Transformant and method for producing carotenoid composition using the same

Abstract

The present invention provides a technique for synthesizing a carotenoid composition by a genetic recombination technique.　A transformant in which a first promoter, an upstream gene of a carotenoid biosynthesis gene including a crtY and crtZ operably linked to the first promoter, a second promoter having higher promoter intensity than the first promoter, and a ZEP gene and a CCS gene operably linked to the second promoter have been introduced into a host cell.

Description

[Technical Field]

[0001] The present invention relates to a transformant and a method for producing a carotenoid composition using the transformant. [Background technology]

[0002] Carotenoids are natural pigments produced by plants, algae, bacteria, etc., and are an important group of compounds that play a variety of physiological roles in living organisms. Many carotenoids have been isolated, and the health benefits of carotenoids, which exist only in trace amounts in nature, have begun to attract attention. However, due to the trace amounts, commercial production of these compounds has been difficult and expensive.

[0003] For this reason, biotechnology research into the inexpensive production of carotenoids using microorganisms has been actively conducted in recent years. The carotenoid biosynthetic pathway and biosynthetic genes in plants had been largely elucidated by 2000. Many attempts have been made to identify upstream genes involved in carotenoid biosynthesis, introduce the identified genes into microorganisms to create transformants, and then produce carotenoids using the transformants.

[0004] Escherichia coli is the microorganism that has been most studied as a host for carotenoid production. E. coli synthesizes isopentenyl diphosphate (IPP) and its isomer, dimethylallyl diphosphate (DMAPP), via the non-mevalonate pathway (MEP pathway), which is the basic metabolic pathway for isoprenoids, including carotenoids (Non-Patent Document 1). The synthesized IPP and DMAPP are metabolized to geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) through sequential condensation reactions mediated by prenyltransferases.

[0005] E. coli is a non-carotenoid-producing bacterium and does not possess the enzyme genes required for carotenoid synthesis after FPP. However, by incorporating carotenoid biosynthesis genes, such as the geranylgeranyl pyrophosphate (GGPP) synthase gene (crtE), the phytoene synthase gene (crtB), the phytoene desaturase gene (crtI) that synthesizes lycopene, the lycopene cyclase gene (crtY) that synthesizes β-carotene, and the β-carotene hydroxylase gene (crtZ) that synthesizes β-cryptoxanthin and zeaxanthin, upstream of the target carotenoid, it is possible to synthesize various carotenoids.

[0006] However, even when carotenoids are found to be producible in this way, simply introducing the carotenoid biosynthesis genes often results in production levels far below practical levels. This is thought to be due to the fact that E. coli can only produce small amounts of FPP, the precursor for carotenoid synthesis. For this reason, pathway engineering aimed at increasing FPP production is being investigated, with the aim of mass-producing carotenoids.

[0007] Pathway engineering approaches can be broadly divided into methods that modify endogenous metabolic pathways, including the MEP pathway, and methods that introduce mevalonate (MVA) pathway genes from heterologous organisms.

[0008] A representative example of the former approach is a method of significantly increasing carotenoid production in Escherichia coli by highly expressing the type 1 IPP isomerase gene (idi) derived from S. cerevisiae or green algae (Haematococcus pluvialis) (Non-Patent Document 2). For example, it has been reported that Escherichia coli into which idi and crtE, crtB, crtI, crtY, and crtZ derived from Pantoea ananatis have been introduced can predominantly produce zeaxanthin (Non-Patent Document 3).

[0009] As a representative example of the latter approach, it has been found that deuterated zeaxanthin can be synthesized from deuterated D-MVA lactone as a substrate using Escherichia coli in which the MVA pathway genes (including the type 2 IPP isomerase gene) derived from the actinomycete Streptomyces sp. CL190 strain are functionally expressed (Non-Patent Document 4). This approach is considered to be a powerful and effective tool in pathway engineering research for carotenoid production using Escherichia coli as a host (Non-Patent Document 5).

[0010] Meanwhile, it has been found that the zeaxanthin epoxidase (ZEP) gene synthesizes antheraxanthin from zeaxanthin, and the capsanthin-capsorubin synthase (CCS) gene synthesizes capsanthin from antheraxanthin (Non-Patent Document 6). However, although these genes have been identified for a long time, there have been no reports of capsanthin being synthesized by introducing them into a host together with other carotenoid biosynthetic genes. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Rohmer, M. et al.: Biochem. J., 295, 517 (1993) [Non-patent document 2] Kajiwara, S. et al.: Biochem. J., 324, 421 (1997) [Non-patent document 3] Takemura, M. et al.: Tetrahedron Letters 56, 6063 (2015). [Non-patent document 4] Kakinuma, K. et al.: J. Am. Chem. Soc., 123, 1238 (2001). [Non-patent document 5] Journal of Bioengineering, Vol. 93, No. 7, 397 (2015). [Non-patent document 6] Bouvier, F. et al. The Plant Journal, 6(1), 45 (1994) Summary of the Invention [Problem to be solved by the invention]

[0012] Unlike lycopene, carotene, cryptoxanthin, and zeaxanthin, capsanthin cannot be synthesized by any of the previously known methods using recombinant microorganisms. In fact, even when the present inventors introduced the CrtE gene, CrtB gene, CrtI gene, CrtY gene, CrtZ gene, ZEP gene, and CCS gene together with the IDI gene into Enterobacter coli, no capsanthin was produced at all.

[0013] An object of the present invention is to provide a technique for producing a composition containing a carotenoid, such as capsanthin, which has not been possible to synthesize until now, by genetic recombination technology. [Means for solving the problem]

[0014] The present inventors came up with the novel idea of ​​controlling the expression levels of only some of the genes in the capsanthin biosynthesis gene cluster so that they are relatively high. Therefore, in order to obtain a relatively large amount of the capsanthin substrate, they constructed a recombinant vector that would relatively increase the expression level of the upstream gene in the capsanthin biosynthesis gene cluster, and transformed it to attempt to synthesize capsanthin. However, this method did not produce any capsanthin, even when idi was incorporated.

[0015] Therefore, we changed our approach and constructed a recombinant vector to increase the expression level of the downstream gene in the capsanthin biosynthesis gene cluster, and unexpectedly found that the transformant was able to produce capsanthin. Furthermore, we tried to express the ZEP gene and CCS gene so that ZEP and CCS indirectly bind via a multimer of a specific affinity protein. NigumiThey also unexpectedly found that when incorporated into a recombinant vector, it is possible to produce a variety of other carotenoids, such as capsorubin and cucurbitaxanthin A. The present invention was completed based on these findings and through further investigations.

[0016] That is, the present invention provides the following aspects of the invention. Item 1. A first promoter, and upstream genes of carotenoid biosynthesis genes including crtY and crtZ operably linked to the first promoter; a second promoter having a promoter strength higher than that of the first promoter, and a ZEP gene and a CCS gene operably linked to the second promoter; is introduced into a host cell, thereby forming a transformant. Item 2. The transformant according to Item 1, wherein the difference in promoter strength between the first promoter and the second promoter is equal to or greater than the difference in promoter strength between the promoter specific to a carotenoid biosynthetic gene and PBAD. Item 3. The transformant according to claim 1 or 2, wherein the upstream genes further include crtI; crtI and crtB; or crtI, crtB, and crtE. Item 4. The transformant according to any one of Items 1 to 3, wherein the first promoter is a promoter specific to a Plac or carotenoid biosynthetic gene. Item 5. The transformant according to any one of Items 1 to 4, wherein the second promoter is PBAD or Ptac. Item 6. Furthermore, an S protein gene, an S tag gene, and a linker gene are introduced into the host cell; the S tag gene is linked to each of the ZEP gene and the CCS gene, Item 6. The transformant according to any one of Items 1 to 5, wherein the linker gene is interposed and linked between the ZEP gene and the S-tag gene and / or between the CCS gene and the S-tag gene. Item 7. The transformant according to Item 6, wherein the S tag encoded by the S tag gene is any one of the following polypeptides (1) and (2): (1) the amino acid sequence shown in SEQ ID NO: 4 Queue? Polypeptides comprising (2) A polypeptide that has a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 4 except for the 12th position, and that specifically binds to S protein. Item 8. The transformant according to any one of Items 1 to 7, wherein the linker gene is interposed and linked at least between the ZEP gene and the S-tag gene. Item 9. The transformant according to any one of Items 1 to 8, wherein the linker gene is interposed between the ZEP gene and the S-tag gene and is linked thereto, but is not interposed between the CCS gene and the S-tag gene. Item 10. A method for producing a carotenoid composition, comprising a step of culturing the transformant according to any one of Items 1 to 9. Item 11. The method according to Item 10, wherein the carotenoid composition contains capsanthin. Item 12. The production method according to Item 10 or 11, wherein the carotenoid composition comprises capsorubin, capsanthin 3'-acetate, capsorubin 3-acetate, cucurbitaxanthin A, capsorubin diacetate, capsanthin 3,6-epoxide, and / or capsanthin 3,6-epoxide 3'-acetate. [Effects of the Invention]

[0017] The present invention makes it possible to produce compositions containing carotenoids, such as capsanthin, that have not previously been synthesized using genetic engineering techniques. While the mechanism by which this effect is achieved is unclear, it is believed to be as follows: CCS has been found to not only synthesize capsanthin from antheraxanthin, but also to have a side activity in synthesizing β-carotene from lycopene upstream in the carotenoid biosynthetic pathway. Therefore, when the upstream substrate is increased, much of the CCS function is utilized to catalyze the reaction synthesizing β-carotene from lycopene, preventing it from functioning in capsanthin synthesis. By relatively reducing the expression levels of zeaxanthin and genes upstream of it, and increasing the expression levels of the ZEP and CCS genes downstream of the genes involved in zeaxanthin synthesis, it is believed that excessive accumulation of zeaxanthin and its upstream intermediates can be suppressed, thereby enabling CCS, which previously could not be activated to synthesize capsanthin, to function for the first time. [Brief explanation of the drawings]

[0018] [Figure 1] The carotenoid biosynthetic pathway and the enzymes that mediate it are shown. [Figure 2] FIG. 1 is a schematic diagram of the recombinant vector prepared in Comparative Example 1. [Figure 3] FIG. 1 is a schematic diagram of the recombinant vectors prepared in Comparative Example 2 (Comparative Example 2-1 and Comparative Example 2-2). [Figure 4] 1 shows HPLC chromatograms of the carotenoid compositions obtained in Comparative Example 2 (Comparative Example 2-1 and Comparative Example 2-2). [Figure 5] FIG. 1 is a schematic diagram of the recombinant vector prepared in Example 1. [Figure 6] 1 is an HPLC chromatogram of the carotenoid composition obtained in Example 1. [Figure 7] 1 shows a UPLC chromatogram (a) of the carotenoid composition obtained in Example 1, an absorption spectrum of capsanthin (b), and a mass spectrum of capsanthin (c). [Figure 8]FIG. 1 is a schematic diagram of the recombinant vectors prepared in Example 2 (Example 2-1 and Example 2-2). [Figure 9] 1 shows HPLC chromatograms of the carotenoid compositions obtained in Example 2 (Example 2-1 and Example 2-2). [Figure 10] 1 shows the amount of capsanthin produced in the carotenoid compositions obtained in Example 2 (Example 2-1 and Example 2-2). [Figure 11] FIG. 1 is a schematic diagram of the recombinant vectors prepared in Example 3 (Example 3-1 to Example 3-3). [Figure 12] 10 is a schematic diagram showing a cooperative system of CCS and ZEP using S protein, constructed in the expression system according to Example 3 (Example 3-1 to Example 3-3). [Figure 13] 1 shows the analysis results of the carotenoid compositions obtained in Example 3 (Example 3-1 to Example 3-3). [Figure 14] 1 shows the analytical results of the carotenoid composition obtained in Example 3 (Example 3-2). DETAILED DESCRIPTION OF THE INVENTION

[0019] 1. Transformants The transformant of the present invention is characterized in that a first promoter, upstream genes of carotenoid biosynthetic genes including crtY and crtZ operably linked to the first promoter, a second promoter having a stronger promoter strength than the first promoter, and ZEP and CCS genes operably linked to the second promoter are introduced into a host cell, thereby expressing carotenoid biosynthetic enzymes in the host cell and enabling the production of a carotenoid composition.

[0020] 1-1. Carotenoid biosynthetic enzymes and the genes encoding them (carotenoid biosynthetic genes) The carotenoid biosynthesis pathway and the enzymes that mediate it are shown in Figure 1. Figure 1 also shows an example of carotenoid biosynthesis using recombinant Escherichia coli.

[0021] CrtE is a GGPP synthase that synthesizes geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP), and hereinafter, the gene encoding CrtE will be referred to as "crtE."

[0022] CrtB is a phytoene synthase that synthesizes phytoene from GGPP, and hereinafter, the gene encoding CrtB will be referred to as "crtB."

[0023] CrtI is a phytoene desaturase that synthesizes lycopene from phytoene, and hereinafter, the gene encoding CrtI will be referred to as "crtI."

[0024] CrtY is a lycopene cyclase that synthesizes β-carotene from lycopene, and hereinafter, the gene encoding CrtY will be referred to as "crtY."

[0025] CrtZ is a β-carotene hydroxylase that synthesizes β-cryptoxanthin and zeaxanthin from β-carotene, and hereinafter, the gene encoding CrtZ will be referred to as "crtZ."

[0026] ZEP is a zeaxanthin epoxidizing enzyme that synthesizes antheraxanthin and violaxanthin from zeaxanthin.

[0027] CCS is a capsanthin / capsorubin synthase known to synthesize capsanthin and capsorubin from antheraxanthin and violaxanthin, respectively. Furthermore, according to the findings of the present inventors, CCS is also a synthase that synthesizes capsanthin 3'-acetate and cucurbitaxanthin A from antheraxanthin, and capsorubin 3-acetate, capsorubin diacetate, capsanthin 3,6-epoxide, and capsanthin 3,6-epoxide 3'-acetate from violaxanthin.

[0028] The above-mentioned genes encoding carotenoid biosynthetic enzymes are known genes, and their specific sequences can be appropriately obtained from official databases or the like.

[0029] The organisms from which these carotenoid biosynthetic genes are derived are not particularly limited, and can be selected without particular limitation from organisms that produce the carotenoids corresponding to these genes. Specific organisms of origin, regardless of whether they are plants or microorganisms, include the genus Capsicum (e.g., Capsicum annuum), the genus Lilium (e.g., Lilium lancifolium), the genus Arabidopsis (e.g., Arabidopsis thaliana), the genus Zea (e.g., Zea mays), the genus Prunus (e.g., Prunus armeniaca), the genus Ipomoea (e.g., Ipomoea nil), the genus Gentiana (e.g., Gentiana lutea), the genus Marchantia (e.g., Marchantia polymorpha), the genus Pantoea (e.g., Pantoea ananas), the genus Agrobacterium (e.g., Agrobacterium aurantiacum), Haemamatococcus, Monoraphidium, Phaffia (Xanthophyllomyces), Paracoccus, Brevundimonas, Gordonia (Rhodococcus), Nostoc, Brevundimonas, and Gloeobacter, with Capsicum, Lilium, and Pantoea being preferred. The genes may be derived from any organism, but are preferably the same organism.

[0030] The carotenoid biosynthetic genes that can be used in the present invention include not only those consisting of nucleotide sequences that can be obtained from official databases and their codon-optimized sequences, but also those consisting of nucleotide sequences similar to the sequences (specifically, similar to the degree equivalent to the similarity of the nucleotide sequences (I-2), (II-2), (III-2) and (I-3), (II-3), (III-3) to the nucleotide sequences (I-1), (II-1), and (III-1) below) as long as they encode enzymes having carotenoid biosynthetic enzyme activity.

[0031] Preferred examples of the base sequences of the carotenoid biosynthesis genes are as follows: (I-1) The nucleotide sequences of the crtE, crtY, crtI, crtB, and crtZ genes shown in DDBJ accession number D90087, (II-1) The nucleotide sequence of the ZEP gene shown in DDBJ accession number XM_016705616, (III-1) The nucleotide sequence of the CCS gene shown in DDBJ accession number X76165, (I-2) A base sequence of a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in (I-1) above, wherein the base sequence encodes an enzyme having GGPP synthase activity that synthesizes geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP), an enzyme having phytoene synthase activity that synthesizes phytoene from GGPP, an enzyme having phytoene desaturase activity that synthesizes lycopene from phytoene, an enzyme having lycopene cyclase activity that synthesizes β-carotene from lycopene, and an enzyme having β-carotene hydroxylase activity that synthesizes β-cryptoxanthin and zeaxanthin from β-carotene, respectively. (II-2) A gene encoding an enzyme having zeaxanthin epoxidation enzyme activity that synthesizes antheraxanthin and violaxanthin from zeaxanthin, wherein the base sequence of a nucleic acid hybridizes under stringent conditions with a nucleic acid having a base sequence complementary to the base sequence shown in (II-1) above; (III-2) A gene encoding an enzyme activity that synthesizes capsanthin and capsorubin from antheraxanthin and violaxanthin, respectively, and also synthesizes capsorubin, capsanthin 3'-acetate, capsorubin 3-acetate, cucurbitaxanthin A, capsorubin diacetate, capsanthin 3,6-epoxide, and capsanthin 3,6-epoxide 3'-acetate, wherein the base sequence of a nucleic acid hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in (III-1) above; (I-3) Genes encoding an enzyme having GGPP synthase activity to synthesize geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP), an enzyme having phytoene synthase activity to synthesize phytoene from GGPP, an enzyme having phytoene desaturase activity to synthesize lycopene from phytoene, an enzyme having lycopene cyclase activity to synthesize β-carotene from lycopene, and an enzyme having β-carotene hydroxylase activity to synthesize β-cryptoxanthin and zeaxanthin from β-carotene, each of which has a homology of 90% or more with a base sequence complementary to the base sequence shown in (I-1) above; (II-3) A gene encoding an enzyme having zeaxanthin epoxidation activity that synthesizes antheraxanthin and violaxanthin from zeaxanthin, wherein the base sequence has 90% or more homology with the base sequence shown in (II-1) above; (III-3) A gene encoding the enzyme activity that synthesizes capsanthin and capsorubin from antheraxanthin and violaxanthin, respectively, and also synthesizes capsorubin, capsanthin 3'-acetate, capsorubin 3-acetate, cucurbitaxanthin A, capsorubin diacetate, capsanthin 3,6-epoxide, and capsanthin 3,6-epoxide 3'-acetate, which base sequence has 90% or more homology with the base sequence shown in (III-1) above.

[0032] In the above (I-2), (II-2), and (III-2), "under stringent conditions" refers to incubation at 50°C to 65°C for 4 hours to overnight in 6xSSC (1xSSC is 0.15M NaCl, 0.015M sodium citrate, pH 7.0) containing 0.5% SDS, 5x Denhartz's (0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400), and 100µg / ml salmon sperm DNA. Hybridization under stringent conditions is specifically performed by the following method: A nylon membrane with an immobilized DNA library or cDNA library is prepared, and the nylon membrane is blocked at 65°C in a prehybridization solution containing 6xSSC, 0.5% SDS, 5x Denhartz's, and 100µg / ml salmon sperm DNA. Then, 32 Each P-labeled probe is added and incubated overnight at 65°C. The nylon membrane is washed in 6xSSC at room temperature for 10 minutes, in 2xSSC containing 0.1% SDS at room temperature for 10 minutes, and in 0.2xSSC containing 0.1% SDS at 45°C for 30 minutes, and then autoradiographed to detect nucleic acids that have specifically hybridized with the probes.

[0033] In the above (I-3), (II-3), and (III-3), the homology may be 90% or more, preferably 95% or more, more preferably 98% or more, even more preferably 98.5% or more, even more preferably 99% or more, even more preferably 99.5% or more, and particularly preferably 99.9% or more. "Homology" is calculated using publicly available or commercially available software with an algorithm for comparing a reference sequence with a query sequence. Specifically, BLAST, FASTA, or GENETYX (manufactured by Genetyx Corporation) can be used, and these can be used with default parameters.

[0034] Furthermore, the carotenoid biosynthetic genes may be incorporated into a recombinant vector in their entirety, or may be incorporated into a recombinant vector with sequences unnecessary for the expression of the target enzyme in the host removed. For example, when the organism from which the carotenoid biosynthetic genes are derived is a plant and the host is a non-plant cell, it is preferable to incorporate the genes into a recombinant vector with the signal sequence removed, from the perspective of further improving the production of capsanthin. The signal sequences of each plant-derived gene can also be easily obtained using official databases or various genetic analysis tools.

[0035] In the following, among the carotenoid biosynthetic genes, crtE, crtB, crtI, crtY, and crtZ are classified as "upstream genes," and the ZEP gene and CCS gene are classified as "downstream genes."

[0036] An upstream gene of a carotenoid biosynthetic gene is operably linked to a first promoter, and a downstream gene of a carotenoid biosynthetic gene is operably linked to a second promoter. That is, the first promoter is a region that controls the transcription of the upstream gene, and the second promoter is a region that controls the transcription of the downstream gene. Operable linkage means that a control factor and the above gene are linked in a state that allows them to operate in a host cell.

[0037] 1-2.Promoter Regarding the relationship between the first promoter and the second promoter, the second promoter is designed so that its promoter strength is greater than that of the first promoter. For example, the promoter strength of the second promoter is preferably at least three times that of the first promoter. Promoter strength refers to the frequency of transcription initiation and is measured, for example, in Pbla units (Deuschle et al., EMBO J., 5:2987-2994 (1986)), Miller units (also known as β-galactosidase units; 5000 times Pbla units (Lanzer et al., PNAS USA, 85, 8973-8977 (1988)), or RPUs (Relative promoter units; Meyer et al., Nature Chemical Biology 15, 196-204 (2019)).

[0038] In the present invention, the first promoter and the second promoter control the expression level of the upstream gene of the carotenoid biosynthesis gene so that it is relatively low and the expression level of the downstream gene is relatively high, thereby enabling the synthesis of carotenoids.

[0039] From the viewpoint of further improving the amount of carotenoid synthesis, it is preferable that the difference in promoter strength between the first promoter and the second promoter is equal to or greater than the difference in promoter strength between a promoter specific to a carotenoid biosynthesis gene (preferably a promoter native to Pantoea ananatis, more preferably a promoter included in DDBJ accession number D90087) and the PBAD promoter, and it is even more preferable that the difference in promoter strength between a promoter specific to a carotenoid biosynthesis gene (preferably a promoter native to Pantoea ananatis, more preferably a promoter included in DDBJ accession number D90087) and the tac promoter.

[0040] The first promoter and the second promoter are not particularly limited, as long as they are designed so that the second promoter has a relatively higher promoter strength, and are appropriately selected depending on the type of host into which the recombinant vector containing these promoters is to be introduced. The first promoter and the second promoter may be natural promoters or artificial promoters.

[0041] Specific examples of promoters used as the first promoter and the second promoter include, in prokaryotes such as Escherichia coli, the lac promoter (Plac), promoters specific to carotenoid biosynthetic genes, the BAD promoter (PBAD), the tac promoter (Ptac), the trc promoter, the T7 promoter, the T7lac promoter, the T5 promoter, the UV5lac promoter, the L8-UV5lac promoter, the cspA promoter, the CAG promoter, the CMV promoter, the RSV promoter, and the like; and in yeast, the GAL1 promoter, the NMT1 promoter, the TEF1 promoter, the ADH1 promoter, the TPI1 promoter, the HXT7 promoter, the TDH3 promoter, the PGK1 promoter, the PYK1 promoter, the CAG promoter, the CMV promoter, the RSV promoter, and the like.

[0042] The carotenoid biosynthetic gene-specific promoter is a promoter inherent in carotenoid biosynthesis. The carotenoid biosynthetic gene-specific promoter can be any promoter that is inherent in the organism from which the carotenoid biosynthetic gene is derived, and can be used as the promoter for the carotenoid biosynthesis gene. For example, when a gene placed under the control of the carotenoid biosynthetic gene-specific promoter is derived from Pantoea ananatis, the carotenoid biosynthetic gene-specific promoter can preferably be a promoter inherent in Pantoea ananatis, more preferably the promoter included in DDBJ accession number D90087.

[0043] From various promoters including these examples, two promoters with different promoter strengths were selected, and the promoter with the lower promoter strength was designated as the first promoter. expensive Furthermore, in order to further improve the amount of carotenoid synthesis, a combination of two promoters can be selected from various promoters including those exemplified above, so that the difference in promoter strength is as described above, and the promoter with the lower promoter strength can be used as the first promoter. expensive Either promoter can be selected as the second promoter.

[0044] More preferably, the first promoter includes promoters specific to Plac and carotenoid biosynthetic genes, and the second promoter includes PBAD and Ptac, more preferably Ptac.

[0045] Furthermore, the expression level is controlled by using the first and second promoters with different promoter strengths as described above, so as to suppress the reaction of CCS synthesizing β-carotene from lycopene. Therefore, the upstream genes expressed by the first promoter only need to include at least crtY and crtZ.

[0046] The other upstream genes may be placed under the control of the first promoter, as with crtY and crtZ, or may be placed under the control of another promoter. Preferably, the other upstream genes are placed under the control of the first promoter. Specific examples of when the other upstream genes are placed under the control of the first promoter include when the upstream genes further include (i) crtI; (ii) crtI and crtB; and (iii) crtI, crtB, and crtE. The IPP isomerase gene (idi) may also be included together with the other upstream genes; that is, (iv) crtI, crtB, crtE, and idi may also be placed under the control of the first promoter.

[0047] The first promoter and the gene placed under its control, and the second promoter and the gene placed under its control may be incorporated into a single expression vector to form a single recombinant vector, or may be incorporated into separate expression vectors to form a first recombinant vector and a second recombinant vector, respectively.

[0048] 1-3. Genes for the coordinated expression of CCS and ZEP via S protein In addition to capsanthin / capsorubin synthesis activity, CCS also possesses the lycopene β-cyclase activity of CrtY. Due to this dual activity of CCS, even if the CCS gene is efficiently expressed in E. coli, it may cooperate with the phytoene desaturase CrtI rather than ZEP, resulting in a reaction in which lycopene is received as a substrate from CrtI and β-carotene is synthesized. Therefore, to further increase the amount of reaction product produced by CCS by selectively allowing CCS to react cooperatively with ZEP, it is preferable that the CCS and ZEP genes be introduced into host cells in a manner that expresses the S protein-mediated CCS and ZEP cooperative system, as described below. A schematic diagram of this cooperative system is shown in Figure 12.

[0049] In an aspect of the present invention in which the cooperative system of CCS and ZEP mediated by S protein is expressed, an S protein gene, an S tag gene, and a linker gene are further introduced into a host cell, the S tag gene is linked to each of the ZEP gene and the CCS gene, and the linker gene is interposed and linked between the ZEP gene and the S tag gene and / or between the CCS gene and the S tag gene.

[0050] The S tag and S protein are polypeptides that form a single RNase A molecule, acting as a specific affinity tag and protein. The N-terminal 20-amino acid fragment obtained by cleaving RNase A with satilisin is the S tag, and the remaining fragment of approximately 100 amino acids is the S protein. The S tag and S protein can noncovalently bind to each other to restore the original higher-order structure, resulting in RNase activity (Lopez-Alonso, JP, Bruix, M., Font, J., Ribo, M., Vilanova, M., Rico, M., Gotte, G., Libonati, M., Gonzalez, C. and Laurents, DV (2006) J. Biol. Chem. 281, 9400-9406).

[0051] S protein and RNase A have the property that some of these molecules swap their C-terminal β-strands between molecules to form multimers such as homodimers, homotrimers, and homomultimers of more than three, as shown in Figure 12.

[0052] On the other hand, the ZEP gene and CCS gene linked to an S-tag gene are expressed in host cells as S-tag fused ZEP and S-tag fused CCS, respectively. Furthermore, since a linker gene is interposed and linked between the ZEP gene and the S-tag gene and / or between the CCS gene and the S-tag gene, either or both of the S-tag fused ZEP and S-tag fused CCS expressed in host cells are in the form of a fusion protein with a linker between the S-tag and the enzyme. The schematic diagram in Figure 1 shows examples in which both the S-tag fused ZEP and the S-tag fused CCS are expressed in the form of a fusion protein with a linker between the S-tag and the enzyme ("S tag + linker + CCS" and "S tag + linker + ZEP" in the diagram).

[0053] When such S-tagged ZEP and S-tagged CCS are coexpressed with S protein, a complex is formed in which the fused ZEP and fused CCS are indirectly bound to the S protein multimer via the S-tag, as shown in Figure 12. The formation of such a complex brings ZEP and CCS into close physical proximity within the host cell, allowing CCS to selectively react cooperatively with ZEP. In other words, CCS can smoothly receive metabolites produced by ZEP. This allows for increased production of carotenoids produced by CCS metabolism. More specifically, the production of capsanthin can be improved, and / or the production of capsorubin, capsanthin 3'-acetate, capsorubin 3-acetate, cucurbitaxanthin A, capsorubin diacetate, capsanthin 3,6-epoxide, and / or capsanthin 3,6-epoxide 3'-acetate can be improved.

[0054] As mentioned above, S protein is known as a cleavage product of approximately 100 amino acids by the RNase A satilisin, and therefore its amino acid sequence is publicly known (specifically, SEQ ID NO: 2), and the nucleotide sequence of the S protein gene is also publicly known (specifically, SEQ ID NO: 1). Examples of nucleotide sequences of S protein genes that can be used in the present invention include the following:

[0055] (IV-1) the base sequence shown in SEQ ID NO: 1; (IV-2) A gene encoding a protein that forms a multimer and specifically binds to an S tag, wherein the base sequence of a nucleic acid hybridizes under stringent conditions with a nucleic acid having a base sequence complementary to the base sequence shown in (IV-1) above; and (IV-3) A gene encoding a protein that forms a multimer and specifically binds to an S tag, the base sequence having 90% or more homology with the base sequence shown in (IV-1) above.

[0056] The "stringent conditions" in (IV-2) above are the same as those described in (I-2), (II-2), and (III-2) above. The "homology" and preferred examples thereof in (IV-3) above are the same as those described in (I-3), (II-3), and (III-3) above.

[0057] As mentioned above, the S tag is known as a 20-amino acid cleavage product at the N-terminus by the RNase A enzyme satilisin, and therefore its amino acid sequence is publicly known (specifically, the amino acid sequence in which position 12 of SEQ ID NO: 4 is histidine instead of phenylalanine), and the nucleotide sequence of the S protein gene is also publicly known (specifically, the nucleotide sequence in which positions 34 to 36 of SEQ ID NO: 3 are histidine-encoding instead of phenylalanine-encoding).

[0058] Examples of S tags that can be used in the present invention include any of the following: (1) A polypeptide consisting of a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 4 (2) A polypeptide having a sequence identity of 90% or more, preferably 95% or more, other than the 12th amino acid, with the amino acid sequence shown in SEQ ID NO: 4, and specifically binding to S protein.

[0059] In addition, in the amino acid sequence shown in SEQ ID NO: 4, the histidine at position 12 in the wild-type S tag has been substituted with phenylalanine in order to suppress the effect on RNase activity caused by the S tag binding to the S protein in host cells.

[0060] In the polypeptide (2) above, the sequence identity to the amino acid sequence shown in SEQ ID NO: 4 refers to the sequence identity calculated by comparing the sequence with the amino acid sequence shown in SEQ ID NO: 4. Furthermore, "sequence identity" refers to the amino acid sequence identity value obtained using the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, pp. 247-250, 1999) of BLASTPACKAGE [sgi32-bit edition, Version 2.0.12; available from the National Center for Biotechnology Information (NCBI)]. The parameters may be set to a gap insertion cost value of 11 and a gap extension cost value of 1.

[0061] The base sequence of the S tag gene that can be used in the present invention is not particularly limited as long as it is a base sequence that encodes the above-mentioned S tag, but preferred examples include the following.

[0062] (V-1) the nucleotide sequence shown in SEQ ID NO: 3 (i.e., the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 4), (V-2) A gene encoding a peptide that specifically binds to S protein, which has a homology of 90% or more, preferably 95% or more, to the base sequence shown in (V-1) above (excluding the base sequence at positions 34 to 36 of SEQ ID NO: 3).

[0063] The "homology" in (V-2) above is the same as that described in (I-3), (II-3), and (III-3) above.

[0064] In addition, positions 34 to 36 of SEQ ID NO: 3 in the S-tag genes (V-1) to (V-2) above have been substituted from the histidine-encoding sequence present in the wild-type S-tag to a phenylalanine-encoding sequence in order to suppress the effect on RNase activity caused by the expressed S-tag binding to the S-protein in the host cell.

[0065] Furthermore, the binding position of the S-tag gene is not particularly limited, but from the viewpoint of minimizing the effect on the activity of the enzyme fused to the S-tag, the S-tag can be designed to be bound to the N-terminus of the enzyme (ZEP and / or CCS).

[0066] The linker gene is not particularly limited as long as it provides an appropriate distance between the S-tag and the enzyme in the fusion protein, thereby ensuring flexibility between the CCS and ZEP in the complex. A preferred sequence for the linker (peptide) in the fusion protein is one in which nonpolar and polar amino acids are alternately linked, from the viewpoint of eliminating polarity bias in the linker (peptide) itself and suppressing undesired adsorption, and a preferred combination of nonpolar and polar amino acids is glycine and serine. The length of the linker (peptide) can be, for example, 15 to 25 amino acids, preferably 18 to 22 amino acids.

[0067] As described above, the linker gene may be located at least between the ZEP gene and the S-tag gene or between the CCS gene and the S-tag gene, but is preferably located at least between the ZEP gene and the S-tag gene, and particularly preferably only between the ZEP gene and the S-tag gene (not between the CCS gene and the S-tag gene).

[0068] 1-4.Other genes In addition to the above sequences, the recombinant vector may contain regulatory sequences other than the first promoter and the second promoter. Such regulatory sequences are located upstream (5' side) of, within, or downstream (3' side) of the sequence of an upstream gene of a carotenoid biosynthesis gene and / or the sequence of a downstream gene of a carotenoid biosynthesis gene, and affect transcription, RNA processing, stability, etc. Regulatory sequences include translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, stem-loop structures, etc.

[0069] 1-5. Expression vector Expression vectors that can be used include those constructed for genetic recombination from plasmids, viruses, phages, transposons, IS elements, phasmids, cosmids, and the like that can replicate autonomously in a host. Examples of such expression vectors include pET, pLG, pACYC, pBR, pUC, pKC, pRep, pHS, pKK, pDHE, pPLc, pMBL, pUR, pIN, λgt, pBdCI, pUB, pC, pBD; pSA, pAJ, YEp, YRp, YIp, pYAC, pCDM, pMT2PC, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and herpesvirus vectors. The expression vectors underlying the first and second recombinant vectors may be the same or different. Furthermore, the expression vector may be used in an appropriate combination with the host cell. For example, when Escherichia coli is used as the host cell, a preferred example is the combination of pACYC vector and pUC vector with the JM101(DE3) Escherichia coli strain.

[0070] 1-6.Host cells The host is not particularly limited as long as the recombinant vector is stable, is capable of autonomous replication, and is capable of expressing the traits of the foreign gene, and may be a carotenoid-producing cell or a non-carotenoid-producing cell. Preferred examples of the host include bacteria belonging to the genus Escherichia, such as Escherichia coli, the genus Bacillus, such as Bacillus subtilis, and the genus Pseudomonas, such as Pseudomonas putida; yeasts (e.g., Saccharomyces cerevisiae, S. pombe, Candida utilis, Pichia pastoris, and Xanthophyllomyces dendrorhous), but may also be animal cells, insect cells, or plants. Among these, a particularly preferred host is Escherichia coli.

[0071] 1-7. Preparation of transformants The transformant of the present invention can be obtained by introducing a recombinant vector into a host, and the conditions for introducing the recombinant vector into the host may be appropriately set depending on the type of host, etc. When the host is a bacterium, examples of the methods include the heat shock method, a method using competent cells treated with calcium ions, and the electroporation method. When the host is a yeast, examples of the methods include the electroporation method, the spheroplast method, and the lithium acetate method. When the host is an animal cell, examples of the methods include the electroporation method, the calcium phosphate method, and the lipofection method. When the host is an insect cell, examples of the methods include the calcium phosphate method, the lipofection method, and the electroporation method. When the host is a plant cell, examples of the methods include the electroporation method, the Agrobacterium method, the particle gun method, and the PEG method.

[0072] Whether or not the recombinant vector has been incorporated into the host can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like.

[0073] When confirming by PCR whether or not a recombinant vector has been incorporated into a host, for example, the recombinant vector may be isolated and purified from a transformant.

[0074] For example, when the host is a bacterium, isolation and purification of the recombinant vector can be carried out using the lysate obtained by lysing the bacteria, for example, by treating the bacteria with a lytic enzyme such as lysozyme, optionally in combination with a protease, other enzymes, and a surfactant such as sodium lauryl sulfate (SDS).

[0075] Furthermore, physical disruption methods such as freeze-thawing, ultrasonic irradiation, and French press treatment may be combined. DNA can be isolated and purified from the lysate by, for example, an appropriate combination of deproteinization treatments using phenol and protease, ribonuclease treatment, alcohol precipitation, and commercially available kits.

[0076] DNA can be cleaved using conventional methods, for example, restriction enzyme treatment. For example, type II restriction enzymes that act on specific nucleotide sequences can be used. DNA can be ligated to an expression vector using, for example, DNA ligase.

[0077] The isolated and purified DNA is then cleaved with a specific restriction enzyme and subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., and stained with ethidium bromide and SYBR Green solution, etc., to detect a band length pattern that can be used to confirm transformation.

[0078] Transformation can also be confirmed by PCR using the isolated and purified DNA as a template and designed primers. The PCR amplification products can be subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., and stained with ethidium bromide and SYBR Green solution, etc., to detect the amplified products as bands, thereby confirming transformation.

[0079] Alternatively, PCR can be performed using primers pre-labeled with fluorescent dyes, etc. to detect the amplified products. Furthermore, a method can be employed in which the amplified products are bound to a solid phase such as a microplate, and the amplified products are confirmed by fluorescence, enzyme reactions, etc.

[0080] 2. Method for producing carotenoid composition The carotenoid composition of the present invention can be obtained by a production method including a step of culturing the transformant of the present invention.

[0081] The culture conditions for the transformant may be appropriately determined taking into consideration the nutritional and physiological properties of the host, but liquid culture is preferred. For industrial production, aeration and agitation culture is preferred.

[0082] The nutrient sources used in the medium may be those required for the growth of the transformant.The carbon source may be any assimilable carbon compound, such as molasses, glucose, fructose, maltose, sucrose, lactose, starch, milk sugar, glycerol, and pyruvic acid.

[0083] The nitrogen source may be any assimilable nitrogen compound, and examples thereof include natural ingredients such as corn steep liquor, peptone (meat peptone, casein peptone, soybean peptone, etc.), extracts (meat extract, yeast extract), etc.; ammonium salts such as ammonium acetate, ammonium chloride, ammonium sulfate, etc.; and amino acids such as glutamic acid, aspartic acid, glycine, etc.

[0084] In addition to carbon and nitrogen sources, for example, salts of phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron, manganese, zinc, and the like, as well as specific vitamins, may be used as needed.

[0085] The culture temperature can be appropriately set within a range in which the transformant of the present invention can grow and the transformant can produce the polypeptide of the present invention, and is preferably about 20 to 40° C., and more preferably about 30 to 37° C. The culture may be completed at an appropriate time when the polypeptide of the present invention reaches its maximum yield, and the culture time may be, for example, 24 to 200 hours, and preferably about 60 to 90 hours.

[0086] Methods for recovering the bacterial cells after the completion of the culture include filtration of the culture medium, decantation, centrifugation, etc. The recovered bacterial cells can be treated with water, a sodium chloride solution, dimethylformamide, etc., as necessary.

[0087] The carotenoid composition obtained by the production method of the present invention typically contains capsanthin, and preferably further contains capsorubin, capsanthin 3'-acetate, capsorubin 3-acetate, cucurbitaxanthin A, capsorubin diacetate, capsanthin 3,6-epoxide, and / or capsanthin 3,6-epoxide 3'-acetate.

[0088] A suitable organic solvent can be used to extract the carotenoid composition from the fungus body. Examples of the organic solvent include methanol, ethanol, isopropyl alcohol, acetone, hexane, diethyl ether, tetrahydrofuran, methyl ethyl ketone, methyl isobutyl ketone, dichloromethane, chloroform, dimethylformamide, dimethyl sulfoxide, methyl acetate, and ethyl acetate. One or more of these solvents can be selected. Among these organic solvents, acetone, methanol, and diethyl ether are preferred.

[0089] The carotenoid composition fraction obtained as described above may be concentrated and dried as it is as a composition, or each carotenoid can be purified, concentrated, and dried.

[0090] As a purification method, for example, it can be carried out by appropriately combining methods such as gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography, etc. As a drying method, freeze drying, vacuum drying, spray drying, etc. can be mentioned.

Examples

[0091] Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples. In the following examples and comparative examples, the promoters used are Plac, the promoter specific to the carotenoid biosynthesis gene (a promoter inherent to Pantoea ananatis, specifically, the promoter included in DDBJ accession number D90087), PBAD, or Ptac. The relationship of promoter strength is Plac < the promoter specific to the carotenoid biosynthesis gene < PBAD < Ptac, and the promoter strength of PBAD is 3 times or more the promoter strength of the promoter specific to the carotenoid biosynthesis gene.

[0092] [Comparative Example 1] In this comparative example, pACHP-Zea shown in Figure 2 was constructed as the first recombinant vector to be incorporated, and pUC-Plac-CaCCS-CaZEP shown in Figure 2 was constructed as the second recombinant vector. These recombinant vectors were designed such that the expression level of the upstream gene was relatively high by the promoter specific to the carotenoid biosynthesis gene (derived from Pantoea ananatis), and the expression level of the downstream gene was relatively low by Plac. The outlines of the first recombinant vector and the second recombinant vector of this comparative example are shown in Table 1.

[0093]

Table 1

[0094] The first recombinant vector, pACHP-Zea, is a vector for zeaxanthin synthesis used in the report of Non-Patent Document 3. pACHP-Zea is a vector containing the Haematococcus pluvialis gene in the pACYC vector. i It is constructed by inserting the idi gene from P. alis (DDBJ accession number AB019034) and a group of genes required for zeaxanthin biosynthesis (the crtE, crtY, crtI, crtB, and crtZ genes from Pantoea ananatis (DDBJ accession number D90087)).

[0095] The second recombinant vector, pUC-Plac-CaCCS-CaZEP, was constructed by placing the ZEP gene derived from Capsicum annuum (hereinafter referred to as "CaZEP"; DDBJ accession number XM_016705616) and the CCS gene derived from Capsicum annuum (hereinafter referred to as "CaCCS"; DDBJ accession number X76165) downstream of the Plac promoter of the pUC vector.

[0096] Both the first and second recombinant vectors were introduced into Escherichia coli JM109 strain by the heat shock method.

[0097] After the introduction of the recombinant vector, a single colony from the LB medium was transferred to 5 mL of LB liquid medium and cultured with shaking at 200 rpm at 37°C for 16 hours. 100 μL of the culture was transferred to 10 mL of TB liquid medium and cultured with shaking at 200 rpm at 30°C for 24 hours. 10 μL of isopropyl-β-D-galactopyranoside (IPTG) was added to induce gene expression, and the culture was continued for an additional 48 hours. Chloramphenicol (30 mg / L) and kanamycin (40 mg / L) were also added as antibiotics to the medium.

[0098] One mL of the resulting culture medium was centrifuged at 8,000 g for 1 minute, and the supernatant was removed to recover the cell fraction. The cell fraction was suspended in 1% sodium chloride solution and centrifuged at 8,000 g for 1 minute. The supernatant was removed and the cells were recovered. 0.5 mL of acetone and 1 mL of diethyl ether / hexane (1:1 (volume basis)) were added to the recovered cells and vigorously mixed using a vortex mixer. 1 mL of water was added, mixed, and allowed to stand for 5 minutes. The upper layer was collected in a 1.5 mL centrifuge tube and dried using a vacuum centrifugal concentrator.

[0099] The resulting dried product was subjected to carotenoid separation using an Aquity H-class UPLC (Waters). The column used was an Aquity UPLC BEH C18 column (2.1 x 100 mm, 1.7 μm, Waters). The mobile phase A was acetonitrile:ultrapure water (85:15 by volume), and the mobile phase B was acetonitrile:methanol (65:35 by volume). The mobile phase A:mobile phase B was run with a gradient of 100:0 to 0:100 over 8 minutes. The carotenoids were then separated by holding the ratio at 0:100 for 5 minutes.

[0100] As a result, no peak with the same elution time as the capsanthin standard was confirmed.

[0101] [Comparative Example 2 (Comparative Example 2-1 and Comparative Example 2-2)] In this comparative example, pAC-HiEBIY-Z-CaZEP shown in Figure 3 was constructed as the first recombinant vector to be incorporated, and pUC-CaCCS (the case where this second recombinant vector was used is referred to as Comparative Example 2-1) or pUC-LlCCS (the case where this second recombinant vector was used is referred to as Comparative Example 2-2) shown in Figure 3 was constructed as the second recombinant vector. These recombinant vectors were designed so that, of the upstream and downstream genes, the expression level of the ZEP gene was relatively high due to Ptac, and, of the downstream genes, the expression level of the CCS gene was relatively low due to PBAD. An overview of the first and second recombinant vectors of this comparative example is shown in Table 2.

[0102] [Table 2]

[0103] In Comparative Examples 2-1 and 2-2, based on the report in Non-Patent Document 3, Haematococcus pluv i alis-derived idi gene, along with the Pantoea ananatis-derived crtE, crtB, crtI, crtY, and crtZ genes required for violaxanthin biosynthesis, and amino acid residues 64 to 668 of CaZEP (because plant-derived genes often have signal sequences at the N-terminus of the protein that are unnecessary in E. coli. CaZEP 64-668 In Figure 3, it is abbreviated as ZEP * The first recombinant vector, pAC-HiEBIY-Z-CaZEP, was constructed by placing the gene encoding ...

[0104] The prepared first recombinant vector pAC-HiEBIY-Z-CaZEP was introduced into Escherichia coli JM101(DE3) and cultured in the same manner as in Comparative Example 1, and a supernatant was obtained.

[0105] The resulting supernatant was subjected to carotenoid separation using a Hitachi HPLC Chromaster system. The column was an Inertsil ODS-3 (4.6 x 150 mm, 5 μm, GL Sciences) with acetonitrile as mobile phase A and isopropanol as mobile phase B. After pumping at a 100:0 ratio of mobile phase A:mobile phase B for 5 minutes, the ratio was increased from 100:0 to 50:50 (volume-based) over 5 minutes, and the 50:50 ratio was maintained for another 5 minutes to separate the carotenoids.

[0106] The obtained HPLC chromatogram is shown in Figure 4. As shown in Figure 4, it was confirmed that in addition to violaxanthin and antheraxanthin, zeaxanthin, β-carotene, and many other carotenoid intermediates were produced.

[0107] Next, in Comparative Example 2-1 of Comparative Example 2-1 and Comparative Example 2-2, it was investigated whether capsanthin and capsorubin could be produced by co-expressing CaCCS in this carotenoid-producing E. coli strain. The N-terminal chloroplast signal sequence of CaCCS was predicted, and CaCCS with the N-terminal amino acid deleted was used. 41-498 CaCCS 41-498 The second recombinant vector, pUC-CaCCS, was constructed by placing the gene downstream of the PBAD promoter of the pUC vector, and was introduced into the carotenoid-producing E. coli strain described above.

[0108] In addition, in Comparative Example 2-2, which is one of Comparative Examples 2-1 and 2-2, pUC-LlCCS was prepared as a second recombinant vector in the same manner as in Comparative Example 2-1, except that CCS derived from Lilium lancifolium (hereinafter referred to as "LlCCS"; DDBJ accession number GU443955) was introduced instead of CaCCS, and introduced into the above-mentioned carotenoid-producing E. coli strain.

[0109] In Comparative Examples 2-1 and 2-2, after the introduction of the second recombinant vector, a single colony on LB medium was transferred to 3 mL of LB liquid medium and cultured with shaking at 200 rpm at 37°C for 16 hours. 100 μL of the culture was then transferred to 10 mL of TB liquid medium and cultured with shaking at 200 rpm at 30°C for 24 hours. 10 μL of isopropyl-β-D-galactopyranoside (IPTG) was added to induce gene expression, and the culture was continued for an additional 48 hours. Chloramphenicol (30 mg / L) and kanamycin (40 mg / L) were also added as antibiotics to the medium.

[0110] One mL of the resulting culture medium was centrifuged at 8,000 g for 1 minute, the supernatant was removed, and the cell fraction was collected. The cell fraction was suspended in 1% sodium chloride solution and centrifuged at 8,000 g for 1 minute, the supernatant was removed, and the cells were collected. Carotenoids were extracted by adding acetone / methanol (7:3 (volume basis)) to the collected cells, and the mixture was centrifuged at 12,000 g for 2 minutes. The supernatant was used for carotenoid analysis.

[0111] The resulting supernatant was subjected to carotenoid separation using a Hitachi HPLC Chromaster system. The column was an Inertsil ODS-3 (4.6 x 150 mm, 5 μm, GL Sciences), with acetonitrile as mobile phase A and isopropanol as mobile phase B. After pumping at a 100:0 ratio of mobile phase A:mobile phase B for 5 minutes, the ratio was increased from 100:0 to 50:50 (volume-based) over 5 minutes, and then maintained at 50:50 for another 5 minutes.

[0112] As a result, as shown in the results of Comparative Examples 2-1 and 2-2 in Figure 4, when either CaCCS or LlCCS was co-expressed, there was no change in the carotenoids produced, and the production of capsanthin could not be confirmed.

[0113] [Example 1] In this example, pACHP-Zea, shown in Figure 5, was constructed as the first recombinant vector to be incorporated, and pUC-CaCCS-CaZEP, shown in Figure 5, was constructed as the second recombinant vector. These recombinant vectors were designed so that the expression level of the upstream gene was relatively low due to a promoter specific to the carotenoid biosynthesis gene (derived from Pantoea ananatis), and the expression level of the downstream gene was relatively high due to PBAD. An overview of the first and second recombinant vectors used in this example is shown in Table 3.

[0114] [Table 3]

[0115] The first recombinant vector, pACHP-Zea, was prepared in the same manner as in Comparative Example 1. Non-Patent Document 3 reports that in the carotenoid composition of the transformant, β-carotene was not detected and zeaxanthin accounted for the total carotenoids.

[0116] pUC-CaCCS-CaZEP as the second recombination vector was used to 64-668 and CaCCS41-498 was placed downstream of the PBAD promoter of a pUC vector.

[0117] Both the first and second recombinant vectors were introduced into Escherichia coli JM101(DE3) in the same manner as in Comparative Example 1.

[0118] After the introduction of the recombinant vector, a single colony on LB medium was transferred to 3 mL of LB liquid medium and cultured with shaking at 200 rpm at 37°C for 16 hours. 100 μL of the culture was transferred to 10 mL of TB liquid medium and cultured with shaking at 200 rpm at 30°C for 24 hours. 10 μL of isopropyl-β-D-galactopyranoside (IPTG) was added to induce gene expression, and the culture was continued for an additional 48 hours. Chloramphenicol (30 mg / L) and kanamycin (40 mg / L) were also added as antibiotics to the medium.

[0119] One mL of the resulting culture medium was centrifuged at 8,000 g for 1 minute, the supernatant was removed, and the cell fraction was collected. The cell fraction was suspended in 1% sodium chloride solution and centrifuged at 8,000 g for 1 minute, the supernatant was removed, and the cells were collected. Carotenoids were extracted by adding acetone / methanol (7:3 (volume basis)), and the mixture was centrifuged at 12,000 g for 2 minutes. The supernatant was used for carotenoid analysis.

[0120] The resulting supernatant was subjected to carotenoid separation using a Hitachi HPLC Chromaster system. The column was an Inertsil ODS-3 (4.6 x 150 mm, 5 μm, GL Sciences) with acetonitrile as mobile phase A and isopropanol as mobile phase B. After pumping at a 100:0 ratio of mobile phase A:mobile phase B for 5 minutes, the ratio was increased from 100:0 to 50:50 (volume-based) over 5 minutes, and the 50:50 ratio was maintained for another 5 minutes to separate the carotenoids.

[0121] The resulting supernatant (carotenoids) was also subjected to an Aquity H-class UPLC (Waters) column. The column was an Aquity UPLC BEH C18 column (2.1 x 100 mm, 1.7 μm, Waters), and the mobile phase A was acetonitrile:ultrapure water (85:15, by volume) and the mobile phase B was acetonitrile:methanol (65:35, by volume). The gradient was run from 100:0 to 0:100 A:B over 8 minutes, followed by a 5-minute hold at 0:100 A:B. Carotenoids were separated using this program.

[0122] The analysis results are shown in Figure 6. Figure 6 shows the HPLC chromatogram (top) when the first expression vector was expressed alone and the HPLC chromatogram (bottom) when the first expression vector and the second expression vector were co-expressed (Example 1), as well as the absorption spectra of peaks 1, 2, 3, and 5 detected in the latter (Example 1). As shown in Figure 6, a new peak 5 was confirmed by co-expression of the first expression vector and the second expression vector.

[0123] The same supernatant (carotenoid) (sample) was analyzed by UPLC and compared with a standard sample, and the results are shown in Figure 7a. As shown in Figure 7a, a new peak 5 was confirmed by co-expression of the first and second expression vectors (Example 1). The absorption spectrum of peak 5 is shown in Figure 7b. Peak 5 confirmed in the UPLC chromatogram of Figure 7a showed the same retention time and absorption spectrum as capsanthin. Calculation based on the molar extinction coefficient from the peak intensity revealed that the amount of capsanthin produced per culture medium was 292 μg / L.

[0124] LC / MS analysis of peak 5 was performed using a Waters Xevo G2S Q TOF mass spectrometer (Waters) equipped with an Acquity UPLC system. The analytical conditions for time-of-flight mass spectrometry (ESI-TOF-MS) were a capillary voltage of 3.2 kV, a cone voltage of 20 eV, 120 °C, and 30 L / h of nitrogen gas, scanning m / z 100–1,500. MS / MS spectra were recorded on a quadrupole TOF MS / MS instrument using argon at a collision energy of 20 V. UV-VIS absorption spectra were recorded from 200 nm to 600 nm using a photodiode array. HPLC separation was performed on an Acquity 1.7 μm BEH UPLC C18 column (Waters) using a gradient program of acetonitrile:ultrapure water (85:15 by volume) to acetonitrile:methanol (65:35 by volume) (flow rate: 0.4 mL / min, 15 min). The results are shown in Figure 7c. The molecular ion peak shown in Figure 7c was consistent with the calculated molecular weight of capsanthin.

[0125] Also, Peak 5 1 The H NMR (500 MHz) spectrum was measured using a Varian UNITY INOVA 500 spectrometer (Varian Corporation) with TMS as the internal standard and deuterated chloroform, and peak 5 was identified as capsanthin.

[0126] [Example 2 (Example 2-1 and Example 2-2)] In this example, pACHP-Zea, shown in Figure 8, was constructed as the first recombinant vector to be incorporated, and pUC-CaCCS-CaZEP (two types), shown in Figure 8, was constructed as the second recombinant vector. These recombinant vectors were designed so that the expression level of the upstream gene was relatively low due to a promoter specific to the carotenoid biosynthesis gene (derived from Pantoea ananatis), and the expression level of the downstream gene was relatively high due to Ptac. An overview of the first and second recombinant vectors used in this example is shown in Table 4.

[0127] [Table 4]

[0128] The first recombinant vector, pACHP-Zea, was prepared in the same manner as in Comparative Example 1. Non-Patent Document 3 reports that in the carotenoid composition of the transformant, β-carotene was not detected and zeaxanthin accounted for the total carotenoids.

[0129] The second recombinant vector, pUC-CaCCS-CaZEP, is a vector containing CaZEP (without removing the signal sequence) and CaCCS (without removing the signal sequence) or CaCCS. 41-498 and were placed downstream of the Ptac promoter of a pUC vector.

[0130] Both the first and second recombinant vectors were introduced into Escherichia coli JM101(DE3) in the same manner as in Comparative Example 1.

[0131] After introducing the recombinant vector, a single colony from the LB medium was transferred to 0.5 mL of LB liquid medium and cultured with shaking at 1000 rpm at 37°C for 16 hours. 20 μL of the culture was transferred to 2 mL of TB liquid medium and cultured with shaking at 1000 rpm at 30°C for 24 hours. 2 μL of 1 M isopropyl-β-D-galactopyranoside (IPTG) was added to induce gene expression. Culture was continued for an additional 48 hours. Chloramphenicol (30 mg / L) and kanamycin (40 mg / L) were added as antibiotics to the medium.

[0132] One mL of the resulting culture medium was centrifuged at 8,000 g for 1 minute, the supernatant was removed, and the cell fraction was collected. The cell fraction was suspended in 1% sodium chloride solution and centrifuged at 8,000 g for 1 minute, the supernatant was removed, and the cells were collected. Carotenoids were extracted by adding acetone / methanol (7:3 (volume basis)), and the mixture was centrifuged at 12,000 g for 2 minutes. The supernatant was used for carotenoid analysis.

[0133] The resulting supernatant was subjected to carotenoid separation using a Hitachi HPLC Chromaster system. The column was an Inertsil ODS-3 (4.6 x 150 mm, 5 μm, GL Sciences) with acetonitrile as mobile phase A and isopropanol as mobile phase B. After pumping at a 100:0 ratio of mobile phase A:mobile phase B for 5 minutes, the ratio was increased from 100:0 to 50:50 (volume-based) over 5 minutes, and the 50:50 ratio was maintained for another 5 minutes to separate the carotenoids.

[0134] For comparison, the second expression vector was used to express CaCCS. 41-498 The same procedure was carried out using a second expression vector for comparison, which was prepared in the same manner except that the vector was not integrated.

[0135] The analysis results are shown in Figure 9. In Figure 9, the HPLC chromatogram (top) shows the co-expression of the first expression vector and the second expression vector for comparison, the HPLC chromatogram (middle) shows the co-expression of the first expression vector and the second expression vector (incorporating full-length CaCCS: Example 2-2), and the HPLC chromatogram (middle) shows the co-expression of the first expression vector and the second expression vector (incorporating CaCCS with the signal sequence removed). 41-498 The HPLC chromatogram (bottom) shows the results when the first expression vector and the second expression vector (CaCCS with the signal sequence removed: Example 2-1) were co-expressed. 41-498 9 shows the absorption spectra of peaks 1, 2, 3, and 5 detected when the first expression vector and the second expression vector (Example 2-1) were co-expressed. As shown in FIG. 9, a new peak 5, i.e., capsanthin, was confirmed by co-expression of the first expression vector and the second expression vector.

[0136] 10 shows a graph comparing the intensities of peak 5 of capsanthin in FIG. 9. As shown in FIG. 10, the CCS from which the signal sequence was removed was more potent than the case where the second expression vector incorporating the full-length CCS was used (Example 2-2). 41-498When the second expression vector incorporating the above was used (Example 2-1), more capsanthin was produced.

[0137] Furthermore, the amount of capsanthin produced in Peak 5 was calculated based on the molar extinction coefficient from the peak intensity. As a result, the amount of capsanthin produced per culture medium when the second expression vector incorporating the full-length CCS (Example 2-2) was used was 326 μg / L, and when the CCS with the signal sequence removed was used, the amount of capsanthin produced per culture medium was 326 μg / L. 41-498 When the second expression vector incorporating the above was used (Example 2-1), the amount of capsanthin produced per culture medium was 452 μg / L.

[0138] [Example 3 (Example 3-1, Example 3-2, Example 3-3)] In this example, the first recombinant vector used was pACHP-Zea, the same as in Comparative Example 1. Non-Patent Document 3 reports that in the carotenoid composition of pACHP-Zea transformants, β-carotene was not detected and zeaxanthin accounted for the total carotenoids.

[0139] As the second recombinant vector, pUC-S / slC / sZ, pUC-S / sC / slZ, or pUC-S / slC / slZ ("S" stands for S Protein, "s" stands for S-Tag, and "1" stands for linker) shown in Figure 11 was constructed. An overview of the first and second recombinant vectors of this example is shown in Table 5.

[0140] [Table 5]

[0141] As shown schematically in Figure 12, this second recombinant vector was designed so that CCS and ZEP are each expressed as proteins fused with an S-Tag (shown as "S tag + linker + CCS" and "S tag + linker + ZEP," respectively) and then indirectly bind to form a complex (shown as "complex" in Figure 12) via the multimerized S Protein (shown as "multimer" in Figure 12). The second recombinant vector designed in this way is also referred to as having "an S-Tag incorporated."

[0142] The three second recombinant vectors, pUC-S / slC / sZ, pUC-S / sC / slZ, and pUC-S / slC / slZ shown in Figure 11, were designed to have different configurations, with or without linkers between the S-Tag and CCS and / or between the S-Tag and ZEP. By varying the position of the linker insertion within the second recombinant vector containing the S-Tag, three conditions were created that differed in the degree of freedom between the CCS and ZEP after expression.

[0143] Furthermore, the sequence information regarding the construction of the second recombinant vector incorporating the S-Tag is shown in Table 6 below.

[0144] [Table 6]

[0145] As shown in SEQ ID NOs: 8 and 12, the linker is a linker ({GS} 10The S-tag was fused to the N-terminus of CaCCS and CaZEP. The construct used to introduce the S protein, S-tag, etc. was pUClac-ptac-CaCCSM40-CaZEP (pMF541) (Furubayashi, M., Kubo, A., Takemura, M., Otani, Y., Maoka, T., Terada, Y., Yaoi, K., Ohdan, K., Misawa, N. and Mitani, Y. (2021) J. Agric. Food Chem., 69, 5076-5085).

[0146] The first and second recombinant vectors were introduced as described in Example 1. A single colony from the LB medium was then transferred to 3 mL of 2YT liquid medium and cultured for 16 hours at 28°C and 105 rpm using a Yamato Scientific Shaking Bath BW201. 20 μL of the culture was transferred to 2.1 mL of TB liquid medium and cultured for 4 hours at 30°C and 180 rpm using a Kuhner Lab-Therm LT-X shaker. Gene expression was then induced by the addition of 0.1 mM isopropyl-β-D-galactopyranoside (IPTG), and the culture was continued for another 48 hours. Chloramphenicol (30 mg / L) and ampicillin (100 mg / L) were also added to the medium as antibiotics. One mL of the resulting culture medium was centrifuged at 8,000 g for 1 minute, the supernatant was removed, and the cell fraction was collected. The cell fraction was suspended in 1% sodium chloride solution and centrifuged at 8,000 g for 1 minute, the supernatant was removed, and the cells were collected. Carotenoids were extracted by adding methanol and chloroform, and the mixture was centrifuged at 12,000 g for 2 minutes. The supernatant was used for carotenoid analysis.

[0147] The resulting supernatant was subjected to carotenoid separation using a Hitachi HPLC Chromaster system. The column was an Inertsil ODS-3 (4.6 x 150 mm, 5 μm, GL Sciences) with acetonitrile as mobile phase A and isopropanol as mobile phase B. After pumping at a 100:0 ratio of mobile phase A:mobile phase B for 5 minutes, the ratio was increased from 100:0 to 50:50 (volume-based) over 5 minutes, and the 50:50 ratio was maintained for another 5 minutes to separate the carotenoids.

[0148] A graph comparing the peak intensities of each carotenoid in HPLC analysis is shown in Figure 13. In Figure 13, the second recombinant vector pUC-CaCCS used in Example 2-1, which does not have an S-Tag incorporated, 41-498 The results for the case where pUC-CaZEP (indicated as "C / Z" in the figure) was used are also shown. As shown in Figure 13, when the second recombinant vector incorporating the S-Tag was used (Examples 3-1 to 3-3), more capsanthin was accumulated than when the S-Tag was not incorporated (Example 2-1). Furthermore, among the three types of second recombinant vectors incorporating the S-Tag, the amount of capsanthin accumulated was significantly greater when pUC-S / sC / slZ, in which a linker was incorporated between the S-Tag and ZEP, was used (Example 3-2).

[0149] Furthermore, the results calculated from the peak intensity based on the molar extinction coefficient are shown in Table 7. Compared to the amount of capsanthin produced per culture medium when a second recombinant vector without an S-tag was used (Example 2-1), the amount of capsanthin produced per culture medium when a second recombinant vector with an S-tag was used (Examples 3-1 to 3-3) was improved. In particular, when pUC-S / sC / slZ, in which a linker was incorporated between the S-tag and ZEP, was used as the second recombinant vector (Example 3-2), not only was the amount of capsanthin produced per culture medium significantly improved, but the ratio of capsanthin to the total amount of carotenoids shown in Table 7 was also significantly improved.

[0150] [Table 7]

[0151] Furthermore, the carotenoids obtained when pUC-S / sC / slZ was used as the second vector (Example 3-2) were analyzed by UPLC using the same method as in Example 1. The results are shown in Figure 14. As is clear from Figure 14, peaks for capsanthin 3'-acetate, capsanthin 3,6-epoxide, capsanthin 3,6-epoxide 3'-acetate, capsorubin 3-acetate, capsorubin diacetate, and cucurbitaxanthin A were detected. These carotenoids were analyzed by LC / MS using the same method as in Example 1. The molecular ion peaks matched the calculated molecular weights of each carotenoid. Furthermore, the ratios of each carotenoid calculated from the peak intensities of the UPLC analysis are shown in Table 8.

[0152] [Table 8]

[0153] For NMR measurement, carotenoids were separated by silica gel chromatography, with increasing polarity in the following order: hexane, ether:hexane (1:1), and ether:acetone (1:1). Each fraction was collected and concentrated. Then, a silica gel (Cosmosil 5 SL-II) column (10 x 250 mm) was used to separate the carotenoids at a flow rate of 2 ml / min using acetone:hexane (3:7) as the mobile phase. The resulting carotenoid peaks were collected and separated in the same manner as in Example 1. 1 It was used for HNMR measurement. 1 The structural formulas of the carotenoids (capsanthin, capsorubin, cucurbitaxanthin A, and other derivatives) identified as a result of HNMR analysis are shown below.

[0154] [ka] [Sequence List Free Text]

[0155] SEQ ID NO: 3 is a nucleotide sequence in which the 34th to 36th bases of the nucleotide sequence encoding S-Tag have been replaced with a sequence encoding phenylalanine. SEQ ID NO: 4 is the amino acid sequence of S-Tag in which the 12th amino acid is substituted with phenylalanine. SEQ ID NOs: 5 and 6 are the nucleotide sequence and amino acid sequence, respectively, of CaCCS into which an S-Tag has been introduced. SEQ ID NOs: 7 and 8 are the nucleotide sequence and amino acid sequence, respectively, of CaCCS into which an S-Tag has been introduced via a linker. SEQ ID NOs: 9 and 10 are the nucleotide sequence and amino acid sequence, respectively, of CaZEP into which an S-Tag has been introduced. SEQ ID NOs: 11 and 12 are the nucleotide sequence and amino acid sequence, respectively, of CaZEP into which an S-Tag has been introduced via a linker.

Claims

1. a first promoter, and upstream genes of carotenoid biosynthetic genes including crtI, crtB, crtE, crtY, and crtZ operably linked to the first promoter; a second promoter having a promoter strength higher than that of the first promoter, and a ZEP gene and a CCS gene operably linked to the second promoter; is introduced into a host cell, thereby forming a transformant.

2. 2. The transformant according to claim 1, wherein the difference in promoter strength between the first promoter and the second promoter is equal to or greater than the difference in promoter strength between a promoter specific to a carotenoid biosynthetic gene and P BAD.

3. The transformant according to claim 1 or 2, wherein the first promoter is a promoter specific to a Plac or carotenoid biosynthetic gene.

4. The transformant according to any one of claims 1 to 3, wherein the second promoter is P BAD or Ptac.

5. Furthermore, an S protein gene, an S tag gene, and a linker gene are introduced into the host cell; the S-tag gene is linked to each of the ZEP gene and the CCS gene; The transformant according to any one of claims 1 to 4, wherein the linker gene is interposed and linked between the ZEP gene and the S-tag gene and / or between the CCS gene and the S-tag gene.

6. The transformant according to claim 5, wherein the S tag encoded by the S tag gene is any one of the following polypeptides (1) and (2): (1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 (2) A polypeptide that has a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 4 except for the 12th amino acid, and that specifically binds to S protein.

7. The transformant according to claim 5 or 6, wherein the linker gene is interposed and linked at least between the ZEP gene and the S-tag gene.

8. The transformant according to any one of claims 5 to 7, wherein the linker gene is interposed between the ZEP gene and the S-tag gene and is not interposed between the CCS gene and the S-tag gene.

9. A method for producing a carotenoid composition, comprising a step of culturing the transformant according to any one of claims 1 to 8.

10. The method of claim 9 , wherein the carotenoid composition comprises capsanthin.

11. The method according to claim 9 or 10, wherein the carotenoid composition comprises capsorubin, capsanthin 3'-acetate, capsorubin 3-acetate, cucurbitaxanthin A, capsorubin diacetate, capsanthin 3,6-epoxide, and / or capsanthin 3,6-epoxide 3'-acetate.

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