CN116376732A - Saccharomyces cerevisiae engineering strain for high-yield of triglyceride and diglyceride, construction method and application thereof - Google Patents
Saccharomyces cerevisiae engineering strain for high-yield of triglyceride and diglyceride, construction method and application thereof Download PDFInfo
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- CN116376732A CN116376732A CN202310197820.3A CN202310197820A CN116376732A CN 116376732 A CN116376732 A CN 116376732A CN 202310197820 A CN202310197820 A CN 202310197820A CN 116376732 A CN116376732 A CN 116376732A
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- saccharomyces cerevisiae
- engineering strain
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- yel036c
- diglyceride
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Abstract
The invention discloses a saccharomyces cerevisiae engineering strain for high-yield triglyceride and diglyceride, which is obtained by knocking out a YEL036C gene shown in SEQ ID NO.1 in a saccharomyces cerevisiae host strain, wherein the amino acid sequence coded by the YEL036C gene is shown in SEQ ID NO. 2. After 12h of fermentation, the saccharomyces cerevisiae engineering strain constructed by the invention has triglyceride and diglyceride contents of 6.22mg/gDCW and 6.42mg/gDCW which are 4.26 times and 5.68 times of that of the wild strain. The engineering strain disclosed by the invention not only improves the yield of triglyceride and diglyceride, but also has relatively simple fermentation conditions, and the cell wall of the saccharomyces cerevisiae engineering strain is relatively fragile, so that the subsequent extraction of the triglyceride and the diglyceride is easy, the investment cost is reduced, and the saccharomyces cerevisiae engineering strain has a broad development prospect.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and relates to construction and application of a saccharomyces cerevisiae engineering strain, in particular to a saccharomyces cerevisiae engineering strain for high-yield triglyceride and diglyceride, a construction method and application thereof.
Background
Triacylglycerol (TAG) is also called triglyceride, commonly called grease, and is glyceride formed by dehydration condensation of 3 hydroxyl groups of glycerol and 3 fatty acid molecules, and belongs to an ester organic compound. Triglycerides are nonpolar substances which are stored in a non-hydrated form and are the most energy substances with the greatest reserves and capacity in the body, and are also the main components of animal oils and fats and vegetable oils and can be taken through daily diets. Whereas the molecular structure of triglycerides consisting of three long hydrocarbon chains linked to one glycerol molecule is similar to petroleum crude oil, thus making triglycerides an important alternative feedstock for replacing petroleum. Triglycerides are widely present in vegetable oils, animal fats and microalgae,providing a huge raw material resource for converting it into direct fuel. The current ways of producing triglycerides as bio-fuels mainly comprise the following steps: 1. the vegetable oil, mainly palm oil, is mainly produced in southeast Asia and has the largest share in the global vegetable oil, and accounts for 34 percent of the yield and 33 percent of the consumption of all vegetable oils. Palm oil is a potential feedstock for biofuel production due to its low cost and highest yield per hectare. However, the acquisition of vegetable oil affects agriculture and forestry, and the price of vegetable oil is easily affected by various factors such as weather and internal problems of the production country, so that the yield and price are unstable; 2. the waste edible oil and the waste fish oil are utilized for recycling and producing the triglycerides, the raw materials are not influenced by factors such as agriculture, and the environment problem of waste treatment can be solved by recycling the waste oil, but compared with the traditional triglyceride extraction, the waste oil contains a large amount of water, impurities and free fatty acid, so that the viscosity, the acid value and the stability of the waste oil are influenced, and therefore, pretreatment is needed before the conversion process, and the influence of rising cost is caused; 3. the photosynthetic efficiency of the microalgae is about 10-20 times higher than that of other oil crops, and biomass and CO can be produced more quickly 2 Sealing and storing. In addition, it can adapt to changeable waste water source, does not need the cultivated land. Microalgae oils show great potential at the best growth rate. However, it also has some drawbacks, the actual growth rate is lower than the highest observed rate, and the equipment required to culture, harvest and dewater the algae is expensive, so microalgae oils are more expensive than any other oil. The existing biofuel has the advantages of various raw materials, strong carbon reduction capability, low cost as a substitute fuel and the like, and has wide application prospect in the fields of transportation and the like. The bio-fuel oil has large production requirement gap, and the domestic bio-fuel oil in China is mostly used in the field of fine chemical industry.
Diglycerides (DAGs) are a class of glycerides formed from two fatty acid chains and one glycerol molecule via an ester linkage. Diglyceride is an intermediate product of fat metabolism, is an important functional fat and is also an internationally recognized and safe food ingredient. According to the difference of the connection positions of fatty acid acyl groups and hydroxyl groups on a glycerol skeleton, diglycerides are divided into three isomers of 1, 3-diglycerides, 1, 2-diglycerides and 2, 3-diglycerides, wherein the 1, 2-diglycerides and the 2, 3-diglycerides are isomers with each other. Currently 1, 3-diglycerides differ from the unique metabolic pattern of triglycerides, making them of great potential value. The 1, 3-diglyceride can be used as health care grease, medical auxiliary materials, cosmetic auxiliary materials, medicine synthesis intermediates and the like, and has wide market and considerable economic value. 1, 2-diglycerides are used as isomers of 1, 3-diglycerides, and are also widely used in the food industry as important components of edible fats and oils, although they are metabolized in a different manner from 1, 3-diglycerides. Therefore, diglycerides have good commercial value. Naturally occurring diglycerides are rarely obtained mainly by post-production modification techniques of oils and fats. At present, common methods for preparing DAG at home and abroad comprise a chemical method, a biological enzyme method and the like, and the problems of environmental pollution, excessive energy consumption, excessive cost and the like exist at different degrees. Compared with the chemical method for preparing DAG, the biological enzyme method has better results in terms of yield, purity, reaction time, safety and the like. However, to apply the enzymatic method to the industrial production of DAG, further researches and improvements are required, such as excessive catalytic cost, reaction conditions, reactor design, how to obtain high purity DAG after reaction, etc. by using immobilized lipase.
With the completion of sequencing the saccharomyces cerevisiae genome, the physiological characteristics and metabolic regulation mechanism of the saccharomyces cerevisiae are deeply and comprehensively known, and the saccharomyces cerevisiae has become a chassis organism for the establishment of microbial cell factories favored by more and more scientific technicians. At present, a plurality of products, food raw materials, industrial raw materials and the like with extremely high medical value are available, and the saccharomyces cerevisiae engineering strain can be utilized for mass production. Therefore, based on the above research analysis, there is an urgent need in the art to develop a yeast strain that can efficiently produce triglycerides or/and diglycerides.
By searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provide a saccharomyces cerevisiae engineering strain for high-yield triglyceride and diglyceride, a construction method and application thereof.
The technical scheme adopted for solving the technical problems is as follows:
a saccharomyces cerevisiae engineering strain for high-yield triglyceride and diglyceride is obtained by knocking out a YEL036C gene shown in SEQ ID NO.1 in a saccharomyces cerevisiae host strain, and the amino acid sequence coded by the YEL036C gene is shown in SEQ ID NO. 2.
Further, the engineering strain is obtained by knocking out the YEL036C gene shown in SEQ ID NO.1 by using a Cre/loxP recombination system through a homologous recombination method;
wherein the host strain is Saccharomyces cerevisiae strain Saccharomyces cerevisiae BY4741.
The construction method of the saccharomyces cerevisiae engineering strain for high production of triglyceride and diglyceride comprises the following steps:
(1) Designing a primer for knocking out the YEL036C gene from the saccharomyces cerevisiae genome and a sequence thereof according to the YEL036C gene sequence shown in SEQ ID No.1 and a Cre/loxP recombination system;
(2) Using plasmid carrying screening mark in Cre/loxP recombination system as template, using the primer designed in step (1), PCR amplifying DNA fragment for knocking out YEL036C gene from saccharomyces cerevisiae genome, and purifying;
(3) The prepared DNA fragment for knocking out the YEL036C gene from the saccharomyces cerevisiae genome is transferred into saccharomyces cerevisiae Saccharomyces cerevisiae BY4741 by adopting a high-efficiency yeast cell transformation method;
(4) And screening the auxotroph solid yeast culture medium corresponding to the screening mark to obtain the recombinant yeast strain.
Further, the specific steps are as follows:
(1) The amplification primers were designed based on the nucleotide sequence of YEL036C shown in SEQ ID NO.1 and the Cre/loxP recombination system:
(2) Taking Cre/loxP recombinant system plasmid as a template, and carrying out PCR amplification to obtain a DNA fragment for knocking out the YEL036C gene;
(3) After agarose gel electrophoresis analysis is carried out on the PCR product, cutting gel and recovering a target fragment;
(4) Transferring the target fragment into Saccharomyces cerevisiae BY4741 BY adopting a lithium acetate conversion method;
(5) Screening by using a leucine auxotroph solid culture medium, wherein single colonies growing on the leucine auxotroph solid culture medium are candidate colonies;
(6) And (3) carrying out gene sequencing on the point position of the YEL036C in the genome of the candidate colony to determine that the YEL036C is successfully replaced by the screening marker gene, thus obtaining the gene.
Further, the leucine auxotroph solid medium per 1L was: 6.5-7g/L yeast nitrogen source base (YeastNitrogen Base), 0.72-0.84g/L leucine deficiency type amino acid mixture (-Leu Do supplement powder) and 1.7-2.3% glucose, agar 2-3% after sterilization, ddH 2 O constant volume to 1L;
wherein the percentages are mass concentration percentages.
The application of the saccharomyces cerevisiae engineering strain for high-yield triglyceride and diglyceride in the production of the triglyceride and the diglyceride is provided.
The method for producing triglyceride and diglyceride by fermenting the saccharomyces cerevisiae engineering strain comprises the following steps:
coating the saccharomyces cerevisiae engineering strain with high yield of triglyceride and diglyceride on a solid rich culture medium plate through three-area lines, culturing for 48-72h at 25-30 ℃, inoculating 1-2 rings of the activated strain in 3-5mL of liquid rich culture medium, culturing for 14-20h at 25-30 ℃ and 180-220rpm to obtain seed culture solution, inoculating the seed culture solution into the liquid rich culture medium according to the inoculation amount of 8-15%, and culturing for 12-24h at 25-30 ℃ and 180-220rpm to obtain the saccharomyces cerevisiae engineering strain.
Further, per 1L of solid rich medium is: 0.055-0.06g/L adenine sulfate, 8-12g/L yeast extract powder, 16-24g/L peptone, 18-22g/L agar, sterilizing, adding 1.8-2.2% glucose, and adding ddH 2 O constant volume to 1L;
every 1L of liquid rich culture medium is: 0.055-0.06g/L adenine sulfate, 8-12g/L yeast extract powder, 16-24g/L peptone, sterilizing, adding 1.8-2.2% glucose, and adding ddH 2 O constant volume to 1L;
wherein the percentages are mass concentration percentages.
The method for measuring the content of triglyceride and diglyceride in cells of the saccharomyces cerevisiae engineering strain with high triglyceride and diglyceride yield comprises the following specific steps:
(1) After fermentation of the saccharomyces cerevisiae engineering strain is finished, collecting all thalli;
(2) Crushing the collected thalli;
(3) Extracting with organic solvent, and mixing solvent layers;
(4) Extracting with organic solvent for several times, and mixing solvent layers;
(5) Drying the organic solvent layer with nitrogen, and redissolving the extracted substances;
(6) Detecting the obtained substance by using UPLC/MS;
(7) The intracellular triglyceride and diglyceride content was calculated.
The method for determining the growth curve of the saccharomyces cerevisiae engineering strain for high-yield triglyceride and diglyceride comprises the following specific steps:
(1) Inoculating Saccharomyces cerevisiae engineering strain and control strain for producing triglyceride and diglyceride into test tubes of liquid rich culture medium, respectively, and culturing at 25-30deg.C and 200-220 rpm/min for 12-18 hr;
(2) Sequentially adding 30 mu L, 40 mu L and 50 mu L of bacterial liquid into an EP tube containing 1mL of liquid rich culture medium, shaking and mixing uniformly, adding 200 mu L into a 100-pore plate, and taking 200 mu L of liquid rich culture medium as a control; placing the 100 pore plates in a full-automatic growth curve tester, culturing at 25-30 ℃, and measuring absorbance values at 600nm of wavelength every 1 h;
(3) On the abscissa of culture time X, OD 600 The value Y is the ordinate and the growth curve is plotted.
The invention has the advantages and beneficial effects that:
1. the saccharomyces cerevisiae engineering strain is obtained by knocking out the YEL036C gene shown in SEQ ID NO.1 in a saccharomyces cerevisiae host strain, and the strain is prepared into a gene knocking-out tool kit through a Cre/loxP system, and is transformed into the saccharomyces cerevisiae host strain to obtain the saccharomyces cerevisiae engineering strain with high triglyceride and diglyceride yield. After 12h of fermentation, the saccharomyces cerevisiae engineering strain constructed by the invention has triglyceride and diglyceride contents of 6.22mg/gDCW and 6.42mg/gDCW which are 4.26 times and 5.68 times of that of the wild strain. The engineering strain disclosed by the invention not only improves the yield of triglyceride and diglyceride, but also has relatively simple fermentation conditions, and the cell wall of the saccharomyces cerevisiae engineering strain is relatively fragile, so that the subsequent extraction of the triglyceride and the diglyceride is easy, the investment cost is reduced, and the saccharomyces cerevisiae engineering strain has a broad development prospect.
2. The saccharomyces cerevisiae engineering strain provided by the invention has the advantages of high yield, can be fermented by relatively simple method conditions, is easy to realize and control in industry, has low investment cost, and has wide application prospect.
Drawings
FIG. 1 is a diagram showing the amplification verification of a DNA fragment for knocking out YEL036C gene according to the present invention; wherein M: DNA molecular weight standard; lane 1: amplified DNA fragments for knocking out YEL036C gene;
FIG. 2 is a diagram showing PCR verification of YEL036C gene knockout in an engineering strain of Saccharomyces cerevisiae constructed in the present invention; wherein M: DNA molecular weight standard; lane 1: YEL036C gene knockout PCR validation;
FIG. 3 is a graph comparing the yields of triglycerides and diglycerides in Saccharomyces cerevisiae engineering strains constructed in the present invention and wild type Saccharomyces cerevisiae strains; wherein, 1: wild-type saccharomyces cerevisiae strain, 2: an engineering strain of Saccharomyces cerevisiae;
FIG. 4 is a graph showing the growth of an engineered strain of Saccharomyces cerevisiae and a control strain thereof constructed in the present invention.
Detailed Description
The present invention will be further described in detail with reference to examples, but the scope of the present invention is not limited to the examples.
The raw materials used in the invention are conventional commercial products unless specified otherwise, the methods used in the invention are conventional methods in the art unless specified otherwise, and the mass of each substance used in the invention is conventional.
A saccharomyces cerevisiae engineering strain for high-yield triglyceride and diglyceride is obtained by knocking out a YEL036C gene shown in SEQ ID NO.1 in a saccharomyces cerevisiae host strain, and the amino acid sequence coded by the YEL036C gene is shown in SEQ ID NO. 2.
Preferably, the engineering strain is obtained by knocking out the YEL036C gene shown in SEQ ID NO.1 by using a Cre/loxP recombination system through a homologous recombination method;
wherein the host strain is Saccharomyces cerevisiae strain Saccharomyces cerevisiae BY4741.
The construction method of the saccharomyces cerevisiae engineering strain for high production of triglyceride and diglyceride comprises the following steps:
(1) Designing a primer for knocking out the YEL036C gene from the saccharomyces cerevisiae genome and a sequence thereof according to the YEL036C gene sequence shown in SEQ ID No.1 and a Cre/loxP recombination system;
(2) Using plasmid carrying screening mark in Cre/loxP recombination system as template, using the primer designed in step (1), PCR amplifying DNA fragment for knocking out YEL036C gene from saccharomyces cerevisiae genome, and purifying;
(3) The prepared DNA fragment for knocking out the YEL036C gene from the saccharomyces cerevisiae genome is transferred into saccharomyces cerevisiae Saccharomyces cerevisiae BY4741 by adopting a high-efficiency yeast cell transformation method;
(4) And screening the auxotroph solid yeast culture medium corresponding to the screening mark to obtain the recombinant yeast strain.
Preferably, the specific steps are as follows:
(1) The amplification primers were designed based on the nucleotide sequence of YEL036C shown in SEQ ID NO.1 and the Cre/loxP recombination system:
(2) Taking Cre/loxP recombinant system plasmid as a template, and carrying out PCR amplification to obtain a DNA fragment for knocking out the YEL036C gene;
(3) After agarose gel electrophoresis analysis is carried out on the PCR product, cutting gel and recovering a target fragment;
(4) Transferring the target fragment into Saccharomyces cerevisiae BY4741 BY adopting a lithium acetate conversion method;
(5) Screening by using a leucine auxotroph solid culture medium, wherein single colonies growing on the leucine auxotroph solid culture medium are candidate colonies;
(6) And (3) carrying out gene sequencing on the point position of the YEL036C in the genome of the candidate colony to determine that the YEL036C is successfully replaced by the screening marker gene, thus obtaining the gene.
Preferably, the leucine auxotroph solid medium per 1L is: 6.5-7g/L yeast nitrogen source base (YeastNitrogen Base), 0.72-0.84g/L leucine deficiency type amino acid mixture (-Leu Do supplementpowder) and 1.7-2.3% glucose, agar 2-3% after sterilization, ddH 2 O constant volume to 1L;
wherein the percentages are mass concentration percentages.
The application of the saccharomyces cerevisiae engineering strain for high-yield triglyceride and diglyceride in the production of the triglyceride and the diglyceride is provided.
The method for producing triglyceride and diglyceride by fermenting the saccharomyces cerevisiae engineering strain comprises the following steps:
coating the saccharomyces cerevisiae engineering strain with high yield of triglyceride and diglyceride on a solid rich culture medium plate through three-area lines, culturing for 48-72h at 25-30 ℃, inoculating 1-2 rings of the activated strain in 3-5mL of liquid rich culture medium, culturing for 14-20h at 25-30 ℃ and 180-220rpm to obtain seed culture solution, inoculating the seed culture solution into the liquid rich culture medium according to the inoculation amount of 8-15%, and culturing for 12-24h at 25-30 ℃ and 180-220rpm to obtain the saccharomyces cerevisiae engineering strain.
Further, per 1L of solid rich medium is: adenine sulfate 0.055-0.06g/L, yeast extract 8-12g/L, peptone 16-24g/L, agar 18-22g/L, sterilizing, addingGlucose 1.8-2.2% and ddH 2 O constant volume to 1L;
every 1L of liquid rich culture medium is: 0.055-0.06g/L adenine sulfate, 8-12g/L yeast extract powder, 16-24g/L peptone, sterilizing, adding 1.8-2.2% glucose, and adding ddH 2 O constant volume to 1L;
wherein the percentages are mass concentration percentages.
The method for measuring the content of triglyceride and diglyceride in cells of the saccharomyces cerevisiae engineering strain with high triglyceride and diglyceride yield comprises the following specific steps:
(8) After fermentation of the saccharomyces cerevisiae engineering strain is finished, collecting all thalli;
(9) Crushing the collected thalli;
(10) Extracting with organic solvent, and mixing solvent layers;
(11) Extracting with organic solvent for several times, and mixing solvent layers;
(12) Drying the organic solvent layer with nitrogen, and redissolving the extracted substances;
(13) Detecting the obtained substance by using UPLC/MS;
(14) The intracellular triglyceride and diglyceride content was calculated.
The method for determining the growth curve of the saccharomyces cerevisiae engineering strain for high-yield triglyceride and diglyceride comprises the following specific steps:
(1) Inoculating Saccharomyces cerevisiae engineering strain and control strain for producing triglyceride and diglyceride into test tubes of liquid rich culture medium, respectively, and culturing at 25-30deg.C and 200-220 rpm/min for 12-18 hr;
(2) Sequentially adding 30 mu L, 40 mu L and 50 mu L of bacterial liquid into an EP tube containing 1mL of liquid rich culture medium, shaking and mixing uniformly, adding 200 mu L into a 100-pore plate, and taking 200 mu L of liquid rich culture medium as a control; placing the 100 pore plates in a full-automatic growth curve tester, culturing at 25-30 ℃, and measuring absorbance values at 600nm of wavelength every 1 h;
(3) On the abscissa of culture time X, OD 600 The value Y is the ordinate, and the growth curve is drawnA wire.
Specifically, the related preparation and detection are as follows:
example 1 amplification and preparation of DNA fragments for knocking out the yes 036C gene:
the following amplification primers were designed based on the nucleotide sequence of YEL036C and the Cre/loxP recombination system:
YEL036C-CL-KO-F:
5’-TAAGGGTGCAGTAACAGTCATTTGAGCTTGTTGACCTACCAATTAACACAAAAAACA GCTGAAGCTTCGTACGC-3’；
YEL036C-CL-KO-R:
5’-AAACAAAACAAAAAATTAAATTAAATTAAATTAATATTAAATGTCATCATGATTCGC ATAGGCCACTAGTGGATCTG-3’；
PCR reaction system:
fastpfu Buffer 10. Mu.L, 2.5mM dNTPs 4. Mu.L, template DNA final concentration < 1. Mu.g, upstream and downstream primers (10. Mu.M) 2.5. Mu.L each, and->
FastPfu DNApolymerase 0.5-1μL，ddH 2 O was made up to a total volume of 50. Mu.L;
and (3) taking the Cre/loxP recombinant system plasmid as a template, and carrying out PCR amplification to obtain a DNA fragment for knocking out the YEL036C gene. The PCR reaction conditions were: the temperature is 95 ℃ for 2-3min,95 ℃ for 20-30s,55-58 ℃ for 30s,72 ℃ for 1-2min, the cycle is 30-40 times, 72 ℃ for 5-10min, and the temperature is kept at 4 ℃. After agarose gel electrophoresis analysis of the PCR products, the target fragment was recovered by cutting into gel, as shown in FIG. 1.
Example 2 construction of YEL036C knockout saccharomyces cerevisiae engineering strain:
and (3) transforming the obtained gene knockout target fragment into a Saccharomyces cerevisiae BY4741 strain by using a lithium acetate efficient transformation method, screening by using a leucine auxotroph solid yeast culture medium, and enriching single colonies obtained by screening the leucine auxotroph solid yeast culture medium. And (3) sequencing the candidate colony by a gold-only intelligent company, and obtaining the required YeL036C gene knockout saccharomyces cerevisiae engineering strain after correct sequencing. The agarose gel electrophoresis results of the PCR amplified fragments of the sequencing DNA are shown in FIG. 2.
Example 3 fermentation of Saccharomyces cerevisiae engineering strains to produce triglycerides and diglycerides:
(1) Activating the bacterial species
Scribing the saccharomyces cerevisiae engineering strain for producing triglyceride and diglyceride on a solid complete culture medium plate, and culturing for 48 hours at 25 ℃;
(2) Seed culture
Inoculating the activated strain into a test tube filled with 5mL of liquid complete culture medium, and culturing at 25 ℃ and 220rpm for 12 hours to obtain seed culture solution;
the liquid culture medium is as follows: adenine sulfate 0.055g/L, yeast extract 10g/L, peptone 20g/L, and glucose 2%.
(3) Shaking flask fermentation culture
Inoculating the seed culture solution into a triangular flask filled with 200mL of liquid complete culture medium according to the inoculation amount of 2%, and fermenting for 12h at 25 ℃ and 220 rpm;
the fermentation medium is as follows: adenine sulfate 0.055g/L, yeast extract 10g/L, peptone 20g/L, and glucose 2%.
Example 4 determination of triglyceride and diglyceride content in Saccharomyces cerevisiae engineering strains:
(1) Measuring the consistent sediment or volume, and placing the sediment or volume in a 2mL EP tube;
(2) Two small steel balls are added, and 800 mu L of precooled dichloromethane/methanol (3:1) buffer solution is added;
(3) Grinding TissueLyser for 5min, and precipitating in a refrigerator at-20deg.C for 2h or overnight;
(4) Centrifuging at 4deg.C for 15min at 25,000Xg, collecting 650 μL supernatant, placing into new EP tube again at 25000Xg, 4deg.C
Centrifuging for 15min;
(5) Taking 600 mu L of supernatant, freezing and pumping, and re-dissolving with 600 mu L of lipid re-solution (isopropanol: acetonitrile: water=2:1:1);
(6) 25,000Xg, centrifugation at 4℃for 20min;
(7) Transferring 60 mu L of supernatant to a 96-hole micro-pore plate, sealing a film mark, and performing UPLC-MS detection;
as a result, as shown in FIG. 3, it was found from the graph that after 12 hours of fermentation, the triglyceride and diglyceride contents reached 6.22mg/gDCW and 6.42mg/gDCW, respectively, which were 4.26 times and 5.68 times that of the wild-type strain.
Example 5 growth curve of Saccharomyces cerevisiae engineering strain:
(1) Inoculating Saccharomyces cerevisiae engineering strain and control strain for producing triglyceride and diglyceride into test tubes of liquid rich culture medium, respectively, and culturing at 27deg.C and 210rpm/min for 12-18 hr;
(2) Sequentially adding 30 mu L, 40 mu L and 50 mu L of bacterial liquid into an EP tube containing 1mL of liquid rich culture medium, shaking and mixing uniformly, adding 200 mu L into a 100-pore plate, and taking 200 mu L of liquid rich culture medium as a control; placing the 100 pore plates in a full-automatic growth curve tester, culturing at 27 ℃, and measuring absorbance values at 600nm of wavelength every 1 h;
(3) OD with incubation time (X) as abscissa 600 The value (Y) is plotted on the ordinate, and the growth curve is plotted as shown in fig. 4.
The YEL036C gene related to the invention is as follows (sequence table SEQ ID NO. 1):
ATGAAGTATAATAACAGAAAACTCTCGTTCAACCCTACCACAGTAAGTATCGCTGGAACG
TTGCTTACGGTGTTCTTTCTCACAAGACTCGTGCTTTCGTTCTTCTCGATATCGCTATTCCA
GCTGGTAACTTTCCAAGGAATCTTCAAGCCCTATGTTCCAGATTTTAAAAATACTCCCAGC
GTAGAGTTCTACGACCTACGAAATTACCAAGGCAACAAAGATGGTTGGCAACAGGGTGA
CCGCATCTTGTTTTGCGTGCCACTGAGAGATGCTTCTGAGCATCTTCCCATGTTTTTTAAC
CATCTGAATACCATGACATATCCGCACAACCTGATTGATCTGTCGTTTTTGGTGAGTGACT
CTTCGGATAACACCATGGGCGTGTTACTATCCAATCTACAGATGGCGCAATCGCAACAAG
ACAAGTCCAAAAGGTTTGGTAACATCGAAATCTATGAGAAGGACTTCGGCCAAATCATTG
GTCAGTCCTTTTCTGATCGTCACGGGTTTGGCGCCCAGGGCCCCAGAAGAAAGCTGATG
GCCAGGGCTCGTAATTGGCTTGGGTCCGTGGCCCTGAAACCATACCACTCCTGGGTTTAC
TGGAGAGACGTGGATGTCGAAACCATCCCCACTACCATCATGGAGGATCTAATGCATCAC
GACAAGGACGTCATTGTCCCCAACGTCTGGAGACCTTTACCAGACTGGCTGGGAAACAT
TCAACCCTACGATTTAAATTCATGGAAGGAATCAGAAGGGGGTCTTCAATTGGCGGATTC
CCTAGACGAAGATGCTGTCATTGTGGAAGGGTACCCAGAGTACGCCACTTGGAGACCTC
ACCTGGCATACATGAGAGACCCAAACGGTAATCCAGAAGACGAAATGGAATTGGACGGT
ATTGGAGGGGTCTCGATTCTCGCGAAAGCTAAAGTATTCAGAACAGGCTCACACTTTCCT
GCATTCTCCTTTGAAAAGCACGCAGAGACAGAAGCATTCGGTAGGCTCTCCCGCAGGAT
GAATTACAATGTCATCGGCTTGCCCCATTATGTCATCTGGCACATCTACGAACCTTCCAGC
GACGACTTAAAGCACATGGCTTGGATGGCTGAAGAGGAGAAGCGGAAACTAGAAGAAG
AGAGAATTCGCGAATTTTACAACAAGATCTGGGAGATTGGATTTGAAGATGTCAGAGACC
AATGGAACGAAGAAAGAGATTCAATCTTAAAAAACATTGACTCCACTTTGAACAATAAA
GTCACTGTAGACTGGTCTGAAGAGGGAGATGGCTCGGAGCTGGTGGACTCAAAGGGTGA
CTTTGTCTCACCAAACAACCAACAACAACAACAACAGCAACAACAACAACAACAGCAG
CAGCAGCAACAGCAACAGCAACAACAGCTCGACGGCAACCCGCAGGGGAAACCTCTTG
ATGACAACGACAAGAACAAGAAAAAACATCCTAAAGAAGTTCCATTAGACTTCGACCCT
GATAGAAACTAG
the amino acid sequence of YEL036Cp encoded by SEQ ID NO.1 is shown as SEQ ID NO. 2: MKYNNRKLSFNPTTVSIAGTLLTVFFLTRLVLSFFSISLFQLVTFQGIFKPYVPDFKNTPSVEFYDLRNYQGNKDGWQQGDRILFCVPLRDASEHLPMFFNHLNTMTYPHNLIDLSFLVSDSSDNTMGVLLSNLQMAQSQQDKSKRFGNIEIYEKDFGQIIGQSFSDRHGFGAQGPRRKLMARARNWLGSVALKPYHSWVYWRDVDVETIPTTIMEDLMHHDKDVIVPNVWRPLPDWLGNIQPYDLNSWKESEGGLQLADSLDEDAVIVEGYPEYATWRPHLAYMRDPNGNPEDEMELDGIGGVSILAKAKVFRTGSHFPAFSFEKHAETEAFGRLSRRMNYNVIGLPHYVIWHIYEPSSDDLKHMAWMAEEEKRKLEEERIREFYNKIWEIGFEDVRDQWNEERDSILKNIDSTLNNKVTVDWSEEGDGSELVDSKGDFVSPNNQQQQQQQQQQQQQQQQQQQQQQLDGNPQGKPLDDNDKNKKKHPKEVPLDFDPDRN x
Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.
Claims (10)
1. A saccharomyces cerevisiae engineering strain for high-yield triglyceride and diglyceride is characterized in that: the saccharomyces cerevisiae engineering strain is obtained by knocking out a YEL036C gene shown in SEQ ID NO.1 in saccharomyces cerevisiae host bacteria, and the amino acid sequence coded by the YEL036C gene is shown in SEQ ID NO. 2.
2. The saccharomyces cerevisiae engineering strain with high triglyceride and diglyceride yield according to claim 1, wherein the saccharomyces cerevisiae engineering strain is characterized in that: the engineering strain is obtained by knocking out YEL036C gene shown in SEQ ID NO.1 by using a Cre/loxP recombination system through a homologous recombination method;
wherein the host strain is Saccharomyces cerevisiae strain Saccharomyces cerevisiae BY4741.
3. The method for constructing a saccharomyces cerevisiae engineering strain with high triglyceride and diglyceride yield according to claim 1 or 2, wherein the method comprises the following steps: the method comprises the following steps:
(1) Designing a primer for knocking out the YEL036C gene from the saccharomyces cerevisiae genome and a sequence thereof according to the YEL036C gene sequence shown in SEQ ID No.1 and a Cre/loxP recombination system;
(2) Using plasmid carrying screening mark in Cre/loxP recombination system as template, using the primer designed in step (1), PCR amplifying DNA fragment for knocking out YEL036C gene from saccharomyces cerevisiae genome, and purifying;
(3) The prepared DNA fragment for knocking out the YEL036C gene from the saccharomyces cerevisiae genome is transferred into saccharomyces cerevisiae Saccharomyces cerevisiae BY4741 by adopting a high-efficiency yeast cell transformation method;
(4) And screening the auxotroph solid yeast culture medium corresponding to the screening mark to obtain the recombinant yeast strain.
4. A method of construction according to claim 3, wherein: the method comprises the following specific steps:
(1) The amplification primers were designed based on the nucleotide sequence of YEL036C shown in SEQ ID NO.1 and the Cre/loxP recombination system:
(2) Taking Cre/loxP recombinant system plasmid as a template, and carrying out PCR amplification to obtain a DNA fragment for knocking out the YEL036C gene;
(3) After agarose gel electrophoresis analysis is carried out on the PCR product, cutting gel and recovering a target fragment;
(4) Transferring the target fragment into Saccharomyces cerevisiae BY4741 BY adopting a lithium acetate conversion method;
(5) Screening by using a leucine auxotroph solid culture medium, wherein single colonies growing on the leucine auxotroph solid culture medium are candidate colonies;
(6) And (3) carrying out gene sequencing on the point position of the YEL036C in the genome of the candidate colony to determine that the YEL036C is successfully replaced by the screening marker gene, thus obtaining the gene.
5. The construction method according to claim 4, wherein: the leucine auxotroph solid medium per 1L was: 6.5-7g/L yeast nitrogen source, 0.72-0.84g/L leucine deficiency type amino acid mixture and 1.7-2.3% glucose, agar 2-3% and ddH are added after sterilization 2 O constant volume to 1L;
wherein the percentages are mass concentration percentages.
6. Use of an engineered strain of saccharomyces cerevisiae for the high production of triglycerides and diglycerides according to claim 1 or 2, for the production of triglycerides and diglycerides.
7. A method for producing triglycerides and diglycerides by fermentation using the saccharomyces cerevisiae engineering strain according to claim 1 or 2, characterized in that: the method comprises the following steps:
coating the saccharomyces cerevisiae engineering strain with high yield of triglyceride and diglyceride on a solid rich culture medium plate through three-area lines, culturing for 48-72h at 25-30 ℃, inoculating 1-2 rings of the activated strain in 3-5mL of liquid rich culture medium, culturing for 14-20h at 25-30 ℃ and 180-220rpm to obtain seed culture solution, inoculating the seed culture solution into the liquid rich culture medium according to the inoculation amount of 8-15%, and culturing for 12-24h at 25-30 ℃ and 180-220rpm to obtain the saccharomyces cerevisiae engineering strain.
8. The method according to claim 7, wherein: every 1L of solid rich culture medium is: 0.055-0.06g/L adenine sulfate, 8-12g/L yeast extract powder, 16-24g/L peptone, 18-22g/L agar, sterilizing, adding 1.8-2.2% glucose, and adding ddH 2 O constant volume to 1L;
every 1L of liquid rich culture medium is: 0.055-0.06g/L adenine sulfate, 8-12g/L yeast extract powder, 16-24g/L peptone, sterilizing, adding 1.8-2.2% glucose, and adding ddH 2 O constant volume to 1L;
wherein the percentages are mass concentration percentages.
9. A method for determining the intracellular triglyceride and diglyceride content of a saccharomyces cerevisiae engineering strain for high triglyceride and diglyceride production according to claim 1 or 2, wherein the method comprises the following steps: the method comprises the following specific steps:
(1) After fermentation of the saccharomyces cerevisiae engineering strain is finished, collecting all thalli;
(2) Crushing the collected thalli;
(3) Extracting with organic solvent, and mixing solvent layers;
(4) Extracting with organic solvent for several times, and mixing solvent layers;
(5) Drying the organic solvent layer with nitrogen, and redissolving the extracted substances;
(6) Detecting the obtained substance by using UPLC/MS;
(7) The intracellular triglyceride and diglyceride content was calculated.
10. A method for determining the growth curve of a saccharomyces cerevisiae engineering strain for high production of triglycerides and diglycerides according to claim 1 or 2, characterized in that: the method comprises the following specific steps:
(1) Inoculating Saccharomyces cerevisiae engineering strain and control strain for producing triglyceride and diglyceride into test tubes of liquid rich culture medium, respectively, and culturing at 25-30deg.C and 200-220 rpm/min for 12-18 hr;
(2) Sequentially adding 30 mu L, 40 mu L and 50 mu L of bacterial liquid into an EP tube containing 1mL of liquid rich culture medium, shaking and mixing uniformly, adding 200 mu L into a 100-pore plate, and taking 200 mu L of liquid rich culture medium as a control; placing the 100 pore plates in a full-automatic growth curve tester, culturing at 25-30 ℃, and measuring absorbance values at 600nm of wavelength every 1 h;
(3) On the abscissa of culture time X, OD 600 The value Y is the ordinate and the growth curve is plotted.
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