CN116622535A - Saccharomyces cerevisiae engineering strain for high yield of phosphatidylserine, construction method and application - Google Patents
Saccharomyces cerevisiae engineering strain for high yield of phosphatidylserine, construction method and application Download PDFInfo
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- CN116622535A CN116622535A CN202310832456.3A CN202310832456A CN116622535A CN 116622535 A CN116622535 A CN 116622535A CN 202310832456 A CN202310832456 A CN 202310832456A CN 116622535 A CN116622535 A CN 116622535A
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Abstract
The invention belongs to the technical field of genetic engineering, and discloses a saccharomyces cerevisiae engineering strain with high yield of phosphatidylserine, which is obtained by over-expressing YML115C gene shown in SEQ ID NO.1 in saccharomyces cerevisiae host bacteria, wherein the amino acid sequence coded by the YML115C gene is shown in SEQ ID NO. 2. After 24h fermentation, the constructed saccharomyces cerevisiae engineering strain has phosphatidylserine content of 7.94mg/gDCW, which is 4.36 times of that of the wild strain. The engineering strain disclosed by the invention has the advantages that the yield of phosphatidylserine is improved, the fermentation condition is simpler, the investment cost is reduced, and the engineering strain has a wide application 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 with high phosphatidylserine yield, a construction method and application.
Background
Phosphatidylserine (PS), also known as serine phospholipids, diacylglycerol phosphoserine, PS for short, is an important membrane phospholipid present in bacterial, yeast, plant, mammalian cells. Phosphatidylserine (PS) is a new resource food, has wide application in the fields of pharmacy and functional food, can improve Alzheimer's disease of old people, has the effects of protecting nerves and resisting oxidization, and also has the effects of improving memory, relieving tension, relieving depression and the like, and is a brain-specific nutrient substance.
The preparation method of PS mainly comprises an extraction method, a chemical synthesis method and an enzyme conversion method. The extraction method is a method for extracting PS (PS) by using animal and plant tissues as raw materials and chemical solvents, and generally, soybean or animal brain and viscera are used as raw materials, so that the phosphatidylserine obtained by extraction is low in purity and content, and the safety of the phosphatidylserine obtained by extraction cannot be ensured and the extraction cost is too high due to the large amount of organic solvents; the chemical method for synthesizing PS is to connect-OH on 2-C position and 3-C position of glycerin with L-serine to form intermediate compound by using phosphodiester bond, then esterify and oxidize the intermediate compound, and then deprotect the intermediate compound to obtain the product PS. The chemical synthesis method has complicated process flow and high required cost. The biological enzyme conversion method uses phosphatidylcholine with high content in nature and nucleophilic reagent L-serine as raw materials, and uses the transacylation reaction of phospholipase D to generate PS. Compared with the traditional extraction method, the biological enzyme catalysis method has the advantages of simplicity, high efficiency, mild reaction conditions, high yield and the like, but as the reaction is enzymatic, different reaction systems are needed to be considered to promote the reaction to proceed smoothly.
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. Currently, the market demand for phosphatidylserine products is enormous and the market demand is increasing. Therefore, based on the above investigation analysis, there is an urgent need in the art to develop a yeast strain that can efficiently produce phosphatidylserine.
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 provides a saccharomyces cerevisiae engineering strain with high yield of phosphatidylserine, a construction method and application.
The technical scheme adopted for solving the technical problems is as follows:
a saccharomyces cerevisiae engineering strain with high yield of phosphatidylserine is obtained by over-expressing YML115C gene shown in SEQ ID NO.1 in saccharomyces cerevisiae host bacteria, and the amino acid sequence coded by the YML115C gene is shown in SEQ ID NO. 2.
Further, the engineering strain adopts an expression vector pRS426 to overexpress YML115C gene shown in SEQ ID NO.1 during construction;
wherein the Saccharomyces cerevisiae host is Saccharomyces cerevisiae strain Saccharomyces cerevisiae BY4743.
The construction method of the saccharomyces cerevisiae engineering strain with high phosphatidylserine yield comprises the following steps:
the YML115C gene shown in SEQ ID NO.1 was overexpressed in Saccharomyces cerevisiae strain Saccharomyces cerevisiae BY4743.
Further, the engineering strain constructs YML115C fusion gene containing myc-HA tag.
Further, the specific steps are as follows:
(1) Designing a primer according to the nucleotide sequence of the YML115C gene shown in SEQ ID NO.1, and constructing a recombinant plasmid with myc-HA tag for over-expression of the YML115C gene;
(2) Introducing the recombinant plasmid constructed in the step (1) into a saccharomyces cerevisiae strain Saccharomyces cerevisiae BY4743 by a lithium acetate transformation method for expression;
(3) Screening is carried out on a uracil auxotroph solid medium, and single colony grown on the uracil auxotroph solid medium is a saccharomyces cerevisiae engineering strain with high yield of phosphatidylserine.
Further, per 1L uracil auxotroph solid medium was: 6.5-7g/L yeast nitrogen source, 0.72-0.84g/L uracil deficiency type amino acid mixture (DO Supplement-Ura) and 1.7-2.3% glucose, 2-3% agar, ddH, and sterilizing 2 O constant volume to 1L;
wherein the percentages are mass concentration percentages.
The application of the saccharomyces cerevisiae engineering strain with high phosphatidylserine yield in phosphatidylserine production is provided.
The method for producing phosphatidylserine by fermenting the saccharomyces cerevisiae engineering strain comprises the following steps:
coating a saccharomyces cerevisiae engineering strain with high yield of phosphatidylserine on a uracil auxotroph solid culture medium flat plate through three dividing lines, culturing for 48-72h at 25-30 ℃, inoculating 1-2 rings of the activated strain into a uracil auxotroph liquid culture medium filled with 3-5mL of uracil, culturing for 12-20h at 25-30 ℃ and 180-220rpm to obtain a seed culture solution, inoculating the seed culture solution into the uracil auxotroph liquid culture medium according to 2-15% of inoculum size, and culturing for 12-24h at 25-30 ℃ and 180-220rpm to obtain the strain.
Further, per 1L uracil auxotroph solid medium was: 6.5-7g/L yeast nitrogen source, 0.72-0.84g/L uracil deficiency type amino acid mixture (DO Supplement-Ura) and 1.7-2.3% glucose, 2-3% agar, ddH, and sterilizing 2 O constant volume to 1L;
the uracil auxotroph liquid medium per 1L was: 6.5-7g/L yeast nitrogen source, adding uracil deficiency type amino acid mixture (DO Supplement-Ura) 0.72-0.84g/L and glucose 1.7-2.3% after sterilization, and adding ddH 2 O constant volume to 1L;
wherein the percentages are mass concentration percentages.
The method for measuring the content of phosphatidylserine in cells of the saccharomyces cerevisiae engineering strain with high phosphatidylserine yield comprises the following specific steps:
(1) After fermentation of the saccharomyces cerevisiae engineering strain is finished, collecting all thalli;
(2) Freeze-drying and 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 phosphatidylserine content was calculated.
The method for determining the growth curve of the saccharomyces cerevisiae engineering strain with high phosphatidylserine yield comprises the following specific steps:
(1) Respectively inoculating a saccharomyces cerevisiae engineering strain and a control strain (transformed saccharomyces cerevisiae Saccharomyces cerevisiae BY4743 containing pRS 426) for producing phosphatidylserine into test tubes of uracil auxotroph liquid culture medium, and culturing for 12-18h at the temperature of 25-30 ℃ and the speed of 200-220 rpm/min;
(2) Sequentially adding 30 mu L, 40 mu L and 50 mu L of bacterial liquid into an EP tube containing 1mL of uracil auxotroph liquid culture medium, shaking and mixing uniformly, adding 200 mu L into a 100-pore plate, and taking 200 mu L of uracil auxotroph liquid 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 over-expressing YML115C gene shown in SEQ ID NO.1 in saccharomyces cerevisiae host bacteria. After 24h fermentation, the constructed saccharomyces cerevisiae engineering strain has phosphatidylserine content of 7.94mg/gDCW, which is 4.36 times of that of the wild strain. The engineering strain of the invention not only improves the yield of phosphatidylserine, but also has simpler fermentation conditions and reduced investment cost, and has wide development prospect.
2. The saccharomyces cerevisiae engineering strain has the advantage 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.
3. The invention establishes a foundation for the preparation of phosphatidylserine by constructing the high-yield phosphatidylserine strain to efficiently synthesize the phosphatidylserine, and can solve the urgent requirements of the development of foods, health-care products and medicaments in China on the phosphatidylserine, thereby forcefully promoting the development of the food and medicine industry in China.
Drawings
FIG. 1 is a diagram showing the amplification verification of a DNA fragment for over-expressing YML115C gene according to the present invention; wherein M: DNA molecular weight standard; lane 1: amplified DNA fragment of YML115C-myc-HA gene;
FIG. 2 shows recombinant plasmid pRS426-P of the present invention TPI1 -YML115C-myc-HA validation map; wherein M: DNA molecular weight standard; lane 1: pRS426-P TPI1 -YML115C-myc-HA cleavage verification;
FIG. 3 is a graph showing comparison of phosphatidylserine production in Saccharomyces cerevisiae engineering strain constructed in the present invention and control strain; wherein, 1: control 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 with high yield of phosphatidylserine is obtained by over-expressing YML115C gene shown in SEQ ID NO.1 in saccharomyces cerevisiae host bacteria, and the amino acid sequence coded by the YML115C gene is shown in SEQ ID NO. 2.
Preferably, the engineering strain adopts an expression vector pRS426 to overexpress YML115C gene shown in SEQ ID NO.1 during construction;
wherein, the engineering strain constructs YML115C fusion gene containing myc-HA tag during construction; the saccharomyces cerevisiae host strain is saccharomyces cerevisiae strain Saccharomyces cerevisiae BY4743.
The construction method of the saccharomyces cerevisiae engineering strain with high phosphatidylserine yield comprises the following steps:
the YML115C gene shown in SEQ ID NO.1 was overexpressed in Saccharomyces cerevisiae strain Saccharomyces cerevisiae BY4743.
Preferably, the specific steps are as follows:
(1) Designing a primer according to the nucleotide sequence of the YML115C gene shown in SEQ ID NO.1, and constructing a recombinant plasmid with myc-HA tag for over-expression of the YML115C gene;
(2) Introducing the recombinant plasmid constructed in the step (1) into a saccharomyces cerevisiae strain Saccharomyces cerevisiae BY4743 by a lithium acetate transformation method for expression;
(3) Screening is carried out on a uracil auxotroph solid medium, and single colony grown on the uracil auxotroph solid medium is a saccharomyces cerevisiae engineering strain with high yield of phosphatidylserine.
Preferably, the uracil auxotroph solid medium is: 6.5-7g/L yeast nitrogen source, 0.72-0.84g/L uracil deficiency type amino acid mixture (DO Supplement-Ura) and 1.7-2.3% glucose, 2-3% agar, ddH, and sterilizing 2 O constant volume to 1L;
wherein the percentages are mass concentration percentages.
The application of the saccharomyces cerevisiae engineering strain with high phosphatidylserine yield in phosphatidylserine production is provided.
The method for producing phosphatidylserine by fermenting the saccharomyces cerevisiae engineering strain comprises the following steps:
coating a saccharomyces cerevisiae engineering strain with high yield of phosphatidylserine on a uracil auxotroph solid culture medium flat plate through three dividing lines, culturing for 48-72h at 25-30 ℃, inoculating 1-2 rings of the activated strain into a uracil auxotroph liquid culture medium filled with 3-5mL of uracil, culturing for 12-20h at 25-30 ℃ and 180-220rpm to obtain a seed culture solution, inoculating the seed culture solution into the uracil auxotroph liquid culture medium according to 2-15% of inoculum size, and culturing for 12-24h at 25-30 ℃ and 180-220rpm to obtain the strain.
Preferably, the method comprises the steps of,
the uracil auxotroph solid medium per 1L was: 6.5-7g/L yeast nitrogen source, 0.72-0.84g/L uracil deficiency type amino acid mixture (DO Supplement-Ura) and 1.7-2.3% glucose, 2-3% agar, ddH, and sterilizing 2 O constant volume to1L；
The uracil auxotroph liquid medium per 1L was: 6.5-7g/L yeast nitrogen source, adding uracil deficiency type amino acid mixture (DO Supplement-Ura) 0.72-0.84g/L and glucose 1.7-2.3% after sterilization, and adding ddH 2 O constant volume to 1L;
wherein the percentages are mass concentration percentages.
The method for measuring the content of phosphatidylserine in cells of the saccharomyces cerevisiae engineering strain with high phosphatidylserine yield comprises the following specific steps:
(8) After fermentation of the saccharomyces cerevisiae engineering strain is finished, collecting all thalli;
(9) Freeze-drying and 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 phosphatidylserine content was calculated.
The method for determining the growth curve of the saccharomyces cerevisiae engineering strain with high phosphatidylserine yield comprises the following specific steps:
(1) Respectively inoculating a saccharomyces cerevisiae engineering strain and a control strain for producing phosphatidylserine into test tubes of uracil auxotroph liquid culture medium, and culturing for 12-18h under the conditions of 25-30 ℃ and 200-220 rpm/min;
(2) Sequentially adding 30 mu L, 40 mu L and 50 mu L of bacterial liquid into an EP tube containing 1mL of uracil auxotroph liquid culture medium, shaking and mixing uniformly, adding 200 mu L into a 100-pore plate, and taking 200 mu L of uracil auxotroph liquid 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 Value Y is verticalAnd (5) drawing a growth curve according to coordinates.
Specifically, the related preparation and detection are as follows:
EXAMPLE 1 construction of YML115C Gene overexpression vector
Primers were designed based on the nucleotide sequence of YML115C gene
pRS426-P TPI1 -YML115C-myc-HA-F：
5’-CTACAAAAAACACATACAATGGGCATGTTTTTTAATTTAAGGT-3’
YML115C-myc-HA-R：
5’-AAAAGCTGGAGCTCCACCGCGGTGGCGGCCGCTTAGCCCGCATAGTCAGG-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;
PCR amplification was performed using BY4741 genomic DNA as a template to obtain a DNA fragment of YML115C gene with myc-HA tag. The PCR reaction conditions were: the temperature is kept at 95 ℃ for 3min,95 ℃ for 30s,56 ℃ for 30s and 72 ℃ for 2min, and the temperature is kept at 72 ℃ for 10min and 4 ℃ after being circulated for 35 times. After agarose gel electrophoresis analysis of the PCR products, the target fragment was recovered by cutting into gel (see FIG. 1).
EXAMPLE 2 construction of high-yield phosphatidylserine Saccharomyces cerevisiae engineering Strain
By genetic engineering, the myc-HA tagged YML115C fusion gene was combined with P linearized by restriction endonuclease Not I TPI1 Recombinant ligation of pRS426 to obtain recombinant plasmid for double cleavage verification (FIG. 2), sequencing by gold-only company, and naming the correctly sequenced recombinant plasmid as pRS426-P TPI1 -YML115C-myc-HA. The obtained recombinant plasmid is transformed into saccharomyces cerevisiae strain Saccharomyces cerevisiae BY4743, and then high-yield phosphatidyl is obtained by screening uracil auxotroph solid yeast culture mediumSerine saccharomyces cerevisiae engineering strain.
EXAMPLE 3 fermentation production of phosphatidylserine by Saccharomyces cerevisiae engineering Strain
(1) Activating the bacterial species
Scribing the Saccharomyces cerevisiae engineering strain producing phosphatidylserine on a uracil auxotroph solid yeast culture medium plate, and culturing at 30 ℃ for 48 hours;
(2) Seed culture
Inoculating the activated strain into a test tube filled with 5mL uracil auxotroph liquid medium, and culturing at 30deg.C and 220rpm for 12h to obtain seed culture solution;
uracil auxotroph liquid medium: 6.7g/L of yeast nitrogen source base (YeastNitrogen Base), and 0.77g/L of uracil-deficient amino acid mixture (Do supplement (-ura) powder) and 2% of glucose are added after sterilization, wherein the percentages are mass concentration percentages;
(3) Shaking flask fermentation culture
The seed culture was inoculated at 2% into a triangular flask containing 200mL uracil auxotroph liquid medium and fermented at 30℃and 220rpm for 24 hours.
Uracil auxotroph liquid medium is: 6.7g/L of yeast nitrogen source base (YeastNitrogen Base), 0.77g/L of uracil-deficient amino acid mixture (Do supplement (-ura) powder) and 2% of glucose are added after sterilization, wherein the percentages are mass concentration percentages.
Example 4 determination of phosphatidylserine content in Saccharomyces cerevisiae engineering strains:
(1) After the fermentation culture (as described in example 3) was completed, all the cells were collected and freeze-dried;
(2) Weighing 0.2g of dry yeast, placing the dry yeast in a 2mL EP tube, adding two small steel balls, and adding 800 mu L of precooled dichloromethane/methanol buffer solution (volume ratio is 3:1);
(3) Grinding TissueLyser for 5min, and precipitating in a refrigerator at-20deg.C for 2h or overnight;
(4) 25,000Xg, centrifuging at 4deg.C for 15min, collecting 650 μL of supernatant, placing into a new EP tube, centrifuging again at 25000Xg, and centrifuging at 4deg.C for 15min;
(5) Taking 600 mu L of supernatant, freezing and pumping, and re-dissolving with 600 mu L of lipid re-solution (the volume ratio of isopropanol to acetonitrile to water is 2:1:1);
(6) 25,000Xg, centrifugation at 4℃for 20min;
(7) 60 mu L of supernatant is transferred to a 96-well microplate, and is marked by sealing film, and UPLC-MS detection is carried out.
As shown in FIG. 3, it can be seen from the graph that the phosphatidylserine content of the Saccharomyces cerevisiae engineering strain of the present invention reaches 7.94mg/gDCW, respectively, which is 4.36 times the phosphatidylserine content of the wild type strain after 24 hours of fermentation.
Example 5 growth curve of Saccharomyces cerevisiae engineering strain:
(1) Respectively inoculating a saccharomyces cerevisiae engineering strain and a control strain for producing phosphatidylserine into test tubes of uracil auxotroph liquid culture medium, and culturing for 16 hours at the temperature of 27 ℃ and the speed of 210 rpm/min;
(2) Sequentially adding 30 mu L, 40 mu L and 50 mu L of bacterial liquid into an EP tube containing 1mL of uracil auxotroph liquid culture medium, shaking and mixing uniformly, adding 200 mu L into a 100-pore plate, and taking 200 mu L of uracil auxotroph liquid 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.
Uracil auxotroph liquid medium is: yeast nitrogen source base (YeastNitrogen Base) 6.7g/L, uracil deficiency type amino acid mixture (Do supply (-ura) powder) 0.77g/L and glucose 2% were added after sterilization.
Wherein the percentages are mass concentration percentages.
The genes involved in the invention are as follows
The YML115C gene is as follows (SEQ ID NO.1 of the sequence Listing):
ATGGGCATGTTTTTTAATTTAAGGTCAAATATAAAGAAGAAAGCCATGGACAATGGAC
TAAGCCTGCCCATTTCAAGGAACGGTAGCTCGAACAACATCAAGGACAAACGCTCAG
AGCATAACTCCAACTCATTAAAGGGCAAATACAGGTACCAGCCGCGCTCCACACCGT
CTAAATTCCAGCTTACGGTGAGTATCACATCTCTTATTATTATCGCCGTTCTGTCGTTAT
ATCTCTTTATATCATTTCTCTCCGGAATGGGCATTGGTGTATCCACGCAAAATGGTAGG
TCGTTGTTGGGTTCCTCAAAATCCTCCGAAAATTACAAGACTATCGACCTAGAAGATG
AAGAATATTACGACTATGATTTTGAGGATATCGATCCTGAAGTGATTTCAAAATTTGAT
GATGGTGTGCAACATTATCTAATATCACAATTTGGTTCAGAAGTGTTGACTCCCAAGG
ATGATGAAAAATACCAAAGGGAACTCAACATGCTTTTTGATTCCACTGTTGAGGAGTA
CGACCTGTCGAATTTTGAAGGTGCTCCGAATGGATTGGAAACACGTGATCACATTCTT
TTATGCATTCCACTAAGAAACGCTGCGGATGTATTGCCATTAATGTTCAAGCATTTAAT
GAACCTAACCTATCCACACGAGCTGATTGATCTGGCCTTTTTAGTCAGTGATTGCTCA
GAAGGTGACACTACGTTGGATGCTTTAATAGCGTATTCTAGGCACTTACAAAATGGCA
CGTTGTCTCAGATTTTTCAAGAGATTGACGCTGTCATTGATTCGCAAACAAAAGGCAC
CGATAAATTATATCTTAAATATATGGACGAAGGTTATATCAACCGTGTCCACCAGGCATT
TTCACCACCATTCCATGAAAATTATGACAAGCCATTTAGATCAGTACAAATTTTCCAAA
AGGATTTTGGCCAAGTAATTGGACAAGGTTTTAGTGACAGACATGCCGTTAAGGTTC
AAGGTATAAGACGAAAACTAATGGGGAGGGCAAGAAATTGGTTAACTGCCAATGCTT
TGAAACCTTACCACTCATGGGTTTATTGGAGAGATGCTGATGTAGAGCTGTGCCCTGG
TTCAGTCATTCAAGATTTGATGAGCAAAAACTACGATGTTATCGTCCCTAACGTTTGG
AGACCACTACCTACATTTTTGGGAACTGAACAACCATATGATTTGAATTCTTGGATGG
AATCTCAGGAGGCACTAGCATTGGCAAAGACTCTTGACGAAGATGATGTTATTGTAGA
AGGCTATGCAGAATATCCTACGTGGAGAGTTCACTTAGCCTATATCAGAGATGCAGAA
GGTGATCCAAATGAAGCGGTGGACTTGGACGGTGTAGGGGGAGTTTCTATTTTGGCG
AAGGCCAAAATATTTAGAAACGGGGTACAGTTTCCTGCATTTACTTTTGAAAATCATG
CAGAAACAGAAGCATTTGGTAAAATGGCAAAGAAAATGGGGTACAGGGTTGGTGGT
TTACCGCATTATACTATTTGGCATATTTATGAACCGAGCGATGACGATTTGAAGGAAAT
TGCGTCAAGGGAAAGAGAGAAGAGAAGACAATCAGAGTAA
the amino acid sequence of YML115Cp encoded by SEQ ID NO.1 is shown as SEQ ID NO. 2: MGMFFNLRSNIKKKAMDNGLSLPISRNGSSNNIKDKRSEHNSNSLKGKYRYQPRSTPSKFQLTVSITSLIIIAVLSLYLFISFLSGMGIGVSTQNGRSLLGSSKSSENYKTIDLEDEEYYDYDFEDIDPEVISKFDDGVQHYLISQFGSEVLTPKDDEKYQRELNMLFDSTVEEYDLSNFEGAPNGLETRDHILLCIPLRNAADVLPLMFKHLMNLTYPHELIDLAFLVSDCSEGDTTLDALIAYSRHLQNGTLSQIFQEIDAVIDSQTKGTDKLYLKYMDEGYINRVHQAFSPPFHENYDKPFRSVQIFQKDFGQVIGQGFSDRHAVKVQGIRRKLMGRARNWLTANALKPYHSWVYWRDADVELCPGSVIQDLMSKNYDVIVPNVWRPLPTFLGTEQPYDLNSWMESQEALALAKTLDEDDVIVEGYAEYPTWRVHLAYIRDAEGDPNEAVDLDGVGGVSILAKAKIFRNGVQFPAFTFENHAETEAFGKMAKKMGYRVGGLPHYTIWHIYEPSDDDLKEIASREREKRRQSE x
Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various alternatives, variations 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 with high yield of phosphatidylserine is characterized in that: the saccharomyces cerevisiae engineering strain is obtained by over-expressing YML115C gene shown in SEQ ID NO.1 in saccharomyces cerevisiae host bacteria, and the amino acid sequence coded by the YML115C gene is shown in SEQ ID NO. 2.
2. The saccharomyces cerevisiae engineering strain with high yield of phosphatidylserine according to claim 1, wherein the saccharomyces cerevisiae engineering strain is characterized in that: the engineering strain adopts an expression vector pRS426 to overexpress YML115C gene shown in SEQ ID NO.1 during construction;
wherein the Saccharomyces cerevisiae host is Saccharomyces cerevisiae strain Saccharomyces cerevisiae BY4743.
3. The method for constructing the saccharomyces cerevisiae engineering strain with high yield of phosphatidylserine according to claim 1 or 2, which is characterized in that: the method comprises the following steps:
the YML115C gene shown in SEQ ID NO.1 was overexpressed in Saccharomyces cerevisiae strain Saccharomyces cerevisiae BY4743.
4. A method of construction according to claim 3, wherein: the method comprises the following specific steps:
(1) Designing a primer according to the nucleotide sequence of the YML115C gene shown in SEQ ID NO.1, and constructing a recombinant plasmid with myc-HA tag for over-expression of the YML115C gene;
(2) Introducing the recombinant plasmid constructed in the step (1) into a saccharomyces cerevisiae strain Saccharomyces cerevisiae BY4743 by a lithium acetate transformation method for expression;
(3) Screening is carried out on a uracil auxotroph solid medium, and single colony grown on the uracil auxotroph solid medium is a saccharomyces cerevisiae engineering strain with high yield of phosphatidylserine.
5. The construction method according to claim 4, wherein: the uracil auxotroph solid medium per 1L was: 6.5-7g/L yeast nitrogen source, 0.72-0.84g/L uracil deficiency type amino acid mixture and 1.7-2.3% glucose are added after sterilization, 2-3% agar is added, ddH is used 2 O constant volume to 1L;
wherein the percentages are mass concentration percentages.
6. Use of the saccharomyces cerevisiae engineering strain with high yield of phosphatidylserine according to claim 1 or 2 in phosphatidylserine production.
7. A method for producing phosphatidylserine by fermentation using the saccharomyces cerevisiae engineering strain according to claim 1 or 2, wherein: the method comprises the following steps:
coating a saccharomyces cerevisiae engineering strain with high yield of phosphatidylserine on a uracil auxotroph solid culture medium flat plate through three dividing lines, culturing for 48-72h at 25-30 ℃, inoculating 1-2 rings of the activated strain into a uracil auxotroph liquid culture medium filled with 3-5mL of uracil, culturing for 12-20h at 25-30 ℃ and 180-220rpm to obtain a seed culture solution, inoculating the seed culture solution into the uracil auxotroph liquid culture medium according to 2-15% of inoculum size, and culturing for 12-24h at 25-30 ℃ and 180-220rpm to obtain the strain.
8. The method according to claim 7, wherein: the uracil auxotroph solid medium per 1L was: 6.5-7g/L yeast nitrogen source, 0.72-0.84g/L uracil deficiency type amino acid mixture (DO Supplement-Ura) and 1.7-2.3% glucose, 2-3% agar, ddH, and sterilizing 2 O constant volume to 1L;
the uracil auxotroph liquid medium per 1L was: 6.5-7g/L yeast nitrogen source, 0.72-0.84g/L uracil deficiency type amino acid mixture and 1.7-2.3% glucose are added after sterilization, ddH is used 2 O constant volume to 1L;
wherein the percentages are mass concentration percentages.
9. A method for determining the intracellular phosphatidylserine content of a saccharomyces cerevisiae engineering strain with high yield of phosphatidylserine according to claim 1 or 2, wherein: the method comprises the following specific steps:
(1) After fermentation of the saccharomyces cerevisiae engineering strain is finished, collecting all thalli;
(2) Freeze-drying and 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 phosphatidylserine content was calculated.
10. A method for determining the growth curve of a saccharomyces cerevisiae engineering strain with high yield of phosphatidylserine according to claim 1 or 2, wherein the method comprises the following steps: the method comprises the following specific steps:
(1) Inoculating Saccharomyces cerevisiae engineering strain producing phosphatidylserine and control strain, namely transformed Saccharomyces cerevisiae Saccharomyces cerevisiae BY4743 containing pRS426, respectively in test tubes of uracil auxotroph liquid culture medium, and culturing for 12-18h at 25-30 ℃ and 200-220 rpm/min;
(2) Sequentially adding 30 mu L, 40 mu L and 50 mu L of bacterial liquid into an EP tube containing 1mL of uracil auxotroph liquid culture medium, shaking and mixing uniformly, adding 200 mu L into a 100-pore plate, and taking 200 mu L of uracil auxotroph liquid 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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