CN114621883B - Saccharomyces cerevisiae with enhanced sun-screening effect and application thereof - Google Patents

Saccharomyces cerevisiae with enhanced sun-screening effect and application thereof Download PDF

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CN114621883B
CN114621883B CN202210118631.8A CN202210118631A CN114621883B CN 114621883 B CN114621883 B CN 114621883B CN 202210118631 A CN202210118631 A CN 202210118631A CN 114621883 B CN114621883 B CN 114621883B
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saccharomyces cerevisiae
gadusol
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nucleotide sequence
xylose
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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
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Jiangnan University
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Abstract

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

Description

Saccharomyces cerevisiae with enhanced sun-screening effect and application thereof
Technical Field
The invention relates to saccharomyces cerevisiae with a strengthened sun-screening effect and application thereof, and belongs to the field of bioengineering.
Background
Coral reefs are oasis in deserts and provide habitats for a large number of marine organisms. Whitening of coral reefs results in the extinction of a large number of fish and marine organisms, which in turn leads to breakdown of the marine ecosystem. In seawater, low concentrations of chemical sunscreens are sufficient to cause a range of coral diseases and whitening of the coral. Among them, octocrylene, oxybenzone, octyl methoxycinnamate and their similar derivatives are mainly mentioned. About 14,000 tons of sunscreen cream are flushed and stocked in the ocean each year, wherein about 10% of the cream is a sunscreen component such as oxybenzone, and far superconductivity causes the lowest concentration of coral whitening, so that about 15% of coral reefs worldwide and about 40% along the coast are deadly threatened by chemical sunscreens. Because the growing development of corals has extremely high requirements on the environment, scientists predict that the existing coral reefs will be extinct after 50 years. The produced coral and marine environment-friendly biological sun-screening agent can effectively slow down the current tendency of large-area whitening of coral and realize the purpose of protecting the coral. Most of the natural sunscreen ingredients currently on the market, such as sulforaphane, ferulic acid, carotenoids, tetrahydrolycopene, etc., are all of vegetable origin. Many plant-derived substances are not present in the ocean at all, and it is difficult to explain whether or not they affect coral and the ocean environment. Gadusol is a natural sunscreen agent which naturally occurs in a variety of fish eggs such as zebra fish, salmon, sturgeon, and coral reef ecosystems and protects marine organisms from ultraviolet light. It has more reliable safety than natural sun protection ingredients of vegetable origin.
However, the natural yield of gadusol is very low, and only 10-20 mg can be extracted from 1000 roes, which not only limits the large-scale application, but also makes the way of collecting the roe extract product quite unsatisfactory. Therefore, the use of Saccharomyces cerevisiae engineering bacteria to produce gadusol is a viable and relatively low cost industrial approach.
Yeast is an important industrial production strain and has the advantages of high fermentation speed, short growth period, large-scale cultivation and the like. The yeast is used for producing the environment-friendly biological sun-screening agent to replace the chemical sun-screening agent, so that the environmental protection and economic benefit are achieved. In addition, the use of glucose as a carbon source for yeast culture fermentation, its source and food production conflict. 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 can be introduced into yeast cells to widen the carbon source for producing gadusol by the yeast and further reduce the cost.
Disclosure of Invention
The invention uses saccharomyces cerevisiae to produce natural sun-screening component gadosol, the EEVS gene and MT-Ox gene are cloned into high copy plasmid pYEP352 BY carrying out codon optimization, finally, the pYEP352 vector containing the EEVS gene and the MT-Ox gene is transferred into saccharomyces cerevisiae BY4743 strain, and the capability of utilizing xylose is obtained BY introducing Xylulokinase (XK) or xylose reductase/xylitol dehydrogenase (XR/XDH), so that the saccharomyces cerevisiae JN-BYXXXEMO capable of preparing gadosol with low cost is finally obtained.
The invention provides a recombinant saccharomyces cerevisiae JN-BYXXXEMO, which simultaneously expresses EEVS with a nucleotide sequence shown as SEQ ID NO.1, MT-Ox with a nucleotide sequence shown as SEQ ID NO.2, xylose reductase XR with a nucleotide sequence shown as SEQ ID NO.3, xylitol dehydrogenase XDH with a nucleotide sequence shown as SEQ ID NO.4 and xylulokinase XK with a nucleotide sequence shown as SEQ ID NO. 5.
In one embodiment of the invention, the EEVS is derived from zebra fish (NCBI serial No. xm_ 001343386.7); the MT-Ox is derived from zebra fish (NCBI sequence No. NM-001013450.1); the xylose reductase XR is derived from Scheffersomyces stipites (NCBI serial number HM 769331.1); the xylitol dehydrogenase XDH is derived from Scheffersomyces stipites (NCBI serial number HM 769332.1); the xylulokinase XK is derived from Scheffersomyces stipites (NCBI sequence No. XM_ 001387288.1). All gene fragments were synthesized again after codon optimization for the Saccharomyces cerevisiae expression system (Jin Weizhi, suzhou, china).
In one embodiment of the invention, the recombinant Saccharomyces cerevisiae JN-BYXXXEMO uses Saccharomyces cerevisiae BY4743 as an expression host.
In one embodiment of the invention, the recombinant Saccharomyces cerevisiae JN-BYXXXEMO expresses EEVS and MT-Ox using pYEP352 plasmid and expresses xylose reductase XR, xylitol dehydrogenase XDH and xylulokinase XK using pY15TEF1 plasmid.
In one embodiment of the invention, the method for culturing the saccharomyces cerevisiae JN-BYXXXEMO is to inoculate the saccharomyces cerevisiae into a culture medium containing glucose and xylose for culturing;
the culture medium is as follows: YNB-X3A culture medium, natural pH, distilled water, and sterilizing at 115deg.C for 15min.
The YNB-X3A medium: YNB medium 6.7g/L, xylose 20g/L, (NH) 4 ) 2 SO 4 10g/L,30mg/L histidine, 30mg/L lysine, 30mg/L methionine.
The Saccharomyces cerevisiae JN-BYXXXEMO provided by the invention can utilize glucose and xylose to produce gadusol, and when xylose is used as a carbon source, the intracellular gadusol content is 10 times that of a production strain which can only use glucose as the carbon source, and the metabolite of the strain has the capability of absorbing ultraviolet rays.
The invention also provides a method for preparing the gadusol at low cost, which is to add the recombinant saccharomyces cerevisiae JN-BYXXXEMO into a reaction system containing xylose for fermentation to prepare the gadusol.
In one embodiment of the invention, the concentration of xylose in the reaction system is at least: 20g/L.
In one embodiment of the invention, the amount of recombinant s.cerevisiae JN-BYXXXEMO added in the reaction system is at least: 1% (v/v).
In one embodiment of the invention, the fermentation conditions are: shaking culture at 28 deg.c at 200r/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 nucleotide sequence shown as SEQ ID NO.1 and MT-Ox with nucleotide sequence shown as SEQ ID NO.2 are simultaneously expressed in Saccharomyces cerevisiae.
In one embodiment of the invention, the recombinant Saccharomyces cerevisiae uses Saccharomyces cerevisiae BY4743 as the expression host.
In one embodiment of the invention, the recombinant Saccharomyces cerevisiae expresses EEVS and MT-Ox using the pYEP352 plasmid and expresses xylose reductase XR, xylitol dehydrogenase XDH and xylulokinase XK using the pY15TEF1 plasmid.
The invention also provides a method for improving the yield of the production of the gadusol by the recombinant saccharomyces cerevisiae, which simultaneously expresses EEVS with a nucleotide sequence shown as SEQ ID NO.1, MT-Ox with a nucleotide sequence shown as SEQ ID NO.2, xylose reductase XR with a nucleotide sequence shown as SEQ ID NO.3, xylitol dehydrogenase XDH with a nucleotide sequence shown as SEQ ID NO.4 and xylulokinase XK with a nucleotide sequence shown as SEQ ID NO.5 in the saccharomyces cerevisiae.
The invention also provides application of the recombinant saccharomyces cerevisiae in preparation of a product containing gadusol.
Advantageous effects
(1) The invention provides a recombinant saccharomyces cerevisiae which simultaneously expresses EEVS, MT-Ox and xylose reductase XR, xylitol dehydrogenase XDH and xylulokinase XK for synthesizing gadosol, thereby realizing efficient synthesis of gadosol. The gadusol prepared by the recombinant Saccharomyces cerevisiae JN-BYXXXEMO can reach 40.5mg/L.
(2) The introduction of the production route of gadosol (EEVS, MT-Ox) into BY4743 has not been reported to promote the accumulation of biomass in yeast, and the present invention has found that the introduction of the metabolism route of xylose can greatly increase the yield of gadosol in yeast and can significantly reduce the production cost.
Drawings
Fig. 1: gadusol biosynthetic pathway.
Fig. 2: a plasmid containing the gadusol synthesis pathway.
Fig. 3: a construction method of Saccharomyces cerevisiae strain producing natural sunscreen agent gadusol by xylose is provided.
Fig. 4: growth curves of Saccharomyces cerevisiae BY4743 and JN-BYSO.
Fig. 5: growth curves for JN-BYXXEMO and JN-BYXXXEMO.
Fig. 6: HPLC results of Gadusol standard.
Fig. 7: HPLC results of gadusol produced by JN-BYSO.
Fig. 8: HPLC results of gadusol produced by JN-BYXXEMO.
Fig. 9: HPLC results of gadusol produced by JN-BYXXXEMO.
Fig. 10: PCR confirmed that the gadusol production pathway (EEVS+MT-Ox) was successfully introduced into Saccharomyces cerevisiae.
Fig. 11: growth curves of W303 and WYEMO.
Fig. 12: PCR confirmed that the production route of gadosol using xylose was successfully introduced into Saccharomyces cerevisiae, where 1 is: EEVS gene, 2: the full length of the synthetic pathway gene (XK+XR+XDH+EEVS+MT-Ox).
Detailed Description
YNB medium as referred to in the examples below was purchased from Sigma Aldrich; saccharomyces cerevisiae W303 and YIP5 plasmids were stored for laboratory.
The following examples relate to the following media:
LB medium: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride.
YPD medium: yeast extract 10g/L, peptone 20g/L, glucose 20g/L.
YNB-G: YNB medium 6.7g/L, glucose 20g/L, (NH) 4 ) 2 SO 4 10g/L。
YNB-G4A: based on YNB-G medium, 30mg/L histidine, 30mg/L lysine, 30mg/L methionine and 30mg/L leucine were added.
YNB-G3A: based on YNB-G medium, 30mg/L histidine, 30mg/L lysine and 30mg/L methionine were added.
YNB-X: YNB medium 6.7g/L, xylose 20g/L, (NH) 4 ) 2 SO 4 10g/L。
YNB-X3A: based on YNB-X medium, 30mg/L histidine, 30mg/L lysine and 30mg/L methionine were added.
The detection method involved in the following examples is 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: 1% methanol is equal to 20min, and 20-40min is equal to 20min from 1% methanol to 95% methanol, 95% methanol
Flow rate: 0.3mL/min
Detection wavelength: 296nm.
The primers involved in the following examples are as follows:
TABLE 1 primers for PCR of target genes
TABLE 2 primers for linearization of plasmid vectors
TABLE 3 primers for colony PCR
Example 1: construction of recombinant Saccharomyces cerevisiae
(1) Construction of Saccharomyces cerevisiae strain JN-BYEMO:
considering the low success rate of restriction and insertion cloning, the method of Gibson Assembly is adopted to connect the linearization vector and the target gene, and the primer of the vector and the target gene is designed by utilizing Snapgene software, so that the Tm value of the homology arm is more than or equal to 55 ℃.
PCR primers (shown in Table 1) are designed according to the metabolic pathway of gadusol, the target genes EEVS (the nucleotide sequence is shown as SEQ ID NO. 1) and MT-Ox (the nucleotide sequence is shown as SEQ ID NO. 2) are obtained BY PCR, the target genes EEVS and MT-Ox are connected with a vector pYEP352 through Gibson Assembly, primers used for linearizing the plasmid vector are shown in Table 2, a plasmid pYEP352-EEVS-MT-Ox is successfully constructed, the plasmid map is shown as FIG. 2, the construction process is shown as FIG. 3 (the plasmid pYEP352 is abbreviated as plasmid YEP352 in the figure), and pYEP352-EEVS-MT-Ox is transformed into Saccharomyces cerevisiae BY4743 to construct a recombinant Saccharomyces cerevisiae strain JN-EMO for producing gadusol.
(2) Colony PCR primers are designed according to the target gene sequences (shown in table 3), and colony PCR verification is carried out on the saccharomyces cerevisiae JN-BYEMO obtained in the step (1), so that the result shows that the saccharomyces cerevisiae JN-BYEMO obtained in the step (1) contains target genes EEVS and MT-Ox (shown in fig. 10).
Example 2: production of gadusol by fermentation of recombinant Saccharomyces cerevisiae JN-BYSPO
The Gadusol biosynthetic pathway is shown in FIG. 1, and comprises the following steps:
(1) Activation of recombinant Saccharomyces cerevisiae
The Saccharomyces cerevisiae strain JN-BYEMO and the original host Saccharomyces cerevisiae BY4743 which are constructed in the example 1 and used for producing the natural sun-screening agent gadusol are respectively inoculated into YNB-G4A liquid culture medium, and are cultured for 24 hours 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
The recombinant Saccharomyces cerevisiae JN-BYEMO seed solution and Saccharomyces cerevisiae BY4743 seed solution prepared in the step (1) are inoculated into 500mL triangular flasks containing 100mL YPD medium according to an inoculum size of 1% (v/v), shake flask fermentation is carried out at 28 ℃, and growth curves are measured respectively, and the results are shown in FIG. 4 and Table 4.
Table 4: OD of Saccharomyces cerevisiae BY4743 and JN-BYSPO at different times 600 Value of
Time (h) JN-BYEMO/OD 600 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
Under the fermentation system, the OD enters a stable period (culturing for 50 h) 600 About 15 and 96h OD 600 And 20 is achieved. And Saccharomyces cerevisiae BY4743 grows on glucose medium, and OD value can only reach about 2. Therefore, EEVS and MT-Ox are expressed in the Saccharomyces cerevisiae BY4743 at the same time, so that the biomass of the Saccharomyces cerevisiae BY4743 strain in a glucose culture medium can be remarkably improved.
(3) Preparation of gadusol
Inoculating the recombinant saccharomyces cerevisiae JN-BYEMO seed solution prepared in the step (1) into a 500mL triangular flask containing 100mL YPD culture medium according to an inoculum size of 1% (v/v), fermenting for 8 days at 28 ℃ to prepare a recombinant saccharomyces cerevisiae JN-BYEMO fermentation solution, collecting the recombinant saccharomyces cerevisiae JN-BYEMO fermentation solution into a 50mL centrifuge tube, centrifuging at a rotating speed of 6000 Xg, and discarding the supernatant;
using pure methanol to resuspend thalli, crushing Saccharomyces cerevisiae cells by using an ultrasonic crusher, centrifuging 10000 Xg, and taking supernatant; using pure methanol as solvent to extract gadusol in the supernatant; evaporating the solvent methanol in the supernatant by using a rotary evaporator to obtain a concentrated extract;
the concentrated extract was treated with 10mL of ultrapure water (ddH 2 O) dissolving, collecting into a centrifuge tube, freezing in a refrigerator at-80 ℃ for 12 hours, and freeze-drying for preservation.
Since gadusol is a polar molecule, it is very soluble in water and polar organic solvents. The lyophilized sample was reconstituted in 5mmol/L PBS and subjected to detection analysis by High Performance Liquid Chromatography (HPLC) (the results are shown in FIGS. 6 to 7).
The results of the quantitative analysis showed that production of gadosol was carried out using recombinant Saccharomyces cerevisiae JN-BYSO with a yield of 4mg/L (about 1.34mg/g dry cell 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 xylose genes (XR, XDH) of yeast, target genes 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 genes were ligated with vector pY15TEF1 (Biovector Co., LTD) 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, and expressing to finally obtain the recombinant saccharomyces cerevisiae strain JN-BYXXEMO.
The recombinant Saccharomyces cerevisiae is coated on YNB-X3A flat plate and cultured in a constant temperature incubator at 28 ℃ for 1-2 days. The strain was then streaked and colony PCR verified (primers shown in Table 3), and the strain was verified to be successfully constructed, which was designated JN-BYXXEMO.
(2) Activation of recombinant Saccharomyces cerevisiae strain JN-BYXXEMO
Inoculating the constructed Saccharomyces cerevisiae strain JN-BYXXEMO producing the natural sun-screening agent gadusol into YNB-X3A liquid culture medium, and culturing for 24h at 28 ℃.
Preparing recombinant Saccharomyces cerevisiae JN-BYXXEMO seed solution.
(3) Fermentation of recombinant Saccharomyces cerevisiae strain JN-BYXXEMO
Inoculating the recombinant Saccharomyces cerevisiae JN-BYXXEMO seed solution prepared in the step (2) into 500mL conical flask containing 100mL xylose culture medium (YNB-X3A culture medium) according to an inoculum size of 1% (v/v), shake flask culturing at 28deg.C, and simultaneously tracking and measuring OD of the strain 600 Values. The results are shown in Table 5Shown in fig. 5. Meanwhile, according to the steps, the OD of the strain is tracked and measured BY taking the Saccharomyces cerevisiae BY4743 as a reference 600 The value, the result shows that the OD of the strain can not be measured 600 The values, it can be seen that the starting host Saccharomyces cerevisiae BY4743 is unable to grow in xylose medium.
Table 5: OD of recombinant Saccharomyces cerevisiae JN-BYXXEMO in different culture times 600 Value of
The results show that the growth of the recombinant Saccharomyces cerevisiae JN-BYXXEMO strain in xylose medium was superior to that in glucose medium before transformation (96 h OD culture 600 Reaching 20), the OD value of the recombinant Saccharomyces cerevisiae JN-BYXXEMO strain is close to 20 in the stationary phase (cultured for 55 h).
(4) Preparation of gadusol
Inoculating the recombinant saccharomyces cerevisiae JN-BYXXEMO seed solution prepared in the step (2) into a 500mL conical flask containing 100mL xylose culture medium (YNB-X3A culture medium) according to an inoculum size of 1% (v/v), carrying out shake flask culture at 28 ℃ for 8 days, preparing the recombinant saccharomyces cerevisiae JN-BYXXEMO fermentation solution, collecting the recombinant saccharomyces cerevisiae JN-BYXXEMO fermentation solution into a 50mL centrifuge tube, centrifuging at a rotating speed of 6000 Xg, and discarding the supernatant;
using pure methanol to resuspend thalli, crushing Saccharomyces cerevisiae cells by using an ultrasonic crusher, centrifuging 10000 Xg, and taking supernatant; using pure methanol as solvent to extract gadusol in the supernatant; evaporating the solvent methanol in the supernatant by using a rotary evaporator to obtain a concentrated extract;
the concentrated extract was treated with 10mL of ultrapure water (ddH 2 O) dissolving, collecting into centrifuge tube, freezing at-80deg.C for 12 hr, lyophilizing, redissolving the lyophilized sample in 5mmol/L PBS solution, and performing High Performance Liquid Chromatography (HPLC)Detection analysis (as shown in fig. 8).
Analysis by HPLC shows that the product is gadusol, and the recombinant Saccharomyces cerevisiae strain JN-BYXXEMO which simultaneously expresses yeast xylose genes (XR, XDH) and gadusol synthetic genes (EEVS, MT-Ox) can be used for preparing gadusol by taking xylose as a substrate, and under the same condition, the yield of the gadusol produced by the strain reaches 22.5mg/L (about 7.5mg/g dry cell weight).
In combination with the above, it was found that xylose was used as a carbon source instead of glucose, and the biomass of Saccharomyces cerevisiae could be significantly increased in the same time, thereby increasing the yield of gadusol. Thus demonstrating that xylose can replace glucose as a highly efficient carbon source for the production of gadusol.
Example 4: construction of recombinant Saccharomyces cerevisiae JN-BYXXXEMO and preparation of gadusol by fermentation of recombinant Saccharomyces cerevisiae JN-BYXXXEMO
(1) Construction of Saccharomyces cerevisiae strain JN-BYXXXEMO
Primers (shown in Table 1) were designed based on the xylose gene (XR, XDH, XK) sequence, and the target genes XR (nucleotide sequence shown in SEQ ID NO. 3), XDH (nucleotide sequence shown in SEQ ID NO. 4) and XK (nucleotide sequence shown in SEQ ID NO. 5) were obtained by PCR, and the target genes were subjected to Gibson Assembly ligation with the vector pY15TEF1 to construct pY15TEF1-XR-XDH-XK plasmids.
And (3) 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-BYXXXEMO.
Colony PCR primers were designed according to the target gene sequences (as shown in Table 3), and colony PCR verification was performed on the Saccharomyces cerevisiae JN-BYXXXEMO obtained in step (1), and the results show that the Saccharomyces cerevisiae JN-BYXXXEMO obtained in step (1) contains a yeast xylose gene (XR, XDH, XK) (as shown in FIG. 12).
(2) Activation of recombinant Saccharomyces cerevisiae strain JN-BYXXXEMO
Inoculating the constructed Saccharomyces cerevisiae strain JN-BYXXXEMO producing the natural sun-screening agent gadusol into YNB-X3A liquid culture medium, and culturing at 28deg.C for 24 hr.
Preparing recombinant Saccharomyces cerevisiae JN-BYXXXEMO seed solution.
(3) Fermentation of recombinant Saccharomyces cerevisiae strain JN-BYXXXEMO
Inoculating the recombinant Saccharomyces cerevisiae JN-BYXXXEMO seed solution prepared in the step (2) into xylose (YNB-X3A) liquid culture medium according to an inoculum size of 1% (v/v), shake flask culturing at 28deg.C, and simultaneously tracking and measuring OD of the strain 600 Values. 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-BYXXXEMO in different culture times 600 Value 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-BYXXXEMO containing xylose genes (XR, XDH, XK) can grow by utilizing xylose, and the OD value is close to 20 in the stationary phase (55 h of culture).
(4) Preparation of gadusol
Inoculating the recombinant saccharomyces cerevisiae JN-BYXXXEMO seed solution prepared in the step (2) into a xylose (YNB-X3A) liquid culture medium according to an inoculum size of 1% (v/v), carrying out shake flask culture at 28 ℃ for 8 days to obtain recombinant saccharomyces cerevisiae JN-BYXXXEMO fermentation liquor, collecting the recombinant saccharomyces cerevisiae JN-BYXXXEMO fermentation liquor into a 50mL centrifuge tube, centrifuging at a rotating speed of 6000 Xg, and discarding the supernatant;
using pure methanol to resuspend thalli, crushing Saccharomyces cerevisiae cells by using an ultrasonic crusher, centrifuging 10000 Xg, and taking supernatant; using pure methanol as solvent to extract gadusol in the supernatant; evaporating the solvent methanol in the supernatant by using a rotary evaporator to obtain a concentrated extract;
the concentrated extracts were each treated with 10mL of ultrapure water (ddH 2 O) dissolving, collecting into a centrifuge tube in a concentrated manner, freezing in a refrigerator at-80 ℃ for 12 hours, freeze-drying, re-dissolving the freeze-dried sample into 5mmol/L PBS solution, and respectively performing detection analysis by using High Performance Liquid Chromatography (HPLC) (shown in figure 9).
The result shows that after the recombinant saccharomyces cerevisiae JN-BYXXXEMO is cultured, the content of the prepared gadusol reaches 40.5mg/L (about 13.5mg/g dry cell weight);
the yield of the genetically engineered bacteria JN-BYXXEMO into which the XK gene was not introduced was 22.5mg/L (the result of example 3);
whereas the gadusol yield using recombinant Saccharomyces cerevisiae JN-BYSO was only 4mg/L (results of example 2);
the result shows that after XK gene is introduced, the S7P supply is effectively improved, the yield of gadosol is further improved, and the yield is 10 times that of a strain (recombinant Saccharomyces cerevisiae JN-BYEMO) which can only produce gadosol by using glucose.
Meanwhile, the introduction of the production path of gadusol into Saccharomyces cerevisiae BY4743 greatly improves the biomass of the yeast, and the OD value in the stationary phase is increased to 20 and 10 times.
Finally, the saccharomyces cerevisiae JN-BYXXXEMO capable of efficiently utilizing xylose to produce the natural sun-screening agent gadusol is obtained, and the gadusol with high yield can be accumulated in the xylose culture medium.
Meanwhile, the result shows that the production of the precursor substances of the gadusol is provided by utilizing the 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 stationary phase can be reached for 28 hours, which is about 20 hours earlier than that of the pure xylose culture medium for 48 hours.
Comparative example 1:
the specific embodiment is the same as in example 1, except that the original strain Saccharomyces cerevisiae BY4743 is replaced BY Saccharomyces cerevisiae W303, namely: converting plasmid pYEP352-EEVS-MT-Ox into Saccharomyces cerevisiae W303 to construct a recombinant Saccharomyces cerevisiae strain WYEMO for producing gadusol; gadusol was prepared according to the method of example 2.
The results showed that when the original host was changed to Saccharomyces cerevisiae W303, the OD value of the recombinant strain obtained was only 4 (as shown in FIG. 11) when the production of gadusol was performed on glucose fermentation medium, and it was found that biomass accumulation was too small and the production cost was increased in the same time when Saccharomyces cerevisiae W303 was not used.
Comparative example 2:
the specific embodiment is the same as example 1, except that the vector is replaced with YIP5 plasmid, YIP5-EEVS-MT-Ox is constructed, and the recombinant plasmid YIP5-EEVS-MT-Ox is transformed into Saccharomyces cerevisiae BY4743 to construct a recombinant Saccharomyces cerevisiae strain BYYEMO for producing gadusol; gadusol was prepared according to the method of example 2.
The results showed that when the original vector was replaced with YIP5 plasmid, the recombinant strain prepared was produced on glucose fermentation medium with only the following yield: 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 as SEQ ID NO. 5) is replaced with the xylulokinase gene XK having nucleotide sequence shown as SEQ ID NO. 6; recombinant Saccharomyces cerevisiae strain JN-BYXXXEMO-2, gadusol was prepared as described in example 4.
The results show that when the xylulokinase is replaced, the prepared recombinant strain is produced on a xylose fermentation medium, and the production yield is only: 32.8mg/L (about 10.93mg/g dry cell weight).
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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 of Jiangnan
<120> Saccharomyces cerevisiae with enhanced sun-screening effect and application thereof
<130> BAA211505A
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 1413
<212> DNA
<213> artificial sequence
<400> 1
atggaaagac ctggtgaaac ttttactgtt tcttctcctg aagaagttag attgccatct 60
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
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ggtttggaat tgcctgtttg gcatcaagat tttacttttg ctttggttca aaaatctttg 1260
tgtgatagat tgcaacattc tggtggtttg gttagaatgc cattgccaac tggtttgggt 1320
agagctgaaa tttttaatga tactgatgaa ggttctttgt ttagagctta tgaaaaatgg 1380
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<212> DNA
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atgcaaactg ctaaagtttc tgatactcct gttgagttca tcgttgaaca tttgttgaaa 60
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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
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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
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<212> DNA
<213> artificial sequence
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ttaaacaaaa attggaattt tatcccaatc ccatggatca ttaaatctca aattaatatc 60
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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
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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
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<212> DNA
<213> artificial sequence
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ttattcagga ccatcaatca aacatttaac agcaccttta ccagctctaa ccaaatcata 60
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agcgttctct ctaccatttt gataatttgt atcaaaaata ccgacagctg tcttataatc 180
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agggaaagaa acaggaccag cagcattacc gacttgaacg aatctaccac caggagcaat 300
agcatcaaca cccaatttaa tacatggttc agcaccagta cattccaaaa caacatttgg 360
aacattacca ccaaaagctt taatcaattc ttcagaacca ccagttttag aattaaaagt 420
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attagcagtc at 1092
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<212> DNA
<213> artificial sequence
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ttaatgtttc aattcagatt ccattttagc caacatacca acaccattag catattccaa 60
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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 (5)

1. A recombinant Saccharomyces cerevisiae is characterized in that the recombinant Saccharomyces cerevisiae simultaneously expresses a nucleotide sequence shown as SEQ ID NO.1EEVSThe nucleotide sequence is shown as SEQ ID NO.2MT-OxXylose reductase with nucleotide sequence shown as SEQ ID NO.3XRXylitol dehydrogenase with nucleotide sequence shown as SEQ ID NO.4XDHXylulokinase with nucleotide sequence shown as SEQ ID NO.5XKThe recombinant saccharomyces cerevisiae takes saccharomyces cerevisiae BY4743 as an expression host; expression using pYEP352 plasmidEEVSAndMT-Oxexpression of xylose reductase Using pY15TEF1 plasmidXRXylitol dehydrogenaseXDHXylulokinaseXK。
2. A method for preparing the gadosol, which is characterized in that the recombinant saccharomyces cerevisiae described in claim 1 is added into a reaction system containing xylose for fermentation, so as to prepare the gadosol.
3. The method of claim 2, wherein the concentration of xylose in the reaction system is at least: 20g/L.
4. A method for improving the yield of production of gadosol by Saccharomyces cerevisiae is characterized in that the nucleotide sequence shown as SEQ ID NO.1 is expressed in Saccharomyces cerevisiae simultaneouslyEEVSThe nucleotide sequence is shown as SEQ ID NO.2MT-OxXylose reductase with nucleotide sequence shown as SEQ ID NO.3XRXylitol dehydrogenase with nucleotide sequence shown as SEQ ID NO.4XDHXylulokinase with nucleotide sequence shown as SEQ ID NO.5XKObtaining recombinant saccharomyces cerevisiae; the recombinant saccharomyces cerevisiae takes saccharomyces cerevisiae BY4743 as an expression host; expression using pYEP352 plasmidEEVSAndMT-Oxexpression of xylose reductase Using pY15TEF1 plasmidXRXylitol dehydrogenaseXDHXylulokinaseXK。
5. Use of the recombinant s.cerevisiae according to claim 1 for the preparation of a product comprising gadusol.
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