CN116179375A - High-yield epsilon-polylysine and streptomyces hydrochloride genetically engineered bacterium as well as construction method and application thereof - Google Patents

High-yield epsilon-polylysine and streptomyces hydrochloride genetically engineered bacterium as well as construction method and application thereof Download PDF

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CN116179375A
CN116179375A CN202310029098.2A CN202310029098A CN116179375A CN 116179375 A CN116179375 A CN 116179375A CN 202310029098 A CN202310029098 A CN 202310029098A CN 116179375 A CN116179375 A CN 116179375A
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dape
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谭之磊
周东浩
贾士儒
侯颖
裴泽峰
许倍铭
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Tianjin University of Science and Technology
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Abstract

The invention discloses a high-yield epsilon-polylysine and Streptomyces hydrochlorus genetically engineered bacterium, which is obtained by over-expressing succinyldiaminopimelate dissuccinylase gene dapE in diaminopimelate DAP in S.diastatocochromagenes 6# -7, wherein the nucleotide sequence of the over-expressed dapE gene has 83.9% or more of consistency with SEQ ID No. 1. The invention takes streptomyces diastatochromogenes6# -7 as chassis strain, and over-expresses the gene dapE of the starch succinyl diaminopimelate dissuccinylase to obtain the high-yield engineering strain dacE of streptomyces diastatochromogenes of the over-expressed dapE gene. The fermentation level of epsilon-polylysine can be obviously improved, the production strength of the strain can be improved, and the production cost can be reduced by using the high-yield engineering strain streptomyces diastatochromogenes dapE for fermentation production of epsilon-polylysine and hydrochloride thereof.

Description

High-yield epsilon-polylysine and streptomyces hydrochloride genetically engineered bacterium as well as construction method and application thereof
Technical Field
The invention belongs to the technical field of bioengineering, relates to a construction method and application of a genetic engineering strain, and in particular relates to a genetic engineering strain for high-yield epsilon-polylysine and streptomyces hydrochloride thereof, and a construction method and application of the genetic engineering strain.
Background
Epsilon-polylysine (epsilon-Poly-L-lysine, epsilon-PL) is prepared from 25-35L-lysine monomers by reacting alpha-COOH and epsilon-NH 2 A natural amino acid homopolymer obtained by dehydration condensation. The epsilon-polylysine and the hydrochloride thereof have the advantages of wide antibacterial spectrum, good thermal stability and water solubility, safety and innocuity to human bodies, and have development potential in the fields of food preservation and corrosion prevention, and can be used as drug coating, cosmetics, drug carriers and the like.
In order to improve the yield of epsilon-polylysine and reduce the production cost, the prior related research is mostly started from the aspects of fermentation process optimization, mutation breeding and the like. However, there are relatively few reports and studies on the construction of engineering strains with high epsilon-polylysine yield, particularly on the method of overexpressing succinyldiaminopimelate disuccinidase (dapE) in the diaminopimelate pathway (DAP).
By searching, the following publications related to the present patent application are found:
1. a Streptomyces albus gene engineering bacterium, its construction method and application (CN 105441373A, 2015.12.4) discloses a Streptomyces albus PD-4, which is prepared by over-expressing an ammonium transporter gene amtB from S.albus PD-1 genome and improving epsilon-polylysine fermentation level by improving nitrogen source supply.
2. Streptomyces and a method for preparing epsilon-polylysine by the same (CN 110804572A, 2019.12.4) are disclosed, and a Streptomyces and a method for preparing epsilon-polylysine by the same are disclosed, belonging to the field of microbial fermentation. The genome rearrangement strain is produced by carrying out resistance screening on concentration gradient on S.album M-Z18, the yield of fermentation for 192 hours can reach 56.3g/L, but the genome rearrangement method for breeding high-yield strains has the defects of large workload and strong uncertainty.
3. Mao Zhonggui et al (Understanding high ε -poly-l-lysine production by Streptomyces albulus using pH shock strategy in the level of transgenics. Journal of Industrial Microbiology & Biotechnology,2019, 46:1781-1792.) in the literature, employ a pH impact strategy to activate the MprA/B and PepD signal transduction systems of S.albulus M-Z18, forward regulate transcription of ε -polylysine synthase (Pls) genes, increasing the activity of Pls during fermentation; meanwhile, the strategy also promotes the transcription of cytochrome c oxidase, ferritin reductase and ferritin gene, improves the respiratory activity of cells and increases the yield of epsilon-polylysine.
4. In Yamanaka et al (Enhancement ofmetabolic flux toward epsilon-poly-l-lysine biosynthesis by targeted inactivation of concomitant polyene macrolide biosynthesis in Streptomyces albulus Enhancement of metabolic flux toward epsilon-poly-l-lysine biosynthesis by targeted inactivation of concomitant polyene macrolide biosynthesis in Streptomyces album. Journal ofBioscience and Bioengineering,2020,129 (5): 558-564.) the epsilon-polylysine yield was increased by 20% over the starting strain by targeted inactivation of large gene fragments in the possible tetramycin and tetracycline biosynthesis (TTM) gene cluster, while the polyene compound yield in the secondary metabolite was reduced to 0.
By contrast, the present patent application differs essentially from the above-mentioned patent publications in terms of chassis strains and construction strategies.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provides a genetically engineered bacterium for high-yield epsilon-polylysine and streptomyces hydrochloride thereof, and a construction method and application thereof.
The technical scheme adopted for solving the technical problems is as follows:
a high-yield epsilon-polylysine and its hydrochloride streptomycete genetic engineering bacteria, the engineering bacteria are amylase chromogenic streptomycete dapE (Streptomyces diastatochromogenes dapE), the amylase chromogenic streptomycete dapE is obtained by over-expressing succinyl diaminopimelate dissuccinylase gene dapE in S.diastatocochrome 6, the nucleotide sequence of the over-expressed dapE gene has 83.9% or more of the identity with SEQ ID No. 1.
Further, the deposit number of s.diastatocochromenes 6# -7 is: CGMCC No.22261, preservation date: 2021, 4 months 30, address: beijing, chaoyang area, north Chen Xi Lu 1, 3, china academy of sciences microbiological institute, deposit unit: china general microbiological culture Collection center (China Committee for culture Collection).
The construction method of the genetically engineered bacteria for producing epsilon-polylysine and streptomyces hydrochloride with high yield comprises the following steps:
(1) Over-expression gene dapE, wherein the nucleotide sequence of the gene dapE has 83.9% and above consistency with SEQ ID No. 1;
(2) Amplifying or directly synthesizing the dapE gene by a PCR technology;
(3) The over-expression plasmid used in the construction was plasmid pIMEP with the strong promoter erythromycin promoter ermE.
Further, the steps are as follows:
(1) Extracting genome of S.diastatocochromenes 6# -7, using the genome as a template, and amplifying a target gene by adopting a PCR technology, wherein the target gene is succinyldiaminopimelate dissuccinylase gene dapE; or directly synthesizing the dapE nucleotide sequence with the same function of other streptomyces obtained according to NCBI retrieval;
(2) Inserting the target gene dapE obtained in the step (1) between enzyme cutting sites of a plasmid pIMEP integrated with a strong promoter erythromycin promoter ermE to obtain a recombinant plasmid pIMEP carrying the target gene, converting the recombinant plasmid pIMEP into escherichia coli DH5 alpha, and screening to obtain escherichia coli DH5 alpha positive transformants;
(3) Extracting recombinant plasmid pIMEP in E.coli DH5 alpha positive transformant of step (2), namely dapE, firstly transforming the extracted recombinant plasmid pIMEP into E.coli ET12567 (pUZ 8002), coating on LB plate containing 20-25 ng/. Mu.L kanamycin, 30-50 ng/. Mu.L apramycin and 20-25 ng/. Mu.L chloramphenicol, selecting E.coli ET12567 (pUZ 8002) positive transformant, culturing in LB liquid culture medium containing same concentration of three antibiotics of kanamycin, apramycin and chloramphenicol at constant temperature of 37-40 deg.C overnight, transferring into LB liquid culture medium containing same concentration of kanamycin, apramycin and chloramphenicol antibiotic, culturing at 180r/min at 37-40 deg.C until OD 600 Between 0.4 and 0.6, centrifugally collecting bacterial liquid, washing bacterial cells with fresh LB for 2-3 times to remove residual antibiotics, and re-suspending the bacterial cells in LB liquid medium to be placed on ice for standby, so as to obtain treated escherichia coli ET12567 (pUZ 8002) positive transformant cells;
(4) Adding TES buffer solution with pH=8.0 to the plate of S.diastatocochromenes 6# -7 strain producing spore on bennett culture medium, scraping S.diastatocochromenes 6# -7 spore, pouring into a container containing glass beads, breaking spore chain by 180r/min oscillation, filtering to remove mycelium, collecting spore suspension, cooling spore suspension to room temperature after water bath heat shock, adding M3G culture medium, shake culturing at 37-40deg.C for 2-3 hr to germinate spores, centrifuging at 5000r/min for 5min to collect S.diastatocochromenes 6# -7 germinated spores, and re-suspending for use;
(5) Mixing positive transformant E.coli ET12567 (pUZ 8002) of step (3) and S.diastatocochromenes 6# -7 germinated spores of step (4) in equal volume, uniformly coating on a medium containing 5-8mM Mg 2+ After the SFM culture medium is subjected to inversion culture for 14-18h at 30 ℃, the flat plate is covered by sterile water containing nalidixic acid with the concentration of 20-25mg/mL and apramycin with the concentration of 20-25mg/mL, the flat plate is dried and then is subjected to inversion culture for 3-5 days, and positive monoclonal zygotes are screened to obtain the high-yield engineering bacteria.
Further, the construction steps of the step (2) carrying the target gene recombinant plasmid pIMEP are as follows:
(a) Obtaining the target fragment gene: designing a primer sequence dapE-F/dapE-R according to a required gene, introducing EcoR I enzyme cutting sites at two ends of the gene, wherein 6 nucleotides are added at the upstream 5 'end and 6 nucleotides are added at the downstream 5' end of the nucleotide sequence to form a restriction enzyme EcoR I site, and amplifying the target gene dapE in the streptomyces diastatochromogenes 6.7 by PCR; or directly synthesizing a Streptomyces galileo (Streptomyces galilaeus) gene dapE with the same function;
the target gene dapE sequence in the amplified amylase streptomyces chromogenes 6-7 is as follows: SEQ ID No.1;
primer dapE-F: SEQ ID No.2;
primer dapE-R: SEQ ID No.3;
the sequence of the Streptomyces galileo (Streptomyces galileus) gene dapE is: SEQ ID No.8;
(b) Construction of recombinant plasmids: the EcoR I single enzyme digestion plasmid pIMEP is used, the amplified target gene fragment is connected with the single enzyme digestion plasmid pIMEP plasmid after the EcoR I single enzyme digestion to obtain a connection product recombinant plasmid, the connection product recombinant plasmid is transformed into E.coli DH5 alpha competent cells, E.coli DH5 alpha positive transformants are selected for preservation, and the target gene carrying recombinant plasmid pIMEP:: dapE is obtained.
Furthermore, the pIMEP plasmid is constructed by connecting erythromycin promoter ermE in front of a polyclonal site on the basis of pSET 152 plasmid, and the plasmid is a shuttle plasmid of escherichia coli-streptomycete, and can be used for expressing genes in streptomycete;
the formula of the culture medium is as follows:
every 1LM3G medium composition was: (NH) 4 ) 2 SO 4 10 g，KH 2 PO 4 1.36 g，K 2 HPO 4 0.8 5g of yeast extract, regulating the pH to 7.2 by ammonia water, adding water to a volume of 900mL, and adding 100mL of 10 Xglucose mother liquor;
every 1L of M3G medium containing glucose, the fermentation medium is short: when using M3G culture medium for fermentation, 100ml10×glucose mother liquor is added per 900ml M3G; 10 x glucose stock: 50g of glucose was weighed and 1ml of 500 XSO was added 4 ·7H 2 O and 1ml of 20 XMgSO 4 ·7H 2 O, sterilizing after the volume is fixed to 100ml by deionized water;
the composition of the bennett culture medium per 1L is: 10g of glucose, 2g of peptone, 1g of yeast powder, 1g of beef extract and 15-20g of agar, adding water to supplement 1L, and adjusting pH to 7.7 with NaOH;
the composition of each 1L of SFM medium was: 30g of soybean cake powder, 20g of mannitol and 15-20g of agar powder, adding water to supplement 1L, and regulating the pH to 7.2-7.4 by NaOH; wherein, the soybean cake powder is used after the following treatment: adding 800-900mL tap water, boiling for 30-60min, and filtering with gauze.
A method for improving the yield of epsilon-polylysine and hydrochloride thereof by using the genetically engineered bacteria comprises the step of improving the fermentation level of epsilon-polylysine through the fermentation of streptomyces diastatochromogenes dapE.
Further, the method comprises the following steps:
the adopted strain is a genetically engineered strain which is used for over-expressing succinyldiaminopimelate dissuccinylase gene dapE, namely amylase streptomyces chromogenes dapE, the genetically engineered strain is inoculated on a bennett culture medium plate and is cultured at 28-35 ℃ until black gray conidia are produced; then inoculating the spores into a shaking bottle of an M3G culture medium containing glucose, culturing for 30-33 hours at 28-35 ℃ and 180rpm/min, transferring the cultured seed culture solution into the M3G culture medium containing glucose for shaking bottle or fermentation tank fed-batch fermentation, and obtaining the epsilon-polylysine-containing fed-batch fermentation amino acid fermentation liquor.
Further, the feed fermentation comprises the following steps:
initial ph=6.8, control in two stages: in the stage I, the pH is controlled at 6.0 so as to be beneficial to the proliferation of thalli; step II, when the glucose concentration in the fermentation liquor is reduced to 10g/L, naturally reducing the pH to 4.0, then automatically feeding ammonia water with the mass concentration of 12.5% to maintain the pH to 4.0, and simultaneously feeding a mixed liquor of glucose and ammonium sulfate to control the glucose concentration to 10g/L; in the fermentation process, the temperature is controlled at 30 ℃, the dissolved oxygen is controlled at 30%, the ventilation ratio is maintained at 1-2vvm, and stirring is associated with the dissolved oxygen until the yield is highest.
The genetically engineered bacterium is applied to epsilon-polylysine and hydrochloride production thereof.
The beneficial effects obtained by the invention are as follows:
1. the invention takes streptomyces diastatochromogenes 6-7 as chassis strain, over-expresses streptomyces diastatochromogenes 6-7 itself or succinyl diaminopimelate disuccinate gene dapE of heterodiaminopimelate pathway (DAP), and obtains high-yield engineering strain streptomyces diastatochromogenes dapE over-expressing dapE gene. The fermentation level of epsilon-polylysine can be obviously improved, the production strength of the strain can be improved, and the production cost can be reduced by using the high-yield engineering strain streptomyces diastatochromogenes dapE for fermentation production of epsilon-polylysine and hydrochloride thereof.
2. The method of the invention obtains the high-yield engineering strain S.diastatocochromenes dapE (comprising dapE-1 and dapE-2) by overexpressing succinyldiaminopimelate dissuccinylase gene dapE of diaminopimelate pathway (DAP). Experiments prove that under the shake flask fermentation condition, the ability of the S.diastachromene-1 strain to produce epsilon-polylysine is improved by 20.1% compared with that of the original strain S.diastachromene 6# -7 under the same condition; under the fed-batch fermentation condition of a 5L fermentation tank, the capacities of the S.diastatocochromene-1 strain and the S.diastatocochromene-2 strain for producing epsilon-polylysine are respectively improved by 22.4 percent and 25.3 percent under the same condition compared with the capacity of the original strain S.diastatocochromene 6# -7, and excellent strains are provided for epsilon-polylysine and hydrochloride production thereof.
3. The method of the invention improves the production strength of the engineering strain S.diastatocochromene dapE (dapE-1 and dapE-2) unit thalli by 31.8 percent and 44.7 percent respectively compared with the original strain S.diastatocochromene 6# -7; wherein, the conversion rate of the engineering strain S.diastatocochromene dapE-2 glucose is improved by 4.2 percent, and the production cost of epsilon-polylysine and hydrochloride thereof is obviously reduced.
Drawings
FIG. 1 is a diagram showing the PCR product of the gene dapE of succinyldiaminopimelate dissuccinylase with homology arms amplified from the genome of the starting strain Sdiastatochromogens 6-7 according to the present invention; wherein lane M: a 5kb marker; lane 1: amplified dapE with homology arm (1089 bp);
FIG. 2 is a schematic diagram showing construction of a recombinant plasmid of dapE, wherein the pIMEP is constructed based on pIMEP;
FIG. 3 is a diagram showing the verification of the digestion of plasmid extraction of recombinant plasmid positive transformants for construction of pIMEP-dapE (wherein dapE is the S. Diastatochromogens gene) according to the present invention; wherein lane M: a 10kb marker; lane 1: ecoRI single enzyme digestion result of pIMEP-dapE recombinant plasmid, the two bands are 6322bp and 1089bp;
FIG. 4 is a diagram showing the verification of successful binding transfer by using the dapE-1 gene and a part of plasmid sequence, wherein the genome of the positive transformant of the genetically engineered strain S.diastatocochromene dapE-1 is obtained in the present invention; wherein lane M:2kb marker; lane 1: verifying the integrated dapE-1 gene in the genetically engineered strain S.diastatocochromene dapE-1; lane 2: control 6# -7 has no corresponding gene;
FIG. 5 is a graph showing the yield of epsilon-polylysine by shake flask fermentation of a strain of the present invention at 96 hours; wherein 6# -7 is the original strain S.diastatocochromogenes 6# -7, and dapE-1 is the high-yield epsilon-polylysine genetic engineering strain S.diastatocochromogenes dapE-1;
FIG. 6 shows the fed-batch fermentation results of a 5L fermenter of the starting strain S.diastatocochromene 6# -7 and the high-yield engineering strain S.diastatocochromene dapE-1 according to the present invention;
FIG. 7 is a diagram showing the verification of the digestion of plasmid extraction of a recombinant plasmid-positive transformant for constructing pIMEP-dapE (wherein dapE is the Streptomyces galilaeus gene) according to the present invention; wherein lane M: a 10kb marker; lane 1: ecoRI single enzyme digestion result of pIMEP-dapE recombinant plasmid, the two bands are 6322bp and 1080bp;
FIG. 8 shows the fed-batch fermentation results of a 5L fermenter containing the starting strain S.diastatocochromene 6# -7 and the high-yield engineering strain S.diastatocochromene dapE-2 according to the present invention.
Detailed Description
The present invention will be further described in detail with reference to examples, but the scope of the present invention is not limited to the examples.
The raw materials used in the invention are conventional commercial products unless otherwise specified, the methods used in the invention are conventional methods in the art unless otherwise specified, and the mass of each substance used in the invention is conventional.
A high-yield epsilon-polylysine and its hydrochloride streptomycete genetic engineering bacteria, the engineering bacteria are amylase chromogenic streptomycete dapE (Streptomyces diastatochromogenes dapE), the amylase chromogenic streptomycete dapE is obtained by over-expressing succinyl diaminopimelate dissuccinylase gene dapE in S.diastatocochrome 6, the nucleotide sequence of the over-expressed dapE gene has 83.9% or more of the identity with SEQ ID No. 1.
Preferably, the deposit number of S.diastatocochromenes 6-7 is: CGMCC No.22261, preservation date: 2021, 4 months 30, address: beijing, chaoyang area, north Chen Xi Lu 1, 3, china academy of sciences microbiological institute, deposit unit: china general microbiological culture Collection center (China Committee for culture Collection).
The construction method of the genetically engineered bacteria for producing epsilon-polylysine and streptomyces hydrochloride with high yield comprises the following steps:
(4) Over-expression gene dapE, wherein the nucleotide sequence of the gene dapE has 83.9% and above consistency with SEQ ID No. 1;
(5) Amplifying or directly synthesizing the dapE gene by a PCR technology;
(6) The over-expression plasmid used in the construction was plasmid pIMEP with the strong promoter erythromycin promoter ermE.
Preferably, the steps are as follows:
(1) Extracting genome of S.diastatocochromenes 6# -7, using the genome as a template, and amplifying a target gene by adopting a PCR technology, wherein the target gene is succinyldiaminopimelate dissuccinylase gene dapE; or directly synthesizing the dapE nucleotide sequence with the same function of other streptomyces obtained according to NCBI retrieval;
(2) Inserting the target gene dapE obtained in the step (1) between enzyme cutting sites of a plasmid pIMEP integrated with a strong promoter erythromycin promoter ermE to obtain a recombinant plasmid pIMEP carrying the target gene, converting the recombinant plasmid pIMEP into escherichia coli DH5 alpha, and screening to obtain escherichia coli DH5 alpha positive transformants;
(3) Extracting recombinant plasmid pIMEP in E.coli DH5 alpha positive transformant of step (2), namely dapE, firstly transforming the extracted recombinant plasmid pIMEP into E.coli ET12567 (pUZ 8002), coating on LB plate containing 20-25 ng/. Mu.L kanamycin, 30-50 ng/. Mu.L apramycin and 20-25 ng/. Mu.L chloramphenicol, selecting E.coli ET12567 (pUZ 8002) positive transformant, culturing in LB liquid culture medium containing same concentration of three antibiotics of kanamycin, apramycin and chloramphenicol at constant temperature of 37-40 deg.C overnight, transferring into LB liquid culture medium containing same concentration of kanamycin, apramycin and chloramphenicol antibiotic, culturing at 180r/min at 37-40 deg.C until OD 600 Between 0.4 and 0.6, centrifugally collecting bacterial liquid, washing bacterial cells with fresh LB for 2-3 times to remove residual antibiotics, and re-suspending the bacterial cells in LB liquid medium to be placed on ice for standby, so as to obtain treated escherichia coli ET12567 (pUZ 8002) positive transformant cells;
(4) Adding TES buffer solution with pH=8.0 to the plate of S.diastatocochromenes 6# -7 strain producing spore on bennett culture medium, scraping S.diastatocochromenes 6# -7 spore, pouring into a container containing glass beads, breaking spore chain by 180r/min oscillation, filtering to remove mycelium, collecting spore suspension, cooling spore suspension to room temperature after water bath heat shock, adding M3G culture medium, shake culturing at 37-40deg.C for 2-3 hr to germinate spores, centrifuging at 5000r/min for 5min to collect S.diastatocochromenes 6# -7 germinated spores, and re-suspending for use;
(5) Mixing positive transformant E.coli ET12567 (pUZ 8002) of step (3) and S.diastatocochromenes 6# -7 germinated spores of step (4) in equal volume, uniformly coating on a medium containing 5-8mM Mg 2+ After the SFM culture medium is subjected to inversion culture for 14 to 18 hours at the temperature of 30 ℃, the flat plate is covered by sterile water containing nalidixic acid with the concentration of 20 to 25mg/mL and apramycin with the concentration of 20 to 25mg/mL, the flat plate is dried and then is subjected to inversion culture for 3 to 5 days, positive monoclonal zygotes are screened, Obtaining the high-yield engineering bacteria.
Preferably, the construction steps of the step (2) carrying the target gene recombinant plasmid pIMEP are as follows:
(a) Obtaining the target fragment gene: designing a primer sequence dapE-F/dapE-R according to a required gene, introducing EcoR I enzyme cutting sites at two ends of the gene, wherein 6 nucleotides are added at the upstream 5 'end and 6 nucleotides are added at the downstream 5' end of the nucleotide sequence to form a restriction enzyme EcoR I site, and amplifying the target gene dapE in the streptomyces diastatochromogenes 6.7 by PCR; or directly synthesizing a Streptomyces galileo (Streptomyces galilaeus) gene dapE with the same function;
the target gene dapE sequence in the amplified amylase streptomyces chromogenes 6-7 is as follows: SEQ ID No.1;
primer dapE-F: SEQ ID No.2;
primer dapE-R: SEQ ID No.3;
the sequence of the Streptomyces galileo (Streptomyces galileus) gene dapE is: SEQ ID No.8;
(b) Construction of recombinant plasmids: the EcoR I single enzyme digestion plasmid pIMEP is used, the amplified target gene fragment is connected with the single enzyme digestion plasmid pIMEP plasmid after the EcoR I single enzyme digestion to obtain a connection product recombinant plasmid, the connection product recombinant plasmid is transformed into E.coli DH5 alpha competent cells, E.coli DH5 alpha positive transformants are selected for preservation, and the target gene carrying recombinant plasmid pIMEP:: dapE is obtained.
Preferably, the pIMEP plasmid is constructed by connecting erythromycin promoter ermE in front of a multiple cloning site on the basis of pSET 152 plasmid, and the plasmid is a shuttle plasmid of escherichia coli-streptomycete, and can be used for expressing genes in streptomycete;
the formula of the culture medium is as follows:
every 1LM3G medium composition was: (NH) 4 ) 2 SO 4 10 g，KH 2 PO 4 1.36 g，K 2 HPO 4 0.8 5g of yeast extract, regulating the pH to 7.2 by ammonia water, adding water to a volume of 900mL, and adding 100mL of 10 Xglucose mother liquor;
every 1L of M3G culture medium containing glucose, short for shortFermentation medium: when using M3G culture medium for fermentation, 100ml10×glucose mother liquor is added per 900ml M3G; 10 x glucose stock: 50g of glucose was weighed and 1ml of 500 XSO was added 4 ·7H 2 O and 1ml of 20 XMgSO 4 ·7H 2 O, sterilizing after the volume is fixed to 100ml by deionized water;
the composition of the bennett culture medium per 1L is: 10g of glucose, 2g of peptone, 1g of yeast powder, 1g of beef extract and 15-20g of agar, adding water to supplement 1L, and adjusting pH to 7.7 with NaOH;
the composition of each 1L of SFM medium was: 30g of soybean cake powder, 20g of mannitol and 15-20g of agar powder, adding water to supplement 1L, and regulating the pH to 7.2-7.4 by NaOH; wherein, the soybean cake powder is used after the following treatment: adding 800-900mL tap water, boiling for 30-60min, and filtering with gauze.
A method for improving the yield of epsilon-polylysine and hydrochloride thereof by using the genetically engineered bacteria comprises the step of improving the fermentation level of epsilon-polylysine through the fermentation of streptomyces diastatochromogenes dapE.
Preferably, the method comprises the steps of:
the adopted strain is a genetically engineered strain which is used for over-expressing succinyldiaminopimelate dissuccinylase gene dapE, namely amylase streptomyces chromogenes dapE, the genetically engineered strain is inoculated on a bennett culture medium plate and is cultured at 28-35 ℃ until black gray conidia are produced; then inoculating the spores into a shaking bottle of an M3G culture medium containing glucose, culturing for 30-33 hours at 28-35 ℃ and 180rpm/min, transferring the cultured seed culture solution into the M3G culture medium containing glucose for shaking bottle or fermentation tank fed-batch fermentation, and obtaining the epsilon-polylysine-containing fed-batch fermentation amino acid fermentation liquor.
Preferably, the feed fermentation comprises the steps of:
initial ph=6.8, control in two stages: in the stage I, the pH is controlled at 6.0 so as to be beneficial to the proliferation of thalli; step II, when the glucose concentration in the fermentation liquor is reduced to 10g/L, naturally reducing the pH to 4.0, then automatically feeding ammonia water with the mass concentration of 12.5% to maintain the pH to 4.0, and simultaneously feeding a mixed liquor of glucose and ammonium sulfate to control the glucose concentration to 10g/L; in the fermentation process, the temperature is controlled at 30 ℃, the dissolved oxygen is controlled at 30%, the ventilation ratio is maintained at 1-2vvm, and stirring is associated with the dissolved oxygen until the yield is highest.
The genetically engineered bacterium is applied to epsilon-polylysine and hydrochloride production thereof.
Specifically, the preparation and detection of the correlation are as follows:
example 1.S.diastatochromogenes dapE-1 obtaining of the dapE Gene of the engineering Strain
According to the gene design primer sequence dap-F/dap-R, a single-enzyme homologous recombination strategy is adopted to construct a recombinant vector, ecoR I cleavage sites are added at two ends of the gene dapE, wherein EcoR I cleavage sites and 15 nucleotides identical to EcoR I upstream of the original vector pIMEP are added at the 5 'end of the upstream of the nucleotide sequence of the dapE gene to form an upstream homology arm, ecoR I cleavage sites and 15 nucleotides identical to EcoR I downstream of the original vector pIMEP are added at the 5' end of the downstream of the nucleotide sequence of the dapE gene to form a downstream homology arm, and the sequence table SEQ ID No.2 and SEQ ID No.3 are used as upstream and downstream primers, so that the total length 1089bp of the dapE gene in the original strain S.diasonochrogenes 6 and 7 is amplified by PCR, see SEQ ID No.1 of the sequence table.
Wherein the reaction system and reaction conditions of PCR: 2X phanta max buffer. Mu.L, dNTP mix (10 mM) 1. Mu.L, template (20 ng/ul) 1. Mu.L, upstream and downstream primers ((SEQ No.2, SEQ No. 3)) 2. Mu.L (10. Mu.M) each, DMSO 2. Mu.L, phanta max X Super-Fidelity DNAPolymerase. Mu.L, and make up ultrapure water to 50. Mu.L. PCR reaction conditions: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 15s, annealing at 58℃for 15s, extension at 72℃for 80s, total 30 cycles, extension at 72℃for 5min, and termination of the reaction at 4℃to obtain the objective gene dapE. The electrophoretogram of the target gene dapE product amplified by PCR is shown in FIG. 1.
EXAMPLE 2 construction of recombinant plasmid pIMEP containing the dapE Gene
And (3) connecting the PCR amplified dapE gene fragment with the homology arm with linear plasmid pIMEP subjected to EcoR I single enzyme digestion and integrated with the erythromycin promoter ermE with a strong promoter by using a homologous recombination method to obtain a recombinant plasmid pIMEP. Wherein, the homologous recombination system is as follows: linearized vector pIMEP 125.72ng, insert dapE43.56ng,5 XCE II Buffer 4. Mu.L, exnase II 2. Mu.L, supplemented with ultra pure water to 20. Mu.L, homologous recombination conditions: the reaction was carried out at 37℃for 30min and was reduced to 4 ℃. A schematic diagram of the construction of the dapE recombinant plasmid is shown in FIG. 2.
Example 3 recombinant plasmid pIMEP:. DapE transformation of E.coli DH 5. Alpha
Adding the recombinant plasmid pIMEP into a centrifuge tube containing E.coli DH5 alpha competent cells which are melted in an ice bath, flicking the tube wall, uniformly mixing, and carrying out ice bath for 30min. Heat shock at 42 ℃ for 90s followed by an immediate ice bath for 5min (this process does not move). Under aseptic conditions, 900 mu L of LB culture medium is added into a centrifuge tube, and after being stirred and mixed uniformly, the mixture is subjected to shaking culture at 37 ℃ for 45min at 200 r/min. Centrifuge tube 12000r/min for 1min, remove 900. Mu.L supernatant, blow mix the remaining liquid with a pipette, and apply to LB solid plates containing 30. Mu.g/mL apramycin resistance. The LB plate is inversely cultured at 37 ℃ for overnight until single colony is clearly distinguished, meanwhile, colony PCR is carried out on the transformant obtained by screening and the plasmid is extracted by taking the sequence table SEQ ID No.4 and the sequence table SEQ ID No.5 as the upstream primer and the downstream primer, ecoRI single enzyme digestion verification is carried out, and the enzyme digestion verification result is shown in figure 3: the recombinant plasmid pIMEP-dapE was successfully transformed into transformants, and the verified E.coli DH 5. Alpha. Positive transformants were stored.
Example 4 obtaining of high yield engineering Strain
Single colonies of E.coli DH 5. Alpha. Positive transformants were picked up and cultured overnight in 5mL LB liquid medium (containing 30. Mu.g/mL apramycin) with shaking at 37℃220r/min, pIMEP:: dapE recombinant plasmid in E.coli DH 5. Alpha. Positive transformants was extracted according to the conventional method, the recombinant plasmid was chemically transformed into helper strain E.coli ET12567 (pUZ 8002), the transformants were plated on LB plates containing 20 ng/. Mu.L kanamycin, 30 ng/. Mu.L apramycin and 20 ng/. Mu.L chloramphenicol resistance, and cultured upside down at 37℃for 24h. E.coli positive transformants were selected as single colonies in 5mL of LB (containing three antibiotics at the same concentration as in the previous step), cultured overnight at 37℃with shaking at constant temperature, and then transferred to fresh 50mL of LB liquid medium containing three antibiotics at 1% transfer rate (antibiotic concentration as in the previous step),shaking culture at 37deg.C to OD at 180r/min 600 Between 0.4 and 0.6. 40mL of bacterial liquid was collected by centrifugation at 8000r/min for 5min, the bacterial cells were washed 2 times with fresh LB to remove residual antibiotics, resuspended in 1mL of LB and placed on ice for later use, yielding treated E.coli ET12567 (pUZ 8002) positive transformant cells. 10mL of TES buffer solution with pH of 8.0 is added to a plate of S.diastatocochromenes 6-7 strain with good spore growth, spores are scraped off by a sterile inoculating loop, poured into a 250mL triangular flask containing glass beads, oscillated at 30 ℃ for 2 hours at 180r/min to break spore chains, and then filtered by sterile absorbent cotton to remove hyphae and collect spore suspension. The spore suspension is heated in a water bath at 50deg.C for 10min, immediately cooled to room temperature, then 10mLM G culture medium is added, and shaking culture is carried out at 37deg.C for 2h at 180r/min to germinate spores, and S.diastatochromene 6# -7 germinated spores are collected by centrifugation at 5000r/min for 5min and resuspended for use.
Mixing treated E.coli ET12567 (pUZ 8002) positive transformant cells with germinated S.diastatocochromagene6# -7 spores in equal volume, and uniformly coating on a medium containing 5mM Mg 2+ Is on SFM medium. Culturing at 30deg.C in an inverted manner. After 14h of inversion culture, the flat plate is covered by sterile water containing 20mg/mL of nalidixic acid and 20mg/mL of apramycin, and after the flat plate is dried, the inversion culture is continued for 3-5 days, positive monoclonal zygotes are screened, and the high-yield engineering strain S.diastatochromogen dapE-1 is obtained.
Example 5 high yield engineering Strain verification
After the screened and obtained dapE gene overexpression strain is subjected to polylysine fermentation medium shake flask fermentation for 30 hours, genome is extracted, and a pair of verification primers are designed at about 100bp upstream and about 100bp downstream of the dapE gene on a pIMEP plasmid and at the tail of the dapE gene: the specific sequences are SEQ ID No.6 and SEQ ID No.7, respectively, and the result is shown in FIG. 4, in which the dapE gene is successfully incorporated into the genome of 6# -7, and the corresponding band is not obtained in the genome of control 6# -7.
PCR reaction system: 2 Xphantamax buffer 25. Mu.L, dNTP mix (10 mM) 1. Mu.L, template (20 ng/ul) 1. Mu.L, each of the upstream and downstream primers ((10. Mu.M)) 2. Mu.L, DMSO 2. Mu.L, phanta max x Super-Fidelity DNA Polymerase. Mu.L, and ultra-pure water to 50. Mu.L. PCR reaction conditions: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 15s, annealing at 55℃for 15s, extension at 72℃for 80s, 30 cycles, extension at 72℃for 5min, and termination of the reaction at 4 ℃.
EXAMPLE 6 production of epsilon-polylysine by fermentation of highly productive engineering Strain S.diastatocochromene dapE-1
Inoculating high-yield engineering strain S.diastatocochromene-1 on a bennett culture plate, and culturing at 28 ℃ for about 7 days until spores are produced; the spores were then inoculated into 500mL shake flasks containing 100mL of glucose-containing M3G medium and fermented at 28℃for 30h at 180 r/min. Transferring to new 100mL M3G medium containing glucose with inoculation amount of 6% for fermentation until the yield is highest, to obtain fermentation broth containing epsilon-polylysine, wherein the result is shown in figure 5, the yield of high-yield engineering strain S.diastatocochromene dapE-1 epsilon-polylysine is increased by 20.1% compared with that of original strain S.diastatocochromene 6, the glucose conversion rate is increased by 26.0% compared with that of original strain, and the thallus production strength is increased by 43.5% compared with that of original strain.
Or inoculating high-yield engineering strain S.diastatocochromene-1 on a bennett culture plate, and culturing at 28deg.C for about 7 days until spores are produced; the spores were then inoculated into 500mL shake flasks containing 100mL of glucose-containing M3G medium and fermented at 28℃for 30h at 180 r/min. The cultured seed liquid is inoculated into a 5L fermentation tank with 3LM3G fermentation medium at an inoculum size of 10 percent, the initial pH value is 6.8, and the two-stage control is carried out: in the stage I, the pH is controlled at 6.0 so as to be beneficial to the proliferation of thalli; step II, when the glucose concentration in the fermentation liquor is reduced to 10g/L, naturally reducing the pH to 4.0, then automatically feeding ammonia water with the concentration of 12.5% to maintain the pH at about 4.0, and simultaneously feeding a mixed solution (800 g/L,80 g/L) of glucose and ammonium sulfate to control the glucose concentration to about 10g/L so as to be beneficial to the production of products; in the fermentation process, the temperature is controlled at 30 ℃, the dissolved oxygen is controlled at 30%, the ventilation ratio is maintained at 1-2vvm, and stirring is associated with the dissolved oxygen until the yield is highest. As shown in FIG. 6, after 216 hours of fed-batch fermentation in a 5L fermenter, the highest yield of the original strain S.diaastochromogenes 6# -7 epsilon-polylysine was found to be 25.11g/L, and the highest yield of the genetically engineered strain S.diaastochromogenes dapE-1 epsilon-polylysine was found to be 30.73g/L, and it is obvious that the yield of the genetically engineered strain S.diaastochromogenes dapE-1 epsilon-polylysine was increased by 22.4% compared with the yield of the original strain 6# -7, and the cell production strength was increased by 31.8% compared with the original strain.
EXAMPLE 7 construction and use of high yield engineering Strain S.diastatocochromene dapE-2
Construction of a highly productive engineering strain S.diastatocochromene dapE-2 overexpressing the Streptomyces succinyldiaminopimelate disuccinate gene of Streptomyces succinyldiaminopimelate (Streptomyces galiaeus) and its use in fermentative production of epsilon-polylysine, wherein the nucleotide sequence of the Streptomyces succinyldiaminopimelate disuccinate gene of Streptomyces galileo (Streptomyces galilaeus) is SEQ No.8 and SEQ No.8 has 83.9% identity with SEQ No. 1:
the gene of the succinyldiaminopimelate dissuccinylase dapE of the Streptomyces galileo (Streptomyces galilaeus) is directly synthesized according to the nucleotide sequence of SEQ No.8 and is integrated at the EcoR I restriction enzyme cleavage site of the plasmid pIMEP to obtain a recombinant plasmid pIMEP:: dapE.
Adding the recombinant plasmid pIMEP into a centrifuge tube containing E.coli DH5 alpha competent cells which are melted in an ice bath, flicking the tube wall, uniformly mixing, and carrying out ice bath for 30min. Heat shock at 42 ℃ for 90s followed by an immediate ice bath for 5min (this process does not move). Under aseptic conditions, 900 mu L of LB culture medium is added into a centrifuge tube, and after being stirred and mixed uniformly, the mixture is subjected to shaking culture at 37 ℃ for 45min at 200 r/min. Centrifuge tube 12000r/min for 1min, remove 900. Mu.L supernatant, blow mix the remaining liquid with a pipette, and apply to LB solid plates containing 50. Mu.g/mL apramycin resistance. And (3) inversely culturing the LB plate at 40 ℃ overnight until single bacterial colonies are clearly distinguished, simultaneously carrying out bacterial colony PCR (polymerase chain reaction) and plasmid extraction by taking a sequence table SEQ ID No.4 and a sequence table SEQ ID No.5 as upstream and downstream primers, carrying out EcoRI single enzyme digestion verification, and preserving the verified E.coli DH5 alpha positive transformants. The results are shown in FIG. 7, in which the recombinant plasmid pIMEP-dapE has been successfully transformed into a transformant.
Single colonies of the E.coli DH 5. Alpha. Positive transformants were cultured overnight in 5mL LB liquid medium (containing 50. Mu.g/mL apramycin) with shaking at 37℃220r/minThe pIMEP in E.coli DH 5. Alpha. Positive transformants was extracted according to the conventional method by dapE recombinant plasmid, the recombinant plasmid was transferred to helper strain E.coli ET12567 (pUZ 8002), the transformants were plated on LB plates containing 25 ng/. Mu.L kanamycin, 50 ng/. Mu.L apramycin and 25 ng/. Mu.L chloramphenicol resistance, and cultured upside down at 37℃for 24h. E.coli ET12567 (pUZ 8002) positive transformants were selected as single colonies in 5mL LB (containing three antibiotics at the same concentration as in the previous step), cultured overnight at 40℃with shaking at constant temperature, then transferred to fresh 50mL LB liquid medium containing three antibiotics at 1% transfer rate (antibiotic concentration as in the previous step), cultured at 180r/min at 40℃with shaking to OD 600 Between 0.4 and 0.6. 40mL of bacterial liquid was collected by centrifugation at 8000r/min for 5min, the bacterial cells were washed 3 times with fresh LB to remove residual antibiotics, resuspended in 1mL of LB and placed on ice for later use, yielding treated E.coli ET12567 (pUZ 8002) positive transformant cells. 10mL of TES buffer solution with pH of 8.0 is added to a plate of S.diastatocochromenes 6-7 strain with good spore growth, spores are scraped off by a sterile inoculating loop, poured into a 250mL triangular flask containing glass beads, oscillated at 30 ℃ for 2 hours at 180r/min to break spore chains, and then filtered by sterile absorbent cotton to remove hyphae and collect spore suspension. The spore suspension is heated in a water bath at 50deg.C for 15min, immediately cooled to room temperature, then 10mL of M3G culture medium is added, and shaking culture is carried out at 40deg.C for 3h to germinate spores, and S.diastatochromene 6# -7 germinated spores are collected by centrifugation at 5000r/min for 5min and resuspended for later use.
Mixing treated E.coli ET12567 (pUZ 8002) positive transformant cells with germinated S.diastatocochromagene6# -7 spores in equal volume, and uniformly coating on a medium containing 8mM Mg 2+ Is on SFM medium. Culturing at 30deg.C in an inverted manner. After 18h of inversion culture, the flat plate is covered by sterile water containing nalidixic acid with the concentration of 25mg/mL and apramycin with the concentration of 25mg/mL, and after the flat plate is dried, the inversion culture is continued for 3-5 days, positive monoclonal zygotes are screened, and the high-yield engineering strain S.diastatochromogen dapE-2 is obtained.
Inoculating high-yield engineering strain S.diastatocochromene-2 on a bennett culture plate, and culturing at 35 ℃ for about 7 days until spores are produced; the spores were then inoculated into 500mL shake flasks containing 100mL of glucose-containing M3G medium and fermented at 35℃for 33h at 180 r/min. The cultured seed liquid is inoculated into a 5L fermentation tank with 3LM3G fermentation medium at an inoculum size of 10 percent, the initial pH value is 6.8, and the two-stage control is carried out: in the stage I, the pH is controlled at 6.0 so as to be beneficial to the proliferation of thalli; step II, when the glucose concentration in the fermentation liquor is reduced to 10g/L, naturally reducing the pH to 4.0, then automatically feeding ammonia water with the concentration of 12.5% to maintain the pH at about 4.0, and simultaneously feeding a mixed solution (800 g/L,80 g/L) of glucose and ammonium sulfate to control the glucose concentration to about 10g/L so as to be beneficial to the production of products; in the fermentation process, the temperature is controlled at 30 ℃, the dissolved oxygen is controlled at 30%, the ventilation ratio is maintained at 1-2vvm, and stirring is associated with the dissolved oxygen until the yield is highest.
As shown in FIG. 8, the fermentation result of high yield engineering bacteria S.diaastochromogenes dapE-2 fed-batch in a 5L fermenter shows that the maximum yield of epsilon-polylysine of the genetic engineering strain Streptomyces diastatochromogenes dapE-2 is 31.47g/L, the maximum yield of the original strain S.diaastochromogenes 6-7 epsilon-polylysine is 25.11g/L, the yield of epsilon-polylysine of the genetic engineering strain S.diaastochromogenes dapE-2 is obviously improved by 25.3% compared with the yield of the original strain, and the glucose conversion rate and the thallus production strength are respectively improved by 4.2% and 44.7% compared with the original strain.
The related gene sequences used in the present invention may be as follows:
Seq ID No.1
succinyldiaminopimelate disuccinate dapE nucleotide sequence:
atggagcgct ccactcaccc ccgccttgac ctgtcgctgg acgccgccgc actgaccgcg 60
cagctcgtcg acttcccgtc cgagagcggc aacgagaagg acctcgccga cgccgtcgag 120
gaagccctgc gtgccctgcc gcacctgacc gtcgaccggt acggcaacaa cgtcgtcgcc 180
cgcacgcacc tgggccgcgc ggagcgggtg gtgctcgccg gacacctcga caccgtgccg 240
atcgcggaca acgtcccctc ccggctcgac gaggacggcg tcctgtgggg ctgcggcacc 300
tgcgacatga agtcgggggt cgccgtccag ctccggatgg ccgccaccgt ccccgccccc 360
aaccgcgacc tgaccttcgt cttctacgac aacgaagagg tcgccgcgca cctcaacggc 420
ctcggccggg tcgccgacgc ccaccccgac tggctcgcgg gcgacttcgc cgtcctcctg 480
gagccctccg acggccaggt ggagggcggc tgccagggca ccctccgggt cctgctgcgc 540
accagcgggg agcgcgcgca ctccgcacgc agctggatgg gcgccaacgc catccacgcc 600
gcggcccccc tcctggcgac gctcgccgcc tacgagccgc gccgcccggt gatcgacggc 660
ctggagtacc gcgagggcct caacgcggtc cgcatcgagg gcggcgtggc cggcaacgtc 720
gtcccggacg cctgcaccgt caccgtcaac ttccgctacg caccggaccg cacccccgaa 780
gaggcggtgg cccacgtccg cgaggtcttc gcggactgcc ccgtggacga gttcgtcatc 840
gacgaccact ccccgggggc gctgcccggg ctgtcccacc cggccgccca ggcgttcatg 900
acggccgtcg gcggcagcgc gaagcccaag ttcggctgga ccgacgtctc ccgcttcagc 960
gccctcggcg tcccggccgt caactacggc cccggtgagg cgctgctcgc ccacaagaag 1020
gacgagcggg tcgcgatcga ccgcatcgcg cactgcgagg agcggctgcg cgcctggctt 1080
accgcctga 1089
Seq ID No.2
dapE-F: namely: 5' -ATCGGATCCGGTACCGAATTCATGGAGCGCTCCACTCACC-3’ 40
Wherein the underline is EcoRI cleavage site; thickening fonts into upstream homology arms
Seq ID No.3
dapE-R: namely:
Figure BDA0004046348500000101
39
wherein the underline is EcoRI cleavage site; thickening fonts into downstream homology arms
Seq ID No.4
p-F: namely: 5'-TGTAAAACGACGGCCAGT-3' 18 and 5218
Seq ID No.5
p-R: namely: 5'-CAGGAAACAGCTATGAC-3' 17 and 5217
Seq ID No.6
vdapE-F: namely: 5'-TGTAAAACGACGGCCAGT-3' 18 and 5218
Seq ID No.7
vdapE-R: namely: 5'-TCAGGCGGTAAGCCAGGC-3' 18 and 5218
Seq ID No.8
Streptomyces succinyldiaminopimelate disuccinate desuccinate dapE nucleotide sequence of Streptomyces galilaeus:
atggccgaga ccccgcttga cctcacgctg gacgccgctc tgctcaccgc gcagctcgtc 60
gacttcccct ccgagagcgg cacggagaag ccgctcgcgg acgccgtcga gacggccctg 120
cgcacgctgc cgcacctcac ggtggaccgg cacggcaaca acgtgatcgc ccgcacccgc 180
ctgggccgtc ccgagcgggt ggtcctcgcc ggccacatcg acaccgtccc gatcgccggc 240
aacgtcccgt cccggctgga cgcggacggc gtcctgtggg gctgcggcac ctgcgacatg 300
aaggcgggcg tcgccgtcca gctccgcatc gccgccacgg tccccgcccc caaccgggac 360
ctgaccttcg tcttctacga caacgaagag gtcgccgccg acctcaacgg cctcaagcac 420
gtcgccgaga cccacccgga ctggctggag ggcgacttcg cggtcctcct ggagccctcc 480
gacggccagg tcgagggcgg ctgccagggc accctgcggg tcctgctgaa gaccaggggc 540
gagcgggccc actcggcgcg cagctggatg ggctccaacg cgatccacgc ggccgccccg 600
atcctggccc gcctcgccgc gtacgagccg cgccacccgg tgatcgacgg cctggagtac 660
cgcgagggcc tcaacgccgt cggcatcacc gggggagtgg ccggcaacgt catccccgac 720
gagtgcgtcg tgaccgtcaa cttccgctac gcgcccgacc gtacgcccga ggaggccatc 780
gcccacgtcc gtgaggtgtt cgccggctgc ggggtggagg agttcgtcat cgacgaccac 840
agcggggcgg ccctgcccgg actctcgcac cccgccgcgg cggccttcat cgaggccgtg 900
ggcggtaccc cgcagcccaa gtacggctgg accgacgtgt cccgcttctc ggcgctcggt 960
gtgccggccg tcaactacgg tcctggcaac ccgcacttgg cgcacaagcg ggacgagcgg 1020
gtggacaccg cgaagatcct ggcgggggag gagcgactgc gggcctggct gaccgcgtga 1080
Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.

Claims (10)

1. A genetically engineered bacterium for high-yield epsilon-polylysine and streptomyces hydrochloride thereof is characterized in that: the engineering bacteria are amylase streptomyces chromogenes dapE (StreptomycesdiastatochromogenesdapE), the amylase streptomyces chromogenes dapE is obtained by over-expressing succinyldiaminopimelate dissuccinylase gene dapE in diaminopimelate DAP in S.diastatocochromagenes 6# -7, and the nucleotide sequence of the over-expressed dapE gene has 83.9% or more of the same as that of SEQ ID No. 1.
2. The genetically engineered bacterium for high-yield epsilon-polylysine and streptomyces hydrochloride thereof according to claim 1, wherein the genetically engineered bacterium is characterized by: the deposit number of S.diastatocochromagene6# -7 is: cgmccno.22261, date of preservation: 2021, 4 months 30, address: beijing, chaoyang area, north Chen Xi Lu 1, 3, china academy of sciences microbiological institute, deposit unit: china general microbiological culture Collection center (China Committee for culture Collection).
3. The construction method of high-yield epsilon-polylysine and Streptomyces hydrochloride genetically engineered bacteria thereof according to claim 1 or 2, which is characterized by comprising the following steps: the method comprises the following steps:
(1) Over-expression gene dapE, wherein the nucleotide sequence of the gene dapE has 83.9% and above consistency with SEQ ID No. 1;
(2) Amplifying or directly synthesizing the dapE gene by a PCR technology;
(3) The over-expression plasmid used in the construction was plasmid pIMEP with the strong promoter erythromycin promoter ermE.
4. The construction method of the genetically engineered bacteria of high-yield epsilon-polylysine and streptomyces hydrochloride thereof according to claim 3, wherein the construction method is characterized in that: the method comprises the following steps:
(1) Extracting the genome of S.diastatocochromagenes 6# -7, using the genome as a template, and amplifying a target gene by adopting a PCR technology, wherein the target gene is succinyldiaminopimelate dissuccinylase gene dapE; or directly synthesizing the dapE nucleotide sequence with the same function of other streptomyces obtained according to NCBI retrieval;
(2) Inserting the target gene dapE obtained in the step (1) between enzyme cutting sites of a plasmid pIMEP integrated with a strong promoter erythromycin promoter ermE to obtain a recombinant plasmid pIMEP carrying the target gene, converting the recombinant plasmid pIMEP into escherichia coli DH5 alpha, and screening to obtain escherichia coli DH5 alpha positive transformants;
(3) Extracting recombinant plasmid pIMEP in E.coli DH5 alpha positive transformant of step (2), namely dapE, firstly transforming the extracted recombinant plasmid pIMEP into E.coli ET12567 (pUZ 8002), coating on LB plate containing 20-25 ng/. Mu.L kanamycin, 30-50 ng/. Mu.L apramycin and 20-25 ng/. Mu.L chloramphenicol, selecting E.coli ET12567 (pUZ 8002) positive transformant, culturing in LB liquid culture medium containing same concentration of three antibiotics of kanamycin, apramycin and chloramphenicol at constant temperature of 37-40 deg.C overnight, transferring into LB liquid culture medium containing same concentration of kanamycin, apramycin and chloramphenicol antibiotic, culturing at 180r/min at 37-40 deg.C until OD 600 Between 0.4 and 0.6, centrifuging to collect bacterial liquid, washing bacterial cells with fresh LB for 2-3 times to remove residual antibiotics, re-suspending in LB liquid medium, and placing on ice to obtain treated Escherichia coliET12567 (pUZ 8002) positive transformant cells;
(4) Adding TES buffer solution with pH=8.0 to the plate of S.diastatocochromagene6# -7 strain producing spore on bennett culture medium, scraping S.diastatocochromagene6# -7 spore, pouring into a container containing glass beads, breaking spore chain by shaking at 30deg.C and 180r/min, filtering to remove mycelium, collecting spore suspension, cooling to room temperature after heat shock in water bath, adding M3G culture medium, shaking culturing at 37-40deg.C for 2-3 hr to germinate spores, centrifuging at 5000r/min for 5min to collect S.diastatocochromagene6# -7 germinated spores, and re-suspending for use;
(5) Mixing positive transformant E.coli ET12567 (pUZ 8002) of step (3) and S.diastatocochromagene6# -7 germinated spores of step (4) in equal volume, and uniformly coating on a substrate containing 5-8mMMg 2+ After the SFM culture medium is subjected to inversion culture for 14-18h at 30 ℃, the flat plate is covered by sterile water containing nalidixic acid with the concentration of 20-25mg/mL and apramycin with the concentration of 20-25mg/mL, the flat plate is dried and then is subjected to inversion culture for 3-5 days, and positive monoclonal zygotes are screened to obtain the high-yield engineering bacteria.
5. The construction method of the genetically engineered bacteria of high-yield epsilon-polylysine and streptomyces hydrochloride thereof according to claim 4 is characterized in that: the construction steps of the step (2) for carrying the target gene recombinant plasmid pIMEP are as follows:
(a) Obtaining the target fragment gene: designing a primer sequence dapE-F/dapE-R according to a required gene, introducing EcoRI enzyme cutting sites at two ends of the gene, wherein 6 nucleotides are added at the upstream 5 'end and 6 nucleotides are added at the downstream 5' end of the nucleotide sequence to form a restriction enzyme EcoRI site, and amplifying the target gene dapE in streptomyces diastatochromogenes 6-7 by PCR; or directly synthesizing a Streptomyces galileo (Streptomyces galilaeus) gene dapE with the same function;
The target gene dapE sequence in the amplified amylase streptomyces chromogenes 6-7 is as follows: SEQ ID No.1;
primer dapE-F: SEQ ID No.2;
primer dapE-R: SEQ ID No.3;
the sequence of the Streptomyces galileo (Streptomyces galileus) gene dapE is: SEQ ID No.8;
(b) Construction of recombinant plasmids: the EcoRI single enzyme digestion plasmid pIMEP is used for carrying out the connection between the amplified target gene fragment and the single enzyme digestion plasmid pIMEP plasmid after the EcoRI single enzyme digestion, thus obtaining a connection product recombinant plasmid, transforming the recombinant plasmid into E.coli DH5 alpha competent cells, screening E.coli DH5 alpha positive transformants for preservation, thus obtaining the recombinant plasmid pIMEP carrying the target gene, namely dapE.
6. The construction method of the genetically engineered bacteria of high-yield epsilon-polylysine and streptomyces hydrochloride thereof according to claim 5 is characterized in that: the pIMEP plasmid is constructed by connecting an erythromycin promoter ermE in front of a polyclonal site on the basis of a pSET152 plasmid, and the plasmid is an escherichia coli-streptomyces shuttle plasmid and can be used for expressing genes in streptomyces;
the formula of the culture medium is as follows:
every 1LM3G medium composition was: (NH) 4 ) 2 SO 4 10g，KH 2 PO 4 1.36g，K 2 HPO 4 0.8g, yeast extract 5g, pH adjusted to 7.2 with ammonia, water was added to volume to 900mL, and 100mL10 Xglucose stock solution was added;
Every 1L of M3G medium containing glucose, the fermentation medium is short: when the M3G culture medium is used for fermentation, 100ml of 10 multiplied by glucose mother liquor is added to 900ml of M3G; 10 x glucose stock: 50g of glucose was weighed and 1ml of 500 XSO was added 4 ·7H 2 O and 1ml20 XMgSO 4 ·7H 2 O, sterilizing after the volume is fixed to 100ml by deionized water;
the composition of the bennett culture medium per 1L is: 10g of glucose, 2g of peptone, 1g of yeast powder, 1g of beef extract, 15-20g of agar, adding water to supplement 1L, and regulating pH to 7.7 with NaOH;
the composition of each 1LSFM medium was: 30g of soybean cake powder, 20g of mannitol and 15-20g of agar powder, adding water to supplement 1L, and adjusting pH to 7.2-7.4 by NaOH; wherein, the soybean cake powder is used after the following treatment: adding 800-900mL tap water, boiling for 30-60min, and filtering with gauze.
7. A method for improving the yield of epsilon-polylysine and hydrochloride thereof by using the genetically engineered bacterium as claimed in claim 1 or 2, which is characterized in that: the method realizes the improvement of the fermentation level of epsilon-polylysine through the fermentation of streptomyces diastatochromogenes dapE.
8. The method according to claim 7, wherein: the method comprises the following steps:
the adopted strain is a genetically engineered strain which is used for over-expressing succinyldiaminopimelate dissuccinylase gene dapE, namely amylase streptomyces chromogenes dapE, the genetically engineered strain is inoculated on a bennett culture medium plate and is cultured at 28-35 ℃ until black gray conidia are produced; then inoculating the spores into a shaking bottle of an M3G culture medium containing glucose, culturing for 30-33 hours at 28-35 ℃ and 180rpm/min, transferring the cultured seed culture solution into the M3G culture medium containing glucose for shaking bottle or fermentation tank fed-batch fermentation, and obtaining the epsilon-polylysine-containing fed-batch fermentation amino acid fermentation liquor.
9. The method according to claim 8, wherein: the feed fermentation comprises the following steps:
initial ph=6.8, control in two stages: in the stage I, the pH is controlled at 6.0 so as to be beneficial to the proliferation of thalli; step II, when the glucose concentration in the fermentation liquor is reduced to 10g/L, naturally reducing the pH to 4.0, then automatically feeding ammonia water with the mass concentration of 12.5% to maintain the pH to 4.0, and simultaneously feeding a mixed liquor of glucose and ammonium sulfate to control the glucose concentration to 10g/L; in the fermentation process, the temperature is controlled at 30 ℃, the dissolved oxygen is controlled at 30%, the ventilation ratio is maintained at 1-2vvm, and stirring is associated with the dissolved oxygen until the yield is highest.
10. The use of the genetically engineered bacterium of claim 1 or 2 in the production of epsilon-polylysine and its hydrochloride.
CN202310029098.2A 2023-01-09 2023-01-09 High-yield epsilon-polylysine and streptomyces hydrochloride genetically engineered bacterium as well as construction method and application thereof Pending CN116179375A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996041871A1 (en) * 1995-06-13 1996-12-27 Ajinomoto Co., Inc. Process for producing l-lysine by fermentation
US20040092710A1 (en) * 2000-05-04 2004-05-13 Madhavan Nampoothiri K. Nucleotide sequences coding for the cdsA gene
CN108504617A (en) * 2018-04-10 2018-09-07 江南大学 A kind of Escherichia coli recombinant strain and its construction method of high-yield L-lysine
US20210269842A1 (en) * 2018-10-05 2021-09-02 Ajinomoto Co., Inc. Method for Producing Target Substance by Bacterial Fermentation

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996041871A1 (en) * 1995-06-13 1996-12-27 Ajinomoto Co., Inc. Process for producing l-lysine by fermentation
US20040092710A1 (en) * 2000-05-04 2004-05-13 Madhavan Nampoothiri K. Nucleotide sequences coding for the cdsA gene
CN108504617A (en) * 2018-04-10 2018-09-07 江南大学 A kind of Escherichia coli recombinant strain and its construction method of high-yield L-lysine
US20210269842A1 (en) * 2018-10-05 2021-09-02 Ajinomoto Co., Inc. Method for Producing Target Substance by Bacterial Fermentation

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