CN112813090A - Heterologous expression recombinant plasmid of phospholipase D and construction method thereof - Google Patents
Heterologous expression recombinant plasmid of phospholipase D and construction method thereof Download PDFInfo
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- CN112813090A CN112813090A CN202011640263.0A CN202011640263A CN112813090A CN 112813090 A CN112813090 A CN 112813090A CN 202011640263 A CN202011640263 A CN 202011640263A CN 112813090 A CN112813090 A CN 112813090A
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- 230000014509 gene expression Effects 0.000 title claims abstract description 37
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/04—Phosphoric diester hydrolases (3.1.4)
- C12Y301/04004—Phospholipase D (3.1.4.4)
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Abstract
The invention relates to a heterologous expression recombinant plasmid of phospholipase D, and a construction method thereof comprises the steps of artificially synthesizing a plasmid pUC57-STR _ ORI, artificially synthesizing a plasmid pUC57-STR _ PLD, and constructing the recombinant plasmid to finally obtain the recombinant plasmid comprising a Kasop constitutive strong promoter, a signal peptide of streptomyces cinnamomi, a phospholipase D synthetic gene derived from the streptomyces and an ApmR resistance screening gene. The recombinant plasmid obtained in the invention can be successfully expressed in streptomycete, the promoter has strong starting function, the signal peptide can lead the newly synthesized protein to be transferred to a secretion pathway with good effect, and the phospholipase D can be successfully expressed by the phospholipase D synthesis gene segment. The expression element of the gene is synthesized by a plasmid synthesis method, so that a complete recombinant plasmid is formed, the plasmid structure is stable, the transfer into a streptomycete host is convenient, and the successful expression in the streptomycete host is ensured.
Description
Technical Field
The invention belongs to the field of gene recombination, and relates to a heterologous expression recombinant plasmid of phospholipase D and a construction method thereof.
Background
Phosphatidylserine has a small amount in nature and is difficult to extract as a substance having a wide market prospect; the phosphatidylserine in the market is mainly 'phosphatidylserine Leci-PS' (August 28, 2003) produced by degussa corporation, and is mainly extracted from soybeans, so that the yield is low and the price is high.
In recent years, attention has been paid to the production of phosphatidylserine by enzymatic conversion, i.e., the synthesis of phosphatidylcholine and L-serine, which are substrates catalyzed by phospholipase D. The microbial phosphatidylserine synthetase D (hereinafter referred to as PLD) 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 PLD secretion is far lower than the industrial production standard, and the application of the PLD in the industrialization of synthesizing phosphatidylserine is seriously influenced.
It is desired to perform genetic construction and genetic modification by genetic engineering means to obtain a strain capable of heterologously expressing PLD in order to obtain PLD in high yield, and it becomes critical to construct a recombinant plasmid capable of well expressing PLD.
In general, expression vectors are provided with replication elements, such as replicons, that allow for the continued replication of the gene in a heterologous host; control elements, such as promoters and terminators, control the initiation and termination of genes; some of them also have some special elements, such as secretion peptide in signal peptide, which can control the secretion of the final expression product to the outside of cell for production. In addition, expression vectors will typically carry a resistance gene or other marker for screening positive clones during the course of the experiment.
According to the disclosure of the prior patent technology, there have been studies on genetic engineering modification of strains such as bacillus subtilis, for example, CN108795837A, a bacillus subtilis engineered bacterium for efficiently expressing phospholipase D, in which efficient expression of endogenous phospholipase D derived from actinomycetes in bacillus subtilis is disclosed, and studies on protein modification of PLD target gene extracted from streptomyces have been also disclosed, for example, CN111004787A, a streptomyces phospholipase D mutant, modification method and application thereof, wherein the studies on wild-type PLD by means of protein engineering are disclosed; then, according to the research direction and results of the predecessors, a heterologous expression recombinant plasmid of phospholipase D is expected to be obtained, so as to search for a more efficient and more convenient construction method.
Disclosure of Invention
The invention aims to construct a heterologous expression plasmid of phospholipase D by means of genetic engineering for genetic modification, so that the heterologous expression plasmid can be successfully expressed in streptomyces.
The technical scheme adopted by the invention is as follows:
a heterologous expression recombinant plasmid of phospholipase D is disclosed, the plasmid expression original includes Kasop constitutive strong promoter, signal peptide of streptomyces cinnamomi, phospholipase D synthetic gene from streptomyces and apmR resistance screening gene.
Wherein the nucleotide sequence of the Kasop promoter is shown as SEQ ID NO. 1.
Wherein the nucleotide sequence of the signal peptide of the streptomyces cinnamoneum is shown as SEQ ID NO. 2.
Wherein the nucleotide sequence of the streptomyces-derived phospholipase D synthetic gene is shown as SEQ ID NO. 3.
A construction method of heterologous expression recombinant plasmid of phospholipase D comprises the following specific steps:
step one, artificially synthesizing a plasmid pUC57-STR _ ORI, step two, artificially synthesizing a plasmid pUC57-STR _ PLD, step three, and constructing a recombinant plasmid by taking a pOJ260 plasmid as a vector.
The first step is to select a replicon region derived from streptomyces pIJ101 as a streptomyces replication original of a recombinant plasmid, and design enzyme cutting sites at two ends as Spe I; the EcoRV site is ligated to the vector pUC 57.
Designing a constitutive strong promoter Kasop as a promoter of a phospholipase D synthetic gene, selecting a signal peptide Sigcin from streptomyces cinnamomi as a secretion peptide of the phospholipase D synthetic gene, and selecting the phospholipase D synthetic gene from streptomyces as a target gene; and enzyme cutting sites at two ends are designed to be XbaI; the EcoRV site is ligated to the vector pUC 57.
The third step is to cut the plasmid pOJ260 and pUC57-STR _ ORI at the SpeI site, recover and purify the plasmid and then connect the plasmid with T4 DNA Ligase to obtain pOJ260-STR _ ORI; then the plasmid pOJ260-STR _ ORI and the plasmid pUC57-STR _ PLD are cut by enzyme at the XbaI site, recovered and purified, and are connected by T4 Ligase to obtain a heterologous expression recombinant plasmid pTK06 of phospholipase D.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. the recombinant plasmid obtained in the invention can be successfully expressed in streptomycete, the promoter of the recombinant plasmid has strong starting function, the signal peptide can lead the newly synthesized protein to be transferred to a secretion pathway with good effect, and the phospholipase D synthesis gene segment is beneficial to the successful expression of the expressed PLD.
2. The expression element of the synthetic gene of the plasmid synthetic method forms a complete recombinant plasmid, so that the plasmid structure is stable, the plasmid is convenient to transfer into a streptomycete host, and the plasmid tends to be efficiently expressed in the streptomycete host.
3. The invention simultaneously comprises replication initiation elements of two systems of escherichia coli and streptomycete, carries a combined transfer site ORIT of the escherichia coli and the streptomycete, can carry out gene modification in the escherichia coli according to the requirements of gene engineering, transfers successfully constructed plasmids into the streptomycete through combined transfer, and finally carries out expression of phospholipase D in the streptomycete.
Drawings
FIG. 1 is a schematic structural view of a replicon carrying vector plasmid pUC57-STR _ ORI of the present invention,
wherein STR-ORI is a streptomycete replicon;
FIG. 2 is a schematic structural diagram of a vector plasmid pUC57-STR-PLD carrying a target gene of the present invention,
STR-PLD represents the whole expression element of the synthetic gene, wherein Kasop is promoter, Sigcin is signal peptide, and PLDgene is phospholipase D synthetic gene;
FIG. 3 is a map of vector plasmid pOJ260 of the present invention,
wherein oriT is a streptomycete conjugation transfer site, ori is an escherichia coli replication initiation, and apmR is a resistance gene;
FIG. 4 is a map of replicon-added recombinant plasmid pOJ260-STR _ ORI according to the present invention;
wherein oriT is a streptomycete conjugation transfer site, ORI is an escherichia coli replication initiation, STR _ ORI is a streptomycete replicon, and ApmR is a resistance gene;
FIG. 5 is a map of a recombinant plasmid pTK06 with an added gene of interest according to the present invention;
wherein oriT is a streptomycete conjugation transfer site, ORI is an escherichia coli replication initiation, ORI-Str is a streptomycete replicon, ApmR is a resistance gene, promoter is a promoter Kasop, Signal Str _ cin is a Signal peptide, and PLDene is a phospholipase D synthesis gene.
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 construction method of heterologous expression plasmid of phospholipase D adopts the following steps:
the method comprises the following steps: artificially synthesized plasmid pUC57-STR _ ORI
Selecting a replicon region derived from streptomyces pIJ101 as a streptomyces replication original of a recombinant plasmid, and designing enzyme cutting sites at two ends as Spe I; the EcoRV locus is connected to a vector pUC57, wherein the vector pUC57 has Amp resistance genes and is artificially synthesized by Nanjing Kingsler biotechnology limited, wherein the sequence source is NCBI database, and finally, a plasmid PUC57-STR _ ORI carrying streptomyces replication elements is formed as shown in figure 1.
Step two, artificially synthesizing plasmid pUC57-STR _ PLD
Obtaining a plasmid containing a target gene expression original by an artificial synthesis means, wherein the plasmid comprises a constitutive strong promoter Kasop designed as a promoter of a phospholipase D synthetic gene, the sequence of the promoter is shown as SEQ ID NO.1, a signal peptide Sigcin from streptomyces cinnamomi is selected as a secretory peptide of the phospholipase D synthetic gene, the sequence of the signal peptide is shown as SEQ ID NO.2, the phospholipase D synthetic gene from streptomyces is selected as a target gene, and the sequence of the gene is shown as SEQ ID NO. 3; and enzyme cutting sites at two ends are designed to be XbaI; the EcoRV site is connected to a vector pUC57, wherein the vector pUC57 has Amp resistance, and finally a vector plasmid pUC57-STR _ PLD carrying the target gene is formed as shown in FIG. 2.
Step three, constructing recombinant plasmid by taking pOJ260 plasmid as vector
The SpeI site is used for enzyme cutting of plasmid pOJ260 and recovery of a linear fragment, and is regarded as a first linear fragment, wherein a map of the plasmid pOJ260 is shown in figure 3, the SpeI site is used for enzyme cutting of plasmid pUC57-STR _ ORI and recovery of a STR _ ORI fragment, the first linear fragment and the STR _ ORI fragment are purified, the first linear fragment and the STR _ ORI fragment are connected through T4 DNA Ligase, and the pOJ260-STR _ ORI is obtained after transformation and verification of a connection product, and is shown in figure 4.
Digesting the plasmid pOJ260-STR _ ORI by an XbaI site and recovering a linear fragment thereof as a second linear fragment, digesting the plasmid pUC57-STR _ PLD by the XbaI site and recovering a STR _ PLD fragment therein, purifying the second linear fragment and the STR _ PLD fragment, connecting the second linear fragment and the STR _ PLD fragment by T4 DNA Ligase, transforming a connection product and verifying the correctness to obtain a heterologous expression recombinant plasmid pTK06 of phospholipase D, wherein the heterologous expression recombinant plasmid pTK06 is shown in FIG. 5.
The specific experimental operation steps of enzyme digestion, recovery, purification and connection are shown in the second chapter and the third chapter in the fourth edition of molecular cloning experimental guidelines published by 2017 scientific press.
The T4 DNA Ligase used for ligation, the restriction enzymes Spe I and XbaI used for digestion, were purchased from PROMEGA.
Streptomyces strains used in the experiments were purchased from Shanghai, and plasmids pJTU1278 and pOJ260 were purchased from Wuhan vast Ling.
The heterologous expression recombinant plasmid pTK06 of phospholipase D thus obtained is shown in FIG. 5, and the sequence thereof is shown in SEQ ID NO.4, wherein the nucleotide sequence of the Kasop promoter is shown in SEQ ID NO. 1; wherein the nucleotide sequence of the signal peptide of the streptomyces cinnamoneum is shown as SEQ ID NO. 2; wherein the nucleotide sequence of the streptomyces-derived phospholipase D synthetic gene is shown as SEQ ID NO. 3.
Introducing a recombinant plasmid pTK06 for heterologous expression of phospholipase D into streptomycete for expression, fermenting, culturing and measuring enzyme activity, wherein the relative enzyme activity data is good in performance, and the specific results are as follows:
from the above table, it can be seen that the enzyme activity data of the recombinant plasmid is better than that of the original plasmid, i.e., the expression effect of the recombinant plasmid is better, and the recombinant plasmid also has the tendency of realizing high-efficiency expression.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
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tcctgcatgc cgtccatgga gcaggacgag gccggctccg ccccggccga gccgaccggc 1440
gacgtcccgg tcatcgccgt cggcggcctg ggcgtcggca tcaaggagtc cgacccgtcc 1500
tccggctacc acccggacct gccgaccgcc ccggacacca agtgcaccgt cggcctgcac 1560
gacaacacca acgccgaccg cgactacgac accgtcaacc cggaggagaa cgccctgcgc 1620
tccctgatcg cctccgcccg ctcccacgtc gagatctccc agcaggacct gaacgccacc 1680
tgcccgccgc tgccgcgcta cgacatccgc acctacgaca ccctggccgg caagctggcc 1740
gccggcgtca aggtccgcat cgtcgtctcc gacccggcca accgcggcgc cgtcggctcc 1800
ggcggctact cccagatcaa gtccctggac gagatctccg acaccctgcg cacccgcctg 1860
gtcgccctga ccggcgacaa cgagaaggcc tcccgcgccc tgtgcggcaa cctgcagctg 1920
gcctccttcc gctcctccga cgccgccaag tgggccgacg gcaagccgta cgccctgcac 1980
cacaagctgg tctccgtcga cgactccgcc ttctacatcg gctccaagaa cctgtacccg 2040
gcctggct 2048
Claims (8)
1. A heterologous expression recombinant plasmid of phospholipase D is characterized in that the sequence of the heterologous expression recombinant plasmid is shown in SEQ ID No.4, wherein the expression elements comprise a Kasop constitutive strong promoter, a signal peptide of streptomyces cinnamomi, a phospholipase D synthetic gene derived from streptomyces and an ApmR resistance screening gene.
2. The recombinant plasmid for heterologous expression of phospholipase D according to claim 1, wherein the nucleotide sequence of the Kasop promoter is shown in SEQ ID No. 1.
3. The heterologous expression recombinant plasmid for phospholipase D according to claim 1, wherein the nucleotide sequence of the signal peptide of Streptomyces cinnamoneum is shown in SEQ ID No. 2.
4. The heterologous expression recombinant plasmid for phospholipase D of claim 1, wherein the nucleotide sequence of the Streptomyces-derived phospholipase D synthetic gene is shown in SEQ ID NO. 3.
5. A construction method of heterologous expression recombinant plasmid of phospholipase D is characterized by comprising the following specific steps:
step one, artificially synthesizing a plasmid pUC57-STR _ ORI, step two, artificially synthesizing a plasmid pUC57-STR _ PLD, and step three, and constructing a recombinant plasmid based on the pOJ260 plasmid.
6. The method for constructing the heterologous expression recombinant plasmid of phospholipase D according to claim 5, wherein the first step is to select the replicon region derived from Streptomyces pIJ101 as the replication element of the recombinant plasmid and design the enzyme cutting sites at both ends as Spe I; the EcoRV site is ligated to the vector pUC 57.
7. The method for constructing heterologous expression recombinant plasmid for phospholipase D according to claim 5, wherein the second step is to design a constitutive strong promoter Kasop as the promoter of the phospholipase D synthetic gene, select the signal peptide Sigcin derived from Streptomyces cinnamomi as the secretory peptide of the phospholipase D synthetic gene, and select the phospholipase D synthetic gene derived from Streptomyces streptomyces as the target gene; and enzyme cutting sites at two ends are designed to be XbaI; the EcoRV site is ligated to the vector pUC 57.
8. The method for constructing the heterologous expression recombinant plasmid for phospholipase D according to claim 5, wherein the third step is to cleave plasmid pOJ260 and pUC57-STR _ ORI at Spe I site, recover and purify the plasmid, and then connect the plasmid with T4 DNA Ligase to obtain pOJ260-STR _ ORI; then the plasmid pOJ260-STR _ ORI and the plasmid pUC57-STR _ PLD are cut by enzyme at the XbaI site, recovered and purified, and are connected by T4 Ligase to obtain a heterologous expression recombinant plasmid pTK06 of phospholipase D.
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| EP2573188A2 (en) * | 2005-03-02 | 2013-03-27 | Metanomics GmbH | Process for the production of fine chemicals |
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| CN108841770A (en) * | 2018-07-06 | 2018-11-20 | 江南大学 | A kind of bacillus subtilis engineering bacteria that can express phospholipase D |
| CN109456929A (en) * | 2018-12-13 | 2019-03-12 | 江南大学 | It is a kind of produce phospholipase D recombined bacillus subtilis and its application |
| CN110904072A (en) * | 2019-11-21 | 2020-03-24 | 天津科技大学 | Novel phospholipase D and method for preparing functional phospholipid by using same |
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| WO2003052075A2 (en) * | 2001-12-14 | 2003-06-26 | Incyte Genomics, Inc. | Enzymes |
| EP2573188A2 (en) * | 2005-03-02 | 2013-03-27 | Metanomics GmbH | Process for the production of fine chemicals |
| JP2008193953A (en) * | 2007-02-13 | 2008-08-28 | Kanazawa Univ | Development and application of new protein expression system using actinomyces as host |
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