Hydrocarbon synthase gene and use thereof

A hydrocarbon synthase gene encoding protein having excellent capacity to synthesize a hydrocarbon such as alkane and novel functions is provided. The gene encodes a protein comprising an amino acid sequence comprising a motif sequence shown in SEQ ID NO: 1 and having activity of synthesizing a hydrocarbon with a carbon number one less than that of an aldehyde compound from the aldehyde compound.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage of International Application No. PCT/JP2013/054956 filed Feb. 26, 2013, claiming priority based on Japanese Patent Application No. 2012-040141 filed Feb. 27, 2012, the contents of all of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a hydrocarbon synthase gene having novel features and the use thereof.

BACKGROUND ART

There are known microorganisms capable of synthesizing hydrocarbons such as alkane. It is expected that the development of recombinant microorganisms having excellent hydrocarbon synthesis capacity, hydrocarbon synthesis systems using the recombinant microorganisms, and the like would be possible through the isolation/separation of genes involved in hydrocarbon synthesis from such microorganisms having hydrocarbon production capacity. For example, Patent Literature 1 (WO2006/109558) discloses a method for obtaining a hydrocarbon from a culture product, which comprises culturing novel microalgae having hydrocarbon synthesis capacity, such asPseudochoricystis ellipsoidea, or microalgae belonging to the genusPseudochoricystisorChoricystiswhich have hydrocarbon production capacity.

In addition, Patent Literature 2 (JP Patent Publication (Kokai) No. 2010-528627 A) discloses a recombinant yeast obtained by incorporating a gene capable of converting aldehyde into alkane into yeast or the like and a method for producing alkane using the recombinant yeast. Patent Literature 3 (JP Patent Publication (Kohyo) No. 2011-520455 A) discloses an alkane synthase gene and an aldehyde synthase gene fromSynechococcus elongatusand a method for producing alkane and aldehyde using such genes. Patent Literature 4 (JP Patent Publication (Kokai) No. 9-322780 (1997) A) discloses a gene encoding a protein involved in the activity ofArabidopsis thaliana-derived fatty aldehyde decarbonylase and a transformed plant showing an altered epicuticular wax composition obtained using the gene.

However, applied use of the microorganism disclosed in Non-Patent Literature 1 and the fly-derived gene disclosed in Non-Patent Literature 3 at practical level cannot be expected because of low alkane production. In addition, in the cases of the algae disclosed in Non-Patent Literature 2 and Patent Literature 1, the alkane production reaction rate is low, resulting in intracellular accumulation of alkane. For such reasons, low-cost synthesis of alkane cannot be achieved even with the use of the algae disclosed in Non-Patent Literature 2 and Patent Literature 1 because alkane production is time-consuming and a step of purifying alkane from cells must be added. This is problematic. Further, there are no practical examples of successful alkane synthesis even by producing a recombinant with the gene disclosed in Patent Literature 4; such synthesis is not practical because an additional factor (i.e., an unknown gene) is necessary. Furthermore, even if a plant-derived gene is used for a microorganism, the gene might not sufficiently function therein. This is also problematic. In addition, the use of the cyanobacteria-derived alkane synthase gene disclosed in Patent Literature 3 would result in low productivity of alkane synthesis. The use of such gene is almost impractical.

CITATION LIST

Patent Literature

Non Patent Literature

SUMMARY OF INVENTION

Technical Problem

In view of the above circumstances, an object of the present invention is to provide a hydrocarbon synthase gene having excellent capacity to synthesize a hydrocarbon such as alkane and novel functions and the use thereof.

Solution to Problem

As a result of intensive studies in order to achieve the above object, the present inventors have found that a group of proteins comprising a certain motif sequence have excellent activity of synthesizing a hydrocarbon with a carbon number one less than that of an aldehyde compound from the aldehyde compound, and that genes encoding the proteins can be used for hydrocarbon synthesis. This has led to the completion of the present invention.

(1) A gene encoding a protein comprising an amino acid sequence comprising a motif sequence shown in SEQ ID NO: 1 and having activity of synthesizing a hydrocarbon with a carbon number one less than that of an aldehyde compound from the aldehyde compound.

(2) The gene according to (1), wherein the protein further comprises a motif sequence shown in SEQ ID NO: 2 on the C-terminal side of the motif sequence shown in SEQ ID NO: 1.

(3) The gene according to (1), wherein the protein is any of the following (a) to (d):

(a) a protein comprising an amino acid sequence shown in any even-numbered sequence ID number of SEQ ID NOS: 3 to 32 and 65 to 170;

(b) a protein comprising an amino acid sequence derived from the amino acid sequence shown in any even-numbered sequence ID number of SEQ ID NOS: 3 to 32 and 65 to 170 by substitution, deletion, insertion, or addition of one or a plurality of amino acids and having activity of synthesizing a hydrocarbon with a carbon number one less than that of an aldehyde compound from the aldehyde compound;
(c) a protein comprising an amino acid sequence having 70% or more identity to an amino acid sequence shown in any even-numbered sequence ID number of SEQ ID NOS: 3 to 32 and 65 to 170 and having activity of synthesizing a hydrocarbon with a carbon number one less than that of an aldehyde compound from the aldehyde compound; and
(d) a protein encoded by a polynucleotide that hybridizes under stringent conditions to at least a portion of a polynucleotide comprising a sequence complementary to a nucleotide sequence shown in any odd-numbered sequence ID number of SEQ ID NOS: 3 to 32 and 65 to 170 and having activity of synthesizing a hydrocarbon with a carbon number one less than that of an aldehyde compound from the aldehyde compound.
(4) The gene according to (3), wherein the even-numbered sequence ID number of SEQ ID NOS: 3 to 32 is SEQ ID NO: 6 or 12 and the odd-numbered sequence ID number of SEQ ID NOS: 3 to 32 is SEQ ID NO: 5 or 11.
(5) The gene according to (1), which is derived from a microorganism belonging to the genusKlebsiellaor fromEscherichia coli.
(6) An expression vector comprising the gene according to any one of (1) to (5).
(7) A transformant, into which the gene according to any one of (1) to (5) has been introduced.
(8) The transformant according to (7), which is obtained usingEscherichia colior yeast as a host.
(9) A protein encoded by the gene according to any one of (1) to (5).
(10) A method for producing a hydrocarbon, comprising allowing a protein encoded by the gene according to any one of (1) to (5), a coenzyme which is involved in the activity of the protein of synthesizing a hydrocarbon with a carbon number one less than that of an aldehyde compound from the aldehyde compound, and an aldehyde compound that serves as a substrate for the activity of the protein to coexist so as to synthesize a hydrocarbon with a carbon number one less than that of the aldehyde compound.
(11) The method for producing a hydrocarbon according to (10), wherein the protein, the coenzyme, and the aldehyde compound are allowed to coexist by culturing a transformant into which the gene according to any one of (1) to (5) has been introduced in a solution containing the aldehyde compound.
(12) The method for producing a hydrocarbon according to (10), wherein the protein, the coenzyme, and the aldehyde compound are allowed to coexist by mixing an enzyme liquid extracted from a transformant into which the gene according to any one of (1) to (5) has been introduced with a solution containing the aldehyde compound.
(13) The method for producing a hydrocarbon according to (10), wherein the protein, the coenzyme, and the aldehyde compound are allowed to coexist by mixing the protein isolated from a transformant into which the gene according to any one of (1) to (5) has been introduced with a solution containing the aldehyde compound and the coenzyme.
(14) The method for producing a hydrocarbon according to (10), wherein the aldehyde compound is a C11-C21aldehyde compound.
(15) The method for producing a hydrocarbon according to (10), wherein the coenzyme is reduced nicotinamide adenine dinucleotide (NADH).

This specification includes part or all of the contents as disclosed in the specification and/or drawings of Japanese Patent Application No. 2012-040141, which is a priority document of the present application.

Advantageous Effects of Invention

According to the present invention, a hydrocarbon synthase gene having activity of converting an aldehyde compound into a hydrocarbon with a carbon number one less than that of the aldehyde compound superior to that of conventionally known aldehyde decarbonylase genes can be provided. A hydrocarbon can be produced using an aldehyde compound as a substrate with good efficiency at low cost by making use of the hydrocarbon synthase gene of the present invention.

DESCRIPTION OF EMBODIMENTS

The present invention is described in more detail below with reference to the drawings and the Examples.

The hydrocarbon synthesis gene of the present invention is a gene encoding a protein comprising an amino acid sequence comprising a motif sequence shown in SEQ ID NO: 1 and having “activity of synthesizing a hydrocarbon with a carbon number one less than that of an aldehyde compound from the aldehyde compound (hereinafter referred to as “hydrocarbon synthesis activity”).” The term “hydrocarbon synthesis activity” refers to an enzyme activity of synthesizing a hydrocarbon with a carbon number one less than that of an aldehyde compound from the aldehyde compound, which also can be said as enzyme activity of removing a carbonyl group from an aldehyde compound. The hydrocarbon synthesis activity may include a reaction for generating, as by-products, carbon monoxide, carbon dioxide, carbonic acid, formic acid, water, and the like.

Here, the motif sequence shown in SEQ ID NO: 1 is a sequence referred to as an aldehyde dehydrogenases glutamic acid active site. In the amino acid sequence shown in SEQ ID NO: 1, the 1st Xaa corresponds to an amino acid single letter code of L, I, V, M, F, G, or A. The 3rd Xaa corresponds to an amino acid single letter code of L, I, M, S, T, A, or C in the amino acid sequence shown in SEQ ID NO: 1. The 4th Xaa corresponds to an amino acid single letter code of G or S in the amino acid sequence shown in SEQ ID NO: 1. The 6th Xaa corresponds to an amino acid single letter code of K, N, L, or M in the amino acid sequence shown in SEQ ID NO: 1. The 7th Xaa corresponds to an amino acid single letter code of S, A, D, or N in the amino acid sequence shown in SEQ ID NO: 1. The 8th Xaa corresponds to an amino acid single letter code of T, A, P, F, or V in the amino acid sequence shown in SEQ ID NO: 1.

Particularly preferably, the hydrocarbon synthesis gene of the present invention is a gene encoding a protein having hydrocarbon synthesis activity and comprising an amino acid sequence further comprising the motif sequence shown in SEQ ID NO: 2 on the C-terminal side of the motif sequence shown in SEQ ID NO: 1. Here, the motif sequence shown in SEQ ID NO: 2 corresponds to the amino acid sequence of a region that is found to be highly conserved upon multiple alignment analysis of amino acid sequences of a plurality of proteins encoded by a gene from aKlebsiellasp. microorganism which is the hydrocarbon synthesis gene of the present invention. The first Xaa corresponds to an amino acid single letter code of P, A, or F in the amino acid sequence shown in SEQ ID NO: 2. In addition, the 2nd Xaa corresponds to an amino acid single letter code of F, H, or V in the amino acid sequence shown in SEQ ID NO: 2. Further, the 3rd Xaa corresponds to an amino acid single letter code of G or A in the amino acid sequence shown in SEQ ID NO: 2. The 5th Xaa may correspond to any amino acid in the amino acid sequence shown in SEQ ID NO: 2. The 6th Xaa corresponds to an amino acid single letter code of K, G, or R in the amino acid sequence shown in SEQ ID NO: 2. The 7th Xaa may correspond to any amino acid in the amino acid sequence shown in SEQ ID NO: 2. The 10th Xaa may correspond to any amino acid in the amino acid sequence shown in SEQ ID NO: 2. The 11th Xaa corresponds to an amino acid single letter code of G or H in the amino acid sequence shown in SEQ ID NO: 2. The 12th Xaa corresponds to an amino acid single letter code of R, K, or G in the amino acid sequence shown in SEQ ID NO: 2. The 13th Xaa corresponds to an amino acid single letter code of F, D, P, or A in the amino acid sequence shown in SEQ ID NO: 2.

Further, the hydrocarbon synthesis gene of the present invention may be a gene from any organism. For example, the hydrocarbon synthesis gene of the present invention can be identified/isolated from a Gram-negative organism, a Gram-positive organism, a fungus, a plant, or an animal. Examples of a Gram-negative organism includeEscherichia coliandPseudomonas putida. Examples of a Gram-positive organism includeBacillus subtilis, Corynebacterium glutamicum, andLactobacillus reuteri. Examples of a fungus includeSaccharomyces cerevisiae, Candida tropicalis, Debaryomyces hansenii, Pichia pastoris, andAspergillus oryzae. Examples of a plant includeZea maysandArabidopsis thaliana. Examples of an animal includeDrosophila melanogaster, Rattus norvegicus, andHomo sapiens. The hydrocarbon synthesis gene of the present invention can be isolated from such various organisms and used in an adequate manner.

More specifically, an aldehyde dehydrogenase gene encoding a protein comprising the motif sequence shown in SEQ ID NO: 1 can be searched for in a database storing gene information such as the NCBI (National Center for Biotechnology Information) database. The target gene can be identified based on the corresponding accession number as described below.

In addition, genes from theBacillus subtilis168 strain, i.e., NP_388129, NP_389813, NP_390984, NP_388203, NP_388616, NP_391658, NP_391762, NP_391865, and NP_391675, can be identified as the hydrocarbon synthesis gene of the present invention.Corynebacterium glutamicumATCC13032-derived genes, i.e., NP_599351, NP_599725, NP_601988, NP_599302, NP_601867, and NP_601908, can be identified as the hydrocarbon synthesis gene of the present invention. ALactobacillus reuteriDSM20016-derived gene, i.e., YP_001270647, can be identified as a hydrocarbon synthesis gene according to the present invention.

Meanwhile, the aforementioned gene encoding a protein comprising the motif sequence shown in SEQ ID NO: 1 can be identified based on the genome sequence information obtained by elucidating the genome sequence of an organism with an unknown genome sequence that is not registered in a database such as the NCBI database. More specifically, when the genome sequence of theKlebsiellasp. NBRC100048 strain is analyzed according to a standard method, the gene encoding a protein comprising the motif sequence shown in SEQ ID NO: 1 can be identified based on the genome sequence information.

Ten types of genes can be identified as hydrocarbon synthesis genes fromKlebsiellasp. according to the present invention. These ten different genes are designated as gene01 to gene10 for convenience. Table 1 below lists nucleotide sequences of the coding regions of gene01 to gene10 and amino acid sequences encoded by the nucleotide sequences.

The genes fromKlebsiellasp. listed in table 1 encode proteins comprising the motif sequence shown in SEQ ID NO: 2.

Table 2 below lists the nucleotide sequences of the coding regions and amino acid sequences encoded by the nucleotide sequences for 5 types ofEscherichia coliK-12 W3110-derived genes, i.e., BAA14869, BAA14992, BAA16524, BAE77705, and BAA15538, as examples of genes registered with the NCBI database.

Table 3 below lists the nucleotide sequences of the coding regions and amino acid sequences encoded by the nucleotide sequences forCorynebacterium glutamicumATCC13032-derived genes, aLactobacillus reuteriDSM20016-derived gene,Saccharomyces cerevisiae-derived genes,Candida tropicalisMYA-3404-derived genes,Debaryomyces hanseniiCBS767-derived genes,Pichia pastorisGS115-derived genes,Schizosaccharomyces pombe-derived genes,Aspergillus oryzaeRIB40-derived genes, aZea mays-derived gene,Arabidopsis thaliana-derived genes,Drosophila melanogaster-derived genes,Rattus norvegicus-derived genes, andHomo sapiens-derived genes, as examples of the hydrocarbon synthesis genes of the present invention registered with the NCBI database. Here, the “Gene name” column in table 3 contains gene IDs in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

Note that the hydrocarbon synthesis gene of the present invention is not limited to genes identified based on the gene names, the nucleotide sequences, and the amino acid sequences described above.

The hydrocarbon synthesis gene of the present invention may be a gene encoding a protein having hydrocarbon synthesis activity and comprising an amino acid sequence derived from the amino acid sequence shown in any even-numbered sequence ID number of SEQ ID NOS: 3 to 32 and 65 to 170 by substitution, deletion, insertion, or addition of one or a plurality of amino acids. The expression “a plurality of amino acids” used herein means, for example, 2 to 100 amino acids, preferably 2 to 80 amino acids, more preferably 2 to 50 amino acids, and further preferably 2 to 15 amino acids.

In addition, the hydrocarbon synthesis gene of the present invention may be a gene encoding a protein having hydrocarbon synthesis activity and comprising an amino acid sequence having 70% or more, preferably 80% or more, more preferably 85% or more, further preferably 90% or more, and most preferably 98% or more identity to an amino acid sequence shown in any even-numbered sequence ID number of SEQ ID NOS: 3 to 32 and 65 to 170. Here, identity between sequences refers to a value (percentage) of alignment between two amino acid sequences determined using sequence similarity search software such as BLAST, PSI-BLAST, or HMMER at a default setting.

Further, the hydrocarbon synthesis gene of the present invention may be a gene encoding a protein having hydrocarbon synthesis activity which is encoded by a polynucleotide that hybridizes under stringent conditions to at least a portion of a polynucleotide comprising a sequence complementary to a nucleotide sequence shown in any odd-numbered sequence ID number of SEQ ID NOS: 3 to 32 and 65 to 170. Here, the term “stringent conditions” refers to conditions under which namely a specific hybrid is formed, but a non-specific hybrid is never formed. For example, such conditions can be adequately determined with reference to Molecular Cloning: A Laboratory Manual (Third Edition). In practice, stringency can be predetermined based on the temperature and the salt concentration in a solution upon Southern hybridization, and the temperature and the salt concentration in a solution in the step of washing during Southern hybridization. Specifically, stringent conditions include, for example, a sodium concentration of 25 to 500 mM and preferably 25 to 300 mM, and a temperature of 42° C. to 68° C. and preferably 42° C. to 65° C. More specifically, stringent conditions include 5×SSC (83 mM NaCl, 83 mM sodium citrate) and a temperature of 42° C. In addition, the expression “at least a portion of a polynucleotide” means the entire polynucleotide comprising a nucleotide sequence complementary to a certain nucleotide sequence and a continuous portion of the entire polynucleotide comprising the complementary nucleotide sequence.

In addition, it is possible to introduce a mutation into a certain amino acid sequence by altering the nucleotide sequence of the above hydrocarbon synthesis gene by a technique known in the art. It is also possible to introduce a mutation into a nucleotide sequence by a known technique such as the Kunkel method or Gapped duplex method or a method according thereto. For example, a mutation is introduced using a mutagenesis kit using site-directed mutagenesis (e.g., Mutant-K and Mutant-G (both are commercial names, TAKARA Bio)) or an LA PCR in vitro Mutagenesis series kit (trade name, TAKARA Bio). Also, a mutagenesis method may be a method using a chemical mutagen represented by EMS (ethyl methanesulfonate), 5-bromouracil, 2-aminopurine, hydroxylamine, N-methyl-N′-nitro-N nitrosoguanidine, or other carcinogenic compounds or a method that involves radiation treatment or ultraviolet treatment typically using X-rays, alpha rays, beta rays, gamma rays, an ion beam, or the like.

It is possible to confirm whether or not a gene comprising a certain nucleotide sequence encodes a protein having hydrocarbon synthesis activity in the following manner. An expression vector incorporating the gene between, for example, an appropriate promoter and an appropriate terminator is produced, an appropriate host is transformed using the expression vector, and hydrocarbon synthesis activity of a protein to be expressed is assayed. Here, it is possible to assay hydrocarbon synthesis activity in the following manner. The above transformant is cultured using a solution containing an aldehyde compound that serves as a substrate. Then, a hydrocarbon from the aldehyde compound (i.e., a hydrocarbon with a carbon number one less than that of the aldehyde compound serving as a substrate) is analyzed using a gas chromatography system/mass spectrometer. In addition, quantitative assay of hydrocarbon synthesis activity can be carried out by quantitatively determining generated hydrocarbon using a gas chromatography system/mass spectrometer. As an aldehyde compound described in detail below, for example, tetradecanal can be used.

The hydrocarbon synthesis gene of the present invention described above is incorporated into an appropriate expression vector so as to be introduced into a host. The host used herein is not particularly limited as long as it is an organism that can express the hydrocarbon synthesis gene of the present invention. Examples of such host include: bacteria belonging to the genusEscherichiasuch asEscherichia coli; bacteria belonging to the genusBacillussuch asBacillus subtilis; bacteria belonging to the genusPseudomonassuch asPseudomonas putida; bacteria belonging to the genusRhizobiumsuch asRhizobium melilotii; yeast such asSaccharomyces cerevisiae, Schizosaccharomyces pombe, orPichia pastoris; and fungi such as filamentous bacteria.

When a bacterium such asEscherichia coliis used as a host, it is preferable for an expression vector to be autonomously replicable in the bacterium, and at the same time, to be composed of promoters, a ribosome-binding sequence, the above gene, and a transcription termination sequence. Such expression vector may further comprise a gene that controls promoter activity.

Examples ofEscherichia colithat can be used include conventionally known bacterial strains such as theEscherichia coliBL21 (DE3), K12, DH1, and JM109 strains. Specifically, so-called K strains such as the K12 strain and a strain produced from the K12 strain can be used asEscherichia coli. In addition, theBacillus subtilis168 strain and the like can be used asBacillus subtilis.

Any promoter can be used as long as it can be expressed in a host such asEscherichia coli. Examples of a promoter that can be used include:Escherichia coli-derived promoters such as a trp promoter, a lac promoter, a PL promoter, and a PR promoter; and phage-derived promoters such as a T7 promoter. Alternatively, an artificially designed or modified promoter such as a tac promoter may be used.

A method for introducing an expression vector is not particularly limited as long as DNA is introduced into a bacterium. Examples of the method include a method using calcium ions [Cohen, S, N., et al.: Proc. Natl. Acad. Sci., USA, 69:2110-2114 (1972)] and electroporation.

Examples of yeast that can be used as a host include, but are not particularly limited to, yeast belonging to the genusCandidasuch asCandida Shehatae, yeast belonging to the genusPichiasuch asPichia stipitis, yeast belonging to the genusPachysolensuch asPachysolen tannophilus, yeast belonging to the genusSaccharomycessuch asSaccharomyces cerevisiae, and yeast belonging to the genusSchizosaccharomycessuch asSchizosaccharomyces pombe. Saccharomyces cerevisiaeis particularly preferable.

In addition, in order to enhance expression of the hydrocarbon synthesis gene of the present invention, an appropriate promoter having high transcription activity is used. Examples of such promoter that can be used include, but are not particularly limited to, a glyceraldehyde-3-phosphate dehydrogenase gene (TDH3) promoter, a 3-phosphoglycerate kinase gene (PGK1) promoter, and a hyperosmolarity-responsive 7 gene (HOR7) promoter. A pyruvate decarboxylase gene (PDC1) promoter is particularly preferable because it has high capacity to cause high expression of a gene of interest located downstream of the promoter. In addition to the above, a downstream gene can be strongly expressed using a gall promoter, a gal10 promoter, a heat shock protein promoter, an MFα1 promoter, a PHO5 promoter, a GAP promoter, an ADH promoter, an AOX1 promoter, or the like.

In addition, as a method for introducing the above gene, any conventionally known method of yeast transformation can be used. Specific examples of such method that can be carried out include, but are not limited to, an electroporation method (Meth. Enzym., 194, p. 182 (1990)), a spheroplast method (Proc. Natl. Acad. Sci. USA, 75 p. 1929 (1978)), and a lithium acetate method (J. Bacteriology, 153, p. 163 (1983), Proc. Natl. Acad. Sci. USA, 75 p. 1929 (1978), Methods in yeast genetics, 2000 Edition: A Cold Spring Harbor Laboratory Course Manual).

As described above, a recombinant organism into which the hydrocarbon synthesis gene of the present invention has been introduced (e.g., recombinantEscherichia colior recombinant yeast) can synthesize a hydrocarbon from an aldehyde compound if the hydrocarbon synthesis gene is expressed in the presence of the aldehyde compound and a coenzyme such as NADH. For synthesis, NADH can be used as a coenzyme that allows a protein encoded by the hydrocarbon synthesis gene of the present invention to show hydrocarbon synthesis activity. Since NADH is abundantly present in cells, the amount of coenzyme would not be a rate-determining factor of a hydrocarbon synthesis reaction. Therefore, a recombinant organism into which the hydrocarbon synthesis gene of the present invention has been introduced (e.g., recombinantEscherichia colior recombinant yeast) can synthesize a hydrocarbon with excellent reaction efficiency. Either of NADH and NADPH can be used as a coenzyme for a protein encoded by the hydrocarbon synthesis gene of the present invention.

Hydrocarbons that can be synthesized herein include hydrocarbons having chain structures (chain hydrocarbons) and hydrocarbons having cyclic structures (cyclic hydrocarbons). Hydrocarbons having chain structures may have one or more branches. Examples of branches include: an alkyl group such as a methyl group, an ethyl group, a propyl group, or a butyl group (including a tert-butyl group); an alkynyl group; and an alkenyl group. Also, examples of branches include a chloromethyl group, an acetyl group, a 2-pyridyl group, a hydroxyphenyl group, an aminoacetyl group, a methoxy group, a phenoxy group, a methylthio group, and a phenylthio group. Further, a hydrocarbon to be synthesized may be a saturated hydrocarbon (alkane) or an unsaturated hydrocarbon (alkene or alkyne).

Meanwhile, the number of carbons for a hydrocarbon to be synthesized is not particularly limited; however, it is preferably 5 to 20 so that the hydrocarbon is in a liquid form at ordinary temperatures. In addition, the hydrocarbon to be synthesized is preferably a C10-C20saturated hydrocarbon in consideration of the use thereof for diesel fuel, more preferably a C12-C14saturated hydrocarbon, and most preferably a C13saturated hydrocarbon. More specifically, the hydrocarbon to be synthesized is C12dodecane, C13tridecane, C14tetradecane, or the like.

When specific hydrocarbons such as those listed above are synthesized, an appropriate aldehyde compound that serves as a substrate can be selected. That is, since hydrocarbon synthesis activity causes synthesis of a hydrocarbon from an aldehyde compound used as a substrate, an appropriate aldehyde compound can be selected in accordance with the structure of a desired hydrocarbon.

Meanwhile, the hydrocarbon synthesis gene of the present invention also can be used for a method for producing a hydrocarbon in vitro. In one example, a hydrocarbon can be synthesized in vitro using a disruption solution obtained by disrupting a recombinant organism into which the hydrocarbon synthesis gene of the present invention has been introduced (e.g., recombinantEscherichia colior recombinant yeast) or an extract obtained by extracting a fraction containing a protein encoded by the hydrocarbon synthesis gene from the disruption solution. Specifically, in vitro hydrocarbon synthesis can be carried out by adding an aldehyde compound that serves as a substrate (and, if necessary, a coenzyme such as NADH) to the disruption solution or extract. In particular, the disruption solution or extract is rich in a coenzyme such as NADH, and thus it is only necessary to add an aldehyde compound that serves as a substrate to the disruption solution or extract (without the need of adding a coenzyme such as NADH) in most cases. In other words, the use of the hydrocarbon synthesis gene of the present invention enables efficient hydrocarbon synthesis without the need of an expensive coenzyme such as NADPH.

Alternatively, a hydrocarbon can be synthesized in vitro by purifying or roughly purifying a protein encoded by the hydrocarbon synthesis gene of the present invention according to a standard method and mixing the purified or roughly purified protein, an aldehyde compound that serves as a substrate, and a coenzyme such as NADH. Here, NADH can be used as a coenzyme for a protein encoded by the hydrocarbon synthesis gene of the present invention so that the protein shows hydrocarbon synthesis activity. Thus, it is not always necessary to use an expensive coenzyme, i.e., NADPH. This means that when a protein encoded by the hydrocarbon synthesis gene of the present invention is used, in vitro hydrocarbon synthesis can be achieved at low cost using NADH as a less expensive coenzyme.

A synthesized hydrocarbon can be isolated by a standard method. For example, the above-described recombinant yeast is cultured in a medium so as to produce a hydrocarbon. Here, a hydrocarbon is synthesized in a medium and thus it can be isolated from a supernatant fraction obtained by isolating cells from the medium by means of centrifugation or the like. For example, a hydrocarbon can be isolated from a supernatant fraction as follows. An organic solvent such as ethyl acetate or methanol is added to a supernatant fraction. The mixture is sufficiently agitated and separated into an aqueous layer and a solvent layer. Then, a hydrocarbon is extracted from the solvent layer.

EXAMPLES

The present invention is hereafter described in greater detail with reference to the following examples, although the technical scope of the present invention is not limited thereto.

In this Example, the genome sequence of a microorganism having alkane synthesis capacity of theKlebsiellasp. NBRC100048 strain was analyzed by a standard method. Ten types of genes encoding proteins comprising the motif sequence shown in SEQ ID NO: 1 were identified based on the obtained genome sequence information. The 10 types of genes identified herein were designated as gene01 to gene10 and functions thereof were estimated. Table 4 summarizes information about putative functions and sequences of the ten different genes.

In this Example, genes encoding proteins comprising the motif sequence shown in SEQ ID NO: 1 were identified based on the genome information of theE. coliW3110 strain, as well as the 10 types of genes. Particularly in this Example, 5 types of genes listed in Table 5 below were mainly examined from among the identified genes.

Nucleic acid fragments separately containing the above 15 types of genes were PCR-amplified using, as a template, genome DNA of theKlebsiellasp. NBRC100048 strain or theE. coliW3110 strain. Table 6 shows primers used for PCR. DNeasy Blood & Tissue Kits (QIAGEN) were used for genome DNA extraction.

A sequence for homologous recombination (i.e., a sequence homologous to a vector region) was added to each primer. PfuUltra II Fusion HS DNA Polymerase (Stratagene) was also used for PCR. Each PCR-amplified nucleic acid fragment was mixed with a BamHI-treated pUC118 plasmid so as to incorporate the amplified nucleic acid fragment into a vector by homologous recombination. QIAquick PCR Purification Kits (QIAGEN) were used for the purification of PCR products. In-Fusion HD Cloning Kits (Clontech) were used for the ligation of PCR products.

The obtained expression plasmids were used for the transformation ofE. coliJM109. Each ofEscherichia colitransformants was cultured overnight in 1-ml LB medium (ampicillin: 100 μg/ml) at 37° C. and 100 rpm. 3-ml LB medium (ampicillin: 100 μg/ml; Triton X-100:0.1%; IPTG: 0.5 mM; and tetradecanal: 1 mM) was inoculated with the obtained culture liquid to result in 10% culture liquid by volume, followed by culture at 30° C. and 100 rpm for 24 hours.

Cells were harvested from the culture product (room temperature, 6000×g, 5 min). The supernatant (1 ml) was introduced into a glass vial bottle (Agilent Technologies) and subjected to GC/MS analysis so as to detect tridecane synthesized from tetradecanal. An HP7694 Headspace Sampler (Hewlett-Packard) was used for GC/MS analysis. Table 7 shows Headspace Sampler analysis conditions and table 8 shows GC/MS analysis conditions.

FIG. 1shows analysis results obtained using the 10 types of genes fromKlebsiellasp. identified in this Example. As shown inFIG. 1, all of the 10 types of genes fromKlebsiellasp. identified in this Example were found to have hydrocarbon synthesis activity. In addition, proteins encoded by gene02 and gene05 were found to have significantly excellent hydrocarbon synthesis activity.FIG. 2shows a GC/MS analysis chart for a vector control strain and that for a strain into which gene02 has been introduced.

Similarly,FIG. 3shows analysis results obtained using the 5 types of genes from theE. coliW3110 strain identified in this Example. As shown inFIG. 3, all of the 5 types of genes from theE. coliW3110 strain identified in this Example were found to have hydrocarbon synthesis activity. In addition, proteins encoded by BAA14992 and BAA14869 were found to have significantly excellent hydrocarbon synthesis activity.

In this Example, in vitro alkane synthesis was attempted using gene02 specified in Example 1 as a gene encoding a protein having excellent hydrocarbon synthesis activity.

Specifically, recombinantEscherichia coliprepared in Example 1, into which gene02 had been introduced, was cultured overnight in 1-ml LB medium (ampicillin: 100 μg/ml) at 37° C. and 100 rpm. Then, 1-ml LB medium (ampicillin 100 μg/ml, IPTG 0.5 mM) was inoculated with the obtained culture liquid to result in 1% culture liquid by volume, followed by culture at 30° C. and 120 rpm for 6 hours. Next, cells were harvested from the culture product (4° C., 6000×g, 3 minutes). The cells were suspended in 500 μl of phosphate buffer (pH 7.2), following which the cells were disrupted using an ultrasonic disintegrator (4° C., 10 minutes). Subsequently, the obtained disruption solution was centrifuged (4° C., 10000×g, 5 minutes) to collect the supernatant. The collected solution was subjected to enzymatic assay.

An enzymatic reaction was carried out overnight at 30° C. using the reaction composition shown in table 9. In addition, 8 types of C11-C18aldehyde compounds were used in this Example. Here, alkane with a carbon number one less than that of an aldehyde compound is synthesized.

FIG. 4shows the results of quantitative determination for synthesized C10-C17alkanes. As is understood fromFIG. 4, the protein encoded by gene02 was found to have excellent capacity to synthesize alkane, and in particular, C12-C14alkane.FIG. 5shows a GC/MS analysis chart for a vector control strain and that for a strain into which gene02 has been introduced created by assaying C13alkane using a C14aldehyde compound as a substrate.

In this Example, in vitro alkane synthesis was attempted using the purified protein encoded by gene04 identified in Example 1.

Specifically, PCR was performed using, as a template, genome DNA of theKlebsiellasp. NBRC100048 strain prepared in the manner described in Example 1 and a pair of primers (forward primer: accacagccaggatccGCGTTATGCACACCCTGGCCAGCCCGGCGCCCTG (SEQ ID NO: 63); reverse primer: gctcgaattcggatccTCAGAACAGGCCCAGCGGCGCGGTGCCGTAGCT (SEQ ID NO: 64)). The PCR product was ligated to the BamHI site of pRSFduet-1 plasmid (Novagen). A PCR amplification kit, a PCR product purification kit, and a PCR product ligation kit used herein were the same as those used in Example 1.

Next,E. coliBL21 (DE3) was transformed using the obtained expression vector. TransformedEscherichia coliwas cultured overnight in 2-ml LB medium (kanamycin: 20 μg/ml) at 37° C. and 120 rpm. Then, 10-ml LB medium (kanamycin: 20 μg/ml, IPTG 0.5 mM) was inoculated with the obtained culture liquid to result in 1% culture liquid by volume, followed by culture at 37° C. for 5 hours. Cells were harvested from the culture product (4° C., 6000×g, 3 minutes). The cells were suspended in 1 ml of phosphate buffer (pH 7.2) and disrupted using an ultrasonic disintegrator (4° C., 10 minutes).

A His-tag protein was purified from the obtained disruption solution using TALON CellThru Resin (Clontech).FIG. 6shows SDS-PAGE results for a disruption solution ofEscherichia colitransformed with pRSFduet-1 plasmid to which gene04 had been ligated, a disruption solution ofEscherichia colitransformed with pRSFduet-1 plasmid to which gene04 had not been ligated, and a solution containing the purified His-tag protein. InFIG. 6, lane 1 represents the disruption solution ofEscherichia colitransformed with pRSFduet-1 plasmid to which gene04 had not been ligated, lane 2 represents the disruption solution ofEscherichia colitransformed with pRSFduet-1 plasmid to which gene04 had been ligated, and lane 3 represents the solution containing the purified His-tag protein. As shown inFIG. 6, it was revealed that the purified His-tag protein was observed at a position corresponding to a molecular weight of 56.6 kDa of a protein predicted from the nucleotide sequence of gene04.

An alkane synthesis reaction was carried out in vitro using the solution containing the His-tag protein. An enzymatic reaction was carried out overnight at 30° C. using the reaction composition shown in table 10. In addition, tetradecanal was used as an aldehyde compound in this Example. Here, tridecane is synthesized as an alkane.

After the termination of the enzymatic reaction, synthesized alkane was subjected to GC/MS analysis in the manner described in Example 1 or 2.FIG. 7shows GC/MS analysis charts. InFIG. 7, chart (a) is an analysis chart for GC/MS analysis using the above reaction solution composition, chart (b) is an analysis chart for GC/MS analysis in which the His-tag protein eluate was not added to the reaction solution composition, and chart (c) is an analysis chart for GC/MS analysis in which no coenzyme was added to the reaction solution composition. As shown inFIG. 7, in this Example, it was revealed that a purified protein encoded by gene04 (but not a protein in cell extract) has hydrocarbon synthesis activity.

Further, hydrocarbon synthesis activity determined with the use of NADPH as a coenzyme was examined in this Example. Specifically, an enzymatic reaction was carried out in the manner described above except that NADPH was used as a coenzyme. A synthesized alkane was subjected to GC/MS analysis.FIG. 8shows the results. As is understood fromFIG. 8, it was found that both NADPH and NADH can be used as a coenzyme for a protein encoded by gene04. In addition, the protein encoded by gene04 was found to have superior hydrocarbon synthesis activity when NADH is used as a coenzyme compared with that obtained when NADPH is used.

In this Example, alkane synthesis was attempted by allowing yeast to express gene02 specified in Example 1 as a gene encoding a protein having excellent hydrocarbon synthesis activity.

Specifically, PCR was performed using, as a template, genome DNA of theKlebsiellasp. NBRC100048 strain prepared in the manner described in Example 1 and a pair of primers (forward primer: aacaaacaaaggatccaaaaaaATGCGTTATGCACACCCTGGCCAGC (SEQ ID NO: 171); reverse primer: gtcgtattacggatccttaTCAGAACAGGCCCAGCGGCGCGGTG (SEQ ID NO: 172)). PfuUltra II Fusion HS DNA Polymerase (Stratagene) was used for PCR.

PCR-amplified nucleic acid fragments were ligated to the BamHI site of a pESCpgkgap-HIS vector (see WO2012/098662) using an In-Fusion HD Cloning Kit (Clontech). TheSaccharomyces cerevisiaeYPH499 strain was transformed using the obtained expression plasmid. Yeast was transformed in accordance with the protocol provided with a Frozen-EZ Yeast Transformation II Kit (ZYMO RESEARCH).

Next, 1-ml SD-His liquid medium was inoculated with colonies of the obtained yeast transformant, followed by overnight culture at 30° C. (Oriental Giken Inc.: IFM type, 130 rpm). Thereafter, 3-ml SD-His medium (supplemented with 1 mM tetradecanal) was inoculated with the obtained preculture liquid to result in 1% preculture liquid by volume, followed by culture at 30° C. and 100 rpm for 2 days.

After the termination of culture, GC/MS analysis was performed in the manner described in Example 1.FIG. 9shows the analysis results. As shown inFIG. 9, it was found that theKlebsiellasp. NBRC100048 strain-derived gene (gene02) functions as a gene encoding a protein having excellent hydrocarbon synthesis activity even if yeast is used as a host.

In this Example, alkane synthase genes from a variety of organism species were evaluated for alkane synthesis capacity.

The 53 types of genes were amplified using pairs of primers listed in table 11 below.

Among the 53 types of genes listed in table 11, genome DNAs extracted from the corresponding strains were used as templates for theCorynebacterium glutamicumATCC13032-derived genes (NCg10098, NCg10463, NCg12698, NCg10049, NCg12578, and NCg12619), theLactobacillus reuteriDSM20016-derived gene (Lreu_0034), and theSaccharomyces cerevisiae-derived genes (YER073W, YHR037W, YHR039C, YMR169C, YMR170C, YOR374W, YBR006W, YMR110C, and YPL061W). In addition, among the 53 types of genes listed in table 11, artificial genes chemically synthesized based on the amino acid sequences in the KEGG database were used as templates for theCandida tropicalisMYA-3404-derived genes (CTRG_04587, CTRG_01342, and CTRG_00532), theDebaryomyces hanseniiCBS767-derived genes (DEHA2G03740g, DEHA2G22572g, and DEHA2B10384g), thePichia pastorisGS115-derived genes (PAS_chr1-3_0024, PAS_chr2-1_0853, and PAS_chr4_0043), theSchizosaccharomyces pombe-derived genes (SPAC139.05, SPAC1002.12c and SPAC9E9.09c), theAspergillus oryzaeRIB40-derived genes (AOR_1_1204144 and AOR_1_1330014), and theZea mays-derived gene (100284047). Further, among the 53 types of genes listed in table 11, a cDNA library (ATCC77500) purchased from the ATCC (American Type Culture Collection) was used as a template for theArabidopsis thaliana-derived genes (AT1G23800, AT1G74920, AT1G79440, AT2G24270, AT3G24503, AT3G48000, and AT1G54100). Furthermore, among the 53 types of genes listed in table 11, a cDNA library (ATCC87285) purchased from the ATCC (American Type Culture Collection) was used as a template for theDrosophila melanogaster-derived genes (Dmel_CG3752, Dmel_CG7145, Dmel_CG8665, Dmel_CG11140, Dmel_CG31075, Dmel_CG4685, and Dmel_CG9629). Moreover, among the 53 types of genes listed in table 11, a cDNA library (ATCC77403) purchased from the ATCC (American Type Culture Collection) was used as a template for theRattus norvegicus-derived genes (24188 and 641316). Also, among the 53 types of genes listed in table 11, a cDNA library (ATCC77402) purchased from the ATCC (American Type Culture Collection) was used as a template for theHomo sapiens-derived genes (216, 219, 223, 224, 501, and 64577).

All publications, patents, and patent applications cited herein are incorporated by reference in their entirety.