An autonomous evolution system and its applications

Abstract

The present invention discloses an autonomous evolution system, which includes a regulatory element, a mutation module and a fidelity module. The fidelity module includes mismatch repair protein genes mutH, mutL, mutS and helicase gene recG. The mutation module includes mutation-prone genes such as DNA Pol IV gene dinB, genes encoding stress factors rpoS, rpoD, and genes encoding damage response protein recA. The present invention also discloses a method for constructing high-glucose-tolerant valine-producing Escherichia coli. The present invention focuses on genes prone to mutation and mismatch repair genes, uses high-glucose adaptive culture and combines metabolic engineering to screen excellent phenotypes, increases the spontaneous high survival rate of stress cells and the enrichment of forward mutations, and selects an L-valine-producing strain E. coli with good glucose tolerance, enhanced amino acid fermentation ability and strong genetic stability, which can provide a research basis for the selection of E. coli strains with high amino acid production and tolerance to environmental stress.

Description

Technical Field

[0001] The present invention relates to the field of synthetic biology, and particularly relates to an autonomous evolution system and its application. Background Art

[0002] L-valine is an excellent nutritional supplement, mainly produced by microbial fermentation. The production strains of L-valine mainly include Corynebacterium glutamicum, Escherichia coli, and Saccharomyces cerevisiae, etc. The metabolic network of the high-yield L-valine strain C. glutamicum is complex, and it is difficult to transform the strain due to the lack of genetic information. In the system of metabolically engineering L-valine with S. cerevisiae as the host bacterium, there is a lack of strong and strictly regulated promoters, the fermentation cycle is long, the nutritional requirements are complex, it is not suitable for high-density culture, and the nutrient salts and monosaccharide components in the later stage of fermentation increase the separation and purification cost. E. coli has become an excellent model strain for amino acid production due to its clear genetic background, short fermentation cycle, high glucose consumption rate, and mature gene operation tools. Glucose is the main sugar raw material for microbial fermentation to produce L-valine. Insufficient glucose supply during the fermentation process leads to slow cell growth and low product yield, while too high glucose concentration will increase the osmotic pressure of the culture medium, hinder the absorption of glucose and nutrients, and limit cell growth and product synthesis. Wild-type E. coli has poor glucose tolerance and low metabolic synthesis ability, and the inherent complexity of the biological system poses challenges to the new phenotypes of E. coli.

[0003] Industrially, L-valine is mainly produced by microbial fermentation. Obtaining the target phenotype that is tolerant to high sugar stress and highly productive in L-valine has become the bottleneck restricting the large-scale application of L-valine. The existing problems mainly include: (1) Most wild-type strains do not have the ability to produce large amounts of L-valine, and their yields are mainly limited by factors such as strain tolerance; (2) Traditional mutagenesis breeding is the main source of industrial L-valine-producing bacteria, but the genetic backgrounds of the strains obtained by traditional breeding methods are unclear and their genetic characteristics are unstable, and the survival rate of mutagenesis breeding of L-valine-producing strains is low; (3) The synthetic metabolic network of L-valine is complex. Laboriously knocking out or overexpressing genes dependent on the L-valine synthesis pathway will cause damage to cells or increase their burden, and single metabolic engineering transformation leads to limited improvement in product yield. Tolerance engineering to regulate the stress resistance of microbial cell factories can enhance stress defense capabilities by strengthening cell barriers and improve damage repair capabilities by accelerating stress responses. However, due to the complexity of the microbial system's metabolic and regulatory networks, it is difficult to obtain a phenotype with better robustness. Therefore, tolerance evolution engineering is becoming increasingly crucial in screening microbial cell factories with enhanced robustness. Tolerance evolution engineering refers to a method that utilizes the plasticity of the microbial genome, enabling microbial populations to continuously evolve under certain selection pressure conditions. Through precise regulatory mechanisms and complex resistance strategies, microorganisms evolve to survive and adapt to various stress pressures, obtaining beneficial mutations. This method can be used to improve microbial cell growth, increase the concentration, yield, and production intensity of chemicals, and discover unknown biological regulatory mechanisms.

[0004] Previous researchers have used synthetic biology to construct a "quorum sensing" gene circuit in Escherichia coli. LuxI, LuxR, and acyl-homoserine lactone (AHL) play important roles in this circuit. The LuxI enzyme catalyzes the synthesis of AHL molecules, while LuxR is an AHL receptor protein that can activate bacterial quorum sensing transcriptional effects. When the bacterial population density is low, the expression level of LuxR is relatively low, and the synthesized AHL molecules do not accumulate in the cells but quickly diffuse extracellularly. When the bacterial density increases, due to the extracellular AHL concentration gradient, AHL accumulates inside the cells. Once the intracellular AHL concentration reaches a certain threshold, the AHL molecules bind to LuxR. In turn, LuxR activates the PluxI promoter, thereby initiating the expression of target genes. Since AHL can diffuse across the cell membrane, the AHL concentration in the population of bacteria is basically the same, ensuring the synchronous execution of the gene expression program in the bacterial population. Summary of the Invention

[0005] Object of the Invention: The first object of the present invention is to provide an autonomous evolution system. Using E. coli with a clear genetic background as the research model, an increase in the survival rate of E. coli will lead to the evolution of new phenotypes. Improving the survival rate and evolution efficiency of E. coli can enhance the tolerance and production performance of the E. coli cell factory.

[0006] The second object of the present invention is to provide a genetically engineered bacterium or cell including an autonomous evolution system.

[0007] The third object of the present invention is to provide a method for constructing an Escherichia coli resistant to high sugar and producing valine.

[0008] Technical solution: In order to achieve the above invention objects, the present invention provides an autonomous evolution system, including a regulatory element, a mutation module and a fidelity module. The regulatory element includes LuxI, LuxR and a reporter gene; the fidelity module includes one or several of the mismatch repair protein genes mutH, mutL, mutS and the helicase gene recG, and the mutation module includes one or several of the DNAPol IV gene dinB, the genes rpoS, rpoD encoding stress factors, and the gene recA encoding a damage response protein.

[0009] The Gene ID of the gene mutH sequence is 947299; the Gene ID of the gene mutL sequence is 948691; the Gene ID of the gene mutS sequence is 947206; the Gene ID of the gene recG sequence is 948162; the Gene ID of the gene dinB sequence is 944922; the Gene ID of the gene rpoS sequence is 947210; the Gene ID of the gene rpoD sequence is 947567; the Gene ID of the gene recA sequence is 947170.

[0010] Preferably, the mutation module is regulated by the promoter PluxI, and the fidelity module is regulated by the promoter PesaS.

[0011] Preferably, the gene sequence of the fidelity module is as shown in SEQ ID NO: 1.

[0012] Preferably, the gene sequence of the mutation module is as shown in SEQ ID NO: 2.

[0013] The present invention also provides a genetically engineered bacterium or cell including the above autonomous evolution system.

[0014] Preferably, the genetically engineered bacterium is Escherichia coli.

[0015] The present invention also provides the application of the above autonomous evolution system, genetically engineered bacterium or cell in the production of L-valine.

[0016] The present invention also provides a method for constructing an Escherichia coli resistant to high sugar and producing valine, including the following steps:

[0017] (1) Knock out the mismatch repair protein genes mutH, mutL, mutS and the helicase gene recG respectively, and use the glucose concentration gradient as the screening pressure to obtain the candidate genes of the fidelity module;

[0018] (2) Overexpress the DNA Pol IV gene dinB, the genes encoding stress factors rpoS, rpoD, and the gene encoding the damage response protein recA respectively, and use the glucose concentration gradient as the screening pressure to obtain the candidate genes of the mutation module;

[0019] (3) Construct the fidelity module with the candidate genes of the fidelity module described in step (1), and construct the mutation module with the candidate genes of the mutation module described in step (2);

[0020] (4) Construct an L-valine synthesis pathway enzyme expression plasmid;

[0021] (5) Using Escherichia coli as the host bacterium, introduce the regulatory elements described above, the fidelity module and the mutation module described in step (3), and the L-valine synthesis pathway enzyme expression plasmid described in step (4), and you will get it.

[0022] Preferably, the gene sequence of the fidelity module is as shown in SEQ ID NO: 1, and the gene sequence of the mutation module is as shown in SEQ ID NO: 2.

[0023] Preferably, the L-valine synthesis pathway enzyme expression plasmid includes one or more of the genes encoding acetolactate synthase, ketol-acid reductoisomerase, dihydroxyacid dehydratase, and branched-chain amino acid transaminase.

[0024] The present invention also provides a method for producing L-valine, and the method includes fermenting the high-glucose-resistant valine-producing Escherichia coli.

[0025] Based on quorum sensing (QS), the present invention constructs regulatory elements of an autonomous evolution system. The regulatory elements include the recombinant plasmids P15A-LuxI-LuxR and PluxI-mKate2. In P15A-LuxI-LuxR, LuxI and LuxR are regulated by promoters Pi and Pj respectively, and the combination of Pi and Pj optimizes the control of the AHL and LuxR concentrations and the binding ability between the two; the expression intensity of genes is optimized by means of promoter engineering or RBS regulation strategies. Using the promoter PluxI that meets the QS response characteristics and using the gene encoding the far-red fluorescent protein mkate2 as the reporter gene, the recombinant plasmid PluxI-mKate2 is obtained. The constructed recombinant plasmids P15A-LuxI-LuxR and PluxI-mKate2 are transferred into E. coli JM109 by electroporation to obtain recombinant strains.

[0026] The mutH screened under glucose stress was used as a candidate gene, and the candidate gene fragment was amplified from the genome and ligated into a plasmid to obtain a fidelity module; the rpoD screened under glucose stress was used as a candidate gene, and the candidate gene fragment was amplified from the genome and ligated into a plasmid to obtain a mutation module. The fidelity module and the mutation module were regulated by the promoters PesaS and PluxI respectively. The fidelity module and the mutation module were transferred into the above recombinant strain to obtain Escherichia coli EC12-G60 with an autonomous evolution system. The tandem expression dual plasmids (pEtac-ilvBN-ilvC, ptet-ilvE-ilvD) of the L-valine synthesis pathway enzymes were introduced into Escherichia coli EC12-G60 by electroporation, and thus Escherichia coli with high glucose tolerance for valine production was obtained. For the Escherichia coli with an autonomous evolution system obtained in the present invention, in the case of not secreting the signal molecule AHL, the promoter PesaS is activated for transcription and is in a high-fidelity state. When the cells reach the logarithmic growth phase, AHL is secreted and the PesaS promoter is inactivated; AHL binds to LuxR to induce the expression of the genes in the mutation module. When the strain reaches a certain mutation rate and can tolerate high-concentration glucose, the mutation module is transformed into the fidelity module to enrich beneficial mutations. With the help of a glucose stress plate, excellent phenotypes are screened to evaluate the conversion effect. An autonomous evolution system including a fidelity module and a mutation module is constructed based on QS to achieve single-level self-regulation and two-level conversion regulation.

[0027] Advantages: Compared with the prior art, the present invention has the following remarkable advantages: (1) Focusing on genes prone to mutation and mismatch repair genes, high-sugar adaptive culture is used in combination with metabolic transformation to screen excellent phenotypes, increasing the spontaneous high survival rate of stressed cells and the enrichment of positive mutations; an L-valine-producing strain E. coli with good glucose tolerance, enhanced amino acid fermentation ability, and strong passage stability is selected and bred; (2) It can provide a general research basis for the selection and breeding of E. coli strains with high amino acid production and tolerance to environmental stress, breaking through the bottleneck restricting the large-scale production of L-valine, and has important theoretical and practical significance; (3) Microbial cells have the ability to highly adapt to the environment and self-reproduce through self-regulation, and can cause variations through mutation or gene recombination. Under high-concentration glucose conditions, the tolerance of E. coli to high-concentration glucose is enhanced, and E. coli with a significantly increased survival rate is obtained. Description of the Drawings

[0028] Figure 1 It is a schematic diagram of the autonomous evolution system of the present invention;

[0029] Figure 2 It is a schematic diagram of the plasmid structures of pTargetF-N20-mutH, pTargetF-N20-mutL, pTargetF-N20-mutS, and pTargetF-N20-recG;

[0030] Figure 3 Schematic diagrams of plasmid structures of pEtac-dinB, pEtac-rpoS, pEtac-rpoD, and pEtac-recA;

[0031] Figure 4 Schematic diagrams of plasmid structures of pEtac-ilvBN-ilvC and ptet-ilvE-ilvD for tandem expression;

[0032] Figure 5 Schematic diagrams of plasmid structures of pCDFDuet-1-PluxI-rpoD and pEM-PesaS-mutH. Specific implementation manners

[0033] The technical solutions of the present invention will be further described below with reference to the accompanying drawings.

[0034] mutH Gene ID: 947299; mutL Gene ID: 948691; mutS Gene ID: 947206; recG Gene ID: 948162; dinB Gene ID: 944922; rpoS Gene ID: 947210; rpoD Gene ID: 947567; recA Gene ID: 947170.

[0035] Example 1

[0036] In order to obtain high-fidelity genes, with glucose tolerance as the test index, the effects on the survival rate of strains were examined under the conditions of deletion of three genes encoding mismatch repair proteins mutL, mutS, mutH and a helicase gene recG. In this example, the CRISPR / Cas9 knockout method was used. The pTargetF-sgRNA and knockout cassette of the target gene were designed, and the knockout cassette was guided to the target gene through pTargetF-sgRNA to achieve directional knockout. Taking the knockout strain of mutS as an example, the specific steps are as follows:

[0037] (1) Construct the pTargetF-sgRNA(mutS) plasmid: Design amplification primers KZ-sgRNA-A and pTargetF-sgRNA(mutS)-S. Using pTargetF as the template, configure the Prime Star system as shown in Table 1, P1 is KZ-sgRNA -A, P2 is pTargetF-sgRNA(mutS)-S, and the PCR amplification program is shown in Table 2. After gel cutting and recovery, digest with DpnⅠ.

[0038] Table 1 200μL Prime Star system Table 2 PCR program

[0039]

[0040] (2) Construction of the mutS knockout cassette: Amplify the flanking sequences on both sides of the gene mutS to be knocked out, with 500 bp for each of the upstream and downstream sequences, which are used as the left and right arms of the knockout cassette respectively. The left and right arm sequences are fused by fusion PCR to obtain the knockout cassette sequence. The fusion system is shown in Table 3, without adding a template, and the primers are KZ-mutS(u)-S and KZ-mutS(d)-A. The fusion PCR program is shown in Table 4.

[0041] Table 3 50 μL Prime Star fusion system Table 4 Fusion PCR program settings

[0042]

[0043] (3) Introduce the Cas9 plasmid, the mutS knockout cassette, and the pTargetF-sgRNA(mutS) plasmid into competent E. coli JM109 cells, and the knockout cassette fragment replaces the gene mutS to be knocked out on the original genome. Pick single colonies for PCR verification to obtain the single-gene knockout strain E. coli JM109-ΔmutS.

[0044] The construction methods of E. coli JM109-ΔmutH, E. coli JM109-ΔmutL, and E. coli JM109-ΔrecG strains are the same as above. The primers used are shown in Table 7. The schematic diagrams of the pTargetF-sgRNA plasmid structures for different genes are as Figure 2 shown.

[0045] Label the obtained gene knockout strains E. coli JM109-ΔmutH, E. coli JM109-ΔmutL, E. coli JM109-ΔmutS, and E. coli JM109-ΔrecG as EC01, EC02, EC03, and EC04. The process of screening strains with a high-concentration glucose medium is as follows:

[0046] (1) Denote the solid screening media containing 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, and 80 g / L glucose as LB-G30, LB-G40, LB-G50, LB-G60, LB-G70, and LB-G80, and use the LB medium with 0 g / L glucose as the blank control.

[0047] As shown in Table 5, for the single-gene knockout strains, the strains with higher survival rates in LB medium were EC03 and EC04, and the survival rates were both 2.0; in LB-G30 medium, the strain with a higher survival rate was EC01, and the survival rate was 12.5; in LB-G40 medium, the strains with higher survival rates were EC02 and EC04, and the survival rates were 0.7 and 0.8 respectively; in LB-G50 medium, the strains with higher survival rates were EC01 and EC04, and the survival rates were 2.0 and 3.6 respectively; in LB-G60 medium, the strain with a higher survival rate was EC03, and the survival rate was 4.8; in LB-G70 medium, the strains with higher survival rates were EC01 and EC03, and the survival rates were 3.3 and 3.7 respectively.

[0048] Using 30 - 80 g / L of glucose as the screening pressure, taking the higher survival rate as the index, the survival rates of the four single-gene knockout strains EC01, EC02, EC03, and EC04 tolerating glucose were increased by 7.2 times, 5.0 times, 1.8 times, and 5.3 times respectively compared with E. coli JM109.

[0049] The calculation methods of the survival rate and the increased multiple of the survival rate are as follows. Taking EC01 as an example:

[0050] Cultivate Escherichia coli EC01 on the solid screening medium for 5 days, and use the increase rate of the number of glucose-tolerant strains on the medium to represent the survival rate of the strain producing tolerance;

[0051] A: The total number of tolerant colonies grown by E. coli JM109 on the 2nd day; B: The total number of tolerant colonies grown by E. coli JM109 on the 5th day; C: The total number of tolerant colonies grown by EC01 on the 2nd day; D: The total number of tolerant colonies grown by EC01 on the 5th day; The number of increased colonies of E. coli JM109: B - A; The number of increased colonies of EC01: D - C;

[0052] The survival rate of EC01 = (D - C) / 3;

[0053] Since under the condition of LB-G30, EC01 has a higher survival rate than other mutant strains, take the least common multiple of A and C;

[0054] Then the survival rate of EC01 is increased by [A(D - C) - C(B - A)] / (the least common multiple of A and C) compared with E. coli JM109.

[0055] The above results indicate that the four genes, mutH, mutL, mutS, and recG, can all affect the mismatch repair in E. coli JM109 to varying degrees. The average survival rate of the single-gene mutH knockout strain is relatively high and fluctuates less than that of other strains, and this mutant strain has better glucose tolerance. The survival rate of the strain with the mutH gene knocked out is the highest in a relatively high-concentration glucose medium (60 g / L).

[0056] Table 5 Survival rates of single-gene knockout strains and E. coli JM109

[0057]

[0058]

[0059] (2) Through enrichment culture in a high-concentration glucose medium, strains with good adaptability to a high-concentration glucose environment were obtained, strengthening the high-sugar tolerance of the strains. The strains after adaptive culture were streaked and isolated on a high-concentration glucose plate, and finally, well-grown colonies were selected from the 60 g / L glucose plate with the highest concentration and cultured in a shake flask with liquid to obtain strain EC01-G60.

[0060] Table 6 Strains and plasmids used in this example

[0061]

[0062] Table 7 Primers used in this example

[0063]

[0064] Example 2

[0065] In order to obtain genes prone to mutation, using glucose tolerance as the test index, the effects of the DNAPol IV gene dinB, the stress factor-encoding genes rpoS and rpoD, and the damage response protein-encoding gene recA on the survival rate of the strain were examined. Taking pEtac-dinB as an example, the specific construction steps are as follows:

[0066] (1) Using the genomic DNA of E. coli MG1655 as a template, the target gene dinB was amplified with primers KZ-dinB-S and KZ-dinB-A.

[0067] (2) Construct a 200 μL double digestion system as shown in Table 8. Enzyme 1 is EcoR I and Enzyme 2 is Sac I. Place the double digestion system in a PCR instrument at 37 °C and react for 3 h. Recombine the fragment obtained by double digestion of the pEtac vector with its corresponding target fragment dinB according to the system shown in Table 9. Place the constructed 10 μL homologous recombination system in a PCR instrument at 50 °C and react for 30 min to obtain the overexpression plasmid pEtac-dinB.

[0068] Table 8 Construction of a 200 μL double digestion system Table 9 Homologous recombination system

[0069]

[0070] The construction methods of pEtac-rpoS, pEtac-rpoD, and pEtac-recA are the same as that of pEtac-dinB, and the plasmid structures are as Figure 3 shown.

[0071] Transform the recombinant plasmids into E. coli JM109 by chemical transformation method respectively to obtain the recombinant strains with single gene overexpression, namely E. coli JM109-dinB, E. coli JM109-rpoS, E. coli JM109-rpoD, and E. coli JM109-recA, which are named EC05, EC06, EC07, and EC08 respectively. The process of screening strains in high-concentration glucose medium is as follows:

[0072] (1) Denote the obtained recombinant strains with single gene overexpression as EC05, EC06, EC07, and EC08, and denote the solid screening media containing 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, and 80 g / L glucose as LB-G30, LB-G40, LB-G50, LB-G60, LB-G70, and LB-G80, and use the LB medium with 0 g / L glucose as the blank control.

[0073] As shown in Table 10, for E. coli single gene overexpression strains, the strain with a higher survival rate in LB medium is EC07, with a survival rate of 0.8; in LB-G30 medium, the strain with a higher survival rate is EC06, with a survival rate of 1.5; in LB-G40 medium, the strain with a higher survival rate is EC07, with a survival rate of 1.5; in LB-G50 medium, the strain with a higher survival rate is EC08, with a survival rate of 1; in LB-G60 medium, the strain with a higher survival rate is EC07, with a survival rate of 2.3; in LB-G70 medium and LB-G80 medium, the strain with a higher survival rate is EC05, but the growth of E. coli JM109-dinB strain is slow.

[0074] Using glucose at a concentration of 30 - 80 g / L as the screening pressure, the survival rates of the four single-gene overexpression strains EC05, EC06, EC07, and EC08 for glucose tolerance were 2.0-fold, 2.2-fold, 3.5-fold, and 1.0-fold higher than that of E. coli JM109, respectively.

[0075] The calculation methods for the survival rate and the multiple of increased survival rate are as follows. Taking EC05 as an example:

[0076] Cultivate Escherichia coli EC05 on a solid screening medium for 5 days, and use the increase rate of the number of glucose-tolerant strains on the medium to represent the survival rate of the strain to produce tolerance;

[0077] A: The total number of tolerant colonies grown by E. coli JM109 on the 2nd day; B: The total number of tolerant colonies grown by E. coli JM109 on the 5th day; C: The total number of tolerant colonies grown by EC05 on the 3rd day; D: The total number of tolerant colonies grown by EC05 on the 5th day; The increase in the number of E. coli JM109 colonies: B - A; The increase in the number of EC05 colonies: D - C;

[0078] The survival rate of EC05 = (D - C) / 3;

[0079] Since under the LB-G30 condition, EC05 has a higher survival rate than other mutant strains, take the least common multiple of A and C;

[0080] The survival rate of EC05 is increased by [A(D - C) - C(B - A)] / (the least common multiple of A and C) compared with E. coli JM109.

[0081] The above results indicate that the four genes dinB, recA, rpoD, and rpoS can all affect the SOS response in E. coli JM109 to varying degrees. Taking the higher survival rate as an index, E. coli JM109-rpoD appears the most times, with a relatively high average survival rate and smaller fluctuations than other strains. In summary, the single-gene overexpression bacterium with a higher survival rate is E. coli JM109-rpoD, and this mutant strain has better glucose tolerance.

[0082] (2) Through enrichment culture in a high-concentration glucose medium, strains with good adaptability to a high-concentration glucose environment were obtained, strengthening the high-sugar tolerance performance of the strains. Streak and separate the strains after adaptive culture on a high-concentration glucose plate. Finally, select well-grown colonies from the glucose plate with the highest concentration of 60 g / L and perform shake-flask liquid culture to obtain the strain EC07-G60.

[0083] Table 10 Survival rates of each strain in this example

[0084]

[0085] Table 11 Strains and plasmids used in this example

[0086]

[0087] Table 12 Primers used in this example

[0088]

[0089] Example 3

[0090] In the biosynthesis of L-valine by E. coli using glucose, pyruvate is the direct precursor for the formation of valine. The metabolism of pyruvate to synthesize L-valine is the key to the L-valine biosynthesis pathway, and the key enzymes involved are acetolactate synthase (ilvBN), ketol-acid reductoisomerase (ilvC), dihydroxy-acid dehydratase (ilvD), and branched-chain amino acid transaminase (ilvE). Taking Ptet-ilvE-ilvD as an example, the tandem expression plasmid of L-valine synthesis pathway enzymes is as follows:

[0091] (1) Amplify the ilvD gene with primers KZ-ilvD-S and KZ-ilvD-A, and amplify the ilvE gene with KZ-ilvE-S and KZ-ilvE-A.

[0092] (2) Construct a 200 μL double digestion system as shown in Table 8. Place the double digestion system in a PCR instrument at 37 °C for 3 h. Recombine the fragment obtained by double digesting the pEtac vector with the fusion fragment of the ilvD gene and the ilvE gene according to the system shown in Table 9. Place the constructed 10 μL homologous recombination system in a PCR instrument at 50 °C for 30 min to obtain Ptet-ilvE-ilvD.

[0093] The construction method of pEtac-ilvBN-ilvC is the same as that of Ptet-ilvE-ilvD. The structures of pEtac-ilvBN-ilvC and Ptet-ilvE-ilvD are as Figure 4 shown.

[0094] The two plasmids were introduced into EC01-G60 and EC07-G60 that are tolerant to high concentrations of glucose by electroporation to obtain EC09 and EC10 respectively, making E. coli a strain with high L-valine productivity. The recombinant plasmids pEtac-ilvBN-ilvC and ptet-ilvE-ilvD were introduced into the wild-type E. coli JM109 and named EC11 as a blank control.

[0095] EC09, EC10, and EC11 had a fast growth rate and excellent performance within 0 - 12 h. Their growth was retarded from 12 - 48 h and showed a downward trend. The downward trends of EC09 and EC10 were less significant than that of EC11. In the fermentation broth, the initial pH of EC09, EC10, and EC11 was 5.5 - 5.6. As the fermentation time extended, the substrates were gradually consumed, products were gradually generated, and the pH gradually decreased. The pH of the fermentation broth at the end of fermentation was 5.4 for all. When EC09, EC10, and EC11 were fermented in shake flasks for L-valine, the bacteria showed good tolerance to glucose and excellent growth performance. The yields of L-valine reached 6.62 g / L, 5.80 g / L, and 3.58 g / L respectively. At the same time, experiments found that E. coli JM109, EC01 - G60, and EC07 - G60 without the transfer of the pEtac-ilvBN-ilvC and Ptet-ilvE-ilvD dual plasmids hardly produced L-valine.

[0096] In summary, according to indicators such as the survival rate of the strains, the tolerance to glucose, and the growth curve, excellent phenotypes of single-gene overexpression strains producing valine and tolerant to high concentrations of glucose, EC10, and excellent phenotypes of single-gene knockout strains producing valine and tolerant to high concentrations of glucose, EC09, were screened.

[0097] Table 13 Strains and plasmids used in this example

[0098]

[0099] Table 14 Primers used in this example

[0100]

[0101] Example 4

[0102] Construction of Escherichia coli with an autonomous evolution system, the specific steps are as follows

[0103] (1) Construct the regulatory element plasmids P15A-LuxI-LuxR and PluxI-mKate2. The construction method refers to the literature (Kim E-M, Woo HM, Tian T, Yilmaz S, Javidpour P, Keasling JD, Lee TS. Autonomous control of metabolic state by a quorum sensing (QS)-mediated regulator for bisabolene production in engineered E. coli. Metab Eng, 2017, 44: 325-336.). Transform the constructed plasmids P15A-LuxI-LuxR and PluxI-mKate2 into E. coli JM109 by electroporation to obtain recombinant strains, and culture them in a 96-well cell culture plate. Detect the fluorescence and cell growth every 30 min to evaluate the effect of the regulatory elements.

[0104] (2) Construction of the fidelity module pEM-PesaS-mutH: Use the mutH screened under glucose stress in Example 1 as the candidate gene. Amplify PesaS with primers KZ-PesaS-S and KZ-PesaS-A, and amplify the mutH gene with KZ-PesaS-mutH-S and KZ-PesaS-mutH-A. The fusion fragment of PesaS and mutH is homologous recombined with the pEM vector after double digestion to construct the homologous recombination system shown in Table 15. Incubate the constructed 10 μL of the homologous recombination system in a PCR instrument at 50 °C for 30 min to obtain the construction of the fidelity module pEM-PesaS-mutH, and its structure is as Figure 5 shown, and the sequence is as shown in SEQ ID NO: 1:

[0105] Table 15 Homologous recombination system

[0106]

[0107] (3) The construction method of the mutation module pCDFDuet-1-pluxI-rpoD is the same as that of pEM-PesaS-mutH. Use the rpoD screened under glucose stress in Example 2 as the candidate gene. The candidate gene rpoD is amplified from the genome, and rpoD and the promoter PluxI are ligated to the plasmid pCDFDuet-1 to obtain the mutation module pCDFDuet-1-pluxI-rpoD, and its structure is as Figure 5 shown, and the sequence is as shown in SEQ ID NO: 2. The fidelity module and the mutation module are regulated by the promoters PesaS and PluxI respectively. The fidelity module and the mutation module are transferred into the recombinant strain in step (1) to obtain Figure 1Escherichia coli EC12-G60 with an autonomous evolution system as shown. After glucose stress testing, the survival rate of EC12-G60 was 8.3 times higher than that of E. coli JM109 respectively.

[0108] (3) The tandem expression dual plasmid (pEtac-ilvBN-ilvC, ptet-ilvE-ilvD) of L-valine synthesis pathway enzymes was introduced into Escherichia coli EC12-G60 described in step (3) by electroporation method to further obtain Escherichia coli with high glucose tolerance for producing valine. Flask fermentation of L-valine showed that the bacteria had good glucose tolerance and excellent growth performance, and the L-valine yield could reach 8.62 g / L.

[0109] Table 16 Strains and plasmids used in this example

[0110]

[0111] Table 17 Primers used in this example

[0112]

[0113]

[0114] Comparative Example 1

[0115] The formula of the seed medium for L-valine fermentation (per liter) is: glucose 20 g, yeast extract 5 g, (NH4)2SO4 2 g, KH2PO4 2.5 g, MgSO4·7H2O 1 g, FeSO4·7H2O 2 mg, MnSO4·H2O 2 mg, pH 7.0 - 7.2.

[0116] Take 200 μL of E. coli JM109 (purchased from Invitrogen) preserved in a -80°C glycerol tube and inoculate it into a 500 mL Erlenmeyer flask containing 50 mL of the seed medium. Incubate at 37°C and 200 r / min for 12 h, control the OD600 of the bacterial suspension, then take 1 mL and inoculate it into a 500 mL Erlenmeyer flask containing 50 mL of the seed medium, and incubate at 37°C and 120 rpm (reciprocating shaker) for 7 - 8 h.

[0117] Formulation of the shake flask fermentation medium for L-valine fermentation (per liter): glucose 20 g, yeast extract 4 g, tryptone 3 g, sodium citrate 2 g, KH2PO4 2 g, MgSO4·7H2O 2 g, FeSO4·7H2O 10 mg, MnSO4·H2O 10 mg, phenol red 8 mg, VB1 1 mg, VB3 1 mg, VB5 1 mg, VB12 1 mg, VH 1 mg, pH 7.0 - 7.2. Using phenol red as the pH indicator, add 25% NH4OH (v / v) with a micro syringe to keep the pH at 6.5. Intermittently add glucose with a concentration of 60% using a pipette to ensure that the pH remains at 6.5. The seed medium is inoculated into a 500 mL shake flask containing 50 mL of the fermentation medium at an inoculation amount of 15%, and cultured with shaking at 37°C and 200 rpm for 24 - 48 h.

[0118] After culturing with the fermentation medium, monitoring the fermentation broth found that E. coli JM109 fermentation hardly produced L-valine.

Claims

1. An autonomous evolution system, characterized in that, The autonomous evolution system includes a regulatory element, a mutation module, and a fidelity module. The regulatory element includes LuxI , LuxR and a reporter gene; the gene sequence of the fidelity module is shown in SEQ ID NO: 1, and the gene sequence of the mutation module is shown in SEQ ID NO:

2.

2. A genetically engineered bacterium or cell, characterized in that, Introduce the autonomous evolution system described in claim 1.

3. Use of the autonomous evolution system described in claim 1 and the genetically engineered bacterium or cell described in claim 2 in the production of L-valine.

4. A method for constructing a high - glucose - tolerant Escherichia coli producing valine, characterized in that, Comprising the following steps: (1)Knock out the mismatch repair protein gene mutH , mutL , mutS and the helicase gene recG respectively, using the glucose concentration gradient as the screening pressure to obtain the candidate gene mutH of the fidelity module; (2)Overexpress the DNA Pol IV gene respectively dinB , the gene encoding the stress factor rpoS , rpoD , the gene encoding the damage response protein recA , and use the glucose concentration gradient as the screening pressure to obtain the candidate genes of the mutation module rpoD ; (3) The candidate genes of the fidelity module described in step (1) mutH Construct a fidelity module, and the candidate genes of the mutation module described in step (2) rpoD Construct a mutation module; (4) Construct an expression plasmid for the enzymes in the L-valine synthesis pathway; (5) Using Escherichia coli as the host bacterium, introduce the regulatory element described in claim 1, the fidelity module and the mutation module described in step (3), and the expression plasmid for the enzymes in the L-valine synthesis pathway described in step (4), thus obtaining, the gene sequence of the fidelity module is as shown in SEQ ID NO: 1, and the gene sequence of the mutation module is as shown in SEQ ID NO:

2.

5. The construction method of a high-sugar-tolerant valine-producing Escherichia coli according to claim 4, characterized in that, The expression plasmid for the enzymes in the L-valine synthesis pathway comprises one or more of the genes encoding acetolactate synthase, ketol-acid reductoisomerase, dihydroxyacid dehydratase, and branched-chain amino acid transaminase.

6. A method for producing L-valine, characterized in that, The method comprises fermenting the high-sugar-tolerant valine-producing Escherichia coli constructed in claim 4 or 5.

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