., SJ1 1538 in the ack locus, was flanked with functional res sites.

Strain SJ1 1538 was made electroporation competent, and transformed with plasmid pSJ3008, selecting erythromycin resistance (10 microgram/ml) at 30 °C for two days. 30 Transformants were inoculated into liquid MRS medium with erythromycin (10 microgram/ml) for 3 days at 30 °C, these cultures plated on MRS with erythromycin (10 microgram/ml).

When colonies had formed after two days, these were replica plated on MRS with 10 microgram/ml erythromycin, as well as on MRS with 6 microgram/ml chloramphenicol, 35 and all colonies were found to be chloramphenicol-sensitive, indicating loss of the chloramphenicol resistance gene. PCR amplification with primers 692431 and 692432 {supra), amplifying across the ack gene position, confirmed the deletion of the cat gene from the chromosome of such strains.

Example 13: Isopropanol production from Lactobacillus reuteri strain SJ11538 comprising a disruption to an acetate kinase gene (ackA1) via insertion of a chloramphenicol acetyltransferase (cat) gene.

The isopropanol operon expression plasmid pSJ1 1231 (supra) was transformed into SJ1 1538 by electroporation using the procedure B described above. Six of the resulting transformants were individually inoculated into 2 ml MRS medium with 10 microgram/ml erythromycin, and propagated at 37°C overnight in closed eppendorf tubes. A 100 microliter aliquot was then used to inoculate two new sets of 2 ml cultures, which were incubated at 30°C for 3 days. One set consisted of MRS medium with 10 microgram/ml erythromycin, the other set was in addition supplemented with fructose to 0.5 %. Supernatants were harvested and analyzed for iso-propanol, 1 -propanol, acetone, and ethanol content as described herein.

Without fructose addition, the six individual cultures produced the following concentrations of isopropanol (g/l): 0.19; 0.18; 0.19; 0.19; 0.19; 0.20. With fructose addition, the 6 individual cultures produced the following concentrations of isopropanol (g/l): 0.32; 0.29; 0.35; 0.29; 0.29; 0.31 . As previously observed for the akcA1 mutant constructed via recombineering, the addition of fructose is beneficial for the production of isopropanol in MRS medium for the ack deletion mutant.

For comparison, three individual cultures of SJ1 1422, and three individual cultures of SJ1 1423 were included in this experiment. SJ1 1422 and SJ1 1423 are recombineering constructed ackA1 mutants containing the isopropanol operon plasmid pSJ1 1231 , previously described.

Without fructose, the three SJ1 1422 strains produced 0.19; 0.19; and 0.20 g/l isopropanol; and the three SJ1 1423 strains produced 0.22; 0.21 ; and 0.18 g/l isopropanol. With fructose addition, the three SJ1 1422 strains produced 0.32; 0.31 ; and 0.34 g/l isopropanol, and the three SJ 1 1423 strains produced 0.35; 0.37, and 0.27 g/l isopropanol.

Although the foregoing has been described in some detail by way of illustration and example for the purposes of clarity of understanding, it is apparent to those skilled in the art that any equivalent aspect or modification may be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention. The present invention may be further described by the following numbered paragraphs:

[1 ] An isolated recombinant Lactobacillus host cell comprising an active isopropanol pathway, wherein the cell comprises a disruption to an endogenous gene encoding an acetate kinase, and wherein the cell produces a greater amount of isopropanol compared to the cell without the disruption when cultivated under identical conditions.

[2] The host cell of paragraph [1 ], wherein the cell produces a greater amount of isopropanol when cultivated in the presence of fructose (e.g., at least 0.5% or at least 2.0%).

[3] The host cell of paragraph [1 ] or [2], comprising one or more heterologous isopropanol pathway genes selected from: a heterologous thiolase gene; a heterologous CoA- transferase gene; a heterologous acetoacetate decarboxylase gene; and a heterologous isopropanol dehydrogenase gene.

[4] The host cell of any one of paragraphs [1 ]-[3], comprising a heterologous thiolase gene; a heterologous CoA-transferase gene; a heterologous acetoacetate decarboxylase gene; and a heterologous isopropanol dehydrogenase gene. [5] The host cell of any one of paragraphs [1 ]-[4], wherein the cell produces a decreased amount of acetate (e.g., at least 25% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, or at least 90% less) compared to the cell without the disruption when cultivated under identical conditions. [6] The host cell of any one of paragraphs [1 ]-[5], wherein (a) the acetate kinase has at least 60% sequence identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO: 60 or the mature polypeptide thereof; (b) the coding sequence of the gene encoding the acetate kinase hybridizes under at least low, medium, medium-high, high, or very high stringency conditions with the full-length complementary strand of SEQ ID NO: 59; or (c) the coding sequence of the gene encoding the acetate kinase has at least 60% sequence identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO: 59, or the mature polypeptide coding sequence thereof. [7] The host cell of any one of paragraphs [1 ]-[5], wherein the acetate kinase has at least 60% sequence identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO: 60 or the mature polypeptide thereof.

[8] The host cell of any one of paragraphs [1 ]-[5], wherein the acetate kinase comprises or consists of SEQ ID NO: 60 or the mature polypeptide thereof.

[9] The host cell of any one of paragraphs [1 ]-[5], wherein the coding sequence of the gene encoding the acetate kinase hybridizes under at least low, medium, medium-high, high, or very high stringency conditions with the full-length complementary strand of SEQ ID NO: 59.

[10] The host cell of any one of paragraphs [1 ]-[5], wherein the coding sequence of the gene encoding the acetate kinase comprises a polynucleotide has at least 60% sequence identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ I D NO: 59, or the mature polypeptide coding sequence thereof. [1 1 ] The host cell of any one of paragraphs [1 ]-[5], wherein the coding sequence of the gene encoding the acetate kinase comprises or consists of SEQ ID NO: 59 or the mature polypeptide coding sequence thereof.

[12] The host cell of any one of paragraphs [1 ]-[1 1 ], wherein the disruption occurs in the coding sequence of the gene encoding the acetate kinase.

[13] The mutant of any one of paragraphs [1 ]-[1 1 ], wherein the disruption occurs in a promoter sequence of the gene encoding the acetate kinase. [14] The host cell of any one of paragraphs [1 ]-[13], wherein the cell produces a greater amount of isopropanol when cultivated in the presence of 1 ,2-propanediol (e.g., at least 0.5% or at least 2.0%).

[15] The host cell of any one of paragraphs [1 ]-[14], wherein the cell is selected from Lactobacillus plantarum, Lactobacillus fructivorans, and Lactobacillus reuteri. [16] The host cell of paragraph [15], wherein the cell is a Lactobacillus reuteri cell.

[17] The host cell of any one of paragraphs [1 ]-[16], wherein the cell produces at least 25% less (e.g., at least 50% less, at least 60% less, at least 70% less, at least 80% less, or at least 90% less) of the acetate kinase compared to the cell without the disruption when cultivated under identical conditions.

[18] The host cell of any one of paragraphs [1 ]-[17], wherein the endogenous gene is inactivated.

[19] The host cell of any one of paragraphs [1 ]-[18], wherein the cell is capable of producing at least 10% more (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 75%, at least 100%, or at least 200% more) isopropanol compared to the cell without the disruption when cultivated under identical conditions.

[20] The host cell of any one of paragraphs [1 ]-[18], wherein the cell is capable of producing at least 10% more (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 75%, at least 100%, or at least 200% more) isopropanol compared to the cell without the disruption when cultivated for 3 days at 30°C in MRS medium supplemented with 10 μg mL erythromycin and 2% fructose.

[21 ] A method for obtaining the Lactobacillus host cell of any one of paragraphs [1 ]-[20], comprising disrupting an endogenous gene encoding an acetate kinase in a Lactobacillus strain having an active isopropanol pathway.

[22] A method for obtaining the Lactobacillus host cell of any one of paragraphs [1 ]-[20], comprising: (a) providing a Lactobacillus strain; (b) disrupting an endogenous gene encoding an acetate kinase in the Lactobacillus strain; (c) transforming the Lactobacillus strain with one or more heterologous isopropanol pathway genes resulting in the strain having an active isopropanol pathway; and (d) isolating the resulting Lactobacillus host cell.

[23] A method of producing isopropanol, comprising: (a) cultivating the Lactobacillus host cell of any one of paragraphs [1 ]-[20]; and (b) recovering the isopropanol.

Claims

Claims What is claimed is:

1 . A recombinant Lactobacillus host cell comprising an active isopropanol pathway, wherein the cell comprises a disruption to an endogenous gene encoding an acetate kinase, and wherein the cell produces a greater amount of isopropanol compared to the cell without the disruption when cultivated under identical conditions.

2. The host cell of claim 1 , wherein the cell produces a greater amount of isopropanol when cultivated in the presence of fructose (e.g., at least 0.5% or at least 2.0%).

3. The host cell of claim 1 or 2, comprising one or more heterologous isopropanol pathway genes selected from:

a heterologous thiolase gene;

a heterologous CoA-transferase gene;

a heterologous acetoacetate decarboxylase gene; and

a heterologous isopropanol dehydrogenase gene.

4. The host cell of any one of claims 1 -3, comprising a heterologous thiolase gene; a heterologous CoA-transferase gene; a heterologous acetoacetate decarboxylase gene; and a heterologous isopropanol dehydrogenase gene.

5. The host cell of any one of claims 1 -4, wherein the cell produces a decreased amount of acetate (e.g., at least 25% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, or at least 90% less) compared to the cell without the disruption when cultivated under identical conditions.

6. The host cell of any one of claims 1 -5, wherein (a) the acetate kinase has at least 60% sequence identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO: 60; (b) the coding sequence of the gene encoding the acetate kinase hybridizes under at least low, medium, medium-high, high, or very high stringency conditions with the full-length complementary strand of SEQ ID NO: 59; or (c) the coding sequence of the gene encoding the acetate kinase has at least 60% sequence identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO: 59.

7. The host cell of any one of claims 1 -5, wherein the acetate kinase has at least 60% sequence identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO: 60.

8. The host cell of any one of claims 1 -5, wherein the acetate kinase comprises or consists of SEQ ID NO: 60.

9. The host cell of any one of claims 1 -5, wherein the coding sequence of the gene encoding the acetate kinase hybridizes under at least low, medium, medium-high, high, or very high stringency conditions with the full-length complementary strand of SEQ ID NO: 59.

10. The host cell of any one of claims 1 -5, wherein the coding sequence of the gene encoding the acetate kinase comprises a polynucleotide has at least 60% sequence identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ I D NO: 59.

1 1 . The host cell of any one of claims 1 -5, wherein the coding sequence of the gene encoding the acetate kinase comprises or consists of SEQ ID NO: 59.

12. The host cell of any one of claims 1 -1 1 , wherein the disruption occurs in the coding sequence of the gene encoding the acetate kinase.

13. The mutant of any one of claims 1 -1 1 , wherein the disruption occurs in a promoter sequence of the gene encoding the acetate kinase.

14. The host cell of any one of claims 1 -13, wherein the cell produces a greater amount of isopropanol when cultivated in the presence of 1 ,2-propanediol (e.g., at least 0.5% or at least 2.0%).

15. The host cell of any one of claims 1 -14, wherein the cell is selected from Lactobacillus plantarum, Lactobacillus fructivorans, and Lactobacillus reuteri.

16. The host cell of claim 15, wherein the cell is a Lactobacillus reuteri cell.

17. The host cell of any one of claims1 -16, wherein the cell produces at least 25% less (e.g., at least 50% less, at least 60% less, at least 70% less, at least 80% less, or at least 90% less) of the acetate kinase compared to the cell without the disruption when cultivated under identical conditions.

18. The host cell of any one of claims 1 -17, wherein the endogenous gene is inactivated.

19. The host cell of any one of claims 1 -18, wherein the cell is capable of producing at least 10% more (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 75%, at least 100%, or at least 200% more) isopropanol compared to the cell without the disruption when cultivated under identical conditions.

20. The host cell of any one of claims 1 -18, wherein the cell is capable of producing at least 10% more (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 75%, at least 100%, or at least 200% more) isopropanol compared to the cell without the disruption when cultivated for 3 days at 30°C in MRS medium supplemented with 10 μg mL erythromycin and 2% fructose.

21 . A method for obtaining the Lactobacillus host cell of any one of claims 1 -20, comprising disrupting an endogenous gene encoding an acetate kinase in a Lactobacillus strain having an active isopropanol pathway.

22. A method for obtaining the Lactobacillus host cell of any one of claims 1 -20, comprising:

(a) providing a Lactobacillus strain;

(b) disrupting an endogenous gene encoding an acetate kinase in the Lactobacillus strain;

(c) transforming the Lactobacillus strain with one or more heterologous isopropanol pathway genes resulting in the strain having an active isopropanol pathway; and (d) isolating the resulting Lactobacillus host cell. A method of producing isopropanol, comprising:

(a) cultivating the Lactobacillus host cell of any one of claims 1 -20; and

(b) recovering the isopropanol.