# Patent

## Title
Hydrocarbon synthase gene and use thereof

## Abstract

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.

## Background
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 as Pseudochoricystis ellipsoidea, or microalgae belonging to the genus Pseudochoricystis or Choricystis which 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 from Synechococcus elongatus and 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 of Arabidopsis thaliana-derived fatty aldehyde decarbonylase and a transformed plant showing an altered epicuticular wax composition obtained using the gene.
Further, Non-Patent Literature 1 (Process Biochemistry, 41, (2006), pp. 1001-1014) discloses the hydrocarbon synthesis pathway in a microorganism. Non-Patent Literature 2 (Appl. Microbiol. Biotechnol., (2005), 66: pp. 486-496) discloses biosynthesis of hydrocarbons in Botryococcus braunii, which is an alga, as in the case of Patent Literature 1. Patent Literature 3 (Proc. Natl. Acad. Sci., (1994), Vol. 91, pp. 10000-10004) discloses a fly-derived cytochrome P450 gene capable of converting aldehyde into a hydrocarbon ((Z)-9-tricosene).
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


    Patent Literature 1: WO2006/109558
    Patent Literature 2: JP Patent Publication (Kokai) No. 2010-528627 A
    Patent Literature 3: JP Patent Publication (Kohyo) No. 2011-520455 A
    Patent Literature 4: JP Patent Publication (Kokai) No. 9-322780 (1997) A


Non Patent Literature


    Non Patent Literature 1: Process Biochemistry, 41, (2006), pp. 1001-1014
    Non Patent Literature 2: Appl. Microbiol. Biotechnol., (2005), 66: pp. 486-496
    Non Patent Literature 3: Proc. Natl. Acad. Sci., (1994), Vol. 91, pp. 10000-10004


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 genus Klebsiella or from Escherichia 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 using Escherichia coli or 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-C21 aldehyde 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.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a characteristic chart showing results of determining “activity of synthesizing a hydrocarbon with a carbon number one less than that of an aldehyde compound from the aldehyde compound” for transformants prepared using 10 types of genes from Klebsiella sp.
FIG. 2 shows a GC/MS analysis chart for a vector control strain and that for a strain into which gene02 has been introduced.
FIG. 3 is a characteristic chart showing results of determining “activity of synthesizing a hydrocarbon with a carbon number one less than that of an aldehyde compound from the aldehyde compound” for transformants prepared using 5 types of genes from the E. coli W3110 strain.
FIG. 4 is characteristic chart showing results of determining “activity of synthesizing a hydrocarbon with a carbon number one less than that of an aldehyde compound from the aldehyde compound” using different aldehyde compounds as substrates for disruption solution supernatants of transformants into which gene02 from Klebsiella sp. has been introduced.
FIG. 5 shows a GC/MS analysis chart for a vector control strain and that for a strain into which gene02 has been introduced created by assaying C13 alkane using a C14 aldehyde compound as a substrate.
FIG. 6 shows an image indicating SDS-PAGE results for a disruption solution of transformed Escherichia coli and a purified His-tag protein solution.
FIG. 7 shows GC/MS analysis charts indicating in vitro alkane synthesis results obtained using purified His-tag proteins.
FIG. 8 is a characteristic chart showing results of a comparison of alkane synthesis capacity between NADH and NADPH used as coenzymes.
FIG. 9 shows a GC/MS analysis chart of yeast transformants into which gene02 has been introduced.
FIG. 10-1 is the first half of a characteristic chart showing results of determining “activity of synthesizing a hydrocarbon with a carbon number one less than that of an aldehyde compound from the aldehyde compound” for transformants prepared using 53 types of genes from a variety of organism species.
FIG. 10-2 is the second half of a characteristic chart showing results of determining “activity of synthesizing a hydrocarbon with a carbon number one less than that of an aldehyde compound from the aldehyde compound” for transformants prepared using 53 types of genes from a variety of organism species.



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 a Klebsiella sp. 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 include Escherichia coli and Pseudomonas putida. Examples of a Gram-positive organism include Bacillus subtilis, Corynebacterium glutamicum, and Lactobacillus reuteri. Examples of a fungus include Saccharomyces cerevisiae, Candida tropicalis, Debaryomyces hansenii, Pichia pastoris, and Aspergillus oryzae. Examples of a plant include Zea mays and Arabidopsis thaliana. Examples of an animal include Drosophila melanogaster, Rattus norvegicus, and Homo 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.
Specifically, Escherichia coli K-12 W3110-derived genes, i.e., BAE77705, BAA35791, BAA14869, BAA14992, BAA15032, BAA16524, BAE77705, BAA15538, and BAA15073, can be identified as the hydrocarbon synthesis gene of the present invention. In addition, Pseudomonas putida_F1-derived genes, i.e., YP_001268218, YP_001265586, YP_001267408, YP_001267629, YP_001266090, YP_001270490, YP_001268439, YP_001267367, YP_001267724, YP_001269548, YP_001268395, YP_001265936, YP_001270470, YP_001266779, and YP_001270298, can be identified as the hydrocarbon synthesis gene of the present invention.
In addition, genes from the Bacillus subtilis 168 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 glutamicum ATCC13032-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. A Lactobacillus reuteri DSM20016-derived gene, i.e., YP_001270647, can be identified as a hydrocarbon synthesis gene according to the present invention.
Further, Saccharomyces cerevisiae-derived genes, i.e., NP_010996, NP_011904, NP_015264, NP_013828, NP_009560, NP_015019, NP_013893, NP_013892, and NP_011902, can be identified as the hydrocarbon synthesis gene of the present invention. Candida tropicalis MYA-3404-derived genes, i.e., XP_002548035, XP_002545751, XP_002547036, XP_002547030, XP_002550712, XP_002547024, XP_002550173, XP_002546610, and XP_002550289, can be identified as the hydrocarbon synthesis gene of the present invention. Debaryomyces hansenii CBS767-derived genes, i.e., XP_460395, XP_457244, XP_457404, XP_457750, XP_461954, XP_462433, XP_461708, and XP_462528, can be identified as the hydrocarbon synthesis gene of the present invention. Pichia pastoris GS115-derived genes, i.e., XP_002489360, XP_002493450, XP_002491418, XP_002493229, XP_002490175, XP_002491360, and XP_002491779, can be identified as the hydrocarbon synthesis gene of the present invention. Schizosaccharomyces pombe-derived genes, i.e., NP_593172, NP_593499, and NP_594582 can be identified as hydrocarbon synthesis genes according to the present invention. Aspergillus oryzae RIB40-derived genes, i.e., XP_001822148, XP_001821214, XP_001826612, XP_001817160, XP_001817372, XP_001727192, XP_001826641, XP_001827501, XP_001825957, XP_001822309, XP_001727308, XP_001818713, XP_001819060, XP_001823047, XP_001817717, and XP_001821011, can be identified as the hydrocarbon synthesis gene of the present invention.
Furthermore, Zea mays-derived genes, i.e., NP_001150417, NP_001105047, NP_001147173, NP_001169123, NP_001105781, NP_001157807, NP_001157804, NP_001105891, NP_001105046, NP_001105576, NP_001105589, NP_001168661, NP_001149126, and NP_001148092 can be identified as the hydrocarbon synthesis gene of the present invention. Arabidopsis thaliana-derived genes, i.e., NP_564204, NP_001185399, NP_178062, NP_001189589, NP_566749, NP_190383, NP_187321, NP_190400, NP_001077676, and NP_175812, can be identified as the hydrocarbon synthesis gene of the present invention.
Moreover, Drosophila melanogaster-derived genes, i.e., NP_733183, NP_609285, NP_001014665, NP_649099, NP_001189159, NP_610285, and NP_610107 can be identified as the hydrocarbon synthesis gene of the present invention. Rattus norvegicus-derived genes, i.e., NP_001006999, XP_001067816, XP_001068348, XP_001068253, NP_113919, XP_001062926, NP_071609, NP_071852, NP_058968, NP_001011975, NP_115792, NP_001178017, NP_001178707, NP_446348, NP_071992, XP_001059375, XP_001061872, and NP_001128170 can be identified as the hydrocarbon synthesis gene of the present invention. Homo sapiens-derived genes, i.e., NP_036322, NP_001193826, NP_001029345, NP_000684, NP_000680, NP_000683, NP_000681, NP_001071, NP_000687, NP_001180409, NP_001173, NP_000682, NP_000373, NP_001154976, NP_000685, and NP_000686, can be identified as the hydrocarbon synthesis gene of 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 the Klebsiella sp. 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 from Klebsiella sp. 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.











TABLE 1







Gene name
Nucleotide sequence
Amino acid sequence









gene01
SEQ ID NO: 3
SEQ ID NO: 4



gene02
SEQ ID NO: 5
SEQ ID NO: 6



gene03
SEQ ID NO: 7
SEQ ID NO: 8



gene04
SEQ ID NO: 9
SEQ ID NO: 10



gene05
SEQ ID NO: 11
SEQ ID NO: 12



gene06
SEQ ID NO: 13
SEQ ID NO: 14



gene07
SEQ ID NO: 15
SEQ ID NO: 16



gene08
SEQ ID NO: 17
SEQ ID NO: 18



gene09
SEQ ID NO: 19
SEQ ID NO: 20



gene10
SEQ ID NO: 21
SEQ ID NO: 22










The genes from Klebsiella sp. 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 of Escherichia coli K-12 W3110-derived genes, i.e., BAA14869, BAA14992, BAA16524, BAE77705, and BAA15538, as examples of genes registered with the NCBI database.











TABLE 2







Gene name
Nucleotide sequence
Amino acid sequence









BAA14869
SEQ ID NO: 23
SEQ ID NO: 24



BAA14992
SEQ ID NO: 25
SEQ ID NO: 26



BAA16524
SEQ ID NO: 27
SEQ ID NO: 28



BAE77705
SEQ ID NO: 29
SEQ ID NO: 30



BAA15538
SEQ ID NO: 31
SEQ ID NO: 32










Table 3 below lists the nucleotide sequences of the coding regions and amino acid sequences encoded by the nucleotide sequences for Corynebacterium glutamicum ATCC13032-derived genes, a Lactobacillus reuteri DSM20016-derived gene, Saccharomyces cerevisiae-derived genes, Candida tropicalis MYA-3404-derived genes, Debaryomyces hansenii CBS767-derived genes, Pichia pastoris GS115-derived genes, Schizosaccharomyces pombe-derived genes, Aspergillus oryzae RIB40-derived genes, a Zea mays-derived gene, Arabidopsis thaliana-derived genes, Drosophila melanogaster-derived genes, Rattus norvegicus-derived genes, and Homo 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.











TABLE 3








Nucleotide
Ammo acid


Organism species
Accession No.
Gene name
sequence
sequence







Corynebacterium
NP_599351
NCgl0098
SEQ ID NO: 65
SEQ ID NO: 66


glutamicum
NP_599725
NCgl0463
SEQ ID NO: 67
SEQ ID NO: 68


ATCC13032
NP_601988
NCgl2698
SEQ ID NO: 69
SEQ ID NO: 70



NP_599302
NCgl0049
SEQ ID NO: 71
SEQ ID NO: 72



NP_601867
NCgl2578
SEQ ID NO: 73
SEQ ID NO: 74



NP_601908
NCgl2619
SEQ ID NO: 75
SEQ ID NO: 76


Lactobacillus
YP_001270647
Lreu_0034
SEQ ID NO: 77
SEQ ID NO: 78


reuteri DSM20016


Saccharomyces
NP_010996
YER073W
SEQ ID NO: 79
SEQ ID NO: 80


cerevisiae
NP_011902
YHR037W
SEQ ID NO: 81
SEQ ID NO: 82



NP_011904
YHR039C
SEQ ID NO: 83
SEQ ID NO: 84



NP_013892
YMR169C
SEQ ID NO: 85
SEQ ID NO: 86



NP_013893
YMR170C
SEQ ID NO: 87
SEQ ID NO: 88



NP_015019
YOR374W
SEQ ID NO: 89
SEQ ID NO: 90



NP_009560
YBR006W
SEQ ID NO: 91
SEQ ID NO: 92



NP_013828
YMR110C
SEQ ID NO: 93
SEQ ID NO: 94



NP_015264
YPL061W
SEQ ID NO: 95
SEQ ID NO: 96


Candida tropicalis
XP_002550289
CTRG_04587
SEQ ID NO: 97
SEQ ID NO: 98


MYA-3404
XP_002547036
CTRG_01342
SEQ ID NO: 99
SEQ ID NO: 100



XP_002545751
CTRG_00532
SEQ ID NO: 101
SEQ ID NO: 102


Debaryomyces
XP_461708
DEHA2G03740g
SEQ ID NO: 103
SEQ ID NO: 104


hansenii CBS767
XP_462528
DEHA2G22572g
SEQ ID NO: 105
SEQ ID NO: 106



XP_457404
DEHA2B10384g
SEQ ID NO: 107
SEQ ID NO: 108


Pichia pastoris
XP_002489360
PAS_chr1-3_0024
SEQ ID NO: 109
SEQ ID NO: 110


GS115
XP_002491418
PAS_chr2-1_0853
SEQ ID NO: 111
SEQ ID NO: 112



XP_002493450
PAS_chr4_0043
SEQ ID NO: 113
SEQ ID NO: 114


Schizosaccharomyces
NP_593172
SPAC139.05
SEQ ID NO: 115
SEQ ID NO: 116


pombe
NP_593499
SPAC1002.12c
SEQ ID NO: 117
SEQ ID NO: 118



NP_594582
SPAC9E9.09c
SEQ ID NO: 119
SEQ ID NO: 120


Aspergillus oryzae
XP_001821214
AOR_1_1204144
SEQ ID NO: 121
SEQ ID NO: 122


RIB40
XP_001822148
AOR_1_1330014
SEQ ID NO: 123
SEQ ID NO: 124


Zea mays
NP_001150417
LOC100284047
SEQ ID NO: 125
SEQ ID NO: 126


Arabidopsis thaliana
NP_564204
AT1G23800
SEQ ID NO: 127
SEQ ID NO: 128



NP_001185399
AT1G74920
SEQ ID NO: 129
SEQ ID NO: 130



NP_178062
AT1G79440
SEQ ID NO: 131
SEQ ID NO: 132



NP_001189589
AT2G24270
SEQ ID NO: 133
SEQ ID NO: 134



NP_566749
AT3G24503
SEQ ID NO: 135
SEQ ID NO: 136



NP_190383
AT3G48000
SEQ ID NO: 137
SEQ ID NO: 138



NP_175812
AT1G54100
SEQ ID NO: 139
SEQ ID NO: 140


Drosophila
NP_609285
Dmel_CG3752
SEQ ID NO: 141
SEQ ID NO: 142


melanogaster
NP_001189159
Dmel_CG7145
SEQ ID NO: 143
SEQ ID NO: 144



NP_610107
Dmel_CG8665
SEQ ID NO: 145
SEQ ID NO: 146



NP_610285
Dmel_CG11140
SEQ ID NO: 147
SEQ ID NO: 148



NP_733183
Dmel_CG31075
SEQ ID NO: 149
SEQ ID NO: 150



NP_001014665
Dmel_CG4685
SEQ ID NO: 151
SEQ ID NO: 152



NP_649099
Dmel_CG9629
SEQ ID NO: 153
SEQ ID NO: 154


Rattus
NP_071852
24188
SEQ ID NO: 155
SEQ ID NO: 156


norvegicus
NP_001128170
641316
SEQ ID NO: 157
SEQ ID NO: 158


Homo sapiens
NP_000680
216
SEQ ID NO: 159
SEQ ID NO: 160



NP_000683
219
SEQ ID NO: 161
SEQ ID NO: 162



NP_000687
223
SEQ ID NO: 163
SEQ ID NO: 164



NP_000373
224
SEQ ID NO: 165
SEQ ID NO: 166



NP_001173
501
SEQ ID NO: 167
SEQ ID NO: 168



NP_001180409
64577
SEQ ID NO: 169
SEQ ID NO: 170









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 genus Escherichia such as Escherichia coli; bacteria belonging to the genus Bacillus such as Bacillus subtilis; bacteria belonging to the genus Pseudomonas such as Pseudomonas putida; bacteria belonging to the genus Rhizobium such as Rhizobium melilotii; yeast such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Pichia pastoris; and fungi such as filamentous bacteria.
When a bacterium such as Escherichia coli is 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 of Escherichia coli that can be used include conventionally known bacterial strains such as the Escherichia coli BL21 (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 as Escherichia coli. In addition, the Bacillus subtilis 168 strain and the like can be used as Bacillus subtilis. 
Any promoter can be used as long as it can be expressed in a host such as Escherichia 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 genus Candida such as Candida Shehatae, yeast belonging to the genus Pichia such as Pichia stipitis, yeast belonging to the genus Pachysolen such as Pachysolen tannophilus, yeast belonging to the genus Saccharomyces such as Saccharomyces cerevisiae, and yeast belonging to the genus Schizosaccharomyces such as Schizosaccharomyces pombe. Saccharomyces cerevisiae is 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., recombinant Escherichia coli or 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., recombinant Escherichia coli or 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-C20 saturated hydrocarbon in consideration of the use thereof for diesel fuel, more preferably a C12-C14 saturated hydrocarbon, and most preferably a C13 saturated hydrocarbon. More specifically, the hydrocarbon to be synthesized is C12 dodecane, C13 tridecane, C14 tetradecane, 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., recombinant Escherichia coli or 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.
Example 1
In this Example, the genome sequence of a microorganism having alkane synthesis capacity of the Klebsiella sp. 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.










TABLE 4





Gene name
Putative function
Nucleotide sequence
Amino acid sequence







gene01
phenylacetaldehyde: NAD+ oxidoreductase
SEQ ID NO: 3
SEQ ID NO: 4


gene02
phenylacetaldehyde: NAD+ oxidoreductase
SEQ ID NO: 5
SEQ ID NO: 6


gene03
4-aminobutanal: NAD+ 1-oxidoreductase
SEQ ID NO: 7
SEQ ID NO: 8


gene04
aldehyde dehydrogenase
SEQ ID NO: 9
SEQ ID NO: 10


gene05
succinate-semialdehyde: NAD+ oxidoreductase
SEQ ID NO: 11
SEQ ID NO: 12


gene06
succinate-semialdehyde: NAD+ oxidoreductase
SEQ ID NO: 13
SEQ ID NO: 14


gene07
betaine-aldehyde: NAD+ oxidoreductase
SEQ ID NO: 15
SEQ ID NO: 16


gene08
N-succinyl-L-glutamate 5-semialdehyde: NAD+
SEQ ID NO: 17
SEQ ID NO: 18



oxidoreductase


gene09
(S)-lactaldehyde: NAD+ oxidoreductase
SEQ ID NO: 19
SEQ ID NO: 20


gene10
betaine-aldehyde: NAD+ oxidoreductase
SEQ ID NO: 21
SEQ ID NO: 22









In this Example, genes encoding proteins comprising the motif sequence shown in SEQ ID NO: 1 were identified based on the genome information of the E. coli W3110 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.










TABLE 5





Gene name
Putative function
Nucleotide sequence
Amino acid sequence







BAA14869
gamma-Glu-gamma-aminobutyraldehyde
SEQ ID NO: 23
SEQ ID NO: 24



dehydrogenase. NAD(P)H-dependent


BAA14992
phenylacetaldehyde dehydrogenase
SEQ ID NO: 25
SEQ ID NO: 26


BAA16524
succinate-semialdehyde
SEQ ID NO: 27
SEQ ID NO: 28



dehydrogenase, NADP-dependent


BAE77705
aldehyde dehydrogenase B
SEQ ID NO: 29
SEQ ID NO: 30


BAA15538
succinylglutamic
SEQ ID NO: 31
SEQ ID NO: 32



semialdehyde dehydrogenase









Nucleic acid fragments separately containing the above 15 types of genes were PCR-amplified using, as a template, genome DNA of the Klebsiella sp. NBRC100048 strain or the E. coli W3110 strain. Table 6 shows primers used for PCR. DNeasy Blood & Tissue Kits (QIAGEN) were used for genome DNA extraction.











TABLE 6







Se-

Se-




quence

quence


Gene
Forward
ID
Reverse
ID


name
primer
number
primer
number







gene04
cggtacccggggat
SEQ ID
cgactctagaggat
SEQ ID



ccCAATATGCCGCT
NO: 33
ccACCCGAATGGAT
NO: 34



GCGTCTCAACCCTA

TGCGGACTGAGGA




CA





 


gene01
cggtacccggggat
SEQ ID
cgactctagaggat
SEQ ID



ccATCTTGATGTTC
NO: 35
ccCGATTAATATCG
NO: 36



ATCGCGTTACCCT

CACCATCACCGACT






T



 


gene02
cggtacccggggat
SEQ ID
cgactctagaggat
SEQ ID



ccCGCGATGAATAA
NO: 37
ccAGATTGCCCTCC
NO: 38



GGAAAGGGTATGTC

ACAGTAGCGAGAA




CA





 


gene03
cggtacccggggat
SEQ ID
cgactctagaggat
SEQ ID



ccTGGTAACGACGA
NO: 39
ccTGTGACTATTAG
NO: 40



TACCAATCTTAGGG

CGGCCTAACACAC



 


gene05
cggtacccggggat
SEQ ID
cgactctagaggat
SEQ ID



ccAGTAGCGATTAA
NO: 41
ccCATGTGAGCGTT
NO: 42



CAAGGAGACATGCG

GACGGTAAAGAGG




A





 


gene06
cggtacccggggat
SEQ ID
cgactctagaggat
SEQ ID



ccCCCCTGAAGACA
NO: 43
ccTCGCTCCTGTTA
NO: 44



GGAAGCAATTATGC

AAGGCCAATGCAC




AACTC





 


gene07
cggtacccggggat
SEQ ID
cgactctagaggat
SEQ ID



ccTATTCGTCAGCA
NO: 45
ccCCGGTTAAAATA
NO: 46



TTTACCGAACCCA

TGGACTGGAATTTA






CCC



 


gene08
cggtacccggggat
SEQ ID
cgactctagaggat
SEQ ID



ccATCCCTGAGGAG
NO: 47
ccAAAGGAGAGCCC
NO: 48



AAAACTGCATGAGT

GGCGTAGTGATGG




CTGT





 


gene09
cggtacccggggat
SEQ ID
cgactctagaggat
SEQ ID



ccAGCCATGACAGC
NO: 49
ccGTGCCTCAGGCC
NO: 50



ACCCGTTCAACAC

TGCAGATAGACCA



 


gene10
cggtacccggggat
SCO ID
cgactctagaggat
SEQ ID



ccGCATAACCGCAG
NO: 51
ccCCCTTTCTCAGT
NO: 52



AGAGGCTGAGATGG

CGCACCAGTGGT




A





 


BAA14869
cggtacccggggat
SEQ ID
cgactctagaggat
SEQ ID



ccATCTGATAGACG
NO: 53
ccGAGGCTTCGAGA
NO: 54



TGAAACAGGA

ACCACTAC



 


BAA14992
cggtacccggggat
SEQ ID
cgactctagaggat
SEQ ID



ccTGTCACGATTTG
NO: 55
ccACCATGGAACTT
NO: 56



CGGAGCTT

CTTTGACGAAAC



 


BAA16524
cggtacccggggat
SEQ ID
cgactctagaggat
SEQ ID



ccCTTTGAAAACAG
NO: 57
ccCCAGTTAAAGAC
NO: 58



GGATGTAGCGA

CGATGCAC



 


BAE77705
cggtacccggggat
SEQ ID
cgactctagaggat
SEQ ID



ccATACCTCACACC
NO: 59
ccCGACCAGCTTCT
NO: 60



GCAAGGAG

TATATCAGAACAG









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 of E. coli JM109. Each of Escherichia coli transformants 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.







TABLE 7





Headspace Sampler: HP7694 (Hewlett-Packard)




















Zone Temp
Oven
90°
C.




Loop
150°
C.




TR. Line
200°
C.



Event Time
GC cycle time
8.5
min




Vial EQ time
8.5
min




Pressuriz. time
0.5
min




Loop fill time
0.2
min




Loop EQ time
0.2
min




Inject time
1.0
min











Vial Parameter
Shake
High

















TABLE 8





<GC/MS analysis conditions>
















GC/MS:
HP6890/5973 (Hewlett-Packard)


Column:
HP-INNOWAX (Agilent: 19091N-213)


Inlet temperature:
260° C.


Detector temperature:
260° C.


Injection parameter split ratio:
1/20


Carrier gas:
Helium 3.0 ml/min


Oven heating conditions
 60° C. 1 min


Heating to 260° C. at
260° C. 1 min


50° C./min









FIG. 1 shows analysis results obtained using the 10 types of genes from Klebsiella sp. identified in this Example. As shown in FIG. 1, all of the 10 types of genes from Klebsiella sp. 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. 2 shows a GC/MS analysis chart for a vector control strain and that for a strain into which gene02 has been introduced.
Similarly, FIG. 3 shows analysis results obtained using the 5 types of genes from the E. coli W3110 strain identified in this Example. As shown in FIG. 3, all of the 5 types of genes from the E. coli W3110 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.
Example 2
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, recombinant Escherichia coli prepared 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-C18 aldehyde compounds were used in this Example. Here, alkane with a carbon number one less than that of an aldehyde compound is synthesized.







TABLE 9





<Reaction solution composition>


















Phosphate buffer (pH 7.2):
500 μl



Aldehyde:
Final concentration 1 mM



NADH:
Final concentration 1 mM



Disruption solution supernatant:
500 μl










FIG. 4 shows the results of quantitative determination for synthesized C10-C17 alkanes. As is understood from FIG. 4, the protein encoded by gene02 was found to have excellent capacity to synthesize alkane, and in particular, C12-C14 alkane. FIG. 5 shows a GC/MS analysis chart for a vector control strain and that for a strain into which gene02 has been introduced created by assaying C13 alkane using a C14 aldehyde compound as a substrate.
Example 3
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 the Klebsiella sp. 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. coli BL21 (DE3) was transformed using the obtained expression vector. Transformed Escherichia coli was 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. 6 shows SDS-PAGE results for a disruption solution of Escherichia coli transformed with pRSFduet-1 plasmid to which gene04 had been ligated, a disruption solution of Escherichia coli transformed with pRSFduet-1 plasmid to which gene04 had not been ligated, and a solution containing the purified His-tag protein. In FIG. 6, lane 1 represents the disruption solution of Escherichia coli transformed with pRSFduet-1 plasmid to which gene04 had not been ligated, lane 2 represents the disruption solution of Escherichia coli transformed with pRSFduet-1 plasmid to which gene04 had been ligated, and lane 3 represents the solution containing the purified His-tag protein. As shown in FIG. 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.







TABLE 10





<Reaction solution composition>


















Phosphate buffer (pH 7.2):
500 μl



Aldehyde:
Final concentration 1 mM



NADH:
Final concentration 1 mM



His-tag protein eluate:
500 μl










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. 7 shows GC/MS analysis charts. In FIG. 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 in FIG. 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. 8 shows the results. As is understood from FIG. 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.
Example 4
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 the Klebsiella sp. 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). The Saccharomyces cerevisiae YPH499 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. 9 shows the analysis results. As shown in FIG. 9, it was found that the Klebsiella sp. 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.
Example 5
In this Example, alkane synthase genes from a variety of organism species were evaluated for alkane synthesis capacity.
Specifically, the 53 types of genes (Corynebacterium glutamicum ATCC13032-derived genes, a Lactobacillus reuteri DSM20016-derived gene, Saccharomyces cerevisiae-derived genes, Candida tropicalis MYA-3404-derived genes, Debaryomyces hansenii CBS767-derived genes, Pichia pastoris GS115-derived genes, Schizosaccharomyces pombe-derived genes, Aspergillus oryzae RIB40-derived genes, a Zea mays-derived gene, Arabidopsis thaliana-derived genes, Drosophila melanogaster-derived genes, Rattus norvegicus-derived genes, and Homo sapiens-derived genes) listed in table 3 above were evaluated for alkane synthesis capacity in the manner described in Example 1. In addition, the Escherichia coli JM109 strain was used as a host in this Example.
The 53 types of genes were amplified using pairs of primers listed in table 11 below.












TABLE 11





Test


Sequence

Sequence


No.
Gene name
Forward primer
ID number
Reverse primer
ID number




















1
NCgl0098
cggtacccggggatccaaggagatata
SEQ ID
cgactctagaggatccTCAACGTT
SEQ ID




ccATGACGTCGATGAATCTGCCTATTG
NO: 173
TAAGTTCCTCCGCCAAC
NO: 174


 


2
NCgl0463
cggtacccggggatccaaggagatata
SEQ ID
cgactctagaggatccTCACGGCA
SEQ ID




ccATGTCTTTGACCTTCCCAGTAATCA
NO: 175
AAGCGAGGTAACGCACG
NO: 176


 


3
NCgl2698
cggtacccggggatccaaggagatatac
SEQ ID
cgactctagaggatccTCAGAACA
SEQ ID




cATGACTGTCTACGCAAATTCCAGGAA
NO: 177
GTCCGGTTGGGTTTGGA
NO: 178


 


4
NCgl0049
cggtacccggggatccaaggagatata
SEQ ID
cgactctagaggatccCTAGCCGG
SEQ ID




ccATGACTATTAATGTCTCCGAACTAC
NO: 179
CGTAAGGATCCCGGATA
NO: 180


 


5
NCgl2578
cggtacccggggatccaaggagatata
SEQ ID
cgactctagaggatccTTAGCTGC
SEQ ID




ccATGACTGCAACATTTGCTGGAATCG
NO: 181
GCTTGATGCCGATCCAT
NO: 182


 


6
NCgl2619
cggtacccggggatccaaggagatata
SEQ ID
cgactctagaggatccCTACGGCA
SEQ ID




ccATGATCAAACGTCTTCCTTTAGGTC
NO: l83
AAACTTTAAAGATTTTG
NO: 184


 


7
Lreu_0034
cggtacccggggatccaaggagatata
SEQ ID
cgactctagaggatccTTATTGTC
SEQ ID




ccATGGCATATCAAAGTATCAATCCAT
NO: 185
GTGCTTCGTAAATTAGA
NO: 186


 


8
YER073W
cggtacccggggatccGCTTTCTCGCA
SEQ ID
cgactctagaggatccTTATCAAC
SEQ ID




CAAGAGCTGCAG
NO: 187
GAATTGGCTTGTCAATGGCA
NO: 188


 


9
YHR037W
cggtacccggggatccGCTATCAGCAA
SEQ ID
cgactctagaggatccTTATTATT
SEQ ID




GGTGCCTCAAAT
NO: 189
CATAATTCGATGGATATTTG
NO: 190


 


10
YHR039C
cggtacccggggatccGTCCAAGGTCT
SEQ ID
cgactctagaggatccTTACTAGC
SEQ ID




ATCTGAATTCAG
NO: 191
TGGCTTCTTTAGCTAAAGAG
NO: 192


 


11
YMR169C
cggtacccggggatccGCCTACCTTGT
SEQ ID
cgactctagaggatccTTATTATT
SEQ ID




ATACTGATATCG
NO: 193
TATCCAATGAAAGATCCACA
NO: 194


 


12
YMR170C
cggtacccggggatccGCCTACCTTGT
SEQ ID
cgactctagaggatccTTATTAGT
SEQ ID




ATACTGATATCG
NO: 195
TGTCCAAAGAGAGATTTATG
NO: 196


 


13
YOR374W
cggtacccggggatccGTTCAGTAGAT
SEQ ID
cgactctagaggatccTTACTCGT
SEQ ID




CTACGCTCTGCT
NO: 197
CCAATTTGGCACGGACC
NO: 198


 


14
YBR006W
cggtacccggggatccGACTTTGAGTA
SEQ ID
cgactctagaggatccTTATTAAA
SEQ ID




AGTATTCTAAAC
NO: 199
TGCTGTTTGGCAAATTCCCA
NO: 200


 


15
YMR110C
cggtacccggggatccGTCAAACGACG
SEQ ID
cgactctagaggatccTTATCAGG
SEQ ID




TGCCAAAAATAT
NO: 201
AAGAACAATGAGCGTAAATG
NO: 202


 


16
YPL061W
cggtacccggggatccGACTAAGCTAC
SEQ ID
cgactctagaggatccTTATTACA
SEQ ID




ACTTTGACACTG
NO: 203
ACTTAATTCTGACAGCTTTT
NO: 204


 


17
CTRG_04587
cggtacccggggatccaacaaacaaag
SEQ ID
cgactctagaggatcctagtgagt
SEQ ID




gatccaaaaaaATG
NO: 205
cgtattacggatcctta
NO: 206


 


18
CTRG_01342
cggtacccggggatccaacaaacaaag
SEQ ID
cgactctagaggatcctagtgagt
SEQ ID




gatccaaaaaaATG
NO: 205
cgtattacggatcctta
NO: 206


 


19
CTRG_00532
cggtacccggggatccaacaaacaaag
SEQ ID
cgactctagaggatcctagtgagt
SEQ ID




gatccaaaaaaATG
NO: 205
cgtattacggatcctta
NO: 206


 


20
DEHA2G03740g
cggtacccggggatccaacaaacaaag
SEQ ID
cgactctagaggatcctagtgagt
SEQ ID




gatccaaaaaaATG
NO: 205
cgtattacggatcctta
NO: 206


 


21
DEHA2G22572g
cggtacccggggatccaacaaacaaag
SEQ ID
cgactctagaggatcctagtgagt
SEQ ID




gatccaaaaaaATG
NO: 205
cgtattacggatcctta
NO: 206


 


22
DEHA2B10384g
cggtacccggggatccaacaaacaaag
SEQ ID
cgactctagaggatcctagtgagt
SEQ ID




gatccaaaaaaATG
NO: 205
cgtattacggatcctta
NO: 206


 


23
PAS_chr1-3_
cggtacccggggatccaacaaacaaag
SEQ ID
cgactctagaggatcctagtgagt
SEQ ID



0024
gatccaaaaaaATG
NO: 205
cgtattacggatcctta
NO: 206


 


24
PAS_chr2-1_
cggtacccggggatccaacaaacaaag
SEQ ID
cgactctagaggatcctagtgagt
SEQ ID



0853
gatccaaaaaaATG
NO: 205
cgtattacggatcctta
NO: 206


 


25
PAS_chr4_0043
cggtacccggggatccaacaaacaaag
SEQ ID
cgactctagaggatcctagtgagt
SEQ ID




gatccaaaaaaATG
NO: 205
cgtattacggatcctta
NO: 206


 


26
SPAC139.05
cggtacccggggatccaacaaacaaag
SEQ ID
cgactctagaggatcctagtgagt
SEQ ID




gatccaaaaaaATG
NO: 205
cgtattacggatcctta
NO: 206


 


27
SPAC1002.12c
cggtacccggggatccaacaaacaaag
SEQ ID
cgactctagaggatcctagtgagt
SEQ ID




gatccaaaaaaATG
NO: 205
cgtattacggatcctta
NO: 206


 


28
SPAC9E9.09c
cggtacccggggatccaacaaacaaag
SEQ ID
cgactctagaggatcctagtgagt
SEQ ID




gatccaaaaaaATG
NO: 205
cgtattacggatcctta
NO: 206


 


29
AOR_1_1204144
cggtacccggggatccaacaaacaaag
SEQ ID
cgactctagaggatcctagtgagt
SEQ ID




gatccaaaaaaATG
NO: 205
cgtattacggatcctta
NO: 206


 


30
AOR_1_1330014
cggtacccggggatccaacaaacaaag
SEQ ID
cgactctagaggatcctagtgagt
SEQ ID




gatccaaaaaaATG
NO: 205
cgtattacggatcctta
NO: 206


 


31
100284047
cggtacccggggatccaacaaacaaag
SEQ ID
cgactctagaggatcctagtgagt
SEQ ID




gatccaaaaaaATG
NO: 205
cgtattacggatcctta
NO: 206


 


32
AT1G23800
cggtacccggggatccGGCATCAAGAA
SEQ ID
cgactctagaggatccTTATTAGA
SEQ ID




GAGTTTCTTCGC
NO: 207
GCCAGGCAGGGTTCTTGAGG
NO: 208


 


33
AT1G74920
cggtacccggggatccGGCGATTCCGA
SEQ ID
cgactctagaggatccTTATTAGT
SEQ ID




TGCCTACTCGCC
NO: 209
TGGGAGATTTGTACCATCCC
NO: 210


 


34
ATIG79440
cggtacccggggatccGGTAATAGGAG
SEQ ID
cgactctagaggatccTTATCAGT
SEQ ID




CAGCAGCGCGTG
NO: 211
GTCTATTCATATCTCCCAAG
NO: 212


 


35
AT2G24270
cggtacccggggatccGGCCGGGACTG
SEQ ID
cgactctagaggatccTTACTAAC
SEQ ID




GATTGTTTGCTG
NO: 213
CCATAGAGTAAGAAGGTGTA
NO: 214


 


36
AT3G24503
cggtacccggggatccGGAGAACGGCA
SEQ ID
cgactctagaggatccTTATTACA
SEQ ID




AATGCAACGGAG
NO: 215
TCCAAGGGGAATTGTGAAGA
NO: 216


 


37
AT3G48000
cggtacccggggatccGGCGGCTCGTA
SEQ ID
cgactctagaggatccTTATCAGA
SEQ ID




GAGTGTCTTCTC
NO: 217
TCCAGGCAGGCTTATTTAGA
NO: 218


 


38
AT1G54100
cggtacccggggatccGGGTTCGGCGA
SEQ ID
cgactctagaggatccTTACTAAC
SEQ ID




ACAACGAGTACG
NO: 219
CGAAGTTAATTCCTTGCGCT
NO: 220


 


39
Dmel_CG3752
cggtacccggggatccGCTGCGCGTTT
SEQ ID
cgactctagaggatccTTATTAGG
SEQ ID




TGAAGACCGGTG
NO: 221
AGTTCTTCTGGGCAACCTTG
NO: 222


 


40
Dmel_CG7l45
cggtacccggggatccGTTGCGAATGA
SEQ ID
cgactctagaggatccTTATTACT
SEQ ID




TGCGAAGTTCCT
NO: 223
CGCACATGTATGGATAGTTG
NO: 224


 


41
Dmel_CG8665
cggtacccggggatccGGCTCTAAAAA
SEQ ID
cgactctagaggatccTTACTAAT
SEQ ID




TGAGAATCGCAA
NO: 225
ATTCAACTGTGACACACTTG
NO: 226


 


42
Dmel_CG11140
cggtacccggggatccGTTTGACAACG
SEQ ID
cgactctagaggatccTTATCACG
SEQ ID




CGATTAAACCTC
NO: 227
TCCACCAAGATGGTGGGTTC
NO: 228


 


43
Dmel_CG31075
cggtacccggggatccGGCCGATCCCA
SEQ ID
cgactctagaggatccTTATTAAA
SEQ ID




ACGCCAGCCCA
NO: 229
GAAGTTTCATGGTGATGGTC
NO: 230


 


44
Dmel_CG4685
cggtacccggggatccGTGGCGACAGC
SEQ ID
cgactctagaggatccTTATCAGT
SEQ ID




TCAGCGGAGTCG
NO: 231
CGTACTTGAGGTTGCCCATG
NO: 232


 


45
Dmel_CG9629
cggtacccggggatccGTTGGCACAAT
SEQ ID
cgactctagaggatccTTACTACT
SEQ ID




TGAGAAATATTT
NO: 233
CCACATTGAAGACAACACCC
NO: 234


 


46
24188
cggtacccggggatccGTCTTCCCCTG
SEQ ID
cgactctagaggatccTTATTAGG
SEQ ID




CACAGCCTGCAG
NO: 235
AGTTCTTCTGAGATATTTTC
NO: 236


 


47
641316
cggtacccggggatccGCTGCCGCCGG
SEQ ID
cgactctagaggatccTTATTACT
SEQ ID




CTTTGCTTCGCC
NO: 237
GCATGTAGGAGTATCGCCAG
NO: 238


 


48
216
cggtacccggggatccGTCATCCTCAG
SEQ ID
cgactctagaggatccTTATTATG
SEQ ID




GCACGCCAGACT
NO: 239
AGTTCTTCTGAGAGATTTTC
NO: 240


 


49
219
cggtacccggggatccGCTGCGCTTCC
SEQ ID
cgactctagaggatccTTATTACG
SEQ ID




TGGCACCCCGGC
NO: 241
AGTTCTTCTGAGGAACCTTG
NO: 242


 


50
223
cggtacccggggatccGTTTCTCCGAG
SEQ ID
cgactctagaggatccTTATCAAA
SEQ ID




CAGGCCTGGCCG
NO: 243
AAGCAGATTCCACATCACCC
NO: 244


 


51
224
cggtacccggggatccGGAGCTCGAAG
SEQ ID
cgactctagaggatccTTATCAGT
SEQ ID




TCCGGCGGGTCCC
NO: 245
AATATTCTGCCTTGACAAGC
NO: 246


 


52
501
cggtacccggggatccGTGGCGCCTTC
SEQ ID
cgactctagaggatccTTATTACT
SEQ ID




CTCGCGCGCTGT
NO: 247
GAAACTTGATTCCTTGGGCC
NO: 248


 


53
64577
cggtacccggggatccGGCTGGAACAA
SEQ ID
cgactctagaggatccTTATCAGT
SEQ ID




ACGCACTTTTGA
NO: 249
GTTTAACGGTGATGGTTTTG
NO: 250









Among the 53 types of genes listed in table 11, genome DNAs extracted from the corresponding strains were used as templates for the Corynebacterium glutamicum ATCC13032-derived genes (NCg10098, NCg10463, NCg12698, NCg10049, NCg12578, and NCg12619), the Lactobacillus reuteri DSM20016-derived gene (Lreu_0034), and the Saccharomyces 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 the Candida tropicalis MYA-3404-derived genes (CTRG_04587, CTRG_01342, and CTRG_00532), the Debaryomyces hansenii CBS767-derived genes (DEHA2G03740g, DEHA2G22572g, and DEHA2B10384g), the Pichia pastoris GS115-derived genes (PAS_chr1-3_0024, PAS_chr2-1_0853, and PAS_chr4_0043), the Schizosaccharomyces pombe-derived genes (SPAC139.05, SPAC1002.12c and SPAC9E9.09c), the Aspergillus oryzae RIB40-derived genes (AOR_1_1204144 and AOR_1_1330014), and the Zea 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 the Arabidopsis 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 the Drosophila 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 the Rattus 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 the Homo sapiens-derived genes (216, 219, 223, 224, 501, and 64577).
Here, the PCR conditions, the conditions for culturing transformants, and the alkane analysis method are the same as those described in Example 1. FIGS. 10-1 and 10-2 show the alkane analysis results. As shown in FIGS. 10-1 and 10-2, it was revealed that all the 53 types of genes described above have hydrocarbon synthesis activity. In particular, the following genes were found to have remarkably excellent hydrocarbon synthesis activity: NCg10098 (Test No. 1), NCg10049 (Test No. 4), NCg12619 (Test No. 6), YER073W (Test No. 8), YOR374W (Test No. 13), YBR006W (Test No. 14), YMR110C (Test No. 15), CTRG_04587 (Test No. 17), PAS_chr2-1_0853 (Test No. 24), SPAC139.05 (Test No. 26), AOR_1_1204144 (Test No. 29), and Dmel_CG7145 (Test No. 40).
All publications, patents, and patent applications cited herein are incorporated by reference in their entirety.

## Claims

1. A method for producing a hydrocarbon, said method comprising incubating together:
a protein having an activity of synthesizing, from an aldehyde compound, a hydrocarbon with a carbon number one less than that of the aldehyde compound, wherein said protein is selected from the group consisting of (a) and (b):
(a) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 26, 66, 72, 76, 80, 90, 92, 94, 98, 112, 116, 122 and 144; and
(b) a protein comprising an amino acid sequence having 90% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 26, 66, 72, 76, 80, 90, 92, 94, 98, 112, 116, 122 and 144, and having an activity of synthesizing, from an aldehyde compound, a hydrocarbon with a carbon number one less than that of the aldehyde compound;

a coenzyme involved in said activity of said protein, wherein the coenzyme is reduced nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate, and
an aldehyde compound that serves as a substrate for said activity of said protein,
to thereby synthesize a hydrocarbon with a carbon number one less than that of the aldehyde compound.

2. The method for producing a hydrocarbon according to claim 1, wherein the protein, the coenzyme, and the aldehyde compound are incubated together by culturing a transformant, into which a gene encoding said protein has been introduced, in a solution containing the aldehyde compound.

3. The method for producing a hydrocarbon according to claim 1, wherein the protein, the coenzyme, and the aldehyde compound are incubated together by mixing an enzyme liquid, extracted from a transformant into which a gene encoding said protein has been introduced, with a solution containing the aldehyde compound.

4. The method for producing a hydrocarbon according to claim 1, wherein the protein, the coenzyme, and the aldehyde compound are incubated together by mixing the protein, isolated from a transformant into which a gene encoding said protein has been introduced, with a solution containing the aldehyde compound and the coenzyme.

5. The method for producing a hydrocarbon according to claim 1, wherein the aldehyde compound is a C11-C21 aldehyde compound.

6. The method for producing a hydrocarbon according to claim 1, wherein the coenzyme is reduced nicotinamide adenine dinucleotide (NADH).