(en)An object of the present invention is to provide enzymes and a DNA encoding the enzymes that are involved in biosynthesis of trehangelin which has the potential to be a therapeutic agent for photosensitivity disorder and cosmetics, and to provide a method for producing trehangelin by utilizing the enzymes and a recombinant microorganism. The present invention is directed to a protein having an amino acid sequence of SEQ ID NO: 3, 5, 7 or 9, or a protein having an amino acid sequence of SEQ ID NO: 3, 5, 7 or 9 in which one to several amino acids are deleted, substituted, added and/or inserted or an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 3, 5, 7 or 9 and having an enzyme activity involved in biosynthesis of trehangelin; and a DNA encoding said protein.

1.ApplicationNumber: US-201715444914-A
1.PublishNumber: US-2017306371-A1
2.Date Publish: 20171026
3.Inventor: OMURA SATOSHI
TAKAHASHI YOKO
NAKASHIMA TAKUJI
INAHASHI YUKI

4.Inventor Harmonized: OMURA SATOSHI(JP)
TAKAHASHI YOKO(JP)
NAKASHIMA TAKUJI(JP)
INAHASHI YUKI(JP)

5.Country: US
6.Claims:
(en)An object of the present invention is to provide enzymes and a DNA encoding the enzymes that are involved in biosynthesis of trehangelin which has the potential to be a therapeutic agent for photosensitivity disorder and cosmetics, and to provide a method for producing trehangelin by utilizing the enzymes and a recombinant microorganism. The present invention is directed to a protein having an amino acid sequence of SEQ ID NO: 3, 5, 7 or 9, or a protein having an amino acid sequence of SEQ ID NO: 3, 5, 7 or 9 in which one to several amino acids are deleted, substituted, added and/or inserted or an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 3, 5, 7 or 9 and having an enzyme activity involved in biosynthesis of trehangelin; and a DNA encoding said protein.

7.Description:
(en)CROSS-REFERENCE TO RELATED APPLICATIONS
All documents cited herein are entirely incorporated herein by reference. The present application claims priority to Japanese Patent Application No. 2016-042045 filed on 4 Mar. 2016, which is entirely incorporated herein by reference.
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to the field of trehangelin synthesis.
Description of the Related Art
Trehangelin A, trehangelin B and trehangelin C has been discovered from secondary metabolites of an actinomycete Polymorphospora rubra K07-0510 isolated from the roots of an Orchidaceae plant. Trehangelins are compounds in which 1 trehalose molecule and 2 angelic acid molecules are condensed, and have an effect of suppressing light-induced cytotoxicity of pheophorbide a, i.e. a cryoprotection effect. Trehangelin A and trehangelin B do not exhibit toxicity towards animal cells (particularly, human cells), and are safe substances. Further, the trehangelins do not exhibit antimicrobial activity towards Gram-negative bacteria, Gram-positive bacteria and fungi, and are believed to have no effect on indigenous bacteria upon use thereof on human bodies (Japanese Patent Laid-Open No. 2015-024985).
As described above, trehangelins are safe and useful substances and are expected to be applied to medicaments such as therapeutic agents for photosensitivity disorder and cosmetics. Establishment of an inexpensive production method of trehangelin may be of great benefit in the medical field. It is difficult to allow condensation of angelic acid to the arbitrary hydroxy group of trehalose by organic chemistry, and thus synthesis of trehangelin is difficult without an enzyme reaction. However, the synthesis system of trehangelin which may be present in Polymorphospora rubra K07-0510 has not been identified.
SUMMARY OF THE INVENTION
The present invention is aimed to provide enzymes and a DNA sequence encoding the enzymes that is involved in biosynthesis of trehangelin, the potential compound as a therapeutic agent for photosensitivity disorder and cosmetics. Further, the present invention is aimed to provide a method for producing trehangelin by utilizing the enzymes and a microorganism recombined with the DNA having the sequence.
The inventors of the present invention sought to obtain DNAs encoding enzymes involved in trehangelin synthesis in an actinomycete Polymorphospora rubra K07-0510, i.e. a gene encoding 3-ketoacyl-CoA synthase, a gene encoding 3-ketoacyl-CoA reductase, a gene encoding enoyl-CoA hydratase and a gene encoding acyltransferase, examined whether or not a transformant obtained by cloning of the above 4 genes could produce trehangelin, and analysed expression and function of the enzymes in the transformant having the genes encoding the enzymes, thereby completing the present invention. In this way, the inventors of the present invention found novel enzymes involved in biosynthesis of trehangelin from an actinomycete Polymorphospora rubra K07-0510 to complete the present invention.
Thus, the present invention relates to:
(1) A nucleic acid molecule having at least one base sequence selected from following (i) to (iv):
(i) a base sequence of SEQ ID NO: 2, or
a base sequence encoding an amino acid sequence of SEQ ID NO: 3, or
a base sequence encoding an amino acid sequence of SEQ ID NO: 3 in which one to several amino acids are deleted, substituted, added and/or inserted, or a base sequence having 90% or more identity with the base sequence of SEQ ID NO: 2, wherein a protein encoded by the base sequence has 3-ketoacyl-CoA synthase activity;
(ii) a base sequence of SEQ ID NO: 4, or
a base sequence encoding an amino acid sequence of SEQ ID NO: 5, or
a base sequence encoding an amino acid sequence of SEQ ID NO: 5 in which one to several amino acids are deleted, substituted, added and/or inserted, or a base sequence having 90% or more identity with the base sequence of SEQ ID NO: 4, wherein a protein encoded by the base sequence has a 3-ketoacyl-CoA reductase activity;
(iii) a base sequence of SEQ ID NO: 6, or
a base sequence encoding an amino acid sequence of SEQ ID NO: 7, or
a base sequence encoding an amino acid sequence of SEQ ID NO: 7 in which one to several amino acids are deleted, substituted, added and/or inserted, or a base sequence having 90% or more identity with the base sequence of SEQ ID NO: 6, wherein a protein encoded by the base sequence has an enoyl-CoA hydratase activity; and
(iv) a base sequence of SEQ ID NO: 8, or
a base sequence encoding an amino acid sequence of SEQ ID NO: 9, or
a base sequence encoding an amino acid sequence of SEQ ID NO: 9 in which one to several amino acids are deleted, substituted, added and/or inserted, or a base sequence having 90% or more identity with the base sequence of SEQ ID NO: 8, wherein a protein encoded by the base sequence has an acyltransferase activity.
(2) The nucleic acid molecule of (1), comprising all of the base sequences of (i) to (iv) as recited in (1).

(3) The nucleic acid molecule of (2), which is a nucleic acid molecule having

a base sequence of SEQ ID NO: 1, or
a base sequence encoding an amino acid sequence encoded by the base sequence of SEQ ID NO: 1 in which one to several amino acids are deleted, substituted, added and/or inserted, or
a base sequence having 90% or more identity with the base sequence of SEQ ID NO: 1.
(4) The nucleic acid molecule of (1), which is
a nucleic acid molecule having a base sequence of SEQ ID NO: 2, or
a nucleic acid molecule having a base sequence encoding an amino acid sequence of SEQ ID NO: 3, or
a nucleic acid molecule having a base sequence encoding an amino acid sequence of SEQ ID NO: 3 in which one to several amino acids are deleted, substituted, added and/or inserted or a base sequence having 90% or more identity with the base sequence of SEQ ID NO: 2, wherein a protein encoded by the base sequence has a 3-ketoacyl-CoA synthase activity.
(5) The nucleic acid molecule of (1), which is
a nucleic acid molecule having a base sequence of SEQ ID NO: 4, or
a nucleic acid molecule having a base sequence encoding an amino acid sequence of SEQ ID NO: 5, or
a nucleic acid molecule having a base sequence encoding an amino acid sequence of SEQ ID NO: 5 in which one to several amino acids are deleted, substituted, added and/or inserted, or a base sequence having 90% or more identity with the base sequence of SEQ ID NO: 4, wherein a protein encoded by the base sequence has a 3-ketoacyl-CoA reductase activity.
(6) The nucleic acid molecule of (1), which is
a nucleic acid molecule having a base sequence of SEQ ID NO: 6, or
a nucleic acid molecule having a base sequence encoding an amino acid sequence of SEQ ID NO: 7, or a nucleic acid molecule having a base sequence encoding an amino acid sequence of SEQ ID NO: 7 in which one to several amino acids are deleted, substituted, added and/or inserted, or a base sequence having 90% or more identity with the base sequence of SEQ ID NO: 6, wherein a protein encoded by the base sequence has an enoyl-CoA hydratase activity.
(7) The nucleic acid molecule of (1), which is
a nucleic acid molecule having a base sequence of SEQ ID NO: 8,
a nucleic acid molecule having a base sequence encoding an amino acid sequence of SEQ ID NO: 9, or
a nucleic acid molecule having a base sequence encoding an amino acid sequence of SEQ ID NO: 9 in which one to several amino acids are deleted, substituted, added and/or inserted, or a base sequence having 90% or more identity with the base sequence of SEQ ID NO: 8, wherein a protein encoded by the base sequence has an acyltransferase activity.
(8) A protein having an amino acid sequence of SEQ ID NO: 3, or
a protein having an amino acid sequence of SEQ ID NO: 3 in which one to several amino acids are deleted, substituted, added and/or inserted or an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 3, and having a 3-ketoacyl-CoA synthase activity.
(9) A protein having an amino acid sequence of SEQ ID NO: 5, or
a protein having an amino acid sequence of SEQ ID NO: 5 in which one to several amino acids are deleted, substituted, added and/or inserted or an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 5, and having a 3-ketoacyl-CoA reductase activity.
(10) A protein having an amino acid sequence of SEQ ID NO: 7, or
a protein having an amino acid sequence of SEQ ID NO: 7 in which one to several amino acids are deleted, substituted, added and/or inserted or an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 7, and having an enoyl-CoA hydratase activity.
(11) A protein having an amino acid sequence of SEQ ID NO: 9, or
a protein having an amino acid sequence of SEQ ID NO: 9 in which one to several amino acids are deleted, substituted, added and/or inserted or an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 9, and having an acyltransferase activity.
(12) A vector comprising the nucleic acid molecule of any one of (1) to (7).

(13) A transformant comprising the vector of (12).

(14) The transformant of (13), which is Escherichia coli.

(15) A method for producing trehangelin comprising culturing the transformant of (13) or (14) to produce trehangelin.

(16) A method for producing trehangelin at least comprising reacting a protein of any one of (8) to (11) with a substrate.

(17) The method for producing trehangelin of (16), comprising reacting all of the proteins of (8) to (11) with a substrate.

In one aspect, the present invention relates to enzymes involved in trehangelin synthesis and a nucleic acid molecule encoding the enzymes. The term “trehangelin” as used herein means the compound represented by the following general formula:









[wherein, any one of R1 to R3 is a 2-methylbut-2-enoyl group and remaining two of R1 to R3 represent hydrogen atoms; and any one of R4 to R6 is a 2-methylbut-2-enoyl group and remaining two of R4 to R6 represent hydrogen atoms.]
Examples of trehangelins as used herein include trehangelin A, trehangelin B and trehangelin C. Trehangelin A is a compound represented by the following formula I:









Trehangelin B as used herein is a compound represented by the following formula II:









Trehangelin C as used herein is a compound represented by the following formula III:









The “trehangelin synthesis” as used herein means at least one step selected from the following (a) to (d). In other words, trehangelin synthesis comprises one or more (2, 3 or all) steps selected from the following (a) to (d):
(a) reacting acetyl-CoA with methylmalonyl-CoA to produce 2-methylacetoacetyl-CoA; (b) converting 2-methylacetoacetyl-CoA into 3-hydroxy-2-methylbutyryl-CoA; (c) converting 3-hydroxy-2-methylbutyryl-CoA into angelyl-CoA; and (d) reacting angelyl-CoA with trehalose to produce trehangelin.

Therefore, the present invention encompasses one or more (2, 3 or all) methods selected from the following (a) to (d):
(a) a method for reacting acetyl-CoA with methylmalonyl-CoA to produce 2-methylacetoacetyl-CoA;

(b) a method for converting 2-methylacetoacetyl-CoA to 3-hydroxy-2-methylbutyryl-CoA;

(c) a method for converting 3-hydroxy-2-methylbutyryl-CoA to angelyl-CoA; and

(d) a method for reacting angelyl-CoA with trehalose to produce trehangelin.

The “enzymes involved in trehangelin synthesis” as used herein means enzymes catalysing the above steps (a) to (d), and specifically means 3-ketoacyl-CoA synthase (catalysing the step (a)), 3-ketoacyl-CoA reductase (catalysing the step (b)), enoyl-CoA hydratase (catalysing the step (c)), and acyltransferase (catalysing the step (d)).
The enzymes involved in trehangelin synthesis as used herein typically are proteins having amino acid sequences of SEQ ID NO: 3 (3-ketoacyl-CoA synthase), SEQ ID NO: 5 (3-ketoacyl-CoA reductase), SEQ ID NO: 7 (enoyl-CoA hydratase), and SEQ ID NO: 9 (acyltransferase). The enzymes, as used herein, may be proteins having amino acid sequences of SEQ ID NO: 3 (3-ketoacyl-CoA synthase), SEQ ID NO: 5 (3-ketoacyl-CoA reductase), SEQ ID NO: 7 (enoyl-CoA hydratase) or SEQ ID NO: 9 (acyltransferase) in which one to several amino acids are deleted, substituted, added and/or inserted. The phrase “one to several amino acids are deleted, substituted, added and/or inserted” as used herein means any number of amino acids, for example 1 to 20, preferably 1 to 15, more preferably 1 to 10 and still more preferably 1 to 5 are deleted, substituted, added and/or inserted. In addition, the enzymes, as used herein, may be proteins having 60% or more homology or identity or, for example, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more homology or identity with amino acid sequences of SEQ ID NO: 3 (3-ketoacyl-CoA synthase), SEQ ID NO: 5 (3-ketoacyl-CoA reductase), SEQ ID NO: 7 (enoyl-CoA hydratase) and SEQ ID NO: 9 (acyltransferase). The homology or identity of amino acid sequences may be examined by using a tool well known in the art such as BLAST (Basic Local Alignment Search Tool).
As used herein, a “protein having an amino acid sequence in which one to several amino acids are deleted, substituted, added and/or inserted” and a “protein having an amino acid sequence that has 60% or more (and the like) homology” have the same biological activity, i.e. an enzyme activity, of the protein having the original amino acid sequence. The biological activities of these proteins need to have the same nature but are not necessary to have the same degree of activities as that of the protein having the original amino acid sequence. Specifically, a protein having an amino acid sequence of SEQ ID NO: 3 in which one to several amino acids are deleted, substituted, added and/or inserted and a protein having 60% or more (and the like) homology or identity with the amino acid sequence of SEQ ID NO: 3 have a 3-ketoacyl-CoA synthase activity that catalyses the above reaction (a). A protein having an amino acid sequence of SEQ ID NO: 5 in which one to several amino acids are deleted, substituted, added and/or inserted and a protein having a 60% or more or the like homology or identity with the amino acid sequence of SEQ ID NO: 5 have a 3-ketoacyl-CoA reductase activity that catalyses the above reaction (b). A protein having an amino acid sequence of SEQ ID NO: 7 in which one to several amino acids are deleted, substituted, added and/or inserted and a protein having 60% or more (and the like) homology or identity with the amino acid sequence of SEQ ID NO: 7 have an enoyl-CoA hydratase activity that catalyses the above reaction (c). A protein having an amino acid sequence of SEQ ID NO: 9 in which one to several amino acids are deleted, substituted, added and/or inserted and a protein having 60% or more (and the like) homology or identity with the amino acid sequence of SEQ ID NO: 9 have an acyltransferase activity catalysing the above reaction (d). Throughout herein, whether or not a protein has a desired enzyme activity can be confirmed by reacting the protein with a corresponding substrate under the condition that the original protein can catalyse, and by detecting an expected product to be obtained. When even a small amount of the expected product is detected, the protein can be determined to have the enzyme activity.
In another aspect, the present invention relates to a nucleic acid molecule having a base sequence encoding any of the above enzymes. As an specific example, the nucleic acid molecule of the present invention is a nucleic acid molecule having at least one base sequence selected from base sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8. The nucleic acid molecule of the present invention further encompasses a base sequence having 90% or more identity with the base sequence of SEQ ID NO: 2, a base sequence having 90% or more identity with the base sequence of SEQ ID NO: 4, a base sequence having 90% or more identity with the base sequence of SEQ ID NO: 6 and a base sequence having 90% or more identity with the base sequence of SEQ ID NO: 8. The identity can exceed 90% or more, for example 95% or more, 96% or more, 97% or more, 98% or more or 99% or more. The identity of nucleic acid sequences may be examined by using a tool well known in the art such as BLAST (Basic Local Alignment Search Tool).
As used herein, “a nucleic acid molecule having a base sequence that has 90% or more (and the like) identity” encodes a protein having a biological activity of the protein encoded by the original nucleic acid molecule, namely an enzyme activity. Thus, for example, a protein encoded by a base sequence that has 90% or more identity with the base sequence of SEQ ID NO: 2 has a 3-ketoacyl-CoA synthase activity that catalyses the above reaction (a). Similarly, a protein encoded by a base sequence that has 90% or more identity with the base sequence of SEQ ID NO: 4 has a 3-ketoacyl-CoA reductase activity catalysing the above reaction (b). A protein encoded by a base sequence that has 90% or more identity with the base sequence of SEQ ID NO: 6 has an enoyl-CoA hydratase activity catalysing the above reaction (c). A protein encoded by a base sequence that has 90% or more identity with the base sequence of SEQ ID NO: 8 has an acyltransferase activity catalysing the above reaction (d).
It is sufficient that the nucleic acid molecule of the present invention may have a base sequence encoding at least one enzyme selected from the above described enzymes, alternatively, the nucleic acid molecule may have a base sequence encoding two or more (for example, 2, 3 or all) enzymes. When the nucleic acid molecule of the present invention has a base sequence encoding two or more enzymes, each of base sequences encoding the respective enzymes may be linked via any non-coding region that does not have the initiation codon. Alternatively, when the nucleic acid molecule of the present invention has a base sequence encoding two or more enzymes, the base sequences may encode the enzymes with partial overlap. For example, the base sequence of SEQ ID NO: 1 has all base sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, and the positions 1 to 828 of SEQ ID NO: 1 correspond to the base sequence of SEQ ID NO: 2, the positions 875 to 1900 of SEQ ID NO: 1 correspond to the base sequence of SEQ ID NO: 4, the positions 1905 to 2684 of SEQ ID NO: 1 correspond to the base sequence of SEQ ID NO: 6, and the positions 2681 to 3475 of SEQ ID NO: 1 correspond to the base sequence of SEQ ID NO: 8. In SEQ ID NO: 1, non-coding regions exist between SEQ ID NO: 2 and SEQ ID NO: 4, and between SEQ ID NO: 4 and SEQ ID NO: 6. SEQ ID NO: 6 and SEQ ID NO: 8 partly share the nucleic acid sequence as an overlapping coding region in SEQ ID NO: 1.
As used herein, the “nucleic acid molecule” means DNA, RNA or a mixture of DNA and RNA. The nucleic acid molecule may be modified as far as the molecule can express a desired protein. The present invention also encompasses a nucleic acid molecule having a base sequence that is complementary to a base sequence encoding at least one enzyme selected from the above enzymes.
According to another aspect of the invention, a vector containing a DNA and/or RNA that has a base sequence encoding the enzymes is provided. The vector of the present invention is not particularly limited as far as the vector can express the DNA and/or RNA in a microorganism, and is preferably able to replicate or to be integrated into a chromosome in a host, Escherichia coli or an actinomycete. The vector of the present invention may contain, in addition to the above DNA and/or RNA, a terminator sequence, a promoter region, a ribosome binding sequence and/or a promoter controlling sequence. The vector of the present invention preferably contains a promoter region, and in this case, the promoter preferably locates at a position that allows the promoter to control expression of the DNA and/or RNA. The vector may be either of a phage vector and a plasmid vector, and is preferably a vector expressed in E. coli or an actinomycete. Examples of the vector of the present invention include ZAP Express [produced by Stratagene Corp., Strategies, 5, 58 (1992)], pBluescript II SK(+) [Nucleic Acids Research, 17, 9494 (1989)], Lambda ZAP II (produced by Stratagene Corp.), lambda gt10, lambda gt11 [DNA Cloning, A Practical Approach, 1, 49 (1985)], lambda TriplEx (produced by Clonetech Laboratories, Inc.), lambda ExCell (produced by Pharmacia Corp.), pT7T318U (produced by Pharmacia Corp.), pcD2 [Mol. Gen. Biol., 3, 280 (1983)], pMW218 (produced by Wako Pure Chemical Industries Ltd.), UC118 (produced by Takara Shuzo Co., Ltd.), pEG400 [J. Bac., 172, 2392 (1990)], pQE-30 (produced by Qiagen), pBTrp2, pBTac1, pBTac2 (all marketed by Boehringer Mannheim), pKK233-2 (produced by Pharmacia Corp.), pSE280 (produced by Invitrogen), pGEMEX-1 (produced by Promega Corporation), pQE-8 (produced by Qiagen), pQE-30 (produced by Qiagen), pKYP200 [Agricultural Biological Chemistry, 48, 669 (1984)], pLSA1 [Agricultural Biological Chemistry., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK+, pBluescript II SK- (produced by Stratagene Corp.), pTrS30 (FERMBP-5407), pTrS32 (FERMBP-5408), pGEX (produced by Pharmacia Corp.), pET-3 (produced by Novagen), pET-15b (produced by Novagen), pSupex, pUB110, pTP5, pC194, pUC18 [Gene, 33, 103 (1985)], pUC19 [Gene, 33, 103 (1985)], pSTV28 (produced by Takara Shuzo Co., Ltd.), pSTV29 (produced by Takara Shuzo Co., Ltd.), pUC118 (produced by Takara Shuzo Co., Ltd.), pCold I (produced by Takara Shuzo Co., Ltd.), pEG400 [J. Bacteriol., 172, 2392 (1990)] and the like.
Any promoter can be used as long as that can express in a host, an actinomycete cell. Examples of the promoter include promoters derived from E. coli and phages such as the trp promoter (Ptrp), the lac promoter (Plac), the PL promoter, the PR promoter and the PSE promoter; the SPO1 promoter; the SPO2 promoter; the penP promoter and the like. Artificially designed and modified promoters such as the promoter including two Ptrps in series (Prpx2), the tac promoter, the letI promoter and the lacT7 promoter may also be used.
Any ribosome binding sequence can be used as long as that can be expressed in a host, an actinomycete cell. It is preferable to use a vector in which the distance between the Shine-Dalgarno sequence and the initiation codon is appropriately adjusted (for example, 6 to 18 bases).
According to another aspect of the present invention, a transformant or host cell containing the above vector is provided. Examples of the transformant or host cell include microorganisms belonging to the following genera: Escherichia, Corynebacterium, Brevibacterium, Bacillus, Microbacterium, Serratia, Pseudomonas, Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Arthrobacter, Azobacter, Chromatium, Erwinia, Methylobacterium, Phormidium, Rhodobacter, Rhodopseudomonas, Rhodospirillum, Scenedesmun, Streptomyces, Synnecoccus, Zymomonas and the like. Preferably microorganisms belonging to the following genera may be mentioned: Escherichia, Corynebacterium, Brevibacterium, Bacillus, Pseudomonas, Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Arthrobacter, Azobacter, Chromatium, Erwinia, Methylobacterium, Phormidium, Rhodobacter, Rhodopseudomonas, Rhodospirillum, Scenedesmun, Streptomyces, Synnecoccus, Zymomonas and the like. Examples of the actinomycete include Streptomyces albus, Streptomyces lividans, Streptomyces chromofuscus, Streptomyces exfoliatus and Streptomyces argenteorus . The transformant of the present invention may be any host cell as far as that is capable of efficiently expressing the enzyme protein, and is preferably E. coli , baker's yeast or an actinomycete (genus Streptomyces ), more preferably E. coli or an actinomycete, and the most preferably E. coli.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the result of detection of trehangelin production from an actinomycete transformed with orfA, orfB, orfC and orfD by LC/MS; and
FIG. 2 shows the result of detection of a reaction product of orfB, orfD and orfA by LC/MS.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
(1) Obtaining Nucleic Acid Molecules
The nucleic acid molecule of the present invention may be obtained by PCR. For example, a nucleic acid molecule (for example, DNA) having a base sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 may be obtained by carrying out PCR, in which the chromosomal DNA of an actinomycete Polymorphospora rubra K07-0510 is used as a template with a pair of primers designed to allow amplification of the base sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, utilizing Expand™ High Fidelity PCR System (produced by Roche Life Science) and the like on a DNA Thermal Cycler (produced by Applied Biosystems). The primers preferably have appropriate restriction sites added thereto, in order to facilitate following cloning procedures.
One of the PCR reaction conditions may be the reaction of, for example, 30 cycles of 94° C. for 30 seconds (denaturation), 60° C. for 30 seconds to 1 minute (annealing) and 72° C. for 1 to 3 minutes (extension) followed by the reaction at 72° C. for 7 minutes. The amplified DNA fragments may then be cloned into an appropriate vector capable of amplifying in E. coli . Cloning may be carried out according to the standard method such as those described in Molecular Cloning, Second Edition; Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997) (hereinafter abbreviated as “Current Protocols in Molecular Biology”); and DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995) or with commercially available kits such as SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (produced by Life Technologies Corp.) and ZAP-cDNA Synthesis Kit (produced by Stratagene Corp.).
Any cloning vector may be used as long as that can replicate in E. coli K12, such as phage vectors and plasmid vectors. The expression vector in E. coli may be used as a cloning vector. Specifically, the vectors described above may be used.
A plasmid containing a desired DNA may be obtained from the resulting transformant according to the standard method such as those described in Molecular Cloning, Second Edition; Current Protocols in Molecular Biology; and DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995). According to the above method, a nucleic acid molecule (for example, DNA) having a base sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 can be obtained.
A nucleic acid molecule having a base sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 in which one to several bases are deleted, substituted, added and/or inserted, wherein the base sequence encodes an enzyme protein having a particular activity may be isolated by utilizing, for example, a base sequence of a DNA fragment derived from an actinomycete that has the base sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 to screen a homologue of the DNA from other microorganisms under appropriate conditions. Alternatively, the above described mutated DNA may be prepared according to any methods known in the art such as chemical synthesis, gene engineering techniques and mutagenesis. Specifically, a mutated DNA may be obtained by artificially introducing a mutation into DNA having the base sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8.
For example, DNA having the base sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 may be contacted with an agent that acts as a mutagen, may be irradiated with ultraviolet rays, or may be subjected to gene engineering techniques. Site specific mutagenesis, one of the gene engineering techniques, allows an introduction of a specific mutation at a specific position, and thus is useful, which can be carried out according to the methods described in, for example, Molecular Cloning Second Edition; Current Protocols in Molecular Biology; Nucleic Acids Research, 10, 6487 (1982); Nucleic Acids Research, 12, 9441 (1984); Nucleic Acids Research, 13, 4431 (1985); Nucleic Acids Research, 13, 8749 (1985); Proc. Natl. Acad. Sci. USA, 79, 6409 (1982); Proc. Natl. Acad. Sci. USA, 82, 488 (1985); and Gene, 102, 67 (1991).
(2) Obtaining Vectors, Transformants, and Enzymes
The vector of the present invention may be obtained by incorporating the above described DNA into a vector by standard genetic recombination techniques. The transformant of the present invention may be obtained by introducing the vector into a host cell by a method well known in the art. Further, the protein (enzyme) of the present invention can be obtained by culturing a transformant having a recombinant DNA in which the above DNA is integrated in a medium, and by allowing to produce the enzyme protein of the present invention involved in trehangelin synthesis in the culture medium.
In order to express a DNA fragment containing genes of the enzymes involved in trehangelin synthesis (3-ketoacyl-CoA synthase, 3-ketoacyl-CoA synthase, enoyl-CoA hydratase and acyltransferase) in a host cell, a DNA fragment containing the genes may be subjected to a restriction enzyme digestion or a DNase treatment to obtain a DNA fragment of appropriate length containing the genes, which may be inserted downstream of a promoter in an expression vector, and the obtained expression vector may be introduced into a host cell suitable for the expression vector.
The host cell used herein is not limited as far as the host cell can efficiently express the enzyme protein, and may be the host cell of the present invention as described above. The expression vector may be the vector of the present invention as described above.
A method for introducing a recombinant vector may be any method for introducing DNA into a host cell, for example an actinomycete, and examples of the method include a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], the protoplast method (Japanese Patent Laid-Open No. S63-2483942) and methods described in Gene, 17, 107 (1982) and Molecular & General Genetics, 168, 111 (1979).
A transformant that has a recombinant DNA in which the above DNA is integrated may be cultured in a medium, and 3-ketoacyl-CoA synthase, 3-ketoacyl-CoA synthase, enoyl-CoA hydratase and acyltransferase of the present invention may be produced in the culture medium.
The transformant of the present invention transformed with the enzymes involved in trehangelin synthesis may be cultured according to common culturing methods used for the host cell employed. When the transformant of the present invention is prokaryotes such as E. coli and actinomycetes or eukaryotes such as yeasts, the medium for culturing the microorganisms may be any of natural media or synthetic media as far as the media contains carbon sources, nitrogen sources, inorganic salts and the like that could be used by the microorganisms, and as far as the media allow efficient culture of the transformant. For example, the medium for culturing actinomycetes may be any of natural media or synthetic media as far as the media contain carbon sources, nitrogen sources, inorganic salts and the like that could be used by the microorganisms, and as far as the media allow efficient culture of the transformant.
The carbon source may be the one that could be used by a host microorganism, and includes carbohydrates such as glucose, fructose, sucrose, molasses containing the foregoing, starch and starch hydrolysates; organic acids such as acetic acid and propionic acid; and alcohols such as ethanol and propanol.
The nitrogen source which may be used includes ammonia; ammonium salts of inorganic acids and organic acids such as ammonium chloride, ammonium sulphate, ammonium acetate and ammonium phosphate; other nitrogen-containing compounds, as well as peptone, meat extracts, yeast extract, corn steep liquor, casein hydrolysates, soybean meal and soybean meal hydrolysates, and various fermented cells and digested materials thereof.
The inorganic substance which may be used includes monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulphate, sodium chloride, ferrous sulphate, manganese sulphate, copper sulphate, calcium carbonate and the like.
Culture is carried out under an aerobic condition such as in shake culture or stirred submerged aerobic culture. The culture temperature is preferably 15 to 40° C. and the incubation period is usually 16 hours to 7 days. pH during culture is maintained at 3.0 to 9.0. pH is adjusted with an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia and the like. During culture, an antibiotic such as ampicillin, tetracycline and thiostrepton may be optionally added to the medium.
In culturing an actinomycete transformed with an expression vector containing an inducible promoter, an inducer may be optionally added to the medium. For example, in case of an actinomycete transformed with an expression vector containing the lac promoter, isopropyl-β-D-thiogalactopyranoside (IPTG) and the like may be added to the medium, and when an actinomycete transformed with an expression vector containing the trp promoter is cultured, indoleacrylic acid (IAA) and the like may be added to the medium.
When the enzyme is produced inside of the host cells, the host cells are further homogenized. The obtained enzyme protein is, after disruption of the host cells purified to a desired purity using any combination of well-known protein purification techniques such as salting-out, dialysis, affinity chromatography, ion-exchange chromatography, size exclusion chromatography, organic solvent treatment, and heating treatment. The cells per se, a solution containing homogenate of the cells, or a soluble fraction of the homogenate may also be used as an enzyme provided these have no effect on reactions or reaction products.
In view of purification and manifestation of activity of an enzyme protein, the desired enzyme protein is preferably water-soluble. However, a water-insoluble enzyme protein may be solubilised according to the method known in the art such as addition of a surfactant. The desired protein may be expressed as a fusion protein with another protein (such as GST and TAG); however, a desired protein is preferably not a fusion protein.
(3) Production Method of Trehangelin
Trehangelin may be produced by producing and accumulating trehangelin in a culture medium of a transformant that is introduced the enzymes of the present invention involved in trehangelin synthesis. For example, in using an actinomycete as the host cell, trehalose may optionally be added during culture to a culture medium, although actinomycetes have the trehalose synthetic pathway. The host cells may be cultured according to the method described above for obtaining the enzymes.
Alternatively, trehangelin may be produced by using purified enzymes of the present invention involved in trehangelin synthesis. Thus, the present invention also encompasses a method for synthesizing trehangelin at least comprising reacting angelyl-CoA with trehalose in the presence of an enzyme protein (orfC) having an amino acid sequence of SEQ ID NO: 7 (or an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence of SEQ ID NO: 7 in which one to several amino acids are deleted, substituted, added and/or inserted. The method for synthesizing trehangelin of the present invention may further comprise one to three steps selected from the following (i) to (iii): (i) reacting acetyl-CoA with methylmalonyl-CoA in the presence of an enzyme protein (orfB) having an amino acid sequence of SEQ ID NO: 5 (or an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence of SEQ ID NO: 5 in which one to several amino acids are deleted, substituted, added and/or inserted) to obtain 2-methylacetoacetyl-CoA; (ii) reacting 2-methylacetoacetyl-CoA with NADPH in the presence of an enzyme protein (orfD) having an amino acid sequence of SEQ ID NO: 9 (or an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence of SEQ ID NO: 9 in which one to several amino acids are deleted, substituted, added and/or inserted) to obtain 3-hydroxy-2-methylbutyryl-CoA; and (iii) converting 3-hydroxy-2-methylbutyryl-CoA to angelyl-CoA into the presence of an enzyme protein (orfA) having an amino acid sequence of SEQ ID NO: 3 (or an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence of SEQ ID NO: 3 in which one to several amino acids are deleted, substituted, added and/or inserted). The reaction using the enzymes may be carried out according to the method described hereinbelow for the enzyme activity assay of the protein.
Trehangelin may be isolated and purified from the culture medium of the transformant of the present invention according to the method described in Japanese Patent Laid-Open No. 2015-024985, namely an appropriate combination of separation of cells, extraction, distillation, chromatography and the like.
(4) Enzyme Activity Assay of the Protein
Enzyme activity of the obtained protein may be assayed according to a common enzyme activity assay. A buffer used for a reaction solution of the activity assay may have pH in the range that does not inhibit the activity of the target enzyme, and preferably has pH in the range including an optimal pH.
Any buffer that does not inhibit the enzyme activity and can attain the above pH may be used. The buffer which may be used includes Tris hydrochloride buffer, phosphate buffer, borate buffer, HEPES buffer, MOPS buffer, hydrogen carbonate buffer and the like. The concentration of the buffer may be any concentration as far as it does not inhibit the enzyme activity and is suitably 1 mM to 1 M.
A substrate of the target enzyme is added to the reaction solution. Specifically, in a 3-ketoacyl-CoA synthase activity assay, acetyl-CoA and methylmalonyl-CoA are added. In a 3-ketoacyl-CoA reductase activity assay, 2-methylacetoacetyl-CoA is added. In an enoyl-CoA hydratase activity assay, 3-hydroxy-2-methylbutyryl-CoA is added. In an acyltransferase activity assay, angelyl-CoA is added. The concentration of the substrate may be any concentration as far as it has no effect on the reaction, and is suitably 0.01 mM to 0.2 M of the reaction solution. The enzyme concentration for the reaction is not particularly limited, and is usually in a range of 0.001 mg/ml to 100 mg/ml. It is not always required that the enzyme used is purified to consisting of a single type of protein, and an authentic preparation containing other contaminated proteins may be used as far as they do not interfere with the reaction.
The reaction temperature may be in the range that does not inhibit the activity of the utilized enzyme, and is preferably in the range including an optimal temperature of the enzyme. Thus, the reaction temperature can be 10° C. to 60° C., and more preferably 27° C. to 40° C.
The activity may be detected by a method that can measure a decrease of substrates or an increase of reaction products during the reaction. Examples of such methods include a method that separates and quantifies a target substance optionally by using thin layer chromatography, column chromatography, high-performance liquid chromatography (HPLC) and the like. The reaction product may be identified by a method separating the target substance with thin layer chromatography, column chromatography, HPLC and the like, and then comparing the retention time of the target substance with that of an authentic preparation, or by a method employing a nuclear magnetic resonator or a mass spectrometer.
(5) Production Method of Angelyl-CoA
The present invention is further directed to a method for producing angelyl-CoA by using 3-ketoacyl-CoA synthase, 3-ketoacyl-CoA reductase and/or enoyl-CoA hydratase of the present invention. In this production method, 3-ketoacyl-CoA synthase, 3-ketoacyl-CoA reductase and/or enoyl-CoA hydratase, and three substrates, i.e. acetyl-CoA, methylmalonyl-CoA and reduced nicotinamide adenine dinucleotide phosphate (NADPH) are appropriately added into a solution to allow appropriate reactions, thereby angelyl-CoA may be produced. More specifically, the method for producing angelyl-CoA of the present invention may comprise one to all steps selected from (i) to (iii) described hereinabove for the method for synthesizing trehangelin. It is conceived that esters of angelic acid may be used for antipyretic analgesics, muscle relaxants, sedatives and the like.
The present invention is more specifically described hereinbelow by way of Examples which do not limit the scope of the present invention. All documents cited herein are entirely incorporated herein by reference.
EXAMPLES
(Analytical Method)
In the following Examples, LC/MS analysis was carried out under the following conditions:
Column: Inertsil ODS-4 (produced by GL Sciences Inc.), size: diameter 3.0 mm×length 250 mm, 40° C.
Mobile phase: 2 mM ammonium acetate aqueous solution (A), 2 mM ammonium acetate-containing methanol solution (B), 0-5 minutes 5% B, 5-35 minutes 5-100% B, 35-40 minutes 100% B, flow rate: 0.5 ml/min.
Detection: QSTAR Elite ESI quadruple time-of-flight (Q-TOF) MS instrument (produced by AB Sciex).
[Example 1] Production of Trehangelin from Actinomycete Cloned with DNA of SEQ ID NO: 1
The K07-0510 strain was cultured in YD medium containing 1% yeast extract and 1% glucose at an appropriate temperature (for example, 27° C.) for a few days. After culturing, cells were collected from the resulting culture medium by centrifugation and the chromosomal DNA was isolated and purified from the cells according to the standard method (Molecular Cloning, Second Edition).
Four open reading frames (orfs) on SEQ ID NO: 1 were named as orfA, orfB, orfC and orfD in the order of the base positions. The positions and functions of orfA to D in SEQ ID NO: 1 are as follows:
orfA (SEQ ID NO: 2: positions 1-828 in SEQ ID NO: 1, encoding enoyl-CoA hydratase)

orfB (SEQ ID NO: 4: positions 875-1900 in SEQ ID NO: 1, encoding 3-ketoacyl-CoA synthase)

orfC (SEQ ID NO: 6: positions 1905-2684 in SEQ ID NO: 1, encoding acyltransferase)

orfD (SEQ ID NO: 8: positions 2681-3475 in SEQ ID NO: 1, encoding 3-ketoacyl-CoA reductase)

A recombinant plasmid sufficiently expressing the above four genes was constructed by PCR [Science, 230, 1350 (1985)] as described hereinbelow.
A DNA capable of expressing orfA, orfB, orfC and orfD (hereinafter referred to as “orfABCD”) was amplified by carrying out PCR on a DNA Thermal Cycler (produced by Applied Biosystems) with using the chromosomal DNA of an actinomycete Polymorphospora rubra K07-0510 as a template, a sense primer of SEQ ID NO: 10 having a PstI restriction enzyme site and a ribosome binding sequence at its 5′-terminal, an antisense primer of SEQ ID NO: 11 having a StuI restriction enzyme site at its 5′-terminal, and Taq DNA polymerase (produced by Roche Life Science). The condition of PCR was 30 cycles of 95° C. for 30 seconds, 68° C. for 30 seconds, and 72° C. for 4 minute followed by 72° C. for 10 minutes. The amplified DNA fragment was purified by agarose gel electrophoresis, and digested with restriction enzymes PstI and StuI to obtain a DNA fragment (hereinafter referred to as “orfABCD-containing DNA fragment”) containing a PstI and StuI-treated DNA expressing orfABCD.
pOSV556t [Nat. Chem., 3, 338 (2011)] was digested with restriction enzymes PstI and StuI to obtain a PstI and StuI-treated pOSV556t fragment. The PstI and StuI-treated orfABCD-containing DNA fragment obtained above and the PstI and StuI-treated pOSV556t fragment were mixed and ligated to obtain a recombinant DNA.
The recombinant DNA was used to transform E. coli Top10 according to the standard method, and the transformant was applied on a LB agar medium containing 100 μg/ml ampicillin, and incubated overnight at 37° C. A plasmid containing the recombinant DNA was isolated from the transformant according to the standard method. The recombinant DNA was sequenced to confirm that the DNA was orfABCD, and the plasmid was named as pOSV556-orfABCD.
The pOSV556-orfABCD was introduced into E. coli ET12567/pUZ8002 [Practical Streptomyces Genetics (2000)] according to the standard method to obtain E. coli ET12567/pUZ8002/pOSV556-orfABCD that is resistant to 50 μg/ml kanamycin, 25 μg/ml chloramphenicol and 100 μg/ml ampicillin. The pOSV556-orfABCD was further transferred by conjugation according to the standard method from E. coli ET12567/pUZ8002 to an actinomycete Streptomyces albus J1074 to obtain Streptomyces albus /pOSV556-orfABCD resistant to 50 μg/ml hygromycin.
Streptomyces albus /pOSV556-orfABCD was grown by shake culture in 10 ml of a liquid medium containing 1% yeast extract and 1% glucose at 27° C. for 1 day, which was then added 1 ml of 20% trehalose aqueous solution, and the shake culture was continued at 27° C. for 4 more days. The obtained culture medium was added 10 ml of ethanol and stirred for 1 hour. Ethanol in the extract was then removed by distillation under reduced pressure, and 5 ml of ethyl acetate was added to the obtained aqueous solution, which was thoroughly stirred, and then the ethyl acetate phase was collected. After concentration to dryness, the solid was dissolved in 100 μl of methanol and analysed on LC/MS to confirm production of trehangelin ( FIG. 1 ).
[Example 2] Confirmation of Function of the Enzyme Protein (orfB) Encoded by the Base Sequence of SEQ ID NO: 4
A recombinant plasmid sufficiently expressing a gene encoding 3-ketoacyl-CoA synthase (orfB) was constructed by PCR [Science, 230, 1350 (1985)] as described hereinbelow.
orfB was amplified by carrying out PCR on a DNA Thermal Cycler (produced by Applied Biosystems) with using the chromosomal DNA of an actinomycete Polymorphospora rubra K07-0510 as a template, a sense primer of SEQ ID NO: 12 and having a NdeI restriction enzyme site at its 5′-terminal, an antisense primer of SEQ ID NO: 13 and having a XhoI restriction enzyme site at its 5′-terminal and Taq DNA polymerase (produced by Roche Life Science). PCR was carried out under the conditions of 30 cycles of 95° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1 minute followed by 72° C. for 10 minutes. The amplified DNA fragment was purified by agarose gel electrophoresis, and digested with restriction enzymes NdeI and XhoI to obtain a NdeI and XhoI-treated orfB-containing DNA fragment.
pET-15b (produced by Novagen) was digested with restriction enzymes NdeI and XhoI to obtain a NdeI and XhoI-treated pET-15b fragment. The NdeI and XhoI-treated orfB-containing DNA fragment obtained above and the NdeI and XhoI-treated pET-15b fragment were mixed and ligated to obtain a recombinant DNA.
The recombinant DNA was used to transform E. coli Top10 according to the standard method, and the transformant was applied on a LB agar medium containing 100 μg/ml ampicillin, which was incubated overnight at 37° C. A plasmid containing the recombinant DNA was isolated from the transformant according to the standard method. Sequencing of the obtained DNA showed that the DNA had the base sequence of SEQ ID NO: 4, thereby the recombinant DNA was confirmed to have orfB, and the plasmid was named as pET-15b-orfB.
The pET-15b-orfB was introduced into E. coli BL21 (DE3) having DE3 (produced by Novagen) according to the standard method to obtain BL21(DE3)/pET-15b-orfB resistant to 100 μg/ml ampicillin. BL21(DE3)/pET-15b-orfB was cultured in 200 ml of a LB liquid medium containing 100 μg/ml ampicillin at 37° C. until the turbidity at 600 nm reached 0.5, and then the culture medium was stored in a refrigerator for 2 hours, followed by addition of isopropyl thiogalactoside at a final concentration of 0.5 mM. After an additional culture at 18° C. for 16 hours, the culture supernatant was removed by centrifugation (9000 rpm, 2 min). The cells were suspended in 10 ml of washing buffer [20 mM Tris hydrochloride (pH 8.0), 100 mM NaCl, 50 mM imidazole and 10% glycerol], to which 10 μl of 90 mg/ml phenylmethylsulfonyl fluoride (PMSF) was added, and the cells were disrupted on an ultrasonicator (produced by Wakenyaku Co., Ltd.) on ice. The obtained cell homogenate was centrifuged (10,000 rpm, 20 min, 4° C.) to obtain a supernatant. The supernatant obtained after centrifugation of the cell extract was applied on Ni Sepharose 6 Fast Flow resin (produced by GE Healthcare), and the resin was washed with 20 ml of the washing buffer. An elution buffer [20 mM Tris hydrochloride (pH 8.0), 100 mM NaCl, 500 mM imidazole and 10% glycerol] 5 ml was applied to the column to allow elution. The eluent was concentrated by using an Amicon Ultra Centrifugal Filter (produced by Merck).
Next, it was examined whether the obtained recombinant enzyme protein (OrfB) catalyses production of 2-methylacetoacetyl-CoA from acetyl-CoA and methylmalonyl-CoA by employing the following reaction conditions. A reaction solution (100 μl) containing 100 mM phosphate buffer (pH 7.5), 250 μM acetyl-CoA, 250 μM methylmalonyl-CoA and 2.5 μg orfB was prepared, and subjected to a reaction at 27° C. for 16 hours. After termination of the reaction by adding 100 μl of methanol, it was analysed on LC/MS.
As a result, production of 2-methylacetoacetyl-CoA was observed ( FIG. 2 ). The result indicates that OrfB catalyses production of 2-methylacetoacetyl-CoA from acetyl-CoA and methylmalonyl-CoA, and thus it was confirmed that the obtained enzyme protein was 3-ketoacyl-CoA synthase.
[Example 3] Confirmation of Function of the Enzyme Protein (orfD) Encoded by the Base Sequence of SEQ ID NO: 8
A recombinant plasmid sufficiently expressing a gene encoding 3-ketoacyl-CoA reductase (orfD) was constructed by PCR [Science, 230, 1350 (1985)] as described hereinbelow. OrfD was amplified by carrying out PCR on a DNA Thermal Cycler (produced by Applied Biosystems) with using the chromosomal DNA of an actinomycete Polymorphospora rubra K07-0510 as a template, a sense primer of SEQ ID NO: 14 having a NdeI restriction enzyme site at its 5′-terminal, an antisense primer of SEQ ID NO: 15 having a XhoI restriction enzyme site at its 5′-terminal, and Taq DNA polymerase (produced by Roche Life Science). The condition of PCR was 30 cycles of 95° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 1 minute followed by 72° C. for 10 minutes. The amplified DNA fragment was purified by agarose gel electrophoresis, and digested with restriction enzymes NdeI and XhoI to obtain a NdeI and XhoI-treated orfD-containing DNA fragment.
pET-15b (produced by Novagen) was digested with restriction enzymes NdeI and XhoI to obtain a NdeI and XhoI-treated pET-15b fragment. The NdeI and XhoI-treated orfD-containing DNA fragment obtained above and the NdeI and XhoI-treated pET-15b fragment were mixed and ligated to obtain a recombinant DNA.
The recombinant DNA was used to transform E. coli Top10 according to the standard method, and the transformant was applied on a LB agar medium containing 100 μg/ml ampicillin, which was incubated overnight at 37° C. A plasmid containing the recombinant DNA was isolated from the transformant according to the standard method. DNA Sequencing showed the DNA had base sequence of SEQ ID NO: 8, thereby the recombinant DNA was confirmed to have orfD, and the plasmid was termed pET-15b-orfD.
The pET-15b-orfD was introduced into E. coli BL21 (DE3) having DE3 (produced by Novagen) according to the standard method to obtain BL21(DE3)/pET-15b-orfD resistant to 100 μg/ml ampicillin. BL21(DE3)/pET-15b-orfD was cultured in 200 ml of a LB liquid medium containing 100 μg/ml ampicillin at 37° C. until the turbidity at 600 nm reached 0.5, and then the culture medium was stored in a refrigerator for 2 hours, followed by addition of isopropyl thiogalactoside at a final concentration of 0.5 mM. After an additional culture at 18° C. for 16 hours, the culture supernatant was removed by centrifugation (9000 rpm, 2 minutes). The cells were suspended in 10 ml of washing buffer [20 mM Tris hydrochloride (pH 8.0), 100 mM NaCl, 50 mM imidazole and 10% glycerol], to which 10 μl of 90 mg/ml PMSF was added, and the cells were disrupted on an ultrasonicator (produced by Wakenyaku Co., Ltd.) on ice. The obtained cell homogenate was centrifuged (10,000 rpm, 20 minutes, 4° C.) to obtain a supernatant. The supernatant obtained after centrifugation of the cell extract was applied on Ni Sepharose 6 Fast Flow resin (produced by GE Healthcare), and the resin was washed with 20 ml of the washing buffer. An elution buffer [20 mM Tris hydrochloride (pH 8.0), 100 mM NaCl, 500 mM imidazole and 10% glycerol] 5 ml was applied to the column to allow elution. The eluent was concentrated by using an Amicon Ultra Centrifugal Filter (produced by Merck).
Next, it was examined whether the obtained recombinant enzyme protein (OrfD) catalysed production of 3-hydroxy-2-methylbutyryl-CoA from 2-methylacetoacetyl-CoA by employing the following reaction conditions. A reaction solution (100 μl) containing 100 mM phosphate buffer (pH 7.5), 250 μM acetyl-CoA, 250 μM methylmalonyl-CoA, 500 μM NADPH, 2.5 μg orfB (obtained in Example 2) and 0.8 μg orfD was prepared, and subjected to a reaction at 27° C. for 16 hours. After termination of the reaction by adding 100 μl of methanol, it was analysed on LC/MS.
As a result, production of 3-hydroxy-2-methylbutyryl-CoA was observed ( FIG. 2 ). The result indicates that OrfD catalyses production of 3-hydroxy-2-methylbutyryl-CoA from 2-methylacetoacetyl-CoA, and thus it was confirmed that the obtained enzyme protein was 3-ketoacyl-CoA reductase.
[Example 4] Confirmation of Function of the Enzyme Protein (orfA) Encoded by the Base Sequence of SEQ ID NO: 2
A recombinant plasmid sufficiently expressing a gene encoding enoyl-CoA hydratase (orfA) was constructed by PCR [Science, 230, 1350 (1985)] as described hereinbelow. OrfA was amplified by carrying out PCR on a DNA Thermal Cycler (produced by Applied Biosystems) with using the chromosomal DNA of an actinomycete Polymorphospora rubra K07-0510 as a template, a sense primer of SEQ ID NO: 16 having a NdeI restriction enzyme site at its 5′-terminal, an antisense primer of SEQ ID NO: 17 having a BamHI restriction enzyme site at its 5′-terminal, and Taq DNA polymerase (produced by Roche Life Science). The condition of PCR was 30 cycles of 95° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1 minute followed by 72° C. for 10 minutes. The amplified DNA fragment was purified by agarose gel electrophoresis, and digested with restriction enzymes NdeI and BamHI to obtain a NdeI and BamHI-treated orfA-containing DNA fragment.
pET-15b (produced by Novagen) was digested with restriction enzymes NdeI and BamHI to obtain a NdeI and BamHI-treated pET-15b fragment. The NdeI and BamHI-treated orfA-containing DNA fragment obtained above and the NdeI and BamHI-treated pET-15b fragment were mixed and ligated to obtain a recombinant DNA.
The recombinant DNA was used to transform E. coli Top10 according to the standard method, and the transformant was applied on a LB agar medium containing 100 μg/ml ampicillin, and incubated overnight at 37° C. A plasmid containing the recombinant DNA was isolated from the transformant according to the standard method. Sequencing showed the DNA had the base sequence of SEQ ID NO: 2, thereby the recombinant DNA was confirmed to have orfA, and the plasmid was termed pET-15b-orfA.
The pET-15b-orfA was introduced into E. coli BL21 (DE3) having DE3 (produced by Novagen) according to the standard method to obtain BL21(DE3)/pET-15b-orfA resistant to 100 μg/ml ampicillin. BL21(DE3)/pET-15b-orfA was cultured in 200 ml of a LB liquid medium containing 100 μg/ml ampicillin at 37° C. until the turbidity at 600 nm reached 0.5, and then the culture medium was stored in a refrigerator for 2 hours, followed by addition of isopropyl thiogalactoside at a final concentration of 0.5 mM. After an additional culture at 18° C. for 16 hours, the culture supernatant was removed by centrifugation (9000 rpm, 2 minutes). The cells were suspended in 10 ml of washing buffer [20 mM Tris hydrochloride (pH 8.0), 100 mM NaCl, 50 mM imidazole and 10% glycerol], to which 10 μl of 90 mg/ml PMSF was added, and the cells were disrupted on an ultrasonicator (produced by Wakenyaku Co., Ltd.) on ice. The obtained cell homogenate was centrifuged (10,000 rpm, 20 minutes, 4° C.) to obtain a supernatant. The supernatant obtained after centrifugation of the cell extract was applied on Ni Sepharose 6 Fast Flow resin (produced by GE Healthcare), and the resin was washed with 20 ml of the washing buffer. An elution buffer [20 mM Tris hydrochloride (pH 8.0), 100 mM NaCl, 500 mM imidazole and 10% glycerol] 5 ml was applied to the column to allow elution. The eluent was concentrated by using an Amicon Ultra Centrifugal Filter (produced by Merck).
Next, it was examined whether the obtained recombinant enzyme protein (OrfA) catalysed production of angelyl-CoA from 3-hydroxy-2-methylbutyryl-CoA by employing the following reaction conditions. A reaction solution (100 μl) containing 100 mM phosphate buffer (pH 7.5), 250 μM acetyl-CoA, 250 μM methylmalonyl-CoA, 500 μM NADPH, 2.5 μg orfB (obtained in Example 2), 0.8 μg orfD (obtained in Example 3) and 15.8 μg orfA was prepared and subjected to a reaction at 27° C. for 16 hours. After termination of the reaction by adding 100 μl of methanol, it was analysed on LC/MS.
As a result, production of angelyl-CoA was observed ( FIG. 2 ). The result indicates that OrfA catalyses production of angelyl-CoA from 3-hydroxy-2-methylbutyryl-CoA, and thus it was confirmed that the obtained enzyme protein was enoyl-CoA hydratase.
As described above, it was showed that expression of OrfA, OrfB, OrfC and OrfD in combination produced trehangelin in Example 1. It was also showed that OrfB produced 2-methylacetoacetyl-CoA by reacting acetyl-CoA with methylmalonyl-CoA, that OrfD converted 2-methylacetoacetyl-CoA to 3-hydroxy-2-methylbutyryl-CoA, and that OrfA converted 3-hydroxy-2-methylbutyryl-CoA to angelyl-CoA, in Examples 2 to 4. These show that OrfC produced trehangelin by reacting angelyl-CoA with trehalose in the test system of Example 1, in other words, OrfC had an acyltransferase activity.
[Example 5] Production of Trehangelin in Corynebacteria
Trehangelin was produced in Corynebacterium glutamicum.
(1) Analytical Method
Column: YMC-PACK ODS-AQ 250×4.6 mm S-5 mm, 12 nm, 30° C. Mobile phase: 0.1% formic acid (A), acetonitrile (B), 15% B, 30 minutes, 0.5 mL/min
Detection: Agilent 6224 TOF LC/MS (Agilent Technologies)
(2) Introduction of Trehangelin Biosynthesis Enzyme Genes into Corynebacterium glutamicum
The nucleic acid sequences of SEQ ID NO: 18 and SEQ ID NO: 19 were designed as the sequences of orfAB and orfCD in which codon usage was optimized to Corynebacterium glutamicum . Based on the designed nucleic acid sequences, nucleic acid molecules encoding orfAB and orfCD were artificially synthesized. Then orfABCD were linked and inserted into a vector.
Specifically, orfAB and orfCD were respectively amplified by PCR with using PrimeSTAR Max DNA Polymerase (TAKARA) and using respective primers for orfAB and orfCD, reacting at 98° C. for 1 minute followed by 30 cycles of one cycle reaction at 98° C. for 10 seconds and at 68° C. for 30 seconds. The orfAB primers and orfCD primers used are as follows:







orfAB primer (forward):


(SEQ ID NO: 20)


AGAGGAGACACAACGAGCTCATGTCCGTTTCCCGCGTTG





orfAB primer (reverse):


(SEQ ID NO: 21)


AGCGGAGGTGGTCATCACTTTAGCGAACGCAGTTG





orfCD primer (forward):


(SEQ ID NO: 22)


ATGACCACCTCCGCTCTG





orfCD primer (reverse):


(SEQ ID NO: 23)


CCGATATCCTGCAGGAGCTCTTAGCCCAGGCCGTAGCC.






















The obtained PCR fragments were purified with Gel/PCR extraction kit (Japan). The purified PCR fragments were mixed with a vector digested with SacI, and two gene fragments were ligated using In-Fuion HD Cloning Kit as simultaneously inserting into the vector. The vector used was pYTKA9-PgapA containing the gapA gene promoter. pYTKA9-PgapA was obtained by artificially creating the pBL1 ori, a typical ori for corynebacteria (Santamaria, R., Gil, J. A., Mesase, J. M. and Martin, J. F. (1984) J. Gen. Microbiol. 130, 2237-2246.), which was introduced into pHSG298 vector (Takara Bio Inc.) with the gapA gene promoter (Hasegawa et al., Appl Environ Microbiol. (2012) 78(3): 865-75). The obtained vector containing nucleic acid molecules encoding orfAB and orfCD was used to transform E. coli HST02 in the LB medium containing kanamycin (50 μg/mL). Further, Corynebacterium glutamicum ATCC 13032 was transformed by electroporation to obtain Corynebacterium glutamicum having trehangelin biosynthesis enzyme genes.
(3) Preculture of Corynebacterium glutamicum
A glycerol stock of Corynebacterium glutamicum ATCC 13032 having trehangelin biosynthesis enzyme genes prepared above was added to 5 mL of BHI medium containing 37 g of brain heart infusion in 1000 mL of pure water. Corynebacterium glutamicum was cultured while shaking at 30° C. and 180 rpm for 24 hours to obtain a preculture solution.
(4) Main Culture of Corynebacterium glutamicum
The preculture solution at the amount of 3% was added to a Sakaguchi Flask containing 50 mL of BHI medium, which was added 2.5 mL of 400 g/L glucose solution so that the initial concentration of glucose at the start of the culture became 20 g/L. At 24 and 48 hours after starting the culture, 2.5 mL of the same glucose solution was added. 100 g/L trehalose solution was added 1 mL at the start of the culture, and added 0.25 mL at each of 24 and 48 hours after the start of the culture. In order to adjust pH, 1.25 mL of 20% calcium carbonate solution was added at 4 and 24 hours after the start of the culture. Culture was carried out at 30° C. for 72 hours with shaking at 180 rpm.
(5) Analysis of Culture Supernatant
The culture supernatant at 72 hours after the start of the main culture was analysed by LC/MS, and a peak of trehangelin was detected at the same retention time as the authentic preparation.