JP7194960B2 - Fatty alcohol-synthesizing bacteria and method for producing fatty alcohol - Google Patents

Description

NPMD NITE P-02620

TECHNICAL FIELD The present invention relates to a fatty alcohol-synthesizing bacterium and a method for producing a fatty alcohol. Specifically, the present invention relates to a novel microorganism belonging to the genus Reinekea, a novel polynucleotide possessed by the microorganism, and a method for producing an aliphatic alcohol using the microorganism or the polynucleotide.

Aliphatic alcohols are used in various products such as cosmetics, pharmaceuticals, additives for foods, raw materials for detergents, and fuels. Fatty alcohols are mainly produced using petroleum as a raw material.

On the other hand, in recent years, there have been reports of attempts to introduce a group of genes involved in fatty alcohol synthesis into Escherichia coli and the like to produce fatty alcohols.
For example, in Non-Patent Document 1, thioesterase ('tesA), acyl-CoA synthase (fadD) and fatty acyl-CoA reductase (acr1) derived from Acinetobacter calcoaceticus are overexpressed, and acyl-CoA dehydrogenase (fadE ) has been reported to increase the production of fatty alcohols in Escherichia coli.
In addition, in Non-Patent Document 2, an α-dioxygenase gene derived from Oryza sativa (rice) (which produces an aliphatic aldehyde with one less carbon number from a fatty acid) was introduced into Escherichia coli, and thioesterase (tesA') and An attempt to increase the production of odd-chain aliphatic alcohols by overexpressing aldehyde reductase is described.
In addition, although non-patent document 3 is also an attempt similar to non-patent document 1, thioesterase (tesA') and acyl-CoA synthase (fadD) are overexpressed, and acyl-CoA dehydrogenase (fadE) Attempts have been reported to increase the production of fatty alcohols by introducing a fatty acyl-CoA reductase gene (Maqu — 2220 gene) derived from Marinobacter aquaeolei into Escherichia coli that has been deleted.

Steen EJ, et al., 2010. Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature. 463: 559-562. Cao YX, et al., 2015. Biosynthesis of odd-chain fatty alcohols in Escherichia coli. Metab Eng. 29: 113-123. Liu A, et al., 2013. Fatty alcohol production in engineered E. coli expressing Marinobacter fatty acyl-CoA reductases. Appl Microbiol Biotechnol. 97: 7061-7071.

The productivity of fatty alcohols by microorganisms is thought to be limited by the intracellular accumulation of fatty alcohols. However, in the transformants described in Non-Patent Documents 1 to 3, the intracellular content of fatty alcohols is not so high.
For example, the fatty alcohol content per dry cell weight calculated from the fatty alcohol production amount described in Non-Patent Documents 1 and 2 is about 10% at most. In addition, the fatty alcohol content per dry cell weight calculated from the fatty alcohol production amount described in Non-Patent Document 3 is about 17% at maximum.

Therefore, in order to put the production of fatty alcohols into practical use by a biological method, enzymes and cells with a higher ability to produce fatty alcohols are required.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a technique for producing an aliphatic alcohol by a biological method.

The present invention includes the following aspects.
[1] Reinekea sp. 1-4 strain (NITE P-02620) or its mutant strain.
[2] A polynucleotide selected from the group consisting of (A1) to (A7) below.
(A1) A polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 (A2) One or more amino acids are deleted, substituted, added, or inserted in the amino acid sequence set forth in SEQ ID NO: 2 Polynucleotide (A3) consisting of an amino acid sequence and encoding a polypeptide having fatty alcohol synthesis activity, consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2, and fatty alcohol Polynucleotide encoding a polypeptide having synthetic activity (A4) Polynucleotide consisting of the base sequence set forth in SEQ ID NO: 1 (A5) Deletion, substitution, or deletion of one or more bases in the base sequence set forth in SEQ ID NO: 1 Polynucleotide (A6) consisting of an added or inserted base sequence and encoding a polypeptide having aliphatic alcohol synthesis activity from a base sequence having 80% or more sequence identity with the base sequence set forth in SEQ ID NO: 1 A polynucleotide encoding a polypeptide (A7) consisting of and having aliphatic alcohol synthesis activity, hybridizes under stringent conditions with a polynucleotide consisting of the base sequence set forth in SEQ ID NO: 1, and has aliphatic alcohol synthesis activity A polynucleotide encoding a polypeptide [3] a polynucleotide selected from the group consisting of (B1) to (B7) below.
(B1) A polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 4 (B2) One or more amino acids are deleted, substituted, added, or inserted in the amino acid sequence set forth in SEQ ID NO: 4 When introduced into a cell together with a polynucleotide (phsA) that encodes a polypeptide consisting of an amino acid sequence and consists of the nucleotide sequence set forth in SEQ ID NO: 1, the number of fatty alcohols in cells is lower than that of cells introduced with phsA alone. When the polynucleotide (B3) encodes a polypeptide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4, and is introduced into a cell together with phsA, phsA Polynucleotide (B4) in the base sequence set forth in SEQ ID NO: 3, consisting of the base sequence set forth in SEQ ID NO: 3, which increases the fatty alcohol content of the cells compared to the cells introduced alone When one or more bases are deleted, substituted, added, or inserted into a base sequence, and when introduced into a cell together with phsA, the fatty alcohol content of the cell is compared to the cell introduced with phsA alone. Polynucleotide (B6) consisting of a nucleotide sequence having a sequence identity of 80% or more with the nucleotide sequence set forth in SEQ ID NO: 3, in increasing amounts, and when introduced into cells together with phsA, compared to cells introduced with phsA alone In comparison, when the polynucleotide (B7) hybridizes under stringent conditions with a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 3, and is introduced into cells together with phsA, which increases the fatty alcohol content of the cell. Either of the polynucleotide according to [4] [2] and the polynucleotide according to [3], or A vector containing both.
[5] A cell comprising the polynucleotide of [2].
[6] The cell of [5], further comprising the polynucleotide of [3].
[7] (a) Reinekea sp. 1-4 strain (NITE P-02620) or its mutant strain, and (b) obtaining a fatty alcohol from the culture obtained in step (a). Production method.
[8] (a') culturing the cells according to [5] or [6]; and (b') obtaining an aliphatic alcohol from the culture obtained in step (a'). A method for producing a fatty alcohol, comprising:

INDUSTRIAL APPLICABILITY According to the present invention, a technology for producing fatty alcohols by a biological method is provided.

FIG. 1 shows fatty acid biosynthetic pathways and putative fatty alcohol synthesis reactions by gene products encoded by the phsAB cluster of strains 1-4. GC-MS profile (total ion chromatogram) of fatty alcohols produced by strain 1-4 grown in dR medium is shown (top). Peaks 1-3 and 1′-3′ are the following fatty alcohols; 1, C _14:0 ; 2, C _16:0 ; 3, C _18:0 ; 1′, C _14:1 ; ', C16 _:1 ; 3', _C18:1 . The sample profile is also shown (bottom). Negatively stained transmission electron micrographs of strains 1-4. FIG. 3A is a photograph of a section, and FIG. 3B is a photograph of unsectioned cells. Neighbor-joining phylogenetic tree showing the position of strains 1-4 within the class Gammaproteobacteria based on the 16S rRNA gene sequence (1254 bp). A type strain that is phylogenetically close to strains 1-4 was shown. Bootstrap values (>50%) based on 1000 resamplings are shown at the branch points. When the phylogenetic tree was prepared by the maximum likelihood method, the branch points where the bootstrap value exceeded 60% were indicated by black circles. Amylibacter marinus 2-3 ^T of the class Alphaproteobacteria (GenBank accession no. AB917595) was used for the outgroup. The length of the lower left bar indicates substitutions at a rate of 0.01 per base. In Examples, the structure of the DNA fragment carried by the plasmid used for transformation of Escherichia coli is shown. Gray and black boxes indicate ORF size and orientation. Hatched boxes indicate multiple cloning sites. FIG. 5 shows a GC-MS profile (total ion chromatogram) of fatty alcohols produced by E. coli DH5α into which the plasmid shown in FIG. 5 was introduced. The introduced plasmid (in parenthesis) and the gene contained in this plasmid are shown in the upper right of each graph. Peaks 1-3 and 1′-3′ are the following fatty alcohols; 1, C _14:0 ; 2, C _16:0 ; 3, C _18:0 ; 1′, C _14:1 ; ', C16 _:1 ; 3', _C18:1 . Effect of phsB on fatty alcohol production by phsA in E. coli DH5α. E. coli strains transfected with pBS29SN(phsAB) or pBS29SNX(phsA) were grown to completion in 30 mL of M9 medium supplemented with IPTG and ampicillin. E. coli transfected with pBS29SN were grown for 5-7 days (OD ₆₆₀ =0.418-0.535) and E. coli transfected with pBS29SNX were grown for 6-9 days (OD ₆₆₀ =0.443-0.566). . Each value in the graph represents the mean ± standard error obtained from four independent cultures.

[definition]
As used herein, "aliphatic alcohol" means a compound in which one hydrogen atom of an aliphatic hydrocarbon is substituted with a hydroxy group. Aliphatic alcohols are preferably compounds represented by R—OH (R represents a linear or branched alkyl or alkenyl group). In the above formula R--OH, R is preferably a linear or branched alkyl group or alkenyl group having 14, 16 or 18 carbon atoms. Specific examples of aliphatic alcohols include 1-tetradecanol, 1-tetradecenol, 1-hexadecanol, 1-hexadecenol, 1-octadecanol, 1-octadecenol and the like.

As used herein, the term "fatty alcohol synthesis activity" refers to enzymatic activity that catalyzes a synthesis reaction of fatty alcohols. For example, the fatty alcohol synthesis activity is the activity to catalyze the reaction of reducing acyl-CoA to produce a fatty alcohol (see FIG. 1). For example, a polynucleotide encoding a polypeptide to be evaluated is introduced into E. coli, and the amount of aliphatic alcohol produced in the E. coli is increased, and aliphatic aldehydes are produced as compared with E. coli into which the polynucleotide is not introduced. If the amount of production does not increase, it can be determined that the polypeptide has fatty alcohol synthesis activity. E. coli does not have an enzyme that reduces acyl-ACP (acyl carrier protein)/fatty acid/acyl-CoA to fatty aldehyde or even fatty alcohol, but reduces fatty aldehyde to fatty acid. It has an enzyme that converts it to alcohol. For example, it has been reported that when an acyl-ACP reductase that synthesizes aliphatic aldehydes was expressed in E. coli, both aliphatic aldehydes and aliphatic alcohols were accumulated (Schirmer et al., 2010, Science. 329, 559-562). Therefore, by confirming that the amount of fatty alcohol produced does not increase in addition to the increase in the amount of produced fatty alcohol, the presence or absence of the aliphatic alcohol synthesis activity of the target polypeptide can be evaluated more accurately. . More specifically, a polynucleotide encoding a polypeptide to be evaluated (polynucleotide of interest) is operably linked to a promoter that functions in E. coli to create an expression vector. Next, the expression vector is introduced into Escherichia coli, and the Escherichia coli is cultured until stationary phase. Aliphatic compounds are then extracted from this culture. Aliphatic alcohols and aliphatic aldehydes in the extract were measured, and compared with the E. coli extract into which the target polynucleotide was not introduced, when the aliphatic alcohol increased and the aliphatic aldehyde did not increase is determined to have fatty alcohol synthesis activity. Aliphatic compounds can be extracted from the culture by mixing the cells with n-heptane or the like. Fatty alcohols and aliphatic aldehydes in the extract can be measured, for example, by gas chromatography-mass spectrometry (GC-MS).
It is known that Escherichia coli does not normally produce aliphatic alcohols and aliphatic aldehydes. , usually below the limit of detection. Therefore, the fatty alcohol was below the detection limit in the fatty compound extract of E. coli into which the target polynucleotide was not introduced, and the fatty alcohol was detected in the fatty compound extract of E. coli into which the target polynucleotide was introduced. It is also included if the production of fatty alcohols is increased. In E. coli into which the target polynucleotide has been introduced, the ratio (%, w/w) of the aliphatic alcohol weight to the cell dry weight is, for example, 5% (w/w) or more, preferably 10% (w/w) or more. , more preferably 15% (w/w) or more, is also included when the amount of fatty alcohol produced is increased.
In addition, the aliphatic aldehyde is below the detection limit in the aliphatic compound extract of E. coli into which the target polynucleotide has not been introduced, and the aliphatic aldehyde is in the detection limit of the aliphatic compound extract of E. coli into which the target polynucleotide has been introduced. The cases below are also included if the amount of aliphatic aldehyde produced does not increase. In addition, in E. coli into which the target polynucleotide has been introduced, the ratio (%, w/w) of the aliphatic aldehyde to the cell dry weight is, for example, 5% (w/w) or less, preferably 3% (w/w) or less. , more preferably 1% (w/w) or less, is also included if the amount of aliphatic aldehyde produced does not increase.

As used herein, the term "aliphatic alcohol synthesis-assisting activity" refers to the subject polynucleotide introduced into a cell together with a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 1 (hereinafter sometimes referred to as "phsA"). In some cases, it refers to the activity of the polypeptide encoded by the polynucleotide to increase the fatty alcohol content of cells compared to cells transfected with phsA alone. Whether or not a polypeptide has fatty alcohol synthesis-assisting activity can be confirmed, for example, as follows. An expression vector (I) is constructed comprising a polynucleotide encoding a polypeptide of interest and phsA operably linked to a promoter functional in E. coli. Similarly, an expression vector (II) is constructed that contains pshA and does not contain a polynucleotide encoding a polypeptide of interest. The expression vectors (I) and (II) are introduced into Escherichia coli, and the respective Escherichia coli are cultured until stationary phase. Cells are then collected from each E. coli culture, fatty alcohols are extracted from the cells, and the fatty alcohols are quantified. When the fatty alcohol content of E. coli into which expression vector (I) has been introduced is increased compared to the fatty alcohol content of E. coli into which expression vector (II) has been introduced, the polypeptide is It is judged to have fatty alcohol synthesis-assisting activity.

As used herein, the fatty alcohol content of cells is expressed as the ratio of fatty alcohol weight to dry cell weight (%, w/w). The ratio (%, w/w) of the aliphatic alcohol weight to the dry cell weight can be determined by the following formula. The sample for dry cell weight measurement and the sample for fatty alcohol weight measurement for calculating the proportion of fatty alcohol are usually collected from the same culture. The dry cell weight can be measured by collecting cells from the culture, drying the collected cells, and measuring the weight. Cells can be dried, for example, by reduced pressure drying using an evaporator or the like, heat drying (50° C., etc.), etc. By using these methods, cells can be dried quickly.

As used herein, the term "operably linked" as used in reference to polynucleotides means that the first base sequence is positioned sufficiently close to the second base sequence and the first base sequence is the second base sequence or It means that the region under control of the second base sequence can be affected. For example, that a polynucleotide is operably linked to a promoter means that the polynucleotide is linked so that it is expressed under the control of the promoter.

As used herein, the term “expressible state” means that the polynucleotide is in a state in which it can be transcribed in a cell into which the polynucleotide has been introduced.
As used herein, the term "expression vector" refers to a vector containing a polynucleotide of interest and having a system that renders the polynucleotide of interest expressible in cells into which the vector has been introduced.

As used herein, the term "mutant strain" means a strain in which the DNA of the original strain is naturally or artificially mutated. The artificial method for generating DNA mutation is not particularly limited, and examples include ultraviolet irradiation, radiation irradiation, chemical treatment with nitrous acid and the like, genetic engineering methods such as gene introduction, and the like.

As used herein, "silent mutation" means a genetic mutation that does not change the amino acid sequence of the encoded protein.

As used herein, "stringent conditions" include, for example, the method described in Molecular Cloning-A LABORATORY MANUAL THIRD EDITION (Sambrook et al., Cold Spring Harbor Laboratory Press). Stringent conditions include, for example, 6 x SSC (composition of 20 x SSC: 3 M sodium chloride, 0.3 M citric acid solution, pH 7.0), 5 x Denhardt's solution (composition of 100 x Denhardt's solution: 2 mass% bovine serum albumin, 2% ficoll, 2% polyvinylpyrrolidone), 0.5% SDS, 0.1 mg/mL salmon sperm DNA, and 50% formamide at 42-70°C in a hybridization buffer. Examples include conditions that allow hybridization by incubation at room temperature for several hours to overnight. A washing buffer used for washing after incubation preferably includes a 1×SSC solution containing 0.1% by mass of SDS, more preferably a 0.1×SSC solution containing 0.1% by mass of SDS.

[Reinekea sp. 1-4 strain and its mutant strain]
In one embodiment, the present invention uses Reinekea sp. 1-4 strain (NITE P-02620) or a variant thereof.

(Reinekea sp. 1-4 strain)
Reinekea sp. The 1-4 stock (hereinafter referred to as "1-4 stock") is surface seawater (33°18'N, 134°11'E, depth 0.5m) collected in the coastal area of Muroto City, Kochi Prefecture, Japan. is a marine aerobic heterotrophic bacterium isolated from 1-4 strains, dated March 28, 2018, as accession number NITE P-02620, National Institute of Technology and Evaluation Patent Microorganism Depositary Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) has been deposited with

The mycological properties of strains 1-4 are as follows.
(1) Morphological Properties It is a bacillus (0.3-1.1×0.3-8.2 μm) with one polar flagellum (see FIG. 3) and is motile.

(2) Physiological Properties It grows in the temperature range of 10-30°C (optimal temperature 20-30°C), and does not grow at 4°C and 35°C. Resistant to 12 μg/mL tetracycline and 50 μg/mL gentamicin. 20 μg/mL chloramphenicol and 100 μg/mL kanamycin are not resistant. Decomposes casein, starch, and agar. Chitin does not decompose.

(3) 16s rRNA gene analysis The 16S rRNA gene sequences of strains 1-4 were most similar to those of bacteria belonging to the genus Reinekea. Strain 1-4 showed 95.3% similarity to Reinekea marinisedimentorum DSM 15388 ^T , the type strain of the type species of the genus Reinekea, in almost full-length 16S rRNA gene sequence comparison. In the phylogenetic tree based on the 16S rRNA gene sequence, strains 1-4 formed clusters with bacteria of the genus Reinekea (see Figure 4).
From the above, strain 1-4 was presumed to be a novel bacterium belonging to the genus Reinekea, and Reinekea sp. Deposited as strains 1-4.

Strains 1-4 can be cultured using known media used for culturing marine aerobic heterotrophic bacteria. The medium may be a solid medium or a liquid medium. A known medium containing a carbon source, a nitrogen source, inorganic salts and the like can be used as the medium. Carbon sources include, for example, glucose, dextran, starch, beef extract, pyruvic acid, acetic acid and the like. Nitrogen sources include ammonium salts, nitrates, amino acids, peptones, casein, casamino acids, urea, yeast extract and the like. Examples of inorganic salts include sodium salts such as sodium phosphate, sodium chloride and sodium molybdenum; potassium salts such as potassium phosphate and potassium chloride; calcium salts such as calcium chloride and calcium phosphate; magnesium salts such as magnesium chloride and magnesium sulfate; mentioned. The medium may be seawater (surface seawater, deep water, etc.) added with the carbon source, nitrogen source, inorganic salts, and the like. The pH of the medium is preferably 6-10. Preferable media include dR1 medium, dR medium, dR2A-SW plate, etc., described in Examples below.

When the 1-4 strain is cultured in a liquid medium, static culture or shaking culture may be used, but shaking culture is preferred from the viewpoint of growth efficiency. Furthermore, aeration and agitation culture may be performed.
The culture temperature is preferably 10-30°C, more preferably 20-30°C.

The 1-4 strain is a bacterium isolated as a bacterium that accumulates fatty alcohols intracellularly. The 1-4 strain contains the polynucleotide set forth in SEQ ID NO: 1 and the polynucleotide set forth in SEQ ID NO: 3 as genes involved in fatty alcohol synthesis.
As shown in the examples below, strain 1-4 can intracellularly accumulate about 0.9% of the dry cell weight of fatty alcohols. This amount of accumulation is higher than that of previously reported fatty alcohol-producing bacteria existing in nature. Therefore, strains 1-4 can be used in the method for producing fatty alcohols, which will be described later.

(1-4 mutant strains)
The mutant strain 1-4 is a strain in which the DNA of strain 1-4 is mutated. The mutant strain 1-4 is preferably a strain having a fatty alcohol-producing ability equal to or greater than that of the 1-4 strain. Such mutant strains, for example, when cultured in dR medium until reaching the stationary phase, have a proportion of fatty alcohol relative to the dry cell weight of 0.3% or more, preferably 0.5% or more, more preferably Mutants with 0.6% or more are exemplified.

Aliphatic alcohols produced by strain 1-4 mutants include C ₁₄ alcohols (1-tetradecanol, 1-tetradecenol, etc.), C ₁₆ alcohols (1-hexadecanol, 1-hexadecenol, etc.), and C ₁₈ It preferably contains an alcohol (1-octadecanol, 1-octadecenol, etc.).

Preferred examples of 1-4 strain mutants include 1-4 strain mutant strains introduced with foreign genes; 1-4 strain mutations in which DNA other than the polynucleotide set forth in SEQ ID NO: 1 is mutated Strain; mutant strain 1-4 in which DNA other than the polynucleotide set forth in SEQ ID NO: 1 and the polynucleotide set forth in SEQ ID NO: 3 is mutated; 1-4 mutant strains in which the expression of the polynucleotide described in 1 and / or the polynucleotide described in SEQ ID NO: 3 is enhanced; 1-4 strain mutant strains in which silent mutation occurs; (A1) described later 1-4 mutant strains containing polynucleotides selected from the group consisting of (A7); 1-4 strain mutants containing polynucleotides selected from the group consisting of (B1) to (B7) described later ; 1-4 mutant strains containing polynucleotides selected from the group consisting of (A1) to (A7) described below and polynucleotides selected from the group consisting of (B1) to (B7) described below; mentioned.

The 1-4 strain mutant can be cultured in the same medium and culture conditions as for the 1-4 strain. Mutant strains 1-4 can also be used in the method for producing fatty alcohols, which will be described later.

In a preferred embodiment, the mutant strain 1-4 may be strain 1-4 into which a polynucleotide selected from the group consisting of (A1) to (A7) described below has been introduced. In another preferred embodiment, the mutant strain 1-4 may be strain 1-4 into which a polynucleotide selected from the group consisting of (B1) to (B7) described below has been introduced. In yet another preferred embodiment, the 1-4 mutant strain is a polynucleotide selected from the group consisting of (A1) to (A7) described below, and (B1) to A polynucleotide selected from the group consisting of (B7) may be introduced.
In this case, the polynucleotide to be introduced into strain 1-4 may be functionally linked to, for example, a promoter described in [Vector] below. For example, by functionally linking the polynucleotide to a promoter that induces high expression of the gene in the 1-4 strain and introducing it into the 1-4 strain, the aliphatic alcohol production ability of the 1-4 strain is enhanced. Mutants can be obtained. Examples of such promoters include, but are not limited to, T7 promoter and the like. When using the T7 promoter, it is preferable that the T7 RNA polymerase gene also coexist in the 1-4 strain. In this case, for example, the T7 RNA polymerase gene may be introduced into strain 1-4 along with the gene operably linked to the T7 promoter.
Alternatively, the promoter of any gene possessed by strain 1-4 may be obtained, and the polynucleotide may be functionally linked to the promoter. Examples of the arbitrary gene include a gene containing the base sequence set forth in SEQ ID NO: 1, a gene containing the base sequence set forth in SEQ ID NO: 3, and the like. The promoters of these genes can be obtained by TAIL-PCR or the like using primers designed based on the sequences of SEQ ID NO: 1 or SEQ ID NO: 3, for example.

Introduction of the above polynucleotide into the 1-4 strain can be performed by the method described in [Cells] below.
(A1) ~ (A7) and / or 1-4 mutant strains introduced polynucleotides selected from the group consisting of (B1) ~ (B7), 1 It may be a mutant strain with enhanced ability to produce fatty alcohols compared to the -4 strain. Therefore, it can be suitably used in the method for producing an aliphatic alcohol, which will be described later.

[Polynucleotide]
(Polynucleotide (A))
In one embodiment, the present invention provides a polynucleotide selected from the group consisting of (A1) to (A7) below.
(A1) A polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 (A2) One or more amino acids are deleted, substituted, added, or inserted in the amino acid sequence set forth in SEQ ID NO: 2 Polynucleotide (A3) consisting of an amino acid sequence and encoding a polypeptide having fatty alcohol synthesis activity, consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2, and fatty alcohol Polynucleotide encoding a polypeptide having synthetic activity (A4) Polynucleotide consisting of the base sequence set forth in SEQ ID NO: 1 (A5) Deletion, substitution, or deletion of one or more bases in the base sequence set forth in SEQ ID NO: 1 Polynucleotide (A6) consisting of an added or inserted base sequence and encoding a polypeptide having aliphatic alcohol synthesis activity from a base sequence having 80% or more sequence identity with the base sequence set forth in SEQ ID NO: 1 A polynucleotide encoding a polypeptide (A7) consisting of and having aliphatic alcohol synthesis activity, hybridizes under stringent conditions with a polynucleotide consisting of the base sequence set forth in SEQ ID NO: 1, and has aliphatic alcohol synthesis activity Polynucleotides Encoding Polypeptides

In (A2) above, the number of amino acids to be deleted, substituted, added, or inserted is not particularly limited as long as the resulting polypeptide has aliphatic alcohol synthesis activity. The number of amino acids to be deleted, substituted, added or inserted can be, for example, 1-80, preferably 1-60, more preferably 1-50, even more preferably 1-30. Further examples of the number of amino acids to be deleted, substituted, added, or inserted include 1 to 20, 1 to 10, 1 to 5, and the like.

In (A3) above, the sequence identity is not particularly limited as long as it is 80% or more. The sequence identity is preferably 85% or higher, more preferably 90% or higher, even more preferably 95% or higher. The sequence identity (or homology) between the amino acid sequences was obtained by aligning the two amino acid sequences so that the corresponding amino acids match the most, while inserting gaps in the portions corresponding to insertions and deletions. It is calculated as the ratio of matched amino acids to the entire amino acid sequence excluding gaps in the alignment. Sequence identity between amino acid sequences can be determined using various homology search software known in the art. For example, sequence identity values of amino acid sequences can be obtained by calculation based on alignments obtained by the known homology search software BLASTP.

In (A5) above, the number of bases to be deleted, substituted, added, or inserted is not particularly limited as long as the resulting polynucleotide encodes a polypeptide having aliphatic alcohol synthesis activity. The number of bases to be deleted, substituted, added or inserted can be, for example, 1-180, preferably 1-150, more preferably 1-90. Further examples of the number of bases to be deleted, substituted, added, or inserted include 1 to 60, 1 to 30, 1 to 15, and the like.

In (A6) above, the sequence identity is not particularly limited as long as it is 80% or more. The sequence identity is preferably 85% or higher, more preferably 90% or higher, even more preferably 95% or higher. The sequence identity (or homology) between base sequences can be considered in the same way as the sequence identity between amino acid sequences described in (A3) above.

In (A1) to (A3) above, it may be preferable to select degenerate codons that are frequently used by host cells. For example, the nucleotide sequence of (A1) may be the nucleotide sequence set forth in SEQ ID NO:1. Alternatively, it may be a nucleotide sequence encoding the same amino acid sequence as the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, which is changed to codons frequently used in host cells. Codons can be changed by, for example, known genetic modification techniques or artificial gene synthesis, but are not limited to these.

The polynucleotides (A1) to (A7) above may further contain base sequences encoding various tags, different proteins, linker sequences and the like.

The nucleotide sequence of SEQ ID NO: 1 was identified as a nucleotide sequence encoding fatty alcohol synthase from strain 1-4. As shown in the examples below, by introducing a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 into E. coli, the aliphatic alcohol is reduced to about 19% (w / w) of the dry cell weight of E. coli. I was able to get E. coli to produce it. At this time, no aliphatic aldehyde was detected. Therefore, the nucleotide sequence set forth in SEQ ID NO: 1 is a nucleotide sequence that encodes a polypeptide (SEQ ID NO: 2) having aliphatic alcohol synthesis activity.
FIG. 1 shows the putative fatty alcohol synthesis pathway in strains 1-4. In FIG. 1, the polypeptide (SEQ ID NO: 2) encoded by the base sequence of SEQ ID NO: 1 is represented by "PhsA". The polypeptide encoded by the nucleotide sequence of SEQ ID NO: 1 catalyzes the reaction of reducing acyl-CoA to produce an aliphatic alcohol, as shown in FIG. Most likely (possibly reducing acyl-ACPs and fatty acids to produce fatty alcohols). That is, the polypeptide encoded by the nucleotide sequence of SEQ ID NO: 1 is highly likely to function as a fatty-alcohol-forming fatty acyl-CoA reductase. Hereinafter, the polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 1 may be referred to as "phsA", and the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 may be referred to as "PhsA".

The polynucleotide of (A1) above is a polynucleotide encoding PhsA, and the polynucleotide of (A4) above is phsA. The above polynucleotides (A2), (A3), (A5) to (A7) are phsA mutants that maintain the fatty alcohol synthesis activity of PhsA.
All of the above polynucleotides (A1) to (A7) are polynucleotides encoding polypeptides having aliphatic alcohol synthesis activity. Therefore, by introducing any of these polynucleotides into cells of other species that do not have the ability to produce fatty alcohols, cells having the ability to produce fatty alcohols can be obtained. In addition, even a cell originally having an ability to produce fatty alcohols can be improved in ability to produce fatty alcohols by introducing any of these polynucleotides.

Hereinafter, the polynucleotide selected from the group consisting of (A1) to (A7) is sometimes referred to as "polynucleotide (A)".

(Polynucleotide (B))
In one embodiment, the present invention provides a polynucleotide selected from the group consisting of (B1) to (B7) below.
(B1) A polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 4 (B2) One or more amino acids are deleted, substituted, added, or inserted in the amino acid sequence set forth in SEQ ID NO: 4 Polynucleotide (B3) SEQ ID NO: 4, which encodes a polypeptide consisting of an amino acid sequence and which, when introduced into cells together with phsA, increases the fatty alcohol content of cells compared to cells into which phsA alone has been introduced When encoding a polypeptide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence described in and introduced into a cell together with phsA, compared to cells into which phsA alone was introduced, the fatty acidity of the cell Polynucleotide (B4) consisting of the base sequence set forth in SEQ ID NO: 3, wherein one or more bases are deleted, substituted, or added in the base sequence set forth in SEQ ID NO: 3 (B5); Or, when introduced into a cell together with a polynucleotide (phsA) consisting of an inserted nucleotide sequence and consisting of the nucleotide sequence set forth in SEQ ID NO: 1, compared to cells introduced with phsA alone, fatty alcohol content of cells Polynucleotide (B6) consisting of a nucleotide sequence having a sequence identity of 80% or more with the nucleotide sequence set forth in SEQ ID NO: 3, in increasing amounts, and when introduced into cells together with phsA, compared to cells introduced with phsA alone In comparison, when the polynucleotide (B7) hybridizes under stringent conditions with a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 3, and is introduced into cells together with phsA, which increases the fatty alcohol content of the cell. to increase the fatty alcohol content of cells compared to cells transfected with phsA alone,

In (B2) above, the number of amino acids to be deleted, substituted, added, or inserted is not particularly limited as long as the resulting polypeptide has fatty alcohol synthesis-assisting activity. The number of amino acids to be deleted, substituted, added or inserted can be, for example, 1-60, preferably 1-50, more preferably 1-40, even more preferably 1-30. Further examples of the number of amino acids to be deleted, substituted, added, or inserted include 1 to 20, 1 to 10, 1 to 5, and the like.

In (B3) above, the sequence identity is not particularly limited as long as it is 80% or more. The sequence identity is preferably 85% or higher, more preferably 90% or higher, even more preferably 95% or higher.

In (B5) above, the number of bases to be deleted, substituted, added, or inserted is not particularly limited as long as the resulting polynucleotide encodes a polypeptide having aliphatic alcohol synthesis-assisting activity. The number of bases to be deleted, substituted, added or inserted can be, for example, 1-100, preferably 1-80, more preferably 1-60. Further examples of the number of bases to be deleted, substituted, added, or inserted include 1 to 50, 1 to 30, 1 to 15, and the like.

In (B6) above, the sequence identity is not particularly limited as long as it is 80% or more. The sequence identity is preferably 85% or higher, more preferably 90% or higher, even more preferably 95% or higher.

In (B1) to (B3) above, it may be preferable to select degenerate codons that are frequently used by host cells. For example, the base sequence of (B1) may be the base sequence set forth in SEQ ID NO:3. Alternatively, it may be a nucleotide sequence encoding the same amino acid sequence as the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 3, which is changed to codons frequently used in host cells.

The above polynucleotides (B1) to (B7) may further contain nucleotide sequences encoding various tags, different proteins, linker sequences and the like.

The nucleotide sequence of SEQ ID NO: 3 was identified as the nucleotide sequence of a gene that forms a cluster with phsA from strain 1-4. As shown in Examples described later, introduction of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 into E. coli together with phsA increases the amount of fatty alcohol produced compared to introduction of phsA alone. and was able to produce about 24% of the dry cell weight of fatty alcohols. Therefore, the nucleotide sequence set forth in SEQ ID NO: 3 is a nucleotide sequence that encodes a polypeptide (SEQ ID NO: 4) having aliphatic alcohol synthesis-assisting activity. In addition, when the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 was introduced into E. coli together with phsA, the growth rate of E. coli increased as compared with the introduction of phsA alone.
The polypeptide (SEQ ID NO: 4) encoded by the polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 3 was presumed to potentially have lipase activity from the results of homology search of its amino acid sequence. The above results are consistent with the fact that the polypeptide set forth in SEQ ID NO:4 is a lipase. In the fatty alcohol synthesis pathway shown in FIG. 1, the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 4 is represented by "PhsB". As shown in FIG. 1, PhsB is considered to be a lipase that degrades acylglycerol to produce fatty acids that are precursors of acyl-CoA. Hereinafter, the polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO:3 is sometimes referred to as "phsB", and the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:4 is sometimes referred to as "PhsB".

The polynucleotide of (B1) above is a polynucleotide encoding PhsB, and the polynucleotide of (B4) above is phsB. The polynucleotides (B2), (B3), (B5) to (B7) above are mutants of phsB that maintain the fatty alcohol synthesis-supporting activity of PhsB.
All of the above polynucleotides (B1) to (B7) are polynucleotides encoding polypeptides having fatty alcohol synthesis-assisting activity. Therefore, by introducing any of these polynucleotides in combination with the above-described polynucleotide (A) into cells of other species, cells with improved aliphatic alcohol-producing ability can be obtained.

Hereinafter, the polynucleotide selected from the group consisting of (B1) to (B7) is sometimes referred to as "polynucleotide (B)".

The polynucleotide (A) or polynucleotide (B) is synthesized using a known nucleic acid synthesis method or the like based on the nucleotide sequence of SEQ ID NO: 1 or 3 or the amino acid sequence of SEQ ID NO: 2 or 4. be able to. Alternatively, it can be obtained by amplifying a DNA fragment by PCR, isothermal amplification, or the like, using the DNA of strain 1-4 as a template. Alternatively, DNA fragments may be amplified by PCR, isothermal amplification, or the like, using cDNA obtained by reverse transcription of the mRNA of strain 1-4 as a template. Primers used in PCR, isothermal amplification, etc. can be designed, for example, based on the base sequence of SEQ ID NO: 1 or 3, taking into consideration the melting temperature (Tm value) and the like.

In another aspect, the present invention provides a polypeptide selected from the group consisting of (A1') to (A3') and (B1') to (B3') below.
(A1') a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2; A polypeptide (A3′) consisting of an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence set forth in SEQ ID NO: 2 and having a fatty alcohol synthesis activity and having a fatty alcohol synthesis activity ( B1′) Polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 4 (B2′) Consists of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted in the amino acid sequence set forth in SEQ ID NO: 4 and a polypeptide (B3′) having fatty alcohol synthesis activity, consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4, and having fatty alcohol synthesis activity

Hereinafter, a polypeptide selected from the group consisting of (A1′) to (A3′) above is referred to as “polypeptide (A)”, and a polypeptide selected from the group consisting of (B1′) to (B3′) above is Sometimes referred to as "polypeptide (B)".

The above polypeptide (A) or polypeptide (B) can be synthesized based on the amino acid sequence shown in SEQ ID NO: 2 or 4 using a known peptide synthesis method or the like. Alternatively, strain 1-4 or cells introduced with polynucleotide (A) or polynucleotide (B) can be grown and polypeptide (A) or polypeptide (B) can be obtained from the culture. For example, when the polypeptide (A) or polypeptide (B) is accumulated in cells, the cells are collected by centrifugation, filter filtration, etc., and the polypeptide is purified from the cells using a known enzyme purification technique or the like. Peptide (A) or polypeptide (B) can be isolated and purified. Alternatively, for example, cells are disrupted by osmotic pressure adjustment, a bacteriolytic agent, physical disruption, etc., and polypeptide (A) or polypeptide (B) is isolated from the disrupted cells using a known enzymatic purification technique or the like.・Refining can also be performed. Alternatively, when the polypeptide (A) or the polypeptide (B) is present in the culture solution, such as by allowing the cells to proliferate to the stationary phase and then leaving them for a while, the cells are placed under conditions that spontaneously lyse the cells. Polypeptide (A) or polypeptide (B) may be isolated and purified from the culture solution using a known enzymatic purification technique or the like. Alternatively, polypeptide (A) or polypeptide (B) is secreted, for example, by using cells into which polynucleotide (A) or polynucleotide (B) has been added, such as by adding a secretory signal peptide coding sequence. In some cases, polypeptide (A) or polypeptide (B) may be isolated and purified from the culture supernatant obtained by culturing the cells using a known enzyme purification technique or the like.
The isolation/purification of polypeptide (A) or polypeptide (B) may be carried out to a degree of purification according to the purpose. Other substances may be contained as long as they do not interfere with the activity.

[vector]
In one embodiment, the invention provides a vector comprising the polynucleotide of the above embodiments.
The vector of the present embodiment may contain the polynucleotide (A), may contain the polynucleotide (B), or may contain the polynucleotide (A) and the polynucleotide (B) can be anything. That is, the vector of this embodiment contains either or both of polynucleotide (A) and polynucleotide (B).

The vector of this embodiment is preferably an expression vector containing polynucleotide (A) and/or polynucleotide (B) in an expressible state. In expression vectors, polynucleotide (A) and/or polynucleotide (B) are usually operably linked to a suitable promoter. When the vector of this embodiment contains both the polynucleotide (A) and the polynucleotide (B), the polynucleotide (A) and the polynucleotide (B) may be functionally linked to a single promoter. Alternatively, each may be operably linked to a separate promoter.

A promoter that functions in the host cell can be appropriately selected as the promoter depending on the host cell. Promoters that function in bacteria include, for example, T7 promoter (working in the presence of T7 RNA polymerase), lac promoter, trp promoter, tac promoter, PR promoter or PL promoter derived from λ phage, and the like. Promoters that function in yeast include, for example, yeast glycolytic gene promoter, alcohol dehydrogenase gene promoter, TPI1 promoter, ADH2-4c promoter and the like. Examples of promoters that function in plant cells include cauliflower mosaic virus (CaMV) 35S promoter, nopaline synthase gene promoter (Pnos), maize-derived ubiquitin promoter, rice-derived actin promoter, tobacco-derived PR protein promoter, and the like. be done. Examples of promoters that function in insect cells include polyhedrin promoter, P10 promoter, Autographa californica polyhedrosis basic protein promoter, baculovirus immediate early gene 1 promoter, baculovirus 39K delayed early gene promoter, and the like. be done.

The vector of this embodiment may further contain other functional sequences. For example, the vectors of this embodiment may contain regulatory sequences such as enhancers and terminators, selectable marker sequences such as drug resistance genes, and the like. It may also contain a sequence (transposon sequence, host DNA sequence, etc.) that allows the polynucleotide (A) and/or polynucleotide (B) to be inserted into the host's DNA. In addition, depending on the host, it may contain regulatory regions such as splicing signals (donor site, acceptor site, branch point, etc.), poly A addition signal, ribosome binding sequence and the like. The type of each sequence can be appropriately selected from known ones depending on the host into which the vector is introduced. In addition, when a T7 promoter is used as a promoter and cells that do not express the T7 RNA polymerase gene are used as hosts, the vector of this embodiment may further contain a T7 RNA polymerase gene sequence. In addition, when the vector of the present embodiment has a transposon sequence, a sequence recognized by a transposase is placed on both sides of the polynucleotide (A) and/or the polynucleotide (B) operably linked to the promoter. (sequences in which recombination occurs) are preferably placed.

The type of vector used in this embodiment is not particularly limited, but a plasmid vector or a virus vector is preferred. Examples of plasmid vectors include E. coli-derived plasmids (pBI, pPZP, pSMA, pUC, pBR, pBluescript, etc.), Bacillus subtilis-derived plasmids (pUB110, pTP5, etc.), and yeast-derived plasmids ( Yep13, Yep24, YCp50, etc.) can be preferably used. Viral vectors include phage (λ phage such as λgt11 and λZAP), plant viruses (CaMV, kidney bean golden mosaic virus (BGMV), tobacco mosaic virus (TMV)), insect viruses (e.g., baculovirus), and the like. can do. The type of vector may be appropriately selected depending on the host to be introduced.
The vector of this embodiment may be obtained by inserting the polynucleotide (A) and/or the polynucleotide (B) into the cloning site of a commercially available expression vector. In addition, the vector of the present embodiment may be obtained by inserting the polynucleotide (A) and/or the polynucleotide (B) into the cloning site of the transposon vector of a gene transfer system using a commercially available transposon.

[cell]
In one embodiment, the invention provides a cell comprising polynucleotide (A).

In one aspect, the cell of this embodiment can be produced by introducing the polynucleotide (A) into a desired cell. That is, a cell into which the polynucleotide (A) has been introduced is a suitable example of the cell of this embodiment.

Polynucleotide (A) is preferably introduced into cells in an expressible state. Therefore, the polynucleotide (A) is preferably introduced into the cell in a state operably linked to a promoter. Polynucleotide (A) may be introduced into cells in the form of the vector of the above embodiment. In this case, the vector of the above embodiment is preferably an expression vector or transposon vector containing polynucleotide (A). Alternatively, a DNA fragment containing polynucleotide (A) operably linked to a promoter may be introduced into cells. Alternatively, a DNA fragment containing a polynucleotide (A) operably linked to a promoter and sequences recognized by transposase added to both ends thereof may be introduced into cells.
Cells into which polynucleotide (A) is introduced are not particularly limited, and may be either prokaryotic or eukaryotic cells. Examples of prokaryotic cells include bacterial cells of the genera Exiguobacterium, Streptomyces, Rhodococus, Brevibacillus, Escherichia, and Bacillus. A preferred example is Escherichia coli. Examples of eukaryotic cells include yeast (genus Saccharomyces, etc.), fungal cells, insect cells, animal cells, plant cells, and the like.

The method for introducing polynucleotide (A) into cells is not particularly limited, and a known gene introduction method or the like may be used as appropriate depending on the type of cell. Examples of gene transfer methods include chemically competent cell method, electroporation method, PEG (polyethylene glycol) method, calcium phosphate transfection method, microinjection method, particle gun method, Agrobacterium method and the like. Moreover, depending on the type of cells, polynucleotide (A) can sometimes be introduced into cells by adding a vector or DNA fragment containing polynucleotide (A) to a cell suspension and mixing. The method for introducing polynucleotide (A) into cells may be such a method. The method for introducing polynucleotide (A) into cells is not limited to the above methods, and any method can be used as long as introduction into cells is possible.
The polynucleotide (A) introduced into the cell may exist in the cell as a plasmid or the like, or may exist in the cell in a state of being inserted into the genome of the cell. For example, when polynucleotide (A) is introduced into a cell using a plasmid vector or the like, polynucleotide (A) can exist in the cell as a plasmid.
Further, for example, when a polynucleotide (A) flanked by sequences recognized by a transposase is introduced into a cell, the polynucleotide (A) exists in the cell in a state of being randomly inserted into the genome or plasmid of the cell. can. In this case, it is preferable to introduce a polynucleotide containing the transposase gene sequence or the transposase itself into the cell together with the polynucleotide (A) flanked by sequences recognized by the transposase.
Polynucleotide (A) may also be site-specifically inserted into the cell genome or plasmid. For example, by preparing a DNA fragment in which the polynucleotide (A) is inserted between the genome sequence or plasmid sequence of the target site or a vector containing this DNA fragment, and introducing the DNA fragment or vector into cells, homologous recombination can insert polynucleotide (A) into the target site of the genome or plasmid. In this case, for example, CRISPER / Cas system, transcription activation-like effector nuclease (Transcription Activator-Like Effector Nuclease: TALEN), by genome editing technology using zinc finger nuclease (Zinc Finger Nuclease: ZFN), etc., polynucleotide (A) may be site-specifically inserted into the cell genome or plasmid.
In the above, the polynucleotide (A) is preferably in an expressible state and is operably linked to a promoter. Also, if the cell is a eukaryotic cell, the genome may be either a nuclear genome or an organellar genome.

Cells into which polynucleotide (A) has been introduced can be cultured and proliferated by appropriately selecting a medium according to the type of the cells. When polynucleotide (A) is introduced together with a marker gene such as a drug resistance gene, transformants may be selected using a drug to which the marker gene exhibits resistance.

By introducing polynucleotide (A) into cells in an expressible state, polynucleotide (A) can be expressed and polypeptide (A) can be produced in the cells. Since the polypeptide (A) has the activity of synthesizing fatty alcohols, it can become capable of synthesizing fatty alcohols when the cells do not produce fatty alcohols. When the cell originally produces an aliphatic alcohol, introduction of the polynucleotide (A) can improve the ability to produce the aliphatic alcohol.

The cell of this embodiment preferably contains polynucleotide (B) in addition to polynucleotide (A). Such cells can be produced, for example, by introducing polynucleotide (B) into desired cells in addition to polynucleotide (A). In this case, for example, vectors or DNA fragments containing polynucleotide (A) and polynucleotide (B) can be used. Alternatively, it can be produced by separately introducing a vector or DNA fragment containing the polynucleotide (A) and a vector or DNA fragment containing the polynucleotide (B) into cells. Also in this case, the polynucleotide (A) and the polynucleotide (B) are preferably in an expressible state, and are preferably operably linked to a promoter. For example, when a vector is used, it is preferably an expression vector, a transposon vector, or a vector that is site-specifically inserted into the cell genome or plasmid (described above) as described in [Vector] above. When polynucleotide (A) and polynucleotide (B) are introduced into cells in an expressible state, polynucleotide (A) and polynucleotide (B) are expressed in the cell, and polypeptide (A) and polynucleotide A peptide (B) can be produced. Since the polypeptide (B) has a fatty alcohol synthesis-assisting activity, the ability of the cells to synthesize the fatty alcohol can be further improved compared to when the polypeptide (A) is produced alone.

In another aspect, the cells of this embodiment may be wild-type cells that contain the polynucleotide (A). Examples of such cells include microorganisms belonging to the genus Reinekea and containing the polynucleotide (A). In a preferred embodiment, the microorganism belongs to the genus Reinekea and contains polynucleotide (A) and polynucleotide (B). Polynucleotide (A) or polynucleotide (A) and polynucleotide (B) are preferably contained in the microorganism in an expressible state. As a result, polypeptide (A), or polypeptide (A) and polypeptide (B) are expressed, and the microorganism can have the ability to synthesize fatty alcohols, or improve the ability to synthesize fatty alcohols originally possessed. can.

Whether a cell contains polynucleotide (A) can be determined by amplification of polynucleotide (A) or a fragment thereof by PCR, Southern hybridization method using polynucleotide (A) or a fragment thereof as a probe, and whole genome analysis. It can be confirmed by a known method such as. Whether or not the cell contains the polynucleotide (B) can be similarly confirmed.

[Method for producing fatty alcohol]
In one embodiment, the present invention provides a method for producing fatty alcohols.

(First embodiment)
In one aspect of the production method of the present embodiment, (a) the step of culturing the 1-4 strain or its mutant strain, and (b) the culture obtained in the step (a), to obtain an aliphatic alcohol process.

<Step (a)>
Step (a) is a step of culturing strain 1-4 or its mutants. 1-4 strain or its mutants are the same as those described in the above "Reinekea sp. 1-4 strain and its mutants".

1-4 strain or its mutant strain can be cultured under the conditions exemplified in the above "Reinekea sp. 1-4 strain and its mutant strain". The medium used for culturing in this step is preferably low nutrient, more preferably containing seawater, and more preferably containing deep water. For example, a culture medium obtained by diluting a culture medium for bacteria with seawater (preferably deep water) can be preferably used. Examples of the dilution ratio include 1 to 20 times, 4 to 15 times, 5 to 12 times, and the like. As an example, the dilution ratio is 10 times. Specific examples of suitable media include dR1 medium and dR medium described in Examples. Among them, dR medium is preferred.

The culture method is not particularly limited, and methods generally used for culturing aerobic bacteria can be used. Cultivation may be performed by static culture or shaking culture, but from the viewpoint of growth efficiency, shaking culture is preferable. In the case of stationary culture, a pump or the like may be used to supply air to the culture medium at appropriate times.
Cultivation may be performed by any method of batch culture, fed-batch culture, and continuous culture.

<Step (b)>
Step (b) is a step of obtaining an aliphatic alcohol from the culture obtained in step (a).

This step can be carried out by appropriately selecting an appropriate method according to the properties of the 1-4 strain or its mutants, the state of the bacteria after culture, and the like. For example, when fatty alcohol is stored in the cells, the cells can be collected from the culture and the fatty alcohol can be obtained from the cells. Centrifugation, filter filtration, or the like can be mentioned as a method for recovering the cells from the culture. A filter with a pore size of 0.22 μm is exemplified as a filter used for filtration. Methods for recovering the aliphatic alcohol from the cells include, for example, a method of adding an organic solvent such as n-heptane to the cells, shaking the mixture, and then recovering the organic layer. Moreover, the cells may be crushed before the addition of the organic solvent to the cells, or before or at the same time as the shaking after the addition of the organic solvent. Examples of methods for disrupting bacterial cells include physical treatments such as beads (glass beads, zirconia beads, etc.), mortar, ultrasonic treatment, French press, homogenizer, freeze-thaw treatment; well-known methods such as chemical treatment, and the like. These treatments may be performed singly or in combination of two or more. In addition, when using mutant strains 1-4 that have acquired the ability to secrete fatty alcohol, the fatty alcohol floating in the culture supernatant can be removed without collecting the bacterial cells. It can be collected as it is. In addition, when the aliphatic alcohol is present in a solid state in the culture supernatant, after removing the bacterial cells as necessary, the solid state aliphatic alcohol is recovered by filtering the culture supernatant. You may Alternatively, the aliphatic alcohol may be extracted by directly adding an organic solvent such as n-heptane to the culture, or the aliphatic alcohol may be recovered by distillation of the culture or the culture supernatant.

<Optional process>
The production method of this embodiment may include other steps in addition to steps (a) and (b). The other step is not particularly limited, but is exemplified by a step of purifying an aliphatic alcohol from the extract obtained in step (b). The method for purifying the aliphatic alcohol is not particularly limited, and methods commonly used in the separation and purification of organic compounds can be used in appropriate combination. Examples of purification methods include various chromatographic methods such as thin layer chromatography and liquid chromatography.

(Second embodiment)
In one aspect, the production method of the present embodiment includes (a') culturing the cells of the above embodiment, and (b') obtaining an aliphatic alcohol from the culture obtained in the step (a'). process.

<Step (a')>
Step (a') is a step of culturing the cells of the above embodiment (the cells described in [Cells] above). The medium used for cell culture may be appropriately selected according to the cell type, promoter, and the like. Examples thereof include media containing glucose, pyruvic acid, acetic acid, malic acid, etc. as carbon sources. A specific example of a suitable medium when the cells are E. coli is M9 medium described in the Examples. In addition, depending on the type of promoter used for polynucleotides A and/or B, a compound necessary for inducing transcription of the promoter may be added to the medium.

The culture method may also be appropriately selected according to the cell type, promoter, and the like. For example, when the cells are aerobic bacteria, shaking culture is preferred from the viewpoint of growth efficiency. Cultivation may be performed by any method of batch culture, fed-batch culture, and continuous culture. In addition, when the promoter used for polynucleotide A and/or B is a stimulus-responsive promoter, a stimulus (light, temperature, pH, compound, etc.) necessary for transcription induction of the promoter should be applied during culture. can be

<Step (b')>
Step (b') is a step of obtaining an aliphatic alcohol from the culture obtained in step (a'), and can be performed in the same manner as step (b) in the first embodiment.

<Optional process>
The production method of this embodiment may include other steps in addition to steps (a') and (b'). Other steps include the same steps as those mentioned in the first embodiment.

The method of the present embodiment biologically produces fatty alcohols using cells with high fatty alcohol-producing ability. Therefore, an aliphatic alcohol can be efficiently produced by a simple method.

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[Materials and methods]
(cell line)
Strains 1-4 were isolated from surface seawater (33°18'N, 134°11'E, depth 0.5m) collected in the summer of 2011 in the coastal area of Muroto City, Kochi Prefecture, Japan. Escherichia coli DH5a was obtained from Toyobo and used as a host strain for DNA manipulations and heterologous gene expression.

(Culture medium)
Table 1 shows the media used in the test.

The dR medium was filtered through a filter (0.22 μm, express plus, Millipore) and autoclaved at 110° C. for 2 seconds. Media was supplemented with 0.5 μg/mL Nile Red, 1 mM isopropyl-β-thiogalactopyranoside (IPTG) and 100 μg/mL ampicillin as needed.

(electron microscopy)
For transmission electron microscopy (JEM1200EX; JEOL), strains 1-4 were grown in dR medium at room temperature until growth was observed in turbidity. Cells were negatively stained with 2% uranyl acetate. Cells were pre-fixed with 0.1 M phosphate buffer (pH 7.4) containing 2% glutaraldehyde, post-fixed with ₂ % OsO4, dehydrated with graded ethanol (50-100%), and transferred to Epon812. Embedded and sectioned (80-90 nm). First, it was stained with 2% uranyl acetate and then with a lead stain solution (Sigma-aldrich).

(Analysis of 16S rRNA gene)
Nearly full-length 16S rRNA gene sequences of strains 1-4 were obtained as previously described (Teramoto, M., et al., 2009. Microbiology 155, 3362-3370.). The resulting sequences were aligned with bacterial sequences of the class Gammaproteobacteria available in public databases using CLUSTAL X (ver.2.1). Alignments were manually corrected and gap trimmed as needed. Neighbor-joining method by CLUSTAL X using default parameters (including Kimura's correction) (Saitou, N. & Nei, M. 1987. Mol Biol Evol 4, 406-425.) and MEGA 6.06 (Tamura, K., A phylogenetic tree was constructed from the 1254 bp aligned sequence using the maximum likelihood method (Felsenstein, J. 1981. J Mol Evol 17, 368-376.) according to Mol Biol Evol 30, 2725-2729.). It was prepared and analyzed by the bootstrap method (Felsenstein, J. 1985. Evolution 39, 783-791.) based on 1000 resamplings.

(Production of fatty alcohol and GC-MS analysis)
Strains 1-4 were grown in dR medium at 28° C. with shaking for 37-38 hours. For fatty alcohol quantification, strains 1-4 were grown in approximately 200 mL of dR medium until an OD ₆₀₀ of 0.05-0.06 was reached. E. coli was grown to maturity in M9 medium supplemented with IPTG and ampicillin at 28° C. with shaking.

Approximately 300 μL of concentrated cell suspension of strains 1-4 or 400-620 μL of E. coli culture was used for lipid extraction. To these solutions, 300-400 μL of n-heptane was added, heated at 60° C. for 10 minutes, vortexed for 1 minute, and centrifuged at 30-40° C. for 5 minutes to remove aliphatic compounds in the n-heptane layer. Extracted. The extraction operation with n-heptane was performed three times in total. For samples in which a thick intermediate layer appeared after centrifugation, the samples were freeze-thawed and then centrifuged again. The n-heptane extract thus obtained was dehydrated over sodium sulfate, concentrated by _N2 purge, and subjected to GC-MS analysis as previously described (Teramoto M, et al., 2013. PLoS One 8: e66594.). All samples were analyzed by splitless injection in aliquots of 1 μL.

Based on mass spectra and retention times, the fatty alcohols were identified using the NIST library and fatty alcohol standards (1-tetradecanol, 1-hexadecanol, and 1-octadecanol). Quantitation was performed by comparison with the peak area of the standard. C ₁₄ alcohols (1-tetradecanol and 1-tetradecanol) were compared to 1-tetradecanol preparations. C ₁₆ alcohols (1-hexadecanol and 1-hexadecenol) were compared to 1-hexadecanol preparations. C ₁₈ alcohols (1-octadecanol and 1-octadecanol) were compared to 1-octadecanol preparations.

The cell dry weight was obtained by centrifuging or filtering the culture solution through a filter (0.22 μm, express plus, Millipore) to collect the cells, drying the cells, and measuring the weight.

(Genome sequencing and plasmid construction)
Extraction of total DNA from strains 1-4 was performed in the same manner as previously described (Misawa, N., et al., 1990. J. Bacteriol. 172, 6704-6712.) unless otherwise stated. Cells of line 1-4 were grown in 2 L of dR medium. Cells were suspended in 1 mL of solution I without being treated with TES buffer. Instead of pronase E, proteinase K was used. After the first phenol-chloroform extraction, 500 μL of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) was added to the extract. Using this total DNA, genome sequencing by HiSeq2500 (Illumina) and assembly of the obtained sequences were performed (Takara Bio Inc.). As a result, 269 contigs containing a total of 5.5 Mb were obtained.

Details of the plasmid construction are shown in Table 2. A 5.9 kb DNA fragment containing phsAB was amplified from the total DNA of strain 1-4 using KOD plus Neo (Toyobo).

(Other method)
Motility was confirmed by a bright field microscope (mil-kin; Aqua system, Japan). Growth temperatures of 4°C, 10°C, 15°C, 20°C, 25°C, 30°C, or 35°C were tested by culturing in dMB containing 1.5% (w/v) agar for 3 weeks. Casein degradation, starch degradation and chitin degradation were tested as previously described (Teramoto M, et al., 2015. Int J Syst Evol Microbiol. 65:353-358.). Agar degradation and antibiotic resistance were tested on dR2A-SW plates.

[result]
(Isolation of 1-4 strains)
Muroto surface seawater was plated directly onto DSW1 plates to isolate bacteria. Isolates that accumulate fatty alcohols were identified as Nile Red (Greenspan P, et al., 1985. J Cell Biol. 100: 965-973.; Spiekermann P., et al., 1999. Arch Microbiol. 171: 73). -80.) was screened on DSW1 plates and accumulated compounds were selected by analysis by GC-MS (data not shown). One isolate was found to accumulate fatty alcohols, and 1-hexadecanol was detected as the major fatty alcohol accumulated by that isolate (Figure 2). This isolate was designated strain 1-4.

Strain 1-4 grew better on dMB and dR1 media than on MB and R2A media. Strain 1-4 produced fatty alcohols in dR1 medium but not in dMB (data not shown). This result suggests that the genes involved in fatty alcohol synthesis are expressed under relatively poor nutritional conditions. Strain 1-4 appeared to accumulate higher concentrations of fatty alcohols in cells in dR medium (deep water-based) than in dR1 medium (surface seawater-based) (data not shown). Generally, deep seawater contains more nitrogen and phosphorus and less organic carbon than surface seawater. When grown on dR medium, strain 1-4 accumulated 0.9±0.2% of dry cell weight of C _14,16,18 fatty alcohol. This fatty alcohol content may be the highest content in the cells of organisms in nature. Bacteria of the genus Acinetobacter, when grown in nutritive-rich broth, accumulate fatty alcohols at 0.001% or less of the dry cell weight, and an inorganic medium containing hexadecane (precursor of fatty alcohols) fatty alcohol accumulation was reported to be 0.06% of the dry cell weight, even when grown at .

(General characteristics of 1-4 strains)
Strains 1-4 are rods (0.3-1.1×0.3-8.2 μm) with one polar flagellum (FIG. 3) and are motile. Strain 1-4 grows in the temperature range of 10-30°C (optimal temperature 20-30°C) and does not grow at 4°C and 35°C. Strains 1-4 do not grow in dR1 medium without seawater (seawater replaced with water). Strain 1-4 is resistant to 12 μg/mL tetracycline and 50 μg/mL gentamicin, but not to 20 μg/mL chloramphenicol and 100 μg/mL kanamycin. Strain 1-4 degrades casein, starch, and agar, but not chitin.

Among the type strains, strains 1-4 were most similar to bacteria of the genus Reinekea. The nearly full-length 16S rRNA gene sequence of strain 1-4 had 95.3% similarity to that of Reinekea marinisedimentorum DSM 15388 ^T , the type strain of the type species of the genus Reinekea. This suggests that strains 1-4 belong to the genus Reinekea (Tindall BJ, et al., 2010. Int J Syst Evol Microbiol 60, 249-266.). In the phylogenetic tree based on the 16S rRNA gene sequences, strains 1-4 clustered with bacteria of the genus Reinekea and were included in the order Oceanospirillales (Fig. 4).
FIG. 4 is a phylogenetic tree by the neighbor-joining method showing the position of strains 1-4 within the class Gammaproteobacteria based on the 16S rRNA gene sequence (1254 bp).

(Identification of the fatty alcohol synthesis cluster, phsAB)
In order to identify the fatty alcohol synthesis gene possessed by strain 1-4, the genome of strain 1-4 was sequenced. The gene product of the gene designated phsA had significant homology with fatty acyl-CoA reductases that form fatty alcohols. The gene product of phsA shows 51% amino acid identity to the gene product of Maqu_2507 (accession no. CP000514; Willis RM, et al., 2011. Biochemistry. 50: 10550-10558) and is identical to Acr1 (accession no. AAC45217; Reiser S and Somerville C. 1997. J Bacteriol. 179: 2969-2975.; Steen EJ, et al., 2010. Nature. 463: 559-562.). phsA appeared to be in an operon with a downstream gene termed phsB (Fig. 5). The phsB gene product showed no significant homology to known proteins, but very low homology to lipase. For example, it showed 7% identity and 43% similarity in amino acid sequence to the lipase of Aeromonas hydrophila (Anguita J, et al., 1993. Appl Environ Microbiol. 59: 2411-2417.). Genes encoding acyl-ACP (acyl carrier protein)/fatty acid/acyl-CoA reductase [Maqu_2507 gene (www.genome.jp/kegg/) and acr1 (Reiser S and Somerville C. 1997. J Bacteriol. 179: 2969- 2975.)] do not cluster with the lipase gene or its homologues.

E. coli strains transfected with phsA accumulated _C14,16,18 fatty alcohols but not fatty aldehydes (pBS29SNX in FIG. 6). On the other hand, the E. coli strain carrying the control vector did not accumulate either fatty alcohols or fatty aldehydes (Control vector in FIG. 6). Together with sequence homology, these results suggest that phsA encodes a fatty acyl-CoA reductase that forms fatty alcohols. E. coli strains harboring phsA alone accumulated fatty alcohols at 19% of dry cell weight (phsA in Figure 6). This is the highest fatty alcohol content to date and obtained by expressing only the acyl-ACP/fatty acid/acyl-CoA reductase. Estimating based on reported absorbance (indicative of cell density) or growth conditions, the maximum amount of fatty alcohol accumulated in recombinant E. coli to date can be estimated at ~17% of the dry cell weight, indicating that this Values have been achieved by overexpressing acyl-ACP thioesterase with expression of acyl-ACP/fatty acid/acyl-CoA reductase (Liu A, et al., 2013. Appl Microbiol Biotechnol. 97: 7061-7071. ; Fig. 1).

On the other hand, the lipase activity of phsB was not detected by TLC that observed changes in fatty acid accumulation in E. coli strains transfected with phsB (pBS29SNC) (data not shown). However, E. coli strains transfected with phsAB(pBS29SN) accumulated high concentrations of _C14,16,18 fatty alcohols, 24% of dry cell weight (Fig. 7). This value is higher than that of the E. coli strain into which only phsA was introduced (Fig. 7). Furthermore, the E. coli strain transfected with phsAB (pBS29SN) grew faster than the E. coli strain transfected with phsA (pBS29SNX). These results indicate that the novel gene cluster phsAB favors high fatty alcohol productivity. And this 24% accumulation could be even higher with overexpression of the acyl-ACP thioesterase.
These high accumulations of fatty alcohols and growth promotion by phsB are consistent with the phsB products potentially functioning as lipases. That is, phsB is thought to increase the level of acyl-CoA, which serves as an energy and carbon source through β-oxidation and as a substrate for reductase (Fig. 1).

INDUSTRIAL APPLICABILITY According to the present invention, a technology for producing fatty alcohols by a biological method is provided.

Claims (11)

Reinekea sp. 1-4 strain (NITE P-02620). A polynucleotide selected from the group consisting of (A1) to (A6) below.
(A1) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2; Polynucleotide (A3) encoding a polypeptide having aliphatic alcohol synthesis activity, consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2, and aliphatic Polynucleotide (A4) encoding a polypeptide having alcohol synthesizing activity Polynucleotide (A5) consisting of the base sequence set forth in SEQ ID NO: 1 Deletion of one or several bases in the base sequence set forth in SEQ ID NO: 1, Polynucleotide (A6) consisting of a substituted, added or inserted base sequence and encoding a polypeptide having aliphatic alcohol synthesis activity A base having 90% or more sequence identity with the base sequence set forth in SEQ ID NO: 1 A polynucleotide comprising a sequence and encoding a polypeptide having aliphatic alcohol synthesis activity A polynucleotide selected from the group consisting of (B1) to (B6) below.
(B1) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 4; When introduced into a cell together with a polynucleotide (phsA) that encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and consists of the nucleotide sequence set forth in SEQ ID NO: 1, compared to cells introduced with phsA alone, the aliphatic Polynucleotide (B3) encoding a polypeptide consisting of an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence set forth in SEQ ID NO: 4, and introducing into a cell together with phsA, the alcohol content of which increases, Polynucleotide (B4) Polynucleotide (B5) consisting of the base sequence set forth in SEQ ID NO: 3, the base sequence set forth in SEQ ID NO: 3, which increases the fatty alcohol content of cells compared to cells into which phsA alone is introduced consists of a nucleotide sequence in which one or several bases are deleted, substituted, added, or inserted, and when introduced into a cell together with phsA, compared to cells introduced with phsA alone, the aliphatic of the cell Polynucleotide (B6) with increasing alcohol content, consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence set forth in SEQ ID NO: 3, and introduced into cells together with phsA, introduced alone with phsA A polynucleotide that increases the fatty alcohol content of a cell compared to a cell A vector comprising either or both of the polynucleotide of claim 2 and the polynucleotide of claim 3. A cell comprising the polynucleotide of claim 2 . 6. The cell of claim 5, further comprising the polynucleotide of claim 3. Reinekea sp. Reinekea sp., which has the same 16S rRNA gene sequence as strain 1-4. Mutant strain of strain 1-4 (NITE P-02620). 8. The variant of claim 7, comprising the polynucleotide of claim 2. 8. The variant of claim 7, comprising the polynucleotide of claim 3. (a) Reinekea sp. 1-4 strain (NITE P-02620) or the mutant strain according to any one of claims 7 to 9; the process of obtaining alcohol,
A method for producing a fatty alcohol, comprising: (a') culturing the cells of claim 5 or 6; and (b') obtaining a fatty alcohol from the culture obtained from step (a');
A method for producing a fatty alcohol, comprising:

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