MYCOSPORINE-LIKE AMINO ACID-PRODUCING MICROORGANISM AND METHOD FOR PRODUCTION OF MYCOSPORINE-LIKE AMINO ACIDS BY USING SAME

Abstract

Provided are a mycosporine-like amino acid-producing microorganism and a method for production of mycosporine-like amino acids by using same. The microorganism can produce mycosporine-like amino acids from xylose.

Claims

1. A microorganism comprising: (a) a xylose assimilation enzyme; and (b) a mycosporine-like amino acid biosynthesis enzyme, wherein the xylose assimilation enzyme comprises a conversion enzyme of xylose to xylulose, a xylulose phosphorylase, or a combination thereof, wherein the xylose assimilation enzyme, mycosporine-like amino acid biosynthesis enzyme, or both are exogenous proteins.

2. The microorganism according to claim 1, wherein the conversion enzyme of xylose to xylulose is xylose isomerase (XI), xylose reductase (XR), xylitol dehydrogenase (XDH), or a combination thereof, and the xylulose phosphorylase is xylulokinase (XK).

3. The microorganism according to claim 2, wherein the xylose isomerase is derived from at least one selected from the group consisting of Pichia stipitis, Paraburkholderia sacchari, Actinomyces olivocinereus, Actinoplanes missouriensis, Aerobacter levanicum, Bacillus coagulans, Bacillus sp., Bifidobacterium adolescentis, Bacteroides thetaiotaomicron, Clostridium cellulovorans, Clostridium phytofermentans, Lactobacillus brevis, Lactococcus lactis, Orpinomyces sp., Piromyces sp., Thermus thermophiles, and Vibrio sp., the xylose reductase and the xylitol dehydrogenase are derived from at least one selected from the group consisting of Pichia sp., Aspergillus carbonarius, Candida sp., Kluyveromyces marxianus, Neurospora crassa, Ogataea siamensis, Trichoderma reesei, Zymomonas mobilis, Pachysolen tannophilus, Odontotaenius disjunctus), and Hansenula polymorpha, the xylulokinase (XK) is derived from at least one selected from the group consisting of Pichia stipitis, Arabidopsis thaliana, Bacillus coagulans, Klebsiella pneumonia, Kluyveromyces marxianus, Hansenula polymorpha, Saccharomyces cerevisiae, Thermotoga maritime, Trichoderma reesei, and Zymomonas mobilis.

4. The microorganism according to claim 1, wherein the mycosporine-like amino acid biosynthesis enzyme comprises at least one selected from the consisting of: 2-dimethyl 4-deoxygadusol synthase (DDGS), O-methyltransferase (O-MT), C—N ligase or ATP-grasp ligase, and non-ribosomal peptide synthetase, non-ribosomal peptide synthetase-like enzyme (NRPS-like enzyme), or D-alanine D-alanine ligase.

5. The microorganism according to claim 1, wherein the microorganism is one in which a pentose phosphate pathway is further enhanced.

6. The microorganism according to claim 5, wherein the enhancement of the pentose phosphate pathway is achieved through at least one selected from: an increase in the transketolase activity to produce sedoheptulose 7-phosphate in the pentose phosphate pathway, an increase in the protein activity that regulates NADPH production, and a reduction in the transaldolase activity involved in the conversion reaction between sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate.

7. The microorganism according to claim 1, wherein the microorganism is a yeast, a microorganism of the genus Corynebacterium, or a microorganism of the genus Escherichia.

8. The microorganism according to claim 1, wherein the microorganism is for producing mycosporine-like amino acids from xylose.

9. A composition for producing mycosporine-like amino acids, comprising the microorganism according to claim 1.

10. The composition for producing mycosporine-like amino acids according to claim 9, wherein the microorganism is a yeast, a microorganism of the genus Corynebacterium, or a microorganism of the genus Escherichia.

11. The composition for producing mycosporine-like amino acids according to claim 9, wherein the microorganism is for producing mycosporine-like amino acids from xylose.

12. The composition for producing mycosporine-like amino acids according to claim 9, wherein the mycosporine-like amino acid is at least one selected from the group consisting of mycosporine-2-glycine, palythinol, palythenic acid, deoxygadusol, mycosporine-methylaminethreonine, mycosporine-glycine-valine, palythine, asterina-330, shinorine, porphyra-334, euhalothece-362, mycosporine-glycine, mycosporine-ornithine, mycosporine-lysine, mycosporine-glutamic acid-glycine, mycosporine-methylamine-serine, mycosporine-taurine, palythene, palythine-serine, palythine-serine-sulfate, palythinol, and usujirene.

13. A method for producing mycosporine-like amino acids, comprising culturing the microorganism of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0136] FIG. 1a is a schematic diagram exemplarily showing the shinorine biosynthesis pathway among mycosporine-like amino acids.

[0137] FIG. 1b shows the mycosporine-like amino acid biosynthesis gene clusters of Nostoc punctiforme and Anabaena variabilis.

[0138] FIG. 2a is a graph showing the results of HPLC analysis of the cell extract after culturing yeast strain JHYS10 introduced with plasmids containing DDGS (NpR5600), O-MT (NpR5599), ATP-grasp ligas (NpR5598), and D-ala-D-ala ligase (NpR5597) genes for 48 hours.

[0139] FIG. 2b is a graph showing the synorin production results after culturing the yeast strain JHYS10 for 48 hours in comparison with the wild-type strain (WT-1).

[0140] FIG. 2c is a graph showing the cell growth during the culture of the yeast strain JHYS10 for 48 hours in comparison with the wild-type strain (WT-1).

[0141] FIGS. 3a and 3b are graphs showing the results of tandem mass spectrometry analysis of cell extracts after culturing the yeast strain JHYS10 for 48 hours.

[0142] FIG. 4a is a cleavage map of a vector comprising an expression cassette containing D-ala-D-ala ligase (NpR5597) and DDGS (NpR5600) genes, and an expression cassette containing ATP-grasp ligase (NpR5598) and O-MT (NpR5599) genes.

[0143] FIG. 4b is a graph showing the results of shinorine production of yeast strains JHYS12 and JHYS13 in which the genes were randomly inserted by transformation with the two expression cassettes of FIG. 4a.

[0144] FIG. 4c is a graph showing the copy number of each gene in yeast strains JHYS11, JHYS12, and JHYS13 in which the genes were randomly inserted by transformation with the two expression cassettes of FIG. 4a.

[0145] FIG. 5 is a graph showing the consumption of glucose and/or xylose, the degree of cell growth resulting therefrom, and the result of shinorine production, wherein the glucose and/or xylose is obtained by culturing a JHYS13-2 strain in which a plasmid containing the xylose assimilation genes XYL1, XYL2, and XYL3 genes is introduced into the yeast strain JHYS13, in a medium containing xylose and glucose.

[0146] FIG. 6a is a cleavage map of a vector for constructing a yeast strain in which a gene encoding a xylose assimilation enzyme is inserted into the chromosome by NTS.

[0147] FIG. 6b is a graph showing the consumption of glucose and/or xylose, and the degree of cell growth resulting therefrom, which is obtained by culturing yeast strains JHYS14, JHYS15, and JHYS16 in which the xylose assimilation genes XYL1, XYL2, and XYL3 genes are randomly inserted into the chromosome of the yeast strain JHYS13, in a medium containing xylose and glucose.

[0148] FIG. 6c is a graph showing the results of shinorine production of yeast strains JHYS14, JHYS15, and JHYS16.

[0149] FIG. 7 is a graph showing the consumption of glucose and/or xylose, the degree of cell growth resulting therefrom, and the result of shinorine production, which is obtained by culturing a JHYS17 strain in which the TAL1 gene is deleted in the JHYS16 strain, in a medium containing xylose and glucose.

[0150] FIG. 8a is a graph showing the consumption of glucose and/or xylose and the degree of cell growth resulting therefrom, which is obtained by culturing a JHYS17-3 strain into which the TKL1 gene was introduced into the JHYS17 strain, and a JHYS17-4 strain into which both the STB5 gene and the TKL1 gene were introduced, in a medium containing xylose and glucose.

[0151] FIG. 8b is a graph showing the results of shinorine production obtained by culturing the JHYS17-2 strain, the JHYS17-3 strain, and the JHYS17-4 strain in a medium containing xylose and glucose.

[0152] FIG. 9a is a graph showing the consumption of glucose and/or xylose and the degree of cell growth resulting therefrom, which is obtained by culturing a JHYS17-4 strain in a medium containing xylose and glucose in various ratios.

[0153] FIGS. 9b and 9c are graphs showing the results of shinorine production, which is obtained by culturing a JHYS17-4 strain in a medium containing xylose and glucose in various ratios.

[0154] FIG. 10 is a graph showing the production of by-products (xylitol, ethanol, glycerol, and acetate), which is obtained by culturing a JHYS17-4 strain in a medium containing xylose and glucose in various ratios.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0155] Hereinafter, the present disclosure will be described in more detail with reference to the following Examples. However, these Examples are for illustrative purposes only and are not intended to limit the scope of the disclosure. It will be apparent to those skilled in the art that the Examples described below can be modified without departing from the essential gist of the invention.

[0156] <Introduction of Exogenous Mycosporine-Like Amino Add Biosynthesis Pathway Based on Yeast Strain>

Example 1: Preparation of Vector for Expression in Yeast of Mycosporine-Like Amino Acid Biosyntehsis Gene Derived from Microalgae

[0157] In order to use yeast (Saccharomyces cerevisiae) as a mycosporine-like amino acid production strain, a mycosporine-like amino acid biosynthesis gene derived from Nostoc punctiforme (ATCC 29133), which is one of cyanobacteria, was introduced into a vector for yeast expression. N. punctiforme shinorine biosynthesis involves four enzymatic reactions: 2-dimethyl 4-deoxygadusol synthase (DDGS), O-methyltransferase (O-MT), ATP-grasp ligase, and D-ala-D-ala ligase which use sedoheptulose 7-phosphate (S7P) as a substrate (see FIG. 1a). The genes encoding the four enzymes in the N. punctiforme genome are as follows, respectively (see the left of FIG. 1b): DDGS: NpR5600 (GenBanK Accession No. ACC83905.1; REGION: 6913123 . . . 6914355), O-MT: NpR5599 (GenBanK Accession No. ACC83904.1; REGION: 6912285 . . . 6913118), ATP-grasp ligase: NpR5598 (GenBanK Accession No. ACC83903.1; REGION: 6910776 . . . 6912161), and D-ala-D-ala ligase: NpR5597 (GenBanK Accession No. ACC83902.1; REGION: 6909563 . . . 6910609).

[0158] The primers used for the construction of the plasmid containing the four genes are summarized in Table 4 below:

TABLE-US-00004 TABLE 4 Primer (F: SEQ PCR Template Forward, R: ID Product used Reverse) Sequence(5′.fwdarw.3′) NO NpR5600 Nostoc NpR5600 F GCGGGATCCATGAGTAATGTTCAAGCATCG  1 punctiforme NpR5600 R GCGCTCGAGTCACACTCCCAATAGTTTGG  2 NpR5599 genomic NpR5599 F GCGGGATCCATGACCAGTATTTTAGGACG  3 DNA NpR5599 R GCGCTCGAGTTATACCAAGCGTCTAATCAG  4 NpR5598 (GenBank NpR5598 F GCGGGATCCATGGCACAATCAATCTCTTTA  5 Accession NpR5598 R GCGCTCGAGTAGTCGCCCCCTAATTCC  6 NpR5597 No. NpR5597 F GCGGGATCCATGCCAGTACTTAATATCCTT  7 CP001037.1) NpR5597 R GCGCTCGAGTCAATTTTGTAACACCTTTTTATTA  8 P.sub.TDH3- p413GPD- Univ F2 GACTCGCGCGCGGGAACAAAAGCTGGAGCTC 32 NpR560- NpR5600 GACTACGCGTGCGGCCGCTAATGGCGCGCCATAGGG 33 T.sub.CYC1 Univ R CGAATTGGGTACC P.sub.TDH3- p414GPD- Univ F2 GACTCGCGCGCGGGAACAAAAGCTGGAGCTC 32 NpR5599- NpR5599 Univ R GACTACGCGTGCGGCCGCTAATGGCGCGCCATAGGG 33 T.sub.CYC1 CGAATTGGGTACC P.sub.TEF1- coex414TEF- Univ F2 GACTCGCGCGCGGGAACAAAAGCTGGAGCTC 32 NpR5598- NpR5598 Univ R GACTACGCGTGCGGCCGCTAATGGCGCGCCATAGGG 33 T.sub.GPM1 CGAATTGGGTACC

[0159] The genome of N. punctiforme (GenBank Accession No. CP001037.1) was used as a template, the four gene fragments involved in shinorine biosynthesis were amplified by PCR using the primers, and then they were cleaved with BamHI-XhoI restriction enzymes, and cloned into p413GPD, coex413TEF, p414GPD, and coex414TEF respectively retreated with the same restriction enzyme. The vectors thus constructed were named p413GPD-NpR5600, p414GPD-NpR5599, coex414TEF-NpR5598, and coex413TEF-NpR5597, respectively.

[0160] The constructed vector was amplified thy PCR with Univ F2 (SEQ ID NO: 32)-Univ R primer (SEQ ID NO: 33) as a template to secure the [Promoter-ORF-Terminator] gene fragment, and then cleaved with MauBI-NotI restriction enzymes and successively cloned into coex413TEF-NpR5597 vector cleaved with AscI-NotI, whereby four genes were introduced into one vector, which was named coex413-NpR4. The vectors used and the constructed vectors are summarized in Table 5 below:

TABLE-US-00005 TABLE 5 Plasmid Description coex413TEF CEN/ARS plasmid, HIS3, P.sub.TEF1, T.sub.GPM1 coex414TEF CEN/ARS plasmid, TRP1, P.sub.TEF1, T.sub.GPM1 p413GPD(ATCC ® 87354 ™) CEN/ARS plasmid, HIS3, P.sub.TDH3, T.sub.CYC1 p414GPD(ATCC ® 87356 ™) CEN/ARS plasmid, TRP1, P.sub.TDH3, T.sub.CYC1 p414ADH(ATCC ® 87372 ™) CEN/ARS plasmid, TRP1, P.sub.ADH1, T.sub.CYC1 p416GPD(ATCC ® 87360 ™) CEN/ARS plasmid, URA3, P.sub.TDH3, T.sub.CYC1 p413GPD-NpR5600 CEN/ARS plasmid, HIS3, P.sub.TDH3-NpR5600-T.sub.CYC1 p414GPD-NpR5599 CEN/ARS plasmid, TRP1, P.sub.TDH3-NpR5599-T.sub.CYC1 coex414TEF-NpR5598 CEN/ARS plasmid, TRP1, P.sub.TEF1-NpR5598-T.sub.GPM1 coex413TEF-NpR5597 CEN/ARS plasmid, HIS3, P.sub.TEF1-NpR5597-T.sub.GPM1 coex413-NpR4 CEN/ARS plasmid, HIS3, P.sub.TEF1-NpR5597-T.sub.GPM1, P.sub.TDH3-NpR5600-T.sub.CYC1, P.sub.TEF1-NpR5598-T.sub.GPM1, P.sub.TDH3-NpR5599-T.sub.CYC1

[0161] (In Table 5 above, coex413TEF and coex414TEF were constructed from p413TEF (ATCC® 87362) and p414TEF (ATC® 87364), respectively, with reference to the method described in Korean Unexamined Patent Publication No. 10-2016-0093492)

Example 2: Evaluation of Chlorine-Producing Ability of Strains Through Expression in Vector-Based Yeast of Mycosporine-Like Amino Acid Biosyntehsis Gene

[0162] A heterologous mycosporine-like amino acid biosynthesis pathway gene derived from N. punctiforme was transformed into a wild-type S. cerevisiae strain, CEN.PK2-1C, by the LiAc/SS carrier DNA/PEG method. The wild-type strain used in this Example and the recombinant yeast strain (JHYS10) into which four genes (coex413-NpR4) were introduced are summarized in Table 6 below:

TABLE-US-00006 TABLE 6 Strain Genotype CEN. PK2-1C MATa ura3-52 trp1-289 leu2-3,112 his3 Δ1 MAL2-8.sup.CSUC2 WT-1 CEN. PK2-1C harboring p413TEF JHYS10 CEN. PK2-1C harboring coex413-NpR4

[0163] The yeast strain (JHYS10) expressing WT-1 and coex412-NpR4 was pre-cultured in synthetic complex (SC)-His (including 20 g/L glucose) medium, and then inoculated at OD600=0.2 into 10 mL of the same medium, and cultured in a 100 mL flask under the conditions of 30° C. and 170 rpm for 48 hours. To quantify the concentration of mycosporine-like amino acids, specifically shinorine, two culture media were sampled at 2 mL each. One medium was dried in an oven and then the dry cell weight (DCW) was measured. Another medium was centrifuged to remove the supernatant, and then 1 mL of distilled water and 1.5 mL of chloroform were added to the yeast cells obtained, and then vortexed for 3 minutes to extract intracellular shinorine. After centrifugation, the aqueous layer was isolated and filtered, and used for measuring the intracellular shinorine concentration. An Ultimate3000 HPLC system (Thermo Fisher Scientific) equipped with an Agilent Eclipse XDB-C18 column (5 μm, 4.6×250 mm) was used, the column temperature was maintained at 40° C., and a solvent (water:acetonitrile=95:5) was flowed at a flow rate of 0.5 mL/min. Shinorine was detected with a UV-vis detector at 334 nm.

[0164] The results of the HPLC analysis as described above are shown in FIGS. 2a and 2b. As shown in FIGS. 2a and 2b, shinorine was not produced in WT-1 without introducing the shinorine biosynthetic gene, whereas a trace amount of shinorine was produced in JHYS10 (FIG. 2b: 0.46 mg/L and 0.085 mg/gDCW). Additionally, the OD600 value of the culture during the 48-hour incubation period was measured and shown in FIG. 2c. As shown in FIG. 2c, the two strains (WT-1 and JHYS10) did not show any difference in the cell growth rate.

[0165] In order to confirm that the substance produced from the tested cell extract is shinorine, tandem mass spectrometry analysis (Thermofisher TSQ quantum access max) was performed. The obtained results are shown in FIGS. 3a and 3b. As shown in FIGS. 3a and 3b, it can be confirmed that the wild-type strain harboring p413GPD plasmid (WT-1) did not produce chinorine, whereas the yeast strain (JHYS10) into which the biosynthetic gene of N. punctiforme was introduced produced chinorine. These results show that the functional expression of the shinorine biosynthetic gene of N. punctiforme is possible in heterologous hosts.

Example 3: Construction of a Vector for Delta-Integration for the Introduction of Mycosporine-Like Amino Acid Biosynthesis Gene into the Yeast Chromosome

[0166] Hundreds of retrotransposon sequences known as delta sequences are interspersed in the genome of S. cerevisae. When these delta sequences are targeted to the gene introduction site in the genome, a large number of genes can be randomly and simultaneously introduced into a chromosome. To introduce mycosporine-like amino acid biosynthesis gene into the delta site of the S. cerevisiae chromosome, an integration vector having a delta sequence at both ends was constructed, and the primers used here are summarized in Table 7 below:

TABLE-US-00007 TABLE 7 Primer (F: SEQ Forward, R: ID PCR Poduct Template used Reverse) Sequence(′.fwdarw.3′) NO Delta1 Saccharomyces Delta1 R ATAGCGGCCGCATGTTTATATTC 19 cerevisiae ATTGATCCTATTACA genomic Delta1 F CACATTTCCCCGAAAAGTGCATT 20 DNA (GenBank TAAATTGTTGGAATAGAAATCAA Accession No. CTATC JRIV01000000) Amp-Ori p413GPD vector Amp-Ori F GCACTTTTCGGGGAAATGTG 21 Amp-Ori R CTCAACATTCACCCATTTCTCAAT 22 TTAAATCGCAGGAAAGAACATGT GAG Delta2 Saccharomyces Delta2R TGAGAAATGGGTGAATGTTGAG 23 cerevisiae Delta2F GCGGCTAGCATAAAACGGAATGA 24 genomic GGAATAATC DNA (GenBank Accession No. JRIV01000000) P.sub.TEF1-NpR5597- coex413-NpR4 Promoter up F GCGGCTAGCGAGCTCGGAAACAG 25 T.sub.GPM1-P.sub.TDH3- vector CTATGACCATGA NpR5600-T.sub.CYC1 Univ R GACTACGCGTGCGGCCGCTAATG 33 GCGCGCCATAGGGCGAATTGGGT ACC P.sub.TEF1-NpR5598- coex413-NpR4 Promoter up F GCGGCTAGCGAGCTCGGAAACAG 25 T.sub.GPM1-P.sub.TDH3- vector CTATGACCATGA NpR5599-T.sub.CYC1 Univ R GACTACGCGTGCGGCCGCTAATG 33 GCGCGCCATAGGGCGAATTGGGT ACC

[0167] The obtained PCR product was used as a template, and [Amp-Ori-delta1-PTEF1-NpR5597-TGPM1-PTDH3-NpR5600-TCYC1-delta2] and [Amp-Ori-delta1-PTEF1-NPR5598-TGPM1-PTDH3-NpR5599-TCYC1-delta2] were constructed by an overlapping PCR method, and cloned using the NheI/NodI restriction enzyme site of the pUG6MCS vector (vector obtained by introducing cloning sites PacI, NheI, BamHI, SmaI, EcoRI, ApaI, KpnI, and AscI into pUG6(P30114)). These were named Delta6M-NPR1 and Delta6M-NPR2, respectively. The vectors used for the construction and the constructed vectors are summarized in Table 8 below:

TABLE-US-00008 TABLE 8 Plasmid Description pUG6MCS pUG6 plasmid containing additional restriction enzyme sites Delta6M-NPR1 Delta6M plasmid, P.sub.TEF1-NpR5597-T.sub.GPM1, P.sub.TDH3-NpR5600-T.sub.CYC1 Delta6M-NPR2 Delta6M plasmid, P.sub.TEF1-NpR5598-T.sub.GPM1, P.sub.TDH3-NpR5599-T.sub.CYC1

Example 4: Delta-4-Integration of Mycosporine-Like Amino Acid Biosynthesis Gem

[0168] The Delta6M-NPR1 and Delta6M-NPR2 vectors obtained in Example 1.3 were constructed so that the delta1 and delta2 sequences were exposed at both ends when treated with the SwaI restriction enzyme. Therefore, the Delta6M-NPR1 and Delta6M-NPR2 vectors were treated with SwaI, and the two cassettes containing the NpR5597 and NpR5600 genes or the NpR5598 and NpR5599 genes were mixed, and co-transformed with S. cerevisiae CEN.PK2-1C (see FIG. 4a), and the obtained transformant was selected on YPD medium (10 g/L yeast extract, 20 g/L bacto-peptone, 20 g/L glucose) containing 2 mg/L of G418 (Geneticin).

[0169] A total of 18 transformants were selected. Among these, strains named JHYS12 and JHYS13 were pre-cultured in synthetic complex (SC) (containing 20 g/L glucose) medium, and then inoculated into the same medium of 10 mL at OD600=0.2, and incubated in a 100 mL flask under the conditions of 30° C. and 170 rpm for 48 hours. The amount of shinorine extracted from the cells of JHYS12 and JHYS13 was measured and shown in FIG. 4b. As shown in FIG. 4b, JHYS12 and JHYS13 produced higher amounts of shinorine than cells harboring coex413-NpR4 (JHYS10). More specifically, the shinorine production of the JHYS13 strain was 0.67 mg/L and 0.125 mg/gDCW, which were higher than that of the JHYS12 strain (0.51 mg/L and 0.106 mg/gDCW).

[0170] The copy numbers of the genes NpR5597, NpR5598, NpR5599, and NpR5600 introduced into the chromosomes of the strains named JHYS11. JHYS12, and JHYS13 among the selected transformants was confirmed by qPCR method. To determine the copy number of introduced genes, NpR5600, NpR5599, NpR5598, and NpR5597 were performed using gene-specific primers (see Table 9) and 1×SYBR Green I master mix (Roche Applied Science). qPCR was performed in 45 cycles (at 95° C. for 40 sec, at 60° C. for 20 see, at 72° C. for 20 sec) in LightCycler 480 II System (Roche Applied Science). The ACT1 gene was used as a control. Intersection (Cp) values were processed using LightCycler Software version 1.5 (Roche Applied Science), Expression levels were normalized to the target/reference ratio. Primers used for qPCR are shown in Table 9 below.

TABLE-US-00009 TABLE 9 Primer (F: Forward, SEQ Target gene R: Reverse) Sequence (5′.fwdarw.3′) ID NO NpR5600 NpR5600 qPCR F ATGGCGAAGAGTTGCTTTCC 40 NpR5600 qPCR R GTGTGACCGTAGGCAATGAC 41 NpR5599 NpR5599 qPCR F CCCTCAGAATGGCAGAGGAA 42 NpR5599 qPCR R GACGCACTGTTTCACCATCA 43 NpR5598 NpR5598 qPCR F CTAGCTGCACCTTTGGAACC 44 NpR5598 qPCR R GAGCGAATCCCAGTCAATCG 45 NpR5597 NpR5597 qPCR F AAACTGACGATGGCGACTTG 46 NpR5597 qPCR R ACCAAGGTTGTCCCTTTGGA 47

[0171] The copy number of genes measured as described above is shown in FIG. 4c. As shown in FIG. 4c, it was confirmed that JHYS12 genome (orange) contains 5 copies of NpR5600 and NpR5597, respectively, and contains 2 copies of NpR5599 and NpR5598, respectively, whereas JHYS13 (green) contains 4 copies of NpR5600 and NpR5597, respectively, and 9 and 8 copies of NpR5599 and NpR5598, respectively. In the meantime, it can be confirmed that strain JHYS11 contains only one of these four genes in its chromosome.

[0172] <Construction of a Yeast Strain that can Use Xylose as a Carbon Source and Production of Shinorine Utilizing the Same>

Example 5: Construction of a Vector for Expressing Xylose Assimilation Enzyme

[0173] Since shinorine is produced from S7P, which is an intermediate material in the pentose phosphate pathway, the production of shinorine can be increased through the enhancement of the pentose phosphate pathway (see FIG. 1a). However, since yeast uses most of the glucose to produce pyruvate through glycolysis, it is difficult to enhance the pentose phosphate pathway when glucose is used as a carbon source. On the other hand, when xylose is used as a carbon source, xylulose-5-phosphate, which is a decomposition product of xylose, flows directly into the pentose phosphate pathway, thereby being able to enhance the pentose phosphate pathway.

[0174] Yeast does not naturally ferment xylose, but by introducing a heterologous pathway consisting of xylose isomerase (XI) or xylose reductase/xylitol dehydrogenase (XR/XDH), it can be grown using xylose. XI is the enzyme that consumes most of the xylose in bacteria, and has the advantage of being able to eliminate cofactor imbalance during xylose fermentation. However, since XI is decreased in enzymatic activity when expressed in yeast, the XR/XDH pathway is more often used for xylose fermentation in yeast. Xylose reductase (XR) and xylitol dehydrogenase (XDH) convert xylose to xylitol, which in turn converts xylitol to xylulose. Xylulose is converted to xylulose-5-phosphate (X5P) by xylulokinase (XK), and then, xylulose-5-phosphate is introduced into the pentose phosphate pathway (see FIG. 1a). Therefore, the use of xylose as a carbon source can increase S7P production through the pentose phosphate pathway. By introducing and expressing xylose assimilation genes encoding Pichia stipitis-derived XR (Xyl1), XDH (Xyl2) and XK (Xyl3) into yeast, a yeast strain capable of effectively fermenting xylose can be constructed.

[0175] To develop a xylose fermenting yeast strain, a plasmid capable of expressing XYL1 (encoding xylose reductase (XR)), XYL2 (encoding xylitol dehydrogenase (XDH)) and XYL3 (encoding xylulokinase (XK)) genes derived from Pichia stipitis under the control of the strong promoter PTDH3 were constructed. The primers used for the construction of the plasmid are shown in Table 10 below.

TABLE-US-00010 TABLE 10 Primer (F: SEQ PCR Forward, R: ID Product Template used Reverse) Sequence (5′.fwdarw.3′) NO XYL1 pSR306-X123 XYL1 F GCGGGATCCATGCCTTCTATTAAGTTGA  9 (Pichia stipitis- ACTC derived XYL1, XYL1 R GCGCTCGAGTTAGACGAAGATAGGAAT 10 XYL2, XYL3 CTTGT XYL2 expression plasmid) XYL2 F GCGGGATCCATGACTGCTAACCCTTCCT 11 T XYL2 R GCGCTCGAGTTACTCAGGGCCGTCAAT 12 G XYL3 XYL3 F GCGGGATCCATGACCACTACCCCATTTG 13 XYL3 R GCGCTCGAGTTAGTGTTTCAATTCACTT 14 TCCATC P.sub.TDH3- p416GPD-XYL2 UnivF2 GACTCGCGCGCGGGAACAAAAGCTGGA 32 XYL2- GCTC T.sub.CYC1 UnivR GACTACGCGTGCGGCCGCTAATGGCGC 33 GCCATAGGGCGAATTGGGTACC PTDH3- p416GPD-XYL3 UnivF2 GACTCGCGCGCGGGAACAAAAGCTGGA 32 XYL3- GCTC T.sub.CYC1 UnivR GACTACGCGTGCGGCCGCTAATGGCGC 33 GCCATAGGGCGAATTGGGTACC

[0176] pSR-306-X123 which is Pichia stipites-derived XYL1, XYL2, and XYL3 expression plasmid (Pichia stipites-derived XYL1, XYL2, and XYL3 gene expression cassettes TDH3p-XYL1-TDH3t, PGK1p-XYL2-PGK1t, and TDH3p-XYL3-TDH3p-cloned pSR306 vector) was subjected to PCR using the primers in Table 7, and the XYL1, XYL2, and XYL3 genes were respectively secured from pSR306-X123, treated with BamHI/XhoI restriction enzymes, and cloned into the p16GPD plasmid (ATCC® 87360™) to construct p416GPD-XYL1, p416GPD-XYL2, and p416GPD-XYL3 plasmids. The constructed p16GPD-XYL2 and p416GPD-XYL3 were used as a prototype, and a DNA fragment containing a promoter and a terminator was secured by PCR using the primer pair of SEQ ID NOs: 32 and 33 (see Table 7), treated with MauBI/NodI restriction enzyme, cloned one after another into the p416GPD-XYL1 vector, and finally, a coex416-XYL vector containing all of the XYL1, XYL2, and XYL3 genes was constructed.

Example 6: Evaluation of Xylose-Consuming Ability and Mycosporine-Like Amino Acid-Producing Ability of Yeast Introduced with Xylose Assimilation Enzyme

[0177] The coex416-XYL constructed in Example 5 was transformed into the shinorine-producing strain JHYS13 obtained in Example 4 by the LiAc/SS carrier DNA/PEG method. The recombinant yeast (S. cerevisiae) strains used in this Example are summarized in Table 11 below.

TABLE-US-00011 TABLE 11 Strain Genotype JHYS13 Selected strain from delta-integration of P.sub.TEF1-NpR5597-T.sub.GPM1-P.sub.TDH3-NpR5600-T.sub.CYC1 and P.sub.TEF1-NpR5598-T.sub.GPM1-P.sub.TDH3-NpR5599-T.sub.CYC1 into CEN. PK2-1C JHYS13-1 JHYS13 harboring p416GPD JHYS13-2 JHYS13 harboring coex416-XYL

[0178] The JHYS13 strain transformed with an empty vector p416GPD (JHYS13-1; control) and the JHYS13 strain (JHYS13-2) transformed with coex416-XYL were cultured in various media including xylose and glucose, and the xylose-consuming ability was confirmed. JHYS13-1 and JHYS13-2 strains were pre-cultured into SC-Ura (containing 20 g/L glucose) medium, and then inoculated at OD600=0.2 in 10 mL of SC-Ura (with 20 g/L glucose), SC-Ura (with 10 g/L glucose and 10 g/L xylose), SC-Ura (with 2 g/L glucose and 18 g/L xylose), and SC-Ura (with 20 g/L xylose) medium in a 100 mL flask, and cultured under the conditions of 30° C. and 170 rpm.

[0179] JHYS13-1 and JHYS13-2 strains were cultured in the above four types of media for 120 hours. To measure the concentration of glucose and xylose in the medium, 500 μl of the culture supernatant was collected, filtered through a 0.22-μm syringe filter, and analyzed using HPLC. 5 mM H.sub.2SO.sub.4 was flowed as a solvent into an UltiMate 3000 HPLC system (Thermo Fisher Scientific) equipped with an Aminex HPX-87H column (300 mm×7.8 mm, 5 μm, Bio-Rad) at 60° C. at a flow rate of 0.6 mL/min, and concentrations of glucose and xylose were measured through a refractive index (RI) detector (35° C.). Cell density (OD600 value) of the culture, and shinorine production (titer (mg/L) and content (mg/gDCW) in the cell extract and medium) during culture for 120 hours were measured, and the results are shown in FIG. 5. To quantify the concentration of shinorine, two culture media were sampled at 2 mL each. One medium was dried in an oven and then the dry cell weight (DCW) was measured. Another medium was centrifuged, and then the supernatant was filtered, and the concentration of shinorine secreted into the medium was measured by HPLC. Yeast cells separated from the supernatant were vortexed for 3 minutes after adding 1 mL of distilled water and 1.5 mL of chloroform, and the intercellular shinorine was extracted. After centrifugation, the aqueous layer was isolated and filtered, and used and used for measuring the intracellular shinorine concentration. An Ultimate3000 HPLC system (Thermo Fisher Scientific) equipped with an Agilent Eclipse XDB-C18 column (5 μm, 4.6×250 mm) was used, the column temperature was maintained at 40° C., and a solvent (water:acetonitrile=95:5) was flowed at a flow rate of 0.5 mL/min. Shinorine was detected with a UV-vis detector at 334 nm.

[0180] As shown in FIG. 5, it was confirmed that JHYS13-1 and JHYS13-2 strains showed similar growth rates in a medium containing only glucose (20 g/L), and the production of shinorine was also similar (see A and E of FIG. 5). Additionally, it was confirmed that the JHYS13-1 strain could not consume xylose (see the yellow circle graphs in B, C and D of FIG. 5). On the other hand, it was confirmed that the JHYS13-2 strain could consume xylose (see the yellow triangle graphs in B, C, and D of FIG. 5), and it was confirmed that the higher the consumption of xylose, the higher the production of shinorine (comparison of the yellow triangle graphs of B, C, and D of FIG. 5 with the results of E of FIG. 5). In particular, in the medium containing only xylose (see D of FIG. 5), the consumption rate of xylose was very slow, and in the medium containing a small amount of glucose, the tendency to consume xylose was better (see B and C of FIG. 5). These results show that the use of xylose through the pentose phosphate pathway is effective in increasing the production of shinorine, but the efficiency of using xylose is low, whereby when a certain amount of glucose is contained in the medium, it is more advantageous for promoting cell growth and shinorine production.

Example 7: Construction of NTS-Integration Vector for Introducing Xylose Anabolic Enzyme into Yeast Chromosome

[0181] After confirming the effect of producing shinorine using xylose as a substrate in the same manner as in Example 6, a fermented xylose strain was developed by introducing XYL1, XYL2, and XYL3 genes into chromosomes. The ribosomal DNA (rDNA) region of the XII chromosome in yeast (S. cerevisiae) repeatedly contains 150 impermeable spacers (NTS1 and NTS2) with a size of 9.1 kb. When this sequence is used as a gene introduction sequence, multiple copies of genes can be randomly introduced into a chromosome at the same time. Vectors for introducing XYL1, XYL2, XYL3 (XYL) genes into the NTS site were constructed as follows.

[0182] DNA fragments of NTS1-2a (400 bp) and NTS1-2b (400 bp) were amplified from the yeast genome by PCR with the primers in Table 12 below. The AmpR cassette and the bleOR DNA cassette were secured from the pUG66 vector by PCR using the primers in Table 12 below. Overlapping PCR was performed on the four PCR products thus obtained to construct one linked fragment NTS1-2a-bleOR-AmpR-NTS1-2b. The resulting DNA fragment NTS1-2a-bleOR-AmpR-NTS1-2b was ligated to the XYL1 expression cassette (PTDH3-XYL1-TCYC1) via NheI and Nod sites to construct an NTS66M-XYL1 plasmid. XYL2 was cloned into p414TEF (ATCC® 87364™) using BamHI and XhoI restriction enzymes to construct a p414TEF-XYL2 vector. Additionally, XYL3 was cloned into coex414TPI1 vector (see Korean Unexamined Patent Publication No. 10-2016-0093492) using BamHI and XhoI restriction enzymes to construct p414TPI1-XYL3. This was used as a template, and XYL2 expression cassette (PTEF1-XYL2-TGPM1) and XYL3 expression cassette (PTPI1-XYL3-TTPI1) with MauBI and Nod sites were secured by PCR, and sequentially cloned between the AscI and Nod sites of NTS66M-XYL1, and an NTS66M-XYL plasmid for introducing the NTS site of the XYL1, XYL2, XYL3 (XYL) genes was constructed (see FIG. 6a). The primers used in the PCR are summarized in Table 12 below.

TABLE-US-00012 TABLE 12 Primer (F: SEQ PCR Forward, R: ID Product Template used Reverse) Sequence (5′.fwdarw.3′) NO NTS1-2a Saccharomyces NTS1-2aF CCGAGCGTGAAAGGATTTGCC 30 cerevisiae NTS1-2aR GCGGCTAGCCAACCATTCCATATCTGT 31 genomic TAAG NTS1-2b DNA (GenBank NTS1-2bF GCGAAGTAAATTTTTGGCG 28 Accession No. NTS1-2b AmpR TTTCCCCGAAAAGTGCATTTAAATCTA JRIV01000000) GTTTCTTGGCTTCCTATG AmpR pUG66 Amp-QriF GCACTTTTCGGGGAAATGTG 21 (EUROSCARF) Amp-OriR CTCAACATTCACCCATTTCTCAATTTA 22 AATCGCAGGAAAGAACATGTGAG bleOR pUG66 (P30116) TEF prom F GCCAAAAATTTACTTCGCGCGGCCCGA 27 CATGGAGGCCCAGAAT NTS term R GCGGGCGCGCCGCGGCCGCTAAGGGT 26 TCTCGAGAGCTC P.sub.TEF1- p414TEF-XYL2 UnivF2 GACTCGCGCGCGGGAACAAAAGCTGG 32 XYL2- AGCTC T.sub.GPM1 UnivR GACTACGCGTGCGGCCGCTAATGGCG 33 CGCCATAGGGCGAATTGGGTACC P.sub.TPI1- p414TPI1-XYL3 UnivF2 GACTCGCGCGCGGGAACAAAAGCTGG 32 XYL3- AGCTC T.sub.TPI1 UnivR GACTACGCGTGCGGCCGCTAATGGCG 33 CGCCATAGGGCGAATTGGGTACC

Example 8: NTS-Integration of Xylose Assimilation Enzyme and Evaluation of Xylose-Consuming Ability and Mycosporine-Like Amino Add-Producing Ability of Yeast Strains Produced Therefrom

[0183] The NTS66M-XYL plasmid prepared in Example 7 was treated with a SwaI restriction enzyme, so that the NITS site was exposed at both ends of the DNA (see FIG. 6a). To insert the XYL gen into the rDNA region of the yeast chromosome, the prepared NTS66M-XYL plasmid was cleaved with SwaI, and transformed into JHYS13 strain by LiAc/SS carrier DNA/PEG method, and then selected on YPX (10 g/L yeast extract, 20 g/L bacto peptone and 20 g/L xylose) plates containing 500 mg/L zeocin. The recombinant yeast strains selected in this way are summarized in Table 13 below.

TABLE-US-00013 TABLE 13 Strain Genotype JHYS14 Selected strain from NTS-integration of P.sub.TDH3-XYL1-T.sub.CYC1-P.sub.TEF1-XYL2-T.sub.GPM1-P.sub.TPI1-XYL3-T.sub.TPI1 into CEN. PK2-1C JHYS15 Selected strain from NTS-integration of P.sub.TDH3-XYL1-T.sub.CYC1-P.sub.TEF1-XYL2-T.sub.GPM1-P.sub.TPI1-XYL3-T.sub.TPI1 into CEN. PK2-1C JHYS16 Selected strain from NTS-integration of P.sub.TDH3-XYL1-T.sub.CYC1-P.sub.TEF1-XYL2-T.sub.GPM1-P.sub.TPI1-XYL3-T.sub.TPI1 into CEN. PK2-1C

[0184] The selected strains was inoculated into SC medium (2 g/L glucose, 18 g/L xylose) at an OD600=0.2, and cultured at 30° C. and 170 rpm for 96 hours. The concentration of glucose and xylose in the medium and the cell density (OD600 value) of the culture were measured and shown in FIG. 6b, and the shinorine production (titer (mg/L) and content (mg/gDCW) in the cell extract and medium) during the 96-hour culture was measured and shown in FIG. 6c.

[0185] As shown in FIGS. 6b and 6c, all three strains selected above have xylose-consuming ability and shinorine-producing ability. In particular, the JHYS16 strain showed the highest shinorine production (17.72 mg/L and 8.7 mg/gDCW).

[0186] The JHYS16 strain was deposited with the Korean Culture Center of Microorganisms (KCCM) located in Seodaemun-gu, Seoul, Korea on Nov. 14, 2019 under the Budapest Treaty, and was given an accession number KCCM12628P.

[0187] <Production of Mycosporine-Like Amino Acids Through the Enhancement of the Pentose Phosphate Pathway>

Example 9: Construction of Transaldolase (Tal1) Deletion Strain

[0188] To further improve the production of shinorine in the JHYS16 strain, which was confirmed to have excellent shinorine-producing ability in Example 8, a plan was devised to increase the production of S7P, which is an intermediate product. For this purpose, it was attempted to remove TAL1, which is a transaldolase involved in the conversion reaction between S7P and glyceraldehyde 3-phosphate in the pentose phosphate pathway (see FIG. 1a). The CRISPR-Cas9 system was used to remove TAL1 using the CRISPR-Cas9 system. Specifically, the coding gene and gRNA fragment (PSNR52-structural component-TSUP4) of Cas9 (NP_269215.1) of Streptococcus pyogenes were cloned into p413TEF (ATCC® 87362™) to produce a coex413-Cas9-gRNA plasmid. TAL1 target gRNA (sgRNA) was designed by Yeastriction v0.1 (http://yeastriction.tnw.tudelft.nl), and inserted into coex413-Cas9-gRNA by DpnI-mediated site-directed mutagenesis based on PCR to construct coex413-Cas9-TAL1gRNA. The primers and the prepared plasmids used in the PCR are shown in Tables 14 and 15 below, respectively.

TABLE-US-00014 TABLE 14 Primer (F: SEQ PCR Forward, R: ID Product Template used Reverse) Sequence (5′.fwdarw.3′) NO TAL1 — TAL1_delF TAGTACGATAGTAAAATACTTCTCGAACTC 34 deletion GTCACATATACGTGTACATAGGAAGTATCT cassette TAL1_delR AGAAACGTGCATAAGGACATGGCCTAAAT 35 TAATATTTCCGAGATACTTCCTATGTACAC G coex413- coex413- TAL1 gRNA F AACTAACCCATCATTGATCTGTTTTAGAGC 38 Cas9- Cas9-gRNA TAGAAATAGC TAL1gRNA TAL1 gRNA R AGATCAATGATGGGTTAGTTGATCATTTAT 39 CTTTCACTGC

TABLE-US-00015 TABLE 15 Plasmid Description coex413-Cas9-TAL1gRNA CEN/ARS plasmid, HIS3, P.sub.TDH3-CAS9-T.sub.TPI1, P.sub.SNR52-TAL1gRNA-T.sub.SUP4

[0189] Coex413-Cas9-TAL1gRNA expressing guide RNA (gRNA) targeting the Cas9 gene and TAL1 gene and TAL1 deletion cassette having homologous arms above and below the TAL1 ORF were transformed into JHYS16 cells, and then SC-His (2 wt % glucose), a TAL1-deletion strain was selected. TAL1 deletion was confirmed by PCR (primers used: TAL1_CF primer: CGGGAATAAAAGCGGAACT (SEQ ID NO: 36); TAL1_CR primer: GGTGGITCCGGATGTIT (SEQ ID NO: 37)). After confirming the TAL1 deletion by PCR, it was cultured overnight in YPD (10 g/L yeast extract, 20 g/L bactopeptone and 20 g/L glucose) medium, and the coex413-Cas9-TAL1gRNA plasmid was removed. The Tal1-deletion strain thus obtained was named JHYS17.

Example 10: Evaluation of Mycosporine-Like Amino Add-Producing Ability of Transaldolase (Tal1) Deletion Strain

[0190] The prepared JHYS16 and JHYS17 were inoculated into SC medium (containing 2 g/L glucose and 18 g/L xylose) or SC medium (containing 10 g/L glucose and 10 g/L xylose) at an OD600=0.2, respectively, and cultured under the conditions of 30° C. and 170 rpm for 120 hours. The glucose and xylose concentrations in the medium, the cell density of the culture (OD600 value), and the shinorine production (titer (mg/L) and content (mg/gDCW) in the cell extract and the medium) during the 120 hour-culture were measured and shown in FIG. 7.

[0191] The JHYS17 strain obtained by deleting TAL1 with JHYS16 was cultured in SC medium (containing 2 g/L glucose and 18 g/L xylose). As a result, it was confirmed that severe growth defects were seen in the xylose-rich medium (A of FIG. 7), confirming that TAL1 is essential for xylose assimilation.

[0192] Therefore, the ratio of glucose to xylose was changed to alleviate the growth defects and the culture was performed. JHYS16 and JHYS1 were cultured in SC medium (containing 10 g/L glucose and 10 g/L xylose), and the results are shown in B of FIG. 7. As shown in B of FIG. 7, JHYS16 and JHYS17 showed similar glucose consumption rates, but unlike JHYS16, which consumed most of the medium xylose, JHYS17, which was defective in xylose anabolic activity, consumed only 5.7 g/L xylose. However, JHYS17 produced about 3.3 times higher titer (21.9 mg/L) and 7.2 times higher amount (7.67 mg/gDCW) of shinorine than JHYS16 (see C of FIG. 6). The shinorine production level of JHYS17 was much higher than that produced in JHYS16 cultured in a medium containing 2 g/L glucose and 18 g/L xylose (see C of FIG. 5). Therefore, when TAL1 was deleted, it was confirmed that S7P generated from xylose may be more useful for shinorine biosynthesis, but xylose assimilation action was limited due to the incomplete pentose phosphate pathway.

Example 11: Construction of a Vector for Overexpression of Transcription Regulator Stb5 and Transketolase (Tkl1) in Yeast

[0193] Since JHY17 is defective in the pentose phosphate pathway, enhancing the expression of other genes involved in the pentose phosphate pathway can aid in xylose fermentation and subsequent shinorine production. Therefore, as overexpression target genes, Stb5 (GenBank accession no. JRIV01000036.1) encoding a transcriptional regulatory factor and TKL1 (GenBank accession no. JRIV01000030.1) encoding a transketolase were selected. Stb5 activates genes in the pentose phosphate pathway, including glucose-6-phosphate dehydrogenase (ZWF1), 6-phosphogluconate dehydrogenase (GND1), TKL1 and other NADPH production pathways to produce NADPH, which plays an important role in the oxidative stress response. Tkl1 forms a reversible link between the pentose phosphate pathway and glycolysis which are two major metabolic pathways (see FIG. 1a), which plays an important role when culturing yeast using xylose as a carbon source. To confirm the overexpression effect of STB5 and TKL1, plasmids expressing these genes individually or in combination were constructed. Fragments of each gene were secured by PCR, and the primers used for PCR are shown in Table 16 below.

TABLE-US-00016 TABLE 16 Primer (F: PCR Forward, SEQ ID Product Template used R: Reverse) Sequence (5′.fwdarw.3′) NO TKL1 Saccharomyces TKL1 F GCGGGATCCATGACTCAATTCA 117 cerevisiae CTGACA genomic TKL1 R GCGCTCGAGTTAGAAAGCTTTT 118 DNA (GenBank TTCAAAGGAG Accession No. STB5F GCGGGATCCATGGATGGTCCCA 115 STB5 JRIV01000000) ATTTTG STB5R GCGGTCGACTCATACAAGTTTA 116 TCAACCCAAG P.sub.TDH3- p414GPD- Univ F2 GACTCGCGCGCGGGAACAAAA 332 TKL1-T.sub.CYC1 TKL1 GCTGGAGCTC Univ R GACTACGCGTGCGGCCGCTAAT 333 GGCGCGCCATAGGGCGAATTGG GTACC

[0194] TKL1 was cloned into p414GPD vector (ATCC®87356™) using BamHI and XhoI as restriction enzyme sites so that is was expressed under the control of PTDH3, thereby obtaining p414GPD-TKL1 (Table 16). For overexpression using a strong promoter, STB5 was cloned into the p414ADH vector (ATCC®87372™) to be expressed under the control of the weak promoter PADH1 due to its growth inhibitory effect on yeast strains. STB5 DNA fragment was treated with BamHI, SalI, and p414ADH was treated with BamHI and XhoI, and cloned (p414ADH-STB5). The TKL1 expression cassette (PTDH3-TKL1-TCYC1) having MauBI and Nod sites obtained by PCR using p414GPD-TKL1 as a template was cloned between the Ascl and NotI sites of p414ADH-STB5 to construct a coex414-STB5-TKL1 plasmid. The constructed plasmids are shown in Table 17 below.

TABLE-US-00017 TABLE 17 Plasmid Description p414ADH-STB5 CEN/ARS plasmid, TRP1, P.sub.ADH1-STB5-T.sub.CYC1 p414GPD-TKL1 CEN/ARS plasmid, TRP1, P.sub.TDH3-TKL1-T.sub.CYC1 coex414-STB5-TKL1 CEN/ARS plasmid, TRP1, P.sub.ADH1-STB5-T.sub.CYC1, P.sub.TDH3-TKL1-T.sub.CYC1

Example 12: Evaluation of Mycosporine-Like Amino Add-Producing Ability Through Overexpression in Yeast of Transcription Regulators Stb5 and Tkl1 of the Pentose Phosphate Pathway

[0195] To determine whether the enhancement of the pentose phosphate pathway via additional gene overexpression is effective in increasing shinorine production, JHYS17 strain was transformed with p414ADH-STB5, p414GPD-TKL1, or coex414-STB5-TKL1 plasmids using the LiAc/SS carrier DNA/PEG method. The transformed yeast strains are shown in Table 18 below.

TABLE-US-00018 TABLE 18 Strain Genotype JHYS17 JHYS16 tal1Δ JHYS17-1 JHYS17 harboring p414GPD JHYS17-2 JHYS17 harboring p414ADH-STB5 JHYS17-3 JHYS17 harboring p414GPD-TKL1 JHYS17-4 JHYS17 harboring coex414GPD-STB5-TKL1

[0196] Each of the obtained transformed yeast stains was inoculated into SC-Trp (10 g/L glucose, 10 g/L xylose) medium at OD600=0.2. While culturing under the conditions of 30° C. and 170 rpm for 120 hours, the glucose and xylose concentrations in the medium and the cell density (OD600 value) of the culture were measured and shown in FIG. 8a, and the shinorine production (titer (mg/L) and content (mg/gDCW) in the cell extract and medium) during the 120-hour culture was measured and shown in FIG. 8b.

[0197] As shown in FIG. 8b, when both genes (STB5 and TKL1) were overexpressed, it was effective in increasing shinorine production. JHYS17-2 and JHYS17-3 strains overexpressing STB5 or TKL1 consumed a small amount of xylose as compared with the control JHYS17-1 strain harboring the empty vector, and the shinorine production was also increased (FIG. 8b). The JHYS17-4 strain overexpressing both STB5 and TKL1 had the highest shinorine production, and the consumption of xylose was also high. Since overexpression of these genes was effective in increasing the rate of xylose consumption (FIG. 8a), overexpression of these genes can alleviate the problem of cell growth degradation that occurs during xylose assimilation in the Tal-deficient JHYS17 strain.

Example 13: Search for the Optimal Ratio of Glucose and Xylose in the Medium

[0198] Due to the inefficient utilization of xylose in Tal1-deficient strains, further addition of glucose to the medium can promote cell growth. On the other hand, the important intermediate S7P for shinorine production is provided mainly through xylose assimilation. Therefore, the ratio of glucose to xylose in the medium can be adjusted to obtain appropriate cell growth and maximum shinorine production effect.

[0199] To find the optimal ratio of these carbon sources (glucose and xylose), eight different media containing xylose and glucose in various proportions (see A of FIG. 9a) were inoculated with the JHYS17-4 strain at OD600=0.2 while maintaining the total carbon source concentration at 20 g/L, and cultured at 30° C. and 170 rpm for 120 hours. The cell density (OD600 value) of the culture obtained by the above culture and the concentration of glucose and xylose in the medium were measured and shown in B, C and D of FIG. 9a, respectively, and the shinorine production (titer (mg/L) and content (mg/gDCW) in cell extract and medium) during the 120-hour culture were measured and shown in FIGS. 9b and 9c.

[0200] As a result, as the xylose ratio of the medium increased (i.e., as the glucose ratio decreased), the cell growth rate decreased (B of FIG. 9a). However, as the xylose concentration of the medium increased to 8 g/L, shinorine production increased. When the strain was cultured in medium #5 containing 12 g/L of glucose and 8 g/L of xylose, 12 g/L of glucose and 5.4 g/L of xylose were consumed (C and D in FIG. 9a), and the most shinorine was produced (31.0 mg/L (FIG. 9b) and 9.6 mg/gDCW (FIG. 9c)). Also, the shinorine content was highest when cultured in medium #6 containing 10 g/L glucose and 10 g/L xylose (10.27 mg/gDCW (FIG. 9c)). When the xylose concentration in the medium was higher than 14 g/L, cell growth was inhibited (A of FIG. 9), and the reduced cell growth had a negative effect on shinorine production (FIGS. 9b and 9c).

[0201] Additionally, the content of by-products (xylitol, ethanol, glycerol, and acetate) produced during the culture was measured and shown in FIG. 10. To determine the concentration of xylitol, ethanol, glycerol and acetate in the medium, 500 μl of the culture supernatant was collected and filtered with 0.22-μm syringe filter and then analysis was performed using HPLC. 5 mM H.sub.2SO.sub.4 was flowed as a solvent into an UltiMate 3000 HPLC system (Thermo Fisher Scientific) equipped with an Aminex HPX-87H column (300 mm×7.8 mm, 5 μm, Bio-Rad) at 60° C. at a flow rate of 0.6 mL/min, and the concentration of by-products was measured through a refractive index (RI) detector (35° C.). As can be seen in FIG. 10, ethanol was the main by-product in most media conditions, but xylitol was accumulated as a by-product of xylose assimilation. The accumulation of xylitol can be an important factor that slows the consumption rate of xylose.

[0202] As described above, respective descriptions and embodiments disclosed in the present disclosure can also be applied to other descriptions and embodiments. That is, all combinations of various elements disclosed in the present disclosure fall within the scope of the present disclosure. In addition, the scope of the present disclosure is not limited by the specific description below. In addition, one of ordinary skill in the art can recognize or identify a number of equivalents with regard to certain aspects of the present disclosure only by routine experimentation. Further, such equivalents are intended to be included in the present disclosure.

Accession Number

[0203] Name of depositary institution: Korea Microorganism Conservation Center

[0204] Accession number: KCCM12628P

[0205] Date of deposit: 20191114