WO2008074794A1 - Butanol production in a prokaryotic cell - Google Patents

Abstract

The present invention relates to a prokaryotic cell which does not naturally produce butanol, but which has been made capable of producing butanol by genetic modification of said microorganism and to a process for the production of butanol by fermenting the prokaryotic cell in a suitable fermentation medium according to the present invention.

Description

BUTANOL PRODUCTION IN A PROKARYOTIC CELL

The present invention relates to butanol production in a prokaryotic host cell, and a process for the production of butanol by using a genetically modified prokaryotic host cell.

The acetone/butanol/ethanol (ABE) fermentation process has received considerable attention in the recent years as a prospective process for the production of commodity chemicals, such as butanol and acetone from biomass.

The fermentation of carbohydrates to acetone, butanol, and ethanol by solventogenic Clostridia is well known since decades. Clostridia produce butanol by conversion of a suitable carbon source into acetyl-CoA. Substrate acetyl-CoA then enters into the solventogenesis pathway to produce butanol using six concerted enzyme reactions. The reactions and enzymes catalysing these reactions are listed below:

2 Acetyl-CoA -> Acetoacetyl-CoA + HSCoA (acetyl-CoA acetyl transferase)

Acetoacetyl-CoA + NAD(P)H -> 3-hydroxylbutyryl-CoA + NAD(P)⁺

(3-hydroxybutyryl-CoA dehydrogenase)

3-hydroxylbutyryl-CoA -> Crotonyl-CoA + H₂O (3-hydroxybutyryl-CoA dehydratase) Crotonyl-CoA + NAD(P)H -> Butyryl-CoA + NAD(P)⁺ (butyryl-CoA dehydrogenase)

Butyryl-CoA + NAD(P)H -> Butyraldehyde + CoA + NAD(P)⁺ (butyraldehyde dehydrogenase) Butyraldehyde + NAD(P)H -> Butanol (butanol dehydrogenase )

The formation of butanol requires the conversion of acetyl-CoA into acetoacetyl- CoA by acetyl-CoA acetyltransferase. This reaction is followed by the conversion of acetoacetyl-CoA into 3-hydroxylbutyryl-CoA by 3-hydroxybutyryl-CoA dehydrogenase, which is followed by the conversion of 3-hydroxylbutyryl-CoA into crotonyl-CoA by 3- hydroxybutyryl-CoA dehydratase (also named crotonase) and the conversion of crotonyl-CoA into butyryl-CoA by butyryl-CoA dehydrogenase and followed by the conversion of butyryl-CoA to butyraldehyde by butyraldehyde dehydrogenase, with the final conversion of butyrylaldehyde to butanol by butanol dehydrogenase (Jones, DT. ,

Woods, D. R., 1986, Microbiol. Rev., 50, 484-524).

However, the production of butanol suffers from poor process economics, because the butanol produced is toxic for the microbial cells and thus titers are low. Many studies have been directed to increase the resistance of Clostridia strains against butanol and consequently achieve an increase in titers.

WO98/51813 discloses a method for the production of butanol by fermenting a mutant Clostridium beijerinckii strain resulting in a butanol titer of between 18 and 20 g/l, compared to 9 and 13 g/l of the wild type strain.

US 6,960,465 shows that overexpression of the heat shock proteins in Clostridium acetobutylicum resulted in an increased butanol production yield compared to the wild type strain.

Despite the improvements that have been achieved so far in the ABE fermentation in Clostridia, the maximum achievable yield of butanol in Clostridia is still considerably low and the reliability of the process is still insufficient for a butanol production process on an industrial scale.

Since Clostridia are sensitive to oxygen, C/osfrvc/Za-fermentations need to be operated under strict anaerobic conditions, which makes it difficult to operate such fermentations on a large scale. Moreover, anaerobic fermentations generally result in low biomass concentrations due to the low ATP-gain under anaerobic conditions. Another disadvantage of butanol production in Clostridia is that undesirable by-products like acetone, acetate and butyrate are also produced, which lowers the yield of butanol on carbon. The aim of the present invention is the provision of a microorganism capable of producing butanol which overcomes one or more of the problems described above.

The aim is achieved according to the invention with a prokaryotic host cell which does not naturally produce butanol, but which has been made capable of producing butanol by genetic modification of said host cell. A prokaryotic cell which does not naturally produce butanol may be a wild type prokaryotic cell. Alternatively, a prokaryotic cell which does not naturally produce butanol may not be a wild type cell, but a prokaryotic cell comprising a genetic modification, which is not a genetic modification enabling the cell to produce butanol.

A prokaryotic cell according to the present invention may also be defined as a (recombinant) prokaryotic cell, comprising one or more heterologous nucleotide sequences encoding enzymes enabling the cell to produce butanol.

When the wording butanol is used in the present invention, this refers to n- butanol or 1 -butanol. Preferably, the genetic modification of a prokaryotic cell according to the present invention comprises transformation with one or more nucleic acid constructs comprising one or more nucleotide sequences encoding acetyl-CoA acetyltransferase, 3- hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase, butyryl-CoA dehydrogenase, alcohol dehydrogenase or acetaldehyde dehydrogenase or NAD(P)H- dependent butanol dehydrogenase.

Preferably, the prokaryotic cell according to the present invention expresses upon transformation with one or more nucleic acid constructs one or more of the following nucleotide sequences: a. a nucleotide sequence encoding an acetyl-CoA acetyltransferase, wherein said nucleotide sequence is selected from the group consisting of: i. nucleotide sequences encoding an acetyl-CoA acetyltransferase, said acetyl-CoA acetyltransferase comprising an amino acid sequence that has at least 20%, preferably at least 25, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 96, 97, 98, or 99% sequence identity with the amino acid sequence of SEQ ID NO:1. ii. nucleotide sequences comprising a nucleotide sequence that has at least 15%, preferably at least 20, 25, 30, 40, 50, 55,

60, 65, 70, 75, 80, 85, 90, 92, 95, 96, 97, 98, or 99% sequence identity with the nucleotide sequence of SEQ ID NO:2. iii. nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence (i) or (ii); iv. nucleotide sequences the sequences of which differs from the sequence of a nucleic acid molecule of (ii) or (iii) due to the degeneracy of the genetic code, b. a nucleotide sequence encoding a 3-hydroxybutyryl-CoA dehydrogenase, wherein said nucleotide sequence is selected from the group consisting of: i. nucleotide sequences encoding a 3-hydroxybutyryl-CoA dehydrogenase, said 3-hydroxybutyryl-CoA dehydrogenase comprising an amino acid sequence that has at least 25%, preferably at least 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 96, 97, 98, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 3, ii. nucleotide sequences comprising a nucleotide sequence that has at least 20%, preferably at least 25, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 96, 97, 98, or 99% sequence identity with the nucleotide sequence of SEQ ID NO:4, iii. nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence (i) or (ii); iv. nucleotide sequences the sequences of which differs from the sequence of a nucleic acid molecule of (ii) or (iii) due to the degeneracy of the genetic code, a nucleotide sequence encoding 3-hydroxybutyryl-CoA dehydratase, wherein said nucleotide sequence is selected from the group consisting of: i. nucleotide sequences encoding a 3-hydroxybutyryl-CoA dehydratase, said 3-hydroxybutyryl-CoA dehydratase comprising an amino acid sequence that has at least 30%, preferably at least 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,

90, 92, 95, 96, 97, 98, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 5; ii. nucleotide sequences comprising a nucleotide sequence that has at least 25%, preferably at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 96, 97, 98, or 99% sequence identity with the nucleotide sequence of SEQ ID NO:6; iii. nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence (i) or (ii); iv. nucleotide sequences the sequences of which differs from the sequence of a nucleic acid molecule of (ii) or (iii) due to the degeneracy of the genetic code, d. a nucleotide sequence encoding butyryl-CoA dehydrogenase, wherein said nucleotide sequence is selected from the group consisting of: i. nucleotide sequences encoding a butyryl-CoA dehydrogenase, said butyryl-CoA dehydrogenase comprising an amino acid sequence that has at least 20%, preferably at least 25, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 96, 97, 98, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 7; ii. nucleotide sequences comprising a nucleotide sequence that has at least 15%, preferably at least 20, 25, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 96, 97, 98, or 99% sequence identity with the nucleotide sequence of SEQ ID NO:8; iii. nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence (i) or (ii); iv. nucleotide sequences the sequences of which differs from the sequence of a nucleic acid molecule of (ii) or (iii) due to the degeneracy of the genetic code, e. a nucleotide sequence encoding alcohol dehydrogenase or acetaldehyde dehydrogenase, wherein said nucleotide sequence is selected from the group consisting of: i. nucleotide sequences encoding an alcohol dehydrogenase or acetaldehyde dehydrogenase, said alcohol dehydrogenase or acetaldehyde dehydrogenase comprising an amino acid sequence that has at least 20%, preferably at least 25, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 96, 97, 98, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11 respectively ii. nucleotide sequences comprising a nucleotide sequence that has at least 15%, preferably at least 20, 25, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 96, 97, 98, or 99% sequence identity with the nucleotide sequence of SEQ ID NO: 10 of SEQ ID NO: 12 respectively; iii. nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence (i) or (ii); iv. nucleotide sequences the sequences of which differs from the sequence of a nucleic acid molecule of (ii) or (iii) due to the degeneracy of the genetic code, f. a nucleotide sequence encoding NAD(P)H-dependent butanol dehydrogenase, wherein said nucleotide sequence is selected from the group consisting of: i. nucleotide sequences encoding a NAD(P)H-dependent butanol dehydrogenase, comprising an amino acid sequence that has at least 30%, preferably at least 40, 50, 55, 60, 65,

70, 75, 80, 85, 90, 92, 95, 96, 97, 98, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 13 or

SEQ ID NO: 15; ii. nucleotide sequences comprising a nucleotide sequence that has at least 25%, preferably at least 25, 30, 40, 50, 55, 60,

65, 70, 75, 80, 85, 90, 92, 95, 96, 97, 98, or 99% sequence identity with the nucleotide sequence of SEQ ID NO:14 or

SEQ ID NO 16; iii. nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence (i) or (ii); iv. nucleotide sequences the sequences of which differs from the sequence of a nucleic acid molecule of (ii) or (iii) due to the degeneracy of the genetic code.

Sequence identity and similarity

Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. Usually, sequence identities are compared over the whole length of the sequences compared. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. MoI. Biol. 215:403-410 (1990), publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894). Preferred parameters for amino acid sequences comparison using BLASTP are gap open 10.0, gap extend 0.5, Blosum 62 matrix. Preferred parameters for nucleic acid sequences comparison using BLASTN are gap open 10.0, gap extend 0.5, DNA full matrix (DNA identity matrix).

Hybridising nucleic acid sequences

Nucleotide sequences encoding the enzymes expressed in the cell of the invention may also be defined by their capability to hybridise with the nucleotide sequences of SEQ ID NO.'s 2, 4, 6, 8, 10, 12, 14, 16, or the nucleotide sequence encoding the enzymes of SEQ ID NO's 1 , 3, 5, 7, 9, 1 1 , 13, 15, respectively, under moderate, or preferably under stringent hybridisation conditions. Stringent hybridisation conditions are herein defined as conditions that allow a nucleic acid sequence of at least about 25, preferably about 50 nucleotides, 75 or 100 and most preferably of about 200 or more nucleotides, to hybridise at a temperature of about 65°C in a solution comprising about 1 M salt, preferably 6 x SSC or any other solution having a comparable ionic strength, and washing at 65°C in a solution comprising about 0.1 M salt, or less, preferably 0.2 x SSC or any other solution having a comparable ionic strength. Preferably, the hybridisation is performed overnight, i.e. at least for 10 hours and preferably washing is performed for at least one hour with at least two changes of the washing solution. These conditions will usually allow the specific hybridisation of sequences having about 90% or more sequence identity.

Moderate conditions are herein defined as conditions that allow a nucleic acid sequence of at least 50 nucleotides, preferably of about 200 or more nucleotides, to hybridise at a temperature of about 45°C in a solution comprising about 1 M salt, preferably 6 x SSC or any other solution having a comparable ionic strength, and washing at room temperature in a solution comprising about 1 M salt, preferably 6 x SSC or any other solution having a comparable ionic strength. Preferably, the hybridisation is performed overnight, i.e. at least for 10 hours, and preferably washing is performed for at least one hour with at least two changes of the washing solution. These conditions will usually allow the specific hybridisation of sequences having up to 50% sequence identity. The person skilled in the art will be able to modify these hybridisation conditions in order to specifically identify sequences varying in identity between 50% and 90%.

The nucleotide sequences encoding an acetyl-CoA acetyltransferase, a 3- hydroxybutyryl-CoA dehydrogenase, a 3-hydroxybutyryl-CoA dehydratase, a butyryl- CoA dehydrogenase, an alcohol dehydrogenase or acetaldehyde dehydrogenase and/or NAD(P)H-dependent butanol dehydrogenase may be from prokaryotic or eukaryotic origin. A prokaryotic nucleotide sequence encoding an acetyl-CoA acetyltransferase may for instance be the thiL gene of Clostridium acetobutylicum as shown in SEQ ID. NO: 2. A prokaryotic nucleotide sequence encoding 3- hydroxybutyryl-CoA dehydrogenase may for instance be the hbd gene of Clostridium acetobutylicum as shown in sequence SEQ ID NO: 4. A prokaryotic nucleotide sequence encoding a 3-hydroxybutyryl-CoA dehydratase may for instance be the crt gene of Clostridium acetobutylicum as shown in sequence SEQ ID NO: 6. A prokaryotic nucleotide sequence encoding a butyryl-CoA dehydrogenase may for instance be the bed gene of Clostridium acetobutylicum as shown in sequence SEQ ID NO: 8. A prokaryotic nucleotide sequence encoding alcohol dehydrogenase or acetaldehyde dehydrogenase may for instance be the adhE or adhE'\ gene of Clostridium acetobutylicum as shown in sequence SEQ ID NO: 10 or SEQ ID NO: 12, respectively. A prokaryotic nucleotide sequence encoding NAD(P)H-dependent butanol dehydrogenase may for instance be the bdhA or bdhB gene of Clostridium acetobutylicum as shown in SEQ ID NO: 14 and SEQ ID NO: 16, respectively.

To increase the likelihood that the introduced enzymes are expressed in active form in the prokaryotic cell of the invention, the corresponding encoding nucleotide sequence may be adapted to optimise its codon usage to that of the chosen prokaryotic cell. The adaptiveness of the nucleotide sequences encoding the enzymes to the codon usage of the chosen host cell may be expressed as codon adaptation index (CAI). The codon adaptation index is herein defined as a measurement of the relative adaptiveness of the codon usage of a gene towards the codon usage of highly expressed genes. The relative adaptiveness (w) of each codon is the ratio of the usage of each codon, to that of the most abundant codon for the same amino acid. The CAI index is defined as the geometric mean of these relative adaptiveness values. Non- synonymous codons and termination codons (dependent on genetic code) are excluded. CAI values range from 0 to 1 , with higher values indicating a higher proportion of the most abundant codons (see Sharp and Li, 1987, Nucleic Acids Research 15: 1281-1295; also see: Jansen et al., 2003, Nucleic Acids Res. 31 (8):2242- 51 ). An adapted nucleotide sequence preferably has a CAI of at least 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7.

The prokaryotic cell according to the present invention may be any suitable aerobic or facultative anaerobic microorganism. Preferably, the cell is a facultative anaerobic microorganism. Preferably, the prokaryotic cell belongs to one of the genera selected from the group consisting of Escherichia, Streptomyces, Alcaligenes, Azoarcus, Thauera, Bradyrhyzobium, Brevibacterium, Shewanella, Staphylococcus, Mycobacterium, Brucella, Bordetella, Fusobacterium, Rhodococcus, Geotrichum, Corynebacterium, Bacillus, Lactobacillus, Lactococcus, Streptococcus, Pseudomonas, and Zymomonas. More preferably, the cell is selected from the group consisting of Escherichia coli, Streptomyces lividans, Staphylococcus cohnii, Bacillus subtilis, B. cereus, B. coagulans, B. sphaericus, B. licheniformis, B.amyloliquefaciens, B. megaterium, B. pumilus, B. thuringiensis, Lactobacillus homohiochii, Lactobacillus heterohiochii, Lactobacillus fructivorans, Lactobacillus plantarum, Lactococcus lactis, Streptococcus lactis, Streptococcus amoris, Pseudomonas butanovora, Pseudomonas putida, and Zymomonas mobilis.

Preferably, the cell according to the present invention is a Lactobacillus plantarum comprising one or more of the nucleotide sequences of SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21 , and/or SEQ ID NO 22.

The nucleic acid construct may be a plasmid carrying the genes encoding all six enzymes of the butanol metabolic pathway as described above, or the nucleic acid construct may comprise two or more plasmids carrying the genes encoding the six enzymes of the butanol pathway distributed in any appropriate way. It may be possible that one or more of the enzymes selected from the group consisting of acetyl-CoA acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase, butyryl-CoA dehydrogenase, alcohol dehydrogenase or acetaldehyde dehydrogenase, or NAD(P)H-dependent butanol dehydrogenase are native to the host cell and that transformation with one or more of the nucleotide sequences encoding these enzymes may not be required to confer the host cell the ability to produce butanol. Hence, the host cell according to the present invention may be transformed with any of the nucleotide sequences encoding acetyl-CoA acetyltransferase, 3- hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase, butyryl-CoA dehydrogenase, alcohol dehydrogenase or aldehyde dehydrogenase, or NAD(P)H- dependent butanol dehydrogenase, to confer the host cell the ability to produce butanol. The cell may comprise a single, but preferably comprises multiple copies of each nucleic acid construct or multiple copies of each nucleic acid sequence. Further improvement of butanol production by the cell may be obtained by classical strain improvement or evolutionary approaches such as directed evolution.

The nucleic acid construct may be maintained episomally and thus comprises a sequence for autonomous replication, such as an autosomal replication sequence sequence. Alternatively, each nucleic acid construct may be integrated in one or more copies into the genome of the host cell. Integration into the host cell's genome may occur at random by non-homologous recombination but preferably the nucleic acid construct may be integrated into the host cell's genome by homologous recombination or site specific recombination, as is well known in the art.

In a preferred embodiment the present invention relates to a nucleic acid construct comprising one or more of the nucleotide sequences of SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21 , and/or SEQ ID NO 22.

Usually, the nucleotide sequences encoding acetyl-CoA acetyltransferase, 3- hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase, butyryl-CoA dehydrogenase, alcohol dehydrogenase or acetaldehyde dehydrogenase, or NAD(P)H- dependent butanol dehydrogenase are each operably linked to a promoter that causes sufficient expression of the corresponding nucleotide sequences in the prokaryotic host cell according to the present invention to confer to the cell the ability to produce butanol.

As used herein, the term "operably linked" refers to a linkage of polynucleotide elements (or coding sequences or nucleic acid sequence) in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skilled in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation.

The promoter that could be used to achieve the expression of the nucleotide sequences coding for acetyl-CoA acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase, butyryl-CoA dehydrogenase, alcohol dehydrogenase or aldehyde dehydrogenase, or NAD(P)H-dependent butanol dehydrogenase enzymes may be not native to the nucleotide sequence coding for the enzyme to be expressed, i.e. a promoter that is heterologous to the nucleotide sequence (coding sequence) to which it is operably linked. Preferably, the promoter is homologous, i.e. endogenous to the host cell.

A "strong constitutive promoter" is one which causes mRNAs to be initiated at high frequency compared to a native host cell. Examples of such strong constitutive promoters in Gram-positive microorganisms include, but are not limited to, SP01-26, SP01-15, veg, pyc (pyruvate carboxylase promoter), amyE, USP-45 and pepN. Examples of inducible promoters in Gram-positive microorganisms include, but are not limited to, the IPTG inducible Pspac promoter, the xylose inducible PxylA promoter and pNICE. Examples of constitutive and inducible promoters in Gram-negative microorganisms include, but are not limited to, tac, tet, trp-tet, Ipp, lac, Ipp-lac, laclq, T7, 75, 73, gal, trc, ara, SP6, λ-P_R, and λ-P_L.

The term "homologous" when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain.

The term "heterologous" when used with respect to a nucleic acid (DNA or RNA) or protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous nucleic acids or proteins are not endogenous to the cell into which it is introduced, but has been obtained from another cell or synthetically or recombinantly produced. Heterologous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins.

One or more enzymes of the butanol pathway as described before, may be overexpressed to achieve a sufficient butanol production by the host cell. There are various means available in the art for overexpression of enzymes in the host cell of the invention. In particular, an enzyme may be overexpressed by increasing the copy number of the gene coding for the enzyme in the host cell, e.g. by integrating additional copies of the gene in the host cell's genome, by expressing the gene from an episomal multicopy expression vector or by introducing an episomal expression vector that comprises multiple copies of the gene.

Alternatively, overexpression of enzymes in the host cells of the invention may be achieved by using a promoter that is not native to the sequence coding for the enzyme to be overexpressed, i.e. a promoter that is heterologous to the coding sequence to which it is operably linked.

A prokaryotic cell according to the present invention preferably has a high tolerance to alcohols, such as ethanol, propanol, butanol, isopropanol, isobutanol, isoamyl alcohol, pentanol, hexanol, heptanol, or octanol. Preferably, the host cell according to the invention has a high tolerance to butanol. A high tolerance to butanol is herein defined as the ability of a host cell according to the present invention to grow and/or produce butanol at a butanol concentration of at least 5 g/l, preferably at least

10 g/l, more preferably at least 15 g/l, more preferably at least 20 g/l, more preferably at least 25 g/l, more preferably at least 30 g/l, more preferably at least 40 g/l. A high alcohol tolerance may be naturally present in the host cell or may be introduced or modified by genetic modification. A preferred cell according to the present invention may be able to grow on any suitable carbon source known in the art and convert it to butanol. The host cell may be able to convert directly plant biomass, celluloses, hemicelluloses, pectines, rhamnose, galactose, fucose, maltose, maltodextrines, ribose, ribulose, glucuronic acid, galacturonic acid or starch, starch derivatives, sucrose, lactose and glycerol. A preferred host organism expresses enzymes such as cellulases (endocellulases and exocellulases) and hemicellulases (eg. endo- and exo-xylanases), pectinases and/or amylases to be able to convert cellulose, hemicellulose and/or pectines. Preferably, the host cell is able to convert a carbon source selected from the group consisting of glucose, xylose, arabinose, sucrose, lactose and glycerol into butanol.

In a further aspect, the present invention relates to a process for the production of butanol comprising fermenting a prokaryotic cell according to present invention in a suitable fermentation medium.

The fermentation medium used in the process for the production of butanol may be any suitable fermentation medium which allows growth of a particular prokaryotic cell. The essential elements of the fermentation medium are known to the person skilled in the art and may be adapted to the host cell selected. Preferably, the fermentation medium comprises a carbon source selected from group consisting of plant biomass, celluloses, hemicelluloses, pectines, rhamnose, galactose, fucose, fructose, maltose, maltodextrines, ribose, ribulose, acid, glucuronic acid, galacturonic acid, or starch, starch derivatives, sucrose, lactose, fatty acids, triglycerides and glycerol. Preferably, the fermentation medium also comprises a nitrogen source such as urea, or an ammonium salt such as ammonium sulphate, ammonium chloride, ammoniumnitrate or ammonium phosphate. The fermentation process according to the present invention may be carried out in batch, fed-batch or continuous mode. A separate hydrolysis and fermentation (SHF) process or a simultaneous saccharification and fermentation (SSF) process may also be applied. A combination of these fermentation process modes may also be possible for optimal productivity. A SSF process may be particularly attractive if starch, cellulose, hemiclelluose or pectin is used as a carbon source in the fermentation process, where it may be necessary to add hydrolytic enzymes, such as cellulases, hemicellulases or pectinases to hydrolyse the substrate. The prokaryotic cell used in the process for the production of butanol according to the present invention may be any suitable aerobic or facultative anaerobic microorganism. Preferably, the prokaryotic cell is a facultative anaerobic microorganism. A facultative anaerobic microorganism is preferred because a facultative microorganism can be propagated aerobically to a high cell concentration and butanol can be produced subsequently under anaerobic conditions. This anaerobic phase can then be carried out at high cell density which reduces the fermentation volume required substantially, and minimizes the risk of contamination with aerobic microorganisms. The fermentation process for the production of butanol according to the present invention may be an aerobic or an anaerobic fermentation process, or combination of an aerobic and an anaerobic fermentation process.

An anaerobic fermentation process is herein defined as a fermentation process run in the absence of oxygen or in which substantially no oxygen is consumed, preferably less than 5, 2.5 or 1 mmol/L/h, and wherein organic molecules serve both as electron donor and electron acceptors The fermentation process according to the present invention may also first be run under aerobic conditions and subsequently under anaerobic conditions.

The fermentation process may also be run under oxygen-limited conditions. Alternatively, the fermentation process may first be run under aerobic conditions and subsequently under oxygen-limited conditions. An oxygen-limited fermentation process is a process in which the oxygen consumption is limited by the oxygen transfer from the gas to the liquid. The degree of oxygen limitation is determined by the amount and composition of the ingoing gasflow as well as the actual mixing/mass transfer properties of the fermentation equipment used. Preferably, in a process under oxygen-limited conditions, the rate of oxygen consumption is at least 5.5, more preferably at least 6 and even more preferably at least 7 mmol/L/h.

The production of butanol in the process according to the present invention may occur during the growth phase of the host cell, during the stationary (steady state) phase or during both phases. Butanol production may occur under aerobic and anaerobic conditions. The process for the production of butanol may be run at any suitable temperature, depending on the prokaryotic host cell. It may be possible to run the fermentation process at different temperatures.

The optimum temperature for growth of the host cell may be at or above 20⁰C, 22°C, 25°C, 28°C, 30⁰C, 35°C, 37°C, 40⁰C, 42°C, 45°C, 50⁰C, 55°C, 60⁰C and preferably below 70⁰C. During the production phase of butanol, the optimum temperature may be lower than during the growth phase of the prokaryotic host cell, but may not be essential. The temperature during this phase may be below 45°C, for instance below 42°C, 40⁰C, 37°C, for instance below 35°C, 30⁰C, or below 28°C, 25°C, 22°C or below 20⁰C preferably above 15°C.

The process for the production of butanol according to the present invention may be carried out at any suitable pH, for instance at a pH between 4 and 8, for instance between 4.5 and 7.5, for instance between 5 and 7, for instance between 5.5 and 6.5. Butanol produced in the process according to the present invention preferably is recovered. Recovery of butanol from the fermentation medium may be performed by known methods in the art, for instance by distillation, vacuum extraction, solvent extraction, or pervaporation.

It was found that the process for the production of butanol according to the invention resulted in a butanol concentration of above 2 mg/l fermentation broth, preferably above 5, 10, 20, mg/l, even more preferably above 30 mg//l fermentation broth, preferably above 40 mg/l, more preferably above 50 mg/l, preferably above 60 mg/l, preferably above 70, preferably above 80 mg/l, preferably above 100 mg/l, preferably above 1 g/l, preferably above 5 g/l, preferably above 10 g/l, but usually below 70 g/l.

The following examples are for illustrative purposes only and are not to be construed as limiting the invention.

Description of the figures

Figure 1. Operon comprising 6 genes of the butanol pathway as described in Example 3 for transformation in L. plantarum. Figure 2. Map of plasmid pHB075BuOH, comprising 6 genes of the butanol pathway.

EXAMPLES

Example 1

Butanol production in Bacillus subtilis 1.1. General

Molecular and genetic techniques Standard genetic and molecular biology techniques are generally know in the art and have been previously described (Maniatis et al. (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Miller (1972) Experiments in molecular genetics, or Cold Spring Harbor Laboratory, Cold Spring Harbor). Sambrook and Russel (2001 ) "Molecular Cloning: A Laboratory Manual (3^rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al, eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987).

DNA transformation, PBS1 generalized transduction, and other standard B. subtilis genetic techniques are also generally know in the art and have been described previously (Cutting and Horn (1990) pp. 21-1 A, In Cutting and Harwood (ed.) Molecular biological methods for Bacillus. John Wiley and Sons, New York).

Strains Bacillus subtilis strains of the present invention were derived from strain PY79

(prototroph SPβ^c; Cat. # 1A747, Bacillus Genetic Stock Center (BGSC), The Ohio State University, Columbus, Ohio 43210 USA) and W23 Cat. # 2A3, Bacillus Genetic Stock Center (BGSC). The neomycin-resistance gene (neo), tetracycline-resistance gene (tet), and the chloramphenicol-resistance gene {cat) are obtained from plasmid pBEST501 (Cat. # ECE47, BGSC), pDG1514 (Cat # ECE100, BGSC), and pC194 (Cat# 1 E17 Bacillus Genetic Stock Center). Media

Standard minimal medium (MM) for B. subtilis contained 1X Spizizen salts, 0.04% sodium glutamate, and 0.5% glucose. Standard solid complete medium is Tryptone Blood Agar Broth (TBAB, Difco). Standard liquid complete medium is Veal Infusion-Yeast Extract broth (VY). The compositions of these media are described below or are standard formula described previously (Harwood and Archibald (1990) pp. 1-26 and 545-552 (Appendix 1 ), In Cutting and Harwood (ed.) Molecular biological methods for Bacillus. John Wiley and Sons, New York).

TBAB medium: 33 g Difco Tryptone Blood Agar Broth, qsp 1 L water. Autoclave. VY medium: 25 g Difco Veal Infusion Broth, 5 g Difco Yeast Extract, qsp 1 L water. Autoclave.

Minimum medium (MM): 100 ml 10X Spizizen salts; 10 ml 50% glucose; 1 ml 40% sodium glutamate, qsp 1 L water.

10X Spizizen salts: 140 g K₂HPO₄; 20 g (NH₄)₂SO₄; 60 g KH₂PO₄; 10 g Na₃(citrate)-2H₂O; 2 g MgSO₄JH₂O; qsp 1 L water.

Identification plasmids

Plasmids carrying the different genes are identified by genetic, biochemical, and/or phenotypic means generally known in the art, such as resistance of the transformants to both ampicillin and neomycin/kanamycin antibiotics, PCR diagnostic analysis of the transformant or purified plasmid, restriction analysis of the purified plasmid, DNA sequence analysis of the cloned DNA insert, and protein identification of the expressed genes by PAGE analysis in cell extracts.

HPLC analysis of butanol

Butanol is detected in the culture medium using HPLC. The HPLC analysis is carried out as follows: pre-column: Biorad Microguard Cation H+ cartridge. Column: Biorad Aminex HPX-87H. Mobile phase: 0.01 N H₂SO₄. Precipitation reagent: 3.3N HCIO₄. Rl detection: Waters 410 differential refractometer.

1.2. Cloning of the butanol biosynthesis route in Bacillus subtilis

For introduction of the butanol pathway into B. subtilis, eight Clostridium acetobutylicum genes are cloned:

• thiL encoding acetyl CoA-acetyltransfrase [E. C. 2.3.1.9] (SEQ ID. NO:2) • hbd encoding 3-hydroxybutyryl-CoA dehydrogenase [E. C.1.1.1.1.57] (SEQ ID NO:4)

• crt encoding crotonase or 3-hydroxybutyryl-CoA dehydratase [E. C.4.2.1.55] (SEQ ID NO:6) • bed encoding butyryl-CoA dehydrogenase [E. C.1.3.99.2] (SEQ ID NO: 8)

• adhE or ac//7E1 both encoding alcohol dehydrogenase or acetaldehyde dehydrogenase [E. C.1.1.1.1.2.1.10] (SEQ ID NO: 10 and SEQ ID NO:12)

• bdhB, bdhk both encoding respectively NADH-dependent butanol dehydrogenase A and B [E. C. :1.1.1.-] (SEQ ID NO: 14 and 16) The expression constructs are synthesized at DNA2.0 (Menlo Park CA, USA).

The genes of the butanol pathway are made synthetically whereby unfavourable Clostridium codons are replaced by B. subtilis favoured codons and synthetic ribosome binding sites (Shine and Dalgarno, 1975, Λ/afwe;254:34-38) are introduced just upstream of the translational start codons.

1.2.1. Construction of strains with thiL and hbd expression cassettes The thiL and hbd genes are synthesized as one fragment expressed from the xylose inducible promoter P_xyM. A single ribosomal binding site (RBS) optimized for B. subtilis is introduced in front of each gene (between 7-1 1 bp upstream from the translational start codon and the A nucleotide in the RBS sequence GGAGG) and the cryT rho- independent transcriptional terminater {Ω._cryT) sequence is inserted at least five bp downstream of the translational stop codon of hbd. Next, the B. subtilis W23 xylA promoter (P_xyi_A) including the 5' leader region and the portion of xylA structural gene containing an optimized CRE sequence is synthesized and inserted upstream of RBS- thiL fragment (Hartl et al., 2001 , J Bacteriol 183:2696-2699; Bhavsar et al., 2001 , Appl Environ Microbiol 67:403-410). The B. subtilis W23 xylR repressor gene and tetracycline resistance gene (tet) are inserted upstream of the P_xyι_A thiL-hbd Ω._cryT fragment oriented in the opposite transcriptional direction as the thiL-hbd genes. The entire DNA fragment

thiL-hbd-Ω._cryT) is then inserted between two continuous segments of the amyE gene (amyE-front and amyE-back). Two identical but otherwise unique restriction enzyme cleavage sites of Notl are also included into this cassette, one located at the outside end of the amyE segment (upstream and distal from tet) and a second located between the cryT gene and the amyE segment. Finally the (Notl)-amy£-tef-xy/R-P_XyiA thiL-hbd-Ω._cryT -(Notl )-amyE' DNA fragment is cloned into the E. coli plasmid pJ44 {ori_pACγc, bla) by standard methods.

The resulting plasmid is used to insert a single copy of the tet-xylR-P^ thiL-hbd- Ω_c,y7- cassette into the amyE locus of B. subtilis PY79 by DNA transformation, selecting for colonies resistant to 20 μg/ml tetracycline. The plasmid DNA is first linearized prior to transformation, using a unique restriction enzyme cleavage site within the vector sequences, a high frequency of these transformants containing the single copy tet- xy//?-P_xyi_A thiL-hbd-Ω._cryτ is obtained. This resulted in the recovery of one Tc^r colony named BU1

thiL-hbd-Ω._cryT).

The plasmid is also digested by Notl, and the

thiL-hbd-Ω._cryT - containing fragment is isolated and ligated at high DNA concentration to form concatomer molecules. The ligated molecules are transformed into PY79, and Tc^r transformants are obtained that contained multiple copies of the tet-xylR-P^_A thiL-hbd- Ω_c,y7- cassette at the amyE locus. This results in the recovery of one Tc^r colony named BU2 (Ωamy£::[tef-xy/R-P_xyiA thiL-hbd-Ω._cryτ]_N- Cultures of BU2 are enriched for cells containing multiple copies of the

thiL-hbd-Q._cryτ]u cassette by selecting for strains with higher resistance to tetracycline (up to 120 μg/ml) using method well known in the art (US 5,837,528; US 6,057,136; WO2004/106557).

1.2.2. Construction of strains with crt and bed expression cassettes

The crt and bed genes are synthesized as one fragment expressed from the xylose inducible promoter P_xyw. A single ribosomal binding site (RBS) optimized for B. subtilis is introduced in front of each gene (between 7-1 1 bp upstream from the translational start codon and the A nucleotide in the RBS sequence GGAGG) and the cryT /fϊo-independent transcriptional terminater (Ω_CΛy7-) sequence is inserted at least five bp downstream of the translational stop codon of bed. The B. subtilis W23 xylA promoter (P_xyι_A) including the 5' leader region and the portion of xylA structural gene containing an optimized CRE sequence are synthesized and inserted upstream of RBS- crt fragment. The B. subtilis W23 xylR repressor gene and chloramphenicol resistance gene {cat) are inserted upstream of the P_xyι_A crt-bcd Ω._cryT fragment oriented in the opposite transcriptional direction as the crt-bcd genes. The entire DNA fragment (cat- xy//?-P_Xyi_A crt-bcd-Ω._cryτ) is then inserted between two continuous segments of the lacA gene (/acΛ-front and lacA-back.). Two identical but otherwise unique restriction enzyme cleavage sites of Notl are also included into this cassette, one located at the outside end of the lacA segment (upstream and distal from cat) and a second located between cryT and the lacA segment. Finally the (Notl)-/acΛ -caf-xy//?-P_xyiA crt-bcd-Ω._cry-r(Not\)- lacA' DNA fragment is cloned into the E. coli plasmid {ori_pAcγc, bla) by standard methods.

The resulting plasmid is then used to insert a single copy of the cat-xylR-P^_A crt- bcd-Ω._cryT cassette into the lacA locus of B. subtilis PY79 by DNA transformation, selecting for colonies resistant to 5 μg/ml chloramphenicol. The plasmid DNA is first linearized prior to transformation, using a unique restriction enzyme cleavage site within the vector sequences, a high frequency of these transformants containing the single copy

crt-bcd-Ω._cryT can be obtained. Using such methods, one Cm^r colony is recovered and named BU3

crt-bcd-Ω._cryT). The plasmid is also digested by Notl, and the

crt-bcd-Ω._cryτ- containing fragment is isolated and ligated at high DNA concentration to form concatomer molecules. The ligated molecules are transformed into PY79, and Cm^r transformants are obtained that contained multiple copies of the cat-xylR-P^_A crt-bcd- Ω._cryT cassette at the lacA locus (or xylR locus, if there is enough homology). This results in the a recovery of one Cm^r colony named BU4 (Ω.lacA::[cat-xylR-P_xy\A crt-bcd-Ω._cryτ]u- Cultures of BU4 are enriched for cells containing multiple copies of the

crt- bcd-Ω._cryT cassette by selecting for strains with higher resistance to chloramphenicol (up to 60 μg/ml) using method well known in the art (US 5,837,528, US 6,057,136, WO2004/106557).

1.2.3. Construction of strains with adhE1 and bdhA, adhE and bdhA, adhE1 and bdhB, or adhE and bdhB expression cassettes

The adhE1 and bdhA genes are synthesized as one fragment expressed from the xylose inducible promoter P_xyw. A single ribosomal binding site (RBS) optimized for B. subtilis is introduced in front of each gene (between 7-1 1 bp upstream from the translational start codon and the A nucleotide in the RBS sequence GGAGG) and the cryT /fϊo-independent transcriptional terminater (Ω_CΛy7-) sequence is inserted at least five bp downstream of the translational stop codon of bdhA. Next, the B. subtilis W23 xylA promoter (P_xyi_A) including the 5' leader region and the portion of xylA structural gene containing an optimized CRE sequence is synthesized and inserted upstream of RBS- adhE1 fragment. The B. subtilis W23 xylR repressor gene and neomycin resistance gene (neo) are inserted upstream of the P_xyi_A adhE1-bdhA Ω._cryT fragment oriented in the opposite transcriptional direction as the adhE1-bdhA genes. The entire DNA fragment (neo-xy/R-PχyiA adhE1 -bdhA-Ω._cryT) is then inserted between two continuous segments of the sacB gene (sacS-front and sacS-back). Two identical but otherwise unique restriction enzyme cleavage sites of Notl are also included into this cassette, one located at the outside end of the sacB segment (upstream and distal from neo) and a second located between c/yT and the sacB segment.

The resulting plasmid is used to insert a single copy of the neo-xy/R-P_xyi_A adhE1- bdhA-Ω._cryτ cassette into the sacB locus of B. subtilis PY79 by DNA transformation, selecting for colonies resistant to 2.5 μg/ml neomycin. The plasmid DNA is first linearized prior to transformation, using a unique restriction enzyme cleavage site within the vector sequences, a high frequency of these transformants containing the single copy neo-xy/R-PχyiA adhE1 -bdhA-Ω._cryT is obtained. This resulted in the recovery of one Neo^r colony named BU5 (ΩsacS::neo-xy/R-P_xyι_A adhE1-bdhA-Ω._cryT).

The plasmid is also digested with Notl, and the sacS-neo-xy/R-P_xyι_A adhE1- jbc//7/A-Ω_c,y7-cont.aining fragment is isolated and ligated at high DNA concentration to form concatomer molecules. The ligated molecules transformed into PY79, and Neo^r transformants are obtained that contained multiple copies of the neo-xy/R-P_xyi_A adhE1- bdhA-Ω._cryτ cassette at the sacB locus. Using such methods, one Neo^r colony is recovered and named BU6 (ΩsacS::[neo-xy/R-P_xyι_A adhE1 -bdhA-Ω._cryJ]u. Cultures of BU6 can be enriched for cells containing multiple copies of the [neo-xy/R-P_xyι_A adhE1- bdhA-Ω._cryτ]u cassette by selecting for strains with higher resistance to neomycin (up to 20 μg/ml) using method well known in the art (US 5,837,528; US 6,057,136; WO2004/106557).

The same method is used to make strains with single-copy and multiple copy cassettes with the following genes: adhE-bdhA (BU7 and BU8, respectively, adhE1- bdhB (BU9 and BU 10, respectively), and adhE-bdhB (BU9 and BU 10, respectively). 1.2.4. Combination of single-copy and multi-copy thiL-hbd, crt-bcd, adhE1-bdhA, adhE- bdhA, adhE1-adhB, and adhE-bdhB expression cassettes into individual strains

PBS1 bacteriophage transducing lysates are produced from strains BU1 to BU 10, using standard procedures. These lysates are used to generate B. subtilis strains containing various combinations of single-copy and multiple-copy Clostridium acetobutylicum butanol biosynthetic gene cassettes as described above. To create a strain in which all the C. acetobutylicum butanol genes are in single copy, the PBSI phage lysate prepared on BU1 {Ω.amyE::tet-xylR-P_xy\_A thiL-hbd-Ω._cryT) is used first to infect BU3 (Ω.lacA::cat-xylR-P_xy\_A-crt-bcd-Ω._cryτ) selecting for transductants resistant to 20 μg/ml tetracycline. Using such methods, one Tc^r Cm^r colony is recovered and named BU 1 1 (Ωamy£::tef-xy/R-P_xyi_A thiL-hbd-Ω._cryT, Ω.lacA::cat-xylR-P^_A-crt-bcd-Ω._cryT). In the next step, the PBSI phage lysate prepared on BU5 {Ω.sacB::neo-xylR-P_xy\_A adhE1-bdhA- Ω._cryτ) is used to infect BU1 1 selecting for transductants resistant to 2.5 μg/ml neomycin. Using such methods, one Neo^r Tc^r Cm^r colony is recovered and named BU12 (Ω.amyE::tet-xylR-P_xy\_A thiL-hbd-Ω._cryT, Ω.lacA::cat-xylR-P_xy\_A crt-bcd-Ω._cryT, Ω.sacB::neo- xy//?-P_Xyi_A adhE1-bdhA-Ω._cryT). In the same way, strains are made combining Ω.amyE::tet- xylR-P_xy\_A-thiL-hbd-Ω._cryT and Ω.lacA::cat-xylR-P_xy\_A-crt-bcd-Ω._cryT single copy cassettes with Ω.sacB::neo-xylR-P_xy]A adhE-bdhA-Ω._cryT (BU13), Ω.sacB: neo-xylR-P_xy]A adhE1- bdhB-Ω._cryT (BU14), and Ω.sacB::neo-xylR-P_xyiA adh1-bdhB-Ω._cryT (BU15). Using these same methods as described above, strains with multiple copy cassettes are created resulting in:

BU16 {Ω.amyE::[tet-xylR-P_xy]A-thiL-hbd-Ω._cryT]N, Ω.lacA::[cat-xylR-P_xy]A-crt-bcd- ΩcyrK Ω.sacB::[neo-xylR-P_xy\_A adhE1 -bdhA-Ω._cryτ]N),

BU17 {Ω.amyE::[tet-xylR-P_xy]A-thiL-hbd-Ω._cryT]N, Ω.lacA::[cat-xylR-P_xy]A-crt-bcd- Ω._cryT]N, Ω.sacB::[ neo-xylR-P_xy\_A adhE-bdhA-Ω._cryτ]N),

BU18 {Ω.amyE::[tet-xylR-P_xy]A-thiL-hbd-Ω._cryT]N, Ω.lacA::[cat-xylR-P_xy]A-crt-bcd- ΩcyrK Ω.sacB::[ neo-xylR-P_xy\_A adhE1-bdhB-Ω._cryτ]N), and

BU19 {{Ω.amyE::[tet-xylR-P_xy]A-thiL-hbd-Ω._cryT]N, Ω.lacA::[cat-xylR-P_xy]A-crt-bcd- ΩcyrK Ω.sacB::[ neo-xylR-P_xy\_A adhE-bdhB-Ω._cryτ]N). The expression of these genes is increased by obtaining colonies that are resistant to successively higher levels of chloramphenicol (for thiL-hbd) up to 60 μg/ml, tetracycline up to 120 μg/ml, and neomycin 20 μg/ml. 1.3. Butanol production by genetically modified Bacillus subtilis

1.3.1. Single stage anaerobic fermentation

B. subtilis strains BU12, BU13, BU14, BU15, BU16, BU17, BU18, and BU19, are grown anaerobically at 37°C in 200 ml Spizizen's minimal medium (14 g/l K₂HPO₄; 2 g/l (NhU)₂SO₄; 6 g/l KH₂PO₄; 1 g/l Na₃(citrate)-2H₂O; 0.2 g/l MgSO₄JH₂O) supplemented with 1 % glucose, 1 % sodium pyruvate, 0.2% glutamate, and 100 μg/ml casamino acids. Also added are trace element solutions at final concentrations per liter as follows: MgCI₂ 6H₂O, 125 mg; CaCI₂, 5.5 mg, FeCI₂-6H₂O, 13.5 mg; MnCI₂-4H₂O, 1 mg; ZnCI₂ 1.7 mg; CuCI₂ 2 H₂O, 0.43 mg; CoCI₂ 6H20, 0.6 mg; Na₂MoO₄ 2H₂O, 0.6 mg; NaSeO₄, 0.47 mg. This is done by growing the culture anaerobically in a closed, filled flask (with or without slow stirring). For strains containing multi-copy gene cassettes, tetracycline, neomycin, and chloramphenicol are added to the media at concentrations 50% of the maximum level of resistance. When the cells reach an OD₆₂o_nm of 0.25, gene expression is induced by the addition of 0.2% xylose under anaerobic conditions. Supernatant is collected at the late exponential phase (OD₆₂o_nm > 0.4). After separation of the cells utilizing centrifugation, butanol is detected in the supernatant by HPLC analysis.

1.3.2. Two stage aerobic- anaerobic fermentation

B. subtilis strains BU12, BU13, BU14, BU15, BU16, BU17, BU18, and BU19, are grown aerobically at 37°C under standard conditions in 200 ml Spizizen's minimal medium (14 g/l K₂HPO₄; 2 g/l (NH₄)₂SO₄; 6 g/l KH₂PO₄; 1 g/l Na₃(citrate)-2H₂O; 0.2 g/l MgSO₄-7H₂O) supplemented with 1 % glucose, 0.2% glutamate, and 100 μg/ml casamino acids. Also added are trace element solutions at final concentrations per liter as follows: MgCI₂ 6H₂O, 125 mg; CaCI₂, 5.5 mg, FeCI₂-6H₂O, 13.5 mg; MnCI₂-4H₂O, 1 mg; ZnCI₂ 1.7 mg; CuCI₂ 2 H₂O, 0.43 mg; CoCI₂ 6H20, 0.6 mg; Na₂MoO₄ 2H₂O, 0.6 mg; NaSeO₄, 0.47 mg. For strains containing multi-copy gene cassettes, tetracycline, neomycin, and chloramphenicol are added to the media at concentrations 50% of the maximum level of resistance.

When the cells reach an OD₆₂o_nm of 1.0 or greater, gene expression was induced by collecting the cells by centrifugation, suspending the cells again in the 200 ml of medium described above but supplemented with 1 % sodium pyruvate and 0.2% xylose and no antibiotic (single copy or multiple copy cassette-containing strains). Incubation continued anaerobically in a closed, filled flask (with or without slow stirring). Supernatant is collected at various time points during anaerobic growth, and after separation of the cells by centrifugation, butanol is detected in the supernatant by HPLC analysis.

Example 2 Butanol production in Escherichia coli

General • Oligonucleotides are synthesized by Invitrogen (Carlsbad CA, US).

• DNA sequencing is performed at SEQLAB (Gottingen, Germany) or at Baseclear (Leiden, The Netherlands)

• Restriction enzymes are supplied by Invitrogen or New England Biolabs.

• Used strains: Escherichia coli DH10B electromax competent cells (Invitrogen). Protocol is delivered by manufacturer.

• SDS-PAGE system (Invitrogen)

• NuPAGE Novex Bis-Tris Gels (Invitrogen). SimplyBlue SafeStain Microwave protocol

2.1. Cloning of the butanol biosynthesis route in Escherichia coli

For introduction of the butanol pathway in E. coli, the 8 Clostridium acetobutylicum genes as described under Example 1 are cloned in total

The expression constructs are synthesized at DNA2.0 (Menlo Park CA, USA). The expression vectors are plasmid pJF1 19EH (Fϋrste, et al. (1986, Gene, 48:1 19-131 ) and pACYCtac (Kramer, M, 2000, Untersuchungen zum Einfluss erhohter Bereitstellung von Erythrose-4-Phosphat und Phosphoenolpyruvat auf den Kohlenstofffluss in den Aromatenbiosyntheseweg von Escherichia coli. Berichte des Forschungszentrums Jϋlich, 3824. ISSN 0944-2952, PhD Thesis). In both cases, the expression system uses the IPTG inducible tac promoter and carries the lac repressor (lac\^q gene).

The genes of the butanol pathway are made synthetically whereby unfavourable Clostridium codons are replaced by E. coli favoured codons. In addition a ribosomal binding site optimized for E. coli is introduced in front of each gene. In the expression vector pACYCtac and pJF1 19EH, 3 genes of the butanol pathway are ligated and placed behind the IPTG inducible promoter resulting in plasmids pACYCtac- thiL-hbd-crt (pAC-THC) and pJFλ λ §EH-bcd-adhe1-bdhA (pJF-BE1A), pJF119EH-bcd- adhe-bdhA (pJF-BEA), pJΨλ WEY\-bcd-adhe1-bdhB (pJF-BE1 B), and pJF1 19EH-bcd- adhe-bdhB (pJF-BEB). To select for and confirm the construction of the correct plasmids E. coli DH 1OB is used.

The influence of the in-parallel expression of the butanol biosynthesis genes is investigated within the E. coli host strain LJ1 10 (Zeppenfeld, et al. (2000), J Bacteriol. 182, 4443-4452). E. coli LJ1 10 is transformed with plasmid pAC-THC resulting in E. coli LJ1 10 pAC-THC. This strain is transformed with either pJF-BE1A, pJF-BEA, pJF-BE1 B and pJF-BEB resulting in 4 different E. coli strains named LJ1 10. pAC-THC pJF-BE1A, LJ1 10 pAC-THC pJF-BEA, LJ1 10 pAC-THC pJF-BE1 B, LJ1 10 pAC-THC pJF-BEB.

2.2 Butanol production by genetically modified Escherichia coli

E. coli strains LJ1 10 pAC-THC pJF-BE1A, LJ1 10 pAC-THC pJF-BEA, LJ1 10 pAC-THC pJF-BE1 B, LJ1 10 pAC-THC pJF-BEB are grown anaerobically on 200 ml 2YTG (16 g of Bacto Tryptone/liter, 10 g of yeast extract/liter, 4 g of NaCI/liter, 5 g of glucose/liter) supplemented with 3 mg of nickel chloride/liter, 60 mg of zinc chloride/liter, 200 mg of nitriloacetic acid/liter, 50 mg/l ampicillin, 25 mg/l chloramphenicol at pH 6.8 and 30⁰C.

When the cells have reached an OD₆₂0_nm of 0.5, gene expression is induced by the addition of 100 μM IPTG.

Supernatant is collected in the late exponential phase. After separation of the cells by centrifugation, butanol is detected in the supernatant of the four different mutant £. coli strains by HPLC analysis as described in Example 1.1

3. Butanol production in Lactobacillus plantarum

3.1. Construction of a butanol biosynthetic genecluster for expression in Lactobacillus plantarum

For introduction of the butanol pathway in L. plantarum, 6 Clostridium acetobutylicum genes as described in Example 1 are cloned in total. The alcohol dehydroegnase encoding gene adhE and and butanol dehydrogenase encoding genes bdhB are used.

Sequences were codon optimized for expression in L. plantarum. All 6 genes were placed in one operon in the order thiL hbd crt hbd adhE bdhB including flanking restriction sites Kpnl, Sphl, BspHI (upstream of the start of the first gene thiL) and Xbal, Sacll and Sacl, and (downstream of the last gene bdhB) for cloning into the final expression vectors. To provide ribosomal binding sites 25base pairs from the native C. acetobutylicum sequence were included upstream of the hbd, crt, hbd, adhE, and bdhB genes. In case of sequences considered disadvantageous, (e.g. upstream of crt and bed) minor changes were introduced to optimize the intergenic regions. The ribosomal binding site for the thiL gene is provided by the expression vectors. Single cutting restriction sites were included downstream of each gene (Zral after thiL, Agel after hbd, Apal after crt, MIuI after bed, Acclll after adhE, and Xbal after bdhB, see figure 1 ). The nucleotide sequence of the optimized nucleotide sequences of the thiL, hbd, crt, hbd, adhE, and bdhB genes are given in SeqlD NO: 17 - SeqlD 22 respectively. The operon (Figure 1 ) was created by gene synthesis by Geneart, Regensburg (Germany).

3.2. Construction of expression vectors for Lactobacillus plantarum containing the optimized butanol operon The vectors pNZ7021 (Wegkamp, et al, 2007., Appl. Environ. Microbiol.

73:2673-2681 12), pNZ8148 (Kuipers et al., 1998, J. of Biotechnology 64: 15-21 ) and pHB075 were used for expression in L. plantarum. pHB075 is a low copy number(1-5 in Lactobacilli) plasmid with a theta-type replicon, derived from plL252 (Simon and Chopin, 1988, Biochimie 70: 559-566) and containing the USP promoter from L. lactis (van Asseldonk et al., 1990, Gene 95: 155-160). pNZ7021 and pNZ8148 contain the replicon of pSH71 (Kuipers et al., 1998, J. of Biotechnology 64: 15-21 ) which has a copy number of 100-200 in Lactobacilli. pNZ7021 contains the constitutive pepN promoter (Tan et al., 1992, FEBS Lett. 306: 9-16). The synthetic operon is digested with Kpnl or Sphl and Xbal and the 7,9kb fragment is gel purified. This fragment is ligated to the similarly-digested pNZ7021. After transformation of the ligation mixture into E. coli MC1061 (Casadaban and Cohen, 1980, J. MoI. Biol. 138: 179-207) single colonies growing on selective media supplied with chloramphenicol are screened for the presence of the correct insert by restriction digestion with e.g. Accl (expected fragments: 4282bps, 3067bps, 2578bps, 1072bps, 1 1 bps) and a correct clone is selected. The resulting plasmid is named pNZ7021 BuOH. pNZ8148 contains the nisin inducible promoter pNICE allowing controlled expression in L. plantarum NZ7100 (Serrano et al. 2007, Microb Cell Fact 6: 29). The synthetic operon is digested with BspHI and Xbal and the 7,9kb fragment is gelpurified. This fragment is ligated in pNZ8148 digested with Ncol and Xbal. After transformation of the ligation mixture into E. coli MC1061 (Casadaban and Cohen, 1980, J. MoI. Biol. 138: 179-207) single colonies growing on selective media supplied with chloramphenicol are screened for the presence of the correct insert by restriction digestion with e.g. Accl (expected fragments: 5426bps, 3067bps, 2578bps) and a correct clone is selected. The resulting plasmid is named pNZ8148BuOH. pHB075 contains the USP-45 promoter which is a constitutive promoter of intermediate strength (van Asseldonk et al., 1990, Gene 95: 155-160). The synthetic operon is digested with BspHI and Sacll and the 7,9kb fragment is gel purified. pHB075 is digested with Ncol and Sacll and the 6.5kb fragment is gel purified. The two resulting fragments are ligated and the mixture is transformed into Lactococcus lactis MG1363 (Gasson, MJ. 1993, J. Bacterid. 154: 1-9). Plasmid transformants growing on selective media supplemented with chloramphenicol are characterized by restriction digestion e. g. with BamHI (expected fragments: 6166bps, 5481 bps, 1757bpd, 960bps) and a correct clone is selected. The resulting plasmid is named pHB075BuOH (Figure 2, SEQ ID NO 32).

3.3. Construction of Lactobacillus plantarum strains containing the butanol Biosynthetic Genes The influence of concurrent expression of butanol genes in L. plantarum

NZ7100 (Serrano et. al., 2007, Microb. Cell Fact. 6:29) and L. plantarum VL103 (Ladera et al. 2007, Appl. Environ. Microbiol. 73:1864-72) is investigated. For this purpose L plantarum NZ7100 is transformed with pNZ7021 BUOH, pNZ8148BUOH and pHB075BUOH isolated from E. coli respectively L. lactis according to standard procedures as described in Example 1 , § 1.1. The resulting strains are named L. plantarum NZ7100-pNZ7021 BUOH, NZ7100-pNZ8148BUOH and NZ7100- pHB075BUOH, respectively. In a similar fashion L. plantarum VL103 is transformed with pNZ7021 BUOH and pHB075BUOH isolated from either E. coli or L lactis yielding L plantarum VL103-pNZ7021 BUOH and VL103-pHB075BUOH, respectively.

3.4. Butanol production by modified Lactobacillus plantarum Strains A volume of 100 ml of MRS medium (de Man et. al., 1960, J. Appl. Bacterid.

23:130-135) supplemented with 0.5% to 1 % (wt/vol) glucose and chloramphenicol and contained in a 100 ml bottle, is inoculated with a single colony of L. plantarum NZ7100- pNZ7021 BUOH, L plantarum NZ7100-pUB75BUOH, L plantarum VL103- pNZ7021 BUOH, or L plantarum VL103-pUB75BUOH. The different cultures are incubated statically at 37°C. After appropriate intervals (e.g. 8h, 12h, 16h or 24h), 1 ml samples are removed from the cultures. Biomass is removed by centrifugation and the BuOH concentration in the supernatant is measured by GC as described in § 3.5. 100ml of MRS medium (de Man et. al., 1960, J. Appl. Bacterid. 23:130-135) supplemented with 0.5% to 1 % (wt/vol) glucose and chloramphenicol contained in a 100ml bottle is inoculated with a single colony of L. plantarum NZ7100-pNZ8148BUOH. The culture is statically incubated in an anaerobic jar at 37°C. After overnight incubation (~18h), this culture is diluted 1 :20 into fresh media and incubated at 30⁰C under the same conditions. Once the OD_c o_nU_nU reaches a value of 0.5 (approximately equal to 0.2 gr/L dry weight) nisin is added to a final concentration of 50 ng/ml as described by Pavan et al. 2000, Appl. Environ. Microbiol. 66:4427-32. 1 ml samples are removed from the different cultures after e.g. 0.5h, 2h, and overnight. Biomass is removed by centrifugation and the BuOH concentration in the supernatant is measured by GC as described in

3.5. Butanol analysis by HS-GC

Samples are analysed on a HS-GC equipped with a flame ionisation detector and an automatic injection system. Column J&W DB-1 length 30 m, id 0.53 mm, df 5 μm. The following conditions are used: helium as carrier gas with a flow rate of 5 ml/min. Column temperature was set at 110⁰C. The injector was set at 140⁰C and the detector performed at 300°C. The data were achieved using Chromeleon software. Samples are heated at 60⁰C for 20 min in the headspace sampler. One ml of the headspace volatiles are automatically injected on the column.

Claims

1. A prokaryotic cell which does not naturally produce butanol, but which has been made capable of producing butanol by genetic modification of said prokaryotic cell.

2. A cell according to claim 1 , wherein the genetic modification comprises transformation with one or more nucleic acid construct(s) comprising one or more nucleotide sequences encoding acetyl-CoA acetyltransferase, 3- hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase, butyryl-CoA dehydrogenase, alcohol dehydrogenase or acetaldehyde dehydrogenase or NAD(P)H-dependent butanol dehydrogenase.

3. A prokaryotic cell according to claim 1 or 2, expressing one or more nucleotide sequence(s) selected from the group consisting of: a. a nucleotide sequence encoding an acetyl-CoA acetyltransferase, wherein said nucleotide sequence is selected from the group consisting of: i. nucleotide sequences encoding an acetyl-CoA acetyltransferase, said acetyl-CoA acetyltransferase comprising an amino acid sequence that has at least 20% sequence identity with the amino acid sequence of SEQ ID NO:1. ii. nucleotide sequences comprising a nucleotide sequence that has at least 15% sequence identity with the nucleotide sequence of SEQ ID NO:2. iii. nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence (i) or (ii); iv. nucleotide sequences the sequences of which differs from the sequence of a nucleic acid molecule of (ii) or (iii) due to the degeneracy of the genetic code, b. a nucleotide sequence encoding a 3-hydroxybutyryl-CoA dehydrogenase, wherein said nucleotide sequence is selected from the group consisting of: i. nucleotide sequences encoding a 3-hydroxybutyryl-CoA dehydrogenase, said 3-hydroxybutyryl-CoA dehydrogenase comprising an amino acid sequence that has at least 25% sequence identity with the amino acid sequence of SEQ ID NO: 3, ii. nucleotide sequences comprising a nucleotide sequence that has at least 20% sequence identity with the nucleotide sequence of SEQ ID NO:4, iii. nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence (i) or (ii); iv. nucleotide sequences the sequences of which differs from the sequence of a nucleic acid molecule of (ii) or (iii) due to the degeneracy of the genetic code, c. a nucleotide sequence encoding 3-hydroxybutyryl-CoA dehydratase, wherein said nucleotide sequence is selected from the group consisting of: i. nucleotide sequences encoding a 3-hydroxybutyryl-CoA dehydratase, said 3-hydroxybutyryl-CoA dehydratase comprising an amino acid sequence that has at least 30% sequence identity with the amino acid sequence of SEQ ID NO: 5; ii. nucleotide sequences comprising a nucleotide sequence that has at least 25% sequence identity with the nucleotide sequence of SEQ ID NO:6; iii. nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence (i) or (ii); iv. nucleotide sequences the sequences of which differs from the sequence of a nucleic acid molecule of (ii) or (iii) due to the degeneracy of the genetic code, d. a nucleotide sequence encoding butyryl-CoA dehydrogenase, wherein said nucleotide sequence is selected from the group consisting of: i. nucleotide sequences encoding a butyryl-CoA dehydrogenase, said butyryl-CoA dehydrogenase comprising an amino acid sequence that has at least 20% sequence identity with the amino acid sequence of SEQ ID NO: 7; ii. nucleotide sequences comprising a nucleotide sequence that has at least 15% sequence identity with the nucleotide sequence of SEQ ID NO:8; iii. nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence (i) or (ii); iv. nucleotide sequences the sequences of which differs from the sequence of a nucleic acid molecule of (ii) or (iii) due to the degeneracy of the genetic code, e. a nucleotide sequence encoding alcohol dehydrogenase or acetaldehyde dehydrogenase, wherein said nucleotide sequence is selected from the group consisting of: i. nucleotide sequences encoding an alcohol dehydrogenase or acetaldehyde dehydrogenase, said alcohol dehydrogenase or acetaldehyde dehydrogenase comprising an amino acid sequence that has at least 20% sequence identity with the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11 respectively ii. nucleotide sequences comprising a nucleotide sequence that has at least 15% sequence identity with the nucleotide sequence of

SEQ ID NO:10 of SEQ ID NO: 12 respectively; iii. nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence (i) or (ii); iv. nucleotide sequences the sequences of which differs from the sequence of a nucleic acid molecule of (ii) or (iii) due to the degeneracy of the genetic code, f. a nucleotide sequence encoding NAD(P)H-dependent butanol dehydrogenase, wherein said nucleotide sequence is selected from the group consisting of: i. nucleotide sequences encoding a NAD(P)H-dependent butanol dehydrogenase, comprising an amino acid sequence that has at least 30% sequence identity with the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15; ii. nucleotide sequences comprising a nucleotide sequence that has at least 25% sequence identity with the nucleotide sequence of SEQ ID NO:14 or SEQ ID NO 16; iii. nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence (i) or (ii); iv. nucleotide sequences the sequences of which differs from the sequence of a nucleic acid molecule of (ii) or (iii) due to the degeneracy of the genetic code.

4. A cell according to any one of the claims 1 to 3, characterised in that the host cell is a facultative anaerobic bacterium.

5. A cell according to any one of the claims 1 to 4, wherein the cell belongs to one of the genera Escherichia, Streptomyces, Alcaligenes, Azoarcus, Thauera, Bradyrhyzobium, Brevibacterium, Rodococcus, Geotrichum,

Shewanella, Staphylococcus, Mycobacterium, Brucella, Bordetella, Fusobacterium, Corynebacterium, Bacillus, Lactobacillus, Lactococcus, Streptococcus, Pseudomonas, Zymomonas.

6. A cell according to any one of the claims 1 to 5, wherein the cell is a Bacillus subtilis, Escherichia coli, or Lactobacillus plantarum.

7. A cell according to any one of the claims 1 to 6, which is a Lactobacillus plantarum comprising one or more of the nucleotide sequences of SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21 , and

/ or SEQ ID NO 22.

8. A nucleic acid construct comprising one or more nucleotide sequences of SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21 , and / or SEQ ID NO 22.

9. A cell according to any one of the claims 1 to 7 which is able to convert a carbon source selected from the group consisting of starch, pectines, rhamnose, galactose, fucose, fructose, maltose, maltodextrines, ribose, ribulose, glucuronic acid, galacturonic acid, cellulose, hemicellulose, glucose, xylose, arabinose, sucrose, lactose, fatty acids, triglycerides and glycerol.

10. Process for the production of butanol comprising fermenting a cell as defined in any of the preceding claims in a suitable fermentation medium and optionally recovery of butanol.