CN114621883A

CN114621883A - Saccharomyces cerevisiae with sun-screening strengthening effect and application thereof

Info

Publication number: CN114621883A
Application number: CN202210118631.8A
Authority: CN
Inventors: 伍小艺; 王金晶; 余施雨; 易崇华; 周庭安; 李崎; 钮成拓; 郑飞云; 刘春凤; 许鑫
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-02-08
Filing date: 2022-02-08
Publication date: 2022-06-14
Anticipated expiration: 2042-02-08
Also published as: CN114621883B

Abstract

The invention discloses saccharomyces cerevisiae with a sun-screening strengthening effect and application thereof, and belongs to the field of bioengineering. The invention provides recombinant saccharomyces cerevisiae which simultaneously expresses EEVS, MT-Ox, xylose reductase XR, xylitol dehydrogenase XDH and xylulokinase XK for synthesizing gadusol, thereby realizing the high-efficiency synthesis of gadusol. The content of the gadusol prepared by the recombinant saccharomyces cerevisiae JN-BYXXXAMO can reach 40.5 mg/L. The invention discovers that the introduction of the gadusol production pathway (EEVS, MT-Ox) into BY4743 can greatly improve the yeast biomass, and meanwhile, the introduction of the xylose metabolic pathway can greatly improve the yeast gadusol yield and can obviously reduce the production cost.

Description

Saccharomyces cerevisiae with sun-screening strengthening effect and application thereof

Technical Field

The invention relates to saccharomyces cerevisiae with a sun-screening strengthening effect and application thereof, belonging to the field of bioengineering.

Background

Coral reefs are oases in deserts and provide habitats for numerous marine organisms. The whitening of coral reef results in the extinction of a large amount of fish and marine life, and further leads to the collapse of marine ecosystem. In seawater, low concentrations of chemical sunscreen substances are sufficient to cause a range of coral diseases and coral whitening. Wherein, the compound mainly comprises octocrylene, oxybenzone, octyl methoxycinnamate and similar derivatives thereof. About 14,000 tons of sunscreen cream are washed out and stocked in the ocean every year, wherein about 10 percent of sunscreen components are oxybenzone and the like, and the minimum concentration of the sunscreen components causing coral albinism is far away, so that about 15 percent of coral reefs and about 40 percent of coral reefs along the bank are threatened by chemical sunscreen agents. Since the growth and development of coral have extremely high environmental requirements, scientists predict that the existing coral reef will be extinct after 50 years. The coral and ocean environment-friendly biological sun-screening agent is produced, the trend of large-area whitening of the existing coral can be effectively slowed down, and the purpose of coral protection is realized. Most of the current commercial natural sunscreen ingredients, such as sulforaphane, ferulic acid, carotenoids, tetrahydrolycopene, etc., are of plant origin. Many plant-derived substances are not present in the ocean and it is difficult to explain whether they affect the coral and the ocean environment. Gadusol is a natural sunscreen agent that occurs naturally in the ecosystem of various fish roes such as zebrafish, salmon, sturgeons, and coral reefs, and protects marine life from ultraviolet rays. Compared with natural sunscreen ingredients of plant sources, the sunscreen composition has more reliable safety.

However, the natural yield of gadusol is very low, and only 10-20 mg of gadusol can be extracted from 1000 fish eggs, so that the large-scale application of the gadusol is limited, and the way of extracting products by collecting the fish eggs is not known. Therefore, the production of gadusol by using engineered saccharomyces cerevisiae is a feasible and relatively low-cost industrial approach.

Yeast is an important industrial production strain, and has the advantages of high fermentation speed, short growth cycle, large-scale culture and the like. The yeast is used for producing the environment-friendly biological sunscreen agent to replace a chemical sunscreen agent, thereby realizing the consideration of environmental protection and economic benefit. In addition, the culture fermentation of yeast using glucose as a carbon source conflicts with food production. While lignocellulose is the most abundant renewable resource on earth, glucose, xylose and arabinose are the most abundant sugars in lignocellulose. Xylose is a non-grain carbon source with low cost, and is introduced into yeast cells, so that the carbon source for producing gadusol by yeast can be widened, and the cost can be further reduced.

Disclosure of Invention

The invention uses saccharomyces cerevisiae to produce natural sunscreen component gadusol, clones recombinant EEVS gene and MT-Ox gene into high copy plasmid pYEP352 BY codon optimization of EEVS and MT-Ox gene, finally transfers pYEP352 vector containing EEVS and MT-Ox gene into saccharomyces cerevisiae BY4743 strain, obtains the capability of utilizing xylose BY introducing Xylulokinase (XK) or xylose reductase/xylitol dehydrogenase (XR/XDH), and finally obtains saccharomyces cerevisiae JN-BYXXXXXMO capable of preparing gadusol at low cost.

The invention provides a recombinant saccharomyces cerevisiae JN-BYXXXEMO, which simultaneously expresses EEVS with a nucleotide sequence shown in SEQ ID No.1, MT-Ox with a nucleotide sequence shown in SEQ ID No.2, xylose reductase XR with a nucleotide sequence shown in SEQ ID No.3, xylitol dehydrogenase XDH with a nucleotide sequence shown in SEQ ID No.4 and xylulokinase XK with a nucleotide sequence shown in SEQ ID No. 5.

In one embodiment of the invention, the EEVS is derived from zebrafish (NCBI serial No. XM _ 001343386.7); the MT-Ox is derived from zebrafish (NCBI serial number NM-001013450.1); the xylose reductase XR is derived from Schiffersomyces stipites (NCBI SEQ ID NO: HM 769331.1); the xylitol dehydrogenase XDH is derived from Schiffersomyces stipites (NCBI sequence number HM 769332.1); the xylulokinase XK is derived from Schiffersomyces stipites (NCBI sequence No. XM-001387288.1). All gene fragments were re-synthesized after codon optimization for the Saccharomyces cerevisiae expression system (Jinzhi, Suzhou, China).

In one embodiment of the invention, the recombinant saccharomyces cerevisiae JN-BYXXXEMO takes saccharomyces cerevisiae BY4743 as an expression host.

In one embodiment of the invention, the recombinant Saccharomyces cerevisiae JN-BYXXXEMO uses pYEP352 plasmid to express EEVS and MT-Ox and pY15TEF1 plasmid to express xylose reductase XR, xylitol dehydrogenase XDH and xylulokinase XK.

In one embodiment of the invention, the method for culturing the saccharomyces cerevisiae JN-BYXXXAMO is characterized in that the yeast is inoculated into a culture medium containing glucose and xylose and cultured;

the culture medium is as follows: YNB-X3A medium, pH natural, distilled water prepared, sterilized at 115 ℃ for 15 min.

The YNB-X3A culture medium: YNB medium 6.7g/L, xylose 20g/L, (NH)₄)₂SO₄10g/L, 30mg/L histidine, 30mg/L lysine, 30mg/L methionine.

The saccharomyces cerevisiae JN-BYXXXAMO provided by the invention can produce gadusol by using glucose and xylose, when the xylose is used as a carbon source, the intracellular gadusol content is 10 times of that of a production strain which only can use the glucose as the carbon source, and the metabolite of the saccharomyces cerevisiae JN-BYXXXAMO has the capacity of absorbing ultraviolet rays.

The invention also provides a method for preparing gadusol at low cost, which comprises the step of adding the recombinant saccharomyces cerevisiae JN-BYXXXEMO into a reaction system containing xylose for fermentation to prepare the gadusol.

In one embodiment of the present invention, the concentration of xylose in the reaction system is at least: 20 g/L.

In an embodiment of the invention, the addition amount of the recombinant saccharomyces cerevisiae JN-BYXXXEMO in the reaction system is at least: 1% (v/v).

In one embodiment of the invention, the fermentation conditions are: shaking-culturing at 28 deg.C with rotation speed of 200 r/min.

The invention also provides a method for improving the biomass of the recombinant saccharomyces cerevisiae in a glucose culture medium, which comprises the following steps:

EEVS with a nucleotide sequence shown as SEQ ID NO.1 and MT-Ox with a nucleotide sequence shown as SEQ ID NO.2 are simultaneously expressed in the saccharomyces cerevisiae.

In one embodiment of the invention, the recombinant saccharomyces cerevisiae takes saccharomyces cerevisiae BY4743 as an expression host.

In one embodiment of the invention, the recombinant Saccharomyces cerevisiae uses pYEP352 plasmid to express EEVS and MT-Ox and pY15TEF1 plasmid to express xylose reductase XR, xylitol dehydrogenase XDH and xylulokinase XK.

The invention also provides a method for improving the yield of gadusol produced by recombinant saccharomyces cerevisiae, which simultaneously expresses EEVS with a nucleotide sequence shown in SEQ ID NO.1, MT-Ox with a nucleotide sequence shown in SEQ ID NO.2, xylose reductase XR with a nucleotide sequence shown in SEQ ID NO.3, xylitol dehydrogenase XDH with a nucleotide sequence shown in SEQ ID NO.4 and xylulokinase XK with a nucleotide sequence shown in SEQ ID NO.5 in the saccharomyces cerevisiae.

The invention also provides application of the recombinant saccharomyces cerevisiae in preparation of products containing gadusol.

Advantageous effects

(1) The invention provides recombinant saccharomyces cerevisiae which simultaneously expresses EEVS, MT-Ox, xylose reductase XR, xylitol dehydrogenase XDH and xylulokinase XK for synthesizing gadusol, thereby realizing the high-efficiency synthesis of gadusol. The content of the gadusol prepared by the recombinant saccharomyces cerevisiae JN-BYXXXAMO can reach 40.5 mg/L.

(2) As for the invention, the introduction of the gadosol production pathway (EEVS, MT-Ox) into BY4743 can greatly improve the biomass of the yeast, and meanwhile, the introduction of the xylose metabolic pathway can greatly improve the yield of the yeast gadosol and can obviously reduce the production cost.

Drawings

FIG. 1: the Gadusol biosynthetic pathway.

FIG. 2: a plasmid containing the gadusol synthetic pathway.

FIG. 3: a method for constructing a saccharomyces cerevisiae strain for producing a natural sun-screening agent gadusol by using xylose.

FIG. 4: growth curves of Saccharomyces cerevisiae BY4743 and JN-BYEMO.

FIG. 5: growth curves for JN-BYXXEMO and JN-BYXXEMO.

FIG. 6: HPLC results of Gadusol standard samples.

FIG. 7: HPLC result of gadusol produced by JN-BYEMO.

FIG. 8: HPLC result of gadusol produced by JN-BYXXEMO.

FIG. 9: HPLC result of gadusol produced by JN-BYXXXEMO.

FIG. 10: PCR confirmed the successful introduction of the gadusol production pathway (EEVS + MT-Ox) into s.cerevisiae.

FIG. 11: growth curves for W303 and WYEMO.

FIG. 12: PCR verifies that the xylose-utilizing gadusol production approach is successfully introduced into the saccharomyces cerevisiae, wherein 1 is as follows: EEVS gene, 2 is: the full length of the synthetic pathway gene (XK + XR + XDH + EEVS + MT-Ox).

Detailed Description

The YNB medium referred to in the examples below was purchased from Sigma Aldrich; the saccharomyces cerevisiae W303 and YIP5 plasmids are stored in a laboratory.

The media involved in the following examples are as follows:

LB medium: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride.

YPD medium: 10g/L of yeast extract, 20g/L of peptone and 20g/L of glucose.

YNB-G: YNB medium 6.7g/L, glucose 20g/L, (NH)₄)₂SO₄ 10g/L。

YNB-G4A: 30mg/L histidine, 30mg/L lysine, 30mg/L methionine and 30mg/L leucine were added to the YNB-G medium.

YNB-G3A: 30mg/L histidine, 30mg/L lysine and 30mg/L methionine were added to the YNB-G medium.

YNB-X: YNB medium 6.7g/L, xylose 20g/L, (NH)₄)₂SO₄ 10g/L。

YNB-X3A: 30mg/L histidine, 30mg/L lysine and 30mg/L methionine were added to the YNB-X medium.

The detection methods referred to in the following examples are as follows:

analysis of Gadusol yield:

detection analysis was performed by High Performance Liquid Chromatography (HPLC) under the following conditions:

HPLC conditions:

mobile phase: 5mmol/L PBS-methanol

The method comprises the following steps: isocratic for 1% methanol for 20min, isocratic for 20-40min from 1% methanol to 95% methanol, isocratic for 95% methanol for 20min

Flow rate: 0.3mL/min

Detection wavelength: 296 nm.

The primers involved in the following examples are as follows:

TABLE 1 primers for PCR of genes of interest

TABLE 2 primers used for plasmid vector linearization

TABLE 3 primers for colony PCR

Example 1: construction of recombinant Saccharomyces cerevisiae

(1) Construction of Saccharomyces cerevisiae strain JN-BYEMO:

in consideration of low success rate of restriction and insertion cloning, a Gibson Assembly method is adopted to connect a linearized vector and a target gene, primers of the vector and the target gene are designed by utilizing Snapgene software, and the Tm value of a homologous arm is more than or equal to 55 ℃.

Designing PCR primers according to the metabolic pathway of gadusol (shown in Table 1), obtaining target genes EEVS (nucleotide sequence is shown in SEQ ID NO. 1) and MT-Ox (nucleotide sequence is shown in SEQ ID NO. 2) BY PCR, connecting the target genes EEVS and MT-Ox with a vector pYEP352 through Gibson Assembly, and using the primers for linearization of plasmid vectors as shown in Table 2, successfully constructing pYEP352-EEVS-MT-Ox plasmid, wherein the plasmid map is shown in FIG. 2, the construction process is shown in FIG. 3 (in the figure, the plasmid pYEP352 is abbreviated as plasmid YEp352), transforming the pYEP352-EEVS-MT-Ox into Saccharomyces cerevisiae BY4743, and constructing a recombinant Saccharomyces cerevisiae strain JN-BYEO for producing gadusol.

(2) Colony PCR primers are designed according to a target gene sequence (shown in Table 3), and colony PCR verification is carried out on the saccharomyces cerevisiae JN-BYE obtained in the step (1), and the result shows that the strain saccharomyces cerevisiae JN-BYE obtained in the step (1) contains target genes EEVS and MT-Ox (shown in figure 10).

Example 2: recombinant saccharomyces cerevisiae JN-BYEMO fermentation production gadusol

The Gadusol biosynthetic pathway is shown in FIG. 1, and the specific steps are as follows:

(1) activation of recombinant Saccharomyces cerevisiae

The saccharomyces cerevisiae strain JN-BYEMO and the starting host saccharomyces cerevisiae BY4743 which are constructed in the embodiment 1 and produce the natural sunscreen gadusol are respectively inoculated in YNB-G4A liquid culture medium and cultured for 24h at the temperature of 28 ℃.

And respectively preparing recombinant saccharomyces cerevisiae JN-BYEMO seed liquid and saccharomyces cerevisiae BY4743 seed liquid.

(2) Fermentation of recombinant Saccharomyces cerevisiae

And (2) respectively inoculating the recombinant saccharomyces cerevisiae JN-BYEMO seed liquid and the saccharomyces cerevisiae BY4743 seed liquid prepared in the step (1) into a 500mL triangular flask containing 100mL YPD medium according to the inoculation amount of 1% (v/v), performing shake flask fermentation at 28 ℃, and respectively determining growth curves, wherein the results are shown in a figure 4 and a table 4.

Table 4: OD of Saccharomyces cerevisiae BY4743 and JN-BYEMO at different times₆₀₀Value of

Time (h)	JN-BYEMO/OD₆₀₀	BY4743/OD ₆₀₀
			0	0.010	0.01
12	0.171	0.6
			25	9.637	1.882
29	13.840	1.855
			36	14.093	1.987
58	20.150	1.837
			96	20.517	1.806
120	19.033	1.795
			144	17.650	1.792

In the fermentation system, OD is added in the stationary phase (50 h for culture)₆₀₀Reaches about 15, 96h OD₆₀₀Up to 20. And the saccharomyces cerevisiae BY4743 grows on a glucose culture medium, and the OD value can only reach about 2. Therefore, EEVS and MT-Ox are simultaneously expressed in the saccharomyces cerevisiae BY4743, so that the saccharomyces cerevisiae BY can be obviously improved4743 the biomass of the strain in glucose medium.

(3) Preparation of gadusol

Inoculating the recombinant saccharomyces cerevisiae JN-BYEMO seed liquid prepared in the step (1) into a 500mL triangular flask containing 100mL YPD medium according to the inoculation amount of 1% (v/v), fermenting for 8 days at 28 ℃, preparing recombinant saccharomyces cerevisiae JN-BYEMO fermentation liquid, collecting the recombinant saccharomyces cerevisiae JN-BYEMO fermentation liquid into a 50mL centrifuge tube, centrifuging at the rotating speed of 6000 Xg, and discarding the supernatant;

resuspending the thalli by using pure methanol, crushing saccharomyces cerevisiae cells by using an ultrasonic crusher, centrifuging at 10000 Xg, and taking supernatant; obtaining saccharomyces cerevisiae cells through centrifugation, and then using pure methanol as a solvent to carry out heavy suspension on the cells so as to extract gadusol in the saccharomyces cerevisiae cells; evaporating the solvent methanol in the supernatant to dryness by using a rotary evaporator to prepare a concentrated extract;

the concentrated extract was diluted with 10mL of ultrapure water (ddH)₂O), dissolving, collecting into a centrifuge tube, freezing in a refrigerator at-80 ℃ for 12h, and freeze-drying for preservation.

Since gadusol is a polar molecule, it is very soluble in water and polar organic solvents. And (3) re-dissolving the freeze-dried sample in 5mmol/L PBS solution, and performing detection analysis by using High Performance Liquid Chromatography (HPLC) (the result is shown in figures 6-7).

The quantitative analysis showed that the yield of gadusol produced with recombinant Saccharomyces cerevisiae JN-BYEMO was 4mg/L (about 1.34mg/g cell dry weight).

Example 3: construction of recombinant saccharomyces cerevisiae JN-BYXXEMO and preparation of gadusol by fermentation of recombinant saccharomyces cerevisiae JN-BYXXEMO

The method comprises the following specific steps:

(1) construction of Saccharomyces cerevisiae strain JN-BYXXEMO

Primers (shown in Table 1) were designed based on the sequences of the yeast xylose genes (XR and XDH), the target gene XR (nucleotide sequence shown in SEQ ID NO. 3) and XDH (nucleotide sequence shown in SEQ ID NO. 4) were obtained by PCR, and the target gene was ligated with vector pY15TEF1(Biovector Co., LTD) by Gibson Assembly to construct pY15TEF1-XR-XDH plasmid.

And (3) introducing the constructed plasmid pY15TEF1-XR-XDH into the saccharomyces cerevisiae strain JN-BYEMO prepared in the step (1) of the example 1, expressing, and finally obtaining the recombinant saccharomyces cerevisiae strain JN-BYXXEMO.

Coating YNB-X3A plate on the recombinant Saccharomyces cerevisiae, and culturing in a constant temperature incubator at 28 deg.C for 1-2 days. Then, streak culture and colony PCR verification (primers are shown in Table 3) are carried out on the strain, the success of strain construction is verified, and the strain is named as JN-BYXXEMO.

(2) Activation of recombinant Saccharomyces cerevisiae Strain JN-BYXXEMO

The constructed saccharomyces cerevisiae strain JN-BYXXEMO producing the natural sunscreen gadusol is inoculated in YNB-X3A liquid culture medium and cultured for 24h at the temperature of 28 ℃.

And preparing the recombinant saccharomyces cerevisiae JN-BYXXEMO seed solution.

(3) Fermentation of recombinant Saccharomyces cerevisiae strain JN-BYXXEMO

Inoculating the recombinant saccharomyces cerevisiae JN-BYXXEMO seed liquid prepared in the step (2) into a 500mL conical flask containing 100mL of xylose culture medium (YNB-X3A culture medium) according to the inoculation amount of 1% (v/v), carrying out shake flask culture at 28 ℃, and simultaneously tracking and determining the OD of the strain₆₀₀The value is obtained. The results are shown in table 5 and fig. 5. Meanwhile, according to the steps, the starting host saccharomyces cerevisiae BY4743 is used as a reference, and the OD of the strain is tracked and determined₆₀₀The results show that the OD of the strain could not be determined₆₀₀Value, it can be seen that the starting host Saccharomyces cerevisiae BY4743 cannot grow on xylose medium.

Table 5: OD of recombinant saccharomyces cerevisiae JN-BYXXEMO in different culture times₆₀₀Value of

The results show that the growth of the recombinant Saccharomyces cerevisiae JN-BYXXEMO strain in a xylose medium is better than that of the recombinant Saccharomyces cerevisiae strain in a glucose medium before modificationGrowth conditions (96 h OD culture)₆₀₀And 20) is achieved, the OD value of the recombinant saccharomyces cerevisiae JN-BYXXEMO strain in the stationary phase (cultured for 55h) is close to 20.

(4) Preparation of gadusol

Inoculating the recombinant saccharomyces cerevisiae JN-BYXXEMO seed liquid prepared in the step (2) into a 500mL conical flask containing 100mL of xylose culture medium (YNB-X3A culture medium) according to the inoculation amount of 1% (v/v), carrying out shake flask culture at 28 ℃, after 8 days of culture, preparing recombinant saccharomyces cerevisiae JN-BYXXEMO fermentation liquid, collecting the recombinant saccharomyces cerevisiae JN-BYXXEMO fermentation liquid into a 50mL centrifuge tube, centrifuging at the rotating speed of 6000 Xg, and discarding the supernatant;

resuspending the thallus with pure methanol, crushing Saccharomyces cerevisiae cells with an ultrasonic crusher, centrifuging at 10000 Xg, and taking the supernatant; saccharomyces cerevisiae cells were obtained by centrifugation and resuspended using pure methanol as solvent to extract gadusol from the Saccharomyces cerevisiae cells. Evaporating the solvent methanol in the supernatant to dryness by using a rotary evaporator to prepare a concentrated extract;

the concentrated extract was diluted with 10mL of ultrapure water (ddH)₂O), collecting the mixture into a centrifuge tube, freezing the centrifuge tube in a refrigerator at-80 ℃ for 12h, freeze-drying the centrifuge tube, re-dissolving the freeze-dried sample into 5mmol/L PBS solution, and performing detection analysis by High Performance Liquid Chromatography (HPLC) (shown in figure 8).

The product was identified as gadusol by HPLC analysis, and the preparation of gadusol using xylose as a substrate was achieved using a recombinant Saccharomyces cerevisiae strain JN-BYXXEMO simultaneously expressing yeast xylose genes (XR, XDH) and gadusol synthetic genes (EEVS, MT-Ox), and the production of gadusol by this strain reached 22.5mg/L (about 7.5mg/g dry cell weight) under the same conditions.

In conclusion, the results show that the biomass of the saccharomyces cerevisiae can be obviously improved in the same time by using xylose instead of glucose as a carbon source, so that the yield of the gadusol is improved. Thus, it was demonstrated that xylose can replace glucose as a highly efficient carbon source for the production of gadusol.

Example 4: construction of recombinant saccharomyces cerevisiae JN-BYXXXAMO and preparation of gadusol by fermentation of recombinant saccharomyces cerevisiae JN-BYXXXAMO

(1) Construction of Saccharomyces cerevisiae Strain JN-BYXXXAMO

Primers (shown in Table 1) are designed according to sequences of xylose genes (XR, XDH and XK), a target gene XR (nucleotide sequence is shown in SEQ ID NO. 3), XDH (nucleotide sequence is shown in SEQ ID NO. 4) and XK (nucleotide sequence is shown in SEQ ID NO. 5) are obtained through PCR, and the target gene is subjected to Gibson Assembly with a vector pY15TEF1 to construct pY15TEF1-XR-XDH-XK plasmid.

And (2) introducing the constructed plasmid pY15TEF1-XR-XDH-XK into the saccharomyces cerevisiae strain JN-BYEMO prepared in the step (1) of the example 1 for expression, and finally obtaining the recombinant saccharomyces cerevisiae strain JN-BYXXEMO.

Colony PCR primers are designed according to the target gene sequence (shown in Table 3), and colony PCR verification is carried out on the saccharomyces cerevisiae JN-BYXXXAMO obtained in the step (1), and the result shows that the saccharomyces cerevisiae JN-BYXXXAMO obtained in the step (1) contains yeast xylose genes (XR, XDH and XK) (shown in figure 12).

(2) Activation of recombinant Saccharomyces cerevisiae Strain JN-BYXXXAMO

The constructed saccharomyces cerevisiae strain JN-BYXXXEMO for producing the natural sunscreen gadusol is inoculated in YNB-X3A liquid culture medium and cultured for 24h at the temperature of 28 ℃.

And preparing the recombinant saccharomyces cerevisiae JN-BYXXXAMO seed liquid.

(3) Fermentation of recombinant Saccharomyces cerevisiae Strain JN-BYXXXAMO

Respectively inoculating the recombinant saccharomyces cerevisiae JN-BYXXXAMO seed liquid prepared in the step (2) into a xylose (YNB-X3A) liquid culture medium according to the inoculation amount of 1% (v/v), performing shake flask culture at the temperature of 28 ℃, and simultaneously tracking and determining the OD of the strain₆₀₀The value is obtained. The fermentation time was 8 days, and the results are shown in Table 6 and FIG. 5.

Table 6: OD of recombinant saccharomyces cerevisiae JN-BYXXXAMO in different culture times₆₀₀Value of

Time (h)	JN-BYXXXEMO/OD ₆₀₀
		0	0.010
11	0.048
		24	3.207
29	5.427
		36	12.133
58	22.817
		96	23.967
120	21.967
		144	19.833

The result shows that the modified saccharomyces cerevisiae engineering bacteria JN-BYXXXAMO containing the xylose genes (XR, XDH and XK) can grow by using xylose, and the OD value is close to 20 in the stationary phase (cultured for 55 h).

(4) Preparation of gadusol

Respectively inoculating the recombinant saccharomyces cerevisiae JN-BYXXXAMO seed liquid prepared in the step (2) into a xylose (YNB-X3A) liquid culture medium according to the inoculation amount of 1% (v/v), performing shake flask culture at 28 ℃, culturing for 8 days to obtain recombinant saccharomyces cerevisiae JN-BYXXXAMO fermentation liquid, collecting the recombinant saccharomyces cerevisiae JN-BYXXXAMO fermentation liquid into a 50mL centrifuge tube, centrifuging at the rotating speed of 6000 Xg, and discarding the supernatant;

respectively using pure methanol to resuspend the thalli, crushing saccharomyces cerevisiae cells by using an ultrasonic crusher, centrifuging at 10000 Xg, and taking supernatant; saccharomyces cerevisiae cells were obtained by centrifugation and resuspended using pure methanol as solvent to extract gadusol from the Saccharomyces cerevisiae cells. Evaporating the solvent methanol in the supernatant to dryness by using a rotary evaporator, and respectively preparing concentrated extracts;

the concentrated extracts were each treated with 10mL of ultrapure water (ddH)₂O), collecting the mixture into a centrifuge tube, freezing the centrifuge tube in a refrigerator at-80 ℃ for 12h, freeze-drying the centrifuge tube, re-dissolving the freeze-dried sample into 5mmol/L PBS solution, and performing detection analysis by High Performance Liquid Chromatography (HPLC) respectively (shown in figure 9).

The results showed that the content of gadusol produced after the culture of recombinant Saccharomyces cerevisiae JN-BYXXXAMO reached 40.5mg/L (about 13.5mg/g dry cell weight);

the yield of the genetically engineered bacterium JN-BYXXEMO into which the XK gene has not been introduced is 22.5mg/L (the result of example 3);

the yield of gadusol produced by recombinant Saccharomyces cerevisiae JN-BYEMO was only 4mg/L (results from example 2);

the result shows that the supply of S7P is effectively improved after the XK gene is introduced, the yield of gadusol is further improved, and the yield of the gadusol is 10 times of that of a strain (recombinant saccharomyces cerevisiae JN-BYE) which can only produce gadusol by using glucose.

Meanwhile, the biomass of the yeast is greatly improved BY introducing the gadusol production way into the saccharomyces cerevisiae BY4743, and the OD value 2 in the stationary phase is increased to 20 and is increased BY 10 times.

Finally, the saccharomyces cerevisiae JN-BYXXXAMO capable of efficiently utilizing xylose to produce the natural sunscreen agent gadusol is obtained, and the gadusol with high yield can be accumulated in a xylose culture medium.

Meanwhile, the results show that, as the gadusol is mainly produced in the stable period of yeast growth, organisms are accumulated by using a carbon source which can be quickly utilized by the yeast, such as glucose, and meanwhile, the precursor substance for producing the gadusol is provided by using xylose, so that the fermentation period can be further shortened, and the production efficiency can be improved. The biomass accumulation is carried out by adopting glucose, and the stable period can be reached within 28 hours, which is about 20 hours earlier than the stable period reached by pure xylose culture medium within 48 hours.

Comparative example 1:

the specific implementation mode is the same as that of example 1, except that the starting strain saccharomyces cerevisiae BY4743 is replaced BY saccharomyces cerevisiae W303, namely: transforming the plasmid pYEP352-EEVS-MT-Ox into saccharomyces cerevisiae W303 to construct a recombinant saccharomyces cerevisiae strain WYEMO for producing gadusol; gadusol was prepared as in example 2.

As a result, when the original host was replaced with Saccharomyces cerevisiae W303 and the recombinant strain thus prepared was used to produce gadusol on a glucose fermentation medium, the OD value was only 4 (as shown in FIG. 11), and it was found that the amount of biomass accumulated in the same period of time was too small and the production cost was increased when Saccharomyces cerevisiae W303 was used without the host.

Comparative example 2:

the specific implementation mode is the same as that of example 1, except that the vector is replaced BY YIP5 plasmid to construct YIP5-EEVS-MT-Ox, and the recombinant plasmid YIP5-EEVS-MT-Ox is transformed into Saccharomyces cerevisiae BY4743 to construct a recombinant Saccharomyces cerevisiae strain BYYEMO capable of producing gadusol; gadusol was prepared as in example 2.

The results show that when the original vector is replaced by YIP5 plasmid, the recombinant strain prepared has the following yield when the gadusol is produced on a glucose fermentation medium: 2mg/L (about 0.67mg/g dry cell weight).

Comparative example 3:

the specific embodiment is the same as example 4 except that the xylulokinase gene XK (nucleotide sequence shown in SEQ ID NO. 5) is replaced with the xylulokinase gene XK of nucleotide sequence shown in SEQ ID NO. 6; recombinant Saccharomyces cerevisiae strain JN-BYXXEMO-2, gadusol was prepared according to the method of example 4.

The results show that, when replacing xylulokinase, the recombinant strain prepared was used for the production of gadusol on xylose fermentation medium, with only the following yields: 32.8mg/L (about 10.93mg/g dry cell weight).

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university in south of the Yangtze river

<120> saccharomyces cerevisiae with sun-screening strengthening effect and application thereof

<130> BAA211505A

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 1413

<212> DNA

<213> Artificial sequence

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gttcatagag acaattctac tatggagaat cataataaac aagagactgt tttttctttg 120

gttcaagtta aaggtacttg gaagaggaaa gctggtcaaa atgctaaaca aggtatgaaa 180

ggtagagttt ctcctgctaa aatttatgaa tcttcatctt catctggtac tacttggaca 240

gttgttactc caattacttt tacttatact gttactcaaa caaagaattt gttggatcca 300

tctaatgata ctttgttatt gggtcatatt attgatactc aacaattgga ggctgttaga 360

tcaaatacta aaccattgaa aaggttcatc gtcatggacg aagttgttta taatatttat 420

ggttctcaag ttactgaata tttggaagct agaaatgttt tgtatagaat tttgccatta 480

ccaactacag aagaaaataa atctatggat atggctttga aaattttgga agaagttcat 540

caatttggta ttgatagaag aactgaacca attattgcta ttggtggagg tgtttgtttg 600

gatattgttg gtttggctgc ttctttgtat agaaggagaa ctccatatat tagagttcca 660

actactttgt tgtcttatat tgatgcttct gttggtgcta aaactggtgt taattttgct 720

aattgtaaaa ataaattggg tacttatatt gctcctgttg ctgctttttt ggatagatct 780

tttattcaat ctattccaag aagacatatt gctaatggtt tggctgaaat gttgaaaatg 840

gctttgatga aacatagagg tttgtttgaa ttgttggaag ttcatggtca atttttgttg 900

gattctaaat ttcaatctgc ttctgttttg gaaaatgata gaattgatcc tgcttctgtt 960

tctactagag ttgctattga aactatgttg gaagaattgg ctccaaattt gtgggaagat 1020

gatttggata gattggttga ttttggtcat ttgatttctc cacaattgga aatgaaagtt 1080

ttgcctgctt tgttgcatgg tgaagctgtt aatattgata tggcttatat ggtctatgtc 1140

tcttgtgaaa ttggtttatt gacagaggaa gaaaaattta gaattatttg ttgtatgatg 1200

ggtttggaat tgcctgtttg gcatcaagat tttacttttg ctttggttca aaaatctttg 1260

tgtgatagat tgcaacattc tggtggtttg gttagaatgc cattgccaac tggtttgggt 1320

agagctgaaa tttttaatga tactgatgaa ggttctttgt ttagagctta tgaaaaatgg 1380

tgtgatgaat tgtctactgg ttctccacaa taa 1413

<210> 2

<211> 1656

<212> DNA

<213> Artificial sequence

<400> 2

atgcaaactg ctaaagtttc tgatactcct gttgagttca tcgttgaaca tttgttgaaa 60

gctaaagaaa ttgctgaaaa tcatgcttct attcctgttg aattgagaga taatttgcaa 120

aaagctttgg atattgcttc tggtttggat gaatatttgg aacaaatgtc ttctaaagaa 180

tctgaaccat tgactgaatt gtatagaaaa tctgtttctc atgattggaa taaagttcat 240

gctgatggta aaactttgtt tagattgcct gttacttgta ttactggtca agttgaaggt 300

caagttttga aaatgttggt tcatatgtct aaagctaaaa gagttttgga aattggtatg 360

tttactggtt atggtgcttt gtctatggct gaagctttgc ctgaaaatgg tcaattgatt 420

gcttgtgaat tggaaccata tttaaaagat tttgctcaac caatttttga taaatctcca 480

catggtaaaa aaattactgt taaaactggt cctgctatgg atactttgaa agaattggct 540

gctactggtg aacagttcga catggtcttt attgatgctg acaaacaaaa ctacattaat 600

tactataaat ttttgttaga tcataatttg ttgagaattg atggtgtcat ttgcgtcgat 660

aatactttgt ttaaaggtag agtttattta aaagattctg ttgatgaaat gggtaaagct 720

ttgagagatt ttaatcaatt tgttactgct gatccaagag ttgaacaagt tattattcca 780

ttgagagatg gtttgactat tattagaaga gttccatata ctccacaacc aaattctcaa 840

tctggtactg ttacttatga tgaagttttt agaggtgttc aaggtaaacc tgttttggat 900

agattgagat tggatggtaa agttgcttat gttactggtg ctggtcaagg tattggtaga 960

gcttttgctc atgctttggg tgaagctggt gctaaagttg ctattattga tatggataga 1020

ggtaaagctg aagatgttgc tcatgaattg actttgaaag gtatttcttc tatggctgtt 1080

gttgctgata tctcaaaacc tgatgatgtt cagaagatga tcgatgatat tgttactaaa 1140

tggggtactt tgcatattgc ttgtaataat gctggtatta ataaaaattc tgcttctgaa 1200

gaaacttctt tggaagaatg ggatcaaact tttaatgtta atttgagagg tacttttatg 1260

tgttgtcaag ctgctggtag agttatgttg aaacaaggtt atggtaaaat tattaatact 1320

gcttctatgg cttctttgat tgttccacat ccacaaaaac aattgtctta taatacttct 1380

aaagctggtg ttgttaaatt gactcaaact ttgggtactg aatggattga tagaggtgtt 1440

agagttaatt gtatttctcc tggtattgtt gatactccat tgattcattc tgaatctttg 1500

gaaccattgg ttcaaagatg gttgtctgat attcctgctg gtagattggc tcaagttact 1560

gatttgcaag ctgctgttgt ttatttggct tctgatgctt ctgattatat gactggtcat 1620

aatttggtta ttgaaggtgg tcaatctttg tggtaa 1656

<210> 3

<211> 957

<212> DNA

<213> Artificial sequence

<400> 3

ttaaacaaaa attggaattt tatcccaatc ccatggatca ttaaatctca aattaatatc 60

caatttagca atatcagcaa aatcttgttc atccaaatcg aaagaattga catccttgtt 120

ttccaacaat cttggaacag tattagattt tgggataata gcgatacctc tttgagaaga 180

ccatctcaac aaaacttgag caggagattt accatgttta gcagcaatag ctttaatagt 240

ttcattttca aacaatggag aagtattcaa agctctacct tgattcaatt caacaaaaga 300

ttgtggacca aaagaagaat aagcagtaac agcaataccc ctagattgag caaattcaat 360

caatcttggt tgttgcaaat atggatgatg ttcgacttgc aaaacagatg gtttaatagt 420

agcacctctc aacaaatcca ataacaaagc accaggaaaa ttagaaacac caatagatct 480

aattttacca gctttgacca atttctccaa agctttccaa gtttccaaaa ttggaacatc 540

ttcataatca aaattatcac ctttaccaca ataaaaacca ggtggatatt tttcttccaa 600

tggaacaaat ttgaaagtaa caggaaaatg gatcaaaaac aaatcgacat aatcgacttg 660

caaatcagac aatgttctat tcaaagcttt ttcaacatta tcaggatgat ggtagttatt 720

ccacaattta gaagtcaaaa acaagtcttc ccttttaacg ataccctcgt caatagcttt 780

tttgacacca gctccaacca atttttcatt agcataatct tcagcaccat caaacaatct 840

ataaccagtt ttaatagctc tataaatttg ttctgagcaa gtatcaacat caactttcca 900

acaaccaaaa ccaacagcag gcatatcata accagaattc aatttaatag atggcat 957

<210> 4

<211> 1092

<212> DNA

<213> Artificial sequence

<400> 4

ttattcagga ccatcaatca aacatttaac agcaccttta ccagctctaa ccaaatcata 60

agcttcaata gcatccttga atttatatct atgagtaatt aattgctcaa agtcaattgg 120

agcgttctct ctaccatttt gataatttgt atcaaaaata ccgacagctg tcttataatc 180

attaaaacca tacctaaaag aaccaaacaa agtcaattcc ttcatagcaa agacagtaat 240

agggaaagaa acaggaccag cagcattacc gacttgaacg aatctaccac caggagcaat 300

agcatcaaca cccaatttaa tacatggttc agcaccagta cattccaaaa caacatttgg 360

aacattacca ccaaaagctt taatcaattc ttcagaacca ccagttttag aattaaaagt 420

atgagtagca gcaccaatat ctttagccat tttcaattta ttgtcaaaaa tgtcaacaac 480

aataacaccc ttagcaccaa aagttttagc aacagctgca gccaacaatc caacaggtcc 540

agcaccaaaa acagcaacat aatcaccaaa agcaacagaa cccaatttag aagcatgaac 600

accaacagac aatggttcaa ccaaagcacc caattccaaa gaaacatgat caggcaattt 660

aaccaaaaaa tcttcaggag atttaaaata tttacacaaa gtaccaggtg gatttggttc 720

accttcttta gaatttggag tagcagcaaa agccatatgt ggacacaaat tataatgacc 780

agatttatat tcatcagaaa atctagatgg aataccaggt tcaatagcaa cattatcacc 840

aactttcaaa gaagtaacac ccttaccaac ttgaacaaca gtaccagcag attcatgacc 900

caaaaccatt ggtttagtca aaacaaaatt accaattcta ccatgagcat aaaaatgaat 960

atcagaacca caaataccag tttttttaac ttgaaccaaa acatcagttg gttcagaaat 1020

ttcaggagca tcataagttt caaaagagat atcgtcaatt ttatttaaaa ctaaagaagg 1080

attagcagtc at 1092

<210> 5

<211> 1872

<212> DNA

<213> Artificial sequence

<400> 5

ttaatgtttc aattcagatt ccattttagc caacatacca acaccattag catattccaa 60

ccatttatct ttaacctcga acaaattcat ttcatcagag acttcaaata atctattaat 120

gtattgatca taaccgatcc attccttttt agcctcgcac tcataagacc aagaagcttt 180

ataagcacca cccaaagcac aagcatttgg aatatcaact ttataattac cattaacagg 240

agccaaaata gaacccattt ttctaataat agaaccatta ttagaagcac caccaacata 300

ataacatcta tttggtctag cagtcaaaga ttcaaaagtt tgttttttac catctgtata 360

caaatcacca aattttttaa ctaaatcttg atagacttta tgcaaatcag taccatctcc 420

ttcaggttga ggagatgcag atgaagatgc tgaagaatca ccagatttag acaacattgg 480

accagttctc aatctacaag acaaagtttg agattcaaca atagaagaaa catcatcttc 540

aggttgccaa tttttatcac ccaattcaac atcaacaatt tcattctttg agtttaaaac 600

agatctttta atttgagctg cagcgtttgg aacaatttca cccaatggaa aataaatacc 660

caatttatta ttaaagtcag ttgatttatc taaaatttca ttgaacttat cccaagattt 720

tttatcttca acgttaaact tttcattaac ttcatctcta actttctctc tagccaaaga 780

accattacaa taacaaatca tacccatata atgatcaggc atagttggat gtttaaacaa 840

atgatattga gaagatggtg cataattttt agtaatgatc aaaactgtag ttgatgtacc 900

caatgaaatt aatgcgtcat ttggagccaa tggcaaagaa ataatagtag ccaaattatc 960

accagtgaaa gaatagattt tacaatcagg gttaaatcca tatctagtga caaagtaaga 1020

agcaatatca ccttcagatt cataagtaat tggtttaaca ggacccaatt ttcttttcaa 1080

ttcattaata ccagctctat aaatttcacc atcttgttca acaccatcca attcaggatg 1140

aacaccagca gcaatagcca acaattcttc attaaattct cttttttcaa tatcatacaa 1200

attcatacca caagcatcag cttcttcaat agaagtaatt ctacccaaca aaacagaagc 1260

aacaaaagaa gaaaccaaag aaattctagc agttctattg tatttttcag gtttaaacct 1320

agtagataat tttctaattt gtaatccagt aaatctataa tgagccctag aaccagaaat 1380

atcagccaaa gcatcagcac caataactct ttcaaattct tccaattctt taccagtaga 1440

atgatcttgc caatttggag catgtttaaa agtaaaagca gatctcattt gagaagacaa 1500

agaagattca gcatccaatt cagacaaaac tttttcagca gttctagacc aataaacaga 1560

accatgttgt tgacaagatc cagaaatacc aacaacttta ttgaatggaa aaccatcttt 1620

tttcatatct tcgaaaacat gatccaaagc gtccaaccac atataaacag gagaaataat 1680

agcaccttta gaaatttcat cattgatagc gatgacacct ttttggactg aagaattaat 1740

agaatcaaat tcgacattat aagttttcaa agcagccaaa ttttcatctg tgacgataat 1800

tttcaattgt tgagtagaca aatcaaaacc caagaacaac ttatcaggag catcaaatgg 1860

agttgtagtc at 1872

<210> 6

<211> 2202

<212> DNA

<213> Artificial sequence

<400> 6

atagatccct ggaggatacc cacagacatt actgctacta attcatacca tacttgacgt 60

atatctgcgc atacatatct accccaactt tcatataaaa ttcctagatt tattgcatct 120

tctaatagag tcatttttca gatttttcaa tttccataga aagcatacat tttcatacag 180

cttctatttg ttaatcgacc tgataatttt actagccata tttctttttt tgatttttca 240

cttaatcgac atataaatac tcacgtagtt gacactcaca atgaccacta ccccatttga 300

tgctccagat aagctcttcc tcgggttcga tctttcgact cagcagttga agatcatcgt 360

caccgatgaa aacctcgctg ctctcaaaac ctacaatgtc gagttcgata gcatcaacag 420

ctctgtccag aagggtgtca ttgctatcaa cgacgaaatc agcaagggtg ccattatttc 480

ccccgtttac atgtggttgg atgcccttga ccatgttttt gaagacatga agaaggacgg 540

attccccttc aacaaggttg ttggtatttc cggttcttgt caacagcacg gttcggtata 600

ctggtctaga acggccgaga aggtcttgtc cgaattggac gctgaatctt cgttatcgag 660

ccagatgaga tctgctttca ccttcaagca cgctccaaac tggcaggatc actctaccgg 720

taaagagctt gaagagttcg aaagagtgat tggtgctgat gccttggctg atatctctgg 780

ttccagagcc cattacagat tcacagggct ccagattaga aagttgtcta ccagattcaa 840

gcccgaaaag tacaacagaa ctgctcgtat ctctttagtt tcgtcatttg ttgccagtgt 900

gttgcttggt agaatcacct ccattgaaga ggccgatgct tgtggaatga acttgtacga 960

tatcgaaaag cgcgagttca acgaagagct cttggccatc gctgctggtg tccaccctga 1020

gttggatggt gtagaacaag acggtgaaat ttacagagct ggtatcaatg agttgaagag 1080

aaagttgggt cctgtcaaac ctataacata cgaaagcgaa ggtgacattg cctcttactt 1140

tgtcaccaga tacggcttca accccgactg taaaatctac tcgttcaccg gagacaattt 1200

ggccacgatt atctcgttgc ctttggctcc aaatgatgct ttgatctcat tgggtacttc 1260

tactacagtt ttaattatca ccaagaacta cgctccttct tctcaatacc atttgtttaa 1320

acatccaacc atgcctgacc actacatggg catgatctgc tactgtaacg gttccttggc 1380

cagagaaaag gttagagacg aagtcaacga aaagttcaat gtagaagaca agaagtcgtg 1440

ggacaagttc aatgaaatct tggacaaatc cacagacttc aacaacaagt tgggtattta 1500

cttcccactt ggcgaaattg tccctaatgc cgctgctcag atcaagagat cggtgttgaa 1560

cagcaagaac gaaattgtag acgttgagtt gggcgacaag aactggcaac ctgaagatga 1620

tgtttcttca attgtagaat cacagacttt gtcttgtaga ttgagaactg gtccaatgtt 1680

gagcaagagt ggagattctt ctgcttccag ctctgcctca cctcaaccag aaggtgatgg 1740

tacagatttg cacaaggtct accaagactt ggttaaaaag tttggtgact tgtacactga 1800

tggaaagaag caaacctttg agtctttgac cgccagacct aaccgttgtt actacgtcgg 1860

tggtgcttcc aacaacggca gcattatccg caagatgggt tccatcttgg ctcccgtcaa 1920

cggaaactac aaggttgaca ttcctaacgc ctgtgcattg ggtggtgctt acaaggccag 1980

ttggagttac gagtgtgaag ccaagaagga atggatcgga tacgatcagt atatcaacag 2040

attgtttgaa gtaagtgacg agatgaatct gttcgaagtc aaggataaat ggctcgaata 2100

tgccaacggg gttggaatgt tggccaagat ggaaagtgaa ttgaaacact aaaatccata 2160

atagcttgta tagaggtata gaaaaagaga acgttataga gt 2202

Claims

1. A recombinant Saccharomyces cerevisiae is characterized in that the recombinant Saccharomyces cerevisiae simultaneously expresses EEVS with a nucleotide sequence shown in SEQ ID No.1, MT-Ox with a nucleotide sequence shown in SEQ ID No.2, xylose reductase XR with a nucleotide sequence shown in SEQ ID No.3, xylitol dehydrogenase XDH with a nucleotide sequence shown in SEQ ID No.4 and xylulokinase XK with a nucleotide sequence shown in SEQ ID No. 5.

2. The recombinant Saccharomyces cerevisiae according to claim 1, wherein the recombinant Saccharomyces cerevisiae has Saccharomyces cerevisiae BY4743 as an expression host.

3. The recombinant Saccharomyces cerevisiae yeast according to claim 1 or 2, wherein EEVS and MT-Ox are expressed using pYEP352 plasmid and xylose reductase XR, xylitol dehydrogenase XDH and xylulokinase XK are expressed using pY15TEF1 plasmid.

4. A method for preparing gadusol at low cost is characterized in that the recombinant Saccharomyces cerevisiae as claimed in any of claims 1-3 is added into a reaction system containing xylose for fermentation to prepare gadusol.

5. The method of claim 4, wherein the concentration of xylose in the reaction system is at least: 20 g/L.

6. A method for improving biomass of recombinant saccharomyces cerevisiae in a xylose culture medium is characterized in that EEVS with a nucleotide sequence shown in SEQ ID No.1, MT-Ox with a nucleotide sequence shown in SEQ ID No.2, xylose reductase XR with a nucleotide sequence shown in SEQ ID No.3, xylitol dehydrogenase XDH with a nucleotide sequence shown in SEQ ID No.4 and xylulokinase XK with a nucleotide sequence shown in SEQ ID No.5 are expressed in the saccharomyces cerevisiae at the same time.

7. The method of claim 6, wherein the recombinant Saccharomyces cerevisiae has Saccharomyces cerevisiae BY4743 as an expression host.

8. The method of claim 6 or 7, wherein EEVS and MT-Ox are expressed using pYEP352 plasmid and xylose reductase XR, xylitol dehydrogenase XDH and xylulokinase XK are expressed using pY15TEF1 plasmid.

9. A method for improving the yield of gadusol produced by recombinant Saccharomyces cerevisiae is characterized in that EEVS with a nucleotide sequence shown in SEQ ID NO.1, MT-Ox with a nucleotide sequence shown in SEQ ID NO.2, xylose reductase XR with a nucleotide sequence shown in SEQ ID NO.3, xylitol dehydrogenase XDH with a nucleotide sequence shown in SEQ ID NO.4 and xylulokinase XK with a nucleotide sequence shown in SEQ ID NO.5 are simultaneously expressed in the Saccharomyces cerevisiae.

10. Use of the recombinant Saccharomyces cerevisiae as claimed in any of claims 1 to 3 for the preparation of products containing gadusol.