CN112575023B - A method for highly expressing phospholipase D in Streptomyces and recombinant Streptomyces - Google Patents

Abstract

The invention belongs to the field of genetic engineering, and relates to a method for efficiently expressing phospholipase D in streptomycete, which comprises the following steps of selecting a heterologous efficient expression host of the phospholipase D, screening an optimal source of a phospholipase D synthetic gene, obtaining a phospholipase D gene fragment from streptomycete, connecting a target gene to form a phospholipase D recombinant plasmid, transferring the recombinant plasmid to a host bacterium, fermenting and culturing the recombinant strain, and detecting the enzyme activity of the phospholipase D, so as to obtain the recombinant streptomycete for efficiently expressing the phospholipase D. The recombinant protein heterologous expression is carried out by selecting the phospholipase D from streptomyces, the recombinant protein heterologous expression has natural advantages, the synthesized gene of the phospholipase D is codon optimized, and the Streptomyces lividans SBT5 is selected as a heterologous expression host, so that the recombinant strain has extremely high innovativeness and practicability due to the excellent heterologous expression effect and good extracellular active secretion expression effect.

Description

A method for highly expressing phospholipase D in Streptomyces and recombinant Streptomyces

technical field

The invention belongs to the field of genetic engineering and relates to a method for highly expressing phospholipase D in streptomyces and recombinant streptomyces.

Background technique

In recent years, people have begun to pay attention to the preparation of phosphatidylserine by enzymatic conversion, that is, the synthesis of substrates phosphatidylcholine and L-serine catalyzed by phospholipase D; D, has the activity of transphosphatidyl, can catalyze the synthesis of phosphatidylserine in an aqueous environment, the reaction conditions are mild, the by-products are few, the product yield is high, and the quality is good, it will become the main production method of industrialization in the future; but directly from The catalytic activity of wild bacteria screened in the environment is low, and the secretion of phospholipase D is far below the industrial production standard, which seriously affects the industrial application of phospholipase D in the synthesis of phosphatidylserine.

Then in order to obtain high-yield phospholipase D, it is hoped to carry out gene construction and genetic modification by genetic engineering means to obtain an engineering strain that can efficiently express phospholipase D, and then use the strain to ferment and extract phospholipase D, then construct a kind that can efficiently express phospholipase D. The strain expressing phospholipase D becomes very critical, and how to make the newly constructed recombinant plasmid express well in a heterologous host is also the direction of thinking for constructing a recombinant plasmid.

At present, many scholars have tried to express phospholipase D heterologously. The most commonly used host is Escherichia coli, followed by Streptomyces, yeast and Bacillus subtilis. In order to investigate the amino acid residues that affect the thermal stability of phospholipase D, some scholars transformed the phospholipase D gene from Streptomyces into E. coli for expression, and studied the enzymatic properties; and Bacillus subtilis has a mature heritage operating system , and it is a food-grade strain. Some scholars introduced the phospholipase D gene derived from E.coliK12 into B.subtilis104 for heterologous expression, and detected that the extracellular enzyme activity was only 0.15u/mL; some scholars constructed two The constitutive shuttle plasmid realizes the high-efficiency expression of phospholipase D in Streptomyces, and after 3 days of fermentation, the enzyme activity reaches 58u/mL, while the secretion of phospholipase D in Streptomyces protozoa is only 1.1u/mL; another scholar will The phospholipase D gene derived from Streptomyces was expressed in Pichia pastoris and Yarrowia lipolytica respectively, and the pIC9K expression vector carrying the endogenous signal peptide of the target gene was constructed and transferred into Pichia pastoris. After three days of fermentation, the its enzyme activity.

Through genetic experiments, it is found that commonly used heterologous hosts carrying the target gene, such as Escherichia coli and Bacillus subtilis, have poor in vivo expression or secretion, then find a more suitable heterologous host and make the recombinant plasmid work well Expression has also become a research direction.

Contents of the invention

The purpose of the present invention is to find a method for highly expressing phospholipase D in Streptomyces, so as to determine the high-efficiency expression recombinant strains for the production of phospholipase D in industrial production.

The technical scheme that the present invention adopts is as follows:

A method for highly expressing phospholipase D in streptomyces, comprising the steps of:

Step 1, select a heterologous high-efficiency expression host of phospholipase D, select Streptomyces as the host, and select the phospholipase D gene derived from Streptomyces as the target gene as the transformation object of genetic engineering;

Step 2, screening the best source of phospholipase D synthetic gene, carrying out comparative experiments by measuring enzyme activity, selecting S.antibioticus Streptomyces antibioticus and S.chromofuscus Streptomyces fuvidus as the target strains for obtaining the target gene;

Step 3, obtaining the phospholipase D gene fragment derived from Streptomyces, and obtaining the phospholipase D gene derived from the antibiotic Streptomyces and the phospholipase D gene derived from Streptomyces fusceus by PCR amplification technology;

Step 4, connecting the target gene to form a phospholipase D recombinant plasmid, connecting the phospholipase D gene from the antibiotic Streptomyces to the commonly used expression vector of Streptomyces and the high-efficiency expression vector of Streptomyces to form the first recombinant plasmid and the third recombinant plasmid, and then The phospholipase D gene derived from Streptomyces fusceus is respectively connected to the common expression vector of Streptomyces and the high-efficiency expression vector of Streptomyces to form the second recombinant plasmid and the fourth recombinant plasmid;

Step 5: Conjugate and transfer the recombinant plasmid to the host bacterium, and then obtain the first recombinant strain and the second recombinant strain corresponding to the first recombinant plasmid, the second recombinant plasmid, the third recombinant plasmid, and the fourth recombinant plasmid in sequence by means of conjugative transfer , the third recombinant strain, the fourth recombinant strain;

Step 6, the fermentation culture of the recombinant strain and the detection of phospholipase D enzyme activity, and then find the strain with the best expression effect through the detection of the enzyme activity of the original strain and the recombinant strain, wherein the original strain is S.lividans Streptomyces lividans, Streptomyces lividans, S.antibioticus Streptomyces antibioticus and S.chromofuscus Streptomyces fuvidus; and the recombinant strains are the first recombinant strain, the second recombinant strain, the third recombinant strain and the fourth recombinant strain obtained in Step 5.

Specifically, the PCR amplification technique described in step 3 includes: (1) extracting the genomic DNAs of S.antibioticus antibiotic Streptomyces and S.chromofuscus chromofuscus respectively; Genomic DNA of mold and S.chromofuscus streptomyces fuvidus was used as templates, primers were designed for PCR to obtain the target gene and sequenced for verification, (3) Codon optimization was performed on the obtained target fragment, and then the phospholipase D gene and color Phospholipase D gene of Streptomyces fucoidans origin.

Preferably, the commonly used expression vector of Streptomyces in step 4 is pSET152.

Preferably, the Streptomyces high-efficiency expression vector in step 4 is pMS82.

Preferably, the host bacteria in step five is Streptomyces SBT5.

A recombinant Streptomyces expressing phospholipase D efficiently, using the above method to obtain recombinant plasmid pMS82-PLD _Anti, and then conjugating and transferring the recombinant plasmid pMS82-PLD _Anti to Streptomyces SBT5 to form a recombinant strain.

In summary, owing to adopting above-mentioned technical scheme, the beneficial effect of the present invention is:

1. Select the phospholipase D synthesis gene derived from Streptomyces as the target gene to improve the expression efficiency of phospholipase D; through the comparative experiments of multiple strains, the results show that Streptomyces itself produces phospholipase D with outstanding effect and the best hydrolysis activity , Therefore, the choice of Streptomyces-derived phospholipase D for heterologous expression of recombinant proteins has natural advantages.

2. By using the Streptomyces high-efficiency expression vector as the upstream element of the synthetic gene, which contains the Streptomyces strong promoter and secreted signal peptide, the expression efficiency of phospholipase D is greatly improved; the constitutive strong promoter has a strong effect on the target gene High-efficiency expression is very important, and the addition of a signal peptide with good secretion effect enables the target protein to be directly secreted to the outside of the cell after formation, which simplifies the production process and reduces production costs.

3. Select Streptomyces lividans SBT5 as the heterologous expression host, which further improves the expression efficiency of phospholipase D; through research, it is found that Streptomyces has innovative and outstanding advantages as a host, and its advantages are: the host strain has knocked out three A biosynthetic gene cluster of endogenous antibiotics, with a clear background of secondary metabolism; meanwhile, the self-constructed high-efficiency expression vector performed well in this host bacteria, making the heterologous expression efficiency of phospholipase D much higher than other hosts.

4. According to the codon preference of the host bacteria, the codon optimization of the phospholipase D synthesis gene is carried out, which makes the codon matching between the target gene and the host stronger, and the replication of the target gene in the host and the process of directing protein synthesis More efficient; excellent heterologous expression effect plus good extracellular active secretion expression effect, making this recombinant strain highly innovative and practical.

Detailed ways

In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples.

A method for highly expressing phospholipase D in streptomyces, comprising the steps of:

Step 1: Select a host for heterologous and high-efficiency expression of phospholipase D

Through data query, many scholars have tried to express phospholipase D heterologously. The most commonly used host is Escherichia coli, followed by Streptomyces, yeast and Bacillus subtilis, and the data of phospholipase D enzyme activity are summarized. The form is as follows:

According to the content of the above table, it can be determined that Streptomyces is selected as the host, and the phospholipase D gene derived from Streptomyces is selected as the transformation object of genetic engineering, so as to obtain an engineering strain with relatively high phospholipase D enzyme activity.

Step two, screening the best source of phospholipase D synthesis gene

Streptomyces from different sources were selected, and the enzyme activity levels of phospholipase D were detected with themselves as hosts, and then the expression levels of phospholipase D in three different Streptomyces were compared.

The specific method is: select the bacterial strains of S.lividans lividans lividans, S.antibioticus antibiotic streptomyces and S.chromofuscus viridans streptomyces to ferment and cultivate for 3 days, take the culture supernatant, after ammonium sulfate precipitation and centrifugation, after simple 100 μg of total protein was used for the catalytic activity experiment, and the catalytic activity of the phospholipase D protein derived from S.lividans lividans Streptomyces lividans was compared and found that the catalytic activity of the phospholipase D protein produced by the antibiotic Streptomyces S.antibioticus was higher than high, the results are shown in the table below.

The strains of S.lividans lividans, S.antibioticus and S.chromofuscus were purchased from Shanghai Shenggong.

The results showed that the phospholipase D derived from Streptomyces antibioticus and Streptomyces fusceus both had high enzymatic activity. Considering the unknown of heterologous expression of the target gene and gene mutation and other factors, S.antibioticus antibiotic Streptomyces and S.chromofuscus were selected at the same time. Streptomyces luteus was used as the target strain to obtain the target gene.

Step 3, obtaining the phospholipase D gene fragment derived from Streptomyces

(1) Extraction of Streptomyces genomic DNA from different sources, specifically, S.antibioticus Streptomyces antibioticus and S.chromofuscus Streptomyces chromofuscus were carried out according to the following method:

Suspend an appropriate amount of bacteria in 500 μl lysozyme solution (2%), incubate at 37°C for about 1 hour until the bacteria are completely lysed, add 500 μl alkaline SDS solution (0.3mol/L NaOH, 2% SDS), shake and mix completely, Open the cap of the tube and place it at 70°C for 15 minutes (for plasmids larger than 20kb, it is best to place it at 55°C for 30 minutes), then cool it to room temperature in a water bath, add 100 μl of acidic phenol/chloroform solution, and shake it with a mixer until the liquid is thoroughly mixed Evenly, centrifuge at 12000rpm for 5min, remove the supernatant, discard the white middle layer; repeat extraction with neutral phenol/chloroform until the middle layer is invisible (or very little); add 1/10 volume of Precipitate with 3M NaAc solution and 1 volume of isopropanol for 5 minutes (or 2.2 volumes of absolute ethanol for 1 h), centrifuge at 12000 rpm for 8 min, wash the precipitate twice with 70% ethanol; add a certain amount of TE (ddH2O) after drying The buffer dissolves.

(2) Using Streptomyces genomic DNA as a template, design primers for PCR to obtain the target gene and verify it by sequencing. The specific primers are as follows:

Antimicrobial Streptomyces Upstream and Downstream Primers

Then use Primer STAR mix polymerase to amplify the target fragment to obtain the target fragment.

(3) Carry out codon optimization to the target fragment obtained, adopt Codon Usage Analyzer online codon statistical table processing software (http://bioinformatics.org/codon/cgi-bin/codon.cgi) to carry out optimization design, it makes to The statistics of the codons are displayed in the form of graphs, which makes the phospholipase D genes derived from the antibiotic Streptomyces and Streptomyces fusceus more in line with the codon preference of the host bacteria;

The finally obtained phospholipase D gene sequence derived from the antibiotic Streptomyces is shown in SEQ ID NO.1;

The finally obtained phospholipase D gene sequence derived from Streptomyces fusceus is shown in SEQ ID NO.2.

Step 4, connecting the target gene to form a phospholipase D recombinant plasmid

The specific method is: select Streptomyces commonly used expression vector pSET152 (from the ADDGENE library and purchased from Zhongyuan Biotechnology Company) and Streptomyces high-efficiency expression vector pMS82 (from the ADDGENE library and purchased from Zhongyuan Biotechnology Company), respectively, and two different The source phospholipase D target gene is connected to form a variety of phospholipase D expression plasmids.

Step 5, conjugative transfer of the recombinant plasmid to the host strain SBT5

The host strain SBT5 is from literature: Bai Tingli et al., Construction of efficient heterologous expression host SBT5 of Streptomyces lividans, Journal of Huazhong Agricultural University, January 2014, Volume 33, Issue 1.

The specific method of joint transfer is:

Escherichia coli ET12567/pUZ8002 was used as the donor bacteria for conjugative transfer, and the plasmid pUZ8002 contained oriT conjugative transfer initiation site, which could assist the transfer of the target plasmid.

First, transform the target plasmid into Escherichia coli ET12567/pUZ8002 by means of electrical transformation or chemical transformation, culture Escherichia coli with LB containing corresponding antibiotics until the OD600 is 0.4-0.6, collect the cells, and culture them with LB without antibiotics The base was washed twice to fully remove the antibiotics in the supernatant.

At the same time, suspend the freshly harvested SBT5 Streptomyces spores in a certain volume of 2×YT medium (if the spores are stored in 20% glycerol, centrifuge to remove the glycerol, wash twice with sterile water, and then use a certain volume of Suspended in 2×YT medium), heat shock at 50°C for 10 minutes, and fully cool.

Mix the treated SBT5 Streptomyces spores with Escherichia coli, smear them on MS medium without antibiotics, and culture them at 30°C for 12-16 hours. Pyrimidine covers the plate; 2-3 days after covering, the conjugative transfer can be seen to grow out, and then the recombinant strain is obtained as shown below.

Step 6, fermentation culture of the recombinant strain and detection of phospholipase D enzyme activity

Through the fermentation culture and enzyme activity detection of the original strain and the recombinant strain, the strain with the best expression effect can be found.

Among them, the original strains were S. lividans lividans, S. antibioticus and S. chromofuscus.

The recombinant strains are the first recombinant strain, the second recombinant strain, the third recombinant strain and the fourth recombinant strain obtained in Step 5.

Use the above-mentioned strains to carry out fermentation culture and enzyme activity detection using a general method. The specific method is: Streptomyces inoculated into the fermentation medium in a 30°C incubator for 5-7 days; the bacteria solution was cultured until turbid, centrifuged, and the supernatant was taken; 0.45 um filter membrane to filter the supernatant to further remove impurities; ammonium sulfate precipitated protein overnight. Centrifuge at high speed, discard the supernatant; redissolve the protein, desalt by ultrafiltration. The hydrolytic activity of phospholipase D was determined spectrophotometrically with p-PNP (phosphatidyl p-nitrophenol) as substrate. Phosphatidyl transfer activity was determined by the conversion of PC to phosphatidylethanolamine (PE). The concentration of PC and PE was determined by using chloroform-methanol-water (13:5:0.8 volume ratio) as the developing agent, TLC on GF254 silica gel plate; iodine vapor staining The thin-layer plate uses image processing software to measure the spot area and gray value, and calculates the concentration of phospholipid components. Enzyme protein concentration was determined according to the Bradford method with bovine serum albumin as the standard, and used for PLD specific activity determination. The results obtained are as follows:

According to the data in the above table, it can be seen that the enzyme activity assay values of the recombinant strains are all higher than the enzyme activity assay values of the original strains, which means that the phospholipase D expression effect of the recombinant strains after genetic engineering transformation is better; while the recombinant strains The measured values of enzyme activity of the recombinant strains using Streptomyces high-efficiency expression vectors are higher than those of the recombinant strains using Streptomyces commonly used expression vectors, that is, the expression effects of the third and fourth recombinant strains in the table are better than those of the first The recombinant strain and the second recombination, and the expression effect of the third recombination strain is the best, then the third recombination strain is the genetically engineered recombinant streptomyces with the highest expression of phospholipase D obtained by our screening by this method.

Wherein the third recombinant strain is a recombinant strain formed by conjugatively transferring the recombinant plasmid pMS82-PLDAnti into Streptomyces SBT5, wherein pMS82 is derived from the ADDGENE library and purchased Streptomyces high-efficiency expression vector pMS82 by Zhongyuan Biological Company, wherein PLDAnti is derived from Phospholipase D synthesis gene from S. antibioticus antibiotic Streptomyces spp.

sequence listing

<110> Henan Commercial Science Research Institute Co., Ltd.

Henan Academy of Sciences

<120> A method for highly expressing phospholipase D in Streptomyces and recombinant Streptomyces

<141> 2020-12-31

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1632

<212>DNA

<213> Artificial sequence

<400> 1

atgctgctgc gccaccgcct gcgccgcctg caccgcctga cccgctccgc cgccgtctcc 60

gccgtcgtcc tggccgccct gccggccgcc ccggccttcg ccgccgacac cccgccgacc 120

ccgcacctgg acgccatcga gcgctccctg cgcgacacct ccccggggcct ggagggctcc 180

gtctggcagc gcaccgacgg caaccgcctg gacgccccgg acggcgaccc ggccggctgg 240

ctgctgcaga ccccgggctg ctggggcgac gccggctgca aggaccgcgc cggcacccgc 300

cgcctgctgg acaagatgac ccgcaacatc gccgacgccc gccacaccgt cgacatctcc 360

tccctggccc cgttcccgaa cggcggcttc gaggacgccg tcgtcgacgg cctgaaggcc 420

gtcgtcgccg ccggccactc cccgcgcgtc cgcatcctgg tcggcgccgc cccgatctac 480

cacctgaacg tcgtcccgtc ccgctaccgc gacgagctga tcggcaagct gggcgccgcc 540

gccggcaagg tcaccctgaa cgtcgcctcc atgaccacct ccaagacctc cctgtcctgg 600

aaccactcca agctgctggt cgtcgacggc aagaccgcca tcacctccgg catcaacggc 660

tggaaggacg actacctgga caccgcccac ccggtctccg acgtcgacat ggccctgtcc 720

ggcccggccg ccgcctccgc cggcaagtac ctggacaccc tgtgggactg gacctgccgc 780

aacgcctccg acccggccaa ggtctggctg gccacctcca acggcgcctc ctgcatgccg 840

tccatggagc aggacgaggc cggctccgcc ccggccgagc cgaccggcga cgtcccggtc 900

atcgccgtcg gcggcctggg cgtcggcatc aaggagtccg acccgtcctc cggctaccac 960

ccggacctgc cgaccgcccc ggacaccaag tgcaccgtcg gcctgcacga caacaccaac 1020

gccgaccgcg actacgacac cgtcaacccg gaggagaacg ccctgcgctc cctgatcgcc 1080

tccgcccgct cccacgtcga gatctcccag caggacctga acgccacctg cccgccgctg 1140

ccgcgctacg acatccgcac ctacgacacc ctggccggca agctggccgc cggcgtcaag 1200

gtccgcatcg tcgtctccga cccggccaac cgcggcgccg tcggctccgg cggctactcc 1260

cagatcaagt ccctggacga gatctccgac accctgcgca cccgcctggt cgccctgacc 1320

ggcgacaacg agaaggcctc ccgcgccctg tgcggcaacc tgcagctggc ctccttccgc 1380

tcctccgacg ccgccaagtg ggccgacggc aagccgtacg ccctgcacca caagctggtc 1440

tccgtcgacg actccgcctt ctacatcggc tccaagaacc tgtacccggc ctggctgcag 1500

gacttcggct acatcgtcga gtccccggcc gccgcccagc agctgaagac cgagctgctg 1560

gacccggagt ggaagtactc ccagcaggcc gccgccaccc cggccggctg cccggcccgc 1620

caggccggct ga 1632

<210> 2

<211> 1947

<212>DNA

<213> Artificial sequence

<400> 2

ctagaacacc cagacctgag cactcaccca cgcgtactca gccccgcgca ctcaccccac 60

gcactcagcc ctgagcaccc acccccgcgc actcagccct gagcacccac cccccgcacc 120

cagcgaggcc cgagcgcccc ggaaccggcc cgtgtcctcg acgtaagcgt tccgtgacct 180

caccgcaccg tccacagggg ttcacccggc gttcatttac gcccttcggc gccttcatct 240

catctgccta atttcggcct taccactcct cgcacgccgc cgaattcagg acggcggctc 300

ctggaaggaa ctccctcaag tgaagcttca gcgcatgttg ctgcgccacc gcctgcgccg 360

cctgcaccgc ctgacccgct ccgccgccgt ctccgccgtc gtcctggccg ccctgccggc 420

cgccccggcc ttcgccgcct ccccgacccc gcacctggac tccgtcgagc agaccctgcg 480

ccaggtctcc ccgggcctgg agggctccgt ctgggagcgc accgccggca actccctggg 540

cgcctccgcc ccgggcggct ccgactggct gctgcagacc ccgggctgct ggggcgaccc 600

gtcctgcacc gaccgcccgg gctcccgccg cctgctggac aagaccgcc aggacatcgc 660

ccaggcccgc cagtccgtcg acatctccac cctggccccg ttcccgaacg gcggcttcca 720

ggacgccgtc gtcgccggcc tgaaggaggc cgtcgccaag ggcaaccgcc tgcaggtccg 780

catcctggtc ggcgccgccc cgatctacca cgccaacgtc atcccgtcct cctaccgcga 840

cgagatggtc gcccgcctgg gcccggccgc cgccaacgtc accctgaacg tcgcctccat 900

gaccacctcc aagaccggct tctcctggaa ccactccaag ctggtcgtcg tcgacggcgg 960

ctccgtcatc acctccggca tcaactcctg gaaggacgac tacctggaca ccgcccaccc 1020

ggtcaacgac gtcgacctgg ccctgtccgg cccggccgcc ggctccgccg gccgctacct 1080

ggacaccctg tgggactgga cctgccgcaa caagtcctcc tggtcctccg tctggttcgc 1140

ctcctccaac aacgccggct gcatgccgac cctgccgcgc ccggccgccc cggccggcgg 1200

cggcgacgtc ccggccctgg ccgtcggcgg cctgggcgtc ggcatccgcc agtccgaccc 1260

ggcctccgcc ttcaagccgg tcctgccgac cgccccggac accaagtgcg gcatcggcgt 1320

ccacgacaac accaacgccg accgcgacta cgacaccgtc aacccggagg agtccgccct 1380

gcgcgccctg gtcgcctccg ccaactccca cgtcgagatc tcccagcagg acctgaacgc 1440

cacctgcccg ccgctgccgc gctacgacat ccgcctgtac gacaccctgg ccgccaagct 1500

ggccgccggc gtcaaggtcc gcatcgtcgt ctccgacccg gccaaccgcg gcgccgtcgg 1560

ctccgacggc tactcccaga tcaagtccct gaacgaggtc tccgacgccc tgcgcggccg 1620

cctgaccgcc ctgaccggcg acgagcgcac ctccaaggcc gccatgtgcc agaacctgca 1680

gctggccacc ttccgcgcct ccgacaaggc cacctgggcc gacggcaagc cgtacgccca 1740

gcaccacaag ctggtctccg tcgacgactc cgccttctac atcggctcca agaacctgta 1800

cccgtcctgg ctgcaggact tcggctacgt cgtcgagtcc ccggccgccg ccaaccagct 1860

gaaggactcc ctgctggccc cgcagtggaa gtactcccag gccaccgcca cctacgacta 1920

cgcccgcggc ctgtgccagg cctgatt 1947

Claims (3)

1. a method for highly expressing phospholipase D in streptomyces, is characterized in that, comprises the steps: Step 1, select a heterologous high-efficiency expression host of phospholipase D, select Streptomyces as the host, and select the phospholipase D gene derived from Streptomyces as the target gene as the transformation object of genetic engineering; Step 2, screen the best source of phospholipase D gene, carry out comparative experiments by enzyme activity assay, select S.antibioticus antibiotic Streptomyces and S.chromofuscus brown Streptomyces as the target strains for obtaining the target gene, the source of the antibiotic Streptomyces The phospholipase D gene sequence is shown in SEQ ID NO.1, and the phospholipase D gene sequence derived from Streptomyces fusceus is shown in SEQ ID NO.2; Step 3, obtaining the phospholipase D gene fragment derived from Streptomyces, and obtaining the phospholipase D gene derived from the antibiotic Streptomyces and the phospholipase D gene derived from Streptomyces fusceus by PCR amplification technology; Step 4, connecting the target gene to form a phospholipase D recombinant plasmid, connecting the phospholipase D gene from the antibiotic Streptomyces to the commonly used expression vector of Streptomyces and the high-efficiency expression vector of Streptomyces to form the first recombinant plasmid and the third recombinant plasmid, and then The phospholipase D gene derived from Streptomyces fusceus is respectively connected to the common expression vector of Streptomyces and the high-efficiency expression vector of Streptomyces to form the second recombinant plasmid and the fourth recombinant plasmid. The common expression vector of Streptomyces is pSET152, and the high-efficiency expression vector of Streptomyces is pMS82 ; Step 5: Conjugate and transfer the recombinant plasmid to the host bacterium, and then obtain the first recombinant strain and the second recombinant strain corresponding to the first recombinant plasmid, the second recombinant plasmid, the third recombinant plasmid, and the fourth recombinant plasmid in sequence by means of conjugative transfer , the third recombinant strain, the fourth recombinant strain, the host bacteria is Streptomyces SBT5; Step 6, the fermentation culture of the recombinant strain and the detection of phospholipase D enzyme activity, and then find the strain with the best expression effect through the detection of the enzyme activity of the original strain and the recombinant strain, wherein the original strain is S.lividans Streptomyces lividans, Streptomyces lividans, S.antibioticus Streptomyces antibioticus and S.chromofuscus Streptomyces fuvidus; and the recombinant strains are the first recombinant strain, the second recombinant strain, the third recombinant strain and the fourth recombinant strain obtained in Step 5. 2. a kind of method according to claim 1 highly expresses phospholipase D in streptomyces, it is characterized in that, the PCR amplification technology described in step 3 comprises: (1) S.antibioticus antibiotic streptomyces and Genomic DNA of S.chromofuscus Streptomyces chromofuscus was extracted respectively, (2) using S.antibioticus antibiotic Streptomyces and S.chromofuscus Streptomyces chromofusus genomic DNA as templates respectively, primer PCR was designed to obtain the target gene and sequence verification, (3 ) to optimize the codon of the obtained target fragment, and then obtain the phospholipase D gene derived from the antibiotic Streptomyces and the phospholipase D gene derived from the Streptomyces fusceus. 3. a recombinant streptomyces highly efficient expression phospholipase D, is characterized in that, adopts the method as claimed in claim 1, obtains recombinant plasmid pMS82-PLD _Anti, then recombinant plasmid pMS82-PLD _Anti conjugation is transferred to streptomyces SBT5 The recombinant strain formed within, PLD _Anti is derived from the phospholipase D gene of S.antibioticus Streptomyces antibioticus.