CN110195023B - Saccharomyces cerevisiae strain and application thereof - Google Patents
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Abstract
The invention relates to the technical field of microorganisms, and discloses a saccharomyces cerevisiae strain and application thereof. The invention provides a mutagenic saccharomyces cerevisiae strain for high-yield astaxanthin, the astaxanthin yield of the mutagenic strain is obviously improved relative to that of a starting strain, wherein the astaxanthin yield of shake-flask fermentation is improved by 3.98 times and reaches 65.93mg/L, and the proportion of astaxanthin in carotenoid is improved by 1.48 times and reaches 68.56%. And the maximum astaxanthin yield in a 5L fermentation tank reaches 404.78mg/L through optimizing the fermentation process conditions.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a saccharomyces cerevisiae strain and application thereof.
Background
Astaxanthin (Astaxanthin, C40H52O4, molecular weight 596.84) is a natural carotenoid widely found in microorganisms and marine organisms, and is also the highest-order product of carotenoid synthesis, and can be classified into 3 configurations (3S,3 'S), (3R, 3' R) depending on the presence of two chiral carbons on the β -ionone ring. Astaxanthin has strong oxidation resistance, and can prevent chain reaction by scavenging active oxygen in cells, inhibit lipid peroxidation to protect membrane structure, thereby preventing oxidative damage. Unlike other carotenoids, astaxanthin simultaneously links the inner membrane and the outer membrane of cells and can simultaneously scavenge active oxygen of the inner membrane and the outer membrane of the cells, so that astaxanthin has stronger oxidation resistance than other carotenoids, and the oxidation resistance of astaxanthin is 65 times that of vitamin C, 54 times that of beta-carotene, 10 times that of canthaxanthin, zeaxanthin and lutein and 100 times that of vitamin E. The astaxanthin has unique chemical structure and properties, so that the astaxanthin has the functions of delaying senility, resisting tumors, enhancing immunity, preventing cardiovascular diseases and the like, and has great market value in the industries of food, medicine, health-care products, aquaculture and the like. Second only to beta-carotene and lutein in market size, astaxanthin accounts for 29% of the carotenoids in the third major carotenoid, and is projected to reach $ 25.7 billion in market size in 2025. The wide market demand has led to bright prospects for the research and production of astaxanthin.
At present, astaxanthin is mainly derived from chemical synthesis, algae and Phaffia rhodozyma biosynthesis. Chemically synthesized astaxanthin and natural astaxanthin have different configurations, the antioxidant capacity of the chemically synthesized astaxanthin is only 1/3 of natural astaxanthin, and the safety of different stereoisomers of the synthetic astaxanthin and some potential synthetic intermediates is also concerned. Haematococcus pluvialis (Haematococcus pluvialis) and Phaffia rhodozyma (Xanthophyllomyces dendrorhous) are the major strains for the industrial production of astaxanthin at present. However, haematococcus pluvialis has slow growth cycle and low cell density, so that the economic cost is high. Phaffia rhodozyma is used as a microorganism naturally producing astaxanthin in the nature, but the astaxanthin produced by Phaffia rhodozyma has a (3R, 3' R) configuration and low oxidation resistance, so that the application of the Phaffia rhodozyma in industrial production is limited. Therefore, researchers have turned their eyes to other microbial cell factories for biosynthesis of astaxanthin. In 2017, Kildegaard et al constructed an astaxanthin synthesis pathway in lipolytic yeast, and balanced metabolic flux increased astaxanthin production by increasing the copy number of CrtZ, and finally reached an astaxanthin yield of 54.6mg/L in lipolytic yeast. In 2018, Park et al mapped the truncated BKT gene (trCrBKT) to the cell membrane, overexpressed ispD and ispF genes identified by FVSEAF software, and optimized fermentation conditions to achieve an astaxanthin yield of 385.04mg/L in E.coli in a 5L fermentor.
Saccharomyces cerevisiae, a well-known safe mode microorganism, has clear genetic background and simple molecular operation, and is widely applied to the production of wine brewing, food baking, medicines, health products and the like. Compared with Escherichia coli, it has high content of vitamins and proteins, and can be used as edible medicinal and feed yeast; it has a shorter growth cycle and is easier to culture than algae. Therefore, achieving high astaxanthin yield in saccharomyces cerevisiae would show great competitiveness in carotenoid industrialization. However, the astaxanthin production in Saccharomyces cerevisiae reported by the present disclosure still does not reach the industrial production level. In 2017, by mutating GGPP synthase to increase precursor supply and adjusting copy number balance metabolic flux between rate-limiting enzymes, the task group in HongWei, the astaxanthin yield of Saccharomyces cerevisiae was increased to a higher level of 47.18 mg/L.
Disclosure of Invention
In view of the above, the present invention aims to provide a saccharomyces cerevisiae strain obtained through mutagenesis, such that the astaxanthin yield is significantly improved;
another object of the present invention is to provide the use of the above-mentioned mutagenized strain of Saccharomyces cerevisiae for the production of related chemicals;
another object of the present invention is to provide a method for producing astaxanthin using the above-mentioned mutagenized strain of Saccharomyces cerevisiae.
In order to achieve the above purpose, the invention provides the following technical scheme:
a Saccharomyces cerevisiae strain with the preservation number of CGMCC No.17913, which is preserved in China general microbiological culture Collection center at 6 months and 10 days in 2019, is classified and named as Saccharomyces cerevisiae.
The method takes a saccharomyces cerevisiae SyBE _ Sc14CY10 for producing astaxanthin as a starting strain, physical mutagenesis is carried out on the SyBE _ Sc14CY10 by using a normal-pressure room-temperature plasma mutagenesis technology (ARTP) to obtain a saccharomyces cerevisiae mutation library, then astaxanthin yield is determined by shake flask fermentation and HPLC, and three mutant strains with higher yield are further optimized; then, on the basis of three mutant bacteria with high astaxanthin yield, carrying out chemical mutagenesis (chemical mutagenesis, CM) by using oxidation pressure applied by hydrogen peroxide, continuously mutagenizing 21 times for one period, obtaining a saccharomyces cerevisiae mutation library again, measuring the astaxanthin yield by shake flask fermentation and HPLC, and taking the three mutant bacteria with high yield as a further optimization object; according to the circulation, based on three yeasts with high astaxanthin yield obtained by physical or chemical mutagenesis in each period, physical or chemical mutagenesis is alternately carried out, and finally, a mutagenized saccharomyces cerevisiae strain with high astaxanthin yield is obtained and named as SyBE _ Sc 04030020.
Fermentation tests prove that the yield and the proportion of SyBE _ Sc04030020 astaxanthin are both obviously improved, wherein the yield of astaxanthin obtained by shake-flask fermentation is improved by 3.98 times and reaches 65.93mg/L, and the proportion of astaxanthin in carotenoid is improved by 1.48 times and reaches 68.56%. And the maximum astaxanthin yield in a 5L fermentation tank can reach 404.78mg/L through optimizing the fermentation process conditions. The above yield data were the highest yield among the currently known yields of astaxanthin from the Saccharomyces cerevisiae.
Based on the technical effects, the invention provides the application of the saccharomyces cerevisiae strain in the production of astaxanthin or other chemicals taking astaxanthin as intermediate chemicals; and application in preparing microbial preparation for producing astaxanthin or microbial preparation for producing other chemicals with astaxanthin as intermediate chemical.
The invention provides two methods for producing astaxanthin by fermentation, wherein the first method for producing astaxanthin comprises the steps of inoculating a saccharomyces cerevisiae strain with the preservation number of CGMCC No.17913 into an SC solid culture medium for activation, then transferring the strain into an SC liquid culture medium for preparing a seed culture solution, and finally transferring the seed culture solution into a YPDG fermentation culture medium for fermentation until the cell OD is reached600The astaxanthin synthesis is basically stable and can not be increased any more.
In the specific implementation process, the saccharomyces cerevisiae strain with the preservation number of CGMCC No.17913 is streaked on an SC solid culture medium for activation culture, and then is inoculated on an SC liquid culture medium for culture to OD600When it is 5-8, then press OD6000.2 transfer to SC broth to OD600Preparing seed culture solution from 6.5-7.5, and adjusting final OD600Transferring the cell strain 0.1 to YPDG fermentation medium, fermenting at 30 deg.C and 250rpm with shaking table until cell OD600The astaxanthin synthesis is basically stable and can not be increased any more. Through HPLC detection, the yield of the astaxanthin in the shake flask fermentation is 65.93 mg/L;
the second method for producing astaxanthin is to inoculate the saccharomyces cerevisiae strain with the preservation number of CGMCC No.17913 into an SC solid culture medium for activation, then transfer the strain into an SC liquid culture medium for preparing a seed culture solution, finally transfer the seed culture solution into a YPD fermentation culture medium for fermentation until the cell OD is reached600The astaxanthin synthesis is basically stable and is not increased any more;
in the specific implementation process, the saccharomyces cerevisiae strain with the preservation number of CGMCC No.17913 is streaked on an SC solid culture medium for activation culture, and then is inoculated on an SC liquid culture medium for culture to OD600Transferring to SC liquid culture medium according to the inoculum size of 4% to medium stage of logarithmic growth phase to prepare seed culture solution, transferring the seed culture solution to YPD fermentation medium according to the inoculum size of 10% to perform fermentation tank fermentation until cell OD600The astaxanthin synthesis is basically stable and can not be increased any more.
During fermentation, glucose is added as a carbon source in a fed-batch manner and is controlled within the range of 0-1g/L (no 0), when the thallus growth enters a lag phase, a D- (+) -galactose inducer is added, and simultaneously, the fed-batch of glucose is stopped, and the astaxanthin synthesis phase is carried out; at the moment, the carbon source in the fermentation tank is switched to ethanol, the ethanol is added in a feeding manner, and the concentration of the ethanol in the fermentation tank is controlled to be below 5 g/L;
among the important factors affecting the fermentation biomass is the nitrogen source in the fermentation medium. In the invention, the yeast extract powder is preferably used as a nitrogen source, in order to avoid excessive nitrogen source feeding, the yeast extract powder is fed in an intermittent feeding mode with descending concentration gradient, and a certain volume of yeast extract powder mother liquor is added into a fermentation tank at intervals (the concentration of the yeast extract powder in an initial YPD fermentation medium is 1.0 x). The concrete feeding process is as follows: adding 1.2 times yeast extract powder in 10h, adding 1.0 times yeast extract powder in 65h, adding 0.8 times yeast extract powder in 75h, adding 0.3 times yeast extract powder in 120h, maintaining the concentration at 0.3 times in the later period, and stopping feeding after 180h (cell growth enters into stagnation period). 1.2 times of yeast extract powder is added into each liter when the fermentation is carried out for 10 hours, the total amount is 30g of yeast extract powder, the concentration of mother liquor is 400g/L, and the volume of 30g of yeast extract powder is 75 ml. Cell growth into the arrest phase at 75h, OD600The growth slowed and the nitrogen source addition was reduced to 0.8 x. Fermenting for 120h, slowing the growth of the strain again, and reducing the nitrogen source to 0.3 x.
The fed-batch fermentation test result shows that the yield of the astaxanthin reaches 226.19mg/L, and is improved by 2.43 times compared with the shake flask fermentation yield (65.93 mg/L).
In order to further improve the yield of astaxanthin, the fermentation conditions of the fermentation tank are continuously optimized. Besides the proportioning composition and addition mode of the fermentation medium, the temperature is also a key factor influencing the growth and metabolism of the strain. On the basis of the fed-batch fermentation, the addition of a D- (+) -galactose inducer is taken as a demarcation point, and the fermentation temperature before the addition is 30 ℃, so that the fed-batch fermentation is most beneficial to the cell growth under the condition. When the cell growth enters a lag phase, D- (+) -galactose is added to induce astaxanthin synthesis pathway gene to start expression, the fermentation second stage is carried out, the temperature is reduced to 20 ℃ to facilitate the accumulation of astaxanthin, the final astaxanthin yield reaches 404.78mg/L, and the astaxanthin yield is increased by 5.14 times compared with that of shake flask fermentation.
According to the technical scheme, the mutagenic saccharomyces cerevisiae strain for high-yield astaxanthin is provided, the astaxanthin yield of the mutagenic saccharomyces cerevisiae strain is obviously improved relative to that of a starting strain, wherein the astaxanthin yield of shake-flask fermentation is improved by 3.98 times and reaches 65.93mg/L, and the proportion of astaxanthin in carotenoid is improved by 1.48 times and reaches 68.56%. And the maximum astaxanthin yield in a 5L fermentation tank reaches 404.78mg/L through optimizing the fermentation process conditions.
Biological preservation Instructions
SyBE _ Sc04030020, classification name: saccharomyces cerevisiae is preserved in China general microbiological culture Collection center (CGMCC) for 6 months and 10 days in 2019, and is addressed to Xilu No.1 Hospital 3 of the Kyoho, Beijing, and the microbial research institute of the Chinese academy of sciences, with the preservation number of CGMCC No. 17913.
Drawings
FIG. 1 shows the astaxanthin production by shake flask fermentation of mutagenized strains and starting strains according to the invention;
FIG. 2 is a graph showing the ratio of chemicals in carotenoids fermented in shake flasks by the mutagenized strain and the starting strain of the present invention;
FIG. 3 shows the results of fermentation measurements in a fermenter for mutagenized strains according to the invention;
FIG. 4 shows the results of fermentation measurements in a fermenter with optimized temperature for mutagenized strains of the present invention.
Detailed Description
The invention discloses a saccharomyces cerevisiae strain and application thereof, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the Saccharomyces cerevisiae strains and applications of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the techniques of the present invention may be implemented and applied by modifying or appropriately modifying and combining the Saccharomyces cerevisiae strains and applications described herein without departing from the spirit, scope, and content of the present invention.
The invention is further illustrated by the following examples.
Example 1: test of fermentation in Shake flasks
Fermentation: the mutagenized saccharomyces cerevisiae strain SyBE _ Sc04030020 preserved by glycerol at the temperature of-80 ℃ is streaked on an SC solid culture medium for activation culture, and then is inoculated into 5ml of SC liquid culture medium for culture for 18h to OD600When it is 5-8, then press OD6000.2 transfer to secondary seed medium tubes. OD after 14-16h600After reaching about 7, the seed culture solution is taken and the final OD is determined600Transferring the strain to 50mL YPDG culture medium for fermentation, and shaking and culturing the strain at 30 ℃ and 250rpm for 96 h;
and (4) HPLC detection: adding 500uL of fermented bacterial liquid into a 1.5-mL LEP tube, centrifuging at 12000r for 2min, pouring out the supernatant, adding 1mL of sterile water to resuspend cells, centrifuging, pouring out the supernatant, adding 1mL of 3M HCl to resuspend cells, carrying out boiling water bath for 3min, carrying out ice bath for 3min, repeating for 3 times, carrying out centrifugal water washing twice, discarding the supernatant, adding 1mL of acetone to resuspend cells, adding a small amount of quartz sand to carry out vortex oscillation for 10min, centrifuging the acetone extracting solution at 12000r for 5min, filtering by using a 0.2-micrometer organic filter membrane, and adding 200 mu L of the acetone extracting solution into a sample bottle for HPLC high performance liquid chromatography detection. The astaxanthin standards were diluted with acetone in a gradient to 50mg/L, 25mg/L, 12.5mg/L, 6.25mg/L, 3.125mg/L, respectively. Setting measurement parameters, setting the wavelength as follows: 450nm (lycopene), 470nm (astaxanthin). Column C18. Mobile phase A: b, nitrile: water 9: 1; mobile phase B: methanol: isopropanol-3: 2. The column temperature was 25 ℃ and the sample temperature was 20 ℃.
As a result, as shown in FIGS. 1 and 2, the yield of astaxanthin was 65.93mg/L, which was 3.98 times higher than that of the starting bacteria, and the ratio of astaxanthin to carotenoid was 1.48 times higher, which was 68.56%.
Example 2: fermentation test in 5L fermenter
The fermentation basal medium was YPD medium (1% yeast extract, 2% peptone, 2% glucose) with an initial glucose concentration of 20g/L, and SyBE _ Sc04030020 was subjected to fed-batch fermentation experiments in a laboratory 5L fermentor. First-order seed culture: inoculating 200ul of SyBE _ Sc04030020 glycerobacteria into a test tube filled with 5mL of SC seed culture medium, and culturing at 30 ℃ and 250rpm until OD is reached6006-7; secondary seed culture: transferring the first seed at 4% inoculum size into 250mL shake flask containing 50mL SC medium, culturing at 30 deg.C and 250rpm to mid-logarithmic growth phase (OD)600＝5-6)。
Inoculating in a fermentation tank: at 10% inoculum size (250ml of secondary seed, initial OD)600About 0.5) transferring the secondary seeds to a 5L fermenter containing 2.25L fermentation medium to start fermentation; the initial liquid loading of the fermenter was 2.5L, and the initial medium was YPD containing 2% glucose.
Setting fermentation tank parameters: the fermentation temperature was 30 ℃, pH was controlled at 5.8, aeration was set at 2.5vvm, DO was 40%, the rotation speed was 400-. The whole fermentation process is divided into a strain growth stage and an astaxanthin production stage, and the adding time of the D- (+) -galactose inducer is a demarcation point of the two stages.
In the growth stage of the strain, in order to ensure the continuous supply of the carbon source, glucose is added in a fed-batch manner and is controlled within the range of 0-1g/L, so as to reduce the generation of byproducts as much as possible, because the excessive glucose in the culture medium can be converted into byproducts such as glycerol, ethanol and acetic acid in yeast overflow metabolism, thereby causing the reduction of the cell yield. When the thallus grows into a stationary phase, adding a galactose inducer with the final concentration of 20g/L D- (+) -galactose, stopping feeding glucose, and entering an astaxanthin synthesis stage. At the moment, the carbon source in the fermentation tank is switched to ethanol, the carbon source is added by a feeding pump in a flow adding manner, and the concentration of the ethanol in the fermentation tank is controlled to be below 5 g/L.
In addition, important factors influencing the fermentation biomass are the nitrogen source in the fermentation medium. The method takes yeast extract powder as a nitrogen source, supplements the yeast extract powder into a fermentation tank in an intermittent feeding mode with descending concentration gradient in order to avoid excessive feeding of the nitrogen source, and adds a certain volume of yeast extract powder mother liquor into the fermentation tank at intervals (the concentration of the yeast extract powder in an initial culture medium is 1.0 x). The concrete feeding process is as follows: adding 1.2 times yeast extract powder in 10h, adding 1.0 times yeast extract powder in 65h, adding 0.8 times yeast extract powder in 75h, adding 0.3 times yeast extract powder in 120h, maintaining the concentration at 0.3 times in the later period, and stopping feeding in 180 h. 1.2 times of yeast extract powder is added into each liter when the fermentation is carried out for 10 hours, the total amount is 30g of yeast extract powder, the concentration of mother liquor is 400g/L, and the volume of 30g of yeast extract powder is 75 ml. Cell growth into the arrest phase at 75h, OD600The growth slowed and the nitrogen source addition was reduced to 0.8 x. Fermenting for 120h, slowing the growth of the strain again, and reducing the nitrogen source to 0.3 x.
The fed-batch fermentation test results are shown in FIG. 3, and the final astaxanthin yield of the 5L fermentation tank reaches 226.19mg/L, which is improved by 2.43 times compared with the shake flask fermentation yield (65.93 mg/L).
Example 3: fermentation test of 5L fermentation tank after fermentation temperature optimization
In order to further improve the yield of astaxanthin, the fermentation conditions of the fermentation tank are continuously optimized. In addition to the composition and addition of the fermentation medium, temperature is also a key factor affecting the growth and metabolism of the strain, and therefore the temperature of the fermentation process is optimized on the basis of example 2. The method comprises the following specific implementation steps: the fermentation process is divided into two stages, the temperature of the first stage is controlled to be 30 ℃, and the growth of cells is most facilitated under the condition. When the cell growth enters a lag phase, D- (+) -galactose is added to induce astaxanthin synthesis pathway gene to start expression, the fermentation second stage is carried out, the temperature is reduced to 20 ℃ to facilitate the accumulation of astaxanthin, the fermentation process is shown in figure 4, finally the astaxanthin yield reaches 404.78mg/L, and the yield is improved by 5.14 times compared with that of shake flask fermentation.
Therefore, the astaxanthin-high-yield mutant strain SyBE _ Sc04030020 is optimized in a 5L fermentation tank, and the optimal temperature conditions are controlled as follows: the adding time of the D- (+) -galactose inducer is taken as a boundary point of a strain growth stage and an astaxanthin production stage in the fermentation process, the temperature of the strain growth stage is 30 ℃, and the temperature of the astaxanthin production stage is 20 ℃.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (3)
1. A method for producing astaxanthin is characterized in that a saccharomyces cerevisiae strain with the preservation number of CGMCC No.17913 is inoculated into an SC solid culture medium for activation, then is transferred into an SC liquid culture medium to prepare a seed culture solution, and finally the seed culture solution is transferred into a YPD fermentation culture medium for fermentation in a fermentation tank;
during fermentation, adding glucose as a carbon source in a fed-batch manner, controlling the adding amount to be within the range of 0-1g/L, adding a D- (+) -galactose inducer when the growth of the thalli enters a lag phase, stopping feeding the glucose at the same time, and entering an astaxanthin synthesis phase; at the moment, the carbon source in the fermentation tank is switched to ethanol, the ethanol is added in a feeding manner, and the concentration of the ethanol in the fermentation tank is controlled to be below 5 g/L;
during fermentation, 1.2 times of yeast extract powder is added in 10 hours, 1.0 time of yeast extract powder is added in 65 hours, 0.8 time of yeast extract powder is added in 75 hours, 0.3 time of yeast extract powder is added in 120 hours, the concentration is always maintained at 0.3 time in the later period, and feeding is stopped when cell growth enters a lag phase; the concentration of the 1.2 times yeast extract powder is 400g/L, and the volume is 75 ml;
bacterial strain production taking D- (+) -galactose inducer addition time as fermentation processThe division point of the growth stage and the astaxanthin production stage is that the temperature of the strain in the growth stage is 30 ℃, and the temperature of the astaxanthin production stage is 20 ℃; the whole process is fermented to the cell OD600The astaxanthin synthesis is basically stable and can not be increased any more.
2. The method according to claim 1, wherein the strain of Saccharomyces cerevisiae with the preservation number of CGMCC No.17913 is streaked on SC solid culture medium for activation culture, and then inoculated on SC liquid culture medium for culture to OD600And =6-7, inoculating to an SC liquid culture medium according to the inoculum size of 4% to culture to the middle stage of logarithmic phase to prepare a seed culture solution, and finally inoculating the seed culture solution to an YPD fermentation culture medium according to the inoculum size of 10% to perform fermentation tank fermentation.
3. The method according to claim 1 or 2, wherein the YPD fermentation medium has an initial glucose concentration of 20 g/L.
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| CN113699053B (en) * | 2020-05-20 | 2023-08-11 | 万华化学(四川)有限公司 | Recombinant saccharomyces cerevisiae for producing astaxanthin and application thereof |
| CN113699052B (en) * | 2020-05-20 | 2023-08-11 | 万华化学(四川)有限公司 | Recombinant saccharomyces cerevisiae for producing astaxanthin and application thereof |
| CN111979132B (en) * | 2020-08-20 | 2022-07-26 | 宜昌东阳光生化制药有限公司 | Fermentation medium for high-yield astaxanthin and application thereof |
| CN112481144B (en) * | 2020-12-11 | 2022-04-12 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Yeast mutant strain for producing carotenoid and application of mutant site thereof |
| CN114574376A (en) * | 2022-05-09 | 2022-06-03 | 暨南大学 | Saccharomyces cerevisiae Delta-M3 for high-yield carotenoid production and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962488A (en) * | 2015-07-22 | 2015-10-07 | 天津大学 | Recombinant yeast strain, and construction method and application thereof |
| CN105861538A (en) * | 2016-05-25 | 2016-08-17 | 天津大学 | Recombinant plasmid and recombinant yeast strain and establishing method and application thereof |
-
2019
- 2019-06-27 CN CN201910567660.0A patent/CN110195023B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962488A (en) * | 2015-07-22 | 2015-10-07 | 天津大学 | Recombinant yeast strain, and construction method and application thereof |
| CN105861538A (en) * | 2016-05-25 | 2016-08-17 | 天津大学 | Recombinant plasmid and recombinant yeast strain and establishing method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Astaxanthin overproduction in yeast by strain engineering and new gene target uncovering;Yingjin Yuan等;《Biotechnol Biofuels》;20180823;第11卷;第1-15页，尤其是摘要，第10页右栏第2段，第11页图7，第12页左栏第2段、右栏第3段，第13页左栏第1段 * |
| Highly efficient biosynthesis of astaxanthin in Saccharomyces cerevisiae by integration and tuning of algal crtZ and bkt;Pingping Zhou等;《Appl Microbiol Biotechnol》;20150709;第99卷;第8419-8428页 * |
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