CN112575023A - Method for efficiently expressing phospholipase D in streptomyces and recombinant streptomyces - Google Patents
Method for efficiently expressing phospholipase D in streptomyces and recombinant streptomyces Download PDFInfo
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Abstract
The invention belongs to the field of genetic engineering, and relates to a method for efficiently expressing phospholipase D in streptomycete. The streptomyces-derived phospholipase D is selected for recombinant protein heterologous expression, so that the method has natural advantages, codon optimization is performed on a phospholipase D synthetic gene, the streptomyces lividans SBT5 is selected as a heterologous expression host, and the recombinant strain has extremely high innovation and practicability due to excellent heterologous expression effect and good extracellular activity secretion expression effect.
Description
Technical Field
The invention belongs to the field of genetic engineering, and relates to a method for efficiently expressing phospholipase D in streptomycete and recombinant streptomycete.
Background
In recent years, attention has been paid to the preparation of phosphatidylserine by an enzymatic conversion method, i.e., the synthesis of substrates phosphatidylcholine and L-serine is catalyzed by phospholipase D; the microbial-derived phosphatidylserine synthetase D, hereinafter referred to as phospholipase D or phospholipase D, has the activity of transphosphatidylation, can catalyze and synthesize phosphatidylserine in an aqueous environment, has mild reaction conditions, few byproducts, high product yield and good quality, and can become a main industrial production method in the future; however, the wild bacteria directly screened from the environment have lower catalytic activity, the secretion amount of the phospholipase D is far lower than the industrial production standard, and the application of the phospholipase D in the industrialization of synthesizing phosphatidylserine is seriously influenced.
Therefore, in order to obtain high-yield phospholipase D, genetic construction and genetic modification are required to be carried out by genetic engineering means to obtain an engineering strain capable of efficiently expressing phospholipase D, and then the strain is used for fermentation to extract phospholipase D, so that the construction of a strain capable of efficiently expressing phospholipase D becomes very critical, and how to make a newly constructed recombinant plasmid be well expressed in a heterologous host is also a thought direction for constructing the recombinant plasmid.
At present, a number of scholars have attempted to heterologously express phospholipase D, the more commonly used hosts being E.coli, followed by Streptomyces, yeast and Bacillus subtilis. In order to investigate amino acid residues affecting phospholipase D thermostability, researchers transferred Streptomyces-derived phospholipase D genes into escherichia coli for expression, and studied enzymatic properties; bacillus subtilis has a mature heritage operating system and is a food-grade strain, and scholars introduce a phospholipase D gene from E.coliK12 into B.subtiliss 104 for heterologous expression, and detect that the extracellular enzyme activity is only 0.15 u/mL; the scholars construct two constitutive shuttle plasmids to realize the high-efficiency expression of the phospholipase D in the streptomyces, the enzyme activity reaches 58u/mL after fermentation for 3 days, and the secretion of the original streptomyces phospholipase D is only 1.1 u/mL; another scholars expresses streptomycete-derived phospholipase D genes in pichia pastoris and yarrowia lipolytica respectively, constructs a pIC9K expression vector carrying target gene endogenous signal peptide and transfers the expression vector into the pichia pastoris, and measures the enzyme activity after fermenting for three days.
Through gene experiments, the common heterologous hosts carrying target genes, such as escherichia coli, bacillus subtilis and the like, have poor in-vivo expression effect or poor secretion effect, so that a more suitable heterologous host is found, and the recombinant plasmid can be well expressed, and the research direction is also provided.
Disclosure of Invention
The invention aims to find a method for efficiently expressing phospholipase D in streptomyces to determine a recombinant strain with high expression so as to be applied to industrial production for producing phospholipase D enzyme.
The technical scheme adopted by the invention is as follows:
a method for efficiently expressing phospholipase D in streptomyces comprises the following steps:
selecting a heterologous high-efficiency expression host of phospholipase D, selecting streptomyces as the host, and selecting a phospholipase D gene from streptomyces as a target gene as a modified object of genetic engineering;
screening the optimal source of a phospholipase D synthetic gene, performing a comparison experiment through enzyme activity measurement, and selecting S.antibioticus streptomyces and S.chromofuscus streptomyces chromofuscus as target strains for obtaining a target gene;
step three, obtaining a streptomyces-derived phospholipase D gene fragment, and obtaining a streptomyces-derived phospholipase D gene of the antibiotic and a streptomyces chromofuscus-derived phospholipase D gene by a PCR amplification technology;
connecting target genes to form a phospholipase D recombinant plasmid, respectively connecting phospholipase D genes from streptomyces antibiotics to a streptomyces common expression vector and a streptomyces high-efficiency expression vector to form a first recombinant plasmid and a third recombinant plasmid, and respectively connecting the phospholipase D genes from streptomyces chromofuscus to the streptomyces common expression vector and the streptomyces high-efficiency expression vector to form a second recombinant plasmid and a fourth recombinant plasmid;
step five, the recombinant plasmids are transferred to host bacteria in a conjugal manner, and a first recombinant strain, a second recombinant strain, a third recombinant strain and a fourth recombinant strain which sequentially correspond to the first recombinant plasmid, the second recombinant plasmid, the third recombinant plasmid and the fourth recombinant plasmid are obtained through the conjugal transfer means;
sixthly, carrying out fermentation culture on the recombinant strain and enzyme activity detection of phospholipase D, and finding out the strain with the best expression effect by detecting the enzyme activity of the original strain and the recombinant strain, wherein the original strain is S.lividans streptomyces lividans, S.antibioticus streptomyces and S.chromofuscus streptomyces chromofuscus; and the recombinant strains are the first recombinant strain, the second recombinant strain, the third recombinant strain and the fourth recombinant strain obtained in the fifth step.
Specifically, the PCR amplification technique described in step three includes: (1) respectively extracting genomic DNA of S.antibioticus streptomyces antibioticus and S.chromofuscus streptomyces chromofuscus, 2 respectively taking the genomic DNA of S.antibioticus and S.chromofuscus streptomyces chromofuscus as templates, designing a primer PCR to obtain a target gene, and carrying out sequencing verification, and 3) carrying out codon optimization on the obtained target fragment to further obtain a phospholipase D gene from the streptomyces antibioticus and a phospholipase D gene from the streptomyces chromofuscus.
Preferably, the expression vector commonly used in Streptomyces in step four is pSET 152.
Preferably, the streptomyces high-efficiency expression vector in the fourth step is pMS 82.
Preferably, the host bacterium in the fifth step is streptomyces SBT 5.
The recombinant streptomycete for efficiently expressing the phospholipase D adopts the method to obtain the recombinant plasmid pMS82-PLDAnti，Then the recombinant plasmid pMS82-PLD was addedAntiConjugation was transferred to a recombinant strain formed within Streptomyces SBT 5.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. a streptomycete-derived phospholipase D synthetic gene is selected as a target gene, so that the expression efficiency of the phospholipase D is improved; through a plurality of strain comparison tests, the results show that the effect of producing the phospholipase D by the streptomyces is outstanding, and the hydrolysis activity is best, so that the streptomyces-derived phospholipase D is selected for heterologous expression of the recombinant protein, and the method has natural advantages.
2. The streptomycete high-efficiency expression vector is used as an upstream element of a synthetic gene, and the upstream element contains a streptomycete strong promoter and a secretory signal peptide, so that the expression efficiency of phospholipase D is greatly improved; the constitutive strong promoter is crucial to the high-efficiency expression of a target gene, and the addition of the signal peptide with good secretion effect ensures that the target protein can be directly secreted to the outside of cells after being formed, thereby simplifying the production process and reducing the production cost.
3. The streptomyces lividans SBT5 is selected as a heterologous expression host, so that the expression efficiency of phospholipase D is further improved; researches show that streptomyces has innovation and outstanding superiority as a host, and has the advantages that: three biosynthesis gene clusters of endogenous antibiotics are knocked out by the host bacteria, and the secondary metabolism background is clear; meanwhile, the independently constructed high-efficiency expression vector is excellent in the host bacteria, so that the heterologous expression efficiency of the phospholipase D is far higher than that of other hosts.
4. According to the codon preference of host bacteria, the phospholipase D synthetic gene is subjected to codon optimization, so that the codon matching of the target gene and the host is stronger, and the process of copying the target gene in the host and guiding protein synthesis is more efficient; the excellent heterologous expression effect and the good extracellular activity secretion expression effect ensure that the recombinant strain has extremely high innovation and practicability.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments.
A method for efficiently expressing phospholipase D in streptomyces comprises the following steps:
step one, selecting a heterologous high-efficiency expression host of phospholipase D
Through data query, many scholars try to perform heterologous expression on phospholipase D, the more commonly used host is Escherichia coli, and then streptomyces, yeast and Bacillus subtilis, and the data of measuring the activity of phospholipase D in the host are summarized as follows:
according to the table contents, the streptomyces can be determined to be selected as a host, and simultaneously, the phospholipase D gene from the streptomyces is selected as a modified object of genetic engineering, so that the engineering strain with higher enzyme activity relative to the phospholipase D is obtained.
Step two, screening the optimal source of the phospholipase D synthetic gene
Selecting streptomycetes from different sources, detecting the enzyme activity level of the phospholipase D by taking the streptomycetes as hosts, and comparing the expression levels of the phospholipase D of the three different streptomycetes.
The specific method comprises the following steps: strains of S.lividans Streptomyces lividans, S.antibioticus streptomyces and S.chromofuscus streptomyces chromofuscus are selected and fermented for 3 days, culture solution supernatant is taken, ammonium sulfate is precipitated and centrifuged, 100 mu g of total protein is taken for catalytic activity experiments through simple coarse purification, phospholipase D catalytic activity from S.lividans Streptomyces lividans is taken as a standard, and the catalytic activity of phospholipase D protein generated by S.antibioticus is relatively higher, and the results are shown in the following table.
Strains of S.lividans Streptomyces lividans, S.antibioticus streptomyces and S.chromofuscus Streptomyces chromofuscus are all purchased from Shanghai.
The result shows that the phospholipase D from the antibiotic streptomyces and the streptomyces chromofuscus has higher enzyme activity, the unknowns of heterologous expression of target genes, gene mutation and other factors are considered, and the S.antibioticus streptomyces and the S.chromofuscus chromofuscus are simultaneously selected as target strains for obtaining the target genes.
Step three, obtaining phospholipase D gene fragment from streptomycete
(1) Respectively extracting the genomic DNAs of streptomyces from different sources, specifically carrying out the S.antibioticus streptomyces and S.chromofuscus chromofuscus streptomyces according to the following method:
suspending appropriate amount of thallus in 500 μ L lysozyme solution (2%), incubating at 37 deg.C for about 1hr to completely dissolve thallus, adding 500 μ L alkaline SDS solution (0.3mol/L NaOH, 2% SDS), immediately shaking and mixing completely, opening tube cover, standing at 70 deg.C for 15min (preferably 55 deg.C for plasmid larger than 20kb, 30min), cooling to room temperature in water bath, adding 100 μ L acidic phenol/chloroform solution, shaking with mixer until the liquid is thoroughly mixed, centrifuging at 12000rpm for 5min, removing supernatant, and discarding white middle layer; repeat extraction with neutral phenol/chloroform until no (or very little) intermediate layer is visible; adding 1/10 volume of 3M NaAc solution and 1 volume of isopropanol into the supernatant, precipitating for 5 minutes (or 2.2 volume of absolute ethyl alcohol precipitating for 1 hour), centrifuging at 12000rpm for 8min, and washing the precipitate twice with 70% ethanol; after drying, the mixture was dissolved in a predetermined amount of TE (ddH2O) buffer.
(2) The streptomycete genome DNA is used as a template, a primer PCR is designed to obtain a target gene, and sequencing verification is carried out, wherein specific primers are as follows:
antibiotic streptomycete upstream and downstream primers
The target fragment was then amplified using Primer STAR mix polymerase to obtain the target fragment.
(3) Carrying out Codon optimization on the obtained target fragment, and carrying out optimization design by adopting Codon use Analyzer online Codon statistical table processing software (http:// bioinformatics.org/Codon/cgi-bin/codon.cgi), so that the Codon statistics is displayed in a chart form, and further the phospholipase D genes from streptomyces antibioticus and streptomyces chromofuscus are respectively more accordant with the Codon preference of host bacteria;
the sequence of the phospholipase D gene derived from the antibiotic streptomyces is shown as SEQ ID NO. 1;
the sequence of the phospholipase D gene derived from the streptomyces chromofuscus is shown as SEQ ID No. 2.
Step four, connecting the target gene to form phospholipase D recombinant plasmid
The specific method comprises the following steps: a streptomycete common expression vector pSET152 (from an ADDGENE library purchased from a Zhongyuan biological company) and a streptomycete high-efficiency expression vector pMS82 (from an ADDGENE library purchased from a Zhongyuan biological company) are respectively connected with target genes of phospholipase D from two different sources to form a plurality of phospholipase D expression plasmids.
Step five, the recombinant plasmid is transferred to the host bacterium SBT5 by conjugation
Wherein the host bacterium SBT5 is obtained from the literature: baikili et al, construction of the Streptomyces lividans high-efficiency heterologous expression host SBT5, proceedings of Huazhong university of agriculture, 1 month in 2014, first phase 33.
The specific method of the joint transfer is as follows:
coli ET12567/pUZ8002 was used as a donor for conjugative transfer, where plasmid pUZ8002 contains an oriT conjugative transfer initiation site, capable of facilitating the transfer of the target plasmid.
Firstly, the target plasmid is transferred into Escherichia coli ET12567/pUZ8002 by an electric conversion or chemical conversion method, the Escherichia coli is cultured by LB containing corresponding antibiotics until OD600 is 0.4-0.6, the thallus is collected and washed twice by LB culture medium without antibiotics, and the antibiotics in the supernatant are sufficiently removed.
Meanwhile, freshly harvested spores of SBT5 Streptomyces were suspended in a volume of 2 XYT medium (in the case of spores stored in 20% glycerol, glycerol was removed by centrifugation, washed twice with sterile water and suspended in a volume of 2 XYT medium), heat-shocked at 50 ℃ for 10min and fully cooled.
Mixing the treated SBT5 streptomycete spores and escherichia coli, coating the mixture on an MS culture medium without antibiotics, culturing the mixture for 12 to 16 hours at the temperature of 30 ℃, and covering a flat plate with the antibiotics corresponding to target plasmids and the nalidixic acid or the trimethoxy benzylaminopyrimidine; the conjugal transfersomes were seen to grow out 2-3 days after the covering, and the resulting recombinant strains were as follows.
Step six, fermentation culture of the recombinant strain and detection of phospholipase D enzyme activity
Through fermentation culture and enzyme activity detection of the original strain and the recombinant strain, the strain with the best expression effect is found.
Wherein the original strains are S.lividans Streptomyces lividans, S.antibioticus streptomyces and S.chromofuscus Streptomyces chromofuscus.
The recombinant strains are the first recombinant strain, the second recombinant strain, the third recombinant strain and the fourth recombinant strain obtained in the fifth step.
The strain is adopted to carry out fermentation culture and enzyme activity detection by adopting a general method, and the specific method comprises the following steps: inoculating streptomycete into a fermentation culture medium in an incubator at 30 ℃ for 5-7 days; after the bacterial liquid is cultured to be turbid, centrifuging and taking supernatant; filtering the supernatant with 0.45um filter membrane to further remove impurities; ammonium sulfate precipitated the protein overnight. Centrifuging at high speed, and discarding the supernatant; redissolving protein, and desalting by ultrafiltration. The phospholipase D hydrolysis activity was measured spectrophotometrically using p-PNP (phosphatidyl-p-nitrophenol) as a substrate. The phosphatidyl transferring activity is measured by the reaction of converting PC into Phosphatidylethanolamine (PE), the concentration of PC and PE is determined by TLC of GF254 silica gel plate with chloroform-methanol-water (13:5:0.8 volume ratio) as developing agent; the iodine vapor-stained thin-layer plate was measured for spot area and gray level using image processing software to calculate the phospholipid component concentration. The enzyme protein concentration was measured according to the Bradford method using bovine serum albumin as a standard and used for the PLD specific activity measurement, and the following results were obtained:
according to the data in the table, the enzyme activity determination values of the recombinant strains are higher than the enzyme activity determination values of the original strains, so that the phospholipase D expression effect of the recombinant strains after being modified by genetic engineering is better; and the enzyme activity determination value of the recombinant strain adopting the streptomycete high-efficiency expression vector in the recombinant strain is higher than that of the recombinant strain adopting the streptomycete common expression vector, namely the expression effects of the third recombinant strain and the fourth recombinant strain in the table are better than those of the first recombinant strain and the second recombinant strain, and the expression effect of the third recombinant strain is optimal, so that the third recombinant strain is the genetic engineering recombinant streptomycete which is screened by the method and expresses the phospholipase D with the highest efficiency.
Wherein the third recombinant strain is formed by conjugatively transferring a recombinant plasmid pMS82-PLDanti into Streptomyces SBT5, wherein pMS82 is derived from ADDGENE Bank, Streptomyces high-efficiency expression vector pMS82 purchased from Zhongyuan Bio-Inc., and PLDanti is derived from a phospholipase D synthetic gene of S.antibioticus Streptomyces.
Sequence listing
<110> Enterprise scientific research institute of Henan province, Limited liability company
HENAN ACADEMY OF SCIENCES
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Claims (8)
1. A method for efficiently expressing phospholipase D in streptomyces is characterized by comprising the following steps:
selecting a heterologous high-efficiency expression host of phospholipase D, selecting streptomyces as the host, and selecting a phospholipase D gene from streptomyces as a target gene as a modified object of genetic engineering;
screening the optimal source of a phospholipase D synthetic gene, performing a comparison experiment through enzyme activity measurement, and selecting S.antibioticus streptomyces and S.chromofuscus streptomyces chromofuscus as target strains for obtaining a target gene;
step three, obtaining a streptomyces-derived phospholipase D gene fragment, and obtaining a streptomyces-derived phospholipase D gene of the antibiotic and a streptomyces chromofuscus-derived phospholipase D gene by a PCR amplification technology;
connecting target genes to form a phospholipase D recombinant plasmid, respectively connecting phospholipase D genes from streptomyces antibiotics to a streptomyces common expression vector and a streptomyces high-efficiency expression vector to form a first recombinant plasmid and a third recombinant plasmid, and respectively connecting the phospholipase D genes from streptomyces chromofuscus to the streptomyces common expression vector and the streptomyces high-efficiency expression vector to form a second recombinant plasmid and a fourth recombinant plasmid;
step five, the recombinant plasmids are transferred to host bacteria in a conjugal manner, and a first recombinant strain, a second recombinant strain, a third recombinant strain and a fourth recombinant strain which sequentially correspond to the first recombinant plasmid, the second recombinant plasmid, the third recombinant plasmid and the fourth recombinant plasmid are obtained through the conjugal transfer means;
sixthly, carrying out fermentation culture on the recombinant strain and enzyme activity detection of phospholipase D, and finding out the strain with the best expression effect by detecting the enzyme activity of the original strain and the recombinant strain, wherein the original strain is S.lividans streptomyces lividans, S.antibioticus streptomyces and S.chromofuscus streptomyces chromofuscus; and the recombinant strains are the first recombinant strain, the second recombinant strain, the third recombinant strain and the fourth recombinant strain obtained in the fifth step.
2. The method of claim 1, wherein the PCR amplification technique in step three comprises: (1) respectively extracting genomic DNA of S.antibioticus streptomyces antibioticus and S.chromofuscus streptomyces chromofuscus, 2 respectively taking the genomic DNA of S.antibioticus and S.chromofuscus streptomyces chromofuscus as templates, designing a primer PCR to obtain a target gene, and carrying out sequencing verification, and 3) carrying out codon optimization on the obtained target fragment to further obtain a phospholipase D gene from the streptomyces antibioticus and a phospholipase D gene from the streptomyces chromofuscus.
3. The method for efficiently expressing phospholipase D in Streptomyces according to claim 1, wherein the expression vector commonly used in Streptomyces in step IV is pSET 152.
4. The method for efficiently expressing phospholipase D in Streptomyces according to claim 1, wherein the Streptomyces high-efficiency expression vector of step four is pMS 82.
5. The method for efficiently expressing phospholipase D in Streptomyces according to claim 1, wherein the host bacterium in step five is Streptomyces SBT 5.
6. The method for efficiently expressing the phospholipase D in the streptomyces as claimed in claim 2, wherein the gene sequence of the phospholipase D derived from the streptomyces antibiotics is shown as SEQ ID No. 1.
7. The method for efficiently expressing the phospholipase D in the streptomyces as claimed in claim 2, wherein the gene sequence of the phospholipase D from the streptomyces chromofuscus is shown as SEQ ID No. 2.
8. A recombinant streptomyces for highly expressing phospholipase D, wherein the recombinant plasmid pMS82-PLD is obtained by the method of claim 1Anti，Then the recombinant plasmid pMS82-PLD was addedAntiConjugation was transferred to a recombinant strain formed within Streptomyces SBT 5.
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| EP0435725A1 (en) * | 1989-12-15 | 1991-07-03 | Asahi Kasei Kogyo Kabushiki Kaisha | DNA having the genetic information of phospholipase D and its use |
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| EP0435725A1 (en) * | 1989-12-15 | 1991-07-03 | Asahi Kasei Kogyo Kabushiki Kaisha | DNA having the genetic information of phospholipase D and its use |
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