JP2007074959A - Protein related to alkane hydroxylation, gene encoding the same and method for producing alkanediol by using them - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for hydroxylating various alkanes and alcohols by cloning CYP153-alkane-1-monooxygenase gene, specifying a protein, expressing the gene in a host and constituting a microorganism conversion system by using a recombinant. <P>SOLUTION: This CYP153-alkane-1-monooxygenase, a recombinant vector containing a ferredoxin gene and ferredoxin reductase gene of Acinetobacter sp. OC4 strain and the transformant containing these genes or the recombinant vector are provided. The method for producing the alcohol or alkanediol comprising the hydroxylation of the alkane or alcohol in the presence of the transformant, and the method for producing the protein comprising the processes of culturing the transformant and collecting the protein from the cultured material are also provided. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a protein related to alkane hydroxylation, a gene encoding the same, and a method for producing alkanediol using the same.

  As for yeast belonging to Candida, it is known that α, ω dicarboxylic acid is produced via α, ω alkanediol when cultured with normal carbon as a carbon source (Non-patent Document 1). According to studies in Candida tropicalis, normal alkanes are first oxidized to 1-alcohol. This reaction is catalyzed by cytochrome P450 monooxygenase (CYP52) and cytochrome P450NADPH reductase (Non-patent Document 2). Further, this 1-alcohol is metabolized to carboxylic acid by fatty acid alcohol oxidase (FAO) and fatty acid aldehyde oxidase (Non-patent Document 3). In addition, 1-alcohol, which is such a ω oxidation product, is converted to α, ω alkanediol through the same ω oxidation pathway. It has been suggested that Candida tropicalis FAO-disrupted strain accumulates α, ω-alkanediol (Patent Document 1).

  On the other hand, bacteria capable of metabolizing alkanes metabolize these by the ω-oxidation pathway when cultured with a carbon source as a normal alkane. Normal alkane is first oxidized to 1-alcohol, but this reaction is caused by AlkB type alkane hydroxylase (AH) and its electron transport system, rubredoxin and rubredoxin reductase, or CYP153-alkane-, one of the cytochrome P450 family. It is catalyzed by 1-monooxygenase (CYP153) and its electron transfer system, ferredoxin and ferredoxin reductase (Non-patent Documents 4 and 5). Furthermore, it is known that this 1-alcohol is metabolized to carboxylic acid by alcohol dehydrogenase and aldehyde dehydrogenase (Non-patent Document 6). However, the enzyme derived from bacteria that oxidizes the end of 1-alcohol or 2-alcohol to convert it to α, ω-alkanediol, or metabolizes carbohydrates such as alkane, 1-alcohol, or 2-alcohol to convert α, ω-alkanediol There is no report about bacteria to produce.

In addition, some reports have been made on the subterminal oxidation of linear fatty acids (ω-1 oxidation, ω-2 oxidation, etc.), but the subterminal oxidation of linear alcohols is limited to several studies. Yes. For example, ω-1 oxidation such as 1-dodecanol by P450 derived from liver microsomes of frogs or guinea pigs (Non-patent Documents 7 and 8), ω-1, 1-dodecanol such as 1-dodecanol by P450 derived from Bacillus megaterium, Trioxidation (Non-patent Document 9) has been reported. However, for enzymes that produce 2-α-alkanediol by metabolizing carbohydrates such as 2-alcohol, using 2-alcohol as a substrate and oxidizing its end to convert it to α, ω-1 alkanediol. There is no report.
Shiio I. Microbial production of long-Chain dicarboxylic acids from n-alkanes. Agric. Biol. Chem. 1971 35 2033-2042. Craft DL, Madduri KM, Eshoo M, Wilson CR.Identification and characterization of the CYP52 family of Candida tropicalis ATCC 20336, important for the conversion of fatty acids and alkanes to alpha, omega-dicarboxylic acids.Appl Environ Microbiol. 2003 69 (10 ): 5983-91. Eschenfeldt WH, Zhang Y, Samaha H, Stols L, Eirich LD, Wilson CR, Donnelly MI.Transformation of fatty acids catalyzed by cytochrome P450 monooxygenase enzymes of Candida tropicalis.Appl Environ Microbiol. 2003 69 (10): 5992-9. Japanese translation of PCT publication No. 2002-506650 (P2002-506650A) Fujii T, Narikawa T, Takeda K, Kato J. Biotransformation of various alkanes using the Escherichia coli expressing an alkane hydroxylase system from Gordonia sp.TF6. Biosci Biotechnol Biochem. 2004 68 (10): 2171-7. Maier T, Forster HH, Asperger O, Hahn U. Molecular characterization of the 56-kDa CYP153 from Acinetobacter sp. EB104. Biochem Biophys Res Commun. 2001 286 (3): 652-8. van Beilen JB, Panke S, Lucchini S, Franchini AG, Rothlisberger M, Witholt B. Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes. Microbiology. 2001 147 (6): 1621 -30. Miura Y. ω- and (ω-1) -Hydroxylation of 1-dodecanol by frog liver microsomes Lipids, 1981 16 721-725 Miura Y, Hisaki H, Siems W, and Oda S. Hydroxylation of fatty acids and alcohols by hepatic Microsomal cytochrome P-450 system from the Mongolian gerbil. Lipids, 1987 22 987-993 Miura Y, and Fulco AJ. Ω-1, ω-2 and ω-3 Hydroxylation of long-chain fatty acids, amides and alcohols by a soluble enzymes system from Bacillus megaterium. Biochimica et Biophysica Acta, 1975 388 305-317

  Alkanediol is important as one of plastic raw materials. Although there is a possibility that α, ω alkanediol can be produced by yeast, in order to produce alkanediol more advantageously by bioprocess, a microbial conversion system using bacteria-derived enzyme is desired.

  An object of the present invention is to provide an enzyme capable of producing α, ω-alkanediol from α-alkanemonool, its gene, and a method for producing α, ω-alkanediol from α-alkanemonool using these enzymes.

  The present inventors cloned the CYP153-alkane-1-monooxygenase gene of Gordonia sp. TF6 strain, a microorganism that was previously isolated and found to have alkane hydroxylating ability, and the gene In addition to identifying the protein encoded by Acinetobacter sp. OC4 strain ferredoxin gene and ferredoxin reductase gene together with the ferredoxin reductase gene, it is expressed in excess or deficiency in a host such as E. coli, and the resulting recombinant is used. The present invention has been completed by finding that various alkanes and alcohols can be hydroxylated by the existing microbial conversion system.

The present invention is as follows.
[1] A gene encoding a protein having any of the following amino acid sequences.
(1) an amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing and having CYP153-alkane-1-monooxygenase activity;
(2) An amino acid having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids and having CYP153-alkane-1-monooxygenase activity in the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing Or (3) an amino acid sequence having an amino acid sequence having 80% or more homology with the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing and having CYP153-alkane-1-monooxygenase activity [2] A gene having any of the following base sequences:
(1) a nucleotide sequence having the nucleotide sequence of 646 to 1995 in SEQ ID NO: 2 in the sequence listing and encoding CYP153-alkane-1-monooxygenase;
(2) It has a base sequence having deletion, substitution and / or addition of one to several bases in the base sequence of Nos. 646 to 1995 shown in SEQ ID No. 2 of the sequence listing, and CYP153-alkane-1- A base sequence encoding a protein having monooxygenase activity; or (3) a DNA comprising a base sequence complementary to a DNA comprising a base sequence from 646 to 1995 in SEQ ID NO: 2 in the sequence listing and a stringent [3] a gene derived from a microorganism belonging to the genus Gordonia, which has a base sequence that hybridizes under various conditions and encodes a protein having alkane-1-monooxygenase activity [1] or The gene according to [2].
[4] A recombinant vector comprising the gene according to any one of [1] to [3].
[5] The recombinant vector according to [4], further comprising a gene encoding ferredoxin of Acinetobacter sp. OC4 strain and a gene encoding ferredoxin reductase.
[6] A transformant comprising the gene according to any one of [1] to [3] or the recombinant vector according to [4] or [5].
[7] The transformant according to [6], wherein the host is Escherichia coli.
[8] A method for producing alcohol, comprising hydroxylating a raw material alkane or alcohol to obtain a monool or diol in the presence of the transformant according to [6] or [7].
[9] A recombinant vector comprising the gene according to any one of [1] to [3], a gene encoding ferredoxin of Acinetobacter sp. OC4 strain, and a gene encoding ferredoxin reductase. [8] The production method according to [8].
[10] The production method according to [8] or [9], wherein the starting alcohol is monool and the alcohol produced is a diol.
[11] The production method according to [8] or [9], wherein the raw alcohol is 1-alkanol or 2-alkanol having 6 to 10 carbon atoms.
[12] The starting alcohol is 2-octanol and 1,7-octanediol is obtained as a diol,
Alternatively, the production method according to [8] or [9], wherein the starting alcohol is 1-heptanol and 1,7-heptanediol is obtained as a diol.
[13] A protein having any of the following amino acid sequences.
(1) an amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing and having CYP153-alkane-1-monooxygenase activity;
(2) An amino acid having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids and having CYP153-alkane-1-monooxygenase activity in the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing Or (3) an amino acid sequence having an amino acid sequence having 80% or more homology with the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing and having CYP153-alkane-1-monooxygenase activity [14] The method for producing a protein according to [13], comprising culturing a transformant containing the recombinant vector according to [4] and collecting the protein according to [13] from the culture.

  If the microbial conversion system using the recombinant of the present invention is used, α, ω alkanediol can be produced from α alkane monool using an enzyme system or a microbial system. Furthermore, analysis of the CYP153 alkane hydroxylation mechanism of bacteria, which has been difficult to analyze so far, is facilitated, and enzyme modification and the like are also possible.

Hereinafter, embodiments of the present invention will be described in detail.
The present inventors cloned the CYP153-alkane-1-monooxygenase gene of Gordonia sp. TF6 strain, which has already been reported. Gordonia sp.TF6 strain was deposited as Gordonia sp.TF6 on May 29, 2003 in the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center (FERM). P-19376).

  In addition, the present inventors have already cloned a gene encoding all components of the alkane hydroxylation mechanism of Acinetobacter sp. OC4 strain. The amino acid sequence and base sequence are shown in SEQ ID NOs: 1 and 2 in the sequence listing. Acinetobacter sp. OC4 strain was deposited as Acinetobacter sp. OC4 on August 11, 2005 at the National Institute of Technology and Evaluation of the National Institute of Technology and Evaluation (NITE) P-125).

1. CYP153-alkane-1-monooxygenase-related gene The present inventors obtained a CYP153-alkane-1-monooxygenase of Gordonia sp. TF6 strain and a gene encoding it.

That is, the gene of the present invention is a gene encoding a protein having any of the following amino acid sequences.
(1) an amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing and having CYP153-alkane-1-monooxygenase activity;
(2) An amino acid having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids and having CYP153-alkane-1-monooxygenase activity in the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing Or (3) an amino acid sequence having an amino acid sequence having 80% or more homology with the amino acid sequence described in SEQ ID NO: 1 in the sequence listing and having CYP153-alkane-1-monooxygenase activity

Specific examples of the gene of the present invention include genes having any of the following base sequences.
(1) a nucleotide sequence having the nucleotide sequence of 646 to 1995 in SEQ ID NO: 2 in the sequence listing and encoding CYP153-alkane-1-monooxygenase;
(2) It has a base sequence having deletion, substitution and / or addition of one to several bases in the base sequence of Nos. 646 to 1995 shown in SEQ ID No. 2 of the sequence listing, and CYP153-alkane-1- A base sequence encoding a protein having monooxygenase activity; or (3) a DNA comprising a base sequence complementary to a DNA comprising a base sequence from 646 to 1995 in SEQ ID NO: 2 in the sequence listing and a stringent Sequence encoding a protein having a base sequence that hybridizes under mild conditions and having CYP153-alkane-1-monooxygenase activity

  In the present invention, “CYP153-alkane-1-monooxygenase” refers to an enzyme belonging to the cytochrome P450 family that oxidizes the terminal end of a linear alkane, and “CYP153-alkane-1-monooxygenase activity” refers to a linear alkane. It means the enzyme activity that oxidizes the end of The activity of the protein having “CYP153-alkane-1-monooxygenase activity” and “CYP153-alkane-1-monooxygenase activity” is CYP153-alkane-1-monooxygenase, ferredoxin, ferredoxin reductase, NADPH, and a substrate. The resulting alkane is suspended in a suitable buffer solution, subjected to a conversion reaction at 28 ° C. or the like, and then the produced alcohol is detected by gas chromatography (GC) or the like.

  The range of “1 to several” in “amino acid sequence having 1 to several amino acid deletions, substitutions and / or additions” in the description of CYP153-alkane-1-monooxygenase-related genes is not particularly limited, For example, it means 1 to 20, preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 5, particularly preferably about 1 to 3.

  The homology in the “amino acid sequence having 80% or more homology with the amino acid sequence described in SEQ ID NO: 1” in the description of the CYP153-alkane-1-monooxygenase-related gene is 80% or more. Although not particularly limited, it is preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and particularly preferably 98% or more.

The range of “1 to several” in “base sequence having deletion, substitution and / or addition of one to several bases” in the description of CYP153-alkane-1-monooxygenase-related genes is not particularly limited, For example, it means 1 to 60, preferably 1 to 30, more preferably 1 to 20, further preferably 1 to 10, particularly preferably about 1 to 5.

  In the description of CYP153-alkane-1-monooxygenase-related genes, “hybridizes under stringent conditions” means using DNA as a probe, colony hybridization method, plaque hybridization method, or Southern blot hybridization method For example, using a colony or plaque-derived DNA or a filter on which the DNA fragment is immobilized, in the presence of 0.7 to 1.0 M sodium chloride at 65 ° C. List DNA that can be identified by washing the filter under conditions of 0.1 to 2x SSC (1x SSC solution is 150 mM sodium chloride, 15 mM sodium citrate) after hybridization. Can do. Hybridization can be performed according to the method described in Molecular Cloning 2nd edition.

  Examples of DNA that hybridizes under stringent conditions include DNA having a certain degree of homology with the base sequence of DNA used as a probe, for example, 80% or more, preferably 85% or more, more preferably 90%. More preferably, DNA having homology of 93% or more, particularly preferably 95% or more, and most preferably 98% or more is mentioned.

  The method for obtaining the CYP153-alkane-1-monooxygenase-related gene of the present invention is not particularly limited. Appropriate probes and primers are prepared based on the amino acid sequence set forth in SEQ ID NO: 1 and the nucleotide sequence set forth in SEQ ID NO: 2 in the sequence listing in this specification, and belong to the genus Gordonia using them. The CYP153-alkane-1-monooxygenase-related gene of the present invention can be isolated by screening a microbial DNA library. A DNA library can be prepared by a conventional method from a microorganism expressing the CYP153-alkane-1-monooxygenase-related gene of the present invention.

  The CYP153-alkane-1-monooxygenase-related gene of the present invention can also be obtained by PCR. Using the above-mentioned DNA library derived from a microorganism as a template, PCR is carried out using a pair of primers designed so as to amplify the nucleotide sequences of 646 to 1995 described in SEQ ID NO: 2. PCR reaction conditions can be set as appropriate. For example, one cycle of the reaction process consisting of 94 ° C for 30 seconds (denaturation), 55 ° C for 30 seconds to 1 minute (annealing), and 72 ° C for 2 minutes (extension) As an example, there may be mentioned conditions for carrying out the reaction at 72 ° C. for 7 minutes after 30 cycles. The amplified DNA fragment can then be cloned into a suitable vector that can be amplified in a host such as E. coli.

  The above-described procedures such as probe or primer preparation, DNA library construction, DNA library screening, and target gene cloning are known to those skilled in the art. For example, Molecular Cloning: A laboratory Mannual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989 (hereinafter abbreviated as "Molecular Cloning 2nd Edition"), Current Protocols in Molecular Biology, Supplements 1-38, John Wiley & Sons (1987-1997) (hereinafter "Current Protocol") And abbreviated as “molecular in biology”).

  In addition, a protein having an amino acid sequence having deletion, substitution and / or addition of one to several amino acids in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing and having CYP153-alkane-1-monooxygenase activity A coding gene; and a nucleotide sequence having a deletion, substitution and / or addition of one to several bases in the nucleotide sequence set forth in SEQ ID NO: 2 in the Sequence Listing, and having CYP153-alkane-1-monooxygenase activity For genes having base sequences encoding proteins (hereinafter referred to as mutant genes), chemical synthesis based on the amino acid sequence set forth in SEQ ID NO: 1 and the base sequence information set forth in SEQ ID NO: 2 Can be made by any method known to those skilled in the art, such as genetic engineering techniques or mutagenesis.

  For example, using DNA having the nucleotide sequence of 646 to 1995 in SEQ ID NO: 2 in the sequence listing, a method of contacting with a mutagen agent, a method of irradiating ultraviolet rays, a genetic engineering method, etc. It can be carried out. Site-directed mutagenesis, which is one of the genetic engineering methods, is useful because it can introduce a specific mutation at a specific position. Molecular cloning 2nd edition, Current Protocols in Molecular. It can be performed according to the method described in biology and the like.

The protein of the present invention is a protein having any of the following amino acid sequences.
(1) an amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing and having CYP153-alkane-1-monooxygenase activity;
(2) An amino acid having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids and having CYP153-alkane-1-monooxygenase activity in the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing Or (3) an amino acid sequence having an amino acid sequence having 80% or more homology with the amino acid sequence described in SEQ ID NO: 1 in the sequence listing and having CYP153-alkane-1-monooxygenase activity;

  The protein of the present invention is a protein having CYP153-alkane-1-monooxygenase activity. In the presence of a protein having CYP153-alkane-1-monooxygenase activity, the end of the linear alkane can be oxidized. The CYP153-alkane-1-monooxygenase activity of the protein of the present invention can be measured by the method described above.

  The method for obtaining the protein of the present invention is not particularly limited, and may be a protein synthesized by chemical synthesis or a recombinant protein produced by a gene recombination technique.

  When producing a recombinant protein, first, a gene (DNA) encoding the protein described above in this specification is obtained. The protein of the present invention can be produced by introducing this DNA into an appropriate expression system. The expression of the protein in the expression system will be described later in this specification.

  The gene of the present invention can be used by inserting it into an appropriate vector. The type of vector used in the present invention is not particularly limited. For example, the vector may be a self-replicating vector (for example, a plasmid), or may be integrated into the host cell genome when introduced into the host cell. It may be replicated together with other chromosomes.

  Preferably, the vector used in the present invention is an expression vector. In the expression vector, the gene of the present invention is functionally linked to elements necessary for transcription (for example, a promoter and the like). A promoter is a DNA sequence that exhibits transcriptional activity in a host cell, and can be appropriately selected according to the type of host.

  Promoters that can operate in bacterial cells include the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpa-amylase gene, and the Bacillus amylase gene. Bacillus amyloliquefaciens BAN amylase gene, Bacillus subtilis alkaline protease gene or Bacillus pumilus xylosidase gene promoter, or phage lambda PR or PL promoter of E. coli, lac, trc or tac promoter of E. coli.

  Examples of promoters that can operate in mammalian cells include the SV40 promoter, the MT-1 (metallothionein gene) promoter, or the adenovirus 2 major late promoter. Examples of promoters operable in insect cells include polyhedrin promoter, P10 promoter, autographa caliornica polyhedrossis basic protein promoter, baculovirus immediate early gene 1 promoter, or baculovirus 39K delayed early gene There are promoters. Examples of promoters that can operate in yeast host cells include promoters derived from yeast glycolytic genes, alcohol dehydrogenase gene promoters, TPI1 promoters, ADH2-4c promoters, and the like. Examples of promoters that can operate in filamentous fungal cells include the ADH3 promoter or the tpiA promoter.

  Moreover, the gene of the present invention may be operably linked to an appropriate terminator as necessary. The recombinant vector of the present invention may further have elements such as a polyadenylation signal (for example, derived from SV40 or adenovirus 5E1b region), a transcription enhancer sequence (for example, SV40 enhancer) and the like.

  The recombinant vector of the present invention may further comprise a DNA sequence that allows the vector to replicate in the host cell, an example of which is the SV40 origin of replication (when the host cell is a mammalian cell). .

  The recombinant vector of the present invention may further contain a selection marker. Selectable markers include, for example, genes that lack their complement in host cells such as dihydrofolate reductase (DHFR) or Schizosaccharomyces pombe TPI genes, or such as ampicillin, kanamycin, tetracycline, chloramphenicol, Mention may be made of resistance genes for drugs such as neomycin or hygromycin. Methods for linking the gene of the present invention, promoter, and optionally a terminator and / or secretory signal sequence, respectively, and inserting them into a suitable vector are well known to those skilled in the art.

  The recombinant vector of the present invention may contain a gene encoding ferredoxin of Acinetobacter sp. OC4 strain and a gene encoding ferredoxin reductase in addition to the gene encoding CYP153-alkane-1-monooxygenase. it can. The ferredoxin gene and the ferredoxin reductase gene may be included in the recombinant vector in any state as long as each can be expressed together with the CYP153-alkane-1-monooxygenase gene. As described later, the recombinant vector of the present invention is introduced into a host such as Escherichia coli, and the host is cultured to express the above three genes together to obtain CYP153-alkane-1-monooxygenase activity. be able to.

  The ferredoxin gene and the ferredoxin reductase gene are not limited to the Acinetobacter sp. OC4 strain gene, and for example, a closely related gene such as the Acinetobacter sp. HP2 strain may be used. Is possible. Acinetobacter sp. HP2 strain was deposited as Acinetobacter sp. HP2 on August 11, 2005 at the National Institute of Technology and Evaluation of Microorganisms (NITE). P-124).

  The CYP153-alkane-1-monooxygenase gene used above may be a mutant CYP153-alkane-1-monooxygenase gene other than the gene of SEQ ID NO: 2.

  A transformant can be prepared by introducing the gene or recombinant vector of the present invention into an appropriate host.

  The host cell into which the gene of the present invention or the recombinant vector is introduced may be any cell as long as it can express the gene of the present invention, and examples thereof include bacteria, yeast, fungi and higher eukaryotic cells.

  Examples of bacterial cells include Gram positive bacteria such as Bacillus or Streptomyces or Gram negative bacteria such as E. coli. Transformation of these bacteria may be performed by using competent cells by a protoplast method or a known method.

Production of recombinant protein using the transformant of the present invention and recombinant protein using the same Examples of mammalian cells include HEK293 cells, HeLa cells, COS cells, BHK cells, CHL cells or CHO cells. Methods for transforming mammalian cells and expressing DNA sequences introduced into the cells are also known, and for example, electroporation method, calcium phosphate method, lipofection method and the like can be used.

  Examples of yeast cells include cells belonging to Saccharomyces or Schizosaccharomyces, and examples include Saccharomyces cerevisiae and Saccharomyces kluyveri. Examples of the method for introducing a recombinant vector into a yeast host include an electroporation method, a spheroblast method, and a lithium acetate method.

  Examples of other fungal cells are cells belonging to filamentous fungi such as Aspergillus, Neurospora, Fusarium, or Trichoderma. When filamentous fungi are used as host cells, transformation can be performed by integrating the DNA construct into the host chromosome to obtain a recombinant host cell. Integration of the DNA construct into the host chromosome can be performed according to known methods, for example, by homologous recombination or heterologous recombination.

  When insect cells are used as the host, the recombinant gene transfer vector and baculovirus are co-introduced into the insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected with the insect cells. And protein can be expressed (for example, as described in Baculovirus Expression Vectors, A Laboratory Manua1; and Current Protocols in Molecular Biology, Bio / Technology, 6, 47 (1988), etc.).

  As the baculovirus, for example, Autographa californica nuclear polyhedrosis virus, which is a virus that infects Coleoptera insects, can be used.

  Insect cells include Sf9, Sf21 [Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York, Spodopterafrugiperda ovarian cells. (1992)], HighFive (manufactured by Invitrogen), which is an ovary cell of Trichoplusiani, and the like can be used.

  Examples of the method for co-introducing a recombinant gene introduction vector into insect cells and the baculovirus for preparing a recombinant virus include a calcium phosphate method and a lipofection method.

  The transformant is cultured in an appropriate nutrient medium under conditions that allow expression of the introduced gene. In order to isolate and purify the protein of the present invention from the culture of the transformant, ordinary protein isolation and purification methods may be used.

  For example, when the protein of the present invention is expressed in a dissolved state in the cell, after the culture is completed, the cell is collected by centrifugation, suspended in an aqueous buffer, and then disrupted by an ultrasonic crusher or the like. A cell-free extract is obtained. From the supernatant obtained by centrifuging the cell-free extract, an ordinary protein isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using a resin such as diethylaminoethyl (DEAE) Sepharose, Cation exchange chromatography using a resin such as SP-SepharoseFF (Amersham Biosciences), Resin such as butyl sepharose and phenyl sepharose Using a method such as a hydrophobic chromatography method using gel, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric focusing, etc. Obtainable.

  The present invention uses a transformant in which a CYP153-alkane-1-monooxygenase gene and a ferredoxin gene and ferredoxin reductase gene of Acinetobacter sp. OC4 strain are introduced into one host. It includes a method for producing an alcohol or alkanediol, wherein a monool or diol is obtained by hydroxylating a raw material alkane or alcohol in the presence. A transformant obtained by introducing CYP153-alkane-1-monooxygenase, ferredoxin gene and ferredoxin reductase gene into one host is the aforementioned CYP153-alkane-1-monooxygenase-related gene of the present invention, Acinetobacter sp. ) The ferredoxin gene of OC4 strain (261 to 548 of SEQ ID NOs: 2 and 15) and the ferredoxin reductase gene (1995 to 3188 of SEQ ID NOs: 2 and 16) are commonly used in hosts such as Escherichia coli, Bacillus subtilis, actinomycetes, etc. It can be obtained by introducing by law.

  More specifically, the transformant can be obtained as follows. For example, the electroporation method can be performed as follows. The plasmid into which the foreign gene has been inserted is added to a suspension of competent cells, this suspension is placed in a cuvette dedicated to the electroporation method, and a high voltage electric pulse is applied to the cuvette. Thereafter, the cells are cultured in an SOC medium, and the transformant is isolated on a plate agar medium. Alkane or alcohol is hydroxylated in the presence of the resulting transformant.

  The alkane that can be hydroxylated by the transformant of the present invention is a linear alkane having 5 to 15 carbon atoms, preferably 6 to 10 carbon atoms, or can be a cycloalkane. For example, it can be a cycloalkane having 6 to 8 carbon atoms. Examples of linear alkanes include, for example, n-hexane (1-hexanol), n-heptane (1-heptanol), n-octane (1-octanol), n-nonane (1-nonanol), n-decane. (1-decanol) and the like. Examples of the cycloalkane include cyclohexane (cyclohexanol). In the parentheses are alcohols that are hydroxylated products. The alcohol that can be hydroxylated by the transformant of the present invention is 1-alkanol or 2-alkanol having 6 to 10 carbon atoms. Examples thereof include 1-heptanol (1,7-heptanediol) and 2-octanol (1,7-octanediol). In the parentheses, alkanediol is a hydroxylated product.

Conditions for hydroxylating alkane or alcohol in the presence of the transformant are as follows. First, the CYP153-alkane-1-monooxygenase gene in the transformant, and the ferredoxin gene and ferredoxin reductase gene of Acinetobacter sp. OC4 strain are expressed in the cells. The bacterial cells expressing the enzyme are reacted with alkane or alcohol as a substrate. The reaction temperature with alkane can be appropriately determined in consideration of the optimum temperature of the transformant. In addition, the reaction time with the alkane or alcohol can also be appropriately determined in consideration of the conversion rate of the alkane or alcohol to the monool or diol (the progress of the reaction). Furthermore, the reaction with the alkane or alcohol can be carried out either batchwise or continuously. For the reaction with alkane or alcohol, it is preferable to further add Fe necessary for alkane or alcohol hydroxylation activity. Fe can be supplied, for example, by adding a compound such as iron sulfate heptahydrate (FeSO ₄ .7H ₂ O) to the reaction system.

  Isolation and purification of the produced alcohol (monool or diol) can be performed, for example, by solvent extraction and distillation. However, there is no intention to be limited to the solvent extraction method and the distillation method. The solvent extraction method and the distillation method can be performed, for example, as follows. An organic solvent such as ethyl acetate or alkane is added to the reaction solution and stirred to extract the alcohol. At this time, it is preferable to add salts (such as sodium chloride) or acids (such as hydrochloric acid) to the reaction solution to increase the ionic strength of the reaction solution, because the extraction rate of alcohol into an organic solvent is improved. Thereafter, the target alcohol can be purified by distillation using the difference between the boiling points of the solvent used and the alcohol.

[Example]
Hereinafter, the present invention will be described in more detail with reference to examples.
Example 1
Cloning of CYP153 alkane CYP153-alkane-1-monooxygenase gene of Gordonia sp. TF6 strain Gordonia sp. TF6 strain (FERM P-19376) at 28 ° C using L-broth After culturing for 3 days, chromosomal DNA was prepared using ISOPLANT (NIPPON GENE). Next, primer Aox-F1 and primer Aox-R1 were prepared (Table 1). Using these and LA Taq (TaKaRa), PCR reaction was performed using genome DNA of TF6 strain as a template. The reaction conditions were 25 cycles of 98 ° C for 20 seconds, 55 ° C for 30 seconds, and 68 ° C for 30 seconds. As a result, a DNA fragment of about 300 bp was amplified. As a result of sequencing, the protein encoded by the gene present here was homologous to CYP153-alkane-1-monooxygenase. Therefore, Inverse PCR was performed to obtain and sequence the peripheral region of this gene fragment (Ochman, H., Gerber, AS, and Hartl, DL Genetic applications of an inverse polymerase chain reaction. Genetics, 120, 621-623 (1988). )). The chromosomal DNA of TF6 strain was digested with restriction enzyme Mlu I, and self-ligation reaction was performed using T4DNA ligase. Using this as a template DNA, PCR was performed using primer Aox-F2 and primer Aox-R2 (Table 1) prepared from the base sequence of a DNA fragment of about 300 bp. The reaction conditions were 98 ° C 20 seconds, 68 ° C 5 minutes 30 cycles. These PCR products were ligated to pT7BlueT-vector (Novagen) for DNA sequencing.

  In order to further sequence the surrounding region of about 2.5 Kbp of DNA revealed here, Inverse PCR was performed again. PCR was performed using the primer Aox-F3 and the primer Aox-R3 (Table 1), using the TF6 strain chromosomal DNA digested with the restriction enzyme BamHI and subjected to self-ligation as the template DNA. The reaction conditions were 98 ° C for 20 seconds and 68 ° C for 30 minutes for 30 minutes. These PCR products were ligated to pT7 BlueT-vector for DNA sequencing.

  The DNA sequence 3485 bp revealed here is shown in SEQ ID NO: 2 in the sequence listing. On this DNA fragment, there was a CYP153-alkane-1-monooxygenase gene aoxA in which the encoded protein showed 66% homology to CYP153-alkane-1-monooxygenase of Alcanivorax borkumensis.

Example 2
Expression of the gene encoding CYP153-alkane-1-monooxygenase of Gordonia sp. TF6 strain and 1? Bacterial transformation of alkanol Gordonia sp. Encodes ferredoxin and ferredoxin reductase of Acinetobacter sp. OC4 strain (NITE P-125), together with gene (aoxA) encoding CYP153-alkane-1-monooxygenase of Gordonia sp. TF6 strain A plasmid for expressing the gene to be expressed in E. coli was constructed.

  First, a plasmid carrying a gene (ACCESSION No. AB221118) encoding CYP153-alkane-1-monooxygenase, ferredoxin and ferredoxin reductase of the Acinetobacter sp. OC4 strain already reported was prepared. Primer N-NcoF and Primer N-HindR were prepared (Table 1). Using these and LA Taq (TaKaRa), PCR was performed using the genome DNA of Acinetobacter sp. OC4 strain as a template. The reaction conditions were 98 ° C. for 20 seconds and 68 ° C. for 30 minutes for 30 cycles. As a result, a DNA fragment of about 3200 bp was amplified and digested with restriction enzymes NcoI and HindIII. This was linked to the NcoI-HindIII site of pTrcHisA (Invitrogen) as pAfrNoxA.

  Using the plasmid prepared here as a template, the gene encoding ferredoxin and ferredoxin reductase of Acinetobacter sp. OC4 strain was used as an electron transport system to prepare an expression plasmid for aoxA expression in E. coli. Primers Aox-F4 and Aox-R4 were prepared (Table 1), and using these and LA Taq (TaKaRa), PCR was performed using the TF6 strain genomic DNA as a template. The reaction conditions were 98 ° C. for 20 seconds and 68 ° C. for 30 minutes for 30 cycles. As a result, a DNA fragment of about 1368 bp was amplified and digested with restriction enzymes XbaI and HpaI (Fragment A). On the other hand, Primer Aox-F5 and Primer Aox-R5 were prepared (Table 1), and these and LA Taq (TaKaRa) were used, and PCR reaction was performed using pAfrNoxA as a template. The reaction conditions were 98 ° C for 20 seconds and 68 ° C for 7 minutes for 23 cycles. As a result, a DNA fragment of about 5 Kbp was amplified, blunted using BLK Kit (TaKaRa) (Fragment B), and further digested with restriction enzyme XbaI (Fragment C). PAfrAoxA was obtained by concatenating Fragment A and Fragment C to each other. Fragment B was self-ligated to construct plasmid pBlue that expresses only the genes encoding ferredoxin and ferredoxin reductase. The gene layout of pAfrAoxA is shown in Figure 1 together with the gene layout of pAfrNoxA. The base sequence of the pAfrAoxA gene is shown in SEQ ID NO: 2.

  In the gene for pAfrAoxA (SEQ ID NO: 2), the gene encoding ferredoxin is 261 to 548 (549 to 551 is a stop codon), and the gene encoding ferredoxin reductase is 1995 to 3188 (3189 to 3191) Is a stop codon). The gene encoding ferredoxin is shown in SEQ ID NO: 15, and the gene encoding ferredoxin reductase is shown in SEQ ID NO: 16.

Example 3
Gordonia sp. TF6 strain CYP153-alkane-1-monooxygenase-encoding gene (aoxA) and Acinetobacter sp. OC4 strain ferredoxin and ferredoxin reductase-encoding genes were expressed in E. coli. Microbial conversion of various alkanes.

pAfrAoxA and pBlue were introduced into E. coli TOP10 (Invitrogen), respectively. Each single colony of these strains was inoculated into 3 ml of L-medium containing ampicillin sodium (150 μg / ml) and cultured at 28 ° C. for 24 hours, and then the cells were collected and 0.2 M phosphate buffer ( A suspension obtained by adding 1.5 ml of pH 7.0) was used as a cell suspension. To 1.5 ml of these cell suspensions, 15 μl of 20% glycerol solution, 1.5 μl of 100 mg / ml FeSO ₄ .7H ₂ O, and 500 μl of various alkanes were added, and cultured with shaking in a centrifuge tube at 28 ° C. for 19 hours. 300 μl of 2N HCl, a small amount of NaCl, and 500 μl of various alkanes were added, shaken at 28 ° C. for 10 minutes, and then centrifuged at 28 ° C. and 3,500 rpm, and the supernatant was analyzed by gas chromatography-massspectrometry (GC-MS). The apparatus used was a Turbomass Gold GC-MS apparatus manufactured by Perkin Elmer, and the operating conditions were as follows. The reaction products (alcohols) were identified from their coincidence by comparing their retention times and mass spectra with commercial standards.

Column: TC-WAX (GL Sciences capillary column, inner diameter 0.25 mm, length 30 m, film thickness 0.25 μm)
Carrier gas: Helium (20 ml / min, 19: 1 split)
Column temperature: 120 ° C to 160 ° C at 5 ° C / min, 180 ° C at 10 ° C / min, 7 min at 180 ° C, 230 ° C at 10 ° C / min, 3 min at 230 ° C Injector temperature: 260 ° C
Detector temperature: 250 ℃
Sample injection volume: 1 μl

  The quantification of the reaction product was carried out using a GC-17A GC apparatus manufactured by Shimadzu under the same conditions as GC-MS (a detector using a hydrogen flame ionization detector). The results are shown in Table 2. The E. coli TOP10 pAfrAoxA strain converted a straight chain alkane having 5 to 15 carbon atoms and a cycloalkane having 5 to 8 carbon atoms into the corresponding alcohol. On the other hand, not all alkanols were detected in the microbial conversion reaction using the negative control E. coli TOP10pBlue strain.

The amount of product in the alkane phase was measured by gas chromatography analysis.

Example 4
Production of 1,7-heptanediol by hydroxylation of 2-heptanol Gordonia sp. Acinetobacter sp. With gene encoding a CYP153-alkane-1-monooxygenase (aoxA) of TF6 strain Genes encoding ferredoxin and ferredoxin reductase of OC4 strain were expressed in E. coli, and microbial conversion reaction of 1-heptanol was attempted using this strain. pAfrAoxA and pBlue were introduced into E. coli JM109 (TaKaRa), respectively. Each single colony of these strains was inoculated into 3 ml of L-medium containing ampicillin sodium (150 μg / ml) and cultured at 28 ° C. for 24 hours, and then the cells were collected and 0.2 M phosphate buffer (pH 7 .0) A suspension obtained by adding 1.5 ml was used as a cell suspension.

To 1.5 ml of the cell suspension, 1.5 μl of 100 mg / ml FeSO ₄ .7H ₂ O was added, and 1.5 μl of 1-heptanol was further added, followed by shaking culture at 28 ° C. for 24 hours. After adding 300 μl of 2N HCl, a small amount of sodium chloride, and 1 ml of ethyl acetate, and stirring, the supernatant centrifuged at 28 ° C. and 3,500 rpm was analyzed by GC-MS. The apparatus used was a Turbomass Gold GC-MS apparatus manufactured by Perkin Elmer, and the operating conditions were as follows. The reaction products (alkanediols) were identified by comparing their retention times and mass spectra with commercial standards and their agreement.

Column: TC-WAX (GL Sciences capillary column, inner diameter 0.25 mm, length 30 m, film thickness 0.25 μm)
Carrier gas: Helium (20 ml / min, 19: 1 split)
Column temperature: 120 ° C to 160 ° C at 5 ° C / min, 180 ° C at 10 ° C / min, 7 min at 180 ° C, 230 ° C at 10 ° C / min, 10 min at 230 ° C Injector temperature: 260 ° C
Detector temperature: 250 ℃
Sample injection volume: 1 μl

  The quantification of the reaction product was carried out using a GC-17A GC apparatus manufactured by Shimadzu under the same conditions as GC-MS (a detector using a hydrogen flame ionization detector). In E.coli JM109 pAfrNoxA strain, 2.5 μg / ml 1,7 in the extract? Is heptanediol 4.9 μg / ml 1,7? In E. coli JM109 pAfrAoxA strain? Heptanediol was obtained. 1,7 for E.coli JM109 pBlue stock? Heptanediol was not obtained. The results of GC-MS analysis of the converted product using E. coli JM109 pAfrAoxA strain are shown in FIG.

Example 5
Production of 1,7-octanediol by hydroxylation of 2-octanol Gordonia sp. Ferredoxin of Acinetobacter sp. OC4 strain with gene encoding CYP153-alkane-1-monooxygenase of TF6 strain (aoxA) And a gene encoding ferredoxin reductase were expressed in E. coli, and microbial conversion of 2-octanol was attempted using this strain. pAfrAoxA and pBlue were introduced into E. coli TOP10 (Invitrogen), respectively. Each single colony of these strains was inoculated into 3 ml of L-medium containing ampicillin sodium (150 μg / ml), cultured at 28 ° C. for 24 hours, and further added to 50 ml of L-medium containing ampicillin sodium (150 μg / ml). Inoculated and cultured at 28 ° C. for 24 hours. The cells were collected and suspended by adding 5 ml of 0.2 M phosphate buffer (pH 7.0) to prepare a cell suspension.

Add 1.5 μl of 100 mg / ml FeSO ₄ · 7H ₂ O and 15 μl of 20% glycerol to 1.5 ml of the cell suspension, and then add 5 μl of 2-octanol diluted 10-fold with DMSO, and culture with shaking at 28 ° C. for 24 hours. did. After adding 300 μl of 2N HCl, a small amount of sodium chloride, and 1 ml of ethyl acetate, and stirring, the supernatant centrifuged at 28 ° C. and 3,500 rpm was analyzed by GC-MS. The apparatus used was a Turbomass Gold GC-MS apparatus manufactured by Perkin Elmer, and the operating conditions were as follows. The reaction products (alkanediols) were identified from their coincidence by comparing their retention time and mass spectrum with the NIST Mass Spectral Database.

Column: TC-WAX (GL Sciences capillary column, inner diameter 0.25 mm, length 30 m, film thickness 0.25 μm)
Carrier gas: Helium (20 ml / min, 19: 1 split)
Column temperature: 120 ° C to 160 ° C at 5 ° C / min, 180 ° C at 10 ° C / min, 7 min at 180 ° C, 230 ° C at 10 ° C / min, 10 min at 230 ° C Injector temperature: 260 ° C
Detector temperature: 250 ℃
Sample injection volume: 1 μl

  The reaction product was quantified using a Shimadzu GC-17A GC instrument under the same conditions as GC-MS. Calculation was performed from a calibration curve of octanediol (detection using a flame ionization detector). In E.coli TOP10 pAfrAoxA strain, 157 μg / ml 1,7 in the extract? Octanediol was obtained. E.coli TOP10 pBlue stock is 1,7? Octanediol was not obtained. The results of GC-MS analysis of the converted product using E. coli TOP10 pAfrAoxA strain are shown in FIG.

  The present invention is useful in the field of producing alkanediols.

The gene arrangement | positioning figure of pAfrAoxA is shown. The result of the GC-MS analysis of the transformant in Example 4 using the E. coli JM109 pAfrAoxA strain is shown. The result of the GC-MS analysis of the transformant in Example 5 using the E. coli TOP10 pAfrAoxA strain is shown.

Claims (14)

A gene encoding a protein having any of the following amino acid sequences.
(1) an amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing and having CYP153-alkane-1-monooxygenase activity;
(2) An amino acid having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids and having CYP153-alkane-1-monooxygenase activity in the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing Or (3) an amino acid sequence having an amino acid sequence having 80% or more homology with the amino acid sequence described in SEQ ID NO: 1 in the sequence listing and having CYP153-alkane-1-monooxygenase activity A gene having any of the following base sequences:
(1) a nucleotide sequence having the nucleotide sequence of 646 to 1995 in SEQ ID NO: 2 in the sequence listing and encoding CYP153-alkane-1-monooxygenase;
(2) It has a base sequence having deletion, substitution and / or addition of one to several bases in the base sequence of Nos. 646 to 1995 shown in SEQ ID No. 2 of the sequence listing, and CYP153-alkane-1- A base sequence encoding a protein having monooxygenase activity; or (3) a DNA comprising a base sequence complementary to a DNA comprising a base sequence from 646 to 1995 in SEQ ID NO: 2 in the sequence listing and a stringent Sequence encoding a protein having a base sequence that hybridizes under mild conditions and having alkane-1-monooxygenase activity The gene according to claim 1 or 2, which is a gene derived from a microorganism belonging to the genus Gordonia. A recombinant vector comprising the gene according to any one of claims 1 to 3. The recombinant vector according to claim 4, further comprising a gene encoding ferredoxin of Acinetobacter sp. OC4 strain and a gene encoding ferredoxin reductase. A transformant comprising the gene according to any one of claims 1 to 3 or the recombinant vector according to claim 4 or 5. The transformant according to claim 6, wherein the host is Escherichia coli. A method for producing alcohol, comprising hydroxylating a raw material alkane or alcohol to obtain a monool or diol in the presence of the transformant according to claim 6 or 7. A transformant comprising a recombinant vector comprising the gene according to any one of claims 1 to 3, a gene encoding ferredoxin of Acinetobacter sp. OC4 strain and a gene encoding ferredoxin reductase Item 9. The manufacturing method according to Item 8. The production method according to claim 8 or 9, wherein the raw alcohol is monool and the alcohol produced is a diol. The production method according to claim 8 or 9, wherein the raw alcohol is 1-alkanol or 2-alkanol having 6 to 10 carbon atoms. The raw material alcohol is 2-octanol and 1,7-octanediol is obtained as the diol,
Alternatively, the raw material alcohol is 1-heptanol, and 1,7-heptanediol is obtained as a diol. A protein having any of the following amino acid sequences.
(1) an amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing and having CYP153-alkane-1-monooxygenase activity;
(2) An amino acid having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids and having CYP153-alkane-1-monooxygenase activity in the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing Or (3) an amino acid sequence having an amino acid sequence having 80% or more homology with the amino acid sequence described in SEQ ID NO: 1 in the sequence listing and having CYP153-alkane-1-monooxygenase activity A method for producing a protein according to claim 13, comprising culturing a transformant comprising the recombinant vector according to claim 4 and collecting the protein according to claim 13 from the culture.