WO2018138966A1 - Transformant and use thereof - Google Patents

Abstract

Provided are: a transformant capable of expressing a gene encoding an exogenous isoprenoid biosynthetic enzyme; and a method for producing isoprenoid or an isoprenoid precursor using the transformant. The method uses a transformant capable of expressing an isoprenoid biosynthetic enzyme gene with a promoter sequence obtained from oleaginous yeast.

Description

Transformant and use thereof

The present invention relates to a transformant and a method for producing an isoprenoid precursor or isoprenoid using the transformant.

About 40,000 kinds of isoprenoids (also called terpenoids) have been isolated from various organisms such as animals and microorganisms so far, forming the most diverse group of compounds in nature. The isoprenoid contains many industrially useful compounds such as compounds used as pharmaceuticals, agricultural chemicals, functional foods or fragrances. Since many of these compounds accumulate only in trace amounts in nature, development of production technology using genetic recombination technology is underway.

Under these circumstances, progress in recombinant DNA technology is making it possible to produce target metabolites more efficiently using microorganisms that have not been used as eukaryotic hosts. One example is oleaginous yeast, which is a generic term for a series of yeasts that have the ability to produce and accumulate as much as 20% of lipids in biomass. These yeasts are expected to have high potential activity in the isoprenoid biosynthetic pathway due to their high lipid-producing ability. By further strengthening the isoprenoid biosynthetic pathway of this oleaginous yeast, it can be expected that isoprenoid compounds will be produced at high yields.

In the production of isoprenoid compounds, the mevalonate pathway plays an especially important role. It has been reported in Saccharomyces cerevisiae that the hydroxymethylglutaryl coenzyme A reductase gene constituting the mevalonate pathway is one of the rate-limiting enzymes of the mevalonate pathway.

Improvement of the mevalonate pathway using the hydroxymethylglutaryl coenzyme A reductase gene has been studied in budding yeast and fission yeast (Schizosaccharomyces pombe), which are conventional yeasts. GAPDH, GAL1, PGK1, TDH3, TEF2, It is reported that the biosynthesis of isoprenoids and the like downstream of the mevalonate pathway is promoted by expressing the hydroxymethylglutaryl coenzyme A reductase gene under the control of the NMT1 gene promoter (Non-Patent Document). 1, Non-Patent Document 2 and Non-Patent Document 3). On the other hand, in non-conventional yeasts, hydroxymethylglutaryl coenzyme A reduction is performed under the control of TEF gene promoter or GAP gene promoter in Yarrowia lipolitica and Candida utilis, which are one of oily yeasts. It has succeeded in increasing the productivity of isoprenoids by expressing an enzyme gene, and the production amount of lycopene is increased 4.2 times under the control of the TEF gene promoter and 3.9 times under the control of the GAP gene promoter. It has been reported (Non-patent document 4 and Non-patent document 5). As explained above, there is very little information on promoters that can be used in oleaginous yeast.

At present, genome base sequences are disclosed in various yeasts such as budding yeast and fission yeast. The genome base sequence of Lipomyces starkeyi, which is one kind of oleaginous yeast, is also available in the Joint Genome Institute (http: //jgi.doe.gov/)

K. Allen G. Donald et. Al., Appl Environment Microbiol, 1997, Vol. 63 (9) P.3341-3344 Chikara Ohto et. Al., Appl Microbiol Biotechnol, 2009, Vol. 82, P.837-845 Bing Cheng et. Al., Appl Biochem Biotechnol, 2010, Vol. 160, P. 523-531 Falk Matthaus et. Al., Appl Enviroment Microbiol, 2014, Vol. P.1660-1669 Hiroshi shimada et. Al., Appl Enviroment Microbiol, 1998, Vol. 64, P.2676-2680 Nikolai A. Shevchuk et. Al., Nucleic Acids Research, 2004, Vol. 32 (2) e19 Christopher H. Calvey et. Al., Current Genetics, 2014, 60 (3) P.223-230

Depending on the eukaryotic host (eg, oleaginous yeast), information on promoters and terminators that control gene expression is extremely lacking, and expression control sequences suitable for expressing genes encoding isoprenoid biosynthetic enzymes are prepared. There was a need to do.

One aspect of the present invention is to provide a transformant capable of expressing a gene encoding an exogenous isoprenoid biosynthetic enzyme, and a method for producing an isoprenoid precursor or isoprenoid using the transformant.

The present inventors have intensively studied to solve the above-mentioned problems. As a result, the genes of oleaginous yeast expressed in the lipid production medium were examined, and the expression control sequences of the genes that showed relatively high expression in the lipid production medium were found. The present inventors have obtained and searched for an expression control sequence suitable for the expression of an isoprenoid biosynthetic enzyme gene using these expression control sequences as an index of the production amount of isoprenoids, thereby completing the present invention.

The present invention includes the inventions described in the following (1) to (9).
(1) A polynucleotide construct comprising, in a eukaryotic host, a gene encoding an isoprenoid biosynthetic enzyme and at least one polynucleotide selected from the group consisting of the following (a) to (d) upstream of the gene: A transformant characterized in that is introduced:
(A) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 or a pyruvate kinase promoter sequence or a part thereof;
(B) hybridizes under stringent conditions with a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 or a polynucleotide comprising a promoter sequence of pyruvate kinase, or a portion complementary thereto, and promoter activity A polynucleotide having
(C) consisting of a polynucleotide or part thereof in which one or several bases are substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 1 or the promoter sequence of pyruvate kinase, and A polynucleotide having promoter activity;
(D) A polynucleotide comprising a nucleotide sequence described in SEQ ID NO: 1 or a polynucleotide having 90% or more identity with the promoter sequence of pyruvate kinase or a part thereof and having promoter activity.

(2) The polynucleotide construct further comprises at least one polynucleotide selected from the group consisting of the following (e) to (h) downstream of the gene encoding the isoprenoid biosynthetic enzyme: The transformant according to (1), characterized by:
(E) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 2 or a terminator sequence of pyruvate kinase or a part thereof;
(F) hybridizing under stringent conditions with a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 2 or a polynucleotide comprising the terminator sequence of pyruvate kinase, or a nucleotide sequence complementary thereto, and terminator activity Or a portion thereof having
(G) in the nucleotide sequence of SEQ ID NO: 2 or the terminator sequence of pyruvate kinase, consisting of a polynucleotide or part thereof in which one or several bases are substituted, deleted, inserted and / or added, and A polynucleotide having terminator activity;
(H) a polynucleotide having a terminator activity, comprising a polynucleotide having 90% or more identity with the nucleotide sequence of SEQ ID NO: 2 or the terminator sequence of pyruvate kinase or a part thereof.

(3) a polynuclear host comprising a gene encoding an isoprenoid biosynthetic enzyme and at least one polynucleotide selected from the group consisting of the following (a ′) to (d ′) upstream of the gene in a eukaryotic host: A transformant, characterized in that a nucleotide construct has been introduced:
(A ′) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 5 or a phosphoketolase promoter sequence or a portion thereof;
(B ′) hybridizes under stringent conditions to a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 5 or a polynucleotide comprising a phosphoketolase promoter sequence or a part thereof and a nucleotide sequence complementary thereto, and has promoter activity A polynucleotide having;
(C ′) a nucleotide sequence as set forth in SEQ ID NO: 5 or a phosphoketolase promoter sequence, consisting of a polynucleotide or part thereof in which one or several bases are substituted, deleted, inserted and / or added, and a promoter An active polynucleotide;
(D ′) a polynucleotide comprising a nucleotide sequence described in SEQ ID NO: 5 or a polynucleotide having 90% or more identity with the promoter sequence of phosphoketolase or a part thereof, and having promoter activity.

(4) The polynucleotide construct further includes at least one polynucleotide selected from the group consisting of the following (e ′) to (h ′) downstream of the gene encoding the isoprenoid biosynthetic enzyme. The transformant according to (3), characterized in that:
(E ′) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 6 or a phosphoketolase terminator sequence, or a portion thereof;
(F ′) hybridizes under stringent conditions with a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 6 or a phosphoketolase terminator sequence or a polynucleotide comprising a base sequence complementary thereto, and has a terminator activity; A polynucleotide having;
(G ′) a nucleotide sequence described in SEQ ID NO: 6 or a terminator sequence of phosphoketolase, wherein the terminator comprises a polynucleotide or a part thereof, wherein one or several bases are substituted, deleted, inserted and / or added. An active polynucleotide;
(H ′) A polynucleotide having a terminator activity, comprising a polynucleotide having a nucleotide sequence of SEQ ID NO: 6 or a phosphoketolase terminator sequence of 90% or more, or a part thereof, and a part thereof.

(5) The transformant according to any one of (1) to (4), wherein the eukaryotic host is an oleaginous yeast.

(6) The transformant according to (5), wherein the oleaginous yeast is Lipomyces starkeyi.

(7) The isoprenoid biosynthetic enzyme is a mevalonate pathway enzyme, carotenoid biosynthetic enzyme, monoterpene biosynthetic enzyme, sesquiterpene synthase, triterpene synthase, or diterpene synthase, (1) The transformant according to any one of (6) to (6).

(8) The transformant according to (7), wherein the mevalonate pathway enzyme is hydroxymethylglutaryl coenzyme A reductase (HMG-CoA Redactase, HMGR).

(9) a culture step of culturing the transformant according to any one of (1) to (8);
A method for producing an isoprenoid precursor or an isoprenoid, comprising a recovery step of recovering a target substance from a culture medium after culture and / or the transformant.

According to one aspect of the present invention, there is an effect that an isoprenoid precursor and an isoprenoid can be efficiently produced using a eukaryotic host such as an oleaginous yeast.

It is a figure which shows the result of the gene expression level analysis by real-time PCR in the Example of this application. It is a figure which shows the construction method of the shortening type | mold hydroxymethylglutaryl coenzyme A reductase (tHMGR) gene expression vector in the Example of this application. It is a figure which shows the construction method of a lycopene biosynthesis vector (pUC-lyc) in the Example of this application. It is a figure which shows the comparison result of the production amount of lycopene in the expression control strain | stump | stock of the shortening type | mold hydroxymethylglutaryl coenzyme A reductase gene in the Example of this application.

One embodiment of the present invention will be described below, but the present invention is not limited to this. The present invention is not limited to each configuration described below, and various modifications can be made within the scope shown in the claims, and technical means disclosed in different embodiments and examples respectively. Embodiments and examples obtained by appropriately combining them are also included in the technical scope of the present invention. Moreover, all the academic literatures and patent literatures described in this specification are used as references in this specification. Unless otherwise specified in the present specification, “A to B” representing a numerical range is intended to be “A or more and B or less”.

As used herein, the terms “polynucleotide” and “gene” are used interchangeably with “nucleic acid” or “nucleic acid molecule” and are intended to be a polymer of nucleotides. Here, the gene may be in the form of DNA (eg, cDNA or genomic DNA) or in the form of RNA (eg, mRNA). DNA and RNA may be double-stranded or single-stranded. Single-stranded DNA and single-stranded RNA may be a coding strand (sense strand) or a non-coding strand (antisense strand). In addition, the gene may be a chemically synthesized gene or a gene in which the codon usage is changed so that the expression of the encoded protein is improved. Substitutions can be made between codons encoding the same amino acid.

Also, the term “protein” is used interchangeably with “peptide” or “polypeptide”. As used herein, the base and amino acid notation uses the one-letter code or three-letter code defined by IUPAC and IUB as appropriate.

In the present specification, the term “isoprenoid” refers to geranyl diphosphate (also referred to as GDP and GPP) (including various isomers of GPP), farnesyl diphosphate (FDP and PGP) as substrates due to the catalytic activity of isoprenoid biosynthetic enzymes. C _x H _y O _z (also referred to as FPP) (including various isomers of FPP) and / or geranylgeranyl diphosphate (also referred to as GGDP and GGPP) (including various isomers of GGPP) (x Is 10, 15, 20, 30, or 40, y is 14 to 54, and y is 0 to 4). The term “isoprenoid” is intended to mean a compound obtained by modifying the compound with an increase in the number of carbon atoms, a decrease in the number of carbon atoms, or a modification such as oxidation. A dehydration reaction or the like may be carried out in the isoprenoid by an in vivo enzyme reaction such as cyclization of the isoprenoid due to catalytic activity, oxidation and hydroxylation, and the accompanying non-enzymatic reaction, but is not limited thereto. Isoprenoids can take any form, acyclic, monocyclic or polycyclic, but in common have an isoprenoid basic skeleton. Accordingly, any compound having an isoprenoid basic skeleton is encompassed by the concept of isoprenoids herein.

In the present specification, “homology” means a ratio having the same base sequence as a similar base sequence. In addition, “identity” intends a ratio having the same base sequence as the comparative base sequence.

[1. Transformant)
The transformant according to one embodiment of the present invention includes a gene encoding an isoprenoid biosynthetic enzyme and a gene (operably or functionally linked) below (operably or functionally linked) to a eukaryotic host: A polynucleotide construct comprising at least one polynucleotide selected from the group consisting of a) to (d) or (a ′) to (d ′) is introduced.
(A) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 or a pyruvate kinase promoter sequence or a part thereof;
(B) hybridizes under stringent conditions with a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 or a polynucleotide comprising a promoter sequence of pyruvate kinase, or a portion complementary thereto, and promoter activity A polynucleotide having:
(C) consisting of a polynucleotide or part thereof in which one or several bases are substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 1 or the promoter sequence of pyruvate kinase, and A polynucleotide having promoter activity;
(D) A polynucleotide comprising a nucleotide sequence described in SEQ ID NO: 1 or a polynucleotide having 90% or more identity with the promoter sequence of pyruvate kinase or a part thereof and having promoter activity.
(A ′) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 5 or a phosphoketolase promoter sequence or a portion thereof;
(B ′) hybridizes under stringent conditions to a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 5 or a polynucleotide comprising a phosphoketolase promoter sequence or a part thereof and a nucleotide sequence complementary thereto, and has promoter activity A polynucleotide having;
(C ′) a nucleotide sequence as set forth in SEQ ID NO: 5 or a phosphoketolase promoter sequence, consisting of a polynucleotide or part thereof in which one or several bases are substituted, deleted, inserted and / or added, and a promoter An active polynucleotide;
(D ′) a polynucleotide comprising a nucleotide sequence described in SEQ ID NO: 5 or a polynucleotide having 90% or more identity with the promoter sequence of phosphoketolase or a part thereof, and having promoter activity.

In the transformant according to one embodiment of the present invention, a specific gene and a polynucleotide construct are introduced into a eukaryotic host.

As used herein, “eukaryotic host” means a host derived from a eukaryotic organism (for example, a eukaryotic cell or a eukaryotic organism). The eukaryote may be, for example, a non-human eukaryote. Examples of the eukaryotic host include microorganisms that perform alcoholic fermentation such as yeast and acid-resistant microorganisms. Among eukaryotic hosts, oleaginous yeast is preferred. The oleaginous yeast means a yeast that has a higher ability to produce and accumulate lipid than general yeast. If the eukaryotic host is an oleaginous yeast, it is preferable because the potential activity of the isoprenoid biosynthetic pathway is high.

The term “oleaginous yeast” refers to oleaginous microorganisms classified as yeast that can produce oil, ie, can accumulate lipids in excess of about 20% of their dry cell weight (“DCW”). Examples of oleaginous yeast include the genus Yarrowia, the genus Candida, the genus Rhodotorula, the genus Rhodosporidium, the genus Cryptococcus, the genus Trichosporon Examples include, but are not limited to, genera. The ability of yeast to accumulate lipids in excess of about 20% of DCW may be the ability gained through attempts at recombinant genetic engineering or the ability through the natural ability of an organism.

For example, the oily yeast includes Lipomyces starkeyi, Lipomyces embembus, Lipomyces doorenjongii, and Lipomyces kockii, etc .; (Rhodotorula graminis), Rhodotorula glutinis, Rhodotorula mucilaginosa, Rhodotorula aurantiaca, Rhodotorula ulauroca rosula Rhodotorula yeasts such as Rhodosporidium toruloides; Cryptococcus curvatus and Cryptococcus albidus and other Cryptococcus yeasts; some Candida curvata yeasts; Yarrowia lipolitica; Xanthophyllomyces dendrorhous; Etc. Among them, as the oleaginous yeast, Lipomyces starkeyi is preferable.

In the transformant according to one embodiment of the present invention, a gene encoding an isoprenoid biosynthetic enzyme is introduced.

The isoprenoid biosynthetic enzyme is not particularly limited as long as it is an enzyme that biosynthesizes isoprenoid or an isoprenoid precursor, that is, an enzyme having a catalytic action using the isoprenoid or isoprenoid precursor as a substrate, but is not limited to monoterpenes, sesquiterpenes, diterpenes, triterpenes Alternatively, an enzyme that biosynthesizes carotenoids or an enzyme that uses these as a substrate can be mentioned. Specifically, the enzyme may be monoterpene, sesquiterpene, diterpene, triterpene, or carotenoid with various functional groups (eg, —CO—, —OH, —O—, —COH, —COOH, —SH). , -C = C-, -C = C = C-, -O-O-, -NH ₂ , or -C≡C-). More specifically, the enzymes include hydroxymethylglutaryl coenzyme A reductase (HMG-CoA Redactase, HMGR), GGPP synthase, phytoene synthase, phytoene desaturase, lycopene cyclase, β-carotene ketase and β- Carotenoid biosynthetic enzymes such as carotene hydroxylase; monoterpene synthases such as menthol, limonene, geraniol, citronellol, β-myrcene and linalool; betulinic acid synthase, lupeol synthase, glycyrrhizin synthase, squalene synthase, ursolic acid Synthetic enzymes and triterpene biosynthetic enzymes such as oleanolic acid synthase; diterpene biosynthetic enzymes such as stevioside synthase and taxadiene synthase; farnesol synthase, elemol synthase, gel Maclene D synthase, β-elemene synthase, β-caryophyllene synthase, β-eudesmol synthase, α-neocloben synthase, β-cubeben synthase, cedrene synthase, artemisinin synthase, santonin synthase and farnesene synthase And sesquiterpene biosynthetic enzymes. Preferably, the enzyme is a mevalonate pathway enzyme, a carotenoid biosynthetic enzyme, a monoterpene biosynthetic enzyme, a sesquiterpene synthase, a triterpene synthase, or a diterpene synthase, and more preferably, the enzyme is hydroxymethylglutamate. Lyl coenzyme A reductase (HMG-CoA reductase). Since the gene encoding the isoprenoid biosynthetic enzyme is a gene encoding hydroxymethylglutaryl coenzyme A reductase, the mevalonate pathway can be activated and the production amount of the isoprenoid precursor or isoprenoid can be increased. Therefore, it is preferable.

The polynucleotide introduced into the transformant according to one embodiment of the present invention is selected from the group consisting of the following (a) to (d) or (a ′) to (d ′).
(A) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 or a pyruvate kinase promoter sequence or a part thereof;
(B) hybridizes under stringent conditions with a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 or a polynucleotide comprising a promoter sequence of pyruvate kinase, or a portion complementary thereto, and promoter activity A polynucleotide having
(C) consisting of a polynucleotide or part thereof in which one or several bases are substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 1 or the promoter sequence of pyruvate kinase, and A polynucleotide having promoter activity;
(D) A polynucleotide comprising a nucleotide sequence described in SEQ ID NO: 1 or a polynucleotide having 90% or more identity with the promoter sequence of pyruvate kinase or a part thereof and having promoter activity.
(A ′) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 5 or a phosphoketolase promoter sequence or a portion thereof;
(B ′) hybridizes under stringent conditions to a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 5 or a polynucleotide comprising a phosphoketolase promoter sequence or a part thereof and a nucleotide sequence complementary thereto, and has promoter activity A polynucleotide having;
(C ′) a nucleotide sequence as set forth in SEQ ID NO: 5 or a phosphoketolase promoter sequence, consisting of a polynucleotide or part thereof in which one or several bases are substituted, deleted, inserted and / or added, and a promoter An active polynucleotide;
(D ′) a polynucleotide comprising a nucleotide sequence described in SEQ ID NO: 5 or a polynucleotide having 90% or more identity with the promoter sequence of phosphoketolase or a part thereof, and having promoter activity.

In the present specification, the description of “polynucleotide or (or part thereof)” means that the promoter sequence or terminator sequence (these are gene expression regulatory sequences) is only a partial core sequence. This is a description reflecting the finding that it may have promoter activity or terminator activity. In other words, a promoter sequence or terminator sequence is a typical gene sequence, that is, a gene sequence that encodes a protein (ie, a structural gene sequence) must have at least the majority of the sequence, so that a functioning protein cannot be produced. It needs to be understood as a gene sequence different from common sense.

The polynucleotides (a) and (a ′) will be specifically described.

SEQ ID NO: 1 is a promoter sequence of a pyruvate kinase (PYK) gene, and SEQ ID NO: 5 is a promoter sequence of a phosphoketolase (PK) gene.

The polynucleotide consisting of the base sequences described in SEQ ID NOs: 1 and 5 has promoter activity. Therefore, a gene encoding an isoprenoid biosynthetic enzyme such as a shortened hydroxymethylglutaryl coenzyme A reductase can be obtained by introducing a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 5 into a eukaryotic host. It is preferable because expression can be activated.

The specific base sequence of the “pyruvate kinase promoter sequence” is not limited to SEQ ID NO: 1, and the specific base sequence of the “phosphoketolase promoter sequence” is not limited to SEQ ID NO: 5. In this case, the “pyruvate kinase promoter sequence” may be any known pyruvate kinase promoter sequence (for example, a well-known pyruvate kinase promoter sequence in various organisms). The “phosphoketolase promoter sequence” may be any known phosphoketolase promoter sequence (for example, a well-known phosphoketolase promoter sequence in various organisms).

In the present specification, “polynucleotide having promoter activity” functions in any eukaryotic host, and when a base sequence encoding any protein is introduced downstream of the polynucleotide, transcription of the base sequence. Is activated, and any mRNA or protein synthesis (ie, expression) can be confirmed. Examples of means for confirming protein expression include RT-PCR, known mRNA such as Western blot, and protein detection methods. The downstream means a position on the 3 ′ side in the transcription direction, that is, the direction from the 5 ′ side to the 3 ′ side in the sense strand.

The polynucleotides (b) and (b ′) are each composed of a base sequence shown in SEQ ID NOs: 1 and 5, or a base sequence complementary to a polynucleotide comprising a promoter sequence of pyruvate kinase and a promoter sequence of phosphoketolase, respectively. To a polynucleotide or a part thereof under stringent conditions and has promoter activity.

Such a polynucleotide is obtained by a general hybridization technique using as a probe a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 5, or a pyruvate kinase promoter sequence or phosphoketolase promoter sequence, or a part thereof. be able to.

If the polynucleotides (b) and (b ′) are polynucleotides having promoter activity, the expression of genes encoding isoprenoid biosynthetic enzymes such as a shortened hydroxymethylglutaryl coenzyme A reductase is activated. This is preferable.

The polynucleotides (b) and (b ′) above are oligonucleotides that specifically hybridize to a gene comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 5, or a pyruvate kinase promoter sequence or a phosphoketolase promoter sequence. It can be synthesized by PCR technology as a primer. By such hybridization or PCR, a polynucleotide having high homology with the nucleotide sequence of SEQ ID NO: 1 or 5, or a polynucleotide comprising a pyruvate kinase promoter sequence or a phosphoketolase promoter sequence can be isolated.

The isolation of the polynucleotide is not particularly limited, but is preferably performed by hybridization under stringent conditions. In the present specification, the term “stringent conditions” refers to conditions under which double-stranded polynucleotides specific to so-called base sequences are formed and non-specific double-stranded polynucleotides are not formed. The above stringent conditions are preferably hybridization conditions in which the hybridization temperature is 37 ° C. in the presence of 50% formamide, or hybridization conditions with similar stringency. Hybridization conditions with higher stringency are preferred because polynucleotides with higher homology can be isolated. Examples of such hybridization conditions include hybridization conditions in which the hybridization temperature is 42 ° C. in the presence of 50% formamide, and further hybridization conditions having a high stringency include 65 ° C. in the presence of 50% formamide. Can be mentioned.

The homology of the isolated polynucleotide with the nucleotide sequence set forth in SEQ ID NO: 1 or 5, or the pyruvate kinase promoter sequence or phosphoketolase promoter sequence is preferably 70% or more, more preferably It is 80% or more, more preferably 90% or more. In addition, the homology of the base sequence of a gene is determined by the gene analysis program, BLAST (http://blast.genome.ad.jp) or FASTA (http://fasta.genome.ad.jp/SIT/FASTA.html) Etc. can be determined.

In the polynucleotides (c) and (c ′) above, 1 or several bases are substituted in the nucleotide sequences set forth in SEQ ID NOs: 1 and 5, or the promoter sequence of pyruvate kinase and the promoter sequence of phosphoketolase, respectively. It consists of a deleted or inserted and / or added polynucleotide or a part thereof, and has promoter activity.

The polynucleotides (c) and (c ′) are functionally equivalent to the nucleotide sequence shown in any of SEQ ID NOs: 1 and 5, or a gene having a pyruvate kinase promoter sequence and a phosphoketolase promoter sequence, respectively. As long as it has promoter activity, its specific sequence is not limited. Here, the number of bases that may be substituted, deleted, inserted and / or added is not limited as long as the function is not lost, but is replaced by a known introduction method such as site-directed mutagenesis. , A number that can be deleted, inserted and / or added, within 50 bases, preferably within 40 bases, more preferably within 30 bases, more preferably within 20 bases, more preferably Is within 10 bases, more preferably within 5 bases, and even more preferably within 3 bases. Further, in the specification, “mutation” mainly means a mutation artificially introduced by site-directed mutagenesis or the like, but may be a naturally occurring similar mutation.

The polynucleotides (d) and (d ′) are polynucleotides having 90% or more identity with the nucleotide sequences of SEQ ID NOS: 1 and 5, respectively, or the pyruvate kinase promoter sequence and the phosphoketolase promoter sequence. Or it consists of a part thereof and has promoter activity.

The identity of the polynucleotides (d) and (d ′) above with the nucleotide sequences set forth in SEQ ID NOs: 1 and 5, or the promoter sequence of pyruvate kinase and the promoter sequence of phosphoketolase may be 90% or more. Preferably, it is 95% or more, more preferably 98% or more. In addition, the identity of the base sequence of a gene is determined by the gene analysis program, BLAST (http://blast.genome.ad.jp) or FASTA (http://fasta.genome.ad.jp/SIT/FASTA.html) Etc. can be determined.

The polynucleotide construct further comprises the following (e) to (h) or (e ′) to (equivalently operably or operably linked) downstream of the gene encoding the isoprenoid biosynthetic enzyme. h ′) may contain at least one polynucleotide selected from the group consisting of h ′).
(E) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 2 or a terminator sequence of pyruvate kinase or a part thereof;
(F) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 or the terminator sequence of pyruvate kinase, and has terminator activity Or part of it;
(G) in the nucleotide sequence of SEQ ID NO: 2 or the terminator sequence of pyruvate kinase, consisting of a polynucleotide or part thereof in which one or several bases are substituted, deleted, inserted and / or added, and A polynucleotide having terminator activity;
(H) a polynucleotide having a terminator activity, comprising a polynucleotide having 90% or more identity with the nucleotide sequence of SEQ ID NO: 2 or the terminator sequence of pyruvate kinase or a part thereof.
(E ′) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 6 or a phosphoketolase terminator sequence, or a portion thereof;
(F ′) hybridizes under stringent conditions with a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 6 or a phosphoketolase terminator sequence or a polynucleotide comprising a base sequence complementary thereto, and has a terminator activity; A polynucleotide having;
(G ′) a nucleotide sequence described in SEQ ID NO: 6 or a terminator sequence of phosphoketolase, wherein the terminator comprises a polynucleotide or a part thereof, wherein one or several bases are substituted, deleted, inserted and / or added. An active polynucleotide;
(H ′) A polynucleotide having a terminator activity, comprising a polynucleotide having a nucleotide sequence of SEQ ID NO: 6 or a phosphoketolase terminator sequence of 90% or more, or a part thereof, and a part thereof.

The genes (e) and (e ′) will be specifically described.

SEQ ID NO: 2 is a terminator sequence of pyruvate kinase (PYK) gene, and SEQ ID NO: 6 is a terminator sequence of phosphoketolase (PK) gene.

The polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 2 or 6 has terminator activity. Therefore, expression of a gene encoding an isoprenoid biosynthetic enzyme such as a shortened hydroxymethylglutaryl coenzyme A reductase can be achieved by introducing a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 or 6 into a eukaryotic host. This is preferable because it can be optimized.

The specific base sequence of the “terminator sequence of pyruvate kinase” is not limited to SEQ ID NO: 2, and the specific base sequence of the “terminator sequence of phosphoketolase” is not limited to SEQ ID NO: 6. In this case, the terminator sequence of pyruvate kinase may be any known terminator sequence of pyruvate kinase (for example, a known terminator sequence of pyruvate kinase in various organisms). The term “phosphoketolase terminator sequence” may be any known phosphoketolase terminator sequence (for example, a known phosphoketolase terminator sequence in various organisms).

In the present specification, “polynucleotide having terminator activity” functions in any eukaryotic host, and when the polynucleotide is introduced downstream of the base sequence encoding any protein, transcription of the base sequence is terminated. It means that As a means for confirming that transcription has been completed, a reporter gene may be introduced downstream of the polynucleotide, and it may be confirmed that the reporter gene is not expressed.

The polynucleotides (f) and (f ′) are each composed of a base sequence shown in SEQ ID NOs: 2 and 6, or a base sequence complementary to a polynucleotide comprising a terminator sequence of pyruvate kinase and a terminator sequence of phosphoketolase, respectively. And a terminator activity.

Such a polynucleotide can be obtained by a general hybridization technique using a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 2 or 6 or a part thereof as a probe.

If the genes (f) and (f ′) have a terminator activity, the expression of a gene encoding an isoprenoid biosynthetic enzyme such as a shortened hydroxymethylglutaryl coenzyme A reductase is optimized. This is preferable.

The isolation of the polynucleotide is not particularly limited, but is preferably performed by hybridization under stringent conditions. The above stringent conditions are preferably hybridization conditions in which the hybridization temperature is 37 ° C. in the presence of 50% formamide, or hybridization conditions with similar stringency. Hybridization conditions with higher stringency are preferred because polynucleotides with higher homology can be isolated. Examples of such hybridization conditions include hybridization conditions in which the hybridization temperature is 42 ° C. in the presence of 50% formamide, and further hybridization conditions having a high stringency include 65 ° C. in the presence of 50% formamide. Can be mentioned.

The homology of the isolated polynucleotide with the nucleotide sequence set forth in SEQ ID NO: 2 or 6, or the terminator sequence of pyruvate kinase or the terminator sequence of phosphoketolase is preferably 70% or more, more preferably It is 80% or more, more preferably 90% or more.

In the polynucleotides (g) and (g ′) above, one or several bases are substituted in the nucleotide sequences shown in SEQ ID NOs: 2 and 6, or the terminator sequence of pyruvate kinase and the terminator sequence of phosphoketolase, respectively. It consists of a deleted, inserted and / or added polynucleotide and has terminator activity.

The polynucleotides (g) and (g ′) are functionally equivalent to the gene having the nucleotide sequence shown in any of SEQ ID NOs: 2 and 6, or the terminator sequence of pyruvate kinase and the terminator sequence of phosphoketolase, respectively. The specific sequence is not limited as long as it has a terminator activity. Here, the number of bases that may be substituted, deleted, inserted and / or added is not limited as long as the function is not lost, but substitution or deletion may be performed by a known introduction method such as site-directed mutagenesis. This number refers to the number that can be deleted, inserted and / or added, and is within 50 bases, preferably within 40 bases, more preferably within 30 bases, still more preferably within 20 bases, more preferably 10 bases. It is within a base, More preferably, it is within 5 bases, More preferably, it is within 3 bases. Further, in the specification, “mutation” mainly means a mutation artificially introduced by site-directed mutagenesis or the like, but may be a naturally occurring similar mutation.

The polynucleotides (h) and (h ′) are polynucleotides having 90% or more identity to the nucleotide sequences of SEQ ID NOs: 2 and 6, or the terminator sequence of pyruvate kinase and the terminator sequence of phosphoketolase, respectively. And has a terminator activity.

The identity of the polynucleotides (h) and (h ′) above with the nucleotide sequence of SEQ ID NO: 2 or 6, or the terminator sequence of pyruvate kinase or the terminator sequence of phosphoketolase may be 90% or more. Preferably, it is 95% or more, more preferably 98% or more. In addition, the identity of the base sequence of a gene is determined by the gene analysis program, BLAST (http://blast.genome.ad.jp) or FASTA (http://fasta.genome.ad.jp/SIT/FASTA.html) Etc. can be determined.

Preferred combinations of a polynucleotide having promoter activity and a polynucleotide having terminator activity are combinations of the above (a) to (d) and (e) to (h), or (a ′) to (d As long as it is selected from a combination of ') and (e') to (h '), there is no particular limitation. When the polynucleotide having promoter activity consists of the base sequence described in SEQ ID NO: 1, it is preferable that the polynucleotide having terminator activity consists of the base sequence described in SEQ ID NO: 2. In addition, when the polynucleotide having promoter activity consists of the base sequence described in SEQ ID NO: 5, the polynucleotide having terminator activity is preferably composed of the base sequence described in SEQ ID NO: 6.

Each of the polynucleotides consisting of the base sequences described in SEQ ID NOs: 1, 2, 5 and 6 was obtained as a DNA fragment having promoter activity or terminator activity of the oleaginous yeast gene. Therefore, the expression control sequence according to an embodiment of the present invention has homology to the oleaginous yeast or DNA having the promoter activity or terminator activity of these genes in the genus Lipomyces, or such promoter sequence or terminator sequence. Further, it may be a polynucleotide having the present promoter activity or the present terminator activity. Such polynucleotides include hybridization techniques using a probe consisting of at least a part of the base sequence set forth in any one of SEQ ID NOs: 1, 2, 5, and 6, and oligonucleotide probes that hybridize to the base sequence. It can be obtained from yeast using the PCR technique used. Furthermore, DNA that hybridizes to each polynucleotide comprising the base sequence set forth in any of SEQ ID NOs: 1, 2, 5, and 6 under the stringent conditions described above can be selected. The promoter sequence or terminator sequence according to one embodiment of the present invention may be a part of such various forms of polynucleotide as long as it has the promoter activity or the terminator activity. The promoter sequence and terminator sequence according to one embodiment of the present invention may be genomic DNA or chemically synthesized DNA.

The polynucleotide construct into which the transformant according to one embodiment of the present invention has been introduced is functionally composed of a gene encoding an isoprenoid biosynthetic enzyme and a specific polynucleotide encoding a gene expression control sequence. It is preferable that it is connected.

In the present specification, “gene expression control sequence (also simply referred to as expression control sequence)” refers to a sequence having promoter activity or terminator activity. In the present specification, “functional linkage” refers to a bond that causes the expression of DNA encoding a linked protein to be under the influence or control of the expression control sequence.

The form of the polynucleotide construct is not particularly limited, and plasmid (DNA), virus (DNA), virus (RNA), bacteriophage (DNA), retrotransposon (DNA), artificial chromosome (YAC, PAC, BAC, MAC, etc.) ) To a vector selected according to the introduced form of the foreign gene (extrachromosomal or intrachromosomal) or the type of eukaryotic host. That is, the present polynucleotide construct may contain DNA as a vector in any of these forms in addition to DNA comprising gene expression control sequences. The polynucleotide construct preferably takes the form of a plasmid vector or a viral vector. As the plasmid vector, for example, a vector well known in the art such as a prokaryotic vector, a eukaryotic vector, an animal cell vector and a plant cell vector can be used. The present polynucleotide construct may be retained in the cytoplasm or outside the host chromosome in a eukaryotic host, or may be retained in the host chromosome.

The polynucleotide construct may contain a DNA encoding a desired protein (hereinafter also referred to as a coding DNA) operably linked to the promoter sequence and the terminator sequence. Further, if the promoter sequence is functional, the terminator sequence is not necessarily required. The coding DNA may include not only cDNA but also a DNA sequence that is transcribed but not translated.

The polynucleotide construct preferably contains a DNA sequence for homologous recombination for integrating the promoter sequence and the coding DNA into the host chromosome by homologous recombination. By including such a DNA sequence, the integration of these DNA sequences can be achieved at a desired site of the host chromosome, and the desired gene can be destroyed at the same time. The DNA sequence for homologous recombination is, for example, a DNA sequence that is homologous to the DNA sequence at or near the target site into which the present DNA sequence is to be introduced in the host chromosome. The DNA sequence for homologous recombination has a sequence that is homologous to at least one position of the target gene or a DNA sequence in the vicinity thereof, and preferably a sequence that is homologous to at least two positions in the target gene or the vicinity thereof. I have. For example, two DNA sequences for homologous recombination are set as DNA sequences that are homologous to the DNA upstream and downstream of the target site on the chromosome, and between these DNA sequences for homologous recombination, It is preferred to link the promoter sequence and the coding DNA.

The polynucleotide construct preferably contains a selection marker in order to confirm whether or not the gene has been introduced into a eukaryotic host, and whether or not the gene has been reliably expressed in the eukaryotic host. Although it does not specifically limit as a selection marker, Well-known various selection marker genes including a drug resistance gene, an auxotrophic gene, etc. can be used. As a selection marker gene, for example, a nurseosricin resistance gene, a kanamycin resistance G418 gene, a hygromycin resistance gene, a neomycin resistance gene, and the like can be used.

The method for introducing the polynucleotide construct into an appropriate eukaryotic host is not particularly limited. For example, transformation method, transfection method, conjugation method, protoplast fusion, electroporation method, lipofection method, lithium acetate method, particle gun method, calcium phosphate precipitation method, Agrobacterium method, PEG method or direct microinjection method, etc. The polynucleotide construct can be introduced into a suitable eukaryotic host by any of a variety of suitable means. After the introduction of the polynucleotide construct, the transformant introduced with the polynucleotide construct is preferably cultured in a selective medium.

In the transformant introduced with the polynucleotide construct, the promoter DNA, terminator DNA, coding DNA, etc., which are the components of the polynucleotide construct, are present on the chromosome or on extrachromosomal factors (including artificial chromosomes). become. When a DNA construct for homologous recombination that can achieve homologous recombination is introduced into the polynucleotide construct described above, the promoter DNA and the terminator DNA are functionally linked to the DNA at desired positions on the host chromosome. A transformant carrying the encoded DNA is obtained. In addition, when a construct that does not retain DNA for homologous recombination is introduced, the promoter DNA and the terminator DNA retain the coding DNA functionally linked to the DNA at random positions on the host chromosome. A transformant is obtained.

[2. Method for producing isoprenoid precursor or isoprenoid)
The method for producing an isoprenoid precursor or isoprenoid (also referred to herein as an isoprenoid compound) according to an embodiment of the present invention includes a culturing step for culturing the transformant, a culture medium after culturing, and / or the transformation. And a recovery step of recovering the target substance from the body.

Examples of the isoprenoid precursor include mevalonic acid (MVA), phosphomevalonic acid (PMVA), diphosphomevalonic acid (DPMVA), isopenterdiphosphate (IPP), dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP).

Examples of isoprenoids include various isoprenoids such as monoterpenes, polyterpenes, sesquiterpenes, diterpenes, triterpenes, and carotenoids.

Monoterpenes are not particularly limited, and examples thereof include menthol, limonene, geraniol, citronellol, β-myrcene, and linalool.

The polyterpene is not particularly limited, and an example thereof is isoprene rubber.

Sexeterpenes are not particularly limited, and examples thereof include farnesol, elemol, gelmacrene D, β-elemene, β-caryophyllene, β-eudesmol, α-neocloben, β-cubeben, cedrene, artemisinin, santonin, and farnesene. it can.

Diterpenes are not particularly limited, and examples include taxanes (eg, baccatin III, 10 deacetylbaccatin III, and paclitaxel) and steviosides (eg, stevioside, rebaudioside A, and rebaudioside C). Can be mentioned.

Triterpenes are not particularly limited, and examples thereof include squalene, ursolic acid, glycyrrhizin, β-amylin, lupeol, oleanolic acid, and ginsenosides (for example, 20 (R) -ginsenoside Rb3, ginsenoside Rb2, and ginsenoside Rc). Can do.

The carotenoid is not particularly limited, and examples thereof include carotenes (for example, α-carotene, β-carotene, γ-carotene, δ-carotene, lycopene, and tolylene) and xanthophylls (for example, lutein, zeaxanthin, canthaxanthin, fucoxanthin, Astaxanthin, anthaxanthin, capsanthin, capsorubin, retinol, retinal, retinoic acid and violaxanthin).

The isoprenoid preferably represents a carotenoid. More preferably, the carotenoid is lycopene.

In the above culturing step, the transformant may be cultured using a solid medium (that is, solid culture), or may be cultured using a liquid medium (that is, liquid culture). From the viewpoint of ease of adjustment of the culture scale of the transformant (in other words, production scale of isoprenoid compound) and reduction in cost of production of isoprenoid compound, it is preferable to culture using a liquid medium. Moreover, if it is culture | cultivation using a liquid medium, the production amount of an isoprenoid compound can be raised dramatically by culturing a transformant continuously.

In culturing a transformant according to an embodiment of the present invention, culture conditions can be selected according to the type of transformant. Such culture conditions are well known to those skilled in the art. Both natural and synthetic media should be used as long as they contain a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by a eukaryotic host and can efficiently culture transformants. Can do. As the carbon source, carbohydrates such as glucose, xylose, glycerol, fructose, sucrose and starch; organic acids such as acetic acid and propionic acid; alcohols such as ethanol can be used. As the nitrogen source, ammonia, ammonium salts of inorganic acids or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, or other nitrogen-containing compounds, peptone, yeast extract and the like can be used. Examples of inorganic substances that can be used include potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

The above culturing step is usually performed at 30 ° C. for 72 to 192 hours under aerobic conditions such as shaking culture or aeration and agitation culture. The culture time is preferably 96 to 190 hours, more preferably 100 to 180 hours, and more preferably 120 to 168 hours. However, one embodiment of the present invention is not limited to these culture times. During the culture period, the pH is preferably maintained at 2.0 to 7.0. The pH during the culture period is preferably pH 3.0 to 7.0, more preferably pH 4.0 to 7.0, and even more preferably pH 5.0 to 7.0. The pH can be adjusted using an inorganic or organic acid, an alkaline solution, or the like. During culture, antibiotics such as G418, hygromycin and noseosricin (clonNAT) can be added to the medium as needed.

The method for producing an isoprenoid compound according to an embodiment of the present invention may have a recovery step of recovering the target substance from the cultured medium and / or the transformant after the above-described culture step. Although the specific configuration of the recovery process is not particularly limited, the recovery process may be a process including an extraction process and / or a purification process described below.

The method for producing an isoprenoid compound according to an embodiment of the present invention may have an extraction step for extracting the isoprenoid compound from the transformant after the above-described culture step. With this configuration, a highly pure isoprenoid compound can be obtained.

In the extraction step, the transformant is contacted with an organic solvent, and the isoprenoid compound produced by the transformant is transferred into the organic solvent. And an isoprenoid compound can be collect | recovered by collect | recovering the said organic solvents. In the extraction step, the isoprenoid compound can also be supercritically extracted using carbon dioxide, methanol, water, or the like.

When the transformant and the organic solvent are contacted, the medium containing the transformant and the organic solvent may be mixed, or the medium containing the transformant is centrifuged to recover the transformant. After suspending the transformant in water, the water and an organic solvent may be mixed. From the viewpoint of obtaining a higher-purity isoprenoid compound, the medium containing the transformant is centrifuged to collect the transformant, and the transformant is suspended in water. Are preferably mixed.

The type of the organic solvent described above may be appropriately selected according to the type of isoprenoid compound. Examples of the organic solvent include n-pentane, n-hexane, ethyl acetate, benzene, toluene, diethyl ether, diisopropyl ether, dioxane, tetrahydrofuran, ethyl methyl ketone, acetone, methanol, ethanol, propanol, butanol, dimethyl sulfoxide, Chloroform, dichloromethane, carbon tetrachloride, acetonitrile, or pyridine may be used alone. Further, a mixture of at least two of these organic solvents may be used.

The method for producing an isoprenoid compound according to an embodiment of the present invention may have a purification step of purifying the isoprenoid compound contained in the extract by chromatography after the extraction step described above. If it is the said structure, a higher purity isoprenoid compound can be acquired.

Examples of the carrier used for separation include silica gel, silver nitrate-added silica gel, octadecyldimethylsilyl-modified silica gel, dimethylsilyl-modified silica gel, octyldimethylsilyl-modified silica gel, propylamino-modified silica gel, cyanopropyl-modified silica gel, and phenyl-modified silica gel. Such as, but not limited to, various chemically modified silica gels such as alumina and activated carbon.

By using the production method according to an embodiment of the present invention, an isoprenoid compound can be produced at a level that is at least 50% higher than the level of the isoprenoid compound in a control cell that does not contain a genetic modification.

Hereinafter, specific examples of the present invention will be described. However, the present invention is not intended to limit the present invention to the following specific examples, and can be implemented in various modes without departing from the gist of the present invention.

In order to solve the above-mentioned problems, the present inventors have conducted earnest research specifically as follows.

Specifically, the present inventors tried to search for the optimum promoter species for the expression of hydroxymethylglutaryl coenzyme A reductase gene in Lipomyces starkeyi, which is a kind of oleaginous yeast. Total RNA was obtained from the lipophilic yeast Lipomyces starkeyi cultured in a lipid production medium. Thereafter, comprehensive gene expression analysis of oleaginous yeast was performed by RNA sequencing, and information on the expression level of the gene expressed in the lipid production medium was obtained. Furthermore, the expression level of each gene was analyzed by a real-time PCR method. Based on the results, a plurality of genes having various expression levels in the lipid production medium were selected, and the promoter sequence and terminator sequence of the gene were isolated. The mevalonate pathway was improved using the isolated expression control sequence (promoter sequence and / or terminator sequence), and an attempt was made to improve lycopene production in an oleaginous yeast strain into which a lycopene biosynthesis gene was introduced.

It has been reported in budding yeast that hydroxymethylglutaryl coenzyme A reductase in the mevalonate pathway is involved in one of the rate-limiting steps of the mevalonate pathway. The influence of the expression of hydroxymethylglutaryl coenzyme A reductase gene on the mevalonate pathway was evaluated using lycopene production as an index, using the promoter sequence and terminator sequence newly searched from oily yeast.

As the hydroxymethylglutaryl coenzyme A reductase gene introduced into the oleaginous yeast, a truncated hydroxymethylglutaryl coenzyme A reductase (tHMGR) gene having a truncated amino terminus was used. The purpose was to suppress a decrease in enzyme activity due to feedback inhibition by using a truncated enzyme gene (Non-patent Document 6).

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

<Example 1. Detection of expressed genes by RNA sequence analysis>
The oleaginous yeast Lipomyces starkey strain was cultured in a lipid production medium at 30 ° C. and 180 rpm for 5 days, and then the cells were collected to prepare total RNA. The lipid production medium is 40 g / l glucose, 0.5 g / l (NH ₄ ) ₂ SO ₄ , 1 g / l KH ₂ PO ₄ , 0.5 g / l MgSO ₄ .7H ₂ O, 0.1 g / l. 1 CaCl ₂ · 2H ₂ O, 0.1 g / l NaCl and 1.5 g / l Yeast extract. For the preparation of RNA, ISOGEN II (manufactured by Nippon Gene) was used. About the obtained total RNA, RNA sequence analysis was outsourced and FPKM value data indicating the expression level of each gene was obtained. From the obtained FPKM value and the genomic base sequence information of the lipomyces starkey strain that had already been decoded, genes expressed in the lipid production medium and their expression levels were listed.

(Gene expression analysis by quantitative PCR)
Genes expressed in the lipid production medium were selected by RNA sequence analysis. The expression level of each gene was confirmed by a real-time PCR method for five genes with high expression intensity among the selected genes. Next, a single-stranded cDNA was prepared by subjecting 0.5 μg of total RNA to reverse transcription using PrimeScript ^™ Reverse Transcriptase (manufactured by Takara Bio Inc.). 1 μl of the prepared cDNA solution was mixed with THUNDERBIRD SYBR qPCR Mix (manufactured by TOYOBO), and quantitative PCR analysis was performed using Stratagene Mx3005P (manufactured by Agilent Technologies). These results are shown in FIG. As shown in FIG. 1, the expression intensity of the phosphoketolase gene is equivalent to 800 times that of the glyceraldehyde 3-phosphate dehydrogenase gene, and the expression intensity of the translation elongation factor gene is equivalent to 600 times. The expression intensity of the histidine kinase gene corresponds to 300 times, the expression intensity of the acetyl coenzyme A carboxylase gene corresponds to 120 times, and the expression intensity of the pyruvate kinase gene corresponds to 120 times.

In order to obtain gene expression control sequences exhibiting various expression activities, promoter sequences and terminator sequences were obtained for the above five genes based on the results of the quantitative PCR analysis described above. Information on the promoter sequence of the gene and information on the expected terminator sequence of 1.0 kb upstream of the start codon and 0.5 kb downstream of the stop codon were obtained. The reverse transcription reaction and real-time PCR were performed according to the protocol of the kit and the measuring device.

<Example 2. Construction of base vector>
Details of the vector construction in this example will be described below with reference to FIGS. 2 and 3, but the vector construction procedure is not limited thereto. FIG. 2 is a diagram showing a method for constructing a truncated hydroxymethylglutaryl coenzyme A reductase (tHMGR) gene expression vector in the examples of the present application. PYK: pyruvate kinase, TEF: translation elongation factor 1, PK: phosphoketolase, HK: histidine kinase, ACC: acetyl coenzyme A carboxylase (Acetyl Co) The promoter sequence and terminator sequence of each gene of -A carboxylase) were introduced into the vector. FIG. 3 is a diagram showing a method for constructing a lycopene biosynthesis vector (pUC-lyc) in the examples of the present application. PGK: Phosphoglycerate kinase, TPI: Triosephosphate isomerase, TEF: Translation elongation factor 1 gene promoter sequence and terminator sequence are used for expression of crtE, crtB and crtI genes. It was. Note that a series of reaction operations in vector construction are standard methods for those skilled in the art, such as cloning by restriction enzyme treatment and ligation, overlap extension PCR cloning (see Non-Patent Document 6), and Hifi DNA cloning kit (NEB). This was performed according to each method. In addition, in a general cloning method, NEB products were used as a series of enzymes. In addition, it is clearly stated in the instruction manual that NEB's use permission does not extend to DNA constructs using this kit.

(Construction of pUC19-Not vector)
In order to delete the BspQI site in the pUC19 vector and introduce a new NotI site, inverse PCR was performed using pUC-Not-F (SEQ ID NO: 7) and pUC-Not-R (SEQ ID NO: 8) as primers. went. Details of each primer sequence used are listed in Table 1. For the PCR reaction, Prime Star HS DNA polymerase (manufactured by Takara Bio Inc.), which is said to have high accuracy of the amplified fragment, was used as an amplification enzyme. A solution was prepared by adding 50 pmol of primer DNA × 10 μl of 5 × concentrated enzyme reaction buffer, 4 μl of 2.5 mM dNTPmix and 1.25 units of Primestar HS DNA polymerase. A thermal cycler PCR (product name: Thermal Cycler Dice ^R Gradient, manufactured by Takara Bio Inc.) was set with 10 ng of pUC19 plasmid DNA and 50 μl of the above solution, and the DNA fragment was amplified using pUC19 plasmid DNA as a template. The reaction conditions of the thermal cycler are as follows: after heat treatment at 94 ° C. for 1 minute, three temperature changes of 98 ° C. for 10 seconds, 55 ° C. for 15 seconds and 72 ° C. for 2 minutes and 40 seconds (extension time 1 minute / 1 kb) 1 cycle, this was repeated 35 cycles, and finally the reaction sample was stored at 4 ° C. 5 μl of this reaction sample was electrophoresed on a 0.8% TAE agarose gel (containing) and immersed in a 0.5 μg / ml ethidium bromide solution. Thereafter, the DNA band was detected by irradiating the gel with ultraviolet rays at 254 nm (ultraviolet irradiator manufactured by Nippon Gene Co., Ltd.) to confirm gene amplification. The amplified DNA fragment was self-ligated to introduce a NotI site onto the vector. The ligation reaction solution was introduced into E. coli competent cells to transform E. coli. DH5α strain (manufactured by Toyobo Co., Ltd.) was used as an E. coli competent cell, and detailed handling was in accordance with the attached protocol. Colony selection was performed using an LB plate containing 50 μg / ml of antibiotic ampicillin, and plasmid DNA was prepared from each selected colony by ethanol precipitation. The detailed manual for the series of operations such as ethanol precipitation and restriction enzyme treatment was in accordance with Molecular Cloning: A Laboratory Manual second edition (Maniatisetal., Cold Spring Harbor Laboratory press. 1989).

(Construction of pUC-G418 vector and pUC-clone NAT vector)
The promoter sequence of the TEF gene (SEQ ID NO: 3) and the terminator sequence of the TDH gene were amplified by PCR using genomic DNA purified from the lipomyces starkey strain as a template. The primers used were pTEF-F1 (SEQ ID NO: 9) and pTEF-R1 (SEQ ID NO: 10) for the promoter sequence of the TEF gene, and tTDH3-F1 (SEQ ID NO: 11) for the terminator sequence of the TDH gene. And tTDH3-R1 (SEQ ID NO: 12) were used. A DNA fragment of a promoter sequence and a terminator sequence was prepared by PCR reaction using primestar HS. Thereafter, all expression control sequences were prepared using Lipomyces starkey strain genomic DNA as a template. For the G418 gene, the G418 gene was amplified by PCR using the pUC19 vector as a template. G418-F (SEQ ID NO: 13) and G418-R (SEQ ID NO: 14) were used as primers. Overlapping PCR was performed using the three amplified fragments, and the promoter sequence and terminator sequence were operably linked to the G418 gene. Overlap PCR was performed according to the method described in Non-Patent Document 6. Primers were designed so that NotI sites were added to both ends of the prepared G418 cassette for ligation to the base vector. Next, the ligated DNA fragment was subcloned into a pCR-BluntII-TOPO vector (Thermo Fisher Scientific). A series of subcloning reaction operations were performed according to a general DNA subcloning method. Details of the DNA subcloning method followed the attached protocol. Next, a base vector pUC-G418 having a G418 selection marker was constructed by ligating the NotI-treated pUC19-NotI and G418 cassettes.

Similarly, for the clonNAT resistance gene, the clonNAT-F (SEQ ID NO: 15) and clonNAT-R (SEQ ID NO: 16) were used as primers to amplify the clonNAT resistance gene by PCR, and then overlap PCR to detect the TEF gene. An expression cassette was constructed in which the promoter sequence and the terminator sequence of the TDH gene were operably linked to the clonNAT resistance gene. The resistance gene expression cassette linked in the same manner as the G418 gene was subcloned into the pCR blunt topo vector, treated with NotI, and cloned into the NotI site of pUC-NotI by a conventional method to construct pUC-cloneNAT (construct details). (See the upper part of FIG. 2).

<Example 3. Construction of expression cassette vector>
(Cloning of expression control sequences (promoter DNA and terminator DNA))
For the genes that showed expression in the lipid production medium in Examples 1 and 2, five genes showing high expression activity were selected, and expression control sequences (promoter DNA (1.5 kb) and terminator DNA (0. 5 kb)) was cloned. The gene resource for obtaining the expression control sequence was isolated by a PCR amplification method using the genomic DNA of the oleaginous yeast Lipomyces starkey strain as a template. This strain was cultured for 2 nights in 5 ml of YPD culture solution, and then genomic DNA was prepared using a genomic DNA preparation kit (product name: Gen Toru-kun (trademark) —for yeast—manufactured by Takara Bio Inc.). The prepared genomic DNA was measured for DNA concentration with a spectrophotometer (product name: UVmini-1240, manufactured by Shimadzu Corporation). The expression control sequence of each gene was obtained by PCR amplification using Primestar HS DNA polymerase using the genomic DNA sequence of Lipomyces starkey strain as a template. The primers used for amplification of the promoter sequence and terminator sequence of each gene are as follows: For the promoter sequence of the pyruvate kinase (PYK) gene expression control sequence, pPYK-F (SEQ ID NO: 17) and pPYK-R ( SEQ ID NO: 18); for the terminator sequence of the pyruvate kinase (PYK) gene expression control sequence, tPYK-F (SEQ ID NO: 19) and tPYK-R (SEQ ID NO: 20); histidine kinase (HK) For the promoter sequence of the gene expression control sequence, pHK-F (SEQ ID NO: 21) and pHK-R (SEQ ID NO: 22); for the terminator sequence of the histidine kinase (HK) gene expression control sequence , THK-F (SEQ ID NO: 23) and tHK-R (SEQ ID NO: 24); Phosphoketola PPK-F (SEQ ID NO: 25) and pPK-R (SEQ ID NO: 26); Phosphoketolase (PK) gene expression control sequence terminator for the promoter sequence of the pho (Phosphoketolase) (PK) gene expression control sequence For the sequence, tPK-F (SEQ ID NO: 27) and tPK-R (SEQ ID NO: 28); for the promoter sequence of the acetoacetyl Co-A carboxylase (ACC) gene expression control sequence PACC-F (SEQ ID NO: 29) and pACC-R (SEQ ID NO: 30); for the terminator sequence of the acetoacetyl co-A carboxylase (ACC) gene expression control sequence, tACC- F (SEQ ID NO: 31) and tACC-R (SEQ ID NO: 32); Translation elongation factor 1 ) (TEF) gene expression control sequence promoter sequence, pTEF-F (SEQ ID NO: 33) and pTEF-R (SEQ ID NO: 34); translation elongation factor 1 (TEF) gene expression control sequence These were tTEF-F (SEQ ID NO: 35) and tTEF-R (SEQ ID NO: 36). Details of each primer sequence are listed in Table 2. Using 50 ng of primer DNA 50 pmol × 2, 5 × concentrated enzyme reaction buffer × 10 μl, 2.5 mM dNTPmix 4 μl and Primestar HS DNA polymerase 1.25 unit Was set in a thermal cycler PCR (product name: Thermal Cycler Dice ^R Gradient, manufactured by Takara Bio Inc.) to obtain a DNA fragment. The reaction condition of the thermal cycler is that after heat treatment at 94 ° C. for 2 minutes, three temperature changes of 98 ° C. for 10 seconds, 55 ° C. for 15 seconds and 72 ° C. for 1 minute 30 seconds are defined as one cycle. The cycle was repeated and finally the reaction sample was stored at 4 ° C. 5 μl of the reaction sample was electrophoresed on a 0.8% TAE agarose gel (containing) and immersed in a 0.5 μg / ml ethidium bromide solution. Subsequently, the DNA band was detected by irradiating the gel with ultraviolet rays at 254 nm (ultraviolet irradiator manufactured by Nippon Gene) to confirm gene amplification.

PUC19-NotI was treated with SmaI restriction enzyme to prepare a vector.

As primers, for the promoter sequence of the PYK gene (SEQ ID NO: 1), for pPYK-F (SEQ ID NO: 17) and pPYK-R (SEQ ID NO: 18), for the terminator sequence of the PYK gene (SEQ ID NO: 2), Using tPYK-F (SEQ ID NO: 19) and tPYK-R (SEQ ID NO: 20), each sequence was amplified by PCR as described above. Using Hifi DNA cloning Kit (manufactured by NEB), the amplified promoter sequence and terminator sequence were cloned so that two BspQI sites were included between the promoter sequence and the terminator sequence in the SmaI site of pUC-G418. Then, pUC-ptPYK was constructed by transformation into DH5α strain. Similarly to the PYK gene, pTFE-F (SEQ ID NO: 33) and pTEF-R (SEQ ID NO: 34) and TEF gene terminator sequence (sequence) are used as primers for the TEF gene promoter sequence (SEQ ID NO: 3), respectively. For number 4), pUC-ptTEF was constructed using tTFE-F (SEQ ID NO: 35) and tTEF-R (SEQ ID NO: 36). As with the PYK gene, pPK-F (SEQ ID NO: 25) and pPK-R (SEQ ID NO: 26) and the terminator sequence of the PK gene (sequence) are used as primers for the promoter sequence of the PK gene (SEQ ID NO: 5), respectively. No. 6), pUC-ptPK was constructed using tPK-F (SEQ ID NO: 27) and tPK-R (SEQ ID NO: 28). Similar to the PYK gene, as a primer, the terminators of pHK-F (SEQ ID NO: 21) and pHK-R (SEQ ID NO: 22), histidine kinase (HK) gene with respect to the promoter sequence of the histidine kinase (HK) gene, respectively. For the sequence, pUC-ptHK was constructed using tHK-F (SEQ ID NO: 23) and tHK-R (SEQ ID NO: 24). Similar to the PYK gene, pACC-F (SEQ ID NO: 29) and pACC-R (SEQ ID NO: 29) were used as primers for the promoter sequence of the acetoacetyl co-A-carboxylase (ACC) gene, respectively. 30) pUC-ptACC using tACC-F (SEQ ID NO: 31) and tACC-R (SEQ ID NO: 32) against the terminator sequence of acetoacetyl Co-A-carboxylase (ACC) gene (For details of each construct of pUC-ptPYK, pUC-ptTEF, pUC-ptPK, pUC-ptHK and pUC-ptACC, refer to the middle of FIG. 2).

Each constructed vector was prepared by an alkali extraction method, and this was subjected to column purification using QIAprep SpinMiniPrep Kit (manufactured by Qiagen). Next, the DNA concentration was measured with a spectrophotometer UltraJP2005-137306A2005.6.2spec3000 (manufactured by Shimadzu Corporation), and the DNA base sequence kit BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems App) Sequencing reaction was performed according to The reaction sample was set in the base sequence analyzer ABIPRISM3100 Genetic Analyzer (PE Applied Biosystems), and the base sequence of the constructed gene cassette was determined. The details of how to use the equipment were in accordance with the manual attached to this device.

(Construction of a vector for controlling expression of a truncated hydroxymethylglutaryl coenzyme A reductase (tHMGR) gene)
Abbreviated hydroxymethylgluta derived from Saccharomyces cerevisiae obtained by obtaining expression control sequences from five genes that showed relatively high expression in culture in a lipid production medium in Example 1 and optimized for the codons for lipomyces starkey A DNA fragment of the ril coenzyme A reductase (tHMGR) gene was prepared by gene synthesis. The synthesized gene fragment was amplified by PCR with Primestar HS DNA polymerase using tHMGR-PYK-F (SEQ ID NO: 37) and tHMGR-PYK-R (SEQ ID NO: 38) as primers. This was ligated using pUC-ptPYK treated with BspQI and Hifi DNA cloning Kit (NEB) (see FIG. 3). Then, the DH5α strain was transformed and selected on an LB agar medium containing 50 mg / L of ampicillin to obtain a clone having pUC-ptPYK-tHMGR (see the lower part of FIG. 2 for details of the construct).

PUC-ptTEF-tHMGR, pUC-ptPK-tHMGR, pUC-ptHK-tHMGR, and pUC-ptACC-tHMGR were constructed in the same manner as pUC-ptPYK-tHMGR. Specifically, a DNA fragment of a shortened hydroxymethylglutaryl coenzyme A reductase gene was used as a template, and a DNA fragment was amplified using the following as a primer: pUC-ptTEF-tHMGR versus tHMGR-TEF- F (SEQ ID NO: 45) and tHMGR-TEF-R (SEQ ID NO: 46); for pUC-ptPK-tHMGR, tHMGR-PK-F (SEQ ID NO: 41) and tHMGR-PK-R (SEQ ID NO: 42); pUC -THMGR-HK-F (SEQ ID NO: 39) and tHMGR-HK-R (SEQ ID NO: 40) for ptHK-tHMGR; tHMGR-ACC-F (SEQ ID NO: 43) for pUC-ptACC-tHMGR And tHMGR-ACC-R (SEQ ID NO: 44). Each amplified DNA fragment was ligated to each of pUC-ptTEF, pUC-ptPK, pUC-ptHK, and pUC-ptACC vectors treated with BspQI, respectively, so that pUC-ptTEF-tHMGR, pUC-ptPK-tHMGR, The pUC-ptHK-tHMGR and pUC-ptACC-tHMGR vectors were constructed. The base sequence of each primer is shown in Table 3, and details of the construction of each vector are shown in the lower part of FIG.

<Example 4. Construction of lycopene biosynthesis vector>
A lycopene biosynthetic pathway gene (crtE, crtB, and crtI) expression cassette from Pantoea ananatis was constructed as follows.

In order to construct a crtE expression cassette, the promoter sequence and terminator sequence of the PGK gene were amplified by Primestar HS DNA polymerase using genomic DNA purified from the Lipomyces starkey strain as a template. Primers used are pPGK-sacI-F (SEQ ID NO: 47) and pPGK-sacI-R (SEQ ID NO: 48) for the promoter sequence of the PGK gene, and tPGK-sacI- for the terminator sequence of the PGK gene. F (SEQ ID NO: 49) and tPGK-sacI-R (SEQ ID NO: 50) were used (see Table 4 for the base sequence of each primer). For the crtE gene, a sequence whose codon was optimized by gene synthesis was prepared, and the above sequence was amplified by PCR using crtE-F (SEQ ID NO: 59) and crtE-R (SEQ ID NO: 60) as primers. Overlapping PCR was performed using the three amplified fragments, and a promoter sequence and a terminator sequence were functionally linked to the crtE gene (see Non-Patent Document 6). For the subsequent vector construction, primers were designed so that SacI sites were added to both ends of the prepared crtE expression cassette (SEQ ID NOs: 47 to 50). After subcloning the ligated DNA fragment into a pCR-bluntII-TOPO vector (manufactured by Life Technology), the DNA sequence was decoded to confirm that it was ligated as designed.

Similarly, for crtB, pTPI-F (SEQ ID NO: 51), pTPI-R (SEQ ID NO: 52), tTPI-F (SEQ ID NO: 53) are used as primers so as to be under the control of the promoter sequence and terminator sequence of the TPI gene. ), Trtpi-R (SEQ ID NO: 54), crtB-F (SEQ ID NO: 61) and crtB-R (SEQ ID NO: 62), a crtB expression cassette in which an expression control sequence and a gene-synthesized crtB are operatively linked Built. For the subsequent vector construction, primers were designed so that SmaI sites were added to both ends of the prepared crtB expression cassette (SEQ ID NOs: 51 to 54). Similarly, for crtI, pTEF-pstI-F (SEQ ID NO: 55), pTEF-pstI-R (SEQ ID NO: 56), tTEF-pstI are used as primers so as to be under the control of the promoter sequence and terminator sequence of the TEF gene. Functions of crtI synthesized with expression control sequence using -F (SEQ ID NO: 57), tTEF-pstI-R (SEQ ID NO: 58), crtI-F (SEQ ID NO: 63) and crtI-R (SEQ ID NO: 64) Ligated crtI expression cassette was constructed. For the subsequent vector construction, primers were designed so that PstI sites were added to both ends of the prepared crtI expression cassette (SEQ ID NOs: 55 to 58). The crtE, crtB and crtI expression cassettes were cloned into the SacI site, SmaI site and PstI site of pUC-G418 according to a conventional method to construct a lycopene biosynthesis vector pUC-lyc (see FIG. 3).

Introducing the lycopene biosynthetic gene into the oleaginous yeast Lipomyces starkey strain was carried out by introducing the gene into the nuclear genome by the lithium acetate method according to the method described in Non-Patent Document 7. Using pUC-lyc as a template and pUC-F (SEQ ID NO: 65) and pUC-R (SEQ ID NO: 66) as primers, a region 11 kbp containing a lycopene biosynthesis gene and a selection marker was amplified by PCR. A DNA fragment used for transformation was prepared. Tks Gflex (Takara Bio Inc.) was used for PCR. A colony showing G418 resistance was obtained by culturing at 30 ° C. for 7 days on a YPD agar medium containing 25 μg / ml of G418, and it was confirmed by colony PCR that the crtE expression cassette, crtB expression cassette and crtI expression cassette were retained. did. KODFx enzyme (TOYOBO) was used for colony PCR, and crtE-F (SEQ ID NO: 59) and crtE-R (with respect to crtE expression cassette) were used as primers for detecting three genes necessary for lycopene biosynthesis. And the crtB-F (SEQ ID NO: 61) and crtB-R (SEQ ID NO: 62) for the crtB expression cassette and the crtI-F (sequence for the crtI expression cassette). No. 63) and crtI-R (SEQ ID NO: 64) were used. Since the lycopene biosynthetic gene-introduced strain has a lighter red color than the wild strain, it can be screened using the color as an index.

<Example 5. Production of transformed yeast>
Introducing the lycopene biosynthetic gene into the oleaginous yeast Lipomyces starkey strain was carried out by introducing the gene into the nuclear genome by the lithium acetate method according to the method described in Non-Patent Document 7. Using pUC-lyc as a template and pUC-F (SEQ ID NO: 65) and pUC-R (SEQ ID NO: 66) as primers, a region 11 kbp containing a lycopene biosynthesis gene and a selection marker was amplified by PCR. A DNA fragment used for transformation was prepared (see Table 5 for the base sequence of each primer). Tks Gflex (manufactured by Takara Bio Inc.) was used for PCR. 50 μl of primer DNA, 25 μl of 2-fold concentrated enzyme reaction buffer and 50 μl of a reaction solution containing 1.25 units of Tks Gflex DNA polymerase were set in a thermal cycler, and 10 ng of pUC-lyc plasmid DNA was used as a template for DNA fragmentation. Was prepared. The thermal cycler reaction conditions are as follows: after heat treatment at 94 ° C. for 1 minute, three temperature changes: 98 ° C. for 10 seconds, 55 ° C. for 15 seconds, 68 ° C. for 5 minutes 30 seconds (extension time 30 seconds / 1 kb) 1 cycle, this was repeated 35 cycles, and finally the reaction sample was stored at 4 ° C. Colonies showing G418 resistance were obtained by culturing for 7 days at 30 ° C. in a YPD agar medium containing 25 mg / l G418, and the crtE gene (0.91 kb), crtB gene (0.93 kb) and crtI gene ( 1.48 kb) was confirmed. KODFx enzyme (TOYOBO) was used for colony PCR, and the following were used as primers for detecting 3 genes necessary for lycopene biosynthesis; crtE-F (SEQ ID NO: 59) for crtE gene And crtE-R (SEQ ID NO: 60), crtB-F (SEQ ID NO: 61) and crtB-R (SEQ ID NO: 62) for the crtB gene, and crtI-F (SEQ ID NO: 63) for the crtI gene crtI-R (SEQ ID NO: 64). Primer DNA 50 pmol × 2, 2 × concentrated enzyme reaction buffer 5 μl, 2 mM dNTPmix 0.4 μl and KOD Fx DNA polymerase 0.1 unit added 10 μl of reaction solution on a thermal cycler, 1 μl of cell suspension 1 μl as template Used to prepare DNA fragments. The thermal cycler reaction conditions are as follows: after heat treatment at 95 ° C. for 5 minutes, three temperature changes: 98 ° C. for 10 seconds, 55 ° C. for 30 seconds, 68 ° C. for 1 minute 30 seconds (extension time 1 minute / 1 kb) 1 cycle, this was repeated 35 cycles, and finally the reaction sample was stored at 4 ° C. Since the lycopene biosynthetic gene-introduced strain has a lighter red color than the wild strain, it can be screened using the color as an index.

Next, a shortened hydroxymethylglutaryl coenzyme A reductase gene was introduced into the strain carrying the lycopene biosynthesis gene. Using the vectors pUC-ptPYK-tHMGR, pUC-ptTEF-tHMGR, pUC-ptPK-tHMGR, pUC-ptHK-tHMGR or pUC-ptACC-tHMGR as a template and primers pUC-F (SEQ ID NO: 65) Using -R (SEQ ID NO: 66), a region 6.5 kbp containing a lycopene biosynthesis gene and a selection marker was amplified by PCR to prepare a DNA fragment used for transformation. Tks Gflex (Takara Bio Inc.) was used for PCR. As described above, gene introduction into nuclear genomic DNA was performed by the lithium acetate method. Transformants were selected on a YPD agar medium containing 25 μg / ml of clonNAT, and the obtained colonies were confirmed to retain the shortened hydroxymethylglutaryl coenzyme A reductase gene by colony PCR. . In colony PCR, tHMGR-PYK-F (SEQ ID NO: 37) and tHMGR-PYK-R (SEQ ID NO: 38) were used as primers, and detection was carried out by electrophoresis as 1.5 kb amplification.

<Example 6. Verification of each promoter by fermentation test>
The amount of lycopene produced was quantified in a gene recombinant in which a shortened hydroxymethylglutaryl coenzyme A reductase gene expression cassette in which a lycopene biosynthetic gene and an expression control sequence of each gene were linked was introduced. As a result, an expression control sequence suitable for expression of the shortened hydroxymethylglutaryl coenzyme A reductase gene was selected. Each strain was inoculated into 2 ml of YPD medium and then precultured for 2 days by shaking culture at 30 ° C. and 180 rpm. Thereafter, 500 μl of the preculture was inoculated into an Erlenmeyer flask containing 50 ml of lipid production medium. Rotational shaking culture at 30 ° C. and 180 rpm was performed for 5 days, and cell pellets were collected from 5 ml of the culture solution. 0.5 ml of DMSO and 2 ml of ethyl acetate were added to the cell pellet and vortexed for 1 minute, and then allowed to stand for 2 hours. Next, 2 ml of a saturated aqueous sodium chloride solution was added and vortexed for 1 minute, and then separated into an organic layer and an aqueous layer by centrifugation at 2000 × G for 5 minutes. Since lycopene is distributed to the organic layer (ethyl acetate), 1 ml of the upper organic layer was recovered, and the recovered organic layer was subjected to HPLC (manufactured by Shimadzu Corporation). Detection of lycopene at an absorption wavelength of 452 nm under the detection conditions on a reverse phase column (COSMSIL 5C18-MS-II 4.6 ID × 150 mm), mobile phase (acetonitrile: ethanol = 7: 3) and flow rate of 1 ml / min. It was. The sample concentration was quantified from a calibration curve using a lycopene preparation with a known concentration (see FIG. 4). FIG. 4 shows the result of comparison of the amount of lycopene produced in the expression-controlled strain of the shortened hydroxymethylglutaryl coenzyme A reductase gene. In FIG. 4, pUC-ptPYK-tHMGR shows the result of expressing the tHMGR gene using the expression control sequence of the pyruvate kinase gene, and pUC-ptTEF-tHMGR is the translation elongation factor (Translation elongation factor). 1) The result of expressing the tHMGR gene using the gene expression control sequence is shown. PUC-ptPK-tHMGR shows the result of expressing the tHMGR gene using the expression control sequence of the phosphoketolase gene. -PtACC-tHMGR shows the result of expressing the tHMGR gene using the expression control sequence of the acetyl coenzyme A carboxylase gene, and pUC-ptHK-tHMGR shows the histidine kinase gene THMGR using the expression control sequence of The result of expressing a gene is shown. As shown in FIG. 4, lycopene production is highest when the expression control sequence of the pyruvate kinase (PYK) gene is used, and then lycopene production when the expression control sequence of the translation elongation factor (TEF) gene is used. It was found that the amount of lycopene produced was high when the expression control sequence of the phosphoketolase (PK) gene was used.

When the inventors expressed a truncated hydroxymethylglutaryl coenzyme A reductase gene in Lipomyces starkey under the control of the promoter sequence and terminator sequence of pyruvate kinase (PYK) gene, Production was found to increase most. This is a result that a person skilled in the art cannot imagine from the relationship between the expression intensity of each promoter and the production amount of the isoprenoid compound. In addition, by expressing the truncated hydroxymethylglutaryl coenzyme A reductase gene under the control of the promoter sequence and terminator sequence of the phosphoketolase (PK) gene, the promoter sequence and terminator sequence of the pyruvate kinase (PYK) gene are expressed. It was found that the production amount of isoprenoid compounds was reduced compared to the case of expressing under control. This indicates that the activity of the isoprenoid biosynthetic pathway can be arbitrarily controlled, and this result is difficult to predict from the data that this gene was the highest expressed in the expression analysis of the phosphoketolase (PK) gene. It was.

In addition, not only in oleaginous yeast but also in eukaryotic hosts, when the promoter sequence of the PYK gene and the promoter sequence of the PK gene are used, the isoprenoid biosynthetic enzyme gene has a considerably high expression level and a considerably high expression level, respectively. Until now, there has been no knowledge that control is possible.

In the transformants of the examples, an expression control sequence suitable for expression to optimize hydroxymethylglutaryl coenzyme A reductase activity, which is the rate-limiting step of the mevalonate pathway, is functionally shortened hydroxymethylglutaryl. A polynucleotide construct linked to the coenzyme A reductase gene has been introduced. Therefore, it was found that culturing the transformants of the Examples increased the activity of the mevalonate pathway, promoted the action of isoprenoid biosynthetic enzymes, and increased the production of isoprenoids.

The present invention can be used in various industries such as pharmaceuticals, feeds, food additives, functional foods, perfumes and biofuels.

Claims (9)

A polynucleotide construct comprising a gene encoding an isoprenoid biosynthetic enzyme and at least one polynucleotide selected from the group consisting of the following (a) to (d) upstream of the gene is introduced into a eukaryotic host: A transformant characterized in that:
(A) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 or a pyruvate kinase promoter sequence or a part thereof;
(B) hybridizes under stringent conditions with a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 or a polynucleotide comprising a promoter sequence of pyruvate kinase, or a portion complementary thereto, and promoter activity A polynucleotide having:
(C) consisting of a polynucleotide or part thereof in which one or several bases are substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 1 or the promoter sequence of pyruvate kinase, and A polynucleotide having promoter activity;
(D) A polynucleotide comprising a nucleotide sequence described in SEQ ID NO: 1 or a polynucleotide having 90% or more identity with the promoter sequence of pyruvate kinase or a part thereof and having promoter activity. The polynucleotide construct further comprises at least one polynucleotide selected from the group consisting of the following (e) to (h) downstream of the gene encoding the isoprenoid biosynthetic enzyme: The transformant according to claim 1:
(E) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 2 or a terminator sequence of pyruvate kinase or a part thereof;
(F) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 or the terminator sequence of pyruvate kinase, and has terminator activity Or part of it;
(G) in the nucleotide sequence of SEQ ID NO: 2 or the terminator sequence of pyruvate kinase, consisting of a polynucleotide or part thereof in which one or several bases are substituted, deleted, inserted and / or added, and A polynucleotide having terminator activity;
(H) a polynucleotide having a terminator activity, comprising a polynucleotide having 90% or more identity with the nucleotide sequence of SEQ ID NO: 2 or the terminator sequence of pyruvate kinase or a part thereof. A polynucleotide construct comprising, in a eukaryotic host, a gene encoding an isoprenoid biosynthetic enzyme and at least one polynucleotide selected from the group consisting of the following (a ′) to (d ′) upstream of the gene: A transformant characterized by being introduced:
(A ′) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 5 or a phosphoketolase promoter sequence or a portion thereof;
(B ′) hybridizes under stringent conditions to a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 5 or a polynucleotide comprising a phosphoketolase promoter sequence or a part thereof and a nucleotide sequence complementary thereto, and has promoter activity A polynucleotide having;
(C ′) a nucleotide sequence as set forth in SEQ ID NO: 5 or a phosphoketolase promoter sequence, consisting of a polynucleotide or part thereof in which one or several bases are substituted, deleted, inserted and / or added, and a promoter An active polynucleotide;
(D ′) a polynucleotide comprising a nucleotide sequence described in SEQ ID NO: 5 or a polynucleotide having 90% or more identity with the promoter sequence of phosphoketolase or a part thereof, and having promoter activity. The polynucleotide construct further comprises at least one polynucleotide selected from the group consisting of the following (e ′) to (h ′) downstream of the gene encoding the isoprenoid biosynthetic enzyme. The transformant according to claim 3:
(E ′) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 6 or a phosphoketolase terminator sequence, or a portion thereof;
(F ′) hybridizes under stringent conditions with a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 6 or a phosphoketolase terminator sequence or a polynucleotide comprising a base sequence complementary thereto, and has a terminator activity; A polynucleotide having;
(G ′) a nucleotide sequence described in SEQ ID NO: 6 or a terminator sequence of phosphoketolase, wherein the terminator comprises a polynucleotide or a part thereof, wherein one or several bases are substituted, deleted, inserted and / or added. An active polynucleotide;
(H ′) A polynucleotide having a terminator activity, comprising a polynucleotide having a nucleotide sequence of SEQ ID NO: 6 or a phosphoketolase terminator sequence of 90% or more, or a part thereof, and a part thereof. The transformant according to any one of claims 1 to 4, wherein the eukaryotic host is an oleaginous yeast. The transformant according to claim 5, wherein the oleaginous yeast is Lipomyces starkeyi. The isoprenoid biosynthetic enzyme is a mevalonate pathway enzyme, a carotenoid biosynthetic enzyme, a monoterpene biosynthetic enzyme, a sesquiterpene synthase, a triterpene synthase, or a diterpene synthase. The transformant of any one of Claims. The transformant according to claim 7, wherein the mevalonate pathway enzyme is hydroxymethylglutaryl coenzyme A reductase (HMG-CoA Reducet, HMGR). A culture step of culturing the transformant according to any one of claims 1 to 8,
A method for producing an isoprenoid precursor or an isoprenoid, comprising a recovery step of recovering a target substance from a culture medium after culture and / or the transformant.

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