JP4331542B2 - Proteins related to alkane hydroxylation and genes encoding them - Google Patents

Description

  The present invention relates to alkane-1-monooxygenase, rubredoxin reductase and two types of rubredoxin genes derived from microorganisms capable of hydroxylating alkanes, proteins encoded by these genes, and uses thereof, particularly these genes. The present invention relates to a method for producing alcohol using a microorganism transformed with the above.

  Alkanes are the most abundant hydrocarbons in crude oil and are also produced by many plants and algae (Non-Patent Document 1). Metabolism of linear alkanes by bacteria is carried out by oxidation of the terminal, and is metabolized to alcohol, aldehyde, and fatty acid (Non-patent Document 1). Many bacteria are known to have the ability to metabolize linear alkanes, but molecular biological analyzes of alkane metabolism mechanisms have not been comparative.

  The most well-analyzed mechanism of linear alkane metabolism is that of Pseudomonas putida GPo1 (Non-patent Document 2). In this case, the initial oxidation is performed by an alkane hydroxylation mechanism composed of three components. Its constituents are the non-heme membrane protein alkane-1-monooxygenase (AlkB) and two soluble proteins involved in electron transfer between it and NADH, rubredoxin (AlkG) and rubredoxin reductase (AlkT). Regarding Gram-positive bacteria, the alkane hydroxylation mechanism of Rhodococcus genus bacteria Q15 and NRRL B-16531 has been studied. Both strains have at least four alkane-1-monooxygenase gene homologs (alkB1, alkB2, alkB3, alkB4) (Non-patent Document 3). Among them, alkB1 and alkB2 homologs are present in the alkane hydroxylation gene cluster, each containing two rubredoxin genes (rubA1 and rubA2 and rubA3 and rubA4), and in the alkB1 cluster, there is a rubredoxin reductase gene (rubB). (Non-Patent Document 3).

  Several alkane hydroxylation machinery genes are expressed in heterologous hosts. Pseudomonas putida An approximately 35-kbp DNA region containing the alkane hydroxylation gene of GPo1 has been isolated, and E. coli and Pseudomonas genus bacteria lacking the ability to metabolize alkane are directly It has been shown by metabolic growth tests that it can metabolize chain alkanes (Non-patent Document 2).

  Furthermore, using this system, Rhodococcus genus bacteria (Non-patent document 3), Alcanivorax borkumensis AP1 (Non-patent document 4), Prauserella rugosa NRRL B-2295 (Non-patent document 4), Expression of alkane-1-monooxygenase genes such as Mycobacterium tuberculosis H37Rv (Non-patent Document 4) has been confirmed.

  Almost all alkane hydroxylation activities in the previous studies are not measured by detection of the reaction product, but by metabolic growth tests of bacterial cells. That is, alkane hydroxylation activity is only measured indirectly. The only reaction product due to alkane hydroxylation activity was detected using a Pseudomonas putida PpS81 deficient in alcohol dehydrogenase introduced with a DNA region of about 29-kbp containing the alkane hydroxylation mechanism gene. (Non-Patent Documents 5 and 6). However, these alkane hydroxylation mechanism gene expression experiments have only studied the nature of the gene product encoded by a long DNA region containing many genes, and have not been clearly analyzed for alkane hydroxylation mechanism genes. Absent.

  An alkane hydroxylation activity test in a test tube is performed by a reconstitution system using AlkB and AlkG expressed and purified by Escherichia coli, and ferredoxin (derived from spinach) (Non-patent Document 6). However, the alkane hydroxylation activity here is measured by an indirect method of measuring the co-oxidation of NADH, and the reaction is considered to be strongly limited by the poor solubility of the substrate and the enzyme.

  On the other hand, industrial production of chemicals and pharmaceuticals using the bacterial alkane hydroxylation mechanism has attracted much attention. Actually, cumene (Non-patent Document 7), 2,5-dimethylhexane (Non-patent Document 8), N- (2-hexylamino-4-phenylimidazol-1-yl) using bacteria having alkane hydroxylating ability -Microbial conversion of hydrocarbons such as acetamide (Non-patent Document 9) has been reported. However, few molecular biological analyzes of these microbial transformations have been made.

Rehm, H. J., and Reiff, I., Mechanisms and occurrence of microbial oxidation of long chain alkanes.Adv. Biochem. Eng., 19, 175-215 (1981). Eggink, G., Lageveen, RG, Altenburg, B., Witholt, B., Controlled and functional expression of the Pseudomonas oleovorans alkane utilizing system in Pseudomonas putida and Escherichia coli.J. Biol. Chem., 262, 17712-17718 (1987). Whyte, LG, Smits, THM, Labbe, D., Witholt, B., Geer, CW, and van Beilen, JB, Gene cloning and characterization of multiple alkane hydroxylase systems in Rhodococcus strains Q15 and NRRL B-16531. Appl. Environ Microbiol., 68, 5933-5942 (2002). Smits, T. H. M., Balada, S. B., Witholt, B., and van Beilen, J. B., Functional analysis of alkane hydroxylase from gram-negative and gram-positive bacteria. J. Bacterial. 184, 1733-1742 (2002). Bosetti, A., van Beilen, JB, Preusting, H., Lageveen, RG, and Witholt, B., Production of primary aliphatic alcohols with a recombinant Pseudomonas strains.Enzyme Microb. Technol., 14, 702-708 (1992) . van Beilen, J. B., Kingma, J., and Witholt, B., Substrate specificity of the alkane hydroxylase of Pseudomonas oleovorans GPo1. Enzyme Microb. Technol., 16, 904-911 (1994). Hou, C. T., Jackson, M., A., Bagby, M. O., and Takagi, M., Microbial oxidation of cumene by octane-grown cells.Appl.Microbiol. Biotechnol., 41, (1994). Matsui, T., and Furuhashi, K. Asymmetric oxidation of isopropyl moieties of aliphatic and aromatic hydrocarbons by Rhodococcus sp. 11B. Biosci. Biotech. Biochem., 59, (1995). Mikolasch, A., Hammer, E., and Schauer, F. Synthesis of imidazole-2-yl amino acids by using cells from alkane-oxidizing bacteria.Appl.Environ.Microbiol., 69, 1670-1679 (2003).

  The object of the present invention is to provide all the components of the alkane hydroxylation mechanism of Gordonia sp.TF6 strain, a microorganism that the present inventors have recently isolated and found to have alkane hydroxylation ability. Cloning the gene to be encoded, specifying the protein encoded by each gene, expressing the gene in a host such as E. coli without excess and deficiency, and constructing a microbial conversion system using the resulting recombinant It is to provide a method for hydroxylating various alkanes.

The present invention is as follows.
[Claim 1]
A gene encoding a protein having any of the following amino acid sequences.
(1) an amino acid sequence having the amino acid sequence of SEQ ID NO: 1 in the sequence listing and having alkane-1-monooxygenase activity;
(2) an amino acid sequence having an alkane-1-monooxygenase activity having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids 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 90 % or more homology to the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing and having alkane-1-monooxygenase activity [Claim 2]
A gene having any of the following base sequences:
(1) a base sequence having the base sequence set forth in SEQ ID NO: 2 in the sequence listing and encoding alkane-1-monooxygenase;
(2) a base sequence having a base sequence having deletion, substitution and / or addition of 1 to several bases in the base sequence described in SEQ ID NO: 2 of the sequence listing and encoding alkane-1-monooxygenase; Or (3) a base sequence that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence set forth in SEQ ID NO: 2 in the sequence listing, and alkane-1-mono A base sequence encoding oxygenase [Claim 3]
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: 3 in the sequence listing and having rubredoxin reductase activity;
(2) an amino acid sequence having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids in the amino acid sequence described in SEQ ID NO: 3 in the sequence listing and having rubredoxin reductase activity; or ( 3) an amino acid sequence having an amino acid sequence having 90 % or more homology to the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing and having rubredoxin reductase activity [Claim 4]
A gene having any of the following base sequences:
(1) a nucleotide sequence having the nucleotide sequence set forth in SEQ ID NO: 4 in the sequence listing and encoding rubredoxin reductase;
(2) a base sequence having a base sequence having deletion, substitution and / or addition of one to several bases in the base sequence set forth in SEQ ID NO: 4 of the sequence listing and encoding rubredoxin reductase; or ( 3) It has a base sequence that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 4 in the sequence listing, and encodes rubredoxin reductase Base sequence [Claim 5]
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: 5 in the sequence listing and having a function of rubredoxin;
(2) an amino acid sequence having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids in the amino acid sequence set forth in SEQ ID NO: 5 in the sequence listing and having a function of rubredoxin; or (3) An amino acid sequence having an amino acid sequence having 90 % or more homology to the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing and having a function of rubredoxin [Claim 6 ]
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: 7 in the sequence listing and having a function of rubredoxin;
(2) an amino acid sequence having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids in the amino acid sequence set forth in SEQ ID NO: 7 in the sequence listing and having a function of rubredoxin; or
(3) An amino acid sequence having an amino acid sequence having a homology of 90% or more with respect to the amino acid sequence described in SEQ ID NO: 7 in the sequence listing and having a function of rubredoxin [Claim 7 ]
A gene having any of the following base sequences:
(1) a nucleotide sequence having the nucleotide sequence set forth in SEQ ID NO: 6 in the sequence listing and encoding rubredoxin;
(2) a nucleotide sequence having a nucleotide sequence having deletion, substitution and / or addition of one to several nucleotides in the nucleotide sequence set forth in SEQ ID NO: 6 in the sequence listing and encoding rubredoxin; or (3) A base sequence that has a base sequence that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence set forth in SEQ ID NO: 6 in the sequence table and encodes rubredoxin [Claim 8]. ]
A gene having any of the following base sequences:
(1) a nucleotide sequence having the nucleotide sequence set forth in SEQ ID NO: 8 in the sequence listing and encoding rubredoxin;
(2) a nucleotide sequence having a nucleotide sequence having deletion, substitution and / or addition of 1 to several nucleotides in the nucleotide sequence set forth in SEQ ID NO: 8 of the sequence listing and encoding rubredoxin; or
(3) a base sequence that encodes rubredoxin, having a base sequence that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence set forth in SEQ ID NO: 8 in the Sequence Listing [Claim 9]
The gene according to any one of claims 1 to 8 , which is a gene derived from a microorganism belonging to the genus Gordonia.
[Claim 10 ]
A protein having any of the following amino acid sequences.
(1) an amino acid sequence having the amino acid sequence of SEQ ID NO: 1 in the sequence listing and having alkane-1-monooxygenase activity;
(2) an amino acid sequence having an alkane-1-monooxygenase activity having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids 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 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing and having alkane-1-monooxygenase activity [Claim 11 ]
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: 3 in the sequence listing and having rubredoxin reductase activity;
(2) an amino acid sequence having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids and having rubredoxin reductase activity in the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing; or ( 3) An amino acid sequence having an amino acid sequence having 90 % or more homology with the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing and having rubredoxin reductase activity [Claim 12 ]
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: 5 in the sequence listing and having a function of rubredoxin;
(2) an amino acid sequence having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids in the amino acid sequence set forth in SEQ ID NO: 5 in the sequence listing and having a function of rubredoxin; or (3) An amino acid sequence having an amino acid sequence having 90 % or more homology with the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing and having a function of rubredoxin [Claim 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: 7 in the sequence listing and having a function of rubredoxin;
(2) an amino acid sequence having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids in the amino acid sequence set forth in SEQ ID NO: 7 in the sequence listing and having a function of rubredoxin; or
(3) An amino acid sequence having an amino acid sequence having a homology of 90% or more with respect to the amino acid sequence described in SEQ ID NO: 7 in the sequence listing and having a function of rubredoxin [Claim 14 ]
A recombinant vector comprising the gene according to any one of claims 1 to 9 .
[Claim 15 ]
A transformant comprising the gene according to any one of claims 1 to 9 or the recombinant vector according to claim 14 .
[Claim 16 ]
A transformant comprising the gene according to claim 1 or 2, the gene according to claim 3 or 4, the gene according to claim 5 or 6, and the gene according to claim 7 or 8 .
[Claim 17 ]
The transformant according to claim 15 or 16 , wherein the host is Escherichia coli.
[Claim 18 ]
Claim 16 is hydroxide linear alkanes or cycloalkanes having a carbon number of 6-8 5-13 carbon atoms in the presence of the transformant according to including Rukoto give alcohol, method for producing the alcohol.
[Claim 19 ]
Culturing the transformant according to claim 15, comprising collecting the protein of any one of claims 10 to 13 from the culture, according to any one of claims 10 to 13 A method for producing a protein.

  By using a microbial conversion system using the recombinant of the present invention, it becomes easy to analyze the alkane hydroxylation mechanism of bacteria, which has been difficult to analyze until now, and enzyme modification and the like are also possible.

  Hereinafter, embodiments of the present invention will be described in detail.

Gene of the present invention The present inventors cloned a gene encoding all components of the alkane hydroxylation mechanism of Gordonia sp. TF6 strain, a newly isolated microorganism. First, acquisition and mycological properties of Gordonia sp. TF6 strain will be described.

  Gordonia sp. TF6 strain is a newly isolated microorganism from soil of tennis courts in Fujisawa City, Kanagawa Prefecture, using n-decane assimilation as an index. Various n-alkanes (carbon number 5, 8, 10, 12, 14, 16). This strain was deposited as Gordonia sp. TF6 on May 29, 2003 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (FERM P-19376). For isolation of this strain and assimilation test of various n-alkanes, 2 ml of liquid medium (in a nero test tube) using 1% n-alkane as the sole carbon source was used. The culture conditions were 28 ° C. and shaking at 220 rpm. The medium composition other than the carbon source is shown below.

(Medium composition)
0.1% (NH ₄ ) ₂ SO ₄
0.01% MgSO ₄・ 7H ₂ O
0.05% KH ₂ PO ₄
0.1% K ₂ HPO ₄
0.5% NaCl
0.1% trace mineral solution (composition of trace mineral solution)
0.01% ZnSO ₄・ 7H ₂ O
0.03% H ₃ BO ₃
0.02% CaCl ₂・ 6H ₂ O
0.2% FeSO ₄・ 7H ₂ O
0.001% CuCl ₂・ 2H ₂ O
0.003% Na ₂ MoO ₄・ 2H ₂ O
0.002% NiCl ₂・ 6H ₂ O
0.003% MnCl ₂・ 4H ₂ O
Since alkane-utilizing microorganisms generally have a gene group of the alkane hydroxylation mechanism involved in the assimilation, the gene group was also cloned in this strain.

1. Alkane-1-monooxygenase-related gene The present inventors selected alkane-1-mono from genes encoding all components of the alkane hydroxylation mechanism of the cloned Gordonia sp. TF6 strain. Oxygenase and the gene encoding it were obtained.

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 of SEQ ID NO: 1 in the sequence listing and having alkane-1-monooxygenase activity;
(2) an amino acid sequence having an alkane-1-monooxygenase activity having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids 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 to the amino acid sequence described in SEQ ID NO: 1 in the sequence listing and having alkane-1-monooxygenase activity Specific examples of the gene of the present invention As such, a gene having any of the following base sequences can be mentioned.
(1) a base sequence having the base sequence set forth in SEQ ID NO: 2 in the sequence listing and encoding alkane-1-monooxygenase;
(2) a base sequence having a base sequence having deletion, substitution and / or addition of 1 to several bases in the base sequence described in SEQ ID NO: 2 of the sequence listing and encoding alkane-1-monooxygenase; Or (3) a base sequence that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence set forth in SEQ ID NO: 2 in the sequence listing, and alkane-1-mono Base sequence encoding oxygenase In the present invention, “alkane-1-monooxygenase” is an enzyme that oxidizes the end of a linear alkane, and “alkane-1-monooxygenase activity” refers to the end of a linear alkane. It means the enzyme activity that oxidizes.

  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 alkane-1-monooxygenase-related genes is not particularly limited. 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 sequence listing” in the explanation of alkane-1-monooxygenase-related genes is particularly limited as long as it is 80% or more. However, it is preferably 85% or more, more preferably 90% or more, still more preferably 95%, particularly preferably 98% or more.
The range of “1 to several” in the “base sequence having deletion, substitution and / or addition of one to several bases” in the description of alkane-1-monooxygenase-related genes is not particularly limited. 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 alkane-1-monooxygenase-related genes, “hybridizes under stringent conditions” means using DNA as a probe, colony hybridization method, plaque hybridization method, Southern blot hybridization method, etc. that means the nucleotide sequence of the obtained DNA by the use.

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 , 90 % or more, more preferably 93% or more, and particularly preferably 95%. %, Most preferably 98% or more homologous DNA.

  The method for obtaining the 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 they are used to belong to the genus Gordonia. The 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 alkane-1-monooxygenase-related gene of the present invention.

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

Preparation of the probes or primers, construction of a DNA library, screening of DNA libraries, as well as operations such as cloning the gene of interest are known to those skilled in the art, for example, Molecular Cloning: A laboratory Mannual, 2 nd Ed,. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989 (hereinafter abbreviated as Molecular Cloning 2nd Edition), Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997) Protocols in Molecular Biology) and the like.

  Moreover, it has an amino acid sequence having deletion, substitution and / or addition of one to several amino acids in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and encodes a protein having alkane-1-monooxygenase activity. A base sequence encoding a alkane-1-monooxygenase having a base sequence having a deletion, substitution and / or addition of one to several bases in the base sequence set forth in SEQ ID NO: 2 in the sequence listing Regarding the genes possessed (hereinafter referred to as “mutant genes”), chemical synthesis, genetic engineering techniques or mutations 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 It can be made by any method known to those skilled in the art, such as induction.

  For example, it can be carried out using a method of bringing a DNA having the base sequence shown in SEQ ID NO: 2 in the Sequence Listing into contact with a drug that becomes a mutagen, a method of irradiating with ultraviolet rays, a genetic engineering method, or the like. 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.

2. Genes associated with rubredoxin reductase The present inventors selected rubredoxin reductase and its gene from among genes encoding all components of the alkane hydroxylation mechanism of the cloned Gordonia sp. TF6 strain. The encoding gene was obtained.

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: 3 in the sequence listing and having rubredoxin reductase activity;
(2) an amino acid sequence having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids in the amino acid sequence described in SEQ ID NO: 3 in the sequence listing and having rubredoxin reductase activity; or ( 3) An amino acid sequence having an amino acid sequence having 50% or more homology with the amino acid sequence described in SEQ ID NO: 3 in the sequence listing and having rubredoxin reductase 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 set forth in SEQ ID NO: 4 in the sequence listing and encoding rubredoxin reductase;
(2) a base sequence having a base sequence having deletion, substitution and / or addition of one to several bases in the base sequence set forth in SEQ ID NO: 4 of the sequence listing and encoding rubredoxin reductase; or ( 3) It has a base sequence that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 4 in the sequence listing, and encodes rubredoxin reductase Base sequence

  In the present invention, “rubredoxin reductase” is an enzyme that reduces rubredoxin, and “rubredoxin reductase activity” means an enzyme activity that reduces rubredoxin.

  The range of “1 to several” in the “amino acid sequence having 1 to several amino acid deletions, substitutions and / or additions” in the description of the rubredoxin reductase-related gene is not particularly limited. It means 20, preferably 1 to 10, more preferably 1 to 7, further preferably 1 to 5, and particularly preferably about 1 to 3.

  The homology in the “amino acid sequence having 50% or more homology to the amino acid sequence described in SEQ ID NO: 3 in the Sequence Listing” in the description of the gene related to rubredoxin reductase is not particularly limited as long as it is 50% or more. , Preferably 70% or more, more preferably 80% or more, particularly preferably 90% or more.

  The range of “1 to several” in the “base sequence having deletion, substitution and / or addition of 1 to several bases” in the description of the gene related to rubredoxin reductase is not particularly limited. It means 60, preferably 1 to 30, more preferably 1 to 20, still more preferably 1 to 10, particularly preferably about 1 to 5.

“Hybridize under stringent conditions” in the description of rubredoxin reductase-related genes means using DNA as a probe and using colony hybridization, plaque hybridization, Southern blot hybridization, etc. that means the nucleotide sequence of the resulting DNA by.

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 , 90 % or more, more preferably 93% or more, and particularly preferably 95%. %, Most preferably 98% or more homologous DNA.

  The method for obtaining the rubredoxin reductase-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: 3 in the sequence listing and the nucleotide sequence set forth in SEQ ID NO: 4 and are used to belong to the genus Gordonia. The rubredoxin reductase-related gene of the present invention can be isolated by screening a microbial DNA library. The DNA library can be prepared by a conventional method from a microorganism expressing the rubredoxin reductase-related gene of the present invention.

  The rubredoxin reductase-related gene of the present invention can also be obtained by PCR. Using the above-described DNA library derived from a microorganism as a template, PCR is carried out using a pair of primers designed so that the base sequence described in SEQ ID NO: 4 can be amplified. PCR reaction conditions can be set as appropriate. For example, one cycle of a reaction step consisting of 94 ° C. for 30 seconds (denaturation), 55 ° C. for 30 seconds to 1 minute (annealing), and 72 ° C. for 2 minutes (extension) For example, after 30 cycles, a condition of reacting at 72 ° C. for 7 minutes can be exemplified. The amplified DNA fragment can then be cloned into a suitable vector that can be amplified in a host such as E. coli.

Preparation of the probes or primers, construction of a DNA library, screening of DNA libraries, as well as operations such as cloning the gene of interest are known to those skilled in the art, for example, Molecular Cloning: A laboratory Mannual, 2 nd Ed,. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989 (hereinafter abbreviated as Molecular Cloning 2nd Edition), Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997) Protocols in Molecular Biology) and the like.

  A gene encoding a protein having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids in the amino acid sequence set forth in SEQ ID NO: 3 of the Sequence Listing and having rubredoxin reductase activity; And a gene having a base sequence having a deletion, substitution and / or addition of 1 to several bases in the base sequence set forth in SEQ ID NO: 4 in the sequence listing and having a base sequence encoding rubredoxin reductase (hereinafter referred to as a gene sequence) These genes are referred to as mutant genes). Based on the amino acid sequence and nucleotide sequence information set forth in SEQ ID NO: 3, any gene known to those skilled in the art such as chemical synthesis, genetic engineering techniques or mutagenesis can be used. Can be produced by a method.

  For example, it can be carried out using a method of contacting a DNA having the base sequence described in SEQ ID NO: 4 of the sequence listing with a drug that becomes a mutagen, a method of irradiating ultraviolet rays, a genetic engineering method, or the like. 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.

3. Genes associated with rubredoxin The present inventors obtained rubredoxin and the gene encoding it from among the genes encoding all components of the alkane hydroxylation mechanism of the cloned Gordonia sp. TF6 strain. .

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: 5 or 7 in the sequence listing and having a function of rubredoxin;
(2) an amino acid sequence having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids in the amino acid sequence set forth in SEQ ID NO: 5 or 7 in the sequence listing and having a function of rubredoxin; or ( 3) An amino acid sequence having an amino acid sequence having a homology of 85% or more to the amino acid sequence described in SEQ ID NO: 5 or 7 in the sequence listing and having a function of rubredoxin

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 set forth in SEQ ID NO: 6 or 8 in the sequence listing and encoding rubredoxin;
(2) a base sequence having a base sequence having deletion, substitution and / or addition of one to several bases in the base sequence set forth in SEQ ID NO: 6 or 8 of the sequence listing and encoding rubredoxin; or (3 ) A nucleotide sequence having a nucleotide sequence that hybridizes under stringent conditions with a DNA comprising a nucleotide sequence complementary to the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 6 or 8 in the sequence listing, and encoding rubredoxin

  In the present invention, “rubredoxin” refers to a protein that transfers electrons from rubredoxin reductase to alkane-1-monooxygenase, and “function of rubredoxin” refers to electrons from rubredoxin reductase as alkane-1- It means the protein function that is transmitted to monooxygenase.

The range of “1 to several” in the “amino acid sequence having 1 to several amino acid deletions, substitutions and / or additions” in the description of rubredoxin-related genes is not particularly limited, but for example , 1 to 5, Particularly preferably, it means about 1 to 3.

  The homology in the “amino acid sequence having 85% or more homology to the amino acid sequence described in SEQ ID NO: 5 or 7 of the sequence listing” in the description of the rubredoxin-related gene is not particularly limited as long as it is 85% or more. More preferably, it is 90% or more, and particularly preferably 95% or more.

The range of “1 to several” in the “base sequence having deletion, substitution and / or addition of 1 to several bases” in the description of rubredoxin-related genes is not particularly limited, but for example , 1 to 10, Particularly preferably, it means about 1 to 5.

“Hybridize under stringent conditions” in the description of rubredoxin-related genes is obtained by using DNA as a probe and using a colony hybridization method, plaque hybridization method, Southern blot hybridization method, or the like. that means the base sequence of DNA.

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 , 90 % or more, more preferably 93% or more, and particularly preferably 95%. %, Most preferably 98% or more homologous DNA.

  The method for obtaining the rubredoxin-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: 5 or 7 and the nucleotide sequence information set forth in SEQ ID NO: 6 or 8 in the sequence listing in this specification, and Gordonia (Gordonia) is prepared using them. ) The rubredoxin-related gene of the present invention can be isolated by screening a DNA library of microorganisms belonging to the genus. A DNA library can be prepared by a conventional method from a microorganism expressing the rubredoxin-related gene of the present invention.

  The rubredoxin-related gene of the present invention can also be obtained by PCR. PCR is performed using a pair of primers designed to amplify the base sequence described in SEQ ID NO: 6 or 8, using the above-described DNA library derived from microorganisms as a template. PCR reaction conditions can be set as appropriate. For example, one cycle of a reaction step consisting of 94 ° C. for 30 seconds (denaturation), 55 ° C. for 30 seconds to 1 minute (annealing), and 72 ° C. for 2 minutes (extension) For example, after 30 cycles, a condition of reacting at 72 ° C. for 7 minutes can be exemplified. The amplified DNA fragment can then be cloned into a suitable vector that can be amplified in a host such as E. coli.

Preparation of the probes or primers, construction of a DNA library, screening of DNA libraries, as well as operations such as cloning the gene of interest are known to those skilled in the art, for example, Molecular Cloning: A laboratory Mannual, 2 nd Ed,. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989 (hereinafter abbreviated as Molecular Cloning 2nd Edition), Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997) Protocols in Molecular Biology) and the like.

  A gene encoding a protein having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids in the amino acid sequence set forth in SEQ ID NO: 5 or 7 in the sequence listing and having a function of rubredoxin; And a gene having a base sequence having a deletion, substitution and / or addition of 1 to several bases in the base sequence in SEQ ID NO: 6 or 8 in the sequence listing and having a base sequence encoding rubredoxin (hereinafter, these For the gene), any method known to those skilled in the art, such as chemical synthesis, genetic engineering techniques or mutagenesis, based on the amino acid sequence and nucleotide sequence information set forth in SEQ ID NO: 5 or 7 Can be produced.

  For example, it can be carried out using a method of contacting a DNA having the base sequence set forth in SEQ ID NO: 6 or 8 in the sequence listing with a mutagen agent, a method of irradiating ultraviolet light, a genetic engineering method, or the like. . 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.

Protein of the present invention The protein of the present invention is a protein having any of the following amino acid sequences.
1- (1) an amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing and having alkane-1-monooxygenase activity;
1- (2) Amino acid having an alkane-1-monooxygenase activity having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids in the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing Or 1- (3) amino acid sequence 2- (3) having an amino acid sequence having 80% or more homology to the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing and having alkane-1-monooxygenase activity 1) an amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing and having rubredoxin reductase activity;
2- (2) An amino acid sequence having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids in the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing and having rubredoxin reductase activity; Or 2- (3) amino acid sequence having an amino acid sequence having 50% or more homology to the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing and having rubredoxin reductase activity 3- (1) sequence listing An amino acid sequence having the function of rubredoxin, having the amino acid sequence set forth in SEQ ID NO: 5 or 7;
3- (2) an amino acid sequence having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids in the amino acid sequence set forth in SEQ ID NO: 5 or 7 in the sequence listing and having a function of rubredoxin; Or 3- (3) an amino acid sequence having an amino acid sequence having 85% or more homology to the amino acid sequence set forth in SEQ ID NO: 5 or 7 in the sequence listing and having a function of rubredoxin

  The protein of the present invention is a protein having alkane-1-monooxygenase activity, a protein having rubredoxin reductase activity, or a protein having the function of rubredoxin. In the presence of a protein having alkane-1-monooxygenase activity, the end of a linear alkane can be oxidized. In the presence of a protein having rubredoxin reductase activity, rubredoxin can be reduced. In the presence of a protein having the function of rubredoxin, electrons from rubredoxin reductase can be transferred to alkane-1-monooxygenase.

  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. By introducing this DNA into an appropriate expression system, the protein of the present invention can be produced. The expression of the protein in the expression system will be described later in this specification.

Recombinant vector of the present invention 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 alpha-amylase gene, and the Bacillus amylo gene. Bacillus amyloliquefaciens BAN amylase gene, Bacillus subtilis alkaline protease gene or Bacillus pumilus xylosidase gene promoter, or phage lambda P _R or P _L promoters, lac of E.coli, such as trp or tac promoter.

  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.

Production of transformant of the present invention and recombinant protein using the transformant A transformant can be produced by introducing the gene or recombinant vector of the present invention into a suitable 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.

  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, Spodoptera frugiperda ovarian cells (1992)], HighFive (manufactured by Invitrogen), which is an ovary cell of Trichoplusia ni, 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 the calcium phosphate method and the 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-Sepharose FF (Amersham Biosciences), butyl sepharose, phenyl sepharose, etc. Purified samples using methods such as hydrophobic chromatography using resins, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, and electrophoretic methods such as isoelectric focusing alone or in combination Can be obtained.

Method for Producing Alcohol The present invention uses a transformant in which all genes of alkane-1-monooxygenase, two rubredoxins and rubredoxin reductase are introduced into one host, and in the presence of this transformant, alkane is used. It includes a method for producing alcohol, which comprises hydroxylating.
The transformant in which all genes of alkane-1-monooxygenase, two rubredoxins and rubredoxin reductase were introduced into one host is the aforementioned alkane-1-monooxygenase-related gene of the present invention, two rubredoxin-related genes. And a rubredoxin reductase-related gene can be obtained by introducing the gene into a host such as Escherichia coli, Bacillus subtilis, actinomycetes or the like by a conventional method.

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.
The alkane 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 13 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 conditions for hydroxylating alkane in the presence of the transformant are as follows.

  First, the gene group of the alkane hydroxylation mechanism in the transformant is expressed in the microbial cells by an inducer such as IPTG. The bacterial cells expressing the enzyme are reacted with alkane as a substrate. The reaction temperature with alkane can be appropriately determined in consideration of the optimum temperature of the transformant. Also, the reaction time with the alkane can be appropriately determined in consideration of the conversion rate of the alkane to the alcohol (degree of reaction progress) and the like. Furthermore, the reaction with the alkane can be carried out either in batch mode or continuous mode. For the reaction with alkane, it is preferable to further add Fe necessary for alkane hydroxylation activity.

  Isolation and purification of the produced alcohol can be performed by, for example, a solvent extraction method and a distillation method. 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.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

[Example]
Hereinafter, the present invention will be described in more detail with reference to examples.
Example 1
Cloning of the gene encoding the alkane hydroxylation mechanism of Gordonia sp. TF6 strain Gordonia sp. TF6 strain (FERM P-19376) was cultured at 28 ° C for 3 days using L-broth Chromosomal DNA was prepared using ISOPLANT (NIPPON GENE). Next, Theo.HM et al. (Smits, THM, Rothlissberger, M., Witholt, B., and van Beilen, JB, Molecular screening for alkane hydroxylase genes in gram-negative and gram-positive strains.Environ. Microbiol. 1 , 307-317 (1999)), TS2S primer and Deg1RE primer were prepared (Table 2). Using these and LA Taq (TaKaRa), PCR reaction was performed using TF6 strain genomic DNA as a template. The reaction conditions were 25 cycles of 98 ° C 20 sec., 40 ° C 1 min., 68 ° C 1 min. As a result, a DNA fragment of about 550 bp was amplified. As a result of sequencing, the protein encoded by the gene present here was homologous to the alkane-1-monooxygenase encoded by alkB2. 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 enzymes SalI or BamHI, and self-ligation reaction was performed using T4 DNA ligase, respectively. Using this as a template DNA, PCR was performed using primers Inv1 primer and Inv2 primer (Table 2) prepared from the base sequence of a DNA fragment of about 550 bp. The reaction conditions were 98 ° C for 20 sec. And 68 ° C for 4 min. For 30 cycles. These PCR products were ligated to pT7 Blue T-vector (Novagen) for DNA sequencing.

  In order to further sequence the peripheral region of about 2 Kbp of DNA revealed here, Inverse PCR was performed again. PCR was performed using primers Inv3 primer and Inv4 primer (Table 2), using TF6 strain chromosomal DNA digested with restriction enzyme MluI and subjected to self-ligation as template DNA. The reaction conditions were 98 ° C 20 sec., 68 ° C 3 min., 30 cycles. These PCR products were ligated to pT7 Blue T-vector for DNA sequencing.

  The DNA sequence 3491 bp revealed here is shown in FIG. Alkane hydroxylation mechanism, alkB2, rubA3, rubA4, and rubB, showing high homology to Rhodococcus erythropolis NRRL B-16531 were present on this DNA fragment (Table 3). These genes are clustered and are thought to be transcribed by a promoter located upstream of alkB2.

Example 2
Expression of the gene encoding the alkane hydroxylation mechanism of Gordonia sp. TF6 strain and microbial conversion of n-octane. Codes the components of the alkane hydroxylation mechanism of the cloned Gordonia sp. TF6 strain. We tried to express alkB2, rubA3, rubA4 and rubB in E. coli. Expression plasmids pAL526, pAL520, pAL501, pAL502, pAL503, and pAL026 were constructed (Table 4), and these and pTrcHisA were introduced into E. coli TOP10 (Invitrogen). Each of these strains was cultured in L-medium containing ampicillin sodium (100 μg / ml) at 28 ° C for 17 hours, and 1 ml of this culture was added to 50 ml L-medium containing ampicillin sodium (100 μg / ml). Inoculated into a 250 ml Erlenmeyer flask. After culturing at 28 ° C. for 3 hours, isopropyl-β-D-thiogalactopyranoside (IPTG) (final concentration 0.1 mM) was added and further cultured for 5 hours. The bacterial cells were collected and suspended by adding 3 ml of 0.2M phosphate buffer (pH 7.0).

Cell suspension 1.5 ml of 10% glycerol solution 100 μl, 100 mg / ml FeSO 4 .7H 2 O 1.5μl, 100 mM IPTG 1.5μl, 50 mg / ml sodium ampicillin 1.5 [mu] l, and n- octane 500 [mu] l In addition, the cells were cultured with shaking in a centrifuge tube at 28 ° C. for 19 hours. After adding 300 μl of 2N HCl and 500 μl of n-octane and shaking for 10 minutes at 28 ° C., the supernatant centrifuged at 28 ° C. and 3,500 rpm was analyzed by gas chromatography-mass spectrometry (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 FIG. E. coli TOP10 pAL526 strain converted n-octane to 1-octanol. Transformation of E. coli TOP10 transformed with pAL526 as a plasmid using plasmid pAL520, plasmid pAL501, plasmid pAL502, plasmid pAL503, or plasmid pAL503, where n-octane is converted to 1-octanol as 100 The relative conversion ability of E. coli TOP10 is shown. The arrow in the figure indicates the alkane hydroxylase ORF. A bold line shows the DNA region which each plasmid has. As shown in Table 4, each x represents a point where Cys is changed to a stop codon.
When one of the rubredoxins was destroyed (pAL501, pAL502), the n-octane conversion activity was about 1/5 that of the case of having both (pAL526). Moreover, when both rubredoxins were destroyed (pAL503), the conversion activity was hardly detectable. On the other hand, when rubredoxin reductase was destroyed (pAL520), the conversion activity was hardly reduced. This is probably because the reductase of E. coli functions nonspecifically.

Example 3
Microbial transformation of various alkanes using Escherichia coli expressing alkane hydroxylation mechanism of Gordonia sp. TF6 strain.
A cell suspension expressing the alkane hydroxylation mechanism of Gordonia sp. TF6 strain was prepared in the same manner as in the previous section, and 100 μl of 10% glycerol solution, 100 mg / ml was added to 1.5 ml of this cell suspension. FeSO ₄ · 7H ₂ O 1.5 μl, 100 mM IPTG 1.5 μl, 50 mg / ml ampicillin sodium 1.5 μl, and various alkanes 500 μl were added, and cultured with shaking in a centrifuge tube at 28 ° C. for 19 hours. After adding 300 μl of 2N HCl and 500 μl of various alkanes, shaking at 28 ° C. for 10 minutes, the supernatant centrifuged at 28 ° C. and 3,500 rpm was analyzed by GC-MS and GC according to Example 2 described above. . The results are shown in Table 5. The E.coli TOP10 pAL526 strain was converted from linear alkanes having 5 to 13 carbon atoms, cyclohexane and cyclooctane to the corresponding alcohols. On the other hand, not all alkanols were detected in the microbial conversion reaction using the negative control E. coli TOP10 pTrcHisA strain.

2 shows the DNA sequence (3491 bp) revealed in Example 1. 2 shows the DNA sequence (3491 bp) revealed in Example 1. 2 shows the DNA sequence (3491 bp) revealed in Example 1. 2 shows the DNA sequence (3491 bp) revealed in Example 1. 2 shows the DNA sequence (3491 bp) revealed in Example 1. The result of GC-MS performed in Example 2 is shown. Escherichia coli TOP10 transformed with pAL526 as a plasmid is transformed with plasmid pAL520, plasmid pAL501, plasmid pAL502, plasmid pAL503, or plasmid pAL503 when the conversion ability of n-octane to 1-octanol is defined as 100. The relative conversion ability of transformed E. coli TOP10 is shown.

Claims (19)

A gene encoding a protein having any of the following amino acid sequences.
(1) an amino acid sequence having the amino acid sequence of SEQ ID NO: 1 in the sequence listing and having alkane-1-monooxygenase activity;
(2) an amino acid sequence having an alkane-1-monooxygenase activity having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids 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 90 % or more homology to the amino acid sequence described in SEQ ID NO: 1 in the sequence listing and having alkane-1-monooxygenase activity A gene having any of the following base sequences:
(1) a base sequence having the base sequence set forth in SEQ ID NO: 2 in the sequence listing and encoding alkane-1-monooxygenase;
(2) a base sequence having a base sequence having deletion, substitution and / or addition of 1 to several bases in the base sequence described in SEQ ID NO: 2 of the sequence listing and encoding alkane-1-monooxygenase; Or (3) a base sequence that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence set forth in SEQ ID NO: 2 in the sequence listing, and alkane-1-mono Base sequence encoding oxygenase 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: 3 in the sequence listing and having rubredoxin reductase activity;
(2) an amino acid sequence having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids in the amino acid sequence described in SEQ ID NO: 3 in the sequence listing and having rubredoxin reductase activity; or ( 3) An amino acid sequence having an amino acid sequence having 90 % or more homology with the amino acid sequence described in SEQ ID NO: 3 in the sequence listing and having rubredoxin reductase activity A gene having any of the following base sequences:
(1) a nucleotide sequence having the nucleotide sequence set forth in SEQ ID NO: 4 in the sequence listing and encoding rubredoxin reductase;
(2) a base sequence having a base sequence having deletion, substitution and / or addition of one to several bases in the base sequence set forth in SEQ ID NO: 4 of the sequence listing and encoding rubredoxin reductase; or ( 3) It has a base sequence that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 4 in the sequence listing, and encodes rubredoxin reductase Base sequence 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: 5 in the sequence listing and having a function of rubredoxin;
(2) an amino acid sequence having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids in the amino acid sequence set forth in SEQ ID NO: 5 in the sequence listing and having a function of rubredoxin; or (3) An amino acid sequence having an amino acid sequence having 90 % or more homology with the amino acid sequence described in SEQ ID NO: 5 in the sequence listing and having a function of rubredoxin 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: 7 in the sequence listing and having a function of rubredoxin;
(2) an amino acid sequence having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids in the amino acid sequence set forth in SEQ ID NO: 7 in the sequence listing and having a function of rubredoxin; or
(3) An amino acid sequence having an amino acid sequence having 90% or more homology to the amino acid sequence described in SEQ ID NO: 7 in the sequence listing and having a function of rubredoxin A gene having any of the following base sequences:
(1) a nucleotide sequence having the nucleotide sequence set forth in SEQ ID NO: 6 in the sequence listing and encoding rubredoxin;
(2) a nucleotide sequence having a nucleotide sequence having deletion, substitution and / or addition of one to several nucleotides in the nucleotide sequence set forth in SEQ ID NO: 6 in the sequence listing and encoding rubredoxin; or (3) A base sequence that encodes rubredoxin, having a base sequence that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence set forth in SEQ ID NO: 6 in the sequence listing A gene having any of the following base sequences:
(1) a nucleotide sequence having the nucleotide sequence set forth in SEQ ID NO: 8 in the sequence listing and encoding rubredoxin;
(2) a nucleotide sequence having a nucleotide sequence having deletion, substitution and / or addition of 1 to several nucleotides in the nucleotide sequence set forth in SEQ ID NO: 8 of the sequence listing and encoding rubredoxin; or
(3) a base sequence that encodes rubredoxin, having a base sequence that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence set forth in SEQ ID NO: 8 in the Sequence Listing The gene according to any one of claims 1 to 8 , which is a gene derived from a microorganism belonging to the genus Gordonia. A protein having any of the following amino acid sequences.
(1) an amino acid sequence having the amino acid sequence of SEQ ID NO: 1 in the sequence listing and having alkane-1-monooxygenase activity;
(2) an amino acid sequence having an alkane-1-monooxygenase activity having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids 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 90% or more homology to the amino acid sequence described in SEQ ID NO: 1 in the sequence listing and having alkane-1-monooxygenase activity 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: 3 in the sequence listing and having rubredoxin reductase activity;
(2) an amino acid sequence having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids in the amino acid sequence described in SEQ ID NO: 3 in the sequence listing and having rubredoxin reductase activity; or ( 3) An amino acid sequence having an amino acid sequence having 90 % or more homology with the amino acid sequence described in SEQ ID NO: 3 in the sequence listing and having rubredoxin reductase activity 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: 5 in the sequence listing and having a function of rubredoxin;
(2) an amino acid sequence having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids in the amino acid sequence set forth in SEQ ID NO: 5 in the sequence listing and having a function of rubredoxin; or (3) An amino acid sequence having an amino acid sequence having 90 % or more homology with the amino acid sequence described in SEQ ID NO: 5 in the sequence listing and having a function of rubredoxin 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: 7 in the sequence listing and having a function of rubredoxin;
(2) an amino acid sequence having an amino acid sequence having a deletion, substitution and / or addition of one to several amino acids in the amino acid sequence set forth in SEQ ID NO: 7 in the sequence listing and having a function of rubredoxin; or
(3) An amino acid sequence having an amino acid sequence having 90% or more homology to the amino acid sequence described in SEQ ID NO: 7 in the sequence listing and having a function of rubredoxin A recombinant vector comprising the gene according to any one of claims 1 to 9 . A transformant comprising the gene according to any one of claims 1 to 9 or the recombinant vector according to claim 14 . A transformant comprising the gene according to claim 1 or 2, the gene according to claim 3 or 4, the gene according to claim 5 or 6, and the gene according to claim 7 or 8 . The transformant according to claim 15 or 16 , wherein the host is Escherichia coli. Claim 16 is hydroxide linear alkanes or cycloalkanes having a carbon number of 6-8 5-13 carbon atoms in the presence of the transformant according to including Rukoto give alcohol, method for producing the alcohol. Culturing the transformant according to claim 15, comprising collecting the protein of any one of claims 10 to 13 from the culture, according to any one of claims 10 to 13 A method for producing a protein.

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