CN118109383A - Recombinant bacterium for producing ectoin and construction method and application thereof - Google Patents

Recombinant bacterium for producing ectoin and construction method and application thereof Download PDF

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CN118109383A
CN118109383A CN202410508317.XA CN202410508317A CN118109383A CN 118109383 A CN118109383 A CN 118109383A CN 202410508317 A CN202410508317 A CN 202410508317A CN 118109383 A CN118109383 A CN 118109383A
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袁强
郭帅印
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Shandong Juntai Pharmaceutical Co ltd
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Abstract

The invention provides recombinant bacteria for producing ectoin, a construction method and application thereof, and belongs to the technical field of biology. According to the invention, E.coli K12 MG1655 is taken as an initial strain, the ectoin synthesis gene ectABC is directly integrated into the genome of the initial strain, and the ectoin synthesis related genes pk, ppc, pyc, aspC, aspA, gdh, ask (M68V) and asd are continuously integrated into a lysA, thrA, iclR, argA, proB knockout strain at specific sites, so that recombinant bacteria with high yield of ectoin are obtained, stable and efficient synthesis of ectoin is realized, and thus the actual production requirements are effectively met.

Description

Recombinant bacterium for producing ectoin and construction method and application thereof
Technical Field
The invention belongs to the technical field of biology, and relates to recombinant bacteria for producing ectoin, a construction method and application thereof.
Background
The statements in this section merely provide background information related to the present disclosure and may not necessarily constitute prior art.
The molecular weight of the ectoine, also known as tetrahydropyrimidine, is 142.16. Scientists in 1985 have found a microorganism that can survive under extreme conditions, the main reason for this being the ability to survive under high salt, high temperature and high radiation conditions is the ability to produce a characteristic substance, which is ectoin (tetrahydropyrimidine), as a protective agent. As a protective agent, the tetrahydropyrimidine can assist cells to maintain the osmotic pressure inside and outside the cells and improve the capacity of the cells to adapt to extreme environments, so that the tetrahydropyrimidine is widely applied to raw materials of cosmetics and has the effects of repairing skin and wounds.
The tetrahydropyrimidine can be produced by extreme microorganisms, but the process is harsh, the yield is extremely low, and meanwhile, the difficulty and the cost of chemical synthesis are seriously increased due to the chiral problem of the tetrahydropyrimidine. With the advent of synthetic biology technology, emerging technologies have allowed great play in the engineering and utilization of microorganisms. Patent CN201510410080.2 uses E.coli W3110 as starting bacteria, freely expresses ectABC genes from halomonas elongata, deletes genes lysA, thrA, iclR, simultaneously overexpresses lysC and ppc genes, uses glucose as a substrate for fermentation for 20-28 h, and the yield of tetrahydropyrimidine reaches 12-18 g, patent CN 116904416A uses escherichia coli BL21 as a host, and series of plasmids such as pET as vectors, and the like, and the L-diaminobutyrate aminotransferase (EctBT S/T49A/Y293M), the L-diaminobutyrate acetyltransferase (EctA) and the tetrahydropyrimidine synthase (EctC) are expressed in a heterogenous manner in cells, and an optimal expression vector and a gene combination mode are obtained through optimizing the copy number of plasmids, and the expression level of the three enzymes is regulated through different levels of RBS so as to further optimize the enzyme activity ratio of the three enzymes in the body. The optimal recombinant bacteria are used as biocatalysts for whole cell transformation, and the maximum yield of the tetrahydropyrimidine reaches 87.36g/L, etc. Although genetic engineering is reported to construct a strain for producing ectoin, no strain modified on genome is found at present, and the yield of the strain is low at present and cannot meet the actual production requirement.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a recombinant bacterium for producing ectoin, and a construction method and application thereof. Specifically, the invention takes escherichia coli as chassis bacteria, integrates the ectoin synthetic gene ectABC directly into the genome of the original strain, combines different integration sites, and realizes stable and efficient product synthesis. After the culture for 72 hours in a 5L fermentation tank, the yield of the ectoin can reach 142g/L. Based on the above results, the present invention has been completed.
Specifically, the technical scheme of the invention is as follows:
in a first aspect of the present invention, there is provided a recombinant bacterium for producing ectoin, which is obtained by overexpressing an ectoin synthesis-related gene and a glutamate dehydrogenase gene gdh in a chassis bacterium while knocking out construction of an ectoin synthesis-competitive pathway-related gene, a glutamate metabolism-related gene and an isocitrate lyase inhibitor gene iclR in the chassis bacterium;
Wherein the ectoin synthesis related genes comprise at least one of a pyruvate kinase synthesis gene pk, a phosphoenolpyruvate carboxylase synthesis gene ppc, a pyruvate carboxylase synthesis gene pyc, an aspartate aminotransferase synthesis gene aspC, an aspartase synthesis gene aspA, an aspartokinase synthesis gene ask (M68V), an aspartate semialdehyde dehydrogenase synthesis gene asd and a tetrahydropyrimidine synthesis enzyme gene cluster ectABC;
The gene related to the competitive pathway of the ectoin synthesis comprises at least one of lysine synthesis pathway gene lysA and threonine synthesis pathway gene thrA;
The glutamic acid metabolism related gene comprises at least one of arginine synthesis pathway gene argA and proline synthesis pathway gene proB;
Preferably, the nucleic acid sequence of the tetrahydropyrimidine synthase gene cluster ectABC is shown as SEQ ID NO. 1;
The nucleic acid sequence of the aspartokinase synthesis gene ask (M68V) is shown in SEQ ID NO. 2;
The nucleic acid sequence of the pyruvate kinase synthetic gene pk is shown as SEQ ID NO. 3;
The nucleic acid sequence of the phosphoenolpyruvate carboxylase synthetic gene ppc is shown in SEQ ID NO. 4;
the nucleic acid sequence of the pyruvic acid carboxylase synthetic gene pyc is shown in SEQ ID NO. 5;
The nucleic acid sequence of the asparatic acid aminotransferase synthetic gene aspC is shown in SEQ ID NO. 6;
The nucleic acid sequence of the aspartase synthesis gene aspA is shown in SEQ ID NO. 7;
the nucleic acid sequence of the aspartic semialdehyde dehydrogenase synthetic gene asd is shown in SEQ ID NO. 8;
the nucleic acid sequence of the glutamate dehydrogenase gene gdh is shown in SEQ ID NO. 9;
Wherein, the gene IDs of lysine synthesis pathway gene lysA, threonine synthesis pathway gene thrA, isocitrate lyase inhibitor gene iclR, arginine synthesis pathway gene argA and proline synthesis pathway gene proB are 947313, 945803, 948524, 947289 and 946425, respectively.
The chassis bacteria can be escherichia coli, and in a specific embodiment of the invention, the recombinant bacteria take escherichia coli E.coli K12 MG1655 as the chassis bacteria.
In a specific embodiment of the invention, the overexpressed gene in the recombinant bacterium is integrated into the genome of the chassis bacterium. In practice, there are many patent public Bucloduofactor high-producing strains, such as patent CN116904379A, CN106754603A, CN112961815A, etc. to express freely the gene or cluster ectABC of the gene, the exogenous gene is expressed in a free form in a carrier form, however, the separation instability of the carrier is easily caused in the fermentation screening-free process of the exogenous gene, so that the exogenous gene is lost, and the protein expression is reduced or stopped. The invention is modified on genome, which can avoid the defect of unstable expression of exogenous plasmid.
The gene insertion/integration effectively increases the copy number of gene expression and enhances the gene expression level, thereby enhancing the overall metabolic flow intensity and achieving the purpose of efficiently accumulating the final product. According to the selection of the escherichia coli gene integration site, the gene expression intensity is influenced by the escherichia coli gene integration site according to the document （Goormans A R, Snoeck N, Decadt H,et al. Comprehensive study onEscherichia coligenomic expression: Does position really matter? Metabolic engineering, 2020, 62:10-19.）, the effect that metabolic flux can smoothly flow to the final product is considered from small to large in ymgF_ycgH＜cspF_quuQ＜ybfC_ybfQ＜yeeJ_yeeL＜ykgA_ykgQ＜dadX_cvrA＜ypjC_ileY＜ileY_ygaQ＜ykgH_betA＜thrW_ykfN＜yjip_yjiR,, the gene expression level is enhanced from top to bottom in sequence when exogenous genes are integrated, and upstream intermediate products are catalyzed and synthesized to flow to the final product, i.e. the ectoin as much as possible. The inserted/integrated genes are uniformly distributed at each position, so that the transcription and translation burden of the microorganism can be effectively reduced, the stability of the whole genome structure of the microorganism is ensured, and the yield of the ectoin is improved.
In a specific embodiment of the invention, the over-expressed genes are sequentially integrated into different gene integration sites of the E.coli K12 MG1655 genome;
Specifically, the gene integration sites include yjip _yjir, dadx_cvra, cspf_quq, ypjc_iley, thrw_ykfn, iley_ygaq, ykga_ykgq, yeej_ yeeL, and ybfC _ ybfQ; and the over-expressed genes ectABC, aspC, gdh, aspA, asd, ask (M68V), pyc, ppc, pk are sequentially integrated into the gene integration sites yjip _yjiR, dadX_cvrA, cspF_quQ, ypjC_ileY, thrW_ykfN, ileY_ygaQ, ykgA_ykgQ, yeeJ_ yeeL and ybfC _ ybfQ. The gene integration mode can be carried out by adopting crispr-cas9 editing technology and/or lambda-Red homologous recombination technology.
The recombinant strain obtained by the invention is constructed by regulating and controlling related genes on an exendin synthesis path (figure 1) through a genetic engineering means to obtain the recombinant strain with high exendin yield. The recombinant strain takes glucose as a substrate, efficiently synthesizes the exendin, and has the highest yield of 142g/L in a 5L fermentation tank, which is obviously higher than the yield of other existing engineering strains producing the exendin.
Thus, in a second aspect of the invention, there is provided the use of a recombinant bacterium as described above for the production of ectoin.
In a third aspect of the present invention, there is provided a method for producing exendin, comprising fermenting and culturing the recombinant bacterium to express exendin, and isolating and purifying the exendin. Specifically, the culture medium used for the fermentation culture strain contains glucose, and can be LB culture medium or YEB culture medium.
In some embodiments, the specific methods of fermentation culture comprise:
Inoculating the recombinant bacteria into 80-150mL of liquid LB culture medium, culturing overnight at 37 ℃ with a 220r shaking table to obtain seed liquid, and transferring the seed liquid into a fermentation tank containing a fermentation culture medium according to 2% of inoculum size; fermenting at 36-38deg.C and 350-500 r; when the fermentation OD reaches about 8-10, adjusting ventilation capacity, tank pressure or rotating speed to control dissolved oxygen to about 35%, measuring the glucose concentration of the fermentation liquid, wherein the glucose concentration is lower than 3g/L, adding a feed culture medium, and keeping the glucose concentration at 1-3 g/L;
Preferably, the fermentation medium contains 6 g/L KH2PO4, 16.4 g/L K2HPO4, 5 g/L(NH4)2SO4, 1.1 g/L citric acid, 1 g/L MgSO 4, 10 g/L yeast powder, 7 g/L glucose, 0.1 g/L vitamin B1, 2 mL/L trace elements;
The feed medium contains 500g/L glucose, 7g/L MgSO 4 and 1mL/L trace elements;
Optionally, the trace element comprises 10 g/L FeSO4·7H2O, 1.53 g/L CaCl2, 2.2 g/L ZnSO4·7H2O, 1g MnSO4·4H2O, 1 g/L CuSO4·5H2O, 0.1 g/L (NH4)6Mo7O24·4H2O, 0.2 g/L Na2B4O7·10H2O, 1g/L NiCl2, 1g/L H3BO3, 10 mL/L HCl,HCl% concentration of 37%.
When the fermentation medium is prepared, firstly, trace elements are dissolved in hydrochloric acid, and then deionized water is added to fix the volume to adjust the pH to 7.0.
In a fourth aspect of the present invention, there is also provided a construction method for producing recombinant escin, the construction method comprising: coli e.coli k12 MG1655 was used as a chassis fungus, which was transformed as follows:
a1 Knocking out at least one of lysine synthesis pathway gene lysA, threonine synthesis pathway gene thrA, isocitrate lyase inhibitor gene iclR, arginine synthesis pathway gene argA and proline synthesis pathway gene proB in chassis bacteria;
a2 Integration of at least one of the exogenous gene pyruvate kinase synthesis gene pk, phosphoenolpyruvate carboxylase synthesis gene ppc, pyruvate carboxylase synthesis gene pyc, aspartate aminotransferase synthesis gene aspC, aspartase synthesis gene aspA, aspartokinase synthesis gene ask (M68V), aspartate semialdehyde dehydrogenase synthesis gene asd, tetrahydropyrimidine synthesis gene cluster ectABC.
Wherein the nucleic acid sequence of the tetrahydropyrimidine synthase gene cluster ectABC is shown as SEQ ID NO. 1;
The nucleic acid sequence of the aspartokinase synthesis gene ask (M68V) is shown in SEQ ID NO. 2;
The nucleic acid sequence of the pyruvate kinase synthetic gene pk is shown as SEQ ID NO. 3;
The nucleic acid sequence of the phosphoenolpyruvate carboxylase synthetic gene ppc is shown in SEQ ID NO. 4;
the nucleic acid sequence of the pyruvic acid carboxylase synthetic gene pyc is shown in SEQ ID NO. 5;
The nucleic acid sequence of the asparatic acid aminotransferase synthetic gene aspC is shown in SEQ ID NO. 6;
The nucleic acid sequence of the aspartase synthesis gene aspA is shown in SEQ ID NO. 7;
the nucleic acid sequence of the aspartic semialdehyde dehydrogenase synthetic gene asd is shown in SEQ ID NO. 8;
the nucleic acid sequence of the glutamate dehydrogenase gene gdh is shown in SEQ ID NO. 9;
Wherein, the gene IDs of the lysine synthesis pathway gene lysA, threonine synthesis pathway gene thrA, isocitrate lyase inhibitor gene iclR, arginine synthesis pathway gene argA and proline synthesis pathway gene proB are 947313, 945803, 948524, 947289 and 946425 respectively.
Specifically, the invention takes E.coli K12 MG1655 as chassis bacteria, knocks out endogenous iclR gene to obtain strain EcMG1655-001, delta iclR;
Knocking out an endogenous gene argA on the basis of EcMG1655-001 of DeltaiclR to obtain a strain EcMG1655-002 of DeltaiclR and DeltaargA;
Knocking out endogenous gene proB to obtain strains EcMG1655-003 based on EcMG1655-002 DeltaiclR, deltaargA and DeltaproB;
Knocking out endogenous gene lysA on the basis of EcMG1655-003 of DeltaiclR, deltaargA and DeltaproB to obtain strains EcMG1655-004 of DeltaiclR, deltaargA, deltaproB and DeltalysA;
Knocking out an endogenous gene thrA on the basis of EcMG1655-004, ΔiclR, ΔargA, ΔproB and ΔlysA to obtain strains EcMG1655-005, wherein ΔiclR, ΔargA, ΔproB, ΔlysA and ΔthrA;
Strains EcMG1655-006 were obtained by inserting/integrating a partial sequence of the gene cluster of foreign gene ectABC based on ΔiclR, ΔargA, ΔproB, ΔlysA, ΔthrA, ectABC/yjip _ yjiR;
Inserting/integrating exogenous gene aspC on the basis of DeltaiclR, deltaargA, deltaproB, deltalysA, deltathrA, ectABC/yjip _ yjiR to obtain strain EcMG1655-007, deltaiclR, deltaargA, deltaproB, deltalysA, deltathrA, ectABC/yjip _ yjiR, aspC/dadX _ cvrA;
Inserting/integrating exogenous gene gdh on the basis of EcMG 1655:1655-007, ΔiclR, ΔargA, ΔproB, ΔlysA, ΔthrA, ectABC/yjip _ yjiR, aspC/dadX _ cvrA to obtain strain EcMG 1655:1655-008, ΔiclR, ΔargA, ΔproB, ΔlysA, ΔthrA, ectABC/yjip _ yjiR, aspC/dadX _ cvrA, gdh/cspF _ quuQ;
Inserting/integrating exogenous gene aspA based on EcMG 1655:1655-008 [ delta ] iclR, delta argA, delta proB, delta lysA, delta thrA, ectABC/yjip _ yjiR, aspC/dadX _ cvrA, gdh/cspF _ quuQ to obtain strain EcMG1655-009: ΔiclR, ΔargA, ΔproB, ΔlysA, ΔthrA, ectABC/yjip_yjiR, aspC/dadX_cvrA, gdh/cspF_quuQ, aspA/ypjC_ileY;
Inserting/integrating exogenous gene asd based on EcMG1655-009: ΔiclR,ΔargA,ΔproB,ΔlysA,ΔthrA, ectABC/yjip_yjiR, aspC/dadX_cvrA, gdh/cspF_quuQ, aspA/ypjC_ileY to obtain strain EcMG1655-010: ΔiclR, ΔargA, ΔproB, ΔlysA, ΔthrA, ectABC/yjip_yjiR, aspC/dadX_cvrA, gdh/cspF_quuQ, aspA/ypjC_ileY, asd/thrW_ykfN;
Inserting/integrating exogenous gene ask (M68V) based on EcMG1655-010: ΔiclR, ΔargA, ΔproB, ΔlysA, ΔthrA, ectABC/yjip_yjiR, aspC/dadX_cvrA, gdh/cspF_quuQ, aspA/ypjC_ileY, asd/thrW_ykfN to obtain strain EcMG1655-011: ΔiclR, ΔargA, ΔproB, ΔlysA, ΔthrA, ectABC/yjip_yjiR, aspC/dadX_cvrA, gdh/cspF_quuQ, aspA/ypjC_ileY, asd/thrW_ykfN, ask(M68V)/ileY_ygaQ;
Inserting/integrating exogenous gene pyc based on EcMG1655-011: ΔiclR, ΔargA, ΔproB, ΔlysA, ΔthrA, ectABC/yjip_yjiR, aspC/dadX_cvrA, gdh/cspF_quuQ, aspA/ypjC_ileY, asd/thrW_ykfN, ask(M68V)/ileY_ygaQ to obtain strain EcMG1655-012: ΔiclR, ΔargA, ΔproB, ΔlysA, ΔthrA, ectABC/yjip_yjiR, aspC/dadX_cvrA, gdh/cspF_quuQ, aspA/ypjC_ileY, asd/thrW_ykfN, ask(M68V)/ileY_ygaQ, pyc/ykgA_ykgQ;
Inserting/integrating exogenous gene ppc based on EcMG1655-012: ΔiclR, ΔargA, ΔproB, ΔlysA, ΔthrA, ectABC/yjip_yjiR, aspC/dadX_cvrA, gdh/cspF_quuQ, aspA/ypjC_ileY, asd/thrW_ykfN, ask(M68V)/ileY_ygaQ, pyc/ykgA_ykgQ to obtain strain EcMG1655-013: ΔiclR, ΔargA, ΔproB, ΔlysA, ΔthrA, ectABC/yjip_yjiR, aspC/dadX_cvrA, gdh/cspF_quuQ, aspA/ypjC_ileY, asd/thrW_ykfN, ask(M68V)/ileY_ygaQ, pyc/ykgA_ykgQ, ppc/yeeJ_yeeL;
Inserting/integrating exogenous gene pk based on EcMG1655-013: ΔiclR,ΔargA,ΔproB,ΔlysA,ΔthrA,ectABC/yjip_yjiR,aspC/dadX_cvrA, gdh/cspF_quuQ, aspA/ypjC_ileY, asd/thrW_ykfN, ask(M68V)/ileY_ygaQ, pyc/ykgA_ykgQ, ppc/yeeJ_yeeL to obtain strain EcMG1655-014: ΔiclR, ΔargA, ΔproB, ΔlysA, ΔthrA, ectABC/yjip_yjiR, aspC/dadX_cvrA, gdh/cspF_quuQ, aspA/ypjC_ileY, asd/thrW_ykfN, ask(M68V)/ileY_ygaQ, pyc/ykgA_ykgQ, ppc/yeeJ_yeeL, pk/ybfC_ybfQ.
The one or more of the above technical solutions have the following beneficial effects:
According to the technical scheme, E.coliK12 MG1655 is taken as chassis fungus, the ectoin synthetic gene ectABC is directly integrated into the genome of the original strain, and different integration sites are combined, so that stable and efficient product synthesis is realized. After being cultured for 48-72 hours in a 5L fermentation tank, the maximum yield of the ectoin can reach 142g/L, and the method has good practical application value.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a schematic diagram of the metabolic pathways of recombinant bacteria producing ectoin according to the present invention.
FIG. 2 is a schematic diagram of the principle of gene knockout in example 1 of the present invention.
FIG. 3 is a schematic diagram showing the principle of gene insertion/integration in example 2 of the present invention.
FIG. 4 is a liquid chromatogram of example 3 of the present invention.
FIG. 5 is a graph showing statistics of yields of exendin in example 3 of the present invention.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
It should be noted that the present invention is not exhaustive and is well known to those skilled in the art. The experimental procedures described in detail below, without specifying specific conditions, are generally described in accordance with conventional conditions or according to the conditions recommended by the manufacturer, with reference to the "molecular cloning Experimental guidelines" (m.r. Green), (j. Sambrook (Joseph Sambrook) principal, fourth edition), pathophysiological experiments, online databases, etc.
In the following examples, materials, reagents and the like used were obtained from commercial sources unless otherwise specified.
The construction method of the ectoin engineering strain EcMG1655-014: ΔiclR,ΔargA,ΔproB,ΔlysA,ΔthrA,ectABC/yjip_yjiR,aspC/dadX_cvrA, gdh/cspF_quuQ, aspA/ypjC_ileY, asd/thrW_ykfN, ask(M68V)/ileY_ygaQ,pyc/ykgA_ykgQ, ppc/yeeJ_yeeL, PK/ybfC_ybfQ is briefly described as follows:
According to the invention, an engineering strain takes escherichia coli MG1655 as an initial strain, a crispr-cas9 combined lambda-Red recombination technology is adopted, strain transformation is carried out by referring to genome GenBank: NC_000913 and metabolic pathways, and genes lysA, thrA, iclR, argA, proB are respectively knocked out (a specific method can refer to doi: 10.1093/abbs/gmab 036); the gene clusters of the genes pk, ppc, pyc, aspC, aspA, gdh, ask (M68V), asd, ectABC were then integrated into the E.coli MG1655 genome, respectively, and the insertion/integration site selection reference (doi: 10.1016/j.ymben.2020.07.007) was used, for example, as described in reference (doi: 10.1093/abbs/gmab 036). The metabolic pathways of the obtained engineering strain are shown in figure 1.
In the present invention, the genbank number of the target gene and detailed information are shown in Table 1.
Table 1: target gene and information thereof
And (5) continuing the table:
Our references (Ye, j.w., & Chen, g.q. (2021)) integrate exogenous genes into the corresponding sites, as in the knockout principle, we prepared overlapping extension PCR products carrying exogenous genes, and then gene editing by CRISPR/Cas 9. The reference has 11 sites of integration without great influence on the growth of microorganisms, the gene expression intensity is ymgF_ycgH,cspF_quuQ,ybfC_ybfQ,yeeJ_yeeL,ykgA_ykgQ,dadX_cvrA,ypjC_ileY,ileY_ygaQ,ykgH_betA,thrW_ykfN,yjip_yjiR, from small to large, genes are sequentially integrated into corresponding sites according to the tetrahydropyrimidine anabolic flow, the gene pk is integrated into ybfC-ybfQ, the gene PPC is integrated into yeeJ-yeeL, the gene pyc is integrated into ykgA-ykgQ, the gene aspC is integrated into dadX-cvrA, the gene aspA is integrated into ypjC-ileY, the gene ask (M68V) is integrated into ileY-ygaQ, the gene aspC is integrated into ykgH-betA, the gene asd is integrated into thrW-ykfN, the gene ectABC is integrated into yjip-yjiR and the gene gdh is integrated into cspF-quuQ, and thus the gene recombinant engineering bacteria for producing the tetrahydropyrimidine are constructed.
Principle of knocking out or inserting/integrating expressed genes in the present invention:
The pEcgRNA plasmid with the complementary sequence of the cas9 cutting target spot, pEcCas plasmid with the cas9 system and the lambda RED system and Donor DNA (the upstream and downstream homologous arms of the knocked-out gene, the inserted gene and the upstream and downstream homologous arms thereof) are transformed into a host strain together, and under the guide action of sgRNA, cas9 plays a cutting function, and the Donor DNA is recombined into the genome of the host strain by means of the lambda RED system to obtain the target strain. Therefore, the efficiency of homologous recombination can be improved, the off-target effect can be avoided, and the target strain can be obtained efficiently. Schematic diagrams of the knockout principle and the insertion principle are shown in fig. 2 and 3, respectively.
In the invention, plasmids used for gene editing are purchased, and specific function and sequence information can be directly obtained through addgene websites: pEcgRNA (Addgene number: 166581), pEcCas (Addgene number: 73227), methods of use reference articles Li, Q., Sun, B., Chen, J., Zhang, Y., Jiang, Y. U.,&Yang, S. (2021). A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing inEscherichia coli.Acta Biochimica et Biophysica Sinica, 53(5), 620-627.
The preparation method of competent cells of the escherichia coli and the plasmid transformation method refer to experimental five in the fourteenth five planning life science innovative characteristic teaching material molecular biology experiment 2 nd edition of common university: preparation and transformation of E.coli competent cells, "authors: li Feng, chen Zixuan.
In the present invention, the strain obtained after one of the genes is knocked out or inserted/integrated is used as the starting strain of the next gene knocked out/inserted gene bacterium, and the corresponding plasmids are transferred into the specific strain and the corresponding relationship of the plasmids are shown in the following table 2.
Table 2: corresponding relation of strain and plasmid transformation
And (5) continuing the table:
And (5) continuing the table:
And (5) continuing the table:
And (5) continuing the table:
The invention will now be described in further detail with reference to the following specific examples, which should be construed as illustrative rather than limiting.
Example 1
EcMG 1655-001. DELTA.iclR Strain construction method:
1. Gene knockout is performed by using CRISPR/Cas9 gene editing technology, and target PAM is designed through an online website CHOPCHOP. Reference genome Escherichia coli K-12 substr MG1655, accession Number NC-000913.
The knockdown gene and corresponding target sequences are shown in Table 3 below, and the sgRNA primer sequences are shown in Table 4.
Table 3: knockout gene and target sequence thereof
Table 4: target-corresponding sgRNA primer sequence
2. Obtaining Donor DNA fragments
The homologous arm upstream and downstream primers of the knockout gene iclR were designed by NCBI using the MG1655 genome as a template.
Wherein, the upstream homology arm up-iclR-F: gacgaccattttgtctacagttctctgag (SEQ ID NO.25)、up-iclR-R: ttctggcgggcagaggcaatattctgcccatcatac (SEQ ID NO.26); and the downstream homology arm primer ：dw-iclR-F: gaatattgcctctgcccgccagaaaaaggacagtctcttttttctgtatcgtg (SEQ ID NO.27)、dw-iclR-R: ggtgttggtttcgatgatatccgcgc (SEQ ID NO.28).
The reference genome GenBank NC-000913 primer design was performed, and the E.coli MG1655 genome was used as a template, and PCR was performed with the primers up-iclR-F/up-iclR-R and dw-iclR-R, respectively, using KOD-Plus-Neo as the polymerase, and the PCR program was 3min pre-denatured at 95 ℃,10 s annealed at 58 ℃, 1min extended at 68 ℃, 5min extended at 68 ℃,10 s denatured at 95 ℃,10 s annealed at 58 ℃, 1min extended at 68℃and 25 cycles were performed. Then, the product recovery was performed by the purification kit to perform the next overlap extension PCR, 100ng of each purified product was mixed and added to the polymerase, the PCR procedure was 95℃pre-denatured for 3min,95℃denatured for 10s, 58℃annealed for 10s,68℃extended for 1min, 68℃final extended for 5min,95℃denatured for 10s, 58℃annealed for 10s,68℃extended for 1min, and then the product recovery was performed by the purification kit to perform 10 cycles as a Donor DNA-iclR for use.
3. Construction of knockout plasmid pEc-sgiclR
The SgRNA primer in Table 4 was used to carry out whole plasmid PCR using pEcgRNA plasmid as a template and KOD-Plus-Neo (KOD-401) as a polymerase, the PCR procedure was performed for 3min at 95℃for 10s for denaturation at 95℃for 10s for annealing at 58℃for 10s for 1min for extension at 68℃for 5min for final extension at 68℃for 10s for denaturation at 95℃for 10s for annealing at 58℃for 3min for 25 cycles, then the template was removed by digestion with nuclease DpnI, the product was recovered by a purification kit, and the PCR product was transformed into cloned strain DH 5. Alpha. To obtain recombinant plasmids such as pEc-sgiclR after sequencing.
Wherein, ADDGENE ID: 73227 of pEcCas plasmid contains CRISPR-Cas9 expression frame and lambda RED expression frame. ADDGENE ID:166581 of pEcgRNA plasmid, which can insert and express sgRNA.
4. Obtaining of knockout Strain EcMG 1655-001. Delta. IclR
Mixing the 50ng pEcCas plasmid, the 50ng pEcCas-sgiclR plasmid and 100ng Donor DNA-iclR, converting into E.coliK12 MG1655 competent strain by an electrotransformation method, wherein the strain is derived from Shanghai Weidi organism DL2030S, regulating an electrotransformation instrument, setting the parameters to be 2.5 kV voltage and 200 omega resistance, immediately adding an SOC recovery culture medium to incubate for 1h at 37 ℃ after electric shock, coating a kanamycin and spectinomycin double-antibody LB solid plate, culturing overnight at 37 ℃ in a constant temperature box, and identifying positive clone by colony PCR.
Colony PCR primers and sequences are shown in Table 5.
Table 5: PCR identification primer for positive clone colony of knocked-out genetically engineered bacterium
5. The obtained positive monoclonal strain is activated to prepare competent strain which is used as the starting strain of the next knockout gene.
By adopting the method, strains EcMG-001 for knocking out the iclR are obtained in sequence, wherein the ΔiclR is the strain; strain EcMG-002 with iclR+argA knocked out, ΔiclR, ΔargA; strains EcMG-003 of which iclR+argA+proB are knocked out, wherein the strains comprise DeltaiclR, deltaargA and DeltaproB; strains EcMG-004, ΔiclR, ΔargA, Δprob, ΔlysA, were deleted from iclR+argA+prob+lysA; the strain EcMG-005 in which iclR, deltaargA, deltaproB, deltalysA and DeltathrA were deleted was selected.
Wherein the Donor DNA primers are as follows:
up-argA-F: gggttactcaacgcgaactgcttata，SEQ ID NO.39；
up-argA-R: atcgcggcacacctctttgcatgattattcgaaattagtgt，SEQ ID NO.40；
dw-argA-F: gcaaagaggtgtgccgcgatgaaaatcgtcggatgcgacatgcgtaac，SEQ ID NO.41；
dw-argA-R: gtacgaaggtcgaccggtgatgattgc，SEQ ID NO.42；
up-proB-F: tcagttgatcgttaatttgtgtttc，SEQ ID NO.43；
up-proB-R: ctgctccgattctctgccattcaattttaggaaaaatgatatc，SEQ ID NO.44；
dw-proB-F: gaatggcagagaatcggagcaggctgatgctggaacaaatg，SEQ ID NO.45；
dw-proB-R: ggatcaccgcattaccggttttcaggc，SEQ ID NO.46；
up-lysA-F: tctatttctgacgccgaagatcgtctgc，SEQ ID NO.47；
up-lysA-R: gagtttgttctgcggttagtcgctggttgcatgatgacttgcctccag，SEQ ID NO.48；
dw-lysA-F: gcgactaaccgcagaacaaactccagataagtgcttttttatg，SEQ ID NO.49；
dw-lysA-R: gggataacgtgccagaaagggttg，SEQ ID NO.50；
up-thrA-F: aatatgtctctgtgtggattaaaaaaag，SEQ ID NO.51；
up-thrA-R: accatgggttgttacctcgttacctttggtcgaaaaaaaaagc，SEQ ID NO.52；
dw-thrA-F: gtaacgaggtaacaacccatggttaaagtttatgccccggcttc，SEQ ID NO.53；
dw-thrA-R: gtgccacgttgtcgtaatgaatgctgc，SEQ ID NO.54。
Example 2
EcMG1655-006 ΔiclR, ΔargA, ΔproB, ΔlysA, ΔthrA, ectABC/yjip _ yjiR strain obtaining methods:
1. Amplification of Donor DNA
The genes were constructed into the vector pETet a-1 between the multiple cloning sites NdeI and XhoI by Huada genes according to the gene numbers in Table 1, respectively, and the corresponding vector was designated as pETet28a- PK,pETet28a- PPC,pETet28a- pyc,pETet28a- aspC,pETet28a- aspA,pETet28a- ask(M68V),pETet28a- asd,pETet28a- ectABC,pETet28a- gdh.
The reference genome GenBank: NC-000913 primer design was performed using the E.coli K12 MG1655 genome as a template, the primers up-PK-F/up-PK-R and dw-PK-F/dw-PK-R were used for PCR, the corresponding vector pETet a-PK was used as a template, and the primer it-PK-F/it-PK-R was used for PCR. The polymerase used was KOD-Plus-Neo, and 25 cycles of the PCR procedure were performed for 3min of pre-denaturation at 95 ℃,10 s annealing at 58 ℃, 1min extension at 68 ℃, 5min final extension at 68 ℃,10 s denaturation at 95 ℃,10 s annealing at 58 ℃, 1min extension at 68 ℃. Then, the product was recovered by a purification kit to perform the next overlap extension PCR, 100ng of each purified product was mixed and added to the polymerase, the PCR procedure was 95℃for 3min,95℃for 10s, 58℃for 10s,68℃for 1min, 68℃for 5min,95℃for 10s, 58℃for 10s,68℃for 1min, and then 10 cycles were performed by a purification kit to recover the product as Donor DNA-PK (upstream homology arm-insert/integrate gene fragment-downstream homology arm) for use.
According to this method, the primer sequences used for Donor DNA-ppc、Donor DNA-pyc、Donor DNA-aspC、Donor DNA-aspA、Donor DNA-ask(M68V) 、Donor DNA-asd、Donor DNA-ectABC、Donor DNA-gdh, were obtained sequentially as follows:
up-PK-F: gtgttatagctgattgcgaaatacaagc，SEQ ID NO.55；
up-PK-R: caattcccctatagtgagtcgtattatcaattgttgaacttaaaattcgaattatttag，SEQ ID NO.56；
it-PK-F: taatacgactcactataggggaattg，SEQ ID NO.57；
it-PK-R: cattcgccaatccggatatagttc，SEQ ID NO.58；
dw-PK-F: gaactatatccggattggcgaatggatactacttattgtctaaagctgtttttc，SEQ ID NO.59；
dw-PK-R: cggcaaaaatagtcaatagtaggatgcctgac，SEQ ID NO.60；
up-PPC-F: gccccaggaaaaacggcggctg，SEQ ID NO.61；
up-PPC-R: caattcccctatagtgagtcgtattaacatcgaacgcgattctggcggtg，SEQ ID NO.62；
it-PPC-F: taatacgactcactataggggaattg，SEQ ID NO.63；
it-PPC-R: cattcgccaatccggatatagttc，SEQ ID NO.64；
dw-PPC-F: gaactatatccggattggcgaatggactatgatcattttcggtgagtactg，SEQ ID NO.65；
dw-PPC-R: cgtgaggcgggttttccagcagtcaggc，SEQ ID NO.66；
up-pyc-F: gatatccgtcttaaaattcttcatac，SEQ ID NO.67；
up-pyc-R: caattcccctatagtgagtcgtattataataatgtaggactgaaacctctc，SEQ ID NO.68；
it-pyc-F: taatacgactcactataggggaattg，SEQ ID NO.69；
it-pyc-R: cattcgccaatccggatatagttc，SEQ ID NO.70；
dw-pyc-F: gaactatatccggattggcgaatgatgaattttttgcataagtgata，SEQ ID NO.71；
dw-pyc-R: ccgctggtaacactctcatttttaatc，SEQ ID NO.72；
up-aspC-F: cagagccaggcaaatggcagcag，SEQ ID NO.73；
up-aspC-R: caattcccctatagtgagtcgtattacagtattcatgatgcgggctttttgc，SEQ ID NO.74；
it-aspC-F: taatacgactcactataggggaattg，SEQ ID NO.75；
it-aspC-R: cattcgccaatccggatatagttc，SEQ ID NO.76；
dw-aspC-F: gaactatatccggattggcgaatggatatgaagcatgagagttacctc，SEQ ID NO.77；
dw-aspC-R: gaaagagcacggtttccagtgccac，SEQ ID NO.78；
up-aspA-F: ttaattgttctccagctcccataatg，SEQ ID NO.79；
up-aspA-R: caattcccctatagtgagtcgtattagctcactgatgataagtgagtac，SEQ ID NO.80；
it-aspA-F: taatacgactcactataggggaattg，SEQ ID NO.81；
it-aspA-R: cattcgccaatccggatatagttc，SEQ ID NO.82；
dw-aspA-F: gaactatatccggattggcgaatggaacagagaatagttcagtgatttg，SEQ ID NO.83；
dw-aspA-R: gcgaaaactcttcagctaatgtaataaag，SEQ ID NO.84；
up-ask(M68V)-F: gtaagcaatttgcccgcttggc，SEQ ID NO.85；
up-ask(M68V)-R: caattcccctatagtgagtcgtattattatattattagatacaaaatc，SEQ ID NO.86；
it-ask(M68V)-F: taatacgactcactataggggaattg，SEQ ID NO.87；
it-ask(M68V)-R: cattcgccaatccggatatagttc，SEQ ID NO.88；
dw-ask(M68V)-F: gaactatatccggattggcgaatgctttattacattagctgaagagttttc，SEQ ID NO.89；
dw-ask(M68V)-R: gtatctcgttgataaacctatg，SEQ ID NO.90；
up-asd-F: catattcgtgaacacggcacacaac，SEQ ID NO.91；
up-asd-R: caattcccctatagtgagtcgtattaagtagataagattgatcttcgttg，SEQ ID NO.92；
it-asd-F: taatacgactcactataggggaattg，SEQ ID NO.93；
it-asd-R: cattcgccaatccggatatagttc，SEQ ID NO.94；
dw-asd-F: gaactatatccggattggcgaatggaccaaaaaggcctgatagggcttcg，SEQ ID NO.95；
dw-asd-R: ctgacacgtctgctggaacagcac，SEQ ID NO.96；
up-ectABC-F: gttggagttgatccaaaagcatattc，SEQ ID NO.97；
up-ectABC-R: caattcccctatagtgagtcgtattacatcgccacgttccagcctgaattaag，SEQ ID NO.98；
it-ectABC-F: taatacgactcactataggggaattg，SEQ ID NO.99；
it-ectABC-R: cattcgccaatccggatatagttc，SEQ ID NO.100；
dw-ectABC-F: gaactatatccggattggcgaatgcagcaaagataaaacgagtcac，SEQ ID NO.101；
dw-ectABC-R: cagtaaaagtattgcaccaggcctg，SEQ ID NO.102；
up-gdh-F: ctttcttcaggaacgtgtgtatag，SEQ ID NO.103；
up-gdh-R: caattcccctatagtgagtcgtattatcatcccgggactcatgtctgttaac，SEQ ID NO.104；
it-gdh-F: taatacgactcactataggggaattg，SEQ ID NO.105；
it-gdh-R: cattcgccaatccggatatagttc，SEQ ID NO.106；
dw-gdh-F: gaactatatccggattggcgaatggattcaacattaccattgccccatttaaag，SEQ ID NO.107；
dw-gdh-R: gtattgcgcgaagtggtgaaacactac，SEQ ID NO.108。
2. design target and sgRNA sequence
Gene knockout is performed by using CRISPR/Cas9 gene editing technology, and target PAM is designed through an online website CHOPCHOP. Reference genome Escherichia coli K-12 substr MG1655, accession Number NC-000913.
The knockdown gene and corresponding target sequences are shown in Table 6 below, and the sgRNA primer sequences are shown in Table 7.
Table 6: target sequence and insertion site of insertion/integration gene
Table 7: insertion/integration of gene-corresponding sgRNA sequences
And (5) continuing the table:
3. construction of an insertion/integration plasmid
The SgRNA primer in Table 7 was used to carry out whole plasmid PCR using pEcgRNA plasmid as a template and KOD-Plus-Neo (KOD-401) as a polymerase, the PCR procedure was performed for 3min at 95℃for 10s for denaturation at 95℃for 10s for annealing at 58℃for 10s for 1min for extension at 68℃for 5min for final extension at 68℃for 10s for denaturation at 95℃for 10s for annealing at 58℃for 3min for 25 cycles, then the template was removed by digestion with nuclease DpnI, the product was recovered by a purification kit, and the PCR product was transformed into cloned strain DH 5. Alpha. To obtain recombinant plasmids such as pEc-sgpk after sequencing.
4. Genome insertion/integration of exogenous genes
Mixing the 50ng pEcCas plasmid, 50ng pEc-sgpk plasmid and 100ng Donor DNA-PK, converting the mixture into E.coliK12 MG1655 competent strain by an electrotransformation method, regulating an electrotransformation instrument to set the parameters to be 2.5 kV of voltage and 200 omega of resistance, immediately adding an SOC recovery culture medium to incubate for 1h at 37 ℃ after electric shock, coating a kanamycin and spectinomycin double-resistant LB solid plate, culturing overnight at 37 ℃ in a constant temperature box, and identifying positive clone by colony PCR and then preserving bacteria. Colony PCR primers and sequences are shown in Table 8.
Table 8: PCR identification primer for inserting/integrating positive clone colony of genetically engineered bacterium
And (5) continuing the table:
And (5) continuing the table:
And (5) continuing the table:
By the method, strains EcMG1655-006 with the functions of knockout of iclR, argA, proB, lysA and thrA and integration of over-expression ectABC are obtained in sequence, wherein the strains comprise DeltaiclR, deltaargA, deltaproB, deltalysA, deltathrA and ectABC/yjip _ yjiR; the strain EcMG1655-007 in which iclR, deltaargA, deltaproB, deltalysA, deltathrA, ectABC/yjip _ yjiR and aspC/dadX _ cvrA are knocked out and ectABC +aspC are integrated; the strain EcMG1655-008 which knocks out iclR+argA+proB+lysA+thrA and integrates ectABC +aspC+gdh is ΔiclR, ΔargA, ΔproB, ΔlysA, ΔthrA, ectABC/yjip _ yjiR, aspC/dadX _ cvrA, gdh/cspF _ quuQ; knocking out iclr+arga+prob+lysa+thra and integrating strain EcMG1655-009: ΔiclR,ΔargA,ΔproB,ΔlysA,ΔthrA, ectABC/yjip_yjiR,aspC/dadX_cvrA, gdh/cspF_quuQ,aspA/ypjC_ileY; which overexpresses ectABC +aspc+gdh+aspa, knocking out iclr+arga+prob+lysa+thra and knocking out iclr+arga+proba+prob+lysa+thra by integrating strain EcMG1655-010: ΔiclR,ΔargA,ΔproB,ΔlysA,ΔthrA, ectABC/yjip_yjiR,aspC/dadX_cvrA, gdh/cspF_quuQ,aspA/ypjC_ileY, asd/thrW_ykfN; which overexpresses ectABC +aspc+gdh+aspa+asd and knocking out iclr+arga+prob+lysa+thra by integrating strain EcMG1655-011: ΔiclR,ΔargA,ΔproB,ΔlysA,ΔthrA,ectABC/yjip_yjiR,aspC/dadX_cvrA, gdh/cspF_quuQ, aspA/ypjC_ileY, asd/thrW_ykfN, ask(M68V)/ileY_ygaQ; which overexpresses ectABC +aspc+gdh+aspa+asd+ask (M68V) and the iclR+argA+proB+lysA+thrA was knocked out by integrating strain EcMG1655-012: ΔiclR,ΔargA,ΔproB,ΔlysA,ΔthrA,ectABC/yjip_yjiR,aspC/dadX_cvrA, gdh/cspF_quuQ,aspA/ypjC_ileY, asd/thrW_ykfN, ask(M68V)/ileY_ygaQ, pyc/ykgA_ykgQ; overexpressing ectABC +aspC+gdh+aspA+asd+ask (M68V) +pyc and the iclR+argA+proB+lysA+lysA+thrA was knocked out by integrating strain EcMG1655-013: ΔiclR,ΔargA,ΔproB,ΔlysA,ΔthrA,ectABC/yjip_yjiR,aspC/dadX_cvrA, gdh/cspF_quuQ,aspA/ypjC_ileY, asd/thrW_ykfN, ask(M68V)/ileY_ygaQ,pyc/ykgA_ykgQ, ppc/yeeJ_yeeL; overexpressing ectABC +aspC+gdh+aspA+asd+ask (M68V) +pyc+ppc and the ectABC +aspC+gdh+aspA+asd+ask (M68V) +pyc+ppc+pk was knocked out by integrating strain EcMG1655-014: ΔiclR,ΔargA,ΔproB,ΔlysA,ΔthrA,ectABC/yjip_yjiR,aspC/dadX_cvrA, gdh/cspF_quuQ, aspA/ypjC_ileY, asd/thrW_ykfN, ask(M68V)/ileY_ygaQ, pyc/ykgA_ykgQ, ppc/yeeJ_yeeL, pk/ybfC_ybfQ.
Example 3
The method for producing the ectoin comprises the following steps:
1. Preparation of culture medium
LB medium: 10g of NaCl,10g of peptone, 5g of yeast powder, adding deionized water to 1L, adjusting the pH to 7.0-7.2, packaging and sterilizing. TB Medium deionized water was added to 900 ml, tryptone 12 g, yeast powder 24 g, and glycerol 4 ml to shake the vessel to completely dissolve the solutes, and steam sterilized under high pressure 20 min. 100ml sterile phosphate buffer was added when the solution cooled to 60 ℃ or below. The preparation method of the phosphate buffer solution comprises the following steps: 2.31 g KH 2PO4 and 12.54 g K 2HPO4 are dissolved with 90 ml deionized water, and after complete dissolution, the volume is fixed to 100ml with deionized water and the solution is steam sterilized at high pressure to 20 min.
2. Shake flask fermentation test
The engineering strain is streaked on LB solid medium, cultured and activated overnight at 37 ℃, the monoclonal is selected and cultured in 100mL shake flask with 20mL LB medium, the seed solution is transferred into 500mL shake flask with 100mL TB medium according to 1% inoculation amount by shaking table overnight at 37 ℃ and 220r shake flask as seed solution, and the monoclonal is cultured overnight at 37 ℃. The supernatant was then centrifuged using centrifuge 12000r and subjected to liquid phase analysis, and the chromatogram was shown in FIG. 4.
3. 5L fermentation tank feed supplement fermentation
Inoculating recombinant bacteria into 500ml shake flask with 100ml LB culture medium, culturing overnight at 37deg.C with 220r shaking table as seed liquid, transferring the seed liquid into 5L fermentation tank containing 2.5L fermentation medium containing 6 g/L KH2PO4, 16.4 g/L K2HPO4, 5 g/L (NH4)2SO4, 1.1 g/L citric acid, 1 g/L MgSO 4, 10 g/L yeast powder, 7g/L glucose, 0.1 g/L vitamin B1, 2 mL/L microelements according to 2% inoculum size; the trace elements contain 10 g/L FeSO4·7H2O, 1.53 g/L CaCl2, 2.2 g/L ZnSO4·7H2O, 1g MnSO4·4H2O, 1 g/L CuSO4·5H2O, 0.1 g/L (NH4)6Mo7O24·4H2O, 0.2 g/L Na2B4O7·10H2O, 1 g/L NiCl2, 1g/L H3BO3, 10 mL/L HCl, HCl with the concentration of 37%, firstly, the trace elements are dissolved in hydrochloric acid, and then deionized water is added to fix the volume to adjust the pH value to 7.0; the feed medium contained 500g/L glucose, 7g/L MgSO 4, 1mL/L trace elements. Continuous feed culture fermentation is carried out at 30 ℃ for 48-72 h, the yield of the liquid phase analysis tetrahydropyrimidine reaches 142g/L at the highest, and the yield graph is shown in figure 5.
It should be noted that the above examples are only for illustrating the technical solution of the present invention and are not limiting thereof. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can make modifications and equivalents to the technical solutions of the present invention as required, without departing from the spirit and scope of the technical solutions of the present invention.

Claims (10)

1. A recombinant bacterium for producing ectoin, which is characterized in that the recombinant bacterium is obtained by over-expressing ectoin synthesis related genes and glutamate dehydrogenase genes gdh in chassis bacteria and simultaneously knocking out ectoin synthesis competition pathway related genes, glutamate metabolism related genes and isocitrate lyase inhibitor genes iclR in the chassis bacteria;
Wherein the ectoin synthesis related genes comprise at least one of a pyruvate kinase synthesis gene pk, a phosphoenolpyruvate carboxylase synthesis gene ppc, a pyruvate carboxylase synthesis gene pyc, an aspartate aminotransferase synthesis gene aspC, an aspartase synthesis gene aspA, an aspartokinase synthesis gene ask (M68V), an aspartate semialdehyde dehydrogenase synthesis gene asd and a tetrahydropyrimidine synthesis enzyme gene cluster ectABC;
The gene related to the competitive pathway of the ectoin synthesis comprises at least one of lysine synthesis pathway gene lysA and threonine synthesis pathway gene thrA;
The glutamic acid metabolism related gene comprises at least one of arginine synthesis pathway gene argA and proline synthesis pathway gene proB.
2. The recombinant bacterium according to claim 1, wherein the nucleic acid sequence of the tetrahydropyrimidine synthase gene cluster ectABC is shown in SEQ ID No. 1;
The nucleic acid sequence of the aspartokinase synthesis gene ask (M68V) is shown in SEQ ID NO. 2;
The nucleic acid sequence of the pyruvate kinase synthetic gene pk is shown as SEQ ID NO. 3;
The nucleic acid sequence of the phosphoenolpyruvate carboxylase synthetic gene ppc is shown in SEQ ID NO. 4;
the nucleic acid sequence of the pyruvic acid carboxylase synthetic gene pyc is shown in SEQ ID NO. 5;
The nucleic acid sequence of the asparatic acid aminotransferase synthetic gene aspC is shown in SEQ ID NO. 6;
The nucleic acid sequence of the aspartase synthesis gene aspA is shown in SEQ ID NO. 7;
the nucleic acid sequence of the aspartic semialdehyde dehydrogenase synthetic gene asd is shown in SEQ ID NO. 8;
the nucleic acid sequence of the glutamate dehydrogenase gene gdh is shown in SEQ ID NO. 9.
3. The recombinant bacterium according to claim 1, wherein gene IDs of the lysine synthesis pathway gene lysA, threonine synthesis pathway gene thrA, isocitrate lyase inhibitor gene iclR, arginine synthesis pathway gene argA and proline synthesis pathway gene proB are 947313, 945803, 948524, 947289 and 946425, respectively.
4. The recombinant bacterium of claim 1, wherein the chassis bacterium is e.coli K12 MG1655.
5. The recombinant bacterium of claim 1, wherein the overexpressed gene in the recombinant bacterium is integrated into the genome of the chassis bacterium; the gene integration mode is carried out by adopting crispr-cas9 editing technology and/or lambda-Red homologous recombination technology.
6. The recombinant bacterium according to claim 1, wherein the tetrahydropyrimidine synthase gene cluster ectABC, the aspartate aminotransferase synthase gene aspC, the glutamate dehydrogenase gene gdh, the aspartate synthase gene aspA, the aspartate semialdehyde dehydrogenase synthase gene asd, the aspartate kinase synthase gene ask (M68V), the pyruvate carboxylase synthase gene pyc, the phosphoenolpyruvate carboxylase synthase gene ppc, and the pyruvate kinase synthase gene pk are sequentially integrated into the gene integration sites yjip _yjir, dadx_cvra, cspf_quq, ypjc_iley, thrw_ykfn, iley_ygaq, ykga_ygq, yeej_ yeeL, and ybfC _ ybfQ.
7. Use of the recombinant bacterium of any one of claims 1-6 for the production of ectoin.
8. A method of producing ectoin, the method comprising: fermenting and culturing the recombinant bacterium of any one of claims 1-6, thereby expressing ectoin; and separating and purifying the ectoin.
9. A method of constructing a recombinant bacterium for producing ectoin, the method comprising: coli e.coli K12 MG1655 was used as a chassis fungus, which was transformed as follows:
a1 Knocking out at least one of lysine synthesis pathway gene lysA, threonine synthesis pathway gene thrA, isocitrate lyase inhibitor gene iclR, arginine synthesis pathway gene argA and proline synthesis pathway gene proB in chassis bacteria;
a2 Integration of at least one of the exogenous gene pyruvate kinase synthesis gene pk, phosphoenolpyruvate carboxylase synthesis gene ppc, pyruvate carboxylase synthesis gene pyc, aspartate aminotransferase synthesis gene aspC, aspartase synthesis gene aspA, aspartokinase synthesis gene ask (M68V), aspartate semialdehyde dehydrogenase synthesis gene asd, tetrahydropyrimidine synthesis gene cluster ectABC.
10. The construction method according to claim 9, wherein the nucleic acid sequence of the tetrahydropyrimidine synthase gene cluster ectABC is shown in SEQ ID No. 1;
The nucleic acid sequence of the aspartokinase synthesis gene ask (M68V) is shown in SEQ ID NO. 2;
The nucleic acid sequence of the pyruvate kinase synthetic gene pk is shown as SEQ ID NO. 3;
The nucleic acid sequence of the phosphoenolpyruvate carboxylase synthetic gene ppc is shown in SEQ ID NO. 4;
the nucleic acid sequence of the pyruvic acid carboxylase synthetic gene pyc is shown in SEQ ID NO. 5;
The nucleic acid sequence of the asparatic acid aminotransferase synthetic gene aspC is shown in SEQ ID NO. 6;
The nucleic acid sequence of the aspartase synthesis gene aspA is shown in SEQ ID NO. 7;
the nucleic acid sequence of the aspartic semialdehyde dehydrogenase synthetic gene asd is shown in SEQ ID NO. 8;
the nucleic acid sequence of the glutamate dehydrogenase gene gdh is shown in SEQ ID NO. 9;
The gene IDs of the lysine synthesis pathway gene lysA, threonine synthesis pathway gene thrA, isocitrate lyase inhibitor gene iclR, arginine synthesis pathway gene argA and proline synthesis pathway gene proB are 947313, 945803, 948524, 947289 and 946425 respectively.
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