JP2017055703A - Shyobunol synthetase, polypeptide, gene, and recombinant cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel shyobunol synthetase and a gene thereof.SOLUTION: The present invention provides an isolated shyobunol synthase comprising the protein of (a), (b) or (c) below and a functional fragment thereof: (A) a protein consisting of a specific amino acid sequence; (B) a protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, or added in the specific amino acid sequence and having an activity of converting farnesyl diphosphate to shyobunol; and (c) a protein having an amino acid sequence showing a homology of 80% or more with the specific sequence and having an activity of converting farnesyl diphosphate to shyobunol.SELECTED DRAWING: Figure 1

Description

  The present invention relates to shovenol synthase, polypeptides, genes, and recombinant cells.

  Sesquiterpene is a generic name for compounds that follow the isoprene rule with 15 carbons, using farnesyl diphosphate (FPP) in which three molecules of isopentenyl diphosphate (IPP) are condensed as biosynthetic precursors. Currently, over 3000 types of sesquiterpenes are known. Sesquiterpenes have a fragrance like roses and citrus fruits, and are often used in perfumes. It also acts as a physiologically active substance and is used in pharmaceuticals.

  Shyobunol, a kind of sesquiterpene, is useful as a fragrance, a cosmetic, a pharmaceutical product and an intermediate thereof, and a resin raw material.

  The following pathways can be considered as the biosynthesis pathway of shobunol. First, geranyl diphosphate (GPP) is biosynthesized from isopentenyl diphosphate (IPP) by the action of geranyl diphosphate (GPP) synthase. Is done. Subsequently, farnesyl diphosphate (FPP) is biosynthesized from geranyl diphosphate (GPP) by the action of farnesyl diphosphate (FPP) synthase. Then, shobunol is biosynthesized from farnesyl diphosphate (FPP) by the action of shovenol synthase. However, to date, no shobunol synthase has been identified.

  Examples of sesquiterpene synthases other than shobunol synthase include those disclosed in Patent Documents 1 to 3.

JP2013-63063A JP 2011-125272 A JP 2011-55721 A

  In view of the above-mentioned present situation, an object of the present invention is to provide an isolated shownol synthase and its gene, and a recombinant cell into which the gene is introduced and capable of producing shownol.

One aspect of the present invention is an isolated shobunol synthase comprising the following protein (a), (b) or (c).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2,
(B) a protein comprising an amino acid sequence in which 1 to 20 amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 and having an activity of converting farnesyl diphosphate into shovenol,
(C) a protein having an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having an activity of converting farnesyl diphosphate into shobunol.

  Another aspect of the present invention is a polypeptide having a partial sequence of the above-described shobunol synthase, which has an activity of converting farnesyl diphosphate into shovenol.

  Another aspect of the present invention is an isolated gene encoding the above-described shovenol synthase or polypeptide.

  Another aspect of the present invention is a recombinant cell having the above gene and capable of producing shovenol by expressing the gene.

  Preferably, the recombinant cell is a bacterium or yeast.

  Preferably, the gene is exogenous to the host cell.

  Preferably, the gene is endogenous to the host cell.

  According to the present invention, a novel shobunol synthase and its gene are provided. Furthermore, according to the present invention, it is possible to produce shobunol using recombinant cells.

It is explanatory drawing showing the biosynthetic pathway from the chemical structure of a shobunol and a farnesyl diphosphate. It is the photograph showing the root and leaf of the vetiver (Vetiveria zizanioides) used in the Example. It is a chromatogram showing the result of gas chromatography. It is a mass spectrum showing the result of mass spectrometry.

  Hereinafter, embodiments of the present invention will be described. In the present invention, the term “gene” can be replaced by the term “nucleic acid” or “DNA”.

One aspect of the present invention is an isolated shobunol synthase comprising the following protein (a), (b) or (c).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2,
(B) a protein comprising an amino acid sequence in which 1 to 20 amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 and having an activity of converting farnesyl diphosphate into shovenol,
(C) a protein having an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having an activity of converting farnesyl diphosphate into shobunol.

  SEQ ID NO: 1 is a base sequence of a gene (cDNA) encoding a shovenol synthase isolated from vetiver (Vetiveria zizanioides) by the present inventors, and a corresponding amino acid sequence. SEQ ID NO: 2 is an excerpt of only the amino acid sequence of SEQ ID NO: 1. That is, SEQ ID NO: 2 represents the amino acid sequence of schivenol synthase derived from vetiver.

  The shovenol synthase of the present invention comprises an amino acid sequence in which 1 to 20 amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and converts farnesyl diphosphate into shovenol. A protein having the activity of The number of amino acids deleted, substituted or added is 1 to 20, preferably 1 to 15, more preferably 1 to 10, and still more preferably 1 to 5.

  The shovenol synthase of the present invention includes a protein having an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having an activity of converting farnesyl diphosphate into shovenol. To do. The homology with the amino acid sequence represented by SEQ ID NO: 2 is 80% or more, preferably 85% or more, more preferably 90% or more, and further preferably 95% or more.

  The present invention includes a functional fragment of Shobunol synthase comprising the protein (a), (b) or (c). The functional fragment of schovenol synthase refers to a polypeptide having a partial sequence of schovenol synthase, which has the activity of schovenol synthase (the activity of converting farnesyl diphosphate to shobunol). . For example, a polypeptide of the above-mentioned schovenol synthase and having an activity of converting farnesyl diphosphate into schovenol is a functional fragment of schovenol synthase.

  FIG. 1 shows the chemical structure of shobunol and the biosynthetic pathway from farnesyl diphosphate. The shovenol synthase of the present invention catalyzes the reaction shown in FIG.

  The present invention includes an isolated gene that encodes the above-mentioned shovenol synthase or a functional fragment thereof.

  The present invention includes a recombinant cell having the above gene and capable of producing shobunol by expressing the gene.

  The gene introduced into the host cell may be a gene that the host cell does not originally have, that is, an exogenous gene, or a gene that the host cell originally has, that is, an endogenous gene. Also good. The latter corresponds to so-called self-cloning.

The host cell is not particularly limited and may be a prokaryotic cell or a eukaryotic cell. Examples of prokaryotic cells include bacteria and actinomycetes. Examples of eukaryotic cells include yeast, filamentous fungi, eukaryotic microalgae, plant cells, and animal cells.
Of these, bacteria and yeast are particularly preferably used as host cells.

A method for introducing a gene into a host cell is not particularly limited, and may be appropriately selected depending on the type of the host cell. For example, a vector that can be introduced into a host cell and can express an integrated gene can be used.
For example, when the host cell is a prokaryotic organism such as a bacterium, the vector can be replicated in the host cell or can be integrated into a chromosome, and can be transcribed into the inserted gene (nucleic acid, DNA). Those containing a promoter can be used. For example, it is preferable to construct a series of components comprising a promoter, a ribosome binding sequence, the above gene, and a transcription termination sequence in the host cell using the vector.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, this invention is not limited only to these Examples.

  In this example, the base sequence of the vetiver-derived schovenol synthase gene was determined, recombinant Escherichia coli into which the gene was introduced was prepared, and the recombinant Escherichia coli was cultured to perform biosynthesis of schovenol.

(1) Analysis of transcripts of vetiver and determination of the base sequence of schovenol synthase gene (VzTPS1) The root and leaf tissues (FIG. 2) of vetiver zizanioides were cut out and frozen in liquid nitrogen. Total RNA was extracted using a bead crusher (FastPrep 24, MP Biomedicals) and FastRNA Pro Green Kit (MP Biomedicals). Using the Agilent 2100 Bioanalyzer (Agilent Technology) and the Agilent RNA6000 Nano Kit (Agilent Technology), RNA quality was quantitatively evaluated by RNA Integrity Number (RIN), and RIN = Eight or more samples were subjected to subsequent experiments.

  5 μg each of total RNA obtained from roots and leaves was provided to Hokkaido System Science, and RNA sequence analysis, sequence data mixing assembly, and homology search using BlastX were performed. As a result of sequencing and mixed assembly, 177,472 contig sequences were obtained. As a result of homology search, a gene fragment showing 52% homology with Zea mays terpene synthase 7 (Genbank accession DAA38747) and 54%, Aegilops tauschii (+)-delta-cadinene synthase (Genbank accession EMT11542) ( SEQ ID NO: 1) was found. This gene fragment was named VzTPS1. VzTPS1 contained a 1521 bp open reading frame (ORF) and encoded a protein predicted to have 506 amino acid residues, a molecular weight of 58.3 kDa, and a pI = 5.8. The deduced amino acid sequence (SEQ ID NO: 2) of VzTPS1 contained a motif conserved in many terpene synthases, such as RxR and DDxxD. Further, it was predicted that the N-terminal amino acid sequence of VzTPS1 does not contain a plastid translocation sequence.

(2) Preparation of Shobunol synthase gene (VzTPS1) expression plasmid (pVzTPS1)
The deduced amino acid sequence of VzTPS1 (SEQ ID NO: 2) was provided to GenScript, and a VzTPS1 gene optimized for E. coli codon usage was prepared by artificial synthesis (SEQ ID NO: 3). At this time, a restriction enzyme NdeI recognition sequence was added to the 5 ′ end so as to include the initiation methionine codon ATG, and a restriction enzyme XhoI recognition sequence was added immediately below the stop codon. The prepared plasmid containing the artificially synthesized gene was digested with NdeI and XhoI, and the obtained DNA fragment was ligated to the NdeI and XhoI sites of pRSFDuet-1 plasmid (Novagen). This plasmid was named pVzTPS1.

(3) Synthesis of Shobunol by E. coli expression system using plasmid pVzTPS1
pVzTPS1 plasmid was introduced into Escherichia coli together with pAC-Mev plasmid according to the method described in Harada H, Yu F, Okamoto S, Kuzuyama T, Utsumi R, Misawa N 2009, 81: 915-925, and a liquid medium supplemented with dodecane Cultivation, production and extraction of the product were carried out. As a result of analyzing 1 μL of the extracted dodecane layer with a gas chromatograph mass spectrometer (GC-MS), an E. coli-derived sample into which pVzTPS1 plasmid was introduced was found to be an E. coli-derived sample into which pRSFDuet-1 was introduced as a control. A plurality of new peaks that could not be obtained appeared (FIG. 3). As a result of mass spectrum analysis of the peak with the longest retention time of 37.5 minutes (FIG. 2, peak 1), it was shown that this peak coincided with the spectrum of Shobunol (FIG. 4).
From the above results, it was shown that VzTPS1 is an enzyme gene that specifically synthesizes shobunol using farnesyl diphosphate as a substrate.

Claims (7)

An isolated shobunol synthase comprising the following protein (a), (b) or (c).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2,
(B) a protein comprising an amino acid sequence in which 1 to 20 amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 and having an activity of converting farnesyl diphosphate into shovenol,
(C) a protein having an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having an activity of converting farnesyl diphosphate into shobunol.   A polypeptide having a partial sequence of the shobunol synthase according to claim 1, which has an activity of converting farnesyl diphosphate into shobunol.   An isolated gene encoding the shovenol synthase of claim 1 or the polypeptide of claim 2.   A recombinant cell comprising the gene according to claim 3 and capable of producing shobunol by expressing the gene.   The recombinant cell according to claim 4, which is a bacterium or a yeast.   The recombinant cell according to claim 4 or 5, wherein the gene is exogenous to the host cell.   The recombinant cell according to claim 4 or 5, wherein the gene is endogenous to the host cell.