CN112575023B - 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, 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
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 enzymatic conversion of phosphatidylserine, i.e., the synthesis of the substrates phosphatidylcholine and L-serine by phospholipase D; the phosphatidylserine synthetase D from microorganism, hereinafter called phospholipase D or phospholipase D for short, has activity of transferring phosphatidyl, can catalyze and synthesize phosphatidylserine in an aqueous phase environment, has mild reaction conditions, few byproducts, high product yield and good quality, and becomes a main production method for future industrialization; however, the wild bacteria directly screened from the environment have low catalytic activity, and the secretion amount of the phospholipase D is far lower than the industrial production standard, so that the application of the phospholipase D in the industrialization of synthesizing phosphatidylserine is seriously affected.
Then, in order to obtain a high yield of phospholipase D, it is desired to perform genetic construction and genetic engineering to obtain an engineering strain capable of expressing phospholipase D efficiently, and then to extract phospholipase D by fermentation using the strain, it becomes critical to construct a strain capable of expressing phospholipase D efficiently, and how to allow a newly constructed recombinant plasmid to be expressed well in a heterologous host, which is the thinking direction of constructing the recombinant plasmid.
At present, many scholars have tried heterologous expression of phospholipase D, and more commonly used hosts are e.coli, followed by streptomyces, yeast and bacillus subtilis. In order to investigate amino acid residues affecting the thermostability of phospholipase D, a learner transferred the phospholipase D gene derived from Streptomyces into E.coli for expression and studied the enzymatic properties; the bacillus subtilis has a mature heritage operating system and is a food-grade strain, and a learner introduces a phospholipase D gene from E.coli K12 into B.subtilis104 for heterologous expression, so that the extracellular enzyme activity is detected to be only 0.15u/mL; two constitutive shuttle plasmids are also constructed by a scholars, so that efficient expression of phospholipase D in streptomycete is realized, and after 3 days of fermentation, the enzyme activity reaches 58u/mL, and the secretion amount of the original streptomycete phospholipase D is only 1.1u/mL; in addition, the scholars express the phospholipase D gene from streptomyces in pichia pastoris and yarrowia lipolytica respectively, construct pIC9K expression vector carrying the endogenous signal peptide of the target gene, transfer the vector into pichia pastoris, and measure the enzyme activity after three days of fermentation.
Through gene experiments, the conventional heterologous hosts carrying the target genes, such as escherichia coli, bacillus subtilis and the like, have poor internal expression effect or poor secretion effect, so that finding more suitable heterologous hosts and enabling the recombinant plasmids to express well also becomes a research direction.
Disclosure of Invention
The present invention aims to find a method for efficiently expressing phospholipase D in Streptomyces to determine a recombinant strain for efficient expression for use in industrial production of phospholipase D enzyme.
The technical scheme adopted by the invention is as follows:
a method for highly expressing phospholipase D in streptomyces comprising the steps of:
selecting a heterologous efficient expression host of phospholipase D, selecting streptomycete as a host, and simultaneously selecting a phospholipase D gene from streptomycete as a target gene as a modification object of genetic engineering;
step two, screening the optimal source of the phospholipase D synthetic gene, and performing a comparison experiment through enzyme activity measurement, wherein S.antiiotics antibiotic streptomycete and S.chromofuscus brown streptomycete are selected as target strains for obtaining target genes;
step three, obtaining a phospholipase D gene fragment from streptomyces, and obtaining a phospholipase D gene from streptomyces antibiotics and a phospholipase D gene from streptomyces chroogomphus through a PCR amplification technology;
step four, connecting target genes to form a phospholipase D recombinant plasmid, respectively connecting phospholipase D genes from streptomyces antibiotics with 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 phospholipase D genes from streptomyces chrooformis with a streptomyces common expression vector and a 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 joint way, and a first recombinant strain, a second recombinant strain, a third recombinant strain and a fourth recombinant strain which are sequentially corresponding to the first recombinant plasmid, the second recombinant plasmid, the third recombinant plasmid and the fourth recombinant plasmid are obtained through the joint transfer way;
step six, fermenting and culturing the recombinant strain and detecting the enzyme activity of phospholipase D, and detecting the enzyme activities of the original strain and the recombinant strain to further find out the strain with the best expression effect, wherein the original strain is S.lividans streptomyces lividans, S.antieticus streptomyces antibiotics and S.chromofuscus streptomyces chrous; the recombinant strains are the first recombinant strain, the second recombinant strain, the third recombinant strain and the fourth recombinant strain obtained in the step five.
Specifically, the PCR amplification technique described in step three includes: extracting genome DNA of S.antiiotic streptomyces antibiotics and S.chromofuscus streptomyces, respectively, (2) designing a primer PCR to obtain a target gene and sequencing and verifying by taking the genome DNA of S.antiiotic streptomyces antibiotics and S.chromofuscus streptomyces as templates, and (3) carrying out codon optimization on the obtained target fragment to obtain phospholipase D genes from streptomyces antibiotics and phospholipase D genes from streptomyces chrofuscus.
Preferably, the streptomyces species commonly used expression vector in step four is pSET152.
Preferably, the streptomycete high-efficiency expression vector in the fourth step is pMS82.
Preferably, the host bacterium in the fifth step is Streptomyces SBT5.
Recombinant streptomycete capable of efficiently expressing phospholipase D is adopted to obtain recombinant plasmid pMS82-PLD by the method Anti， Recombinant plasmid pMS82-PLD Anti The conjugation was transferred to recombinant strains formed within Streptomyces SBT5.
In summary, due to the adoption of the technical scheme, the beneficial effects of the invention are as follows:
1. selecting a phospholipase D synthesis gene from streptomyces as a target gene, and improving the expression efficiency of phospholipase D; the result of a plurality of strain comparison tests shows that the streptomycete has outstanding effect of generating phospholipase D and the hydrolysis activity is the best, so that the streptomycete-derived phospholipase D is selected for heterologous expression of recombinant protein, and the streptomycete-derived phospholipase D has natural advantages.
2. The expression efficiency of the phospholipase D is greatly improved by taking the streptomycete high-efficiency expression vector as an upstream original of a synthetic gene, wherein the streptomycete high-efficiency expression vector comprises a streptomycete strong promoter and a secretion type signal peptide; the constitutive strong promoter is crucial to the efficient expression of the target gene, and meanwhile, the addition of the signal peptide with good secretion effect enables the target protein to be directly secreted out of cells after being formed, so that the production process is simplified, and the production cost is reduced.
3. The Streptomyces lividans SBT5 is selected as a heterologous expression host, so that the expression efficiency of the phospholipase D is further improved; the research shows that the streptomycete has innovative and outstanding superiority as a host, and has the advantages that: the host bacteria knocks out three biosynthesis gene clusters of endogenous antibiotics, and the secondary metabolism background is clear; meanwhile, the autonomously constructed high-efficiency expression vector is excellent in expression 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 the host bacteria, the codon optimization is carried out on the phospholipase D synthetic gene, 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 make the recombinant strain have extremely high innovation and practicability.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent.
A method for highly expressing phospholipase D in streptomyces comprising the steps of:
step one, selecting a heterologous high efficiency expression host for phospholipase D
Through data query, many scholars tried heterologous expression of phospholipase D, more commonly used hosts were E.coli, followed by Streptomyces, yeast and Bacillus subtilis, and the data for measurement of phospholipase D enzyme activity were summarized as follows:
according to the table contents, it is found that the engineering strain with higher relative phospholipase D enzyme activity can be obtained by determining to select streptomycete as a host and simultaneously selecting the phospholipase D gene from streptomycete as an engineering object of genetic engineering.
Step two, screening the optimal source of phospholipase D synthesis gene
And selecting streptomyces from different sources, respectively taking the streptomyces as a host to detect the enzyme activity level of the phospholipase D, and comparing the expression levels of the phospholipase D of the three different streptomyces.
The specific method comprises the following steps: strains of S.lividans streptomyces plumbaginensis, S.antiiotics streptomyces antibiotics and S.chromofuscus streptomyces chroogorum are selected, the strains are fermented and cultured for 3 days, a culture solution supernatant is taken, ammonium sulfate is precipitated and centrifuged, 100 mug total protein is taken as a catalytic activity experiment through simple coarse purification, the catalytic activity of phospholipase D from S.lividans streptomyces plumbaginensis is taken as a standard, and the result is shown in the following table.
Strains of S.lividans Streptomyces lividans, S.antieticus Streptomyces antibiotics and S.chromofusus brown mold were all purchased from Shanghai.
The result shows that the phospholipase D from the streptomyces antibiotics and the streptomyces chroofaciens has higher enzyme activity, and the factors such as unknowing of the heterologous expression of the target gene, gene mutation and the like are considered, and S.antiiotics streptomyces antibiotics and S.chromofuscus chroomes are selected as target strains for obtaining the target gene.
Step three, obtaining a phospholipase D gene fragment from streptomyces
(1) The extraction of genome DNA of different sources of streptomyces is carried out by respectively extracting S.antiiotics antibiotics streptomyces and S.chromofuscus brown streptomyces according to the following method:
suspending appropriate amount of thallus in 500 μl lysozyme solution (2%), incubating at 37deg.C for about 1hr until complete lysozyme, adding 500 μl alkaline SDS solution (0.3 mol/L NaOH,2% SDS), immediately shaking and mixing completely, opening the tube cover, standing at 70deg.C for 15min (preferably 55 deg.C for plasmid larger than 20kb for 30 min), cooling to room temperature in water bath, adding 100 μl acidic phenol/chloroform solution, shaking with mixer until liquid thoroughly mixed, centrifuging at 12000rpm for 5min, removing supernatant, and discarding white middle layer; repeated extractions with neutral phenol/chloroform until no (or very little) intermediate layer is visible; adding 1/10 volume of 3M NaAc solution and 1 times volume of isopropanol into the supernatant to precipitate for 5 minutes (or 2.2 times volume of absolute ethanol to precipitate for 1 h), centrifuging at 12000rpm for 8 minutes, and washing the precipitate twice with 70% ethanol; after drying, a certain amount of TE (ddH 2O) buffer was added for dissolution.
(2) The Streptomyces genomic DNA is used as a template, a primer PCR is designed to obtain a target gene, and sequencing verification is carried out, wherein the specific primer is as follows:
upstream and downstream primers of streptomyces antibiotics
Then, the target fragment was amplified using Primer STAR mix polymerase to obtain the target fragment.
(3) Optimizing the obtained target fragment by adopting Codon Usage Analyzer online codon statistics table processing software (http:// bioinformation information. Org/codon/cgi-bin/codon. Cgi), and displaying the statistics of the codons in a form of a graph so as to ensure that the phospholipase D genes derived from the streptomyces antibiotics and the streptomyces chroogorum are more in line with the codon preference of host bacteria;
the final obtained phospholipase D gene sequence of the streptomyces antibiotics source is shown as SEQ ID NO. 1;
the final sequence of the obtained phospholipase D gene from the streptomyces chrofuscus is shown as SEQ ID NO. 2.
Step four, connecting target genes to form phospholipase D recombinant plasmid
The specific method comprises the following steps: the common expression vector pSET152 of streptomycete (purchased from an ADDGENE library through a medium-source biological company) and the efficient expression vector pMS82 of streptomycete (purchased from an ADDGENE library through a medium-source biological company) are respectively connected with two different source phospholipase D target genes to form a plurality of phospholipase D expression plasmids.
Step five, the recombinant plasmid is transferred to host bacterium SBT5 in a joint way
Wherein the host strain SBT5 is from literature: bai Tingli and the like, and the volume 33 first period of the university of China agricultural university report, 1 month in 2014.
The specific method for the joint transfer is as follows:
coli ET12567/pUZ8002 is used as donor bacteria for conjugal transfer, wherein plasmid pUZ8002 contains oriT conjugal transfer initiation site, which can assist transfer of target plasmid.
Firstly, transferring the target plasmid into Escherichia coli ET12567/pUZ8002 by using an electrotransformation or chemical transformation method, culturing the Escherichia coli to OD600 of 0.4-0.6 by using LB containing corresponding antibiotics, collecting thalli, washing twice by using LB culture medium without antibiotics, and fully removing the antibiotics in the supernatant.
Meanwhile, freshly harvested spores of Streptomyces SBT5 were suspended in a volume of 2 XYT medium (if spores stored in 20% glycerol were removed by centrifugation, washed twice with sterile water, and suspended in a volume of 2 XYT medium), heat-shocked at 50℃for 10min, and cooled well.
Mixing the treated SBT5 streptomycete spores with escherichia coli, coating on an MS culture medium without antibiotics, culturing for 12-16 hours at the temperature of 30 ℃, and covering a flat plate with the antibiotics corresponding to the target plasmid and naphthalenedone acid or trimethoprim An; the growth of the conjugant was seen 2-3 days after the coverage, and then the recombinant strain was obtained as follows.
Step six, fermenting and culturing recombinant strain and detecting phospholipase D enzyme activity
The strain with the best expression effect is found out through fermentation culture and enzyme activity detection of the original strain and the recombinant strain.
Wherein the original strain is S.lividans Streptomyces lividans, S.antieticus Streptomyces antibiotic and S.chromofulcus Streptomyces chroomofuscus.
The recombinant strains are the first recombinant strain, the second recombinant strain, the third recombinant strain and the fourth recombinant strain obtained in the step five.
The strain is adopted for fermentation culture and enzyme activity detection by adopting a general method, and the specific method is as follows: inoculating Streptomyces into a fermentation medium at 30deg.C in an incubator for 5-7 days; culturing the bacterial liquid until the bacterial liquid is turbid, centrifuging, and taking a supernatant; filtering the supernatant with a 0.45um filter membrane to further remove impurities; the protein was precipitated over night with ammonium sulfate. Centrifuging at a high speed, and discarding the supernatant; and re-dissolving the protein, and performing ultrafiltration and desalination. Phospholipase D hydrolysis activity was spectrophotometrically determined using p-PNP (phosphatidyl para-nitrophenol) as substrate. The phosphatidyl transfer activity was determined by the reaction of PC to Phosphatidylethanolamine (PE), the PC and PE concentrations were measured by chloroform-methanol-water (13:5:0.8 volume ratio) as developing agent, GF254 silica gel plate TLC; the iodine vapor stained lamina plate was used to measure the spot area and gray scale values using image processing software to calculate the phospholipid component concentration. Enzyme protein concentration was measured using Bradford method with bovine serum albumin as standard and used for PLD specific activity measurement, and the following results were obtained:
according to the data in the table, the enzyme activity measurement values of the recombinant strain are higher than those of the original strain, so that the phospholipase D expression effect of the recombinant strain after genetic engineering is better; the enzyme activity measurement values of the recombinant strain adopting the efficient expression vector of streptomycete are higher than those of the recombinant strain adopting the common expression vector of streptomycete, 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 genetically engineered recombinant streptomycete which is screened by the method and is used for expressing phospholipase D most efficiently.
The third recombinant strain is a recombinant strain formed by transferring recombinant plasmid pMS82-PLDAnti into Streptomyces SBT5 in a joint way, wherein pMS82 is derived from an ADDGENE library, and a Streptomyces high-efficiency expression vector pMS82 is purchased by a medium-source biological company, and PLDAnti is derived from a phospholipase D synthetic gene of S.anti-biological antibiotic Streptomyces.
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Claims (3)
1. A method for highly expressing phospholipase D in streptomyces, comprising the steps of:
selecting a heterologous efficient expression host of phospholipase D, selecting streptomycete as a host, and simultaneously selecting a phospholipase D gene from streptomycete as a target gene as a modification object of genetic engineering;
screening an optimal source of a phospholipase D gene, and performing a comparison experiment through enzyme activity measurement, wherein S.anti-biological streptomyces and S.chromofuscus brown streptomyces are selected as target strains for obtaining target genes, the sequence of the phospholipase D gene from the streptomyces is shown as SEQ ID NO.1, and the sequence of the phospholipase D gene from the brown streptomyces is shown as SEQ ID NO. 2;
step three, obtaining a phospholipase D gene fragment from streptomyces, and obtaining a phospholipase D gene from streptomyces antibiotics and a phospholipase D gene from streptomyces chroogomphus through a PCR amplification technology;
step four, connecting target genes to form a phospholipase D recombinant plasmid, respectively connecting a phospholipase D gene derived from streptomyces antibiotics with a streptomyces common expression vector and a streptomyces high-efficiency expression vector to form a first recombinant plasmid and a third recombinant plasmid, respectively connecting a phospholipase D gene derived from streptomyces chrofuscus with a streptomyces common expression vector and a streptomyces high-efficiency expression vector to form a second recombinant plasmid and a fourth recombinant plasmid, wherein the streptomyces common expression vector is pSET152, and the streptomyces high-efficiency expression vector is pMS82;
step five, the recombinant plasmid is transferred to host bacteria in a joint way, and the first recombinant strain, the second recombinant strain, the third recombinant strain and the fourth recombinant strain which are sequentially corresponding to the first recombinant plasmid, the second recombinant plasmid, the third recombinant plasmid and the fourth recombinant plasmid are obtained through the joint transfer way, wherein the host bacteria are Streptomyces SBT5;
step six, fermenting and culturing the recombinant strain and detecting the enzyme activity of phospholipase D, and detecting the enzyme activities of the original strain and the recombinant strain to further find out the strain with the best expression effect, wherein the original strain is S.lividans streptomyces lividans, S.antieticus streptomyces antibiotics and S.chromofuscus streptomyces chrous; the recombinant strains are the first recombinant strain, the second recombinant strain, the third recombinant strain and the fourth recombinant strain obtained in the step five.
2. The method for highly expressing phospholipase D in streptomyces according to claim 1, wherein the PCR amplification technique in step three comprises: extracting genome DNA of S.antiiotic streptomyces antibiotics and S.chromofuscus streptomyces, respectively, (2) designing a primer PCR to obtain a target gene and sequencing and verifying by taking the genome DNA of S.antiiotic streptomyces antibiotics and S.chromofuscus streptomyces as templates, and (3) carrying out codon optimization on the obtained target fragment to obtain phospholipase D genes from streptomyces antibiotics and phospholipase D genes from streptomyces chrofuscus.
3. A recombinant Streptomyces capable of efficiently expressing phospholipase D, which is characterized in that a recombinant plasmid pMS82-PLD is obtained by the method of claim 1 Anti， Recombinant plasmid pMS82-PLD Anti Conjugation of recombinant strains, PLD, transferred into Streptomyces SBT5 Anti Phospholipase D gene from s.
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Citations (2)
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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 |
| CN102286440A (en) * | 2011-07-22 | 2011-12-21 | 天津科技大学 | Preparation of high-activity phospholipids enzyme D and cell surface display phospholipids enzyme D yeast whole cell catalysts |
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| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
| CN102286440A (en) * | 2011-07-22 | 2011-12-21 | 天津科技大学 | Preparation of high-activity phospholipids enzyme D and cell surface display phospholipids enzyme D yeast whole cell catalysts |
Non-Patent Citations (1)
| Title |
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| Comparison of the expression of phospholipase D from Streptomyces halstedii in different hosts and its over-expression in Streptomyces lividans;Xinyi Tao 等;FEMS Microbiology Letters;第366卷;全文 * |
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