CN111593029A - Gamma-polyglutamic acid synthetase complex with improved catalytic activity and encoding gene thereof - Google Patents
Gamma-polyglutamic acid synthetase complex with improved catalytic activity and encoding gene thereof Download PDFInfo
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Abstract
The invention discloses a gamma-polyglutamic acid synthetase complex with improved catalytic activity and a coding gene thereof, belonging to the field of genetic engineering and enzyme engineering. The invention adopts an in vitro evolution method to directionally transform a gamma-polyglutamic acid synthetase complex PgsBCAE derived from Bacillus subtilis IFO3336 of natto, and screens to obtain a gamma-polyglutamic acid synthetase complex PgsB with improved catalytic activitymCmAmEm. Compared with PgsBCAE, the gamma-polyglutamate synthetase complex PgsB provided by the inventionmCmAmEmThe catalytic activity of synthesizing the gamma-polyglutamic acid is improved by 10.67 times. The gamma-polyglutamic acid synthetase complex with improved catalytic activity can realize high-yield biosynthesis of gamma-polyglutamic acid, and has great application potential in production of gamma-polyglutamic acid.
Description
Technical Field
The invention belongs to the field of gene engineering and enzyme engineering, and particularly relates to a gamma-polyglutamic acid synthetase complex with improved catalytic activity and a coding gene thereof.
Background
Gamma-polyglutamic acid (gamma-PGA) is polyanionic polypeptide molecules formed by condensing D-glutamic acid and L-glutamic acid monomers through amido bonds formed by alpha-amino and gamma-carboxyl. The molecular weight of the compound is generally between 100 and 1000kDa, and the compound is equivalent to about 500 to 5000 glutamic acid monomers. The gamma-polyglutamic acid is a water-soluble and biodegradable high polymer material, has good biocompatibility, low immunogenicity, no toxicity and no pollution, and the degradation product glutamic acid monomer can be absorbed and utilized by human bodies without toxic or side effects. Therefore, the gamma-polyglutamic acid has potential application value in the fields of cosmetics, biomedicine, environmental protection and the like.
The production method of gamma-polyglutamic acid includes chemical synthesis method, direct extraction method and microbial fermentation method. Compared with chemical synthesis and direct extraction, the microbial fermentation method for producing the gamma-polyglutamic acid has the obvious advantages of easy control of the production process, stable fermentation yield, high extraction rate, convenience for large-scale production and the like. The research on the production of the gamma-polyglutamic acid by a microbial fermentation method at home and abroad comprises screening microorganisms for high yield of the gamma-polyglutamic acid, mutagenizing and breeding strains for high yield of the gamma-polyglutamic acid, optimizing the fermentation conditions of the strains to obtain the high yield of the gamma-polyglutamic acid, constructing genetic engineering bacteria for high yield of the gamma-polyglutamic acid and the like. With the development of molecular biology technology, the molecular modification research of gamma-polyglutamic acid synthetase and the construction research of high-yield gamma-polyglutamic acid genetic engineering bacteria by using the molecular biology technology become new research hotspots.
For the synthesis of Bacillus subtilis IFO3336, 4 key enzymes are required for the synthesis of gamma-polyglutamic acid, namely PgsB, PgsC, PgsA and PgsE. These four key enzymes together constitute the gamma-polyglutamate synthetase complex PgsBCAE. And four genes pgsB, pgsA, pgsC and pgsE encoding the gamma-polyglutamic acid synthetase complex PgsBCAE constitute a gene cluster and belong to the same operon.
The invention adopts an in vitro evolution method to carry out molecular modification on a gamma-polyglutamic acid synthetase complex PgsBCAE derived from Bacillus subtilis IFO3336 of natto, and combines a high-throughput screening technology to obtain a mutant of the gamma-polyglutamic acid synthetase complex with improved catalytic activity, thereby realizing high-yield biosynthesis of gamma-polyglutamic acid.
Disclosure of Invention
In order to solve the problems in the prior art, improve the catalytic activity of a gamma-polyglutamic acid synthetase complex and realize the high-yield biosynthesis of gamma-polyglutamic acid, the invention provides the gamma-polyglutamic acid synthetase complex with improved catalytic activity and a coding gene thereof.
The invention provides a gamma-polyglutamic acid synthetase complex PgsB with improved catalytic activitymCmAmEmCharacterized in that gamma-polyglutamic acid synthetase PgsBmThe amino acid sequence of (a) is as shown in SEQ ID NO: 1 is shown in the specification; gamma-polyglutamic acid synthetase PgsCmThe amino acid sequence of (a) is as shown in SEQ ID NO: 2 is shown in the specification; gamma-polyglutamic acid synthetase PgsAmThe amino acid sequence of (A) is shown as SEQ ID NO: 3 is shown in the specification; gamma-polyglutamic acid synthetase PgsEmThe amino acid sequence of (a) is as shown in SEQ ID NO: 4, respectively.
The invention also provides a method for obtaining the gamma-polyglutamic acid synthetase complex PgsBmCmAmEmThe method of (1), wherein the amino acid sequence of SEQ ID NO: 1 of gamma-polyglutamic acid synthetase PgsBmIs ammoniaThe amino acid sequence is shown as SEQ ID NO: 6, the gamma-polyglutamic acid synthetase PgsB is an initial sequence, and the 89 th serine is mutated into aspartic acid, the 110 th serine is mutated into cysteine, the 151 th aspartic acid is mutated into asparagine, the 188 th lysine is mutated into glutamic acid, and the 375 th alanine is mutated into serine; SEQ ID NO: 2, a gamma-polyglutamic acid synthetase PgsCmIs represented by an amino acid sequence shown as SEQ ID NO: 7, taking gamma-polyglutamic acid synthetase PgsC as an initial sequence, and mutating lysine at the 23 th position into isoleucine and glutamic acid at the 104 th position into arginine; SEQ ID NO: 3, and a gamma-polyglutamic acid synthetase PgsAmIs represented by an amino acid sequence shown as SEQ ID NO: the gamma-polyglutamic acid synthetase PgsA shown in 8 is a starting sequence, and lysine at the 24 th position is mutated into leucine, lysine at the 45 th position is mutated into glutamine, and glutamic acid at the 90 th position is mutated into alanine; SEQ ID NO: 4 of gamma-polyglutamic acid synthetase PgsEmIs represented by an amino acid sequence shown as SEQ ID NO: 9 is used as an initial sequence, and the 21 st proline is mutated into serine.
The present invention also provides a method for encoding the gamma-polyglutamic acid synthetase complex PgsB with improved catalytic activitymCmAmEmThe gene of (1); the nucleotide sequence of the gene is shown as SEQ ID NO: 5, respectively.
The invention also provides a bacillus subtilis genetic engineering bacterium for high yield of gamma-polyglutamic acid.
The construction method of the bacillus subtilis genetic engineering bacteria comprises the following specific steps: the method of artificial synthesis or PCR amplification is adopted to obtain the coded gamma-polyglutamic acid synthetase complex PgsBmCmAmEmThe gene of (1); the obtained gene was ligated with vector pSTOP1622 to construct recombinant vector pSTOP1622-pgsBmCmAmEm(ii) a Transforming the recombinant vector into Bacillus subtilis WB600 to obtain the Bacillus subtilis WB600/pSTOP1622-pgsBmCmAmEm。
The invention adopts an in vitro evolution method to carry out the in vitro evolution of the Bacillus subtilis bud derived from nattoCarrying out directional modification on a gamma-polyglutamic acid synthetase complex PgsBCAE of Bacillus subtilis IFO3336, and screening to obtain a gamma-polyglutamic acid synthetase complex PgsB with improved catalytic activitymCmAmEm. The invention relates to a gamma-polyglutamic acid synthetase complex PgsBmCmAmEmThe gene is transferred into Bacillus subtilis WB600 to obtain genetically engineered bacterium Bacillus subtilis WB600/pSTOP1622-pgsBmCmAmEm. The invention has the advantages that: genetically engineered bacterium Bacillus subtilis WB600/pSTOP1622-pgsBmCmAmEmThe yield of the gamma-polyglutamic acid is 43.938g/L, which is improved by 10.67 times, and the average molecular weight of the produced gamma-polyglutamic acid is 621kDa, which is improved by 1.49 times. Namely, compared with PgsBCAE, the gamma-polyglutamate synthetase complex PgsB provided by the inventionmCmAmEmThe catalytic activity of synthesizing the gamma-polyglutamic acid is improved by 10.67 times. The gamma-polyglutamic acid synthetase complex with improved catalytic activity can realize high-yield biosynthesis of gamma-polyglutamic acid, and has great application potential in production of gamma-polyglutamic acid.
Drawings
FIG. 1 is a graph of a gamma-polyglutamic acid standard.
Detailed Description
The gamma-polyglutamic acid synthetase complex with improved catalytic activity and the encoding gene thereof according to the present invention will be described in further detail with reference to the following specific examples.
The experimental conditions are as follows:
1. bacterial strains and vectors
Coli DH5 alpha (from TaKaRa), Bacillus subtilis WB600 (stored in the laboratory), Bacillus subtilis expression vector pSTOP1622 (from MoBiTec).
2. Enzymes and other biochemical reagents
KOD-Plus-neo DNA polymerase was purchased from Toyobo, DNA restriction endonuclease was purchased from Fermentase, DNA agarose gel recovery kit, plasmid extraction kit E.Z.N.A. was purchased from Omega Bio-tek, gamma-polyglutamic acid was purchased from Sigma-Aldrich Sigma Aldrich trade company, and other chemical reagents were made in China or imported analytically pure.
3. Culture medium
LB medium (g/L): tryptone 10, yeast extract 5, NaCl 10, pH 7.0. The screening medium was LB medium containing 50. mu.g/mL ampicillin.
Seed medium (g/L): glucose 10, yeast extract 5, KH2PO40.5、K2HPO40.5、MgSO40.1，pH7.2。
Shake tube fermentation medium (g/L): glucose 10, yeast extract 5, L-glutamic acid 5, KH2PO40.5、K2HPO40.5、MgSO40.1，pH 7.2。
Shake flask fermentation medium (g/L): glucose 40, yeast extract 20, L-glutamic acid 20, KH2PO40.5、K2HPO40.5、MgSO40.1，pH 7.2。
Batch fermentation medium on tank (g/L): glucose 80, yeast extract 40, L-glutamic acid 80, KH2PO40.5、K2HPO40.5、MgSO40.1、NaCl 10，pH 7.2。
Supplemented fermentation medium (g/L): and 500 of glucose.
The molecular cloning and protein detection techniques used in the present invention are conventional in the art. The techniques not described in detail in the following examples were performed in accordance with the relevant portions of the following experimental manuals. Green MR, Sambrook J.molecular cloning: a Laboratory Manual [ M ]. New York: Cold spring harbor Laboratory Press, 2012.
Example 1 construction and fermentation culture of genetically engineered bacterium Bacillus subtilis WB600/pSTOP1622-pgsBCAE
(1) Construction of expression vector pSTOP1622-pgsBCAE and genetically engineered bacterium Bacillus subtilis WB600/pSTOP1622-pgsBCAE
According to GenBank accession number AB016245.1 of a gamma-polyglutamic acid synthetase gene derived from Bacillus subtilis IFO3336, searching and obtaining a gene sequence, and handing the gene sequence to Shanghai Bo probiotic science and technology company, wherein the gene sequence codes the gamma-polyglutamic acid synthetase complex PgsBCAE pgsBCAE.
Designing PCR primers P1 and P2 (Table 1) according to the nucleotide sequence of pgsBCAE, carrying out PCR amplification by using synthetic gene pgsBCAE as a template and P1 and P2 as primers to obtain an amplification product, wherein the PCR amplification system is 10 × buffer I5 mu L, dNTP 5 mu L, MgSO45. mu.L of each primer, 2. mu.L of each primer, 1. mu. L, KOD-Plus-neo DNA polymerase 2. mu. L, ddH2O28. mu.L. The PCR amplification conditions were: 5min at 98 ℃; 20sec at 98 ℃, 40sec at 60 ℃ and 2min at 74 ℃ for 30 cycles; at 74 ℃ for 10 min. The amplified product is subjected to double enzyme digestion by Bgl II and BamH I and is connected to a vector pSTOP1622 subjected to the same double enzyme digestion treatment, so that an expression vector pSTOP1622-pgsBCAE is constructed.
The expression vector pSTOP1622-pgsBCAE is transferred into Bacillus subtilis WB600 to obtain the genetically engineered bacterium Bacillus subtilis WB600/pSTOP 1622-pgsBCAE.
TABLE 1 primers for vector construction
Note: the underlined part is the cleavage site of the restriction enzyme.
(2) Fermentation culture of genetically engineered bacterium Bacillus subtilis WB600/pSTOP1622-pgsBCAE
The seed culture conditions of the genetic engineering bacteria are as follows: the seed culture medium is adopted, and a 250mL triangular flask is used for culturing, wherein the liquid loading of the culture medium is 20mL, the culture temperature is 37 ℃, the rotation speed is 200rpm, and the culture time is 24 h.
The shake flask fermentation culture conditions of the genetic engineering bacteria are as follows: a fermentation medium was used, and the culture was carried out in a 250mL Erlenmeyer flask, in which the medium contained 25mL of liquid, the inoculum size was 3%, the culture temperature was 37 ℃ and the rotation speed was 200 rpm. When cultured to the thallusOD600nmWhen 1 is reached, xylose with a final concentration of 0.5% is added, and the induction time is 48 h.
Fermentation culture test of genetically engineered bacteria Bacillus subtilis WB600/pSTOP1622-pgsBCAE takes Bacillus subtilis WB600 and Bacillus subtilis WB600/pSTOP1622 as negative controls.
(3) Extraction and quantification of gamma-polyglutamic acid in fermentation liquor
Extracting gamma-polyglutamic acid in fermentation liquor: diluting the fermentation liquid by 10 times, centrifuging at 12000 Xg for 10min, collecting supernatant, adding 4 times of glacial ethanol, mixing, and standing at 4 deg.C overnight. Centrifuging the sample at 12000 Xg for 10min, discarding the supernatant, and dissolving the obtained precipitate with deionized water to obtain the gamma-polyglutamic acid sample.
And (3) determining the concentration of the gamma-polyglutamic acid in the sample by adopting a high performance liquid chromatography, and calculating the concentration of the gamma-polyglutamic acid in the fermentation liquor according to the extraction method of the gamma-polyglutamic acid in the fermentation liquor. The genetically engineered bacterium Bacillus subtilis WB600/pSTOP1622-pgsBCAE is cultured by shake flask fermentation, and the yield of the gamma-polyglutamic acid is 2.418 g/L. The yield of gamma-polyglutamic acid of the negative control Bacillus subtilis WB600 is 0.017g/L, and the yield of gamma-polyglutamic acid of the negative control Bacillus subtilis WB600/pSTOP1622 is 0.018 g/L.
Example 2 in vitro directed evolution of the Gamma-polyglutamate synthetase Complex PgsBCAE
(1) In vitro directed evolution of gamma-polyglutamic acid synthetase PgsB
An error-prone PCR technology and a large-primer whole-plasmid PCR technology are adopted to construct a PgsB-related random mutation genetic engineering bacterium library, and then a high-throughput screening method is combined to screen out the genetic engineering bacterium with improved synthesis yield of the gamma-polyglutamic acid from the random mutation genetic engineering bacterium library. DNA sequencing is carried out on the recombinant vector carried by the genetic engineering bacteria, the mutation site of the PgsB is identified, and the mutant PgsBm of the gamma-polyglutamate synthetase PgsB with improved catalytic activity is obtained. The specific operation steps are as follows.
Construction of a random mutation gene engineering bacterium library related to PgsB: firstly, an error-prone PCR technology is adopted to obtain a random mutation library of the gene pgsB. Recombinant vector pSTOP1622-pgsBCAE is taken as a template and introducedThe substance is P3 and P4. the error-prone PCR reaction system is 10 × buffer I5 mu L, dATP 2 mu L, dGTP 2 mu L, dTTP 8 mu L, dCTP 8 mu L, MgSO45. mu.L of each primer, 2. mu.L of each primer, 1. mu. L, KOD-Plus-neoDNA polymerase 2. mu. L, ddH2O13 mu L error-prone PCR amplification conditions comprise that the temperature is 98 ℃ for 5min, the temperature is 98 ℃ for 20sec, the temperature is 60 ℃ for 40sec, the temperature is 74 ℃ for 2min, 30 cycles are carried out, the temperature is 74 ℃ for 10min, the PCR amplification product of the current round is used as a large primer in the next round of large primer full-plasmid PCR reaction, then the large primer full-plasmid PCR technology is adopted to carry out full-plasmid amplification on the recombinant vector pSTOP1622-pgsBCAE to obtain a random mutation recombinant vector library containing the gene pgsB, the recombinant vector pSTOP1622-pgsBCAE is used as a template, the primer is the amplification product of the previous round of PCR, and the large primer full-plasmid PCR reaction system is 10 × buffer I5 mu L, dNTP 5 mu L, MgSO45 μ L, primer 5 μ L, template 1 μ L, KOD-Plus-neo DNA polymerase 2 μ L, ddH2O28 mu L large primer whole plasmid PCR reaction conditions comprise that after the PCR amplification product of the round is treated by Dpn I enzyme, Escherichia coli DH5 α competent cells are transformed, the transformed Escherichia coli DH5 α competent cells are coated on an LB plate containing ampicillin, the transformed Escherichia coli DH5 α competent cells are cultured for 18h at 37 ℃, all transformants are scraped from the transformed plate by using a sterile coating rod and inoculated in an LB liquid culture medium containing ampicillin, the transformed Escherichia coli DH5 α competent cells are cultured overnight at 37 ℃ and 200rpm, the overnight culture is centrifuged for 3min at 00 12000 × g, thallus precipitates are collected, plasmids are extracted, a random mutation recombinant vector library containing a gene pgsB random mutation library is obtained, the obtained recombinant vector library is transformed into Bacillus subtilis WB600 competent cells, the Bacillus subtilis competent cells are coated on the LB plate containing ampicillin, and the Bacillus subtilis competent cells are cultured for 24h at 37 ℃, and all single colonies on the transformation plate jointly form a random mutation gene engineering bacterium library related to PgsB.
High-throughput screening of genetic engineering bacteria libraries: inoculating the mutant and monocloning in the fermentation medium of a shake tube. The conditions for fermenting and culturing the shake bacteria tube are as follows: culturing with 50mL shake tube, wherein the liquid volume of culture medium is 5mL, the culture temperature is 37 deg.C, the rotation speed is 200rpm, and when the culture is carried out until thallus OD600nmWhen 1 is reached, xylose with a final concentration of 0.5% is added, and the induction time is 48 h. After the culture is finished, taking 1mL of fermentation liquor, anddiluting the fermentation liquor by 10 times, centrifuging at 12000 × g for 10min, taking 1mL of supernatant, adding 4mL of glacial ethanol into the supernatant, uniformly mixing, standing at 4 ℃ overnight, centrifuging the sample at 12000 × g for 10min, discarding the supernatant, dissolving the obtained precipitate with 10mL of deionized water to obtain a gamma-polyglutamic acid sample, putting 200 mu L of the sample into a 96-hole enzyme label plate, and measuring the light absorption value A of the sample at 216nm216nm. Concentration of gamma-polyglutamic acid standard and A thereof216nmThe concentration range of 0-200 mug/mL presents a positive linear relationship, the standard curve of the gamma-polyglutamic acid is shown in figure 1, and the linear regression equation of the standard curve is as follows: a. the216nmNot rate [0.0061 × gamma-polyglutamic acid concentration (μ g/mL)]+0.0034，R20.9997. According to the linear regression equation and the absorbance value A of the sample at 216nm216nmAnd calculating the concentration of the gamma-polyglutamic acid sample. According to the extraction method of the gamma-polyglutamic acid in the fermentation liquor, the concentration of the gamma-polyglutamic acid in the fermentation liquor is calculated to be 100 times of that of the gamma-polyglutamic acid sample.
In the high-throughput screening experiment of a genetic engineering bacteria library, the genetic engineering bacteria Bacillus subtilis WB600/pSTOP1622-pgsBCAE is used as a control. The genetically engineered bacterium Bacillus subtilis WB600/pSTOP1622-pgsBCAE is cultured by bacteria shaking tube fermentation, and the yield of the gamma-polyglutamic acid is 1.182 g/L. The genetic engineering bacteria with the highest gamma-polyglutamic acid yield are obtained by screening according to the method, and the gamma-polyglutamic acid yield is 4.255 g/L. Compared with the genetically engineered bacterium Bacillus subtilis WB600/pSTOP1622-pgsBCAE, the yield of the gamma-polyglutamic acid is improved by 3.60 times.
And (3) sending the recombinant vector carried by the screened genetic engineering bacteria to Shanghai Bo probiotic science and technology limited for DNA sequencing, wherein the sequencing result shows that the mutation sites of the PgsB are as follows: serine (Ser) at the 89 th position is mutated into aspartic acid (Asp), serine (Ser) at the 110 th position is mutated into cysteine (Cys), aspartic acid (Asp) at the 151 th position is mutated into asparagine (Asn), lysine (Lys) at the 188 th position is mutated into glutamic acid (Glu), and alanine (Ala) at the 375 th position is mutated into serine (Ser). The recombinant vector was named pSTOP1622-pgsBmCAE, wherein the corresponding genetically engineered bacterium is Bacillus subtilis WB600/pSTOP1622-pgsBmCAE。
(2) In vitro directed evolution of gamma-polyglutamic acid synthetase PgsC
The method of in vitro directed evolution of PgsC refers to the method of in vitro directed evolution of PgsB. The specific differences include: the primers used in the error-prone PCR reaction system are P5 and P6; the template used in the large primer whole plasmid PCR reaction system is pSTOP1622-pgsBmCAE。
In the high-throughput screening experiment of a genetic engineering bacteria bank, the genetic engineering bacteria Bacillus subtilis WB600/pSTOP1622-pgsBmCAE is a control. Genetically engineered bacterium Bacillus subtilis WB600/pSTOP1622-pgsBmThe yield of the gamma-polyglutamic acid is 4.255g/L when the CAE is fermented and cultured by a shake tube. The genetic engineering bacteria with the highest gamma-polyglutamic acid yield are obtained by screening according to a high-throughput screening method of a genetic engineering bacteria library, and the gamma-polyglutamic acid yield is 5.149 g/L. And genetically engineered bacterium Bacillus subtilis WB600/pSTOP1622-pgsBmCompared with CAE, the yield of gamma-polyglutamic acid is improved by 1.21 times.
And (3) sending the recombinant vector carried by the screened genetic engineering bacteria to Shanghai Bo probiotic science and technology limited for sequencing, wherein the sequencing result shows that the PgsC mutation sites in the mutant are as follows: lysine (Lys) at position 23 is mutated into isoleucine (Ile), and glutamic acid (Glu) at position 104 is mutated into arginine (Arg). The recombinant vector was named pSTOP1622-pgsBmCmAE, the corresponding genetically engineered bacterium is Bacillus subtilis WB600/pSTOP1622-pgsBmCmAE。
(3) In vitro directed evolution of gamma-polyglutamic acid synthetase PgsA
The in vitro directed evolution method of PgsA is referred to the in vitro directed evolution method of PgsB. The specific differences include: the primers used in the error-prone PCR reaction system are P7 and P8; the template used in the large primer whole plasmid PCR reaction system is pSTOP1622-pgsBmCmAE。
In the high-throughput screening experiment of a genetic engineering bacteria bank, the genetic engineering bacteria Bacillus subtilis WB600/pSTOP1622-pgsBmCmAE is control. Genetically engineered bacterium Bacillus subtilis WB600/pSTOP1622-pgsBmCmAE is cultured by fermentation in a shake tube, and the yield of gamma-polyglutamic acid is 5.149g/L. The genetic engineering bacteria with the highest gamma-polyglutamic acid yield are obtained by screening according to a high-throughput screening method of a genetic engineering bacteria library, and the gamma-polyglutamic acid yield is 9.216 g/L. And genetically engineered bacterium Bacillus subtilis WB600/pSTOP1622-pgsBmCmCompared with AE, the yield of gamma-polyglutamic acid is improved by 1.79 times.
Sending the screened mutant to Shanghai probiotic science and technology limited for sequencing, wherein the sequencing result shows that the mutation sites of PgsA in the mutant are as follows: lysine (Lys) at position 24 is mutated into leucine (Leu), lysine (Lys) at position 45 is mutated into glutamine (Gln), and glutamic acid (Glu) at position 90 is mutated into alanine (Ala). The recombinant vector was named pSTOP1622-pgsBmCmAmE, the corresponding genetically engineered bacterium is Bacillus subtilis WB600/pSTOP1622-pgsBmCmAmE。
(4) In vitro directed evolution of gamma-polyglutamic acid synthetase PgsE
The method of in vitro directed evolution of PgsE refers to the method of in vitro directed evolution of PgsB. The specific differences include: the primers used in the error-prone PCR reaction system are P9 and P10; the template used in the large primer whole plasmid PCR reaction system is pSTOP1622-pgsBmCmAmE。
In the high-throughput screening experiment of a genetic engineering bacteria bank, the genetic engineering bacteria Bacillus subtilis WB600/pSTOP1622-pgsBmCmAmE is a control. Genetically engineered bacterium Bacillus subtilis WB600/pSTOP1622-pgsBmCmAmE is cultured by fermenting in a shake tube, and the yield of the gamma-polyglutamic acid is 9.216 g/L. The genetic engineering bacteria with the highest gamma-polyglutamic acid yield are obtained by screening according to a high-throughput screening method of a genetic engineering bacteria library, and the gamma-polyglutamic acid yield is 12.165 g/L. And genetically engineered bacterium Bacillus subtilis WB600/pSTOP1622-pgsBmCmAmCompared with E, the yield of the gamma-polyglutamic acid is improved by 1.32 times.
Sending the screened mutant to Shanghai Bo probiotic science and technology limited for sequencing, wherein the sequencing result shows that the PgsE mutation sites in the mutant are as follows: mutation of proline (Pro) at position 21 to serineAcid (Ser). The recombinant vector was named pSTOP1622-pgsBmCmAmEmThe corresponding genetically engineered bacterium is Bacillus subtilis WB600/pSTOP1622-pgsBmCmAmEm。
Example 3 verification of highly productive gamma-polyglutamic acid strains
The genetically engineered bacterium Bacillus subtilis WB600/pSTOP1622-pgsB constructed in example 2 was fermented by batch-fed fermentationmCmAmEmAnd (5) carrying out fermentation verification. The genetically engineered bacterium Bacillus subtilis WB600/pSTOP1622-pgsBCAE is used as a reference. The seed culture conditions of the genetic engineering bacteria are as follows: the seed culture medium is adopted, and a 500mL triangular flask is used for culturing, wherein the liquid loading of the culture medium is 50mL, the culture temperature is 37 ℃, the rotation speed is 200rpm, and the culture time is 24 h. The batch-fed batch fermentation conditions of the genetically engineered bacteria are as follows: inoculating seeds into a 5L full-automatic fermentation tank by adopting a tank batch fermentation culture medium, wherein the liquid loading amount is 2L, the stirring rotation speed is 400rpm, the culture temperature is 37 ℃, the ventilation volume is 1.5vvm, and the pH is controlled to be not less than 7.2 by using ammonia water. When the glucose concentration in the culture medium is lower than 5g/L, feeding the fermentation culture medium, and maintaining the glucose concentration on the tank within the range of 3-8 g/L. When cultured to the OD of the cells600nmWhen the concentration reaches 15%, xylose with the final concentration of 0.5% is added, and the fermentation is finished after 48h of culture. Extraction and quantification of gamma-polyglutamic acid in the fermentation broth were performed with reference to example 1, and the average molecular weight of gamma-polyglutamic acid was measured by gel permeation chromatography. The yield of the gamma-polyglutamic acid of the genetically engineered bacterium Bacillus subtilis WB600/pSTOP1622-pgsBCAE is 4.117g/L, and the average molecular weight of the produced gamma-polyglutamic acid is 418 kDa; genetically engineered bacterium Bacillus subtilis WB600/pSTOP1622-pgsBmCmAmEmThe yield of the gamma-polyglutamic acid is 43.938g/L, which is improved by 10.67 times, and the average molecular weight of the produced gamma-polyglutamic acid is 621kDa, which is improved by 1.49 times.
The above description is only for the preferred embodiment of the present invention, and not intended to limit the present invention, and any changes or substitutions that can be easily conceived by one skilled in the art within the technical scope of the present invention are also within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope defined by the claims.
Sequence listing
<110> institute of microbiology of academy of sciences of Jiangxi province
<120> a complex of gamma-polyglutamic acid synthetase with improved catalytic activity and its coding gene
<160>10
<170>SIPOSequenceListing 1.0
<210>1
<211>393
<212>PRT
<213> Artificial sequence
<400>1
Met Trp Leu Leu Ile Ile Ala Cys Ala Val Ile Leu Val Ile Gly Ile
1 5 10 15
Leu Glu Lys Arg Arg His Gln Lys Asn Ile Asp Ala Leu Pro Val Arg
20 25 30
Val Asn Ile Asn Gly Ile Arg Gly Lys Ser Thr Val Thr Arg Leu Thr
35 40 45
Thr Gly Ile Leu Ile Glu Ala Gly Tyr Lys Thr Val Gly Lys Thr Thr
50 55 60
Gly Thr Asp Ala Arg Met Ile Tyr Trp Asp Thr Pro Glu Glu Lys Pro
65 70 75 80
Ile Lys Arg Lys Pro Gln Gly Pro Asp Ile Gly Glu Gln Lys Glu Val
85 90 95
Met ArgGlu Thr Val Glu Arg Gly Ala Asn Ala Ile Val Cys Glu Cys
100 105 110
Met Ala Val Asn Pro Asp Tyr Gln Ile Ile Phe Gln Glu Glu Leu Leu
115 120 125
Gln Ala Asn Ile Gly Val Ile Val Asn Val Leu Glu Asp His Met Asp
130 135 140
Val Met Gly Pro Thr Leu Asn Glu Ile Ala Glu Ala Phe Thr Ala Thr
145 150 155 160
Ile Pro Tyr Asn Gly His Leu Val Ile Thr Asp Ser Glu Tyr Thr Glu
165 170 175
Phe Phe Lys Gln Lys Ala Lys Glu Arg Asn Thr Glu Val Ile Ile Ala
180 185 190
Asp Asn Ser Lys Ile Thr Asp Glu Tyr Leu Arg Lys Phe Glu Tyr Met
195 200 205
Val Phe Pro Asp Asn Ala Ser Leu Ala Leu Gly Val Ala Gln Ala Leu
210 215 220
Gly Ile Asp Glu Glu Thr Ala Phe Lys Gly Met Leu Asn Ala Pro Pro
225 230 235 240
Asp Pro Gly Ala Met Arg Ile Leu Pro Leu Ile Ser Pro Ser Glu Pro
245 250 255
Gly His Phe ValAsn Gly Phe Ala Ala Asn Asp Ala Ser Ser Thr Leu
260 265 270
Asn Ile Trp Lys Arg Val Lys Glu Ile Gly Tyr Pro Thr Asp Asp Pro
275 280 285
Ile Ile Ile Met Asn Cys Arg Ala Asp Arg Val Asp Arg Thr Gln Gln
290 295 300
Phe Ala Asn Asp Val Leu Pro Tyr Ile Glu Ala Ser Glu Leu Ile Leu
305 310 315 320
Ile Gly Glu Thr Thr Glu Pro Ile Val Lys Ala Tyr Glu Glu Gly Lys
325 330 335
Ile Pro Ala Asp Lys Leu His Asp Leu Glu Tyr Lys Ser Thr Asp Glu
340 345 350
Ile Met Glu Leu Leu Lys Lys Ser Met His Asn Arg Val Ile Tyr Gly
355 360 365
Val Gly Asn Ile His Gly Ser Ala Glu Pro Leu Ile Glu Lys Ile His
370 375 380
Glu Tyr Lys Val Lys Gln Leu Val Ser
385 390
<210>2
<211>149
<212>PRT
<213> Artificial sequence
<400>2
Met Phe Gly Ser Asp Leu Tyr Ile Ala Leu Ile Leu Gly Val Leu Leu
1 5 10 15
Ser Leu Ile Phe Ala Glu Ile Thr Gly Ile Val Pro Ala Gly Leu Val
20 25 30
Val Pro Gly Tyr Leu Gly Leu Val Phe Asn Gln Pro Val Phe Ile Leu
35 40 45
Leu Val Leu Leu Val Ser Leu Leu Thr Tyr Val Ile Val Lys Tyr Gly
50 55 60
Leu Ser Lys Phe Met Ile Leu Tyr Gly Arg Arg Lys Phe Ala Ala Met
65 70 75 80
Leu Ile Thr Gly Ile Val Leu Lys Ile Ala Phe Asp Phe Leu Tyr Pro
85 90 95
Ile Val Pro Phe Glu Ile Ala Arg Phe Arg Gly Ile Gly Ile Ile Val
100 105 110
Pro Gly Leu Ile Ala Asn Thr Ile Gln Lys Gln Gly Leu Thr Ile Thr
115 120 125
Phe Gly Ser Thr Leu Leu Leu Ser Gly Ala Thr Phe Ala Ile Met Phe
130 135 140
Val Tyr Tyr Leu Ile
145
<210>3
<211>380
<212>PRT
<213> Artificial sequence
<400>3
Met Lys Lys Glu Leu Ser Phe His Glu Lys Leu Leu Lys Leu Thr Lys
1 5 10 15
Gln Gln Lys Lys Lys Thr Asn Leu His Val Phe Ile Ala Ile Pro Ile
20 25 30
Val Phe Val Leu Met Phe Ala Phe Met Trp Ala Gly Gln Ala Glu Thr
35 40 45
Pro Lys Val Lys Thr Tyr Ser Asp Asp Val Leu Ser Ala Ser Phe Val
50 55 60
Gly Asp Ile Met Met Gly Arg Tyr Val Glu Lys Val Thr Glu Gln Lys
65 70 75 80
Gly Ala Asp Ser Ile Phe Gln Tyr Val Ala Pro Ile Phe Arg Ala Ser
85 90 95
Asp Tyr Val Ala Gly Asn Phe Glu Asn Pro Val Thr Tyr Gln Lys Asn
100 105 110
Tyr Lys Gln Ala Asp Lys Glu Ile His Leu Gln Thr Asn Lys Glu Ser
115 120 125
Val Lys Val Leu Lys Asp Met Asn Phe Thr Val Leu Asn Ser Ala Asn
130 135 140
Asn His Ala Met Asp Tyr Gly Val Gln Gly Met Lys Asp Thr Leu Gly
145 150 155 160
Glu Phe Ala Lys Gln Asn Leu Asp Ile Val Gly Ala Gly Tyr Ser Leu
165 170 175
Ser Asp Ala Lys Lys Lys Ile Ser Tyr Gln Lys Val Asn Gly Val Thr
180 185 190
Ile Ala Thr Leu Gly Phe Thr Asp Val Ser Gly Lys Gly Phe Ala Ala
195 200 205
Lys Lys Asn Thr Pro Gly Val Leu Pro Ala Asp Pro Glu Ile Phe Ile
210 215 220
Pro Met Ile Ser Glu Ala Lys Lys His Ala Asp Ile Val Val Val Gln
225 230 235 240
Ser His Trp Gly Gln Glu Tyr Asp Asn Asp Pro Asn Asp Arg Gln Arg
245 250 255
Gln Leu Ala Arg Ala Met Ser Asp Ala Gly Ala Asp Ile Ile Val Gly
260 265 270
His His Pro His Val Leu Glu Pro Ile Glu Val Tyr Asn Gly Thr Val
275 280 285
Ile Phe Tyr Ser Leu Gly Asn Phe Val Phe Asp Gln Gly Trp Thr Arg
290 295 300
Thr Arg Asp Ser Ala Leu Val Gln Tyr His Leu Lys Lys Asn Gly Thr
305 310 315 320
Gly Arg Phe Glu Val Thr Pro Ile Asp Ile His Glu Ala Thr Pro Ala
325 330 335
Pro Val Lys Lys Asp Ser Leu Lys Gln Lys Thr Ile Ile Arg Glu Leu
340 345 350
Thr Lys Asp Ser Asn Phe Ala Trp Lys Val Glu Asp Gly Lys Leu Thr
355 360 365
Phe Asp Ile Asp His Ser Asp Lys Leu Lys Ser Lys
370 375 380
<210>4
<211>55
<212>PRT
<213> Artificial sequence
<400>4
Met Lys Phe Val Lys Ala Ile Trp Pro Phe Val Ala Val Ala Ile Val
1 5 10 15
Phe Met Phe Met Ser Ala Phe Lys Phe Asn Asp Gln Leu Thr Asp Gln
20 25 30
Glu Lys Gln Lys Ile Asp Met Glu Met Asn Lys Ile Gln Gln Gln Glu
35 40 45
Glu Pro Val Asn Ala Asn Lys
50 55
<210>5
<211>2989
<212>DNA
<213> Artificial sequence
<400>5
atgtggttac tcattatagc ctgtgctgtc atactggtca tcggaatatt agaaaaacga 60
cgacatcaga aaaacattga tgccctccct gttcgggtga atattaacgg catccgcgga 120
aaatcgactg tgacaaggct gacaaccgga atattaatag aagccggtta caagactgtt 180
ggaaaaacaa caggaacaga tgcaagaatg atttactggg acacaccgga ggaaaagccg 240
attaaacgga aacctcaggg gccggatatt ggagagcaaa aagaagtcat gagagaaaca 300
gtagaaagag gggctaacgc gattgtctgt gaatgcatgg ctgttaaccc agattatcaa 360
atcatctttc aggaagaact tctgcaggcc aatatcggcg tcattgtgaa tgttttagaa 420
gaccatatgg atgtcatggg gccgacgctt aatgaaattg cagaagcgtt taccgctaca 480
attccttata atggccatct tgtcattaca gatagtgaat ataccgagtt ctttaaacaa 540
aaagcaaaag aacgaaacac agaagtcatc attgctgata actcaaaaat tacagatgag 600
tatttacgta aatttgaata catggtattc cctgataacg cttctctggc gctgggtgtg 660
gctcaagcac tcggcattga cgaagaaaca gcatttaagg gaatgctgaa tgcgccgcca 720
gatccgggag caatgagaat tcttccgctg atcagtccga gcgagcctgg gcactttgtt 780
aatgggtttg ccgcaaacga cgcttcttct actttgaata tatggaaacg tgtaaaagaa 840
atcggttacc cgaccgatga tccgatcatc atcatgaact gccgcgcaga ccgtgtcgat 900
cggacacagc aattcgcaaa tgacgtattg ccttatattg aagcaagtga actgatctta 960
atcggtgaaa caacagaacc gatcgtaaaa gcctacgaag aaggcaaaat tcctgcagac 1020
aaactgcatg atctagagta taagtcaaca gatgaaatta tggaattgtt aaagaaaagt 1080
atgcacaacc gtgtcatata tggcgtcggc aatattcatg gttccgcaga gcctttaatt 1140
gaaaaaatcc acgaatacaa ggtaaagcag ctcgtaagct agggggaaat gcagacatgt 1200
tcggatcaga tttatacatc gcactaattt taggtgtact actcagttta atttttgcgg 1260
aaataacagg gatcgtgccg gcaggacttg ttgtaccggg atatttagga cttgtgttta 1320
atcagccggt ctttatttta cttgttttgc tagtgagctt gctcacgtat gtcattgtga 1380
aatacggttt atccaaattt atgattttgt acggacgcag aaaattcgct gccatgctga 1440
taacagggat cgtcctaaaa atcgcgtttg attttctata cccgattgta ccatttgaaa 1500
tcgcaagatt tcgaggaatc ggcatcatcg tgccaggttt aattgccaat accattcaga 1560
aacaaggttt aaccattacg ttcggaagca cgctgctatt gagcggagcg acctttgcta 1620
tcatgtttgt ttactactta atttaatgta aggtgtgtca aacgatgaaa aaagaactga 1680
gctttcatga aaagctgcta aagctgacaa aacagcaaaa aaagaaaacc aatctgcacg 1740
tatttattgc cattccgatc gtttttgtcc ttatgttcgc tttcatgtgg gcgggacaag 1800
cggaaacgcc gaaggtcaaa acgtattctg acgacgtact ctcagcctca tttgtaggcg 1860
atattatgat gggacgctat gttgaaaaag taacggagca aaaaggggca gacagtattt 1920
ttcaatatgt tgcaccgatc tttagagcct cggattatgt agcaggaaac tttgaaaacc 1980
cggtaaccta tcaaaagaat tataaacaag cagataaaga gattcatctg cagacgaata 2040
aggaatcagt gaaagtcttg aaggatatga atttcacggt tctcaacagc gccaacaacc 2100
acgcaatgga ttacggcgtt cagggcatga aagatacgct tggagaattt gcgaagcaaa 2160
atcttgatat cgttggagcg ggatacagct taagtgatgc gaaaaagaaa atttcgtacc 2220
agaaagtcaa cggggtaacg attgcgacgc ttggctttac cgatgtgtcc gggaaaggtt 2280
tcgcggctaa aaagaatacg ccgggcgtgc tgcccgcaga tcctgaaatc ttcatcccta 2340
tgatttcaga agcgaaaaaa catgctgaca ttgttgttgt gcagtcacac tggggccaag 2400
agtatgacaa tgatccaaac gaccgccagc gccagcttgc aagagccatg tctgatgcgg 2460
gagctgacat catcgtcggc catcatccgc acgtcttaga accgattgaa gtatataacg 2520
gaaccgtcat tttctacagc ctcggcaact ttgtctttga ccaaggctgg acgagaacaa 2580
gagacagtgc actggttcag tatcacctga agaaaaatgg aacaggccgc tttgaagtga 2640
caccgatcga tatccatgaa gcgacacctg cacctgtgaa aaaagacagc cttaaacaga 2700
aaaccattat tcgcgaactg acgaaagact ctaatttcgc ttggaaagta gaagacggaa 2760
aactgacgtt tgatattgat catagtgaca aactaaaatc taaataaacg gagtgataaa 2820
gatgaaattt gtcaaagcta tctggccgtt tgttgccgta gccatcgtgt tcatgtttat 2880
gtcagctttt aaattcaatg atcagctgac agatcaggaa aaacagaaga ttgatatgga 2940
aatgaataaa atccaacagc aggaagaacc ggtaaacgcc aataaataa 2989
<210>6
<211>393
<212>PRT
<213>Bacillus subtilis IFO 3336
<400>6
Met Trp Leu Leu Ile Ile Ala Cys Ala Val Ile Leu Val Ile Gly Ile
1 5 10 15
Leu Glu Lys Arg Arg His Gln Lys Asn Ile Asp Ala Leu Pro Val Arg
20 25 30
Val Asn Ile Asn Gly Ile Arg Gly Lys Ser Thr Val Thr Arg Leu Thr
35 40 45
Thr Gly Ile Leu Ile Glu Ala Gly Tyr Lys Thr Val Gly Lys Thr Thr
50 55 60
Gly Thr Asp Ala Arg Met Ile Tyr Trp Asp Thr Pro Glu Glu Lys Pro
65 70 75 80
Ile Lys Arg Lys Pro Gln Gly Pro Ser Ile Gly Glu Gln Lys Glu Val
85 90 95
Met Arg Glu Thr Val Glu Arg Gly Ala Asn Ala Ile Val Ser Glu Cys
100 105 110
Met Ala Val Asn Pro Asp Tyr Gln Ile Ile Phe Gln Glu Glu Leu Leu
115 120 125
Gln Ala Asn Ile Gly Val Ile Val Asn Val Leu Glu Asp His Met Asp
130 135 140
Val Met Gly Pro Thr Leu Asp Glu Ile Ala Glu Ala Phe Thr Ala Thr
145 150 155 160
Ile Pro Tyr Asn Gly His Leu Val Ile Thr Asp Ser Glu Tyr Thr Glu
165 170 175
Phe Phe Lys Gln Lys Ala Lys Glu Arg Asn Thr Lys Val Ile Ile Ala
180 185 190
Asp Asn Ser Lys Ile Thr Asp Glu Tyr Leu Arg Lys Phe Glu Tyr Met
195 200 205
Val Phe Pro Asp Asn Ala Ser Leu Ala Leu Gly Val Ala Gln Ala Leu
210 215 220
Gly Ile Asp Glu Glu Thr Ala Phe Lys Gly Met Leu Asn Ala Pro Pro
225 230 235 240
Asp Pro Gly Ala Met Arg Ile Leu Pro Leu Ile Ser Pro Ser Glu Pro
245 250 255
Gly His Phe Val Asn Gly Phe Ala Ala Asn Asp Ala Ser Ser Thr Leu
260 265 270
Asn Ile Trp Lys Arg Val Lys Glu Ile Gly Tyr Pro Thr Asp Asp Pro
275 280 285
Ile Ile Ile Met Asn Cys Arg Ala Asp Arg Val Asp Arg Thr Gln Gln
290 295 300
Phe Ala Asn Asp Val Leu Pro Tyr Ile Glu Ala Ser Glu Leu Ile Leu
305 310 315 320
Ile Gly Glu Thr Thr Glu Pro Ile Val Lys Ala Tyr Glu Glu Gly Lys
325 330 335
Ile Pro Ala Asp Lys Leu His Asp Leu Glu Tyr Lys Ser Thr Asp Glu
340 345 350
Ile Met Glu Leu Leu Lys Lys Ser Met His Asn Arg Val Ile Tyr Gly
355 360 365
Val Gly Asn Ile His Gly Ala Ala Glu Pro Leu Ile Glu Lys Ile His
370 375 380
Glu Tyr Lys Val Lys Gln Leu Val Ser
385 390
<210>7
<211>149
<212>PRT
<213>Bacillus subtilis IFO 3336
<400>7
Met Phe Gly Ser Asp Leu Tyr Ile Ala Leu Ile Leu Gly Val Leu Leu
1 5 10 15
Ser Leu Ile Phe Ala Glu Lys Thr Gly Ile Val Pro Ala Gly Leu Val
20 25 30
Val Pro Gly Tyr Leu Gly Leu Val Phe Asn Gln Pro Val Phe Ile Leu
35 40 45
Leu Val Leu Leu Val Ser Leu Leu Thr Tyr Val Ile Val Lys Tyr Gly
50 55 60
Leu Ser Lys Phe Met Ile Leu Tyr Gly Arg Arg Lys Phe Ala Ala Met
65 70 75 80
Leu Ile Thr Gly Ile Val Leu Lys Ile Ala Phe Asp Phe Leu Tyr Pro
85 90 95
Ile Val Pro Phe Glu Ile Ala Glu Phe Arg Gly Ile Gly Ile Ile Val
100 105 110
Pro Gly Leu Ile Ala Asn Thr Ile Gln Lys Gln Gly Leu Thr Ile Thr
115 120 125
Phe Gly Ser Thr Leu Leu Leu Ser Gly Ala Thr Phe Ala Ile Met Phe
130 135 140
Val Tyr Tyr Leu Ile
145
<210>8
<211>380
<212>PRT
<213>Bacillus subtilis IFO 3336
<400>8
Met Lys Lys Glu Leu Ser Phe His Glu Lys Leu Leu Lys Leu Thr Lys
1 5 10 15
Gln Gln Lys Lys Lys Thr Asn Lys His Val Phe Ile Ala Ile Pro Ile
20 25 30
Val Phe Val Leu Met Phe Ala Phe Met Trp Ala Gly Lys Ala Glu Thr
35 40 45
Pro Lys Val Lys Thr Tyr Ser Asp Asp Val Leu Ser Ala Ser Phe Val
50 55 60
Gly Asp Ile Met Met Gly Arg Tyr Val Glu Lys Val Thr Glu Gln Lys
65 70 75 80
Gly Ala Asp Ser Ile Phe Gln Tyr Val Glu Pro Ile Phe Arg Ala Ser
85 90 95
Asp Tyr Val Ala Gly Asn Phe Glu Asn Pro Val Thr Tyr Gln Lys Asn
100 105 110
Tyr Lys Gln Ala Asp Lys Glu Ile His Leu Gln Thr Asn Lys Glu Ser
115 120 125
Val Lys Val Leu Lys Asp Met Asn Phe Thr Val Leu Asn Ser Ala Asn
130 135 140
Asn His Ala Met Asp Tyr Gly Val Gln Gly Met Lys Asp Thr Leu Gly
145 150 155 160
Glu Phe Ala Lys Gln Asn Leu Asp Ile Val Gly Ala Gly Tyr Ser Leu
165 170 175
Ser Asp Ala Lys Lys Lys Ile Ser Tyr Gln Lys Val Asn Gly Val Thr
180 185 190
Ile Ala Thr Leu Gly Phe Thr Asp Val Ser Gly Lys Gly Phe Ala Ala
195 200 205
Lys Lys Asn Thr Pro Gly Val Leu Pro Ala Asp Pro Glu Ile Phe Ile
210 215 220
Pro Met Ile Ser Glu Ala Lys Lys His Ala Asp Ile Val Val Val Gln
225 230 235 240
Ser His Trp Gly Gln Glu Tyr Asp Asn Asp Pro Asn Asp Arg Gln Arg
245 250 255
Gln Leu Ala Arg Ala Met Ser Asp Ala Gly Ala Asp Ile Ile Val Gly
260 265 270
His His Pro His Val Leu Glu Pro Ile Glu Val Tyr Asn Gly Thr Val
275 280 285
Ile Phe Tyr Ser Leu Gly Asn Phe Val Phe Asp Gln Gly Trp Thr Arg
290 295 300
Thr Arg Asp Ser Ala Leu Val Gln Tyr His Leu Lys Lys Asn Gly Thr
305 310 315 320
Gly Arg Phe Glu Val Thr Pro Ile Asp Ile His Glu Ala Thr Pro Ala
325 330 335
Pro Val Lys Lys Asp Ser Leu Lys Gln Lys Thr Ile Ile Arg Glu Leu
340 345 350
Thr Lys Asp Ser Asn Phe Ala Trp Lys Val Glu Asp Gly Lys Leu Thr
355 360 365
Phe AspIle Asp His Ser Asp Lys Leu Lys Ser Lys
370 375 380
<210>9
<211>55
<212>PRT
<213>Bacillus subtilis IFO 3336
<400>9
Met Lys Phe Val Lys Ala Ile Trp Pro Phe Val Ala Val Ala Ile Val
1 5 10 15
Phe Met Phe Met Pro Ala Phe Lys Phe Asn Asp Gln Leu Thr Asp Gln
20 25 30
Glu Lys Gln Lys Ile Asp Met Glu Met Asn Lys Ile Gln Gln Gln Glu
35 40 45
Glu Pro Val Asn Ala Asn Lys
50 55
<210>10
<211>2989
<212>DNA
<213>Bacillus subtilis IFO 3336
<400>10
atgtggttac tcattatagc ctgtgctgtc atactggtca tcggaatatt agaaaaacga 60
cgacatcaga aaaacattga tgccctccct gttcgggtga atattaacgg catccgcgga 120
aaatcgactg tgacaaggct gacaaccgga atattaatag aagccggtta caagactgtt 180
ggaaaaacaa caggaacaga tgcaagaatg atttactggg acacaccgga ggaaaagccg 240
attaaacgga aacctcaggg gccgagtatt ggagagcaaa aagaagtcat gagagaaaca 300
gtagaaagag gggctaacgc gattgtcagt gaatgcatgg ctgttaaccc agattatcaa 360
atcatctttc aggaagaact tctgcaggcc aatatcggcg tcattgtgaa tgttttagaa 420
gaccatatgg atgtcatggg gccgacgctt gatgaaattg cagaagcgtt taccgctaca 480
attccttata atggccatct tgtcattaca gatagtgaat ataccgagtt ctttaaacaa 540
aaagcaaaag aacgaaacac aaaagtcatc attgctgata actcaaaaat tacagatgag 600
tatttacgta aatttgaata catggtattc cctgataacg cttctctggc gctgggtgtg 660
gctcaagcac tcggcattga cgaagaaaca gcatttaagg gaatgctgaa tgcgccgcca 720
gatccgggag caatgagaat tcttccgctg atcagtccga gcgagcctgg gcactttgtt 780
aatgggtttg ccgcaaacga cgcttcttct actttgaata tatggaaacg tgtaaaagaa 840
atcggttacc cgaccgatga tccgatcatc atcatgaact gccgcgcaga ccgtgtcgat 900
cggacacagc aattcgcaaa tgacgtattg ccttatattg aagcaagtga actgatctta 960
atcggtgaaa caacagaacc gatcgtaaaa gcctacgaag aaggcaaaat tcctgcagac 1020
aaactgcatg atctagagta taagtcaaca gatgaaatta tggaattgtt aaagaaaagt 1080
atgcacaacc gtgtcatata tggcgtcggc aatattcatg gtgccgcaga gcctttaatt 1140
gaaaaaatcc acgaatacaa ggtaaagcag ctcgtaagct agggggaaat gcagacatgt 1200
tcggatcaga tttatacatc gcactaattt taggtgtact actcagttta atttttgcgg 1260
aaaaaacagg gatcgtgccg gcaggacttg ttgtaccggg atatttagga cttgtgttta 1320
atcagccggt ctttatttta cttgttttgc tagtgagctt gctcacgtat gtcattgtga 1380
aatacggttt atccaaattt atgattttgt acggacgcag aaaattcgct gccatgctga 1440
taacagggat cgtcctaaaa atcgcgtttg attttctata cccgattgta ccatttgaaa 1500
tcgcagaatt tcgaggaatc ggcatcatcg tgccaggttt aattgccaat accattcaga 1560
aacaaggttt aaccattacg ttcggaagca cgctgctatt gagcggagcg acctttgcta 1620
tcatgtttgt ttactactta atttaatgta aggtgtgtca aacgatgaaa aaagaactga 1680
gctttcatga aaagctgcta aagctgacaa aacagcaaaa aaagaaaacc aataagcacg 1740
tatttattgc cattccgatc gtttttgtcc ttatgttcgc tttcatgtgg gcgggaaaag 1800
cggaaacgcc gaaggtcaaa acgtattctg acgacgtact ctcagcctca tttgtaggcg 1860
atattatgat gggacgctat gttgaaaaag taacggagca aaaaggggca gacagtattt 1920
ttcaatatgt tgaaccgatc tttagagcct cggattatgt agcaggaaac tttgaaaacc 1980
cggtaaccta tcaaaagaat tataaacaag cagataaaga gattcatctg cagacgaata 2040
aggaatcagt gaaagtcttg aaggatatga atttcacggt tctcaacagc gccaacaacc 2100
acgcaatgga ttacggcgtt cagggcatga aagatacgct tggagaattt gcgaagcaaa 2160
atcttgatat cgttggagcg ggatacagct taagtgatgc gaaaaagaaa atttcgtacc 2220
agaaagtcaa cggggtaacg attgcgacgc ttggctttac cgatgtgtcc gggaaaggtt 2280
tcgcggctaa aaagaatacg ccgggcgtgc tgcccgcaga tcctgaaatc ttcatcccta 2340
tgatttcaga agcgaaaaaa catgctgaca ttgttgttgt gcagtcacac tggggccaag 2400
agtatgacaa tgatccaaac gaccgccagc gccagcttgc aagagccatg tctgatgcgg 2460
gagctgacat catcgtcggc catcatccgc acgtcttaga accgattgaa gtatataacg 2520
gaaccgtcat tttctacagc ctcggcaact ttgtctttga ccaaggctgg acgagaacaa 2580
gagacagtgc actggttcag tatcacctga agaaaaatgg aacaggccgc tttgaagtga 2640
caccgatcga tatccatgaa gcgacacctg cacctgtgaa aaaagacagc cttaaacaga 2700
aaaccattat tcgcgaactg acgaaagact ctaatttcgc ttggaaagta gaagacggaa 2760
aactgacgtt tgatattgat catagtgaca aactaaaatc taaataaacg gagtgataaa 2820
gatgaaattt gtcaaagcta tctggccgtt tgttgccgta gccatcgtgt tcatgtttat 2880
gccagctttt aaattcaatg atcagctgac agatcaggaa aaacagaaga ttgatatgga 2940
aatgaataaa atccaacagc aggaagaacc ggtaaacgcc aataaataa 2989
Claims (4)
1. Gamma-polyglutamic acid synthetase complex PgsB with improved catalytic activitymCmAmEmCharacterized in that gamma-polyglutamic acid synthetase PgsBmThe amino acid sequence of (a) is as shown in SEQ ID NO: 1 is shown in the specification; gamma-polyglutamic acid synthetase PgsCmThe amino acid sequence of (a) is as shown in SEQ ID NO: 2 is shown in the specification; gamma-polyglutamic acid synthetase PgsAmThe amino acid sequence of (a) is as shown in SEQ ID NO: 3 is shown in the specification; gamma-polyglutamic acid synthetase PgsEmThe amino acid sequence of (a) is as shown in SEQ ID NO: 4, respectively.
2. A method for encoding the gamma-polyglutamic acid synthetase complex PgsB as claimed in claim 1mCmAmEmThe gene of (1), wherein the nucleotide sequence of the gene is as shown in SEQ ID NO: 5, respectively.
3. A vector or a genetically engineered bacterium carrying the gene of claim 2.
4. A method for obtaining the complex PgsB of the gamma-polyglutamic acid synthetase in claim 1mCmAmEmThe method of (1), wherein the amino acid sequence of SEQ ID NO: 1 of gamma-polyglutamic acid synthetase PgsBmIs represented by an amino acid sequence shown as SEQ ID NO: 6, the gamma-polyglutamic acid synthetase PgsB is an initial sequence, and the 89 th serine is mutated into aspartic acid, the 110 th serine is mutated into cysteine, the 151 th aspartic acid is mutated into asparagine, the 188 th lysine is mutated into glutamic acid, and the 375 th alanine is mutated into serine; SEQ ID NO: 2, a gamma-polyglutamic acid synthetase PgsCmIs represented by an amino acid sequence shown as SEQ ID NO: 7, taking gamma-polyglutamic acid synthetase PgsC as an initial sequence, and mutating lysine at the 23 th position into isoleucine and glutamic acid at the 104 th position into arginine; SEQ ID NO: 3, and a gamma-polyglutamic acid synthetase PgsAmIs represented by an amino acid sequence shown as SEQ ID NO: the gamma-polyglutamic acid synthetase PgsA shown in 8 is a starting sequence, and lysine at the 24 th position is mutated into leucine, lysine at the 45 th position is mutated into glutamine, and glutamic acid at the 90 th position is mutated into alanine; SEQ ID NO: 4 of gamma-polyglutamic acid synthetase PgsEmIs represented by an amino acid sequence shown as SEQ ID NO: 9 is used as an initial sequence, and the 21 st proline is mutated into serine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010478733.1A CN111593029B (en) | 2020-05-29 | 2020-05-29 | Gamma-polyglutamic acid synthetase complex with improved catalytic activity and encoding gene thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1539018A (en) * | 2001-08-10 | 2004-10-20 | Surface expression vectors having pgs BCA the gene coding poly-gamma-glutamate synthetase, and method for expression of target protein at the surface of microorganism using the vector | |
| WO2005012902A1 (en) * | 2003-07-31 | 2005-02-10 | Genefield, Inc. | Method of screening useful protein |
| CN101578361A (en) * | 2005-06-17 | 2009-11-11 | 米克罗比亚精密工程股份有限公司 | Improved amino acid and metabolite biosynthesis |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1539018A (en) * | 2001-08-10 | 2004-10-20 | Surface expression vectors having pgs BCA the gene coding poly-gamma-glutamate synthetase, and method for expression of target protein at the surface of microorganism using the vector | |
| WO2005012902A1 (en) * | 2003-07-31 | 2005-02-10 | Genefield, Inc. | Method of screening useful protein |
| CN101578361A (en) * | 2005-06-17 | 2009-11-11 | 米克罗比亚精密工程股份有限公司 | Improved amino acid and metabolite biosynthesis |
Non-Patent Citations (3)
| Title |
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| WANG, LY等: "Poly-gamma-glutamic acid productivity of Bacillus subtilis BsE1 has positive function in motility and biocontrol against Fusarium graminearum", 《JOURNAL OF MICROBIOLOGY》 * |
| YI-HUANG HSUEH等: "Poly-γ-glutamic Acid Synthesis, Gene Regulation, Phylogenetic Relationships, and Role in Fermentation", 《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》 * |
| 吉美萍: "γ- 聚谷氨酸的基因克隆与序列分析", 《基因组学与应用生物学》 * |
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