CN117821476A - Saline-alkali tolerant pantothenate synthase gene and expression vector and application thereof - Google Patents
Saline-alkali tolerant pantothenate synthase gene and expression vector and application thereof Download PDFInfo
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Abstract
The invention relates to a salt and alkali tolerant pantothenate synthase gene and an expression vector and application thereof. A salt and alkali tolerant pantothenate synthase gene having the sequence <210>1, or <210>2, or <210>3, or <210>4, or <210>5, or <210>6. The invention also discloses a saline-alkali resistant expression vector and application thereof. The invention provides a salt and alkali tolerant pantothenate synthase gene, an expression vector and application thereof, and provides the salt and alkali tolerant pantothenate synthase gene and an expression vector constructed by using the gene, wherein the salt and alkali tolerant performance of transgenic microorganisms can be obviously improved after the gene is over-expressed, and the invention provides important reference value for constructing salt and alkali tolerant microbial cell factories.
Description
Technical Field
The invention belongs to the technical field of microbial genetic engineering, and particularly relates to a saline-alkali tolerant pantothenate synthase gene, an expression vector and application thereof.
Background
With the increasing maturity of synthetic biology and metabolic engineering technologies, microbial cell factories have been widely used for industrial production of substances including terpenes, organic acids, biofuels, sugar substitutes, proteins, and the like. As the microorganism chassis which is the most deeply studied and widely used at present, the escherichia coli and the yeast have clear genetic background and abundant gene manipulation tools, so that the escherichia coli and the yeast are greatly and wonderful in the research and industrial fields. However, the limited robustness of such microorganisms determines high demands on nutrients, sterilization conditions and temperatures in the actual production process, resulting in increased production costs and energy consumption. The improvement of the microorganism robustness through metabolic engineering means is a potential requirement for the control of industrial production cost and the improvement of efficiency.
Environmental pollution is a major challenge in today's world, and life and industrial pollutant emissions are the major contributors to these problems. Therefore, how to environmentally friendly and harmless various pollutants and even turn waste into wealth are key problems to be solved urgently. Microorganisms are the most widely used organisms in nature, and adapt to evolution for a long time in a living environment to obtain unique physiological metabolic characteristics, such as catabolism of pollutants, reduction of heavy metal pollution and the like. Unfortunately, in practical applications, most microorganisms that acquire degradation capability cannot adapt to the complex physicochemical environment of the contaminant; in contrast, microorganisms that survive in a contaminated environment have difficulty in efficiently degrading the contaminants. This greatly limits the effective use of microorganisms in environmental control.
The salt-tolerant/alkalophilic microorganism has a unique response mechanism to a saline-alkaline and hypertonic environment. The analysis of the response mechanisms can obtain high-efficiency saline-alkali response elements, and the elements are integrated into the genome of the target strain through metabolic engineering means, so that the performance of the elements in a saline-alkali environment can be effectively improved. The saline-alkali tolerant microorganism is highly tolerant to high saline-alkali environment, and is the chassis cell successfully developed first by the next generation industrial biotechnology.
Soil salinization is one of main abiotic stress factors affecting agriculture and forestry production and causing agriculture and forestry biomass reduction, and the excavation of new genes responding to saline-alkali stress has important strategic significance in improving saline-alkali soil utilization and promoting sustainable development of crops. The strain in industrial fermentation can be subjected to multiple environmental stresses, and compared with industrial production of model microorganisms, the saline-alkali tolerant microorganisms can perform open and continuous fermentation under the conditions of non-sterilization and disinfection, so that the fermentation yield is improved, the cost is reduced, the process is simplified, and the industrial development is promoted.
Along with the further excavation of the biosynthesis capacity of the saline-alkali tolerant microorganisms and the development of chassis transformation and transformation of the saline-alkali tolerant microorganisms by a genetic engineering technology, the application scenes of the saline-alkali tolerant microorganisms are gradually expanded, and more commercial values are brought.
Disclosure of Invention
A first object of the present invention is to provide a salt and alkali tolerant pantothenate synthase gene having the sequence <210>1, or <210>2, or <210>3, or <210>4, or <210>5, or <210>6. The pantothenate synthase gene (panC) is a universal saline-alkali resistant gene element, and can improve the saline-alkali resistance of a microbial cell factory.
Further, the sequence of the encoded amino acid of the pantothenate synthase gene is <210>7, or <210>8, or <210>9, or <210>10, or <210>11, or <210>12.
Further, the pantothenate synthase gene also includes the sequence <210>1, or <210>2, or <210>3, or <210>4, or <210>5, or <210>6 codon-optimized, as <210>13, or <210>14, or <210>15, or <210>16, or <210>17, or <210>18.
The second object of the present invention is to provide an expression vector for a salt and alkali tolerant pantothenate synthase gene, which has the aforementioned pantothenate synthase gene.
Furthermore, the expression vector is panC-PET28a vector, and is expressed in microorganisms to improve the saline-alkali resistance.
The third object of the present invention is to provide the use of the aforementioned pantothenate synthase gene or the aforementioned expression vector in microorganisms.
Further, the expression vector is transformed into a receptor host cell, and then whether the saline-alkali tolerance of the host cell is improved is screened and identified.
The expression vector is constructed by adopting the pantothenate synthase gene.
Still further, the host comprises bacteria;
the pantothenate synthase gene has the sequence <210>1.
Still further, the process for identifying whether the saline-alkali tolerance of the host cell is improved is as follows: and (3) screening and identifying by using a marker gene according to the screening markers on the vector, and determining the saline-alkali tolerance of the host microorganism.
Still further, in the identification process, the selection marker on the vector is kanamycin sulfate.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides pantothenate synthase (panC) genes, an expression vector and application thereof, wherein the expression vector is constructed by utilizing the genes, and the pantothenate synthase (panC) genes are expressed in transgenic microorganisms to obtain saline-alkali tolerant gene microorganisms. Specific: the obtained panC gene can be expressed in the escherichia coli, the saline-alkali tolerance of the escherichia coli can be obviously improved, the saline-alkali resistance of the escherichia coli can be obviously improved, and meanwhile, the escherichia coli strain which is beneficial to industrial production in a high saline-alkali environment is obtained.
Drawings
FIG. 1 is a graph showing the results of pET-P19panC transformation of E.coli BL21 (DE 3) (A), E.coli S17-1. Lambda. Pir (B) and E.coli Dh5α (C).
FIG. 2 is a diagram showing the result of PCR of a transformant bacterial liquid; lanes 1 and 4 show pET-P19panC transformed E.coli Dh5α, lanes 2 and 5 show pET-P19panC transformed E.coli BL21 (DE 3), and lanes 3 and 6 show pET-P19panC transformed E.coli S17-1. Lambda. Pir.
FIG. 3 is a graph showing the growth states of E.coli BL21 (DE 3) carrying pET-P19panC in saline-alkali environments at pH8, 6.5% (left panel) and 7% (right panel). A. B is a control group, namely wild escherichia coli and escherichia coli with empty plasmid pET28a respectively; c is experimental group, and the induced genetically engineered bacteria.
FIG. 4 is a graph showing the growth of E.coli S17-1. Lambda. Pir carrying pET-P19panC in saline-alkaline environments at pH8, naCl6% (left panel) and 6.5% (right panel). A. B is a control group, namely wild escherichia coli and escherichia coli with empty plasmid pET28a respectively; c is experimental group, and the induced genetically engineered bacteria.
FIG. 5 is a graph showing the growth of E.coli Dh5α carrying pET-P19panC in saline-alkaline environments at pH8, naCl6% (left panel) and 6.5% (right panel). A. B, C is a control group, which is wild E.coli, E.coli with empty plasmid pET28a and non-induced engineering bacteria; d is an experimental group, and is a genetically engineered bacterium after induction.
FIG. 6 is a graph showing the growth of E.coli BL21 (DE 3) carrying pET-P19panC in pH8, naCl6% liquid medium. WT is wild E.coli BL21 (DE 3); EV is E.coli BL21 (DE 3) with empty plasmid pET28 a; ppB is a genetically engineered bacterium.
FIG. 7 is a graph showing the growth of E.coli S17-1. Lambda. Pir carrying pET-P19panC in a 6% NaCl solution at pH 8. WT is wild E.coli S17-1 lambda pir; EV is E.coli S17-1 lambda pir with empty plasmid pET28 a; ppS is a genetically engineered bacterium.
FIG. 8 is a graph showing growth of E.coli Dh5α carrying pET-P19panC in pH8, naCl6% liquid medium. WT is wild E.coli Dh5α; EV is the Escherichia coli Dh5α with empty plasmid pET28 a; ppD is a genetically engineered bacterium.
Detailed Description
In order to further illustrate the saline-alkali tolerant pantothenate synthase gene, the expression vector and the application thereof, which achieve the aim of the expected invention, the following detailed description refers to the saline-alkali tolerant pantothenate synthase gene, the expression vector and the application thereof, the specific implementation, the structure, the characteristics and the efficacy thereof according to the present invention by combining with the preferred examples. In the following description, different "an embodiment" or "an embodiment" do not necessarily refer to the same embodiment. Furthermore, the particular features, structures, or characteristics of one or more embodiments may be combined in any suitable manner.
The following will describe the salt and alkali tolerant pantothenate synthase gene, its expression vector and application in further detail with reference to specific examples:
the invention provides a universal salt and alkali resistant gene element-pantothenic acid synthase gene (panC) for the salt and alkali resistance of microorganisms; and constructs an expression vector: panC-PET28a is expressed in the transgenic microorganism, so that the salt tolerance of the transgenic microorganism is improved. Through the over-expression of the gene, the salt tolerance of the microorganism is improved, and the microorganism with obviously enhanced salt tolerance is finally obtained. The technical scheme of the invention is as follows:
a salt and alkali tolerant pantothenate synthase gene has the sequence <210>1, or <210>2, or <210>3, or <210>4, or <210>5, or <210>6. The pantothenate synthase gene (panC) is a universal saline-alkali resistant gene element, and can improve the saline-alkali resistance of a microbial cell factory.
In the technical scheme, the gene also comprises all sequences of the panC gene sequence subjected to codon optimization.
Preferably, the pantothenate synthase gene has an amino acid sequence of <210>7, or <210>8, or <210>9, or <210>10, or <210>11, or <210>12.
An expression vector of a salt and alkali tolerant pantothenate synthase gene, said expression vector having said pantothenate synthase gene.
Preferably, the expression vector is panC-PET28a vector, and is expressed in microorganisms to improve the saline-alkali resistance.
In the above technical scheme, constructing a vector and transforming the vector into a receptor microorganism, the preferred vector is P19panC-pET28a + A carrier.
The pantothenate synthase gene described above, or the expression vector described above, is used in microorganisms.
Preferably, the expression vector is transformed into a receptor host cell, and then whether the saline-alkali tolerance of the host cell is improved is screened and identified.
The expression vector is constructed by adopting the pantothenate synthase gene.
The technical scheme comprises the following steps: more specific operations are:
(1) experimental materials and selection: the mode of origin of the panC gene and the recipient host to be transformed are chosen.
(2) Cloning of panC Gene: cloning of the panC gene can be performed by chemical synthesis or biosynthesis. When a biosynthesis method is used, cloning of the panC gene is performed using the whole gene of <210>1, or <210>2, or <210>3, or <210>4, or <210>5, or <210>6 (preferably, the gene of Bacillus haynesii P19, that is, <210> 1) as a template, and cloning of the cloned product is performed.
(3) Construction of vectors and transformation to receptors: the primers are designed to realize the connection of panC genes into vectors by using a homologous recombination technology, and the recombinant vectors are transformed into host receptors.
(4) Identification of transformation results and determination of the saline-alkali tolerance of organisms; screening and identifying by using a marker gene according to the screening marker on the carrier, and determining the saline-alkali tolerance of organisms.
Further preferred, the host comprises a bacterium;
the pantothenate synthase gene has the sequence <210>1. Namely, in the selected microbial material, the bacillus marinus P19 (Bacillus haynesii P19) is sourced, and the host is bacteria.
In the above technical solution, as a preferred solution for the application of the panC gene sequence related to the microbial saline-alkali tolerance according to the present invention, the following applies: in cloning of the panC gene, the CDS sequence of the panC gene was determined using Bacillus haynesii P19 whole genome information.
Further preferably, the process for identifying whether the saline-alkali tolerance of the host cell is improved is as follows: and (3) screening and identifying by using a marker gene according to the screening markers on the vector, and determining the saline-alkali tolerance of the host microorganism.
Further preferably, in the identification process, the selection marker on the carrier is kanamycin sulfate.
In the above technical scheme, the screening marker on the carrier is kanamycin sulfate, so that the kanamycin sulfate can be used for screening and identifying transformation results.
Example 1.
The pantothenic acid synthase gene (panC) which is a salt and alkali tolerant gene element is mined from salt and alkali tolerant microorganisms by utilizing a multi-group chemical combination technology. The element of the salt and alkali tolerant gene is panC, and the gene exists in salt and alkali tolerant bacteria. The panC gene sequence and the amino acid sequence are specifically as follows:
in Bacillus marinus (Bacillus haynesii) P19, the panC gene size was 867bp, the panC gene sequence was <210>1, the codon-optimized sequence of the panC gene sequence was <210>13, and the amino acid sequence was <210>7.
In Micrococcus luteus (Micrococcus luteus) R17, the panC gene size is 1032bp, the panC gene sequence is <210>2, the sequence of the panC gene sequence after codon optimization is <210>14, and the amino acid sequence is <210>8.
In Enterobacter cloacae (Enterobacter cloacae) RS35, the panC gene is in the size of 522 p, the panC gene sequence is <210>3, the codon-optimized sequence of the panC gene sequence is <210>15, and the amino acid sequence is <210>9.
In Brevibacterium lactobacilli (Brevibacterium strain) G20, the panC gene size is 635bp, the panC gene sequence is <210>4, the sequence of the panC gene sequence after codon optimization is <210>16, and the amino acid sequence is <210>10.
In halomonas (halomonas genes) TD01, the panC gene size is 884bp, the panC gene sequence is <210>5, the sequence of the panC gene sequence after codon optimization is <210>17, and the amino acid sequence is <210>11.
In the halomonas (Halomonas campaniensis LS) LS21, the panC gene size was 870bp, the panC gene sequence was <210>6, the codon-optimized sequence of the panC gene sequence was <210>18, and the amino acid sequence was <210>12.
Example 2.
The embodiment provides a method for synthesizing and constructing a carrier of a saline-alkali tolerance gene panC of a saline-alkali tolerant microorganism, which comprises the following steps:
(1) The P19panC CDS sequence is obtained by taking the whole genome of Bacillus haynesii P panC (gene sequence is <210> 1) as an information template, and panC is synthesized by a chemical synthesis method.
(2) The panC gene element is cloned to an expression vector pET28a through NcoI and HindIII digestion + Finally obtaining the over-expression vector P19panC-PET28a + A carrier.
(3) Adopting the same steps of steps (1) - (2), respectively adopting Micrococcus luteusR panC (gene sequence is<210>2) Enterobacter cloacaeRS35panC (Gene sequence is<210>3) Brevibacterium casei G20panC (Gene sequence is<210>4) Halomonasbluephagenesis TD01panC (Gene sequence is<210>5) Halomonas campaniensis LS21panC (Gene sequence is<210>6) The whole genome of (2) is used as an information template to respectively obtain R17panC-PET28a + Carrier, RS35panC-PET28a + Vector, G20panC-PET28a + Carrier, TD01panC-PET28a + Carrier, LS21panC-PET28a + A carrier.
Example 3.
This example was performed using the P19panC-PET28a constructed in example 2 + The vector was transformed into E.coli BL21 (DE 3), E.coli Dh5α and E.coli S17-1λpir by the heat shock transformation method (FIG. 1). The resulting transformants were verified by bacterial liquid PCR using panC specific primers, followed by detection by 1% agarose gel electrophoresis and observation by means of a gel electrophoresis imager (FIG. 2).
Wherein the primer design, amplification system, reaction procedure are shown in tables 1-3 below:
TABLE 1panC specific primers
TABLE 2 bacterial liquid PCR reaction system
TABLE 3 bacterial liquid PCR reaction procedure
Example 4.
Diluting the bacterial liquid after the induction of the escherichia coli BL21 (DE 3) carrying pET-P19panC to 10 -2 At pH8, naCl6.5% and 7% saline-alkali plate medium culture, each cell inoculation amount of 30 u L, results are shown in figure 3. The result shows that the control group has no bacteria colony growth and the experimental group engineering bacteria colony has good growth vigor.
E.coli S17-1 lambda pir carrying pET-P19panC is diluted to 10 after induction -2 Saline-alkali plate medium with pH8, naCl6% and 6.5% is used for culturing, and each cell is inoculated with 30 mu LThe results are shown in FIG. 4. The result shows that the control group has no bacteria colony growth and the experimental group engineering bacteria colony has good growth vigor.
Diluting the bacterial liquid after induction of the Escherichia coli Dh5α carrying pET-P19panC to 10 -2 At pH8, naCl 3 The results of the 6% and 6.5% saline-alkali plate medium cultures with 30. Mu.L of each cell inoculum size are shown in FIG. 5. The result shows that the control group has no bacteria colony growth and the experimental group engineering bacteria colony has good growth vigor.
Example 5.
Picking positive clones, culturing overnight, collecting overnight culture bacterial liquid, inoculating to kan-containing strain containing 1% of inoculating amount + In LB shake flask medium to be OD 600 When reaching 0.6-0.8, 0.1mM IPTG was added (no IPTG was added to the control group) and induced to grow for 3 hours at 30℃and 220 rpm. Dividing the bacterial liquid into two groups, and diluting the bacterial liquid to 10 -2 The growth was observed every 4h in a 6% nacl, ph=8lb liquid medium inoculated at 1% inoculum size.
The growth curve of E.coli BL21 (DE 3) carrying pET-P19panC in pH8, 6% NaCl liquid medium is shown in FIG. 6. The result shows that the growth amount of engineering bacteria E.coli BL21 (DE 3) -pET28a-P19panC carrying the plasmid containing the target gene P19panC exceeds that of wild E.coli BL21 (DE 3) of a control group and E.coli BL21 (DE 3) -pET28a carrying an empty vector at the 10 th hour.
The growth curve of E.coli S17-1. Lambda. Pir carrying pET-P19panC in pH8, 6% NaCl liquid medium is shown in FIG. 7. The result shows that the growth amount of engineering bacteria E.coli S17-1 lambda pir-pET28a-P19panC carrying the target gene P19panC at 32h exceeds that of wild E.coli S17-1 lambda pir of a control group and E.coli S17-1 lambda pir-pET28a carrying an empty vector.
The growth curve of E.coli Dh5α carrying pET-P19panC in pH8, 6% NaCl liquid medium is shown in FIG. 8. The result shows that the growth amount of the engineering bacteria E.coli Dh5α -pET28a-P19panC carrying the target gene P19panC at 32h exceeds that of the wild E.coli Dh5α of the control group and E.coli Dh5α -pET28a carrying the empty vector.
It will be appreciated in connection with the embodiments of the invention thatCloning saline-alkali tolerant gene element panC found in saline-alkali tolerant bacteria to expression vector pET-28a + The strain is transformed into host cells such as escherichia coli BL21 (DE 3), escherichia coli S17-1 lambda pir, escherichia coli Dh5α and the like for induced expression, and the result shows that the host cells have higher saline-alkali tolerance than wild strains in saline-alkali environment. In addition, it has been found that E.coli promotes pantothenic acid synthesis while increasing saline-alkali tolerance. The invention excavates the stress-resistant element of the saline-alkali tolerant microorganism and can obviously improve the stress resistance of the host by heterologous expression in other hosts. The invention provides important reference value for constructing saline-alkali tolerant microbial cell factories.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the embodiment of the present invention in any way, but any simple modification, equivalent variation and modification of the above embodiment according to the technical substance of the embodiment of the present invention still fall within the scope of the technical solution of the embodiment of the present invention.
Claims (10)
1. A salt and alkali tolerant pantothenate synthase gene, characterized in that the sequence of the pantothenate synthase gene is <210>1, or <210>2, or <210>3, or <210>4, or <210>5, or <210>6.
2. The pantothenate synthase gene according to claim 1, characterized in that,
the pantothenate synthase gene has an amino acid sequence of <210>7, or <210>8, or <210>9, or <210>10, or <210>11, or <210>12.
3. The pantothenate synthase gene according to claim 1, characterized in that,
the pantothenate synthase gene also includes the sequence <210>1, or <210>2, or <210>3, or <210>4, or <210>5, or <210>6 codon-optimized, as <210>13, or <210>14, or <210>15, or <210>16, or <210>17, or <210>18.
4. An expression vector for a salt and alkali tolerant pantothenate synthase gene, wherein said expression vector comprises the pantothenate synthase gene of claim 1.
5. The expression vector of claim 4, wherein the vector comprises a nucleotide sequence,
the expression vector is panC-PET28a vector, and is expressed in microorganisms to improve the saline-alkali resistance.
6. Use of the pantothenate synthase gene of any one of claims 1-3, or the expression vector of any one of claims 4-5, in a microorganism.
7. The use according to claim 6, wherein,
transforming the expression vector of any one of claims 4-5 into a recipient host cell, and then screening and identifying whether the saline-alkali tolerance of the host cell is improved.
The expression vector is constructed by adopting the pantothenate synthase gene according to claim 1.
8. The use according to claim 7, wherein,
the pantothenate synthase gene has a sequence of <210>1;
the host includes bacteria.
9. The use according to claim 7, wherein,
the process for identifying whether the saline-alkali tolerance of the host cells is improved is as follows: and (3) screening and identifying by using a marker gene according to the screening markers on the vector, and determining the saline-alkali tolerance of the host microorganism.
10. The use according to claim 9, wherein,
in the identification process, the screening mark on the carrier is kanamycin sulfate.
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