MX2008004086A - Fermentive production of four carbon alcohols - Google Patents

Abstract

Methods for the fermentive production of four carbon alcohols is provided. Specifically, butanol, preferably 1-butanol is produced by the fermentive growth of a recombinant bacterium expressing a 1- butanol biosynthetic pathway.

Description

FOUR CARBON ALCOHOL FERMENTIVE PRODUCTION Field of the Invention The invention relates to the field of industrial microbiology and the production of alcohols. More specifically, 1-butane is produced via industrial fermentation of a recombinant microorganism.

BACKGROUND OF THE INVENTION Butanol is an important industrial chemical, useful as a fuel additive, as a feed material chemical in the plastics industry, and as a food grade extraction solvent in the food and flavoring industry. Every year 10 to 12 thousand million pounds of butanol are produced by petrochemical means and the need for this chemical will probably increase. Methods for the chemical synthesis of 1-butanol are known, such as the Oxo Process, the Reppe Process, and the hydrogenation of crotonaldehyde (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCH Verlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719). These processes use starting materials derived from petrochemicals and are generally expensive and not environmentally friendly. The production of 1-butanol from Ref. 191032 of raw materials derived from plants could minimize greenhouse gas emissions and could represent an advance in the technique. The methods for producing 1-butanol by biotransformation of other organic chemicals are also known. For example, Muramoto et al. (JP63017695) describes a method for the production of alcohols, including butanol, from aldehydes using Pseudomonas strains. Additionally, Kuehnle et al. (EP 1149918) describes a process for preparing 1-butanol and 2-butanol by the oxidation of hydrocarbons by several strains of Rhodococcus ruber. Methods for producing butanol by fermentation are also known, where the most popular process produces a mixture of acetone, 1-butanol and ethanol and is referred to as the ABE processes (Blaschek et al., U.S. Pat.

No. 6,358,717). The fermentation of acetone-butanol-ethanol (ABE) by Clostridium um acetobutylicum is one of the oldest known industrial fermentations, and the trajectories and genes responsible for the production of these solvents have been reported (Girbal et al., Trends in Biotechnology 16: 11-16 (1998)) . The current fermentation, however, has been quite complicated and difficult to control. ABE fermentation has been continuously declining since the 1950s, and almost all ethanol is now produced via petrochemical routes, as described above. In a Typical ABE fermentation, butyric, propionic, lactic and acetic acids are first produced by C. acetobutylicum, the culture pH drops and undergoes a metabolic "butterfly" change, and 1-butanol, acetone, isopropanol and ethanol are then formed. In conventional ABE fermentations, the yield of 1-butanol from glucose is low, typically around 15 percent and rarely exceeds 25 percent. Accordingly, the concentration of 1-butanol in conventional ABE fermentations is usually less than 1.3 percent. Attempts to maximize the production of 1-butanol from the ABE process by the removal of all other solvent by-products have not been totally successful, and consequently, the process produces significant amounts of acetone which is not useful as a gasoline additive. A process for the fermentative production of butanol where 1-butanol is the only product could represent an advance in the technique. There is a need, therefore, for a cost-effective, environmentally responsible process for the production of 1-butanol as a single product. The present invention addresses the need through the discovery of a recombinant microbial production host expressing a biosynthetic pathway of 1-butanol.

Brief Description of the Invention The invention provides a recombinant microorganism having a biosynthetic pathway of modified 1-butanol. The modified microorganism can be used for the commercial production of 1-butanol. Therefore, the invention provides a recombinant microbial host cell comprising at least one DNA molecule encoding a polypeptide that catalyzes a conversion of substrate to product selected from the group consisting of: a) acetyl-CoA to acetoacetyl-CoA b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA c) 3-hydroxybutyryl-CoA to crotonyl-CoA d) crotonyl-CoA to butyryl-CoA e) butyryl-CoA to butyraldehyde and f) butyraldehyde to 1-butanol; wherein at least one DNA molecule is heterologous to the microbial host cell and wherein the microbial host cell produces 1-butanol. In another embodiment the invention provides a method for the production of 1-butanol comprising: i) providing a recombinant microbial host cell comprising at least one DNA molecule encoding a polypeptide that catalyzes a conversion of substrate to product selected from the group consists of: a) acetyl-CoA to acetoacetyl-CoA b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA c) 3-hydroxybutyryl-CoA to crotonyl-CoA d) crotonyl-CoA to butyryl-CoA e) butyryl-CoA to butyraldehyde and f) Butyraldehyde to 1-butanol; wherein at least one DNA molecule is heterologous to the microbial host cell; and ii) contacting the host cell of (i) with a fermentable carbon substrate under conditions whereby 1-butane is produced.

Brief Description of the Figures and Sequences The invention can be more fully understood from the following detailed description, figure, and accompanying sequence descriptions, which form a part of this application. Figure 1 shows the biosynthetic path of 1-butanol. The steps labeled "a", "b", "c", "d", "e", and "f" represent the substrate to product conversions described below. The following sequences are according to 37 C.F.R. 1.821-1.825 ("Requirements for Patent Applications Containing Descriptions of Nucleotide Sequences and / or Amino Acid Sequences - the Sequence Rules ") and are consistent with the World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the EPO and PCT sequence listing requirements (Rules 5.2 and 49.5 (a-bis), and Section 208 and Annex C of the Administrative Instructions.) The symbols and format used for the amino acid and nucleotide sequence data comply with the rules described in 37 CFR §1.822.

Table 1 Summary of SEQ ID Numbers of Genes and Proteins SEQ ID NOs: 17-44 are the nucleotide sequences of oligonucleotide primers used to amplify the genes of the biosynthetic pathway of 1-butanol. SEQ ID NOs: 45-72 are the nucleotide sequences of oligonucleotide primers used for sequencing. SEQ ID NOs: 73-75 are the nucleotide sequences of oligonucleotide primers used to construct the transformation vectors described in Example 9.

SEQ ID NO: 76 is the nucleotide sequence of the codon optimized CAC0462 gene, referred to herein as CaTER. SEQ ID NO: 77 is the codon optimized EgTER gene nucleotide sequence, referred to herein as EgTER (opt). SEQ ID NO: 78 is the nucleotide sequence of the optimized codon ald gene, referred to herein as ald (opt). SEQ ID NO: 79 is the nucleotide sequence of plasmid pFP988. SEQ ID NOs: 80-127, 160-185, and 190-207 are the nucleic acid sequences of cloning primers, sequencing, or selection of PCR used for the cloning, sequencing, or selection of the biosynthetic pathway genes of 1-butanol described herein, and are more fully described in Tables 4 and 5. SEQ ID NO: 156 is the nucleotide sequence of the cscBKA gene cluster. SEQ ID NO: 157 is the amino acid sequence of sucrose hydrolase (CscA). SEQ ID NO: 158 is the amino acid sequence of D-fructokinase (CscK). SEQ ID NO: 159 is the amino acid sequence of sucrose permease (CscB).

SEQ ID NO: 186 is the nucleotide sequence of the optimized codon tery gene described in example 17. SEQ ID NO: 187 is the amino acid sequence of butyl-CoA dehydrogenase (ter) encoded by the codon tery gene optimized (SEQ ID NO: 186). SEQ ID NO: 188 is the nucleotide sequence of the optimized codon aldy gene described in example 17. SEQ ID NO: 189 is the amino acid sequence of butyraldehyde dehydrogenase (ald) encoded by the optimized codon aldy gene ( SEQ ID NO: 188). SEQ ID NO: 208 is the nucleotide sequence of the model DNA used in Example 14.

Detailed Description of the Invention The present invention relates to methods for the production of 1-butanol using recombinant microorganisms. The present invention needs a number of commercial and industrial needs. Butanol is an important industrial chemical base with a variety of applications, where its potential as a fuel or fuel additive is particularly significant. Although only a four-carbon alcohol, butanol has an energy content similar to that of gasoline and can be mixed with any fossil fuel. Butanol is favored as a fuel or fuel additive since it produces only C02 and little or nothing S0X or N0X when burned in the standard internal combustion engine. Additionally butanol is less corrosive than ethanol, the most preferred fuel additive to date. In addition to its usefulness as a biofuel or fuel additive, butanol has the potential for shocking hydrogen distribution problems in the emerging fuel cell industry. Fuel cells today are plagued with safety interests associated with the transportation and distribution of hydrogen. Butanol can easily be reformed for its hydrogen content and can be distributed through existing gas stations with the purity required for either fuel cells or vehicles. Finally, the present invention produces ethanol from carbon sources derived from plants, avoiding the negative environmental impact associated with the standard petrochemical processes for the production of butanol. The following definitions and abbreviations will be used for the interpretation of the claims and the specification. The term "invention" or "present invention" as used herein is a non-limiting term and is not intended to refer to any single modality of the particular invention but encompasses all modalities possible as described in the specification and the claims. "ABE" is the abbreviation of the fermentation process of Acetone-Butanol-Ethanol. The term "biosynthetic pathway of 1-butanol" means the path of the enzyme to produce 1-butanol from acetyl-coenzyme A (acetyl-CoA). The term "acetyl-CoA acetyltransferase" refers to an enzyme that catalyzes the conversion of two molecules of acetyl-CoA to acetoacetyl-CoA and coenzyme A (CoA). The preferred acetyl-CoA acetyltransferases are acetyl-CoA acetyltransferases with substrate preferences (reaction in the forward direction) for a short chain acyl-CoA and acetyl-CoA and are classified as E.C. 2.3.1.9 [Enzyme Nomencla ture 1992, Academic Press, San Diego]; although, enzymes with a wider substrate range (E.C. 2.3.1.16) will also be functional. Acetyl-CoA acetyltransferases are available from a number of sources, for example, Escherichia coli (Gene Bank Nos: NP_416728 (SEQ ID NO: 129), NC_000913 (SEQ ID NO: 128), NCBI amino acid sequence (National Center of Biotechnology Information), nucleotide sequence NCBI), Clostridium um acetobutylicum (Genes Bank: NP_349476.1 (SEQ ID NO: 2), NC_003030 (SEQ ID NO: 1), NP_149242 (SEQ ID NO: 4 ), NC 001988 (SEQ ID NO: 3)), Bacillus subtilis (We of Gene Bank: NP_390297 (SEQ ID NO: 131), NC_000964 (SEQ ID NO: 130)), and Saccharomyces cerevisiae (We of Bank of Genes: NP_015297 (SEQ ID N0.133), NC_001148 (SEQ ID N0 .132)). The term "3-hydroxybutyryl-CoA dehydrogenase" refers to an enzyme that catalyzes the conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA. 3-Hydroxybutyryl-CoA dehydrogenases can be reduced to nicotinamide adenine-dependent dinucleotide (NADH), with a substrate preference for (S) -3-hydroxybutyryl-CoA or (R) -3-hydroxybutyryl-CoA and are classified as EC 1.1.1.35 and E.C. 1.1.1.30, respectively. Additionally, 3-hydroxybutyryl-CoA dehydrogenases can be reduced to nicotinamide adenine-dependent dinucleotide phosphate (NADPH), with a substrate preference for (S) -3-hydroxybutyryl-CoA or (R) -3-hydroxybutyryl-CoA and they are classified as EC 1.1.1.157 and E.C. 1.1.1.36, respectively. 3-Hydroxybutyryl-CoA dehydrogenases are available from a number of sources, eg, C. acetobutylicum (Genes Bank Nos: NP_349314 (SEQ ID NO: 6), NC_003030 (SEQ ID NO: 5)), B. subtilis (We from Bank of Genes: AAB09614 (SEQ ID NO: 135), U29084 (SEQ ID NO: 134)), Ralstonia eutropha (We from Gene Bank: YP_294481 (SEQ ID NO: 137), NC_007347 (SEQ ID NO. : 136)), and eutrophus alkalines (We from Bank of Genes: AAA21973 (SEQ ID NO: 139), J04987 (SEQ ID NO: 138)). The term "crotonane" refers to an enzyme that catalyzes the conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA and H20. The crotonanes may have a substrate preference for (S) -3-hydroxybutyryl-CoA or (R) -3-hydroxybutyryl-CoA and are classified as E.C. 4.2.1.17 and E.C. 4.2.1.55, respectively. The crotonases are available from a number of sources, for example, E. Coli (US Genes Bank: NP_415911 (SEQ ID NO: 141), NC_000913 (SEQ ID NO: 140)), C. acetobutylicum (US Genes Bank: NP_349318 (SEQ ID NO: 8), NC_003030 (SEQ ID NO: 6)), B. subtilis (We from Gene Bank: CAB13705 (SEQ ID NO: 143), Z99113 (SEQ ID NO: 142)), and Aeromonas caviae (We from Bank of Genes: BAA21816 (SEQ ID NO: 145), D88825 (SEQ ID NO: 144)). The term "butyryl-CoA dehydrogenase" refers to an enzyme that catalyzes the conversion of crotonyl-CoA to butyryl-CoA. The butyryl-CoA dehydrogenases may be either NADH-dependent or NADPH-dependent and are classified as E.C. 1.3.1.44 and E.C. 1.3.1.38, respectively. Butyryl-CoA dehydrogenases are available from a number of sources, for example, C. acetobutyli cum (Gene Bank Nos: NP_347102 (SEQ ID NO: 10), NC_003030 (SEQ ID NO: 9)), Euglena gracilis (Nos. from Gene Bank: Q5EU90 (SEQ ID NO: 147), AY741582 (SEQ ID NO: 146)), Streptomyces collinus (We of Gene Bank: AAA92890 (SEQ ID NO: 149), U37135 (SEQ ID NO: 148) ), and Streptomyces coelicolor (Nos de Banco de Genes: CAA22721 (SEC) ID N0.151), AL939127 (SEQ ID NO.150)). The term "butyraldehyde dehydrogenase" refers to an enzyme that catalyzes the conversion of butyryl-CoA to buriraldehyde, using NADH or NADPH as a co-factor. The butyraldehyde dehydrogenases with a preference for NADH are known as E.C. 1.2.1.57 and are available from, for example, Clostridi um beij erinckii (Gene Bank Nos: AAD31841 (SEQ ID N0: 12), AF157306 (SEQ ID NO: 11)) and C. acetobutylicum (Genes Bank Nos : NP_149325 (SEQ ID NO: 153), NC_001988 (SEQ ID NO: 152)). The term "butanol dehydrogenase" refers to an enzyme that catalyzes the conversion of butyraldehyde to 1-butanol, using either NADH or NADPH as co-factor. Butanol dehydrogenases are available from, for example, C. acetobutylicum (Gene Bank Nos: NP_149325 (SEQ ID NO: 153), NC_001988 (SEQ ID NO: 152); note: this enzyme possesses both aldehyde and alcohol dehydrogenase activity ); NP_349891 (SEQ ID NO: 14), NC_003030 (SEQ ID NO: 13), and NP_349892 (SEQ ID NO: 16), NC_003030 (SEQ ID NO: 15)) and E. coli (US Genes Bank: NP_417484 ( SEQ ID NO: 155), NC_000913 (SEQ ID NO: 154)). The term "an facultative anaerobe" refers to a microorganism that can grow in both aerobic and anaerobic environments. The term "carbon substrate" or "substrate of "fermentable carbon" refers to a carbon source capable of being metabolized by host organisms of the present invention and particularly carbon sources selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and one-carbon substrates or mixtures thereof. "Gene" refers to a fragment of nucleic acid that is capable of being expressed as a specific protein, optionally including regulatory sequences that precede (non-coding sequences 51) and follow (3 'non-coding sequences) to the coding sequence. The "native gene" refers to a gene as found in nature with its own regulatory sequences.The "chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together In nature, therefore, a chimeric gene can comprise regulatory sequences and coding sequences that can be erivan of different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a different way than that found in nature. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "heterologous" or "foreign" gene refers to a gene not normally found in the host organism, but which is introduced into the body Host by transfer. Foreign genes can include native genes inserted into a non-native organism, or chimeric genes. A "transgene" is a gene that has been introduced into the genome by a transformation procedure. As used herein the term "coding sequence" refers to a DNA sequence encoding a specific amino acid sequence. "Suitable regulatory sequences" refer to nucleotide sequences located in the 5 'direction (non-coding sequences 51), within, or in the 3' direction (3 'non-coding sequences) of a coding sequence, and which influence transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and hairpin structure. The term "promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3 'to a promoter sequence. The promoters can be derived in their entirety from different promoters found in nature, or still comprise segments of synthetic DNA. It is understood by those experts in the art that different promoters can direct the expression of a gene in different cell types or tissues, or at different stages of development, or in response to different physiological or environmental conditions. Promoters, which cause a gene to be expressed in most cell types, are often referred to as "constitutive promoters". It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been fully defined, DNA fragments of different lengths may have identical promoter activity. The term "operably linked" refers to the association of nucleic acid sequences in a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence when it is capable of affecting the expression of this coding sequence (ie, that the coding sequence is under the transcriptional control of the promoter). The coding sequences can be operably linked to regulatory sequences in sense or anti-sense orientation. The term "expression", as used herein, refers to the transcription and stable accumulation of sense (mRNA) or anti-sense RNA derived from the nucleic acid fragment of the invention. The expression can also refer to the translation of mRNA into a polypeptide. As used herein the term "transformation" refers to the transfer of a nucleic acid fragment in a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" or "recombinant" or "transformed" organisms. The terms "plasmid", "vector" and "cassette" refer to an extra chromosomal element that frequently carries genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA fragments. Such elements may be autonomously replicated sequences, integrating genome sequences, phage or single or double stranded nucleotide, DNA or RNA sequences, derived from any source, in which a number of nucleotide sequences have been linked or recombined into one construct only one which is capable of introducing a promoter fragment and DNA sequence for a gene product selected together with the appropriate 3 'untranslated sequence in a cell. The "transformation cassette" refers to a specific vector that contains a foreign gene and that has elements besides the foreign gene that facilitate the transformation of a particular host cell. "Cassete de expresión" refers to a specific vector that contains a foreign gene and that has elements besides the foreign gene that allow the improved expression of this gene in a foreign host. As used herein the term "codon degradation" refers to the nature in the genetic code that allows variation of the nucleotide sequence without effecting the amino acid sequence of a coded polypeptide. The skilled artisan is very aware of the "codon of choice" exhibited by a specific host cell in use of nucleotide codons to specify a given amino acid. Therefore, when a gene for enhanced expression is synthesized in a host cell, it is desirable to design the gene so that its codon usage frequency approximates the frequency of the preferred codon use of the host cell. The term "optimized codon" when referring to genes or regions encoding nucleic acid molecules for transformation of several hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect codon usage typical of the host organism without altering the polypeptide encoded by the DNA. The molecular cloning and standard recombinant DNA techniques used herein are well known in the art and are described by Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Labora tory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) (after "Maniatis"); and by Silhavy, T.J., Bennan, M.L. and Enquist, L.W., Experiments with the Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1984); and by Ausubel, F. M. et al., Curren t Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987).

The Biosynthetic Pathway of 1-Butanol Microorganisms using carbohydrate use the trajectory of Embden-Meyerhof-Parnas (EMP), the trajectory of Entner-Doudoroff and the pentose phosphate cycle as the central metabolic pathways to provide energy and cellular precursors for growth and maintenance. These trajectories have in common the intermediate glyceraldehyde-3-phosphate and, finally, pyruvate, is formed directly or in combination with the EMP path. Subsequently, pyruvate is transformed to acetyl-coenzyme A (acetyl-CoA) via a variety of media, including reaction with the complex of pyruvate dehydrogenase, pyruvate-formate lyase, and pyruvate-ferredoxin oxide-reductase. Acetyl-CoA serves as a key intermediary, for example, in the generation of fatty acids, amino acids and secondary metabolites. The combined reactions of conversion of sugar to acetyl-CoA produce energy (e.g., adenosine-5'-triophosphate, ATP) and reduced equivalents (e.g., reduced nicotinamide adenine dinucleotide, NADH, and reduced nicotinamide adenine dinucleotide phosphate, NADPH ). NADH and NADPH must be recycled to their oxidized forms (NAD + and NADPp respectively). In the presence of inorganic electron acceptors (eg, 02, N03 ~ and S042"), the reduced equivalents can be used to increase the energy well, alternatively, a reduced carbon by-product can be formed. ethanol and 1-butanol resulting from carbohydrate fermentation are examples of the latter.This invention makes possible the production of 1-butanol from carbohydrate sources with recombinant microorganisms by providing a biosynthetic pathway of complete 1-butanol from acetyl-CoA to 1-butanol, as shown in figure 1. This biosynthetic trajectory, generally lacking in the microbial community due to the absence of genes or the lack of appropriate gene regulation, comprises the following conversions from substrate to product: a) acetyl- CoA to acetoacetyl-CoA, as catalyzed for example by acetyl-CoA acetyltransferase; b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA, as catalyzed for example by 3-hydroxybutyryl-CoA dehydrogenase; c) 3-hydroxybutyryl-CoA to crotonyl-CoA, as catalyzed for example by crotonane; d) Crotonyl-CoA to butyryl-CoA, as catalyzed for example by butyryl-CoA dehydrogenase; e) butyryl-CoA to butyraldehyde, as catalyzed for example by butyraldehyde dehydrogenase; and f) Butyraldehyde to 1-butanol, as catalyzed for example by butanol dehydrogenase. The trajectory does not require ATP and generates NAD + and / or NADPp, therefore, it balances with the central metabolic pathways that generate acetyl-CoA. The ability of natural organisms to produce 1-butanol by fermentation is rare and is most prominently exemplified by Clos tridi um beij erínckií and Clostridíum acetobutylicum. The organization of genes and gene regulation by Clostridium um acetobutyli cum has been described (L. Girbal and P. Soucaille, Trends in Biotechnology 216: 11-16 (1998)). However, many of these enzyme activities are also associated with alternate trajectories, for example, hydrocarbon utilization, fatty acid oxidation, and polyhydroxyalkanoate metabolism. Therefore, in the proportion of a recombinant pathway from acetyl-CoA to 1-butanol, there are a number of choices to meet the stages of individual reactions, and the person skilled in the art will be able to use publicly available sequences to construct the relevant trajectories. A listing of a representative number of genes known in the art and useful in the construction of the biosynthetic pathway of 1-butanol are listed below in Table 2.

Table 2 Sources of Trajectory Genes of 1-Butanol Microbial hosts for the production of 1-Butanol Microbial hosts for the production of 1-butanol can be selected from bacteria, cyanobacteria, filamentous fungi and yeasts. The microbial host used for the production of 1-butanol is preferably tolerant to 1-butanol so that the yield is not limited by the toxicity of butanol. Microbes that are metabolically active at high titers of 1-butanol are not well known in the art. Although butanol-tolerant mutants have been isolated from Solventogenic Clostridia, little information is available regarding butanol tolerance of other bacterial strains. potentially useful Most studies on the comparison of alcohol tolerance in bacteria suggest that butanol is more toxic than ethanol (de Cavalho et al., Microsc Res. Tech. 64: 215-22 (2004) and Kabelitz et al. , FEMS Microbiol, Lett 220: 223-227 (2003)). Tomas et al. (J. Bacteriol 186: 2006-2018 (2004)) reports that the yield of butanol during fermentation in Clostridium acetobutyli cum can be limited by the toxicity of butanol. The primary effect of butanol on Clostridium to ketobutylicum is the interruption of membrane functions (Hermann et al., Appi r Environ.Microbiol.50: 1238-1243 (1985)). The microbial hosts selected for the production of 1-butanol are preferably tolerant to 1-butanol and are capable of converting carbohydrates to 1-butanol. Criteria for the selection of suitable microbial hosts include the following: intrinsic tolerance to 1-butanol, high rate of glucose utilization, availability of genetic tools for gene manipulation, and the ability to generate stable chromosomal alterations. Suitable host strains with a tolerance for 1-butanol can be identified by selection based on the intrinsic tolerance of the strain. The intrinsic tolerance of microbes to 1-butanol can be measured by determining the concentration of 1-butanol which is responsible for 50% of inhibition of the growth rate (IC50) when it grows in a minimal medium. IC50 values can be determined using methods known in the art. For example, the microbes of interest can grow in the presence of various amounts of 1-butanol and the growth rate is monitored by measuring the optical density at 600 nanometers. The folded time can be calculated from the logarithmic part of the growth curve and used as a measure of the growth rate. The concentration of 1-butanol which produces 50% growth inhibition can be determined from a graph of the percentage of growth inhibition against the concentration of 1-butanol. Preferably, the host strain should have an IC50 for 1-butanol greater than about 0.5% w / v. The microbial host for the production of 1-butanol should also use glucose at a high speed. Most microbes are capable of using carbohydrates. However, certain environmental microbes can not use carbohydrates for high efficiency, and therefore could not be suitable hosts. The ability to genetically modify the host is essential for the production of any recombinant microorganism. The technology mode of gene transfer can be by electroporation, conjugation, transduction or natural transformation. A wide Interfering plasmid-conjugated hosts and drug-resistant markers is available. The cloning vectors are adapted to host organisms based on the nature of the antibiotic resistance markers that can function in this host. The microbial host also has to be manipulated to inactivate the competition trajectories for carbon flux by suppressing several genes. This requires the availability of either transposons to direct the inactivation or chromosomal integration vectors. Additionally, the production host should be amenable to chemical mutagenesis so that mutations to improve tolerance to intrinsic 1-butanol can be obtained. Based on the criteria described above, suitable microbial hosts for the production of 1-butanol include, but are not limited to, members of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, La ctobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula and Sa ccharomyces. Preferred hosts include: Escherichia coli, Al caligenes eutrophus, Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas putida, Lactobacillus plan tarum, In terococcus faeci um, In terococc? S gallinari um, Enterococcus faecalis, Bacillus subtílis and Saccharomyces cerevisiae.

Production Host Construction Recombinant organisms containing the necessary genes that will encode the enzymatic path for the conversion of a fermentable carbon substrate to 1-butanol can be constructed using techniques well known in the art. In the present invention, the genes encoding the enzymes of the biosynthetic pathway of 1-butanol, ie acetyl-CoA acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonane, butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase, and butanol dehydrogenase, are They can isolate from several sources, as described above. Methods for obtaining desired genes from a bacterial genome are common and well known in the molecular biology art. For example, if the gene sequence is known, suitable genomic libraries can be created by restriction of endonuclease digestion and can be selected with probes complementary to the desired gene sequence. Once the sequence is isolated, the DNA can be amplified using standard primer directed amplification methods such as polymerase chain reaction (Mullis, U.S. Patent No. 4,683,202) to obtain suitable DNA quantities for the transformation using appropriate vectors. Codon optimization tools for expression in a heterologous host are readily available. Some tools for codon optimization are available based on the GC content of the host organism. The GC content of some exemplary microbial hosts is given in Table 3.

Table 3 GC Content of Microbial Hosts Once the relevant pathway genes are identified and isolated they can be transformed into suitable expression hosts by means well known in the art. Vectors or cassettes useful for the transformation of a variety of host cells are common and commercially available from companies such as EPICENTRE® (Madison, Wl), Invitrogen Corp. (Carisbad, CA), Stratagene (La Jolla, CA), and New England Biolabs, Inc. (Beverly, MA). Typically, the vector or cassette contains sequences that direct the transcription and translation of the relevant gene, a selectable marker, and sequences that allow autonomous replication or chromosomal integration. Suitable vectors comprise a 5 'region of the gene which hosts transcriptional initiation controls and a 3' region of the ADB fragment which controls the transcriptional termination. Both control regions can be derived from homologous genes to the transformed host cell, although it will be understood that such control regions can also be derived from genes that are not native to the specific species chosen as the production host. The initiation control regions or promoters, which are useful to drive the expression of the relevant pathway coding regions in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genetic elements is suitable for the present invention including, but not limited to, CYCl, HIS3, GAL1, GALI O, ADH1, PGK, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI , CUP1, FBA, GPD, and GPM (useful for expression in Saccharomyces); A0X1 (useful for expression in Pichia); and lac, ara, tet, trp, IPL, IPR, TI, tac, and trc (useful for expression in Escheri chia coli, Alcaligenes, and Pseudomonas); the amy, apr, npr promoters and several phage promoters useful for expression in Bacill us subtilis, Bacill us li cheniformis, and Paeniba cill us macerans; nisA (useful for expression of Gram-positive bacteria, Eichenbaum et al., Appl. Environ Microbiol. 64 (8): 2763-2769 (1998)); and the synthetic Pll promoter (useful for expression in Lactobacillus planum tarum, Rud et al., My Crobiology 152: 1011-1019 (2006)). Termination control regions can also be derived from several genes native to the preferred hosts. Optionally, a termination site may be unnecessary, however, it is more preferred if it is included. Certain vectors are capable of replicating in a wide range of host bacteria and can be transferred by conjugation. The complete and annotated sequence of pRK404 and three related vectors pRK437, pRK442, and pRK442 (H) are available. These derivatives have proven to be valuable tools for genetic manipulation in Gram-negative bacteria (Scott et al., Plasmid 50 (l): 74-79 (2003)). Various plasmid derivatives of broad-range host Inc P4 plasmid RSF1010 are also available with promoters that can function in a range of Gram-negative bacteria. Plasmid pAYC36 and pAYC37, have promoters active in conjunction with multiple cloning sites to allow heterologous gene expression in Gram-negative bacteria. Chromosomal gene replacement tools are also widely available. For example, a thermosensitive variant of the broad host interval replicon pWVIOl has been modified to construct a plasmid pVE6002 which can be used to create gene replacement in a range of Gram-positive bacteria (Maguin et al., J. Bacteriol. (17): 5633-5638 (1992)). Additionally, in vitro transposomes are available to create random mutations in a variety of genomes from commercial sources such as EPICENTRE®. The expression of the biosynthetic pathway of 1-butanol in several preferred microbial hosts is described in more detail below.

Expression of the biosynthetic pathway of 1-butanol in E. coli Vectors or cassettes useful for the transformation of E. coli are common and commercially available from the companies listed above. For example, genes from the biosynthetic pathway of 1-butanol can be isolated from several strains of Clostridium um, cloned into a modified pUC19 vector and transformed into E. coli.

NM522, as described in Example 11. Expression of the biosynthetic pathway of 1-butanol in several other strains of E. coli is described in example 13.

Expression of the biosynthetic trajectory of 1-butanol in Rhodococcus erythropolis A series of vector transporters of E. coli - Rhodococcus is available for expression in R. erythropolis, including, but not limited to pRhBR17 and pDA71 (Kostichka et al., Appl. Microbiol. Biotechnol 62: 61-68 (2003)). Additionally, a series of promoters is available for heterologous gene expression in R. erythropolis (see for example Nakashima et al., Appl.

Send. Microbiol. 70: 5557-5568 (2004), and Tao et al., Appl. Microbiol. Biotechnol. 2005, DOI 10.1007 / s00253-005-0064). The disruption of the target gene of chromosomal genes in R. Erythropolis can be created using the method described by Tao et al., supra, and Brans et al. (Appl. Envir. Microbiol. 66: 2029-2036 (2000)). The heterologous genes required for the production of 1-butane, as described above, can be cloned initially into pDA71 or PRhBR71 and transformed into E. coli. The vectors can then be transformed into JR. erythropolis by electroporation, as described by Kostichka et al., supra. The recombinants can grow in Synthetic medium containing glucose and the production of 1-butanol can be continued using methods known in the art.

Expression of the biosynthetic pathway of 1-butanol in Bacillus Subtilis Methods for gene expression and creation of mutations in B. Subtilis are also well known in the art. For example, genes from the biosynthetic pathway of 1-butanol can be isolated from several strains of Clostridium, cloned into a modified pUC19 vector and transformed into Ba cill us subtilis BE1010, as described in example 12. Additionally, all six genes of the 1-biosynthetic trajectory can be divided into two operons for expression, as described in example 14. The first three genes of the trajectory (thl, hbd, and crf) were integrated into the chromosome of Ba cillus subtilis BE1010 (Payne and Jackson, J. Bacteriol 173: 2278-2282 (1991)). The last three genes (EgTER, ald, and bdhB) were cloned into expression plasmids and transformed into the Bacill us strain carrying the integrated 1-butanol genes.

Expression of the biosynthetic pathway of 1-butanol in Bacillus licheniformis Most plasmids and vectors transporters that replicate in B. subtilis can be used to transform B. licheniformis either by protoplast transformation or electroporation. For example, the genes required for the production of 1-butanol can be cloned into derivatives of plasmids pBE20 and PEB60 (Nagarajan et al., Gene 114: 121-126 (1992)). The methods to transform B. li cheniformis are known in the art (for example see Fleming et al. Appl. Environ. My crobiol , 61 (11): 3775-3780 (nineteen ninety five) ) . The plasmids constructed for expression in B. subtilis can also be transformed into B. licheniformis to produce a recombinant microbial host that produces 1-butanol.

Expression of the biosynthetic pathway of 1-butanol in Paenibacillus macerans The plasmids can be constructed as described above for expression in B. subtilis and used to transform Paenibacillus macerans by protoplast transformation to produce a recombinant microbial host that produces 1-butanol.

Expression of the biosynthetic pathway of 1-butanol in Alcaligenes (Ralstonia) eutrophus The methods for gene expression and creation of mutations in Ralstonia eutrophus are known in the art (see for example Taghavi et al., Appl. Environ. Mi crobiol., 60 (10): 3585-3591 (1994)). Genes for the biosynthetic pathway of 1-butanol can be cloned into any of the broad range of host vectors described above, and electroporated to generate recombinants that produce 1-butanol. The trajectory of polyhydroxy butyrate in Ralstonia has been described in detail and a variety of genetic techniques to modify the genome Ralstonia eutrophus is known, and those tools can be applied to modify the biosynthetic pathway of 1-butanol.

Expression of the biosynthetic pathway of 1-butanol in Pseudomonas putida Methods for gene expression in Pseudomonas putida are known in the art (see, for example, Ben-Bassat et al., U.S. Patent No. 6,586,229, which is incorporated in the present for reference). For example, butanol pathway genes can be inserted into pPCUld and this ligated DNA can be electroporated into competent Pseudomonas putida DOT-Tl C5aARl cells to generate recombinants that produce 1-butanol.

Expression of the biosynthetic pathway of 1-butanol in Saccharomyces cerevisiae The methods for the expression of genes in Saccharomyces cerevisiae are known in the art (see for example Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San. Diego, CA) Gene expression in yeast typically requires a promoter, followed by the gene of interest, and a transcriptional terminator. A number of yeast promoters can be used in the construction of expression cassettes for genes encoding the biosynthetic pathway of 1-butanol, including, but not limited to constitutive promoters FBA, GPD, and GPM, and inducible promoters GAL1, GALIO , and CUP1. Suitable transcriptional terminators include, but are not limited to FBAt, GPDt, GPMt, ERGIOt, and GALlt. Suitable promoters, transcriptional terminators, and genes from the biosynthetic pathway of 1-butanol can be cloned into 2 micron (2 μ) plasmids of yeast, as described in example 17.

Expression of the biosynthetic pathway of 1-butanol in Lactobacillus planum tarum The genus Lactobacillus belongs to the Lactobacillales family and many plasmids and vectors used in the transformation of Bacillus subtilis and Streptococcus can be used for lactobacillus. Non-limiting examples of suitable vectors include pAMßl and derivatives thereof (Renault et al., Gene 183: 175-182 (1996); and O'Sullivan et al., Gene 137: 227-231 (1993)); pMBB1 and pHW800, a derivative of pMBB1 (Wyckoff et al., Appl. Environ Microbiol., 62: 1481-1486 (1996)); pMG1, a conjugating plasmid (Tanimoto et al., J. Bacteriol 184: 5800-5804 (2002)); pNZ9520 (Kleerebezem et al., Appl. Environ Microbiol. 63: 4581-4584 (1997)); pAM401 (Fujimoto et al., Appl. Environ Microbiol. 67: 1262-1267 (2001)); and pAT392 (Arthur et al., Antimicrob, Agents Chemother, 38: 1899-1903 (1994)). Several Lactobacillus plantarum plasmids have also been reported (eg, van Kranenburg R, Golic N, Bongers R, Leer RJ, de Vos WM, Siezen RJ, Kleerebezem M. Appl. Environ.Microbiol., 2005 Mar; 71 (3): 1223-1230). For example, the expression of the biosynthetic pathway in Lactobacillus plantarum is described in example 18.

Expression of the biosynthetic trajectory of 1-butanol in Enterococcus faecium, Enterococcus gallinarium, and Enterococcus faecalis The genus Enterococcus belongs to the Lactobacillales family and many plasmids and vectors used in the transformation of Lactobacillus, Bacillus subtilis, and Streptococcus can be used for Enterococcus. Non-limiting examples of suitable vectors include pAMßl and derivatives thereof (Renault et al., Gene 183: 175-182 (1996); and O'Sullivan et al., Gene 137: 227-231 (1993)); pMBBl and pHW800, a derivative of pMBBl (Wyckoff et al., Appl. Environ Microbiol. 62: 1481-1486 (1996)); pMG1, a conjugating plasmid (Tanimoto et al., J. Bacteriol 184: 5800-5804 (2002)); pNZ9520 (Kleerebezem et al., Appl. Environ. My Crobiol 63: 4581-4584 (1997)); pAM401 (Fujimoto et al., Ap 1. Environ Microbiol. 67: 1262-1267 (2001)); and pAT392 (Arthur et al., An timi crob Agents Chemother, 38: 1899-1903 (1994)). The expression vectors for E. faecalis using the nisA gene of Lactococcus can also be used (Eichenbaum et al., Appl. Environ Microbiol. 64: 2763-2769 (1998).) In addition, the gene replacement vectors on the E. faecium chromosome can be used ( Nallaapareddy et al., Appl. Environ Microbiol., 72: 334-345 (2006).) For example, the expression of the biosynthetic pathway of 1-butanol in En terococcus faecalis is described in example 19.

Fermentation Means The fermentation media in the present invention should contain suitable carbon substrates. Suitable substrates may include but are not limited to monosaccharides such as glucose and fructose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures of renewable feedstock such as serum permeate. cheese, corn infusion liquor, beet molasses sugar, and barley malt. Additionally the carbon substrate can also be substrates of a carbon such as carbon dioxide, or methanol for which the metabolic conversion into key biochemical intermediates has been demonstrated. In addition to one and two carbon substrates, methylotrophic organisms are also known to utilize a number of other carbon-containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeast is known to utilize the carbon of methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth Cl Compd., [Int. Symp.], 7th (1993), 415-32. : Murrell, J. Collin, Kelly, Don P. Publisher: Intercept, Andover, UK). Similarly, several species of Candida will metabolize alanine or oleic acid (Suiter et al., Arch. Microbiol., 153: 485-489 (1990)). It is therefore contemplated that the carbon source used in the present invention may comprise a wide variety of carbon-containing substrates and will only be limited by the choice of the organism. Although it is contemplated that all mentioned carbon substrates and mixtures thereof are suitable in the present invention, the preferred carbon substrates are glucose, fructose, and sucrose. In addition to an appropriate carbon source, the fermentation media must contain minerals, salts, cofactors, buffers and other suitable components, known to those skilled in the art, suitable for the growth of crops and promotion of the enzymatic path necessary for the production of 1-butanol.

Culture Conditions Typical cells grow at a temperature in the range of about 25 ° C to about 40 ° C in an appropriate medium. Suitable growth media in the present invention are common commercially prepared media such as Luria Bertani broth (LB), Sabouraud dextrose broth (SD) or yeast medium broth (YM). Other synthetic or defined growth media can also be used and the appropriate medium for growth of the particular microorganism will be known to one skilled in the art of fermentation science or microbiology. The use of known agents to modulate the catabolic repression directly or indirectly, for example, adenosine 2 ': 3'-cyclic monophosphate, can also be incorporated into the fermentation medium. The pH ranges suitable for fermentation are between pH 5.00 to pH 9.0, where pH 6.0 to pH 8.0 is preferred as the initial condition. Fermentations can be carried out under aerobic or anaerobic conditions where the conditions anaerobic or microaerobic are preferred. The amount of 1-butanol produced in the fermentation medium can be determined using a number of methods known in the art, for example, high performance liquid chromatography (HPLC) or gas chromatography (GC).

Industrial Batch Fermentation and Industrial The present process uses a batch fermentation method. A classic batch fermentation is a closed system where the composition of the medium is adjusted at the beginning of fermentation and is not subject to artificial alterations during fermentation. Therefore, at the beginning of fermentation the medium is inoculated with the desired organism or organisms, and fermentation is allowed to occur by adding anything to the system. Typically, however, a "batch" fermentation is batch with respect to the addition of the carbon source and attempts are often made in the control of factors such as pH and oxygen concentration. In batch systems the metabolite and biomass compositions of the system change constantly until the fermentation stops. Within cells of batch cultures are moderated through a static label phase to a high growth logarithm phase and finally to a stationary phase where the rate of growth is slowed or stopped. If they are not treated, the cells in the stationary phase will eventually die. Cells in logarithmic phase are generally responsible for the volume of production of the final product or intermediary. A variation of the standard batch system is the Batch Feed system. The Batch Feeding fermentation processes are also suitable in the present invention and comprise a typical batch system with the exception that the substrate is added in increments when the fermentation progresses. Batch Feeding systems are useful when catabolic repression is able to inhibit cell metabolism and where it is desirable to have limited amounts of substrate in the media. The measurement of the current substrate concentration in batch feed systems is difficult and is therefore estimated on the basis of changes in measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as C02. Batch fermentations and Batch Feeding are common and well known in the art and examples can be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, MA ., or Deshpande, Mukund V., Appl. Biochem. Biotechnol. , 36: 227, (1992), incorporated herein by reference. Although the present invention is carried out in batch mode it is contemplated that the method could be adaptable to continuous fermentation methods. Continuous fermentation is an open system where a defined fermentation medium is continuously added to a bioreactor and an equal amount of conditioned media is simultaneously removed for processing. Continuous fermentation generally keeps the cultures at a constant high density where the cells are mainly in logarithmic phase growth. Continuous fermentation allows the modulation of a factor or any number of factors that affect cell growth or concentration of final product. For example, a method will maintain a limited nutrient such as nitrogen level or carbon source at a fixed rate and allow all other parameters to moderate. In other systems a number of factors that affect growth can be altered continuously while the cell concentration, measured by the turbidity of the medium, remains constant. The continuous systems strive to maintain growth conditions in a stable state and consequently the cell loss due to the medium that is extracted must be balanced against the rate of cell growth in the fermentation. The methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the fermentation rate of product are well known in the industrial microbiology technique and a variety of methods are used. detailed by Brock, supra. It is contemplated that the present invention can be practiced using either batch, batch or continuous processes and that any known mode of fermentation could be suitable. Additionally, it is contemplated that the cells can be immobilized on a substrate as full cell catalysts and subjected to fermentation conditions for production of 1-butanol.

Methods of Isolation of 1-butanol from the Fermentation Medium The bioproduced 1-butanol can be isolated from the fermentation medium using methods known in the art. For example, the solids can be removed from the fermentation medium by centrifugation, filtration, decantation, or the like. Then, 1-butanol can be isolated from the fermentation medium, which has been treated to remove solids as described above, using methods such as distillation, liquid-liquid extraction, or membrane-based separation. Because 1-butanol forms a low boiling point, azeotropic mixing with water, distillation can only be used to separate the mixture to its azotropic composition. The distillation can be used in combination with another separation method to obtain separation around the azeotrope. The methods that can be used in combination with distillation to isolate and purify 1-butanol include, but are not limited to, decanting, liquid-liquid extraction, adsorption, and membrane-based techniques. Additionally, 1-butanol can be isolated using azeotropic distillation using a solvent (see for example Doherty and Malone, Conceptual Design of Distiltion Systems, McGraw Hill, New York, 2001). The 1-butane-water mixture forms a heterogeneous azeotrope so that distillation can be used in combination with decanting to isolate and purify 1-butanol. In this method, the 1-butanol containing fermentation broth is distilled to almost the azeotropic composition. Then, the azeotropic mixture is condensed, and the 1-butanol is separated from the fermentation medium by decantation. The decanted aqueous phase can be returned to the first distillation column as reflux. The decanted organic phase rich in 1-butanol can be further purified by distillation in a second distillation column. The 1-butanol can also be isolated from the fermentation medium using liquid-liquid extraction in combination with distillation. In this method, the 1-butanol is extracted from the fermentation broth using liquid-liquid extraction with a suitable solvent. The organic phase containing 1-butanol is then distilled to remove the 1-butanol from the solvent. Distillation in combination with adsorption can also be used to isolate 1-butanol from the medium of fermentation. In this method, the fermentation broth containing the 1-butanol is distilled to almost the azeotropic composition and then the remaining water is removed by the use of an adsorbent, such as molecular sieves (Aden et al., Lignocell ulosic Biomass to Ethanol Process Design and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzyme tic Hydrolysis for Corn Stover, Report NREL / TP-510-32438, National Renewable Energy Laboratory, June 2002). Additionally, distillation in combination with pervaporation can be used to isolate and purify 1-butanol from the fermentation medium. In this method, the fermentation broth containing the 1-butanol is distilled to almost the azeotropic composition, and then the remaining water is removed by pervaporation through a hydrophilic membrane (Guo et al., J. Membr. Sci. 245 , 199-210 (2004)).

EXAMPLES The present invention is further defined in the following examples. It should be understood that these examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these examples, one skilled in the art can find out the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make several changes and modifications of the invention to adapt it to various uses and conditions.

GENERAL METHODS The techniques of molecular cloning and standard recombinant DNA used in the examples are well known in the art and are described by Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Labora tory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experimen ts wi th Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M. et al. , Current Protocols in Molecular Biology, pub. From Greene Publishing Assoc, and Wiley-lnterscience (1987). Suitable materials and methods for the manufacture and growth of bacterial cultures are well known in the art. Techniques suitable for use in the following examples can be found as described in Manual of Methods for General Bacteriology (Phillipp Gerhardt, RGE Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds), American Society for Microbiology, Washington, DC. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, MA (1989).

All reagents, restriction enzymes and materials used for the growth and maintenance of bacterial cells were obtained from Aldrich Chemicals (Milwaukee, WI), BD Diagnostic Systems (Sparks, MD), Life Technologies (Rockville, MD), or Sigma Chemical Company (St. Louis, MO) unless otherwise specified. The oligonucleotide primers used for cloning in the following examples are given in Table 4. The primers used to sequence or select the cloned genes are given in Table 5. All the oligonucleotide primers were synthesized by Sigma-Genosys (Woodlands, TX).

Table 4 Oligonucleotide Cloning Primers Table 5 Methods for Determining the Concentration of 1-Butanol in Culture Media The concentration of 1-butanol in the culture media can be determined by a number of methods known in the art. For example, a specific high-performance liquid chromatography (HPLC) method uses a Shodex SH-1011 column with a Shodex SH-G precolumn, both purchased from Waters Corporation (Milford, MA), with refractive index (IR) detection. . Chromatographic separation was achieved using 0.01 M H2SO4 as the mobile phase with a flow rate of 0.5 ml / min and a column temperature of 50 ° C. The 1-butanol had a retention time of 52.8 min under the conditions used. Alternatively, the Gas chromatography (GC) methods are available. For example, a specific GC method uses an HP-INNOWax column (30m x 0.53mm di, 1μm film thickness, Agilent Technologies, Wilmington, DE), with a flame ionization detector (FID). The carrier gas was helium at a flow rate of 4.5 ml / min, measured at 150 ° C with constant head pressure; fractionated injector was 1:25 at 200 ° C; Oven temperature was 45 ° C for 1 min, 45 to 220 ° C at 10 ° C / min, and 220 ° C for 5 min; and the FID detection was used at 240 ° C with 26 ml / min of helium gas. The retention time of 1-butanol was 5.4 min. A similar GC method using a Varian CP-WAX 58 (FFAP) CB column (25m x 0.25mm di x 0.2 μm film thickness, Varian, Inc., Palo Alto, CA) was also used. The meaning of the abbreviations is as follows: "s" means second (s), "min" means minute (s), "h" means time (s), "psi" means pounds per square inch, "nm" means nanometers, "d" means day (s) , "μL" means microliter (s), "mL" means milliliter (s), "L" means liter (s), "mm" means millimeter (s), "nm" means nanometer, "mM" means millimolar, " M "means molar," mmol "means millimole (s)," μmol "denotes micromol (s)," g "means gram (s)," μg "means microgram (s) and" ng "means nanogram (s), "PCR" stands for polymerase chain reaction, "OD" means density optics, "OD6oo" means the optical density measured at a wavelength of 600 nm, "OD550" means the optical density measured at a wavelength of 550 nm, "kDa" means kilodaltons, "g" means the gravitation constant , "rpm" means revolutions per minute, "bp" means base pair (s), "kbp" means kilobase pair (s), "% p / v" means percent weight / volume, "% v / v" means one hundred volume / volume, "HPLC" means high performance liquid chromatography, and "GC" means gas chromatography.

EXAMPLE 1 Cloning and Expression of Acetyl-CoA Acetyltransferase The purpose of this example was to express the enzyme acetyl-CoA acetyltransferase, also referred to herein as acetoacetyl-CoA thiolase, in E. coli The thlA gene of acetoacetyl-CoA thiolase was cloned from C. acetobutylicum (ATCC 824) and expressed in E. coli. The thlA gene was amplified from C-gen acetobutylicum (ATCC 824) using PCR, resulting in a 1.2 kbp product. Genomic DNA from Clostridium um to ketobutylicum (ATCC 824) either purchased from the Type Culture Collection American (ATCC, Manassas, VA) or was isolated from cultures of Clostrodíum acetobutylicum (ATCC 824), as described later.

Genomic DNA from Clostridium um to ketobutylicum (ATCC 824) was prepared from anaerobically grown cultures. The Clostridium strain was grown in 10 ml of Clostridial growth medium (Lopez-Contreras et al., Appl. Env.Microbiol. 69 (2), 869-877 (2003)) in fixed and hooked 100 ml war serum bottles ( Warico Glass Inc., Vineland, NJ) in an anaerobic chamber at 30 ° C. The inoculum was a single colony of a 2X YTG plate (Kishii, et al., Thymicrobial Agen ts &Chemotherapy, 47 (1), 77-81 (2003)) grown on a 2.5 L AnaeroPakMR MGC (Mitsubishi Gas Chemical America Inc, New York, NY) at 37 ° C. Genomic DNA was prepared using the Gentra Puregene® kit (Gentra Systems, Inc., Minneapolis, MN; catalog No. D-6000A) with modifications to the manufacturer's instruction (Wong et al., Curren t Microbiology, 32, 349- 356 (1996)). The thlA gene was amplified from Clostridium acetobutylicum genomic DNA (ATCC 824) by PCR using primers N7 and N8 (see Table 4), given as SEQ ID NOs: 21 and 22, respectively. Other PCR amplification reagents were supplied in the manufacturer's kit for example, Kod HiFi DNA Polymerase (Novagen Inc., Madison, Wl; catalog No. 71805-3) and used according to the manufacturer's protocol. The amplification was performed on a Thermocycler GeneAmp 9700 DNA (PE Applied Biosystems, Foster City, CA). For expression studies the technology of Cloning Gateway cloning technology (Invitrogen Corp., Carisbad, CA) was used. The pENTR / SD / D-TOPO input vector allowed for directional cloning and provided a Shine-Dalgarno sequence for the gene of interest. The target vector pDEST14 used a T7 promoter for the expression of the unbranded gene. The forward primer incorporated four bases (CACC) immediately adjacent to the translational start codon to allow directional cloning in pENTR / SD / D-TOPO (Invitrogen) to generate the pENTRSDD-TOPOthlA plasmid. The pENTR construct was transformed into E cells. ToplO coli (Invitrogen) and placed on a plate according to the manufacturer's recommendations. The transformants were grown overnight and the plasmid DNA was prepared g the QIAprep Spin Miniprep kit (Qiagen, Valencia, CA; catalog No. 27106) according to the manufacturer's recommendations. The clones were subjected to sequencing with forward and reverse M12 primers (see Table 5), given as SEQ ID NOs: 45 and 46, respectively, to confirm that the genes were inserted in the correct orientation and to confirm the sequence. The additional sequencing primers, N7SeqF1 and N7SeqR1 (see Table 5), given as SEQ ID Nos: 47 and 48, respectively, were necessary to completely sequence the PCR product. The nucleotide sequence of the open reading structure (ORF) for this gene and the predicted amino acid sequence of the enzyme are given as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. To create an expression clone, the thlA gene was transferred to the pDEST 14 vector by recombination to generate pDEST14thlA. The pDEST14thlA vector was transformed into BL21-A1 cells. The transformants were inoculated in LB medium supplemented with 50 μg / mL ampicillin and grown overnight. An aliquot of the culture overnight was used to inoculate 50 mL of LB supplemented with 50 μg / mL of ampicillin. The culture was incubated at 37 ° C with shaking until the OD60o reached 0.6-0.8. The culture was divided into two 25 mL cultures and arabinose was added to one of the flasks to a final concentration of 0.2% by weight. The negative control flask was not induced with arabinose. The flasks were incubated for 4 h at 37 ° C with shaking. The cells were harvested by centrifugation and the pellets of cells were resuspended in 50 mM MOPS buffer pH 7.0. The cells were interrupted either by sonication or by passage through a French Pressure Cell. The whole cell lysate was centrifuged yielding the supernatant or cell-free extract and the pellet or the insoluble fraction. An aliquot of each fraction (whole-cell lysate, cell-free extract and insoluble fraction) was resupended in SDS charge buffer (MES) (Invitrogen), heated at 85 ° C for 10 min and subjected to SDS-PAGE analysis (NuPAGE 4- 12% Bis-Tris Gel, catalog no. NP0322Box, Invitrogen). A protein of the expected molecular weight of approximately 41 kDa, as deduced from the nucleic acid sequence, was present in the induced culture but not in the non-induced control. The activity of acetoacetyl-CoA thiolase in the cell-free extracts was measured as the degradation of an Mg2 + -acetoacetyl-CoA complex monitoring the decrease in absorbance at 303 nm. Standard assay conditions were 100 mM Tris-HCl pH 8.01, 1 mM DTT (dithiothreitol) and 10 mM MgCl2. The cocktail was equilibrated for 5 min at 37 ° C; then the cell-free extract was added. The reaction was initiated with the addition of 0.05 mM acetoacetyl-CoA plus 0.2 mM CoA. The protein concentration was measured either by the Bradford method or by the Bicinchoninix kit (Sigma, catalog No. BCA-1). Bovine serum albumin (Bio-Rad, Hercules, CA) was used as the standard in both cases. In a typical assay, the specific activity of the ThlA protein in the induced culture was determined to be 16.0 μmol mg "1 min" 1 compared to 0.27 μmol mg "1 min-1 in the non-induced culture.

EXAMPLE 2 Cloning and Expression of Acetyl-CoA Acetyltransferase The purpose of this example was to express the enzyme acetyl-CoA acetyltranserase, also referred to herein as acetoacetyl-CoA thiolase, in E. coli. L. The thlB gene of acetoacetyl-CoA thiolase was cloned from C. acetojbutylicum (ATCC 824) and expressed in E. coli. The thlB gene was amplified from C. acetobutylicum genomic DNA (ATCC 824) using PCR. The thlB gene was cloned and expressed in the same manner as the thlA gene described in example 1. The genomic DNA C. acetobutylicum (ATCC 824) was amplified by PCR using primers N15 and N16 (see table 4), given as SEQ ID. NOs: 27 and 28, respectively, creating a product of 1.2 kbp. The forward primer incorporated four bases (CCAC) immediately adjacent to the codon of translational initiation to allow directional cloning in pENTR / SD / D-TOPO (Invitrogen) to generate the pENTRSDD-TOPOthlB plasmid. The clones were scted to sequencing with forward and reverse M13 primers, given as SEQ ID NOs: 45 and 46 respectively, to confirm that the genes were inserted in the correct orientation and to confirm the sequence. The additional sequencing primers, N15SeqF1 and NldSeqR1 (see Table 5), given as SEQ ID NOs: 49 and 50 respectively, were necessary to completely sequence the PCR product. The nucleotide sequence of the open reading frame (ORF) for this gene and the predicted amino acid sequence of the enzyme are given as SEQ ID NO: 3 and SEQ ID NO: 4, respectively. To create an expression clone, the thlB gene is transferred to the pDEST 14 vector (Invitrogen) by recombination to generate pDEST14thlB. The vector pDEST14thlB was transformed into BL21-A1 cells and the expression of the T7 promoter was induced by the addition of arabinose. A protein of the expected molecular weight of approximately 42 kDa, as deduced from the nucleic acid sequence, was present in the induced culture, but not in the non-induced control. Enzyme assays were performed as described in example 1. In a typical assay, the specific activity of the thlB protein in the induced culture was determined to be 14.9 μmol mg "1 min -i compared to 0.28 μmol mg l min- 1 in the non-induced culture.

EXAMPLE 3 Cloning and Expression of 3-Hydroxybutyryl-CoA Dehydrogenase The purpose of this example was to clone the hbd gene from C. acetobutyli cum (ATCC 824) and express it in E. coli. The hbd gene was amplified from C. acetobutylicum genomic DNA (ATCC 824) using PCR. The hbd gene was cloned and expressed using the method described in example 1. The hbd gene was amplified from genomic DNA C. acetobutylicum (ATCC 824) by PCR using primers N5 and N6 (see table 4), given as SEQ ID NOs: 19 and 20, respectively, creating a product of, 881 bp. The forward primer incorporated four bases (CACC) immediately adjacent to the codon of translational initiation to allow directional cloning in pENTR / SD / D-TOPO (Invitrogen) to generate the pENTRSDD-TOPOhbd plasmid. The clones were scted to sequencing with forward and reverse M13 primers, given as SEQ ID NOs: 45 and 46 respectively, to confirm that the genes were inserted in the correct orientation and to confirm the sequence. The additional sequencing primers, N5SeqF2 and N6SeqR2 (see Table 5), given as SEQ ID NOS: 51 and 52 respectively, were necessary to completely sequence the PCR product. The nucleotide sequence of the open reading frame (ORF) for this gene and the predicted amino acid sequence of the enzyme are given as SEQ ID NO: 5 and SEQ ID NO: 6, respectively. To create an expression clone, the hbd gene was transferred to the pDEST 14 vector (Invitrogen) by recombination to generate pDEST14hbd. The vector pDEST14hbd was transformed into BL21-A1 cells and the expression of the T7 promoter was induced by the addition of arabinose, as described in example 1. A protein of the expected molecular weight of approximately 31 kDa, as deduced from the acid sequence nucleic acid, was present in the induced culture, but was absent in the non-induced control. The activity of hydroxybutyryl-CoA dehydrogenase was determined by measuring the oxidation rate of NADH as measured by the decrease in absorbance at 340 nm. A Standard assay mixture contained 50 mM MOPS, pH 7.0, 1 mM DTT and 0.2 mM ADH. The cocktail was equilibrated for 5 min at 37 ° C and then the cell-free extract was added. The reactions were initiated by adding the substrate, 0.1 mM acetoacetyl-CoA. In a typical assay, the specific activity of the BHBD protein in the induced culture was determined to be 57.4 μmol mg "1 min" 1 compared to 0.885 μmol mg "1 min" 1 in the non-induced culture.

EXAMPLE 4 Cloning and Expression of Crotonane The purpose of this example was to clone the crt gene from C. acetobutyli cum (ATCC 824) and express it in E. coli. The crt gene was amplified from C genomic DNA to cetobutyli cum (ATCC 824) using PCR. The crt gene was cloned and expressed using the method described in example 1. The crt gene was amplified from C genomic DNA to cetobutyli cum (ATCC 824) by PCR using primers N3 and N4 (see table 4), given as SEQ ID NO. NOs: 17 and 18, respectively, creating a product of 794 bp. The forward primer incorporated four bases (CACC) immediately adjacent to the codon of translational initiation to allow directional cloning in pENTR / SD / D-TOPO (Invitrogen) to generate the pENTRSDD-TOPOcrt plasmid. The clones were subjected to sequencing with M13 primers Forward and Reverse, given as SEQ ID NOs: 45 and 46 respectively, to confirm that the genes were inserted in the correct orientation and to confirm the sequence. The nucleotide sequence of the open reading frame (ORF) for this gene and its predicted amino acid sequence are given as SEQ ID NO: 7 and SEQ ID NO: 8, respectively. To create an expression clone, the crt gene was transferred to the pDEST 14 vector (Invitrogen) by recombination to generate pDEST14crt. The vector pDEST14crt was transformed into BL21-A1 cells and the expression of the T7 promoter was induced by the addition of arabinose, as described in example 1. A protein of the expected molecular weight of approximately 28 kDa, as deduced from the acid sequence nucleic acid was present in much greater amounts in the induced culture than in the non-induced control. The crotonin activity was assayed as described by Stern (Methods Enzymol., 559-566 (1954)). In a typical assay, the specific activity of the crotone protein in the induced culture was determined to be 444 μmol mg "1 min" 1 compared to 47 μmol mg "1 min" 1 in the non-induced culture.

EXAMPLE 5 Cloning and Expression of Butyryl-CoA Dehydrogenase The purpose of this example was to express the enzyme butyryl-CoA dehydrogenase, also referred to herein as trans-2-Enoyl-CoA reductase, in E. coli. The CAC0462 gene, a homolog of putative trans-2-enoyl-CoA reductase, was cloned from C. acetobutylicum (ATCC 824) and expressed in E. coli. The CAC0462 gene was amplified from genomic DNA C. acetobutyli cum (ATCC 824) using PCR. The CAC0462 gene was cloned and expressed using the method described in Example 1. The CAC0462 gene was amplified from C. acetobutylicum genomic DNA (ATCC 824) by PCR using primers N17 and N21 (see Table 4), given as SEQ ID NOs: 29 and 30, respectively, creating a product of 1.3 kbp. The forward primer incorporated four bases (CACC) immediately adjacent to the codon of translational initiation to allow directional cloning in pENTR / SD / D-TOPO (Invitrogen) to generate the pENTRSDD-TOPOCAC0462 plasmid. The clones were subjected to sequencing with forward and reverse M13 primers, given as SEQ ID NOs: 45 and 46 respectively, to confirm that the genes were inserted in the correct orientation and to confirm the sequence. The additional sequencing primers, N22SeqFl (SEQ ID NO: 53), N22SeqF2 (SEQ ID NO: 54), N22SeqF3 (SEQ ID NO: 55), N23SeqRl (SEQ ID NO: 56), N23SeqR2 (SEQ ID NO: 57), and N23SeqR3 (SEQ ID NO: 58) (see Table 5), were necessary to completely sequence the PCR product. The nucleotide sequence of the open reading frame (ORF) for this gene and the predicted amino acid sequence of the enzyme are given as SEQ ID NO: 9 and SEQ ID NO: 10, respectively. To create an expression clone, the CAC0462 gene was transferred to the pDEST 14 vector (Invitrogen) by recombination to generate pDEST14CAC0462. The vector pDEST14CAC0462 was transformed into BL21-A1 cells and the expression of the T7 promoter was induced by the addition of arabinose, as described in Example 1. The analysis by SDS-PAGE showed no overexpressed protein of the expected molecular weight in the negative control or in induced culture. The CAC0462 gene from C. acetobutylicum used many rare E. coli codons. To avoid problems with the use of codon plasmid pRARE (Novagen) was transformed into BL21-A1 cells that host the vector pDEST14CAC0462. Expression studies with arabinose oxidation were repeated with cultures carrying the pRARE vector. A protein of the expected molecular weight of approximately 46 kDa was present in the induced culture but not in the non-induced control. The trans-2-enoyl-CoA reductase activity was assayed as described by Hoffmeister et al. (J. Biol. Chem. 280, 4329-4338 (2005)). In a typical assay, the specific activity of the TER protein CAC0462 in the induced culture was determined to be 0.694 μmol mg "1 min" 1 compared to 0.0128 μmol mg "1 min" 1 in the uninduced culture.

EXAMPLE 6 Cloning and Expression of Butyraldehyde Dehydrogenase (Acetylation) The purpose of this example was to clone the ald gene from C. beij erinckii (ATCC 35702) and express it in E. coli. The ald gene was amplified from C. beij erinckii genomic DNA (ATCC 35702) using PCR. The ald gene was cloned and expressed using the method described in example 1. The ald gene was amplified from C. beij erinckii genomic DNA (ATCC 35702) (prepared from anaerobically grown cultures, as described in example 1) by PCR using primers N27 Fl and N28 Rl (see table 4), given as SEQ ID NOs: 31 and 32, respectively, creating a product of 1.6 kbp. The forward primer incorporated four bases (CACC) immediately adjacent to the codon of translational initiation to allow directional cloning in pENTR / SD / D-TOPO (Invitrogen) to generate the pENTRSDD-TOPOald plasmid. The clones were subjected to sequencing with forward and reverse M13 primers, given as SEQ ID NOs: 45 and 46 respectively, to confirm that the genes were inserted in the correct orientation and to confirm the sequence. The additional sequencing primers, N31SeqF2 (SEQ ID NO: 59), N31SeqF3 (SEQ ID NO: 60), N31SeqF4 (SEQ ID NO: 612), N32SeqRl (SEQ ID NO: 72), N31SeqR2 (SEQ ID NO: 62), N31SeqR3 (SEQ ID NO: 63), N31SeqR4 (SEQ ID NO: 64), and N31SeqR5 (SEQ ID NO: 65) (see Table 5), were necessary to completely sequence the PCR product. The nucleotide sequence of the open reading frame (ORF) for this gene and the predicted amino acid sequence of the enzyme are given as SEQ ID NO: 11 and SEQ ID NO: 12, respectively. To create an expression clone, the ald gene was transferred to the pDEST 14 vector (Invitrogen) by recombination to generate pDEST14ald. The vector pDEST14ald was transformed into BL21-A1 cells and the expression of the T7 promoter was induced by the addition of arabinose, as described in example 1. A protein of the expected molecular weight of approximately 51 kDa, as deduced from the acid sequence nucleic acid, was present in the induced culture, but not in the non-induced control. The acylation aldehyde dehydrogenase activity was determined by monitoring the formation of NADH, as measured by the increase in absorbance at 340 nm, as described by Husemann et al. (Appl. Microbiol. Biotechnol.31: 435-444 (1989)). In a typical assay, the specific activity of the Ald protein in the induced culture was determined to be 0.106 μmol mg "1 min" 1 compared to 0.01 μmol mg-1 min "1 in the non-induced culture.

EXAMPLE 7 Cloning and Expression of Butanol Dehydrogenase The purpose of this example was to clone the bdhB gene from C. acetobutyli cum (ATCC 824) and express it in E. coli. The bdhB gene was amplified from C. acetobutylicum genomic DNA (ATCC 824) using PCR. The bdhB gene was cloned and expressed using the method described in Example 1. The bdhB gene was amplified from genomic DNA C. acetobutyli cum (ATCC 824) by PCR using primers Nll and N12 (see Table 4), given as SEQ ID NOs : 25 and 26, respectively, creating a product of 1.2 kbp. The forward primer incorporated four bases (CACC) immediately adjacent to the codon of translational initiation to allow directional cloning in pENTR / SD / D-TOPO (Invitrogen) to generate the pENTRSDD-TOPObdhB plasmid. The translational start codon was also changed from "GTG" to "ATG" by the primer sequence. The clones were subjected to sequencing with forward and reverse M13 primers, given as SEQ ID NOs: 45 and 46 respectively, to confirm that the genes were inserted in the correct orientation and to confirm the sequence. The additional sequencing primers, NllSeqFl (SEQ ID NO: 66), NllSeqF2 (SEQ ID NO: 67), N12SeqRl (SEQ ID NO: 68), and N12SeqR2 (SEQ ID NO: 69), (see Table 5), were necessary to completely sequence the PCR product. The nucleotide sequence of the open reading frame (ORF) for this gene and the predicted amino acid sequence of the enzyme are given as SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

To create an expression clone, the bdhB gene was transferred to the pDEST 14 vector (Invitrogen) by recombination to generate pDEST14bdhB. The vector pDEST14bdhB was transformed into BL21-A1 cells and the expression of the T7 promoter was induced by the addition of arabinose, as described in example 1. A protein of the expected molecular weight of approximately 43 kDa, as deduced from the acid sequence nucleic acid, was present in the induced culture, but not in the non-induced control. The butanol dehydrogenase activity was determined from the oxidation rate of NADH as measured by the decrease in absorbance at 340 nm as described by Husemann and Papoutsakis, supra. In a typical assay, the specific activity of the BdhB protein in the induced culture was determined to be 0.169 μmol mg "1 min" 1 compared to 0.022 μmol mg "1 min" 1 in the non-induced culture.

EXAMPLE 8 Cloning and Expression of Butanol Dehydrogenase The purpose of this example was to clone the bdhA gene from C. to cetobutylicum 824 and express it in E. coli. The bdhA gene was amplified from C. acetobutylicum 824 genomic DNA using PCR. The bdhA gene was cloned and expressed using the method described in example 1. The bdhA gene was amplified from DNA genomic C. acetobutylicum 824 by PCR using primers N9 and N10 (see table 4), given as SEQ ID NOs: 23 and 24, respectively, creating a product of 1.2 kbp. The forward primer incorporated four bases (CACC) immediately adjacent to the codon of translational initiation to allow directional cloning in pENTR / SD / D-TOPO (Invitrogen) to generate the pENTRSDD-TOPObdhA plasmid. The clones, given as SEQ ID NOs: 45 and 46 respectively, to confirm that the genes are inserted in the correct orientation and to confirm the sequence. Additional sequencing primers, N9SeqFl (SEQ ID NO: 70) and NIOSeqRl (SEQ ID NO: 71), (see Table 5) are needed to completely sequence the PCR product. The sequence of the open reading structure (ORF) for this gene and the predicted amino acid sequence of the enzyme are given as SEQ ID NO: 15 and SEQ ID NO: 16, respectively. To create an expression clone, the bdhA gene is transferred to the pDEST 14 vector (Invitrogen) by recombination to generate pDEST14bdhA. The vector pDEST14bdhA is transformed into BL21-AI cells and the expression of the T7 promoter is induced by the addition of arabinose, as described in Example 1. A protein of the expected molecular weight of approximately 43 kDa, as deduced from the sequence of nucleic acid, occurs in the induced culture, but not in the non-induced control.

The butanol dehydrogenase activity was determined from the oxidation rate of NADH as measured by the decrease in absorbance at 340 nm, as described by Husemann and Papoutsakis, supra. In a typical assay, the specific activity of the BdhA protein in the induced culture is determined to be 0.102 μmol mg "1 min" 1 compared to 0.028 μmol mg "1 min" 1 in the non-induced culture.

EXAMPLE 9 Construction of a Transformation Vector for Genes in the Biosynthetic Pathway of 1-butanol - Bottom Path To construct a transformation vector comprising the genes encoding the six stages in the biosynthetic pathway of 1-butanol, the genes they encode The 6 stages in the trajectory are divided into two operons. The upper pathway comprises the first four steps catalyzed by acetyl-CoA acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonane and butyryl-CoA dehydrogenase. The lower pathway comprises the last two steps, catalyzed by butyraldehyde dehydrogenase and butanol dehydrogenase. The purpose of this Example is to construct the lower trajectory operon. The construction of the upper path operon is described in Example 10.

Individual genes are amplified by PCR with primers that incorporate restriction sites for subsequent cloning and forward primers contained in an optimized E. coli ribosome binding site (AAAGGAGG). The PCR products are TOPO cloned in the vector pCR 4Blunt-T0P0 and transformed into ToplO cells of E. coli (Invitrogen). The plasmid DNA of the TOPO cabbages is prepared and the sequence of the genes is verified. T4 DNA ligase and restriction enzymes are used (New England Biolabs, Beverly, MA) in accordance with the manufacturer's instructions. For cloning experiments, the restriction fragments are purified by gel electrophoresis using the QIAquick Gel Extraction Kit (Qiagen). After conformation of the sequence, the genes are subcloned into a modified pUC19 vector as a cloning platform. The vector pUC19 is modified by a HindIII / SapI, creating pUC19dHS. The digested remove the lac promoter adjacent to the MCS (multiple cloning site), which prevents transcription of the operons in the vector. The ald gene is amplified from the ATCC 35702 genomic DNA of C. acetobutylicum by PCR using primers N58 and N59 (see Table 4), provided as SEQ ID NO: 41 and 42, respectively, creating a product of 1.5 kbp. The forward primer incorporated into the Aval and BstEII restriction sites and an RBS (ribosome binding site). He reverse primer incorporated into the Hpal restriction site. The PCR product is cloned into pCRBlunt II-TOPO creating pCRBluntlI. Plasmid DNA is prepared from TOPO clones and the sequence of the genes is checked with the forward M13 primers (SEQ ID NO: 45), Inverse M13 (SEQ ID NO: 46), N31SeqF2 (SEQ ID NO: 59) , N31SeqF3 (SEQ ID NO: 60), N31SeqF4 (SEQ ID NO: 61), N32SeqRl (SEQ ID NO: 72), N31SeqR2 (SEQ ID NO: 62), N31SeqR3 SEQ ID NO: 63), N31SeqR4 (SEQ ID NO. : 64) and N31SeqR5 (SEQ ID NO: 65) (see Table 5). The bdhB gene is amplified from genomic DNA (ATCC 824) of C. acetobutylicum by PCR using primers N64 and N65 (see Table 4), provided as SEQ ID Nos: 43 and 44, respectively, creating a 1.2 kbp product. The forward primer is incorporated in a Hpal restriction site and an RBS. The reverse primer is incorporated as a Pmel restriction site and a Sphl. The PCR product is cloned into pCRBlunt II-TOPO creating pCRBluntlI-bdhB. The DNA plasmid was prepared from TOPO clones and the sequence of the genes was verified with forward primers M13 (SEQ ID NO: 45), Inverse M13 (SEQ ID NO: 46), NllSeqFl (SEQ ID NO: 66), NllSeqF2 (SEQ ID NO: 67), N12SeqRl (SEQ ID NO: 68) and N12SeqR2 (SEQ ID NO: 69) (see Table 5). To construct the lower trajectory operon, the 1.2 kbp Sphl and Hpal fragment from pCRBluntlI-bdhB, a 1.4 kbp Hpal and Sphl fragment from pCRBluntlI-ald, and the Long fragment of an Aval and digested Sphl from pUC19dHS, were ligated together. The three-way ligation created pUC19dHS-ald-bdhB. The pUC19dHS-ald-bdhB vector was digested with BstEII and Pmel releasing a 2.6 kbp fragment that was cloned into pBenBP, a shuttle vector of E. coli-B. subtilis. Plasmid pBenBP was created by modification of the vector pBE93, which is described by Nagarajan, WO 93/24631 (Example 4). The neutral protease promoter from Bacill us amyloliquefacíens (NPR), signal sequence and the phoA gene were removed from pBE93 with an NcoI / HindIII digestion. The NPR promoter was amplified by PCR from pBE93 by primers BenF and BenBPR, provided by SEQ ID NOS: 73 and 75, respectively. The BenBPR primer incorporates BstEII, Pmel and HindIII sites downstream of the promoter. The PCR product was digested with Ncol and HindIII and the fragment was cloned into the corresponding sites in the vector pBE93 to create pBenBP. The lower operon fragment was subcloned into the BstEII and Pmel sites in pBenBP creating pBen-ald-bdhB. Assays for the activity of butyraldehyde dehydrogenase and butanol dehydrogenase were conducted in crude extracts using the methods described above. Both enzymatic activities were demonstrated at levels above the control strain that contains an empty vector.

EXAMPLE 10 (Prophetic) Construction of a Transformation Vector for Genes in the Biosynthetic Pathway of 1-Butanol - Superior Trajectory The purpose of this prophetic Example is to describe how to assemble the superior trajectory operon. The general procedure is the same as described in Example 9. The thIA gene is amplified from the genomic DNA of C. acetobutylicum (ATCC 824) by PCR using a primer pair N44 and N45 (see Table 4), given as SEQ ID NOS: 33 and 34, respectively, creating a product of 1.2 kbp. The forward primer incorporates a Sphl restriction site and a ribosome binding site (RBS). The reverse primer incorporates AscI and PstI restriction sites. The PCR product is cloned into pCR4Blunt-T0P0 creating pCR4Blunt-TOPO-thIA. The plasmid DNA is prepared from the TOPO clones and the sequence of the genes is verified with the forward primers M13 (SEC ID NO: 45), reverse M13 (SEQ ID NO: 46), N7SeqFl (SEQ ID NO: 47) and N7SeqRl (SEQ ID NO: 48) (See Table 5). The hbd gene is amplified from the genomic DNA of C. acetobutylicum (ATCC 824) by PCR using primer pair N42 and N43 (see Table 4), given as SEQ ID NOS: 35 and 36, respectively, creating a product of 0.9 kbp. The forward primer incorporates a Sali restriction site and an RBS. HE reverse primer incorporates a Sphl restriction site. The PCR product is cloned into pCR4Blunt-T0P0 creating pCR4Blunt-T0P0-hbd. The plasmid DNA is prepared from the TOPO clones and the sequence of the genes verified with forward primers M13 (SEQ ID NO: 45), Inverse M13 (SEQ ID NO: 46), N5SeqF2 (SEQ ID NO: 51) and N6SeqR2 ( SEQ ID NO: 52) (See Table 5). The CAC0462 gene is codon optimized for expression in E. coli as primary host and B. subtilis as a secondary host. The new gene called CaTER, provided as SEQ ID NO: 76, is synthesized by Genscript Corp (Piscataway, NJ). The CaTER gene is cloned into the pUC57 vector as a BamHI-Sall fragment and includes an RBS, producing a pUC57-CaTER plasmid. The crt gene is amplified from genomic DNA of C. acetobi tylicum (ATCC 824) by PCR using the primer pair N38 and N39 (see Table 4), given as SEQ ID NOS: 39 and 40, respectively, creating a product of 834 bp . The forward primer incorporates EcoRI and Mlul restriction sites and an RBS. The reverse primer incorporates EcoRI and Mlul restriction sites and an RBS. The reverse primer incorporates a BamHI restriction site. The PCR product is cloned into pCR4Blunt-TOPO, creating pCR4Blunt-TOPO-crt. The plasmid DNA is prepared from the TOPO clones and the sequence of the genes is verified with forward M13 primers (SEQ ID NO: 45) and Inverse M13 (SEQ ID NO: 46) (see table 5). After confirmation of the sequence, the genes were subcloned into a modified pUC19 vector as a cloning platform. The vector pUC19 was modified by a Sphl / Sapl digestion solution, creating pUC19dSS. The digestion solution removes the lac promoter adjacent to the MCS, preventing the transcription of the operons in the vector.

To construct the pCR4 blunt-TOPO-crt operon with superior trajectory, it is digested with EcoRI and BamHI, releasing a crt fragment of 0.8 kbp. The vector pUC19dSS is also digested with EcoRI and BamHI releasing a 2.0 kbp vector fragment. The crt fragment and the vector fragment are ligated together using T4 DNA ligase (New England Biolabs) to form pUC19dSS-crt. The CaTER gene is inserted into pCU19dSS-crt by digesting pUC57-CaTER with BamHI and SalI, releasing a 1.2 kbp CaTER fragment. PUC19dSS-crt is digested with BamHI and SalI and the large vector fragment is ligated with the CaTER fragment, creating pUC19dSS-crt-CaTER. To complete the operon a Salí and Sphl fragment of 884 bp of pCR4 Blunt-TOPO-hbd, a fragment of Sphl and Pstl thIA of 1.2 kb of pCR4 Blunt-TOPO-thlA and the large fragment of a Sali digestion solution and PstI of pUC19dSS-crt-CaTER are linked. The product of the 3-way ligation is pUC19dSS-crt-CaTER-hbd-thIA.

The pUC19dSS-crt-CaTER-hbd-thIA vector is digested with Mlul and AscI which releases a 4.1 kbp fragment which is cloned into a derivative of pBE93 (Caimi, WO2004 / 018645, pp. 39-40) a carrier vector of E. coli-B. subtilis, referred to as pBenMA. Plasmid pBenMA was created by modification of the pBE93 vector. The neutral protease promoter (NRP) Bacillus amyloliquefaciens, signal sequence and the phoA gene are removed from pBE93 with an NcoI / HindIII digestion solution. The NPR promoter is PCR amplified from pBE93 by primers BenF and BenMAR, provided as SEQ ID NOS: 73 and 74, respectively. The BenMAR primer incorporates Mlul, AscI, and HindIII sites downstream of the promoter. The PCR product was digested with Ncol and HindIII and the fragment was cloned into the corresponding sites in the vector pBE93, creating pBenMA. The higher operon fragments were subcloned into the Mlul and AscI sites in pBenMA creating pBen-crt-hbd-CaTER-thlA.

EXAMPLE 11 (Prophetic) Expression of the Biosynthetic Pathway of 1-Butanol in E. coli The purpose of this prophetic Example is to describe how to express the biosynthetic pathway of 1-butanol in E. coli. The pBen-crt-hbd-CATER-thlA and pBen-ald-bdhB plasmids, constructed as described in Examples 10 and 9, respectively, they were transformed into E. coli NM522 (ATCC 47000) and the expression of the genes in each operon was monitored by analysis of SDS-PAGE, enzyme assay and Western analysis. For Westerns, the antibodies were raised to synthetic peptides by Sigma-Genosys (The Woodlands, TX). After confirming the expression of all genes, pBen-ald-bdhB were digested with EcoRI and Pmel to release the NPR promoter-ald-bdhB fragment. The EcoRI digestion solution of the fragment is terminated truncated using the Klenow fragment of DNA polymerase (New England Biolabs, catalog No. M0210S). Plasmid pBen-crt-hbd-CaTER-thlA is digested with Pvull to create a linearized closed-end vector fragment. The vector and the NPR-ald-bdhB fragment are ligated, creating plBl 0.1 and plBl 0.2, which contain the biosynthetic pathway of 1-butanol complete with the NPR promoter-ald-bdhB fragment in opposite orientations. Plasmids plBl 0.1 and plBl 0.2 are transformed into E. coli NM522 and the expression of the genes is monitored as previously described. The strain E. coli NM522 / plBl 0.1 or NM522 / plBl 0.1 is inoculated in a shaker flask of 250 ml containing 50 ml of medium and shaking at 250 rpm and 35 ° C. The medium is composed of: dextrose, 5 g / l; MOPS, 0.05 M; Ammonium sulfate, 0.01 M; potassium phosphate, monobasic, 0.005 M; SIO metal mixture, 1% (v / v); yeast extract, 0.1% (w / v); casamino acids, 0. 1% (p / v); thiamine, 0.1 mg / L; proline, 0.05 mg / L; and biotin 0.002 mg / L, and titrated to pH 7.0 with KOH. The SIO metal mixture contains: MgCl2, 200 mM; CaCl2, 70 mM; MnCl2, 5 mM; FeCl3, 0.1 mM; ZnCl 2, 0.1 mM; thiamine hydrochloride, 0.2 mM; CuSO4, 172 μM; CoCl2, 253 μM; and Na2Mo04, 242 μM. After 18 to 24 h, 1-butanol was detected by CLAR or GC analysis, as described in the General Methods section.

EXAMPLE 12 (Prophetic) Expression of the Biosynthetic Pathway of 1-Butanol in Bacillus subtilis The purpose of this prophetic Example is to describe how to express the biosynthetic pathway of 1-butanol in Bacillus subtilis. The same procedure as described in Example 11 is used. The upper and lower operons constructed as described in Examples 10 and 9, respectively, are used. Plasmids plBl 0.1 and plBl 0.2 are transformed into Bacillus subtilis BE1010 (J. Bacteriol 173: 2278-2282 (1991)) and the expression of the genes in each operon is monitored as described in Example 11. Strain B. subtilis BElOlO / plBl 0.1 or BElOlO / plBl 0. 2 is inoculated in a 250 ml shaker flask containing 50 ml of medium and shake at 250 rpm and 35 ° C for 18 h. The medium is composed of: dextrose, 5 g / l; MOPS, 0.05 M; acid glutamic, 0.02 M; Ammonium sulfate, 0.01 M; potassium phosphate, monobasic buffer, 0.005 M; metal mixture S10 (as described in Example 11), 1% (v / v); yeast extract, 0.1% (w / v); casamino acids, 0.1% (w / v); tryptophan, 50 mg / L; methionine, 50 mg / l; and lysine, 50 mg / l, and titrated at pH 7.0 with KOH. After 18 to 24 hours, 1-butanol was detected by HPLC or GC synthesis, as described in the General Methods section.

EXAMPLE 13 Production of 1-Butanol from Glucose using Recombinant E. coli This Example describes the production of 1-butanol in E. coli. The expression of the genes encoding the 6 stages of the biosynthetic pathway of 1-butanol was divided into three operons. The upper path comprises the first four steps coded by thIA, hbd, crt and EgTER in an operon. The next step, coded by ald, was provided by a second operon. The last stage in the trajectory, coded by yqhD, was provided in a third operon. The production of 1-butanol was demonstrated in E. coli strains comprising all three operons. Unless indicated otherwise in the text, the cloning primers described in this Example are referred to by SEQ ID NO: in Table 4, and the primers of Selection of PCR and sequencing are referred to by SEQ ID NO: in Table 5. Acetyl-OoA acetyltransferase. The th / A gene was amplified from genomic DNA of C. acetobutylicum (ATCC 824) by PCR using a pair of primers N44 and N45 (see Table 4), provided as SEQ ID NOs: 33 and 34, respectively, creating a product of 1.2 kbp. The forward primer incorporates a restriction site Sphl and a ribosome binding site (RBS). The reverse primer incorporates Ascl and Pstl restriction sites. The PCR product was cloned into pCR4Blunt-TOPO (Invitrogen Corp., Carisbad, CA) creating pCR4Blunt-TOPO-thlA. Plasmid DNA was prepared from the TOPO clones and the sequence of the genes was checked with forward M13 primers (SEQ ID NO: 45), reverse M13 (SEQ ID NO: 46), N7SeqFl (SEQ ID NO: 47), and N7SeqRl (SEQ ID NO: 48) (see Table 5). 3-Hydroxybutyryl-CoA dehydrogenase. The hbd gene was amplified from the genomic DNA (ATCC 824) of C. acetobutylicum by PCR using the pair of primers N42 and N43 (see Table 4) providing a SEQ ID NOs: 35 and 36, respectively, creating a product of 0.9 kbp . The forward primer incorporates a Sali restriction site and an RBS. The reverse primer incorporates a Sphl restriction site. The PCR product was cloned into pCR4Blunt-TOPO creating pCR4Blunt-TOPO-hbd. Plasmid DNA was prepared from the TOPO clones and the sequence of genes checked with forward M13 primers (SEQ ID NO: 45), reverse M13 (SEQ ID NO: 46), N5SeqF2 (SEQ ID NO: 51), and N6SeqR2 (SEQ ID NO: 52) (see Table 5) . Crotonin The crt gene was amplified from the genomic DNA of C. acetobutylicum (ATCC 824) by PCR using pair of primers N38 and N39 (see Table 4), provided as SEQ ID NOs: 39 and 40, respectively, creating a product of 834 bp . The forward primer incorporates EcoRI and Mlul restriction sites and an RBS. The reverse primer incorporates a BamHI restriction site. The PCR product was cloned into pCR4Blunt-TOPO creating pCR4Blunt-TOPO-crt. Plasmid DNA was prepared from TOPO clones and the sequence of the genes was checked with forward M13 primers (SEQ ID NO: 45) and reverse M13 (SEQ ID NO: 46) (see Table 5). Butyryl-CoA Dehydrogenase (trans-2-enoyl-CoA reductase). The CAC0462 gene was synthesized for the use of improved codon in E. coli as a primary host and B. subtilis as a secondary host. The new gene (CaTER, SEQ ID NO: 76) was synthesized and cloned by Genscript Corporation (Piscataway, NJ) in the vector pUC57 as a BamHI-Sall fragment and includes an RBS. An alternative gene for butyryl-CoA dehydrogenase from Euglena gracilis (TER, GenBank No. Q5EU90) was synthesized for the use of improved codon in E. coli and Bacillus subtilis.

The gene was synthesized and cloned by GenScript Corporation into pUC57 creating pUC57 :: EgTER. Primers N85 and N86, (SEQ ID NO: 80 and 81 respectively) together with pUC57 :: EgTER as hardened DNA, provided a PCR fragment comprising 1224 bp of pUC57 :: EgTER DNA. The 1224 bp sequence was provided as SEQ ID NO: 77, where bp 1-1218 is the coding sequence (cds) of EgTER (opt). EgTER (opt) is a codon-optimized TER gene, which lacks the normal mitochondrial presequence to be functional in E. coli (Hoffmeister et al., J. Biol. Chem. 280-4329 (2005)). EgTER (opt) was cloned into pCR4Blunt-TOPO and its sequence was confirmed with forward M13 primers (SEQ ID NO: 45) and M13 Inverse (SEQ ID NO: 46). The additional sequencing primers N62SeqF2 (SEQ ID NO: 114), N62SeqF3 (SEQ ID NO: 115), N62SeqF4 (SEQ ID NO: 116), N63SeqRl (SEQ ID NO: 117), N63SeqR2 (SEQ ID NO: 118), N63SeqR3 (SEQ ID NO: 119) and N63SeqR4 (SEQ ID NO: 120) were necessary to completely sequence the PCR product. The EgKE (opt) sequence of 1.2 kbp was then cut with HincII and Pmel and cloned into pET23 + (Novagen) linearized with HincII. The orientation of the EgTER (opt) gene for the promoter was confirmed by the selection of colony PCR with primers T7Primer and N63SeqR2 (SEQ ID NOs: 82 and 118 respectively). The resulting plasmid, pET23 + :: EgTER (opt), was transformed into BL21 (DE3) (Novagen) for expression studies.

The trans-2-enoyl-CoA reductase activity was assayed as described by Hoffmeister et al., J. Biol. Chem. 280: 4329 (2005). In a typical assay, the specific activity of the EgTER protein (opt) in culture BL21 (DE3) / pET23 +:: EgTER (opt) was determined to be 1.9 μmol mg'l min "1 compared to 0.547 μmol mg" 1 min 1 in the non-induced culture The EgTER gene (opt) was then cloned into the vector pTrc99a under the control of the trc promoter The EgTER gene (opt) was isolated as a BamHI / Sall fragment of 1287 bp from pET23 +: : EgTER (opt) The 4.2 kbp pTrc99a vector was linearized with BamHI / Sall The vector and the fragment were ligated creating 5.4 kbp pTrc99a-EgTER (opt) The positive clones were confirmed by colony PCR with Trc99aF primers and N63SeqR3 (SEQ ID NOs: 83 and 119 respectively) producing a product of 0.5 kb Construction of plasmid pTrc99a-ECHT comprising genes encoding acetyl-CoA acetyltransferase (thlA), 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonane (crt) ), and butyryl-CoA dehydrogenase (trans-2-enoyl-CoA reductase, EgTER (opt)). From an operon of four genes comprising the superior trajectory (EgTER (opt), crt, hbd and thlA), pCR4Blunt-TOPO-crt is digested with EcoRI and BamHI that releases a crt fragment of 0.8 kbp. The pUC19dSS vector (described in Example 10) is also digested with EcoRI and BamHI that releases a 2.0 kbp vector fragment. The crt fragment and the vector fragment were ligated together using T4 DNA ligase (New England Biolabs) to form pUC19dSS-crt. The CaTER gene was inserted into pUC19dSS-crt by digesting pUC57-CaTER with BamHI and SalI, which releases a 1.2 kbp CaTER fragment. PUC19dSS-crt was digested with BamHI and SalI and the large vector fragment was ligated with the CaTER fragment, creating pUC19dSS-crt-CaTER. To complete the operon a Salí and Sphl fragment of 884 bp of pCR4Blunt-TOPO-hbd, a fragment of Sphl and Pstl thlA of 1.2 kb of pCR4Blunt-TOPO-thlA and the large fragment of a solution of digestion of Sali and PStl of pUC19dSS-crt-CaTER were ligated. The product of the 3-way ligation was named pUC19dSS-crt-CaTER-hbd-thlA or pUC19dss :: Operon 1. Higher activity of butyryl-CoA dehydrogenase was obtained from pTrc99a-EgTER (opt) than from the CaTER constructs, as well , an operon derived from pTrc99a-EgTER (opt) was constructed. The CaTER gene was removed from pUC19dss :: Operon 1 by digesting with BamHI / Sal 1 and gel that purified the 5327 bp vector fragment. The vector was treated with Klenow and ligated again creating pUC19dss:: Operon 1? CaTer. The 2934-bp crt-hbd-thlA (C-H-T) fragment was then isolated as an EcoRI / Pstl fragment from pUC19dss: Operon 1? CaTer. The C-H- fragment was treated with Klenow to return the blunt ends. The vector pTrc99a-EgTER (opt) was digested with Sali and the ends were treated with Klenow. The blunt end vector and the blunt end C-H-T fragment were ligated to create pTrc99a-E-C-H-T. Colony PCR reactions were performed with primers N62SeqF4 and N5SeqF4 (SEQ ID NOs: 116 and 84 respectively) to confirm the orientation of the insert. Construction of plasmids pBHR T7-ald and pBHR-Ptrc-ald (opt) comprising genes encoding butyraldehyde dehydrogenase (ald and ald (opt)). The PT7-ald operon was subcloned from pDESTl4-ald (Example 6) into the broad host range plasmid pBHR1 (MoBitec, Goettingen, Germany) to create pBHRl PT7-ald. Plasmid pBHRl is compatible with plasmids pUC19 or pBR322 as well as pBHRl PT7-ald can be used in combination with derivatives of pUC19 or pBR322 which carry the superior path operon for the production of 1-butanol in E. coli. Plasmid pDEST14-ald was digested with Bgl II and treated with the Klenow fragment of DNA polymerase to make blunt ends. The plasmid was then digested with EcoRI and the 2,245 bp PT7-ald fragment was gel purified. Plasmid pBHRl was digested with Seal and EcoRI and the 4.883 bp fragment was gel purified. The fragment of PT7-añd was ligated with the pBHR1 vector, creating pBHR T7-ald. PCR amplification of transformant colony with T-ald (BamHI) and B-ald (EgTER) primers (SEQ ID NOs: 85 and 86 respectively) confirmed the expected 1.4 kb PCR product. The restriction mapping of pBHR clones T7-ald with EcoRI and Drdl confirmed the expected fragments of 4.757 and 2.405 bp. For assays of butyraldehyde dehydrogenase activity, plasmid pBHR T7-ald was transformed into BL21StarMR (DE3) cells (Invitrogen) and expression of the T7 promoter was induced by the addition of L-arabinose as described in Example 1. The activity of Acylated aldehyde dehydrogenase was determined by monitoring the formation of NADH, as measured by the increase in absorbance at 340 nm, as described in Example 6. An alternative DNA sequence for the ald gene from Clostridium beijerinckii ATCC 35702 was synthesized (optimized for codon use in E. coli and Bacillus subtilis) and was cloned into pUC57 by GenScript Corporation (Piscataway, NJ), creating the plasmid pUC57-ald (opt). PUC57-ald (opt) was digested with Sacl and SalI to release a 1498 bp fragment comprising the optimized codon gene, ald (opt) and an RBS ready for E. coli. The sequence of the 1498 bp fragment is provided as SEQ ID NO: 78. pTrc99a was digested with Sacl and SalI providing a 4153 bp vector fragment, which was ligated with the ald (opt) fragment of 1498 bp to create pTrc-ald (opt). The expression of the synthetic gene, ald (opt), is under the control of the IPTG-inducible Ptrc promoter. The Ptrc-ald operon (opt) was subcloned into the plasmid pBHRl (MoBitec) broad host range to be compatible with the above pathway plasmid. The fragment of Ptrc-ald (opt) was PCR-amplified from pTrc99A:: ald (opt) with T-Ptrc (BspEl) and B-aldopt (Seal), (SEQ ID NOs: 87 and 88 respectively) that incorporate sites of BspEl and Seal restriction within the corresponding primers. The PCR product was digested with BspEl and Seal. Plasmid pHBR1 was digested with Seal and BspEl and the 4.883 bp fragment was gel purified. The Ptrc-ald fragment (opt) was ligated with the pBHR1 vector, creating pBHR-PcatPtrc-ald (opt). Restriction mapping of the pBHR-PcatPtrc-ald (opt) clones with Seal and BspEl confirmed the expected 4.883 and 1.704 bp fragments. To remove the cat promoter region (Pcat) from the plasmid, the pBHR-PcatPtrc-ald plasmid (opt) was digested with BspEl and Aatll and the 6.172 bp fragment was gel purified. T-BspElAatll and B-BspElAatll (SEQ ID NOs: 89 and 90 respectively) were mixed in a solution containing 50 mM NaCl, 10 mM Tris-HCl, and 10 mM MgCl2 (pH 7.9) at a final concentration of 100 μM and hybridized by incubating at 75 ° C for 5 min and cooled slowly to room temperature. Hybridized oligonucleotides were ligated with the 6.172 bp fragment, creating pBHR-Ptrc-ald (opt). Construction of E. coli strains expressing butanol dehydrogenase (yghD). E. coli contains a native gene (yqhD) which was identified as a 1,3-propanediol dehydrogenase (US Patent No. 6,514,733). The yqhD gene has 40% identity to the adhB gene in Clostridium, a probable butadiene dehydrogenase dependent on NADH. The yqhD gene was placed under the constitutive expression of a variant of the glucose promoter isomerase 1.6G1 (SEQ ID NO: 91) in the E. coli strain MG1655 1.6yghD :: Cm (WO 2004/033646), using Technology? Network (Datsenko and Wanner, Proc. Nati, Acad. Sci. U.S.A. 97: 6640 (2000)). Similarly, the native promoter was replaced by the 1.5GI promoter (WO 2003/089621) (SEQ ID NO: 92), creating strain MG1655 1.6GI-yqhD:: Cm, thereby replacing the 1.6GI promoter. of MG1655 1.6yghD :: Cm with the 1.5GI promoter. A Pl lysate was prepared from MG1655 1.5GI and qhD :: CM and the cassette was moved for expression of strains, MG1655 (DE3), prepared from the E. coli strain MG1655 and a lambda DE3 lysogenization kit (Invitrogen), and BL21 (DE3) (Invitrogen), creating MG1655 (DE3) 1.5GI-yqhD:: Cm and BL21 (DE3) 1.5-yqhD:: Cm, respectively. Demonstration of 1-butanol production from Recombinant E. coli. The E. coli strain MG1655 (DE3) 1.5GI-yqhD:: Cm was transformed with plasmids pTrc99a-ECHT and pBHR T7-ald to produce the strain MG1655 (DE3) 1.5GI-yqhD:: Cm / pTrc99a-ECHT / pBHR T7-ald. Two independent isolates were initially grown in LM medium which contains 50 μg / ml kanamycin and 100 μg / ml carbenicillin. The cells were used to inoculate shaken flasks (approximately 175 mL total volume), containing 15, 50 and 150 ml of TM3a / glucose medium (with appropriate antibiotics) to represent high, medium and low oxygen conditions, respectively. The TM3a / glucose medium contains (per liter): 10 g of glucose, 13.6 g of KH2P04, 2.0 g of citric acid monohydrate, 3.0 g (NH4) 2S0, 2.0 g of MgSO4.7H20, 0.2 g of CaCl2.2H20 , 0.33 g of ferric ammonium citrate, 1.0 mg of thiamine-HCl, 0.50 g of yeast extract, and 10 ml of microelements solution, adjusted to pH 6.8 with NHOH. The solution of microelements contains: citric acid. H20 (4.0 g / L), MnS04.H20 (3.0 g / L), NaCl (1.0 g / L), FeS04.7H20 (0.10 g / L), CoCl2.6H20 (0.10 g / L), ZnS04.7H20 ( 0.10 g / L), CuS04.5H20 (0.10 g / L), H3B03 (0.010 g / L) and Na2Mo04.2H20 (0.010 g / L). The flasks were inoculated to an initial OD600 of < 0.01 units and incubated at 34 ° C with shaking at 300 rpm. The flasks containing 15 and 50 ml of medium were capped with vented plugs; The flasks containing 150 ml were capped with non-vented stoppers to minimize air exchange. The IPTG was added to a final concentration of 0.04 mM; the ODβ of the flasks at the time of addition was > 0.4 units. Approximately 15h after the induction, an aliquot of the broth was analyzed by HPLC (Shodex Sugar column) SH1011) with refractive index detection (Rl) and GC column (Varian CP-WAX 58 (FFAP) and CB, 25 mx 0.25 mm id x 0.2 μm film thickness) with flame ionization detection (FID) by content of 1-butanol, as described in the General Methods section. The results of the 1-butanol determinations are given in Table 6.

Table 6 Production of 1-butanol per E strain. coli MG1655 (DE3) 1.5GI-yqhD:: Cm / pTrc99a-E-C-H-T / pBHR T7-ald.

-The values were determined from the CLAR analysis. Strain suffixes "a" and "b" indicate independent isolates.

The two independent isolates of MG1655 (DE3) 1.6Gl-yqhD:: Cm / pTrc99a-ECHT / pBHR T7-ald, were tested for production of 1-butanol in an identical manner except that the medium contains 5 g / l extract of yeast. The results are shown in Table 7.

Table 7 Production of 1-butanol per strain of E. coli MG1655 (DE3) 1.5GI-yqhD:: Cm / pTrc99a-E-C-H-T / pBHR T7-ald. -quantitative values were determined from the CLAR analysis. - "-" = not detected. Strain suffixes "a" and "b" indicate independent isolates.

The E. coli strain BL21 (DE3) 1.5GI-yqhD was transformed: Cm with plasmids pTrc99a-ECHT and pBHR T7-ald to produce strain BL21 (DE3) 1.5GI-yqhD:: Cm / pTrc99a-ECHT / pBHR T7-ald. Two independent isolates were tested by production of 1-butanol exactly as described above. The results are given in Tables 8 and 9.

Table 8 Production of 1-butanol by E. coli strain BL21 (DE3) 1.5GI- yqhD:: Cm / pTrc99a-E-C-H-T / pBHR T7-ald. -quantitative values were determined from the CLAR analysis. - "-" indicates not detected. "+" indicates qualitative, positive identification by GC with a lower detection limit than with CLAR. Strain suffixes "a" and "b" indicate independent isolates.

Table 9 Production of 1-butanol per E strain. coli BL21 (DE3) 1.5GI- yqhD:: Cm / pTrc99a-E-C-H-T / pBHR T7-ald.

-The values were determined from the CLAR analysis. - "-" indicates not detected. "+" indicates qualitative, positive identification by GC with a lower detection limit than with CLAR. Strain suffixes "a" and "b" indicate independent isolates.

The E. coli strain MG1655 (DE3) 1.5GI-yqhD:: Cm was transformed with plasmids pTrc99a-ECHT and pBHR Ptrc-ald (opt) to produce the strain MG1655 (DE3) 1.5GI-yqhD:: Cm / pTrc99a- ECHT / pBHR-Ptrc-ald (opt). Two independent isolates were initially grown in LM medium containing 50 μg / ml kanamycin and 100 μg / ml carbenicillin. The cells were used to inoculate shaken flasks (approximately 175 mL total volume), containing 50 and 150 mL of TM3a / glucose medium (with appropriate antibiotics). The flasks were inoculated to an initial OD550 of < 0.04 units and incubated as described above, with and without induction. The IPTG was added to a final concentration of 0.04 mM; the OD550 of the flasks at the time of the addition was between 0.6 and 1.2 units. In this case, the induction was not necessarily for the expression of the 1-butanol path gene, because of the permeability of the inducible IPTG promoters and the constitutive nature of the 1.5GI promoter, however, they provide induction with an interval from broader expression. Approximately 15h after the induction, an aliquot of the broth was analyzed by GC with detection of flame ionization by content of 1-butanol, as described above. The results are given in Table 10. For the recombinant strains of E. coli, 1-butanol was produced in all cases; in separate experiments, E. coli strains of native type were shown to produce undetectable 1-butanol (data not shown).

Table 10 Production of 1-butanol per E strain. coli MG1655 1.5GI- yqhD:: Cm / pTrc99a-E-C-H-T / pBHR-Ptrc-ald (opt).

Strain suffixes and "b" indicate independent isolates EXAMPLE 14 Production of 1-Butanol from Glucose using B. recombinant subtillis. This example describes the production of 1-butanol in Ba cillus subtilis. The six genes of the 1-biosynthetic path, which encode six enzymatic activities, were divided into two operons per expression. The first three genes of the trajectory (thk, hbd, and crt) were integrated into the chromosome of Bacillus subtilis BE1010 (Payne and Jackson, J. Bacteriol 173: 2278-2282 (1991)). The last three genes (EgTER, ald, and bdhB) were cloned into an expression plasmid and transformed into the Bacillus strain carrying the integrated 1-butanol genes. Unless indicated otherwise in the text, the cloning primers described in this Example are referenced by SEQ ID NO: in Table 4, and PCR selection and sequencing primers are referenced by their SEQ ID NO: in Table 5. Integration Plasmid. Plasmid pFP988 is a Bacillus integration vector that contains an E. coli replicon from pBR322, a marker of ampicillin antibiotic for selection in E. coli and two sections of homology to the sacB gene on the Ba cillus chromosome, which directs the integration of the vector and intervention sequence by homologous recombination. Between sacB homology regions, there is the Pamy promoter and the signal sequence that can direct the synthesis and secretion of a cloned gene, a His tag and erythromycin as a selectable marker for Bacillus. The Pamy promoter and the signal sequence is from the alpha-amylase of Bacill us amyloliquefaciens. The promoter region also contains the lacO sequence for regulation of expression by a lacl repressor protein. The sequence of pFP988 (6509 bp) is given as SEQ ID NO: 79. Since the 1-butanol pathway genes were expressed in the cytoplasm, the amylase signal sequence was deleted. Plasmid pFP988 was amplified with Pamy / lacO F and Pamy / lacO R primers creating a 317 bp (0.3 kbp) product, which contains the Pamy / lacO promoter. The 5 'end of the Pamy / lacO F primer incorporated a BsrGI restriction site followed by an EcoRI site. The 5 'end of the Pamy / lacO R primer incorporated a BsrGI restriction site followed by a Pmel restriction site. The PCR product was TOPO cloned with pCR4Blunt-TOPO creating pCR4Blunt-TOPO-Pamy / lacO. Plasmid DNA was prepared from night cultures and was allowed to sequence with forward M13 and Inverse M13 primers (SEQ ID NO: 45 and SEQ ID NO: 46, respectively), to ensure no mutation has been introduced into the promoter . A clone of pCR4Blunt-TOPO-Pamy / lacO was digested with BsrGl and the 0.3 kbp fragment was gel purified. The pFP988 vector was digested with BsrGI resulting in suppression of 11 bp of the sacB homology region of 5 'and the removal of the Pamy / lacO promoter and the signal and His tag sequence. The digested vector BsrGI of 6 kbp was gel purified and ligated with the BsrGI Pamy / lacO insert. The resulting plasmids were selected with primers Pamy SeqF2 and Pamy SeqR to determine the orientation of the promoter. The correct clone restored the Pamy / lac promoter to its original orientation and was named pFP988Dss. The cassette with the thl-crt genes was constructed with SOE (splice by opening extension). The genes were amplified using as template pUC19dss:: Operon. The thl primers were Topo TF and Bot TR amplifying a product of 0.9 kbp. The crt primers were Top CF and Bot CR amplifying a product of 1.3 kbp. The two genes were linked by SOE with PCR amplification using Topo TF and Bot CR primers generating a 2.1 kbp product that was cloned TOPO in pCR4Blunt-TOPO creating pCR4Blunt-TOPO-T-C. The clones were admitted for sequencing to confirm the sequence. Plasmid pCR4Blunt-TOPO-T-C was digested with BstEII and Pmel releasing a 2.1 kbp fragment that was gel purified. The insert was treated with Klenow polymerase to blunt the BstEII site. The vector pFP988Dss was digested with Pmel and treated with bovine intestinal alkaline phosphatase (New England BioLabs), to prevent auto-ligation. The 2.1 kpb thl-crt fragment and the pFP988Dss digested were ligated and transformed into TopolO strains of E. coli The transformants were selected by PCR amplification with Pamy SeqF2 and N7SeqR2 for a product of 0.7 kbp, the correct product is called pFP988Dss-T-C. The construction of the thl-crt cassette creates unique Sali and Spel sites between the two genes. To add the hbd gene to the cassette, the hdb gene is subcloned from pCR4Blunt-TOPO-hbd as a Sall / Spel fragment of 0.9 kbp. The vector pFP988Dss-T-C is digested with Sali and Spel and the 8 kbp vector fragment is gel purified. The vector and the hbd insert are ligated and transformed into Topo 10 E cells. coli Transformants are selected by PCR amplification with Pamy SeqF and N3SeqF3 primers for a 3.0 kbd fragment. The resulting plasmid is named pFP988Dss-T-H-C. The Pamy promoter is subsequently replaced with the Pspac promoter of plasmid pMUTIN4 (Vagner et al., Microbiol. 144: 3097-3104 (1998)). The Pspac promoter is purified from pMUTIN4 with Spac F and Spac R primers as a product of 0.4 kbd and TOPO cloned in pCR4Blunt-TOPO. Transformants are selected by PCR amplification with M13 Front and M13 Inverse primers for the presence of an insert 0.5 kbd. Positive clones are provided for sequencing with the same primers. The pCR4Blunt-TOPO-Pspac plasmid is digested with Smal Xhol and the 0.3 kbd fragment is gel purified. The pFP988Dss-T-H-C vector is digested with Smal and Xhol and the 9 kbd vector is isolated by gel purification. The digested vector and the Pspac insert are ligated and transformed into Topo 10 cells of E. coli. Transformants are selected by PCR amplification with SpacF Seq and N7SeqR2 primers. Positive clones yield a product of 0.7 kbd. The plasmid DNA is prepared from positive clones and selected by PCR amplification with SpacF Seq and N3SeqF2 primers. Positive clones yield a PCR product of 3 kbd and are named pFP988DssPspac-T-H-C.

Integration in BE1010 of B. subtilis to form? sacB:: T-H-C:: erm # 28 of B. subtilis comprising exogenous thl, hbd and crt genes. The competent cells of BE1010 B. subtilis are prepared as described in Doyle et al., J. Bacteriol. 144: 957-966 (1980). The competent cells are collected by centrifugation and the cell pellets are resuspended in a small volume of the cell supernatant. Two volumes of the SPII-EGTA medium (Methods for General and Molecular Bacteriology, P. Gerthardt et al., Eds, American Society for Microbiology, Washington, DC (1994)) are added for 1 volume of competent cells. Aliquots of 0.3 ml of the cells are dispersed in test tubes and the plasmid pFP988DssPspac-T-H-C is added to the tubes. Cells are incubated for 30 minutes at 37 ° C with shaking, after which which 0.1 ml of 10% yeast extract is added to each tube and the cells are further incubated for 60 minutes. Transformants are placed for selection in LB erythromycin plates using the double-agar overlay method (Methods for General and Molecular Bacteriology, supra). The transformants are initially selected by PCR amplification with primers Pamy SeqF and N5SeqF3. Positive clones that amplify the expected 2 kbp PCR product are further selected by PCR amplification. If the insertion of the cassette into the chromosome has occurred via a double crossing even if the primer establishes sacB Up and N7SeqR2 and the primer establishes sacB Dn and N4SeqR3 can amplify a product of 1.7 kbp and one of 2.7 kbp respectively. A positive clone is identified and named? SacB:: T-H-C:: erm # 28 of B. subtilis.

Expression of EgTER gene plasmid, ald and bdhB. The three remaining 1-butanol genes are expressed from plasmid pHTO1 (MocBitec). Plasmid pHTOl is a shuttle vector Bacillus-E. coli that replicates via a theta mechanism. The cloned proteins are expressed from the GroEL promoter fused to a lacO sequence. Downstream of lacO is the efficient RBS of the gsiB gene followed by an MCS. The ald gene is amplified by RCO with AF BamHI and AR Aat2 primers using pUCl9dHS-ald-dbhB (described in Example 9) as a template, creating a 1.4 kbp product. The product is TOPO cloned in pCR4-T0P0 and transformed into ToplO cells of E. coli The transformants are selected with forward M13 and forward M13 primers. Positive clones amplify a 1.6 kbp product. Clones are provided for sequencing with forward M13 and reverse M13 primers, N31SeqF2, N31SeqF3, N32SeqF3, N32SeqR2, N32SeqR3 and N32SeqR4. The plasmid is named pCR4T0P0-B / A-ald. The vector pHTOl and the plasmid pCR4T0P0-B / A-ald are both digested with BamHI and Aatll. The 7.9 kbp vector fragment and the 1.4 kbp ald fragment are ligated together to create pHTOl-ald. The ligation is transformed into TopolO cells of E. coli and the transformants are selected by PCR amplification with primers N31SeqFl and HT R for a product of 1.3 kbd. To add the last two stages of the trajectory to the vector pHTOl, two cloning schemes are designed. For both schemes, EgTER and bdhB are amplified together by SOE. Subsequently, the EgTER-bdh fragment is cloned into either pHTOl-ald creating pHTOl-ald-EB or cloned into pCR4-TOPO-B / A-ald creating pCR4-TOPO-ald-EB. The ald-EgTer-bdhB fragment of the TOPO vector is then cloned into pHTOl creating pHTOl-AEB. An EgTER-bdhB fragment is amplified by PCR using forward 1 (E) and reverse 2 (B) primers, using template DNA giving SEQ ID NO: 208. The resulting 2.5 kbp PCR product is TOPO cloned into pCR4Blunt-TOPO, creating pCR4Bunt-TOPO-E-B. The TOPO reaction is transformed into ToplO cells of E. coli Colonies are selected with forward M13 and forward M13 primers by PCR amplification. Positive clones generate a product of 2.6 kbp. The clones of pCR4Blunt-TOPO-E-B are provided by sequencing with forward and reverse M13 primers, N62SeqF2, N62SeqF3, N62SeqF4, N63SeqRl, N63SeqR2, N63SeqR3, NllSeqFl and NllSeqF2, N12SeqRl and N12SeqR2. Plasmid pCR4Blunt-TOPO-E-B is digested with Hpal and Aatll to release a 2.4 kbp fragment. The E-B fragment is treated with Klenow polymerase to blunt the end and then purified by gel. Plasmid pHTOl-ald is digested with Aatll and treated with Klenow polymerase to blunt the ends. The vector is then treated with calf intestinal alkaline phosphatase and purified in gel. The E-B fragment is ligated to the linearized vector pHTOl, transformed into ToplO cells of E. coli, and is selected on LB plates containing 100 μg / ml ampicillin. Transformants are selected by PCR amplification with N3SeqFl and N63SeqRl primers to provide a 2.4 kbp product. The resulting plasmid, pHTOl-ald-EB, is transformed into JM103 cells, a strain of E. coli recAY Plasmids prepared from recA * strains form more multimers than recA * strains. The Bacillus subtilis is transformed more efficiently with plasmid multimers instead of monomers (Methods for General and Molecular Bacteriology, supra). The plasmid DNA is prepared from JM103 and transformed into? SacB:: THC:: erm # 28 of B. subtilis component forming the strain? SacB:: THC:: erm # 28 / pHT01-ald-EB from B. subtilis . The competent cells are prepared and transformed as previously described. Transformants are selected on LB plates containing 5 μg / ml chloramphenicol and selected by PCR colony with the primers N31SeqFl and N63SeqR4 for a product of 1.3 kbp. In the alternative cloning strategy, pCR4Blunt-TOPO-E-B is digested with Hpal and Aatll that releases a 2.4 kbp fragment that is gel purified. The pCR4-TOPO-B / A-ald plasmid is digested with Hpal and Aatll and the 5.4 kbp vector fragment is gel purified. The vector fragment of pCR4-TOPO-B / A-ald is ligated with the Hpal-Aatll E-B fragment creating pCR4-TOPO-ald-EB. The ligation is transformed into E. coli ToplO cells and the resulting transformants are selected by PCR amplification with primers NllSeqF2 and N63SeqR4 for a 2.1 kbp product. The plasmid pCR4-TOPO-ald-EB is digested with BamHI and Aatll and Sphl. The digested BamHI / AatlI releases an ald-EB fragment of 3.9 kbp that is purified by gel. The purpose of the Sphl is digested to cut the remaining vector into the small fragments so that it does not co-migrate in a gel with the insert ald-EB. The pHTOl vector is digested with BamHII and Aatll and the 7.9 kbp vector fragment is gel purified. The vector and fragments of the ald-EB insert are ligated to form the plasmid pHTOl-AEB and transformed into ToplO cells of E. coli. Colonies are selected by PCR amplification with primers N62SeqF4 and HT R for a 1.5 kbp product. The plasmid is prepared and transformed into JM103. Plasmid DNA is prepared from JM103 and transformed into? SacB:: T-H-C :: erm # 28 from competent B. subtilis which forms strain? SacB:: T-H-C :: erm # 28 / pHT01-AEB from B. subtilis. Competent BE1010 cells are prepared and transformed as previously described. Bacillus transformants are selected by PCR amplification with primers N31SeqFl and N63SeqR4 for a product of 1.3 kbp.

Demonstration of 1-butanol production from recombinant B. subtilis. Three independent isolates from each strain of? SacB:: THC:: erm # 28 / pHT01-ald-EB from B. subtilis and? SacB:: THC :: erm # 28 / pHT01-AEB from B. subtilis are inoculated into flasks agitated (approximately 175 mg total volume) containing 15 ml of medium. A BE1010 strain of B. subtilis lacking exogenous 1-butanol, six gene trajectories are also included as a negative control. The medium contained (per liter): 10 ml of 1 M of (NH4) 2S0; 5 ml of 1M of potassium phosphate buffer, pH 7.0; 100 ml of 1M MOPS / KOH buffer, pH 7.0; 20 ml of 1M L-glutamic acid, potassium salt; 10 g of glucose; 10 ml of 5 g / l each of L-methionine, L-tryptophan and L-lysine; 0.1 g each of yeast extract and casamino acids; 20 ml of metal mixture and appropriate antibiotics (5 mg of chloramphenicol and erythromycin for the recombinant strains). The target mixture contains 200 mM MgCl2, 70 mM CaCl2, 5 mM MnCl2, 0.1 mM FeCl3, 0.1 mM ZnCl2, 0.2 mM thiamine hydrochloride, 172 μM CuS04, 253 μM CoCl2 and 242 μM of Na2Mo04. The flasks are inoculated starting from an OD50o of = 0.1 units, sealed with non-ventilated layers, and inoculated at 37 ° C with agitation at approximately 200 rpm. Approximately 24 hours after the inoculation, an aliquot of the broth is analyzed by CLAR (Shodex Sugar SH1011 column) with refractive index (Rl) and GV detection (Varian CP-WAX 58 Column (FFAP) CB, 0.25 mm X 0.2 μm X 25 m) with structure ionization detection (FID) for 1-butanol content, as described in the General Methods section. The results of the 1-butanol determinations are given in Table 11.

Table 11 Production of 1-butanol by strains? SacB:: T-C-C:: erm # 28 / pHT01-ald-EB from B. subtilis and? SacB:: T-H-C:: erm # 28 / pHT01-AEB from B. subtilis * Concentration determined by GC. - the suffixes "a", "b" and "c" of the strain indicate separate isolates.

EXAMPLE 15 Production of 1-butanol from Glucose or Sucrose by Recombinant E. coli To endow MG1655 with E. coli with the ability to use sucrose as the carbon and energy source for production of 1-butanol, a pooling of the gene Use of sucrose (cscBKA) from plasmid pScrl (described below) is subcloned into pBHR-Ptrc-ald (opt) (described in Example 13) in this organism. The sucrose utilization genes (cscA, cscK and cscB) encode a hydrolase of sucrose (CscA), provided as SEQ ID NO: 157, D-fructokinase (CscK), provided as SEQ ID NO: 158 and sucrose permease (CscB), provided as SEQ ID NO: 159. To allow constitutive expression of the three genes from its natural promoter, the sucrose-specific repressor gene, cscR, which regulates the clustering of the gene is not present in the construct.

Cloning and expression of the cluster of the sucrose utilization gene cscBKS in the plasmid pBHR-Ptrc-ald (opt). Clustering of the sucrose utilization gene cscBKA, provided as SEQ ID NO: 156, is isolated from genomic DNA of an E. coli strain using sucrose derived from ATCC 13281 of the E. coli strain. The genomic DNA is digested to completion with BamHI and EcoRI. Fragments having an average size of approximately 4 kbp are isolated from an agarose gel, ligated to plasmid pLitmus28 (New England Biolabs, Beverly, MA), which are then digested with BamHI and EcoRI. The resulting DNA is transformed into TOP10F 'from ultracompetent E. coli (Invitrogen, Carisbad, CA). The transformants are placed on MacConkey agar plates containing 1% sucrose and 100 μg / ml ampicillin and selected for purple colonies. The plasmid DNA is isolated for the purple and sequenced transformants using forward M13 primers (SEQ ID NO: 45), Inverse M13 (SEQ ID NO: 46), scrl (SEQ ID NO.160), scr6 (SEQ ID NO: 161), scr3 (SEQ ID NO: 162) and scr4 (SEQ ID NO: 163). The plasmid containing cscB, cscK and cscA genes (cscBKA) are designated pScrl. Plasmids pSCrl are digested with XhoI and treated with the Klenow fragment of DNA polymerase to make blunt ends. The plasmid is then digested with Agel, and the clustering fragment of the cscBKA 4,179 gene is gel purified. The plasmid pBHR-Ptrc-ald (opt) is prepared as described in Example 13 and digested with Agel and Nael. The resulting pBHR-Ptrc-ald (opt) 6,003 bp fragment is gel purified. The cscBKA fragment is ligated with the pBHR-Ttrc-ald (opt), providing pBHR-Ptrc-ald (opt) -cscAKB. The plasmid pBHR-Ptrc-ald (opt) -cscAKB is transformed into electrocomposite NovaXG cells of E. coli (Novagen, Madison, Wl) and the use of sucrose is confirmed by placing the transformants on McConkey agar plates containing 2% sucrose and 25 μg / ml kanamycin. In the pBHR-Ptrc-ald (opt) -cscAKB construct, the sucrose utilization gene is cloned downstream of Ptrc-ald (opt) as a separate fragment in the order cscA, cscK and cscB. Alternatively, sucrose utilization genes are cloned in the opposite direction in pBHR-Ptrc-ald (opt). The plasmid pBHR-Ptrc-ald (opt) is digested with Seal and Agel, and the fragment pBHT-Ptrc-ald (opt) of 5.971 bp is purify with gel. The cscBKA fragment 4,179 bp, prepared as described above, is ligated with the pBHR-Ptrc-ald fragment (opt), yielding pBHR-Ptrc-ald (opt) -cscBKA. The plasmid pBHR-Ptrc-ald (opt) -cscBKA is transformed into electrocomposite NovaXG cells of E. coli (Novagen, Madison, Wl) and confirmed to use by placing the transformants on McConkey agar plates containing 2% sucrose and 25 μg / ml of kanamycin. In the pBHR-Ptrc-ald (opt) -cscBKA construct, the sucrose utilization genes are cloned as upstream of the separate fragment of Ptrc-ald (opt) in the order cscB, cscK and cscA.

Demonstration of glucose or sucrose 1-butanol production using E. recombinant coli. MG 1655 1.5GI-yqhD:: Cm of strain E. coli (described in Example 13) is transformed with pTrc99a-ECHT plasmids (prepared as described in Example 13) and pBHR-Ptrc-ald (opt) -cscAKB or pBHR-Ptrc-ald (opt) -cscBKA to produce two strains, MG1655 1.5GI-yqhD:: Cm / pTrc99a-ECHT / pBHR-Ptrc-ald (opt) -cscAKB # 9 and MG1655 1.5 GI-yqhD:: Cm / pTRC99a-ECHT / pBHR-Ptrc-ald (opt) -cscBKA # 1. Starting the cultures of the two strains are prepared by growing the cells in the LB medium containing 25 μg / ml kanamycin and 100 μg / ml carbenicillin. These cells are then used to inoculate shaken flasks (approximately 175 ml of total volume) containing 50, 70 and 150 ml of TM3A / glucose medium (with appropriate antibiotics) to represent high medium and low oxygen conditions, respectively, as described in Example 13. A third strain, MG1655 / pScrl of E. coli, grows in TM3a / glucose medium containing 100 μg / ml of carbenicillin, is used as a negative control. For each of the strains, an identical series of flasks was prepared with TM3a / sucrose medium (with appropriate antibiotics). The TM3a / sucrose medium is identical to the TM3a / glucose medium, except that sucrose (10 g / l) replaces the glucose. The flasks were inoculated at an initial OD550 of < 0.03 units and incubated as described in Example 13. With the exception of the negative control flasks, the IPTG was added to the flasks (final concentration of 0.04 mM) when the cultures reached an OD55o between 0.2 and 1.8 units. The cells were harvested with the OD550 of the cultures increased at least 3 times. Approximately 24 hours after the inoculation, an aliquot of the broth was analyzed by CLAR (Shodex Sugar SHlOll column) with refractive index (Rl) detection and GC (HP-INNOWax column, 30 m x 0.53 mm id, 1 μm film thickness) with flame ionization detection (FID) for 1-butanol content, as described in the section General Methods. The concentrations of 1-butanol in crops after growth in the medium containing glucose and sucrose are given in Table 12 and Table 13, respectively. Both recombinant E. coli strains containing the biosynthetic pathway of 1-butanol produce 1-butanol from glucose and sucrose under all oxygen conditions, while the negative control strain does not produce detectable 1-butanol.

Table 12 Production of 1-butanol from glucose by recombinant E. coli strains MG1655, 1.5Gl-yqhD:: Cm / pTrc99a-EC-HT / pHBR-Ptrc-ald (opt) -cscAKB # 9 and MG1655 1.5G1 - yqhD:: Cm / pTrc99a-E-CH-T / pBHR-Ptrc-ald (opt) -cscBKA # l Table 13 Production of 1-butanol from glucose by recombinant E. coli strains EXAMPLE 16 Production of 1-butanol from Sucrose using B. recombinant subtilus This example describes the production of 1-butanol from sucrose using recombinant Bacillus subtilus. Two independent isolates of strain B. subtilis? sacB:: THC:: erm # 28 / pHT01-ald-EB (Example 14), were examined by production of 1-butanol essentially as described in Example 14. The strains were inoculated in shake flasks (approximately 175 mg of total volume) containing either 20 ml or 100 ml of medium to simulate high and low oxygen conditions, respectively. Medium A was exactly as described in Example 14, except that the glucose was replaced with 5 g / L of sucrose. Medium B was identical to the TM3a / glucose medium described in Example 13, except that the glucose is replaced with 10 g / l sucrose and the medium is supplemented with (per L) 10 ml of a solution 5 g / l each of L-methionine, L-tryptophan and L-lysine . The flasks were inoculated at an initial OD550 of < 0.1 units, covered with vented plugs and incubated at 34 ° C with shaking at 300 rpm. Approximately 24 hrs after the inoculation, an aliquot of broth was analyzed by GC (Column HP-INNOWax, 30 mx 0. 53 mm id, 1.0 μm film thickness) with FID detection by 1-butanol content, as described in the General Methods section. The results of the 1-butanol determinations are given in Table 14. The recombinant Bacillus strain containing the biosynthetic pathway of 1-butanol produces detectable levels of 1-butanol under high and low oxygen conditions in both media. Table 14 Production of 1-butanol from glucose by strains of B. subtilis? sacB:: T-H-C:: erm # 28 / pHT01-ald-EB - "- Concentration determined by GC 2" + "indicates qualitative presence of 1-butanol Suffixes of strain" a "and" b "indicate separate isolates EXAMPLE 17 Production of 1-butanol from glucose and sucrose using recombinant Saccharomyces cerevisiae This example describes the production of 1-butanol in the yeast Saccharomyces cerevisiae. One of the six genes encoding the enzymes catalyzed the steps in the biosynthetic pathway of 1-butanol, five were cloned into three compatible yeast plasmids of 2 microns (2 μ) and co-expressed in Saccharomyces cerevisiae. The "superior" trajectory "is defined as the first of the three enzymatic stages, catalyzed by acetyl-CoA acetyltransferase (thIA, thiolase), 3-hydroxybutyryl-CoA dehydrogenase (hbd) and crotonane (crt). as the fourth (butyl-CoA dehydrogenase, ter) and the fifth (butylaldehyde dehydrogenase, ald), enzymatic stages of the trajectory.The last enzymatic stage of the path of 1-butanol is catalyzed by alcohol dehydrogenase, which can be encoded by endogenous yeast genes, eg, adhl and adhll. Gene expression in yeast typically requires a promoter, followed by the gene of interest, and a transcriptional terminator. A number of yeast constitutive promoters are used in the construction of expression cassettes for genes encoding the biosynthetic pathway of 1-butanol, including FBA, GPD and GPM promoters. Some inducible promoters, for example GALI, GALIO, CUP1, were also used in the construction of intermediate plasmids, but not in the final demonstration strain. Several transcriptional terminators were used, including FBAt, GPDt, GPMt, FRGIOt, and GALlt. The genes encoding the biosynthetic pathway of 1-butanol were first subcloned into a yeast plasmid flanked by a promoter and a terminator, which provides expression cassettes for each gene. The expression cassettes were optionally combined into a single vector by opening repair cloning as described below. For example, the three cassettes of the gene encoding the superior path were subcloned into a 2μ plasmid of yeast. The ter and ald genes were each individually expressed in the 2μ plasmids. The co-transformation of the three plasmids into a single yeast strain resulted in a biosynthetic pathway of 1-butanol. Alternatively, several DNA fragments encoding promoters, genes and terminators were directly combined into a single vector by opening repair cloning.

Methods for construction of plasmids and strains in yeast of Saccharomyces cervisiae. Basic yeast molecular biology protocols that include transformation, cell growth, gene expression, open repair recombination, etc., are described in Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthire and Gerald R. Fink (Eds.,) Elsevier Academic Press, San Diego, CA). The plasmids used in this Example were the shuttle vectors of -S. cerevisiae -E. coli pRS423, pRS424, pRS425, and pRS426 (American Type Culture Collection, Rockville, MD), which contain an origin of replication of E. coli (for example, pMBl), a yeast replication origin of 2 μ and a marker for nutritional selection. The selection markers for these four vectors are His3 (vector pRS423), Trpl (vector pRS424), Leu2 (vector pRS425) and Ura3 (vector pRS426). These vectors allow the propagation of the strain in love strains of E. coli and yeast. A haploid strain of yeast BY4741 (MATa his3? L leu2? 0 metl5? 0 ura3? 0) (Research Genetics, Huntsville, AL, also available from ATCC 201388) and a diploid strain BY4743 (MATa / alpha his3? L / his3? L leu2? 0 / leu2? 0 lis2? 0 / LIS2 MET15 / metl5? 0 ura3? 0 / ura3? 0) (Research Genetics, Huntsville, AL, also available from ATCC 201390), were used as hosts for the cloning and expression of the gene. The construction of expression vectors for genes encoding the enzymes of the biosynthetic pathway of 1-butanol were performed either by standard molecular cloning techniques in E. coli or by the open repair recombination method in yeast. The opening repair cloning procedure takes advantage of highly efficient homologous recombination in yeast. Typically, a DNA yeast vector is digested (e.g., at its multiple cloning site) to create an "opening" in its sequence. A number of DNA inserts of interest containing a sequence of > 21 bp in both the 5 'and 3' ends that sequentially overlap each other, with the terms 5 'and 3' of the DNA vector. For example, to construct a yeast expression vector for "Gen X", a yeast promoter and a yeast terminator are selected by the expression cassette. The promoter and terminator are amplified from the yeast genomic DNA, and the X gene is either amplified by PCR from its source organism or obtained from a cloning vector comprising the sequence of Gen X. There is at least , a 21 bp overlap sequence between the 5 'end of the linearized vector and the promoter sequence between the promoter and the X gene, between the X gene and the terminator sequence, and between the terminator and the 3 'end of the linearized vector. The "opening" vector and the DNA insert are then co-transformed into a yeast strain and plated into the minimal marginal SD medium, and the colonies were selected for growth of cultures and mini-preparations for plasmid DNAs. The presence of correct insert combinations can be confirmed by PCR mapping. The ADM plasmid isolated from yeast (usually low in concentration), then can be transformed into a strain of E. coli, for example, TOP10, followed by mini preparations and restriction mapping to further verify the plasmid construct. Finally, the construct can be verified by sequence analysis. The positive plasmid yeast transformants are grown in SD medium to perform the enzyme assays to characterize the activities of the enzymes expressed by the genes of interest. The yeast cultures were grown in YPD complex medium or synthetic minimum reduced medium containing glucose (SD medium) and the appropriate mixtures of compounds that allow the complementation of the nutritional selection markers in the plasmids (Methods in Enzymology, Volume 194 , Guide to Yeast Genetics and Molecular and Cell Biology, 2004, Part A, pp.13-15). The sugar component in the reduced medium SD was 2% glucose. For production of 1-butanol, the yeast cultures were also grown in reduced Medium Synthetic with 2% sucrose (SS medium). By enzymatic activity analysis, a single colony of each strain was scored on a fresh plate containing minimum SD reduced medium and incubated at 30 ° C for 2 days. The cells in this plate were used to inoculate 20 ml of reduced SD medium in a shaken 125 ml flask and grown overnight at 30 ° C, with shaking at 250 rpm. Optical density (OD60o) of the culture was measured overnight, and the culture was diluted to ODeoo = 0.1 in 250 ml of the same medium in a shake flask 1.0 1, and grown at 30 ° C with shaking at 250 rpm to an ODeoo between 0.8 to 1.0. The cells were then collected by centrifugation at 2000 x g for 10 minutes, and resuspended in 20 ml of 50 mM Tris-HCl buffer, pH 8.5. Enzyme assays were performed as described above.

Construction of plasmid pNY102 for co-expression of thA and hbd. A number of dual expression vectors were constructed for the co-expression of the thIA and hbd genes. The ERGIO gene of Sa ccharomyces cerevisiae is a functional ortholog of the thIA gene. Initially, a dual vector of ERGIO and hbd was constructed using the divergent dual promoter of yeast GAL1-GAL10, the GALI terminator (GALlt) and the ERGIO terminator (ERGIOt). The ERGIOt-gene DNA fragment ERGIO, was amplified by PCR from the genomic DNA of Saccharomyces cerevisiae strain BY4743, using primers OT731 (SEQ ID NO: 164) and OT732 (SEQ ID NO: 165). The GALlO-Gall divergent veiling promoter was also amplified by PCR from the genomic DNA BY4743 using primers OT733 (SEQ ID NO: 166) and OT734 (SEQ ID NO: 167). The hbd gene was amplified from the E. coli plasmid pTrc99a-E-C-H-T (described in Example 13), using PCR primers OT735 (SEQ ID NO: 168) and OT736 (SEQ ID NO: 169). GAL lt was amplified from BY4743 genomic DNA using primers OT737 (SEQ ID NO: 170) and OT738 (SEQ ID NO: 171). Four PCR fragments, ergIO-ERGIOt, GAL1-GAL10, hbd, and GALlt promoters were thus obtained with approximately 25 bp of overlapping sequences between each of the adjacent PCR fragments. The GALlt and ERGIO-ERGIOt fragments each contain approximately 25 bp of overlapping sequences with the yeast vector pRS425. To assemble these sequences by gap repair recombination, the DNA fragments were co-transformed into the yeast strain PY4741 (together with the vector pRS425, which was digested with the enzymes BamHI and HindIII.) The colonies were selected from minimal SD plates. -Leu, and the clones with inserts were identified by PCR amplification.The new plasmid was named pNY6 (pRS425. ERGlOt-erglO-GALlO-GALl-hbd-GALlt).

Additional conformation was performed by restriction mapping. Yeast strain BY4741 (pNY6), prepared by transformation of plasmid pNY6 in S. cerevisiae BY4741, showed good Hbd activity but no thiolase activity. Due to the lack of thiolase activity, the ERGIO gene was replaced with the thIA gene by opening repair recombination. The thIA gene was amplified from the E vector. coli pTrc99a-E-C-H-T by PCR using primers OT797 (SEQ ID NO: 172), which adds a restriction site Sphl, and OT798 (SEQ ID NO: 173), which adds an Ascl restriction site. Plasmid pNY6 was digested with restriction enzymes Sphl and Pstl, gel purified and co-transformed in yeast PY4741 together with the thIA PCR product. Due to the 30 bp of overlapping sequences between the thIA PCR product and the digested pNY6, the thIA gene was recombined into pNY6 between the GALIO promoter and the ERGIOt terminator. This plasmid provided pNY7 (pRS425, ERGIOt-thlA-GALlO-GALl-hbd-GALlt), which was verified by PCR and restriction mapping. In a subsequent cloning step based on the aperture repair recombination, the GALIO promoter in pNY7 was replaced with the CUP1 promoter, and the GALI promoter was replaced with the strong GPD promoter. This pNYlO plasmid (pRS425, ERGIOt-thlA-CUPl-GPD-hbd-GALlt), allowed the expression of the thIA gene under CPU1, a copper-inducible promoter, and the expression of the hbd gene under the GPD promoter. The Promoter sequence CUP1 was amplified from PCR from the yeast genomic DNA BY4743 using primers OT806 (SEQ ID NO.174), and OT807 (SEQ ID NO.175). The GPD promoter was amplified from the genomic DNA BY4743 using primers OT808 (SEQ ID NO: 176) and OT809 (SEQ ID NO: 177). The PCR products of the CPU1 and GPD promoters were combined with pNY7 plasmid digested with restriction enzymes Ncol and Sphl. From this stage of opening repair cloning, the plasmid pNY10 was constructed, which was verified by PCR and restriction mapping. Yeast strain BY4741 containing pNYlO, has Hbd activity, but no ThIA activity. The Hbd activity under the GPD promoter was significantly improved compared to the GALl promoter of controlled Hbd activity (1.8 U / mg against 0.40 U / mg). The sequencing analysis revealed that the thIA gene in pNYlO, has a deletion of a base near the 3 'end, which resulted in a truncated protein. This explains the lack of thiolase activity in the strain. Plasmid pNY12 was constructed with the correct thIA gene sequence. The thIA gene was cut from the pTrc99a-E-C-H-T vector by digestion with Sphl and Ascl. The FAB1 promoter was amplified by PCR of BY4743 genomic DNA using primers PT799 (SEQ ID NO: 178) and OT761 (SEQ ID NO: 179), and digested with SalI and Sphl restriction enzymes. The thIA gene fragment and the FAB1 promoter fragment were ligated into the pNYlO plasmid at Ascl and Salí sites, generating plasmid pNY12 (pRS425.ERG10t-thIA-FBAl), which was confirmed by restriction mapping. PNY12 was transformed into yeast strain BY4741 and the resulting transformant showed ThIA activity of 1.66 U / mg. The FBAl promoter fragment of the thIA gene of pNY12 was re-subcloned into pNYlO. The vector pNYlO was cut with the restriction enzyme Acl and ligated with the fragment of the thiA-promoter FBA1 gene digested with Ascl, isolated from the plasmid pNY12. This created a new plasmid with two possible insert orientations. Clones with FBA1 and GPD promoters located adjacent to each other in opposite orientation were chosen and this plasmid was named pNY102. PNY102 (pRS425.ERG10t-thIA-FBAl-GPD-hbd-GALlt), was verified by restriction mapping. Strain DPD5206 was made by transforming pNY102 into yeast strain BY4741. The thIA activity of DPD5206 was 1.24 U / mg and the Hbd activity was 0.76 U / mg.

Construction of pNYll plasmid by expression of crt. The crt gene expression cassette was constructed by combining the GPM1 promoter, the crt gene, and the GPMlt terminator in the pRS426 vector using open repair recombination in yeast. The GPM1 promoter was amplified by PCR from yeast BY4743 genomic DNA using primers PT803 (SEQ ID NO: 180) and OT804 (SEQ ID N0.181). The crt gene was amplified using OT785 PCR primers (SEQ ID.

NO: 182) and OT786 (SEQ ID NO: 183) of the E plasmid. coli pTrc99a-E-C-H-T. The GPMlt terminator was amplified by PCR from yeast genomic DNA BY4743 using PT787 (SEQ ID NO: 184) and OT805 (SEQ ID NO: 185). The yeast vector pRS426 was digested with BamHI and HindIII and gel purified.

This DNA was co-transformed with the PCR products of the GPM1 promoter, the crt gene and the GPM1 terminator in yeast competent cells BY4741. The clones with the correct inserts were verified by PCR and restriction mapping and the yeast strain BY4741 (pNYll: pRS426-GPMl-crt-GPMlt), has a Crt activity of 85 U / mg.

Construction of plasmid pNY103 for co-expression thIA, hbd and crt. For the co-expression of the higher pathway enzymes of 1-butanol, the cassette of the crt gene of pNYll was subcloned into the plasmid pNY102 to create an expression vector hbd, thIA and crt. A 2.347 bp DNA fragment containing the GPM1 promoter, the crt gene, and the GPM1 terminator was cut from the pNYll plasmid with restriction enzymes Sacl and NotI and cloned into the pNY102 vector, which was digested with NotI and partially digested with Sacl, producing the expression vector pNY103 (pRS425, ERGlOt-thlA-FBAl-GPD-hbd-GALlt-GPMlt-crt-GPMl). After the confirmation of the presence of the three cassettes in pNY103 by digestion with HindIII, the plasmid was transformed into the yeast cells BY4743 and the transformed yeast strain was named DPD5200. When grown under standard conditions, the DPD5200 showed ThIA, Hbd and Crt enzyme activities of 0.49 U / mg, 0.21 U / mg and 23.0 U / mg, respectively.

Construction of pNY8 plasmid for ald expression. An optimized codon gene named tery (SEQ ID NO: 186), which encodes the Ter protein (SEQ ID NO: 187), and an optimized codon gene named aldy (SEQ ID NO: 188), which encodes the Ald protein ( SEQ ID NO: 189), were synthesized using Saccharomyces cerevisiae preferred codons. The pTERy plasmid containing the optimized codon ter gene and pALDy containing the optimized codon ald gel was made by DNA2.0 (Palo Alto, CA). To mount pNY8 (pRS426.GPD-ald-GPDt), three insert fragments including a PCR product of the GPD promoter (synthesized from primers OT800 (SEQ ID NO: 190) and OT758 (SEQ ID NO: 191) and genomic DNA BY4743), a fragment of the aldy gene excised from pALDy by digestion with Ncol and Sfil (SEQ ID NO: 188), and a PCR product from the GPD terminator (synthesized from primers OT754 (SEQ ID NO: 192) and OT755 (SEC ID NO: 193), and genomic DNA BY4743), were recombined with pRS426 vector digested with BamHI, HindIII via gap repair recombination cloning. The yeast BY4741 transformation clones were analyzed by PCR mapping. The new plasmid thus constructed, pNY8, was further confirmed by restriction mapping. The yeast transformants BY4741 containing pNY8 were analyzed by Ald activity and the specific activity towards butyryl-CoA was approximately 0.07 U / mg.

Construction of plasmid pNY9 and pNY13 for the expression ter. The optimized codon tery gene was cloned into the pRS426 vector under the control of the FBA1 promoter by open repair cloning. The FBA1 promoter was amplified by PCR from the yeast BY4743 genomic DNA using primers OT760 (SEQ ID NO: 194) and OT792 (SEQ ID NO: 195). The tery gene was obtained by digestion of plasmid pTERy by restriction enzymes Sphl and Notl that resulted in the fragment given as SEQ ID NO: 186. The PCR fragment of the FBA1 terminator was generated by PCR from yeast BY4743 genomic DNA using primers PT791 (SEQ ID NO: 196) and OT765 (SEQ ID NO: 197). Three DNA fragments, the FBA1 promoter, the ter gene and the FBA1 terminator, were combined with the vector digested with BamHI and HindIII pRS426 and transformed into yeast BY4741 by opening repair recombination. The resulting plasmid, pNY9 (pRS425- FBAl-tery-FBAlt), was confirmed by PCR mapping, as well as restriction digestion. The yeast BY4741 transformant of pNY9 produces a Ter activity of 0.26 U / mg. To make the biosynthetic pathway of final 1-butanol, it was necessary to construct a yeast expression strain that contains several plasmids, each with a unique nutritional selection marker. Since the precursor vector pRS426 contains a selection marker Ura, the expression cassette ter was subcloned into the vector pRS423, which contains a His3 marker. A 3.2 kb fragment containing the cassette FBAl-tery-FBAlt was isolated from plasmid pNY9 by digestion with Sacl and Xhol restriction enzymes, and ligated into the vector pRS423 which was cut with these same two enzymes. The new plasmid, pNY13 (pRS423-FBAl-tery-FBAlt), was mapped by restriction digestion. PNY13 was transformed into strain BY4741 and the supernatant was cultured in SD-HIs medium, yielding a strain with an activity Ter of 0.19 U / mg.

Construction of a yeast strain containing biosynthetic pathway genes of 1-butanol for demonstration of 1-butanol production. As described above, yeast strain PDP5200 was constructed by transformation of plasmid pNY103 into strain BY4743 of S. cerevisiae, which allows co-transformation of thIA genes, hbd and crt. Yeast competent PDP5200 cells were prepared as described above, and plasmids pNY8 and pNY13 were co-transformed into DPD5200, generating strain DPD5213. DPD5213 allows simultaneous constitutive expression of five genes in the butanol biosynthetic pathway thIA, hbd, crt, ter and ald. Strain DPD5212 (strain BY4743 from S. cerevisiae was transformed with the empty plasmids pRS425 and pRS426), was used as a negative control. Four independent isolates of strain DPD5213 were grown in minimal reduced medium SD-Ura-Leu-His in the presence of either 2% glucose or 2% sucrose to allow the growth complementation of three plasmids. A single isolate of DPD5212 was grown similarly in an appropriate medium. To demonstrate the production of 1-butanol by aerobic cultures, a single colony of each strain was scratched on a fresh agar plate containing minimal reduced growth medium of SD (containing 2% glucose) or minimum reduced growth medium of SS (containing 2% sucrose) and incubated at 30 ° C for 2 days. Cells from these plates were used to inoculate 20 ml of the minimum reduced medium (either SD or SS) in shake flasks of 125 ml plastic and grown overnight at 30 ° C with shaking at 250 rpm. The optical density (OD60o) of the night culture was measured, the culture was diluted to OD600 of 0.1 in 25 ml of the same medium in a shake flask of 125 ml and was grown at 30 ° C with shaking at 250 rpm. Aliquots of the culture were removed at 24 h and 48 h by GC analysis of production of 1-butanol (HP-INNOWAX column, 30 mx 0.53 mm id, film thickness of 1 μm), with FID detection, as described in the section General Methods. The results of the GC analysis are given in Table 15.

Table 15 Production of 1-butanol from glucose and sucrose by strain S. cerevisiae DPD5213 - "- the independent isolates are indicated by a-d, Concentration determined by GC.

EXAMPLE 18 (Prophetic) Expression of the Biosynthetic Pathway of 1-Butanol in Lactobacillus plantarum The purpose of this prophetic Example is to describe how to express the biosynthetic pathway of 1-butanol in Lactobacillus plan tarum. The six genes of the trajectory of 1-butanol, which encode the activities of six enzymes, are divided into two operons for expression. The first three genes of the trajectory (thl, hbd and crt, which encode the enzymes acetyl-CoA acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase and crotonane, respectively), are integrated into the chromosome of Lactobacillus plantarum by homologous recombination using the method described by Hols et al. (Appl.

Environ. Microbiol. 60: 1401-1413 (1994)). The last three genes (EgTER, ald and bdhB, which encode the butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase and butanol dehydrogenase enzymes, respectively), are cloned into an expression plasmid and transformed into the Lactobacillus strain carrying the 1-butanol genes integrated upper path. Lactobacillus is grown in MRS medium (Difco Laboratories, Detroit, MI) at 37 ° C. Chromosomal DNA is isolated from La ctoba cill us plantarum as described by Moreira et al. (BMC microbiol 5:15 (2005)). Integration The thl-hbd-crt cassette under the control of the synthetic Pll promoter (Rud et al., Microbiology 152: 1011-1019 (2006)), is integrated into the chromosome of Lactobacillus plan tarum ATCC BAA-793 (MCIMB 8826) in the ldhL1 site by homologous recombination. To construct the IdhL integration target vector, a DNA fragment from La ctoba cillus plan tarum (Bank of Gen NC_004567) with homology to IdhL, is amplified by PCR using primers LDH EcoRV F (SEQ ID NO: 198) and LDH AatllR (SEQ ID NO.199). The 1986 bp PCR fragment is cloned into the pCR4Blunt-TOPO and sequenced. The pCR4Blunt-TOPO-ldhLl clone is digested with EcoRV and Aatll, releasing a 1982 bp IdhLl fragment that is gel purified. The integration vector pFP988, described in Example 14, is digested with HindIII and treated with Klenow DNA polymerase to blunt the ends. The linearized plasmid is then digested with Aatll and the fragment of the 2931 bp vector is gel purified. The EcoRV / AatlI IdhLl fragment is ligated with the vector fragment pFP988 and transformed into E. coli ToplO cells. Transformants are selected on LB agar plates containing ampicillin (100 μg / ml) and selected by colony PCR to confirm the construction of pFP988-IdHL. To add a selectable marker to the integration DNA, the Cm gene with its promoter is amplified by PCR from pC194 (Gen Bank NC_002013) with primers Cm F (SEQ ID NO: 200) and Cm R (SEQ ID NO: 201) , amplifying a PCR product of 836 bp. The amplicon is cloned in pCR4Blunt-TOPO and transformed into ToplO cells of E. coli, creating pCR4Blunt-TOPO-Cm. After sequencing to confirm that no errors were introduced by PCR, the Cm cassette is digested from pCR4Blunt-T0P0-Cm as a Mlul / Sawl fragment of 828 bp and is gel purified. The integration vector containing the IdhL homology pFP988-ldhL is digested with Mlul and Swal and the 4740 bp vector fragment is gel purified. The CM cassette fragment is ligated into the vector pFP988-ldhL creating pFP988-DldhL:: Cm. Finally, the thl-hbd-crt cassette of pF988Dss-T-H-C, described in Example 14, is modified to replace the amylase promoter with the synthetic Pll promoter. Then, the complete operon is moved in pFP988-DldhL:: Cm. The Pll promoter is constructed by oligonucleotide annealing with primer PllE (SEQ ID NO: 202) and PllR (SEQ ID NO: 203). The tempered oligonucleotide is gel purified on a 6% Ultra PAGE gel (Embi, Tec, San Diego, CA). Plasmid pFP988Dss-T-H-C is digested with Xhol and Saml and the 9 kbp vector fragment is gel purified. The isolated Pll fragment is ligated with the digested pFP988Dss-T-H-C to create pFP988-Pll-T-H-C. The plasmid pFP988-Pll-T-H-C is digested with Xhol and BamHI and the Pll-T-H-C fragment of 3034 bp is gel purified. PFP988-DldhL:: Cm is digested with Xhol and BamHI and the 5558 bp vector isolated. The upper path operon is linked with the integration vector to create pFP988-DldhL-Pll-THC:: Cm.

Integration of pFP988-DldhL-Pll-THC:: Cm in L. plan tarum BAA-793 to form? LdhLl: T-H-C :: Cm of L. plantarum comprising exogenous genes thl, hbd, and crt. Electrocompetent L cells. tarum plan are prepared as described by Aukrust, T. W., et a. (in: Electroporation Protocols for Microorganisms, Nickoloff, J.A., Ed., Methods in Molecular Biology, Vo. 47, Humana Press, Inc., Totowa, NJ, 1995, pp 201-208). After electroporation, the cells were grown in MRSSM medium (MRS medium supplemented with 0.5 M sucrose and 0.1 M MgCl 2) as described by Aukrust et al., Supra for 2 h at 37 ° C without shaking. Electroporated cells are plated by selection in MRS plates containing chloramphenicol (10 μg / ml) and incubated at 37 ° C. The transformants are initially selected by colony PCR amplification, to confirm integration, and the initial positive clones are then more rigorously selected by PCR amplification with a set of primers.

Expression of EgTER, ald, and bhdB gene plasmids.

The three remaining 1-butanol genes are expressed from the pTRKH3 plasmid (O'Sullivan DJ and Klaenhammer TR, Gene 137: 227-231 (1993)) under the control of the IdhL promoter of L. plantarum (Ferain et al., J. Bacteriol 176: 596-601 (1994)). The IdhL promoter is amplified by PCR from the L genome. plantarum ATCC BAA-793 with primers PIdhL F (SEQ ID NO: 204) and PIdhL R (SEQ ID NO: 205). The PCR product of 369 bp is cloned in pCR4Blunt-TOPO and sequenced. The resulting plasmid, pCR4Blunt-TOPO-PIdhL, is digested with Sacl and BamHI releasing the 359 bp PIdhL fragment. The pHTOl-ald-EB, described in Example 14, is digested with Sacl and BamHI and the 10503 bp vector fragment is recovered by gel purification. The PIdhL fragment and the vector are ligated creating pHTOl-PIdhl-ald-EB. To subclone the cassette ald-EgTER-bdh-promoter IdhL, the pHTOl-PIdhl-ald-EB is digested with Mlul and the ends are treated with Klenow DNA polymerase. The linearized vector is digested with Sali and the 4270 bp fragment containing the PldhL-AEB fragment is gel purified. The plasmid pTRKH3 is digested with Sali and EcoRV and the fragment of the vector purified in gel is ligated with the PldhL-AEB fragment. The plasmid pTRKH3 is digested with SalI and EcoRV and the fragment of the vector purified in gel is ligated with the PIdhL-AEB fragment. The ligation mixture is transformed into E. coli ToplO cells and the transformants are plated onto Brain Heart infusion plates (HBI, Difco Laboratories, Detroit, MI), which contain erythromycin (150 mg / l). The transformants are selected by PCR to confirm the construction of pTRKH3-ald-E-B. The expression plasmid pTRKH3-ald-E-B, is transformed into? LdhLl :: T-H-C :: Cm of L. plan tarum by electroporation, as described above. The? LdhLl :: T-H-C:: Cm of L. plan tarum containing pTRKH3-ald-E-B, is inoculated in a 250 ml shaken flask containing 50 ml of MRS medium plus erythromycin (10 μg / ml) and grown at 37 ° C for 18 to 24 hours without shaking. After 18 h at 24 h, 1-butanol was detected by HPLC or GC analysis, as described in the General Methods section.

EXAMPLE 19 (Prophetic) Expression of the Biosynthetic Pathway of 1-butanol in Enterococcus faecalis The purpose of this prophetic Example is to describe how to express the biosynthetic pathway of 1-butanol in In terococcus faecalis. The complete genome sequence of the Enterococcus faecalis strain V583, which was used as the host strain for 1 expression of the biosynthetic pathway of 1-butanol in this Example, has been established (Paulsen et al., Science 299: 2071- 2074 (2003)).

Plasmid pTRKH3 (O'Sullivan DJ and Klaenhammer TR, Gene 137: 227-231 (1993)), and the shuttle vector Gram-positive of E. coli, was used for expression of the six genes (thIA, hbd, crt, egTER, ald, dbnB) of the 1-butanol path in an operon. PTRKH3 contains a pl5A origin of replication of the E. coli plasmid and the pAMßl replicon, and the two markers for selection of antibiotic resistance, tetracycline resistance and erythromycin resistance. Resistance to tetracycline is only expressed in E. coli, and resistance to erythromycin is expressed in bacteria by both E. coli and Gram positive bacteria. PAMßl plasmid derivatives can be replicated in E. faecalis (Poyart et al., FEMS Microbiol. Lett 156: 193-198 (1007)). The inducible nisA promoter (PnisA), which has been used for efficient control of gene expression by nisin in a variety of Gram-positive bacteria including En terococcus faecalis (Eichenbaum et al., Appl. Environ. Microbiol. 2763-2769 (1998)), is used to control the expression of the six desired genes encoding the enzymes of the biosynthetic pathway of 1-butanol. The linear DNA fragment (215 bp) containing the nisA promoter (Chandrapati et al., Mol.Microbial 46 (2): 467-477 (2002)), is amplified by PCR from Lactococcus genomic DNA la ctis with primers F-PnisA (EcoRV) (SEQ ID NO: 206) and R-PnisA (Pmel BamHI) (SEQ ID NO: 207). The 215 bp PCR fragment is digested with EcoRV and BamHI, and the resulting PnisA fragment is gel purified. The plasmid pTRKH3 is digested with EcoRV and BamHI and the vector fragment is gel purified. The linearized pTRKH3 is ligated into the PnisA fragment. The ligation mixture is transformed into E. coli ToplO cells by electroporation and the transformants are selected after growth overnight at 37 ° C on LB agar plates containing erythromycin (25 μg / ml). The transformants are then screened by colony PCR with primers F-PnisA (EcoRV) and R-PinsA (BamHI) to confirm the correct clone of pTRKH3-PnisA. Plasmid pTRKH3-PnisA is digested with Pmel and BamHI, and the vector is gel purified. Plasmid pHTOl-ald-EgTER-bdhB is constructed as described in Example 14 and is digested with Smal and BamHI, and the ald-EgTER-bdhB fragment of 2.973 bp is gel purified. The ald-EgTER-bdhB fragment of 2.973 bp is ligated into the pTRKH3-PnisA vector at the Pmel and BamHI sites. The ligation mixture is transformed into ToplO cells of E. coli by electroporation and the transformants are selected after incubation at 37 ° C overnight on LB agar plates containing erythromycin (25 μg / ml). The transformants are then selected by colony PCR with forward primer ald N27F1 (SEQ ID NO: 31) and reverse primer bdhB N65 (SEQ ID NO: 44). The resulting plasmid is named pTRKH3-PnisA-ald-EgTER-bdhB (= pTRKH3-A-E-B).

The plasmid pTRKH3-A-E-B is purified from the transformant and used for further cloning of the remaining genes (thIA, hbd, crt) in the BamHI site located downstream of the dbhB gene. The plasmid pTRKH3-A-E-B is digested with BamHI and treated with the Klenow fragment of DNA polymerase to make the ends blunt. The plasmid pFP988-Dss-thIA-hbd-crt (= pFP988Dss-T-H-C) is constructed as described in Example 14 and is digested with Smal and BamHI. The resulting thia-hbd-crt fragment of 2.973 bp is treated with the Klenow fragment of DNA polymerase to make the blunt ends and is gel purified. The thlA-hbd-crt fragment of 2.973 bp is ligated with the linearized pTRKH3-A-E-B. The ligation mixture is transformed into ToplO cells of E. coli by electroporation and the transformants are selected after growth overnight at 37 ° C on LB agar plates containing erythromycin (25 μg / ml). The transformants are then selected by colony PCR with forward primer thIA N7 (SEQ ID NO: 21) and reverse primer crt N4 (SEQ ID NO: 18). The resulting plasmid is named pTRKH3-PnisA-ald-EgTER-bdhB-thIA-hbd-crt (= pTRKH3-A-E-B-T-H-C). Plasmid pTRJH3-AEBTHC is prepared from the E. coli transformants and transformed into electro-competent V583 cells of E. faecalis by electroporation using methods known in the art (Aukrust, TW, et al., In: Electroporation Protocols for Microorganisms; Nickoloff , J.A., Ed .: Methods in Molecular Biology, Vol. 47; Humana Press, Inc., Totowa, NJ. , 1995, pp 217-226), resulting in V583 / pTRKH3-A-E-B-T-H-C from E. faecalis. The second plasmid containing the regulatory genes nisA, nisR and nisk, the resistance gene of specta inomycin add9, and the origin of replication pSH71, is transformed into V583 / pTRKH3-A-E-B-T-H-C of E. F eca l i s by electroporation. The plasmid containing the origin of replication pSH71 is compatible with pAMßl derivatives in E. fa e ca l i s (Eichembaum et al., supra). Double drug-resistant transformants are selected on LB agar plates containing erythromycin (25 μg / ml) and inomycin spectacle (100 μg / ml). Strain V583B of E. The resulting plasmid, which harbors two plasmids, i.e., an expression plasmid (pTRKH3-AEBTHC) and a regulatory plasmid (pSH71 - or sRK), is inoculated into a shaken 250 ml flask containing 50 ml of Todd broth -Hewitt supplemented with yeast extract (0.2%) (Fischetti et al., J. Exp. Med. 161: 1384-1401 (1985)), nisin (20 μg / ml) (Eichenbaum et al., Supra), erythromycin (25 μg / ml), and inomycin spectacle (100 μg / ml). The flask is incubated without shaking at 37 ° C for 18 to 24 hours, after such time, the Production of 1-butanol is measured by CLAR or GC analysis, as described in the General Methods section. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (57)

CLAIMS Having described the invention as above, the contents of the following claims are claimed as property:

1. Recombinant microbial host cell, characterized in that it comprises at least one DNA molecule encoding a polypeptide that catalyzes a conversion of substrate to product selected from the group consisting of: a) acetyl-CoA to acetoacetyl-CoA b) acetoacetyl-CoA at 3- hydroxybutyryl-CoA c) 3-hydroxybutyryl-CoA to crotonyl-CoA d) crotonyl-CoA to butyryl-CoA e) butyryl-CoA to butyraldehyde and f) butyraldehyde to 1-butanol; wherein at least one DNA molecule is heterologous to the microbial host cell and wherein the microbial host cell produces 1-butanol.

2. Host cell according to claim 1, characterized in that the polypeptide that catalyzes a conversion of substrate to acetyl-CoA product to acetoacetyl-CoA is acetyl-CoA acetyltransferase.

3. Host cell according to claim 1, characterized in that the polypeptide that catalyzes a conversion of substrate to product of Acetoacetyl-CoA to 3-hydroxybutyryl-CoA is 3-hydroxybutyryl-CoA dehydrogenase.

Host cell according to claim 1, characterized in that the polypeptide that catalyzes a conversion of substrate to 3-hydroxybutyryl-CoA product to crotonyl-CoA is crotonane.

Host cell according to claim 1, characterized in that the polypeptide that catalyzes a conversion of substrate to product of crotonol-CoA to butyryl-CoA is butyryl-CoA dehydrogenase.

6. Host cell according to claim 1, characterized in that the polypeptide that catalyzes a conversion of substrate to product of butyryl-CoA to butyraldehyde is butyraldehyde dehydrogenase.

Host cell according to claim 1, characterized in that the polypeptide that catalyzes a conversion of substrate to product of butyraldehyde to 1-butanol is butanol dehydrogenase.

Host cell according to claim 1, characterized in that the cell is selected from the group consisting of: a bacterium, a cyanobacterium, a filamentous fungus and a yeast.

9. Host cell according to claim 8, characterized in that the cell is a member of a genus selected from the group consisting of Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alkaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces.

10. Host cell according to claim 9, characterized in that the cell is Escherichia coli.

11. Host cell according to claim 9, characterized in that the cell is Alcaligenes eutrophus.

12. Host cell according to claim 9, characterized in that the cell is Bacillus licheniformis.

13. Host cell according to claim 9, characterized in that the cell is Paenibacillus macerans.

14. Host cell according to claim 9, characterized in that the cell is Rhodococcus erythropolis.

15. Host cell according to claim 9, characterized in that the cell is Pseudomonas putida. •

16. Host cell according to claim 9, characterized in that the cell is Bacill us subtilis.

17. Host cell according to claim 9, characterized in that the cell is Lactobacillus plantarum.

18. Host cell according to claim 9, characterized in that the cell is selected from the group consisting of En terococcus faecium, Enterococcus gallinarium and Enterococcus faecalis.

19. Host cell according to claim 9, characterized in that the cell is Saccharomyces cerevisiae.

20. Host cell according to claim 3, characterized in that the acetyl-CoA acetyltransferase has an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 129, SEQ ID NO: 131, and SEQ ID NO: 133.

21. Host cell according to claim 4, characterized in that the 3-hydroxybutyryl-CoA dehydrogenase has an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 135, SEQ ID NO: 137, and SEQ ID NO: 139

22. Host cell according to claim 5, characterized in that the crotonane has an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 141, SEQ ID NO: 143, and SEQ ID NO: 8. NO: 145

23. Host cell according to claim 6, characterized in that the butyryl-CoA dehydrogenase has an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO. : 151 and SEQ ID NO: 187.

24. Host cell according to claim 7, characterized in that the butyraldehyde dehydrogenase has an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 153, and SEQ ID NO: 189.

25. Host cell according to claim 8, characterized in that the butanol dehydrogenase has an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 153, SEQ ID NO: 155 , and SEQ ID NO: 157.

26. Host cell according to claim 1, characterized in that it is an facultative anaerobe.

27. Method for the production of 1-butanol, characterized in that it comprises: i) providing a recombinant microbial host cell comprising at least one DNA molecule encoding a polypeptide that catalyzes a conversion of substrate to product selected from the group that consists of: a) acetyl-CoA to acetoacetyl-CoA b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA c) 3-hydroxybutyryl-CoA to crotonyl-CoA d) crotonyl-CoA to butyryl-CoA e) butyryl-CoA to butyraldehyde and f) Butyraldehyde to 1-butanol; wherein at least one DNA molecule is heterologous to the microbial host cell; and ii) contacting the host cell of (i) with a fermentable carbon substrate under conditions whereby 1-butane is produced.

28. Method according to claim 27, characterized in that the fermentable carbon substrate is selected from the group consisting of monosaccharides, oligosaccharides, and polysaccharides.

29. Method according to claim 27, characterized in that the carbon substrate is selected from the group consisting of glucose, sucrose, and fructose.

30. Method according to claim 27, characterized in that the conditions whereby 1-butanol is produced are anaerobic.

31. Method according to claim 27, characterized in that the conditions whereby 1-butanol is produced are microaerobic.

32. Method according to claim 27, characterized in that the host cell is contacted with the carbon substrate in minimal medium.

33. Method according to claim 27, characterized in that the polypeptide that catalyzes a conversion of substrate to acetyl-CoA product to acetoacetyl-CoA is acetyl-CoA acetyltransferase.

34. Method according to claim 27, characterized in that the polypeptide that catalyzes a conversion of substrate to acetoacetyl-CoA product to 3-hydroxybutyryl-CoA is 3-hydroxybutyryl-CoA dehydrogenase.

35. Method according to claim 27, characterized in that the polypeptide that catalyzes a conversion of substrate to product of 3-hydroxybutyryl-CoA to crotonol-CoA is crotonane.

36. Method according to claim 27, characterized in that the polypeptide that catalyzes a conversion of substrate to product of crotonyl-CoA to butyryl-CoA is butyryl-CoA dehydrogenase.

37. Method according to claim 27, characterized in that the polypeptide that catalyzes a conversion of substrate to product of butyryl-CoA to butyraldehyde is butyraldehyde dehydrogenase.

38. Method according to claim 27, characterized in that the polypeptide that catalyzes a conversion of substrate to product of butyraldehyde to 1-butanol is butanol dehydrogenase.

39. Method according to claim 27, characterized in that the host cell is selected from the group consisting of: a bacterium, cyanobacteria, a filamentous fungus and a yeast.

40. Method according to claim 39, characterized in that the host cell is a member of a gene selected from the group consisting of Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter , Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces.

41. Method according to claim 40, characterized in that the host cell is Escherichia coli.

42. Method according to claim 40, characterized in that the host cell is Alcaligenes eutrophus.

43. Method according to claim 40, characterized in that the host cell is Bacillus licheniformis.

44. Method according to claim 40, characterized in that the host cell is Paenibacillus macerans.

45. Method according to claim 40, characterized in that the host cell is Rhodococcus erythropolis.

46. Method according to claim 40, characterized in that the host cell is Pseudomonas putida.

47. Method according to claim 40, characterized in that the host cell is Bacillus subtilis.

48. Host cell according to claim 40, characterized in that the cell is Lactobacillus plantarum.

49. Host cell according to claim 40, characterized in that the cell is selected from the group consisting of Enterococcus faecium, Enterococcus gallinarium, and Enterococcus faecalis.

50. Host cell according to claim 40, characterized in that the cell is Saccharomyces cerevisiae.

51. Method according to claim 33, characterized in that the acetyl-CoA acetyltransferase has an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 129, SEQ ID NO: 131, and SEQ ID NO: 133.

52. Method according to claim 34, characterized in that the 3-hydroxybutyryl-CoA dehydrogenase has an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 135, SEQ ID NO: 137, and SEQ ID NO. : 139

53. Method according to claim 35, characterized in that the crotonane has an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 141, SEQ ID NO: 143, and SEQ ID NO: 145.

54. Method according to claim 36, characterized in that the butyryl-CoA dehydrogenase has an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO.151, and SEQ ID NO: 187.

55. Method according to claim 37, characterized in that the butyraldehyde dehydrogenase has an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 153, and SEQ ID NO: 189.

56. Method according to claim 38, characterized in that the butanol dehydrogenase has an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 153, SEQ ID NO: 155, and SEQ. ID NO: 157

57. Method according to claim 27, characterized in that the host cell is an facultative anaerobe.