Genetically engineered bacterium modified based on escherichia coli and method for preparing L-valine by using genetically engineered bacterium

Abstract

The invention provides a genetically engineered bacterium based on Escherichia coli modification and a method for preparing L-valine, the genetically engineered bacterium takes Escherichia coli as a host, the genetically engineered bacterium is genetically modified, and one or both of a leuA gene and an ilvA gene are knocked out and replaced by a gene editing technology to obtain a genetically modified strain VAL2. The L-valine is prepared by culturing on a culture medium, the culture medium is placed in a fermentation tank, the culture medium comprises the strain VAL2 in claim 1, and the production of the L-valine comprises three steps of strain activation, seed culture and fermentation culture. Escherichia coli is used as a starting strain, and a key enzyme of a synthetic pathway of L-leucine and L-isoleucine is subjected to systematic metabolic engineering modification to obtain a genetic engineering strain VAL2 for producing high-purity L-valine, so that the production quantity of the L-valine is increased.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a genetic engineering bacterium based on Escherichia coli transformation and a method for preparing L-valine. Background Art

[0002] Valine (L-Valine) is an important branched-chain amino acid (BCAA) with a wide range of applications in medicine, food, and feed. As one of the essential amino acids for the human body, valine is mainly obtained through external intake. In industry, the production methods of valine mainly include chemical synthesis and microbial fermentation.

[0003] 1. Development of industrial production of valine

[0004] Traditional chemical synthesis methods are gradually being replaced by microbial fermentation methods due to their high cost, complex steps and environmental pollution. Microbial fermentation methods use microorganisms (such as Escherichia coli, Corynebacterium glutamicum, etc.) to undergo metabolic engineering to produce valine through fermentation. In recent years, with the development of synthetic biology and genetic engineering technology, microbial fermentation methods have been significantly improved, the yield and purity of valine have been continuously improved, and the cost has been significantly reduced.

[0005] 2. Anabolic Links between Leucine, Isoleucine, and Valine

[0006] In microorganisms, valine, leucine (L-Leucine) and isoleucine (L-Isoleucine) are synthesized through a common metabolic pathway, which uses pyruvate as the starting material and produces these branched-chain amino acids through a series of enzymatic reactions. Specifically:

[0007] Leucine biosynthesis pathway: pyruvate is used as the starting material, and leucine is finally produced through multiple intermediates and enzymatic reactions. The enzyme encoded by the leuA gene is one of the key enzymes in this pathway.

[0008] Isoleucine biosynthesis pathway: pyruvate and α-ketobutyrate are used as starting materials to generate isoleucine through a series of enzymatic reactions. The enzyme encoded by the ilvA gene is one of the key enzymes in this pathway.

[0009] Valine synthesis pathway: pyruvate is used as the starting material, and valine is finally produced through multiple intermediates and enzymatic reactions.

[0010] Since these three amino acids share part of the metabolic pathway, in the process of producing valine, the synthesis of leucine and isoleucine inevitably competes with valine for intermediates and enzyme resources, thus affecting the yield and purity of valine.

[0011] 3. Challenges in the production of high-purity valine

[0012] In actual production, high-purity valine is difficult to obtain. The main reasons include:

[0013] Metabolic competition: The synthesis of leucine and isoleucine competes with valine for the same intermediates and enzyme resources, resulting in reduced efficiency of valine synthesis.

[0014] Difficulty of separation: The chemical properties of leucine, isoleucine and valine are similar, which makes it difficult to effectively remove leucine and isoleucine during the separation and purification process, resulting in reduced purity of valine.

[0015] In order to solve the above problems, it is urgent to develop a new method to reduce the synthesis of leucine and isoleucine through genetic modification, thereby increasing the yield and purity of valine.

[0016] 4. Deficiencies of existing technologies

[0017] At present, the existing technical methods have certain shortcomings in improving the production and purity of valine. Although certain results have been achieved in the production of valine by fermentation, there is no effective solution for inhibiting the synthesis of leucine and isoleucine. Therefore, how to accurately regulate metabolic pathways through genetic engineering to reduce the synthesis of leucine and isoleucine, thereby increasing the production and purity of valine, is still the focus and difficulty of current research.

[0018] Proposal of the present invention

[0019] Based on the above background, the present invention provides a method for obtaining high-purity L-valine by modifying the metabolic pathway of Escherichia coli. The leucine synthesis pathway gene leuA and the isoleucine synthesis pathway gene ilvA are knocked out by CRISPR-Cas9 technology, the synthesis of leucine and isoleucine is reduced, and the fermentation conditions are optimized, thereby significantly improving the yield and purity of valine. The method of the present invention has the advantages of high efficiency, environmental protection, and low cost, and is suitable for industrial production. Summary of the invention

[0020] The present invention aims to construct a genetically engineered strain of Escherichia coli that can efficiently produce L-valine, and improve the production purity of L-valine by systematically modifying the key leucine and isoleucine synthases that are similar in the L-valine synthesis pathway. The present invention provides a method for producing high-purity L-valine based on the genetically modified Escherichia coli metabolic pathway. The CRISPR-Cas9 technology is used to knock out the leucine synthesis pathway gene leuA and the isoleucine synthesis pathway gene ilvA, thereby reducing the synthesis of leucine and isoleucine and optimizing the production of L-valine.

[0021] In order to achieve the above object, the present invention provides the following technical solutions:

[0022] First, the present invention provides a genetically engineered bacterium and a method for constructing the same. The genetically engineered bacterium uses Escherichia coli as a host, and uses CRISPR-Cas9 technology to knock out the leucine synthesis pathway gene leuA and the isoleucine synthesis pathway gene ilvA, thereby reducing the synthesis of leucine and isoleucine, thereby optimizing the production of L-valine. The host of the genetically engineered bacterium is one of E coli MG1655, Ecoli W3110, E coli BL21 or E. coli BW25113;

[0023] The genetically modified strain is constructed by the following steps: designing specific gRNA sequences for the leucine synthesis pathway gene leuA and the isoleucine synthesis pathway gene ilvA; and introducing the CRISPR-Cas9 plasmid and the gRNA sequence into Escherichia coli MG1655 by electrotransformation.

[0024] Second, the present invention provides a method for producing high-purity L-valine using the genetically engineered bacteria as described above, comprising: culturing the genetically engineered bacteria on a culture medium in a fermentation tank to produce L-valine; and collecting the L-valine from the genetically engineered bacteria and / or the culture medium.

[0025] The beneficial effects of the present invention are as follows:

[0026] The invention uses Escherichia coli as a starting strain, and systematically performs metabolic engineering transformation on key enzymes in the L-leucine and L-isoleucine synthesis pathways to obtain a genetically engineered strain VAL2 for producing high-purity L-valine, thereby improving the production of L-valine. Under the condition of 220 rpm, the strain can produce 18.86 g / L of L-valine in a shake flask fermentation for 16 hours; and can produce 48 g / L of L-valine in a 5 L fermenter in a batch fed fermentation and oxygen-limited fermentation for 24 hours, with a conversion rate of 60.4%, and no significant accumulation of other byproducts. DETAILED DESCRIPTION

[0027] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0028] A method for constructing a genetically engineered bacterium, the method comprising:

[0029] 1 The primers used in the strain construction process are as follows:

[0030] leuA knockout

[0031] Using the E.coli MG1655 genome as a template, upstream homology arm primers (UP-ΔleuA-S, UP-ΔleuA-A) and downstream homology arm primers (DN-ΔleuA-S, DN-ΔleuA-A) were designed according to the upstream and downstream sequences of its leuA gene (NCBI-GeneID: GeneID: 947465), and PCR amplification of its upstream and downstream homology arm fragments was performed. The above fragments were fused by overlapping PCR to obtain the integration fragment of the ΔleuA gene (upstream homology arm-downstream homology arm). The DNA fragment containing the target sequence used to construct the plasmid pGRB-leuA was prepared by annealing primers gRNA-leuA-S and gRNA-leuA-A. The recombinant fragment and plasmid pGRB-leuA were electrotransformed into competent cells of E.coli MG1655, and the plasmid was eliminated after screening positive strains to obtain strain VAL1. The positive strains were verified by PCR and electrophoresis to verify the successful knockout of the leuA gene.

[0032] ilvA knockout

[0033] Using the E. coli MG1655 genome as a template, upstream homology arm primers (UP-ΔilvA-S, UP-ΔilvA-A) and downstream homology arm primers (DN-ΔilvA-S, DN-ΔilvA-A) were designed according to the upstream and downstream sequences of its ilvA gene (NCBI-GeneID: GeneID: 948287), and PCR amplified its upstream and downstream homology arm fragments. The above fragments were fused by overlapping PCR to obtain the integration fragment of the ΔilvA gene (upstream homology arm-downstream homology arm). The DNA fragment containing the target sequence used to construct the plasmid pGRB-ilvA was prepared by annealing primers gRNA-ilvA-S and gRNA-ilvA-A. The recombinant fragment and plasmid pGRB-ilvA were electrotransformed into the competent cells of VAL1, and the plasmid was eliminated after screening the positive strain to obtain the strain VAL2. The positive strain was verified by PCR and electrophoresis to successfully knock out the ilvA gene.

[0034] Specifically, the genetically engineered bacteria uses Escherichia coli as a host, and the genetically engineered bacteria is genetically modified, and one or both of the leuA gene and the ilvA gene are knocked out or replaced by gene editing technology to obtain the genetically modified strain VAL2.

[0035] Design specific gRNA sequences for the leucine synthesis pathway gene leuA and the isoleucine synthesis pathway gene ilvA; introduce the CRISPR-Cas9 plasmid and the gRNA sequence into Escherichia coli MG1655 by electroporation. The host is one of E coli MG1655, Ecoli W3110, E coli BL21 or E. coli BW25113.

[0036] The present invention will be described in more detail below through specific examples. In the following examples:

[0037] Unless otherwise specified, the gene editing method involved in the embodiment of the present invention is carried out with reference to the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9meditated genome editing. Metabolic engineering, 2015, 31: 13-21.). Among them, pREDCas9 carries the elimination system of the gRNA expression plasmid pGRB, the Red recombination system of lambda phage and the Cas9 protein expression system, spectinomycin resistance (working concentration: 100 mg / L), and is cultured at 32°C; pGRB uses pUC18 as a skeleton, including promoter J23100, gRNA-Cas9 binding region sequence and terminator sequence, ampicillin resistance (working concentration: 100 mg / L), and is cultured at 37°C. This method achieves knockout or knock-in of the recombinant fragment by transforming the pGRB plasmid and the recombinant DNA fragment, screening the positive strain, and then eliminating the plasmid. Wherein the recombinant fragment for knocking out is composed of the upstream and downstream homology arms of the gene to be knocked out (upstream homology arms-downstream homology arms), and the recombinant fragment for integration is composed of the upstream and downstream homology arms of the integration site and the gene fragment to be integrated (upstream homology arms-target gene-downstream homology arms). Other related specific operation means such as molecular biology and genetic engineering can be achieved according to the technical manuals, textbooks or literature reports that are easily available to those skilled in the art, and it is not necessary to describe the operation process in detail here. In addition, in the following examples, specific bacterial strains are selected as hosts, thus specific gene integration sites, target genes and primers thereof are selected according to hosts, but it is not meant that the purpose of the present invention can only be achieved by these specific selections, and the scope of the present invention can not be limited by this, and the essence and scope of the present invention are only limited by claims.

[0038] Example 1

[0039] The genetically modified strain VAL2 was used to produce L-valine in a 5L fermenter. The specific method is as follows:

[0040] (1) Activation of bacterial strains: dip the inoculation loop into the bacterial strains in the glycerol tube and inoculate them into the slant culture medium for subculture activation. Place the streaked slant in a 37°C incubator for constant temperature static culture for 20-24 hours to obtain a first-generation activated slant. Scratch the bacterial cells on the first-generation activated slant with the inoculation loop and transfer them to the eggplant-shaped slant for subculture again. Place the streaked slant in a 37°C incubator for constant temperature static culture for 20-24 hours to obtain a second-generation activated slant.

[0041] (2) Seed culture: The activated second-generation strain on the eggplant-shaped slope was washed with sterile water and inoculated into a 5L fermenter for seed culture, and the seed culture solution was fixed to 3L; the temperature was controlled at 37°C, 25% ammonia water was automatically added to control the pH value of the fermentation solution to 6.8-7.2, the initial ventilation volume was controlled to 2L / min, the initial stirring speed was 200r / min, and the dissolved oxygen was maintained at 10-40% for 18h, wherein the seed culture medium composition was: glucose 30g / L, KH2PO41.5g / L, MgSO4·7H2O 0.4g / L, MnSO4·H2O 10mg / L, V B1 0.3 mg / L, V H 0.2mg / L, yeast powder 5g / L, methionine 1g / L, glutamic acid 1g / L, corn steep liquor powder 20g / L (121℃, 20min sterilized separately), soybean meal hydrolyzate 10mL / L, the rest is water, sterilized at 115℃ for 15min;

[0042] (3) Fermentation culture: When the seeds are cultured to an OD of 20-30, the cultured seed solution is inoculated into a fermentation medium at an inoculation rate of 20%, and the initial fermentation liquid volume is fixed to 5 L. The pH of the medium is controlled at 6.8-7.2 by automatically adding 25% ammonia water, the temperature is controlled at 37°C, the dissolved oxygen is controlled at 10-40% by stirring and ventilation, and fermentation is carried out for 23-24 h by adding 800 g / L of glucose solution, wherein the fermentation medium composition is: glucose 80 g / L, (NH4)2SO4 3 g / L, KH2PO4 2.5 g / L, MgSO4·7H2O 2 g / L, FeSO4·7H2O 10 mg / L, MnSO4·H2O 10 mg / L, V B1 0.2mg / L,V H 0.08mg / L, yeast powder 5g / L, methionine 0.7g / L, glutamic acid 5g / L, choline chloride 1g / L, corn powder 16g / L (121℃, 20min sterilized separately), soybean meal hydrolyzate 20mL / L, the rest is water, sterilized at 115℃ for 15min.

[0043] The fermentation process mentioned above uses VAL2 as the strain to carry out the fermentation culture mentioned above;

[0044] Samples were taken every 2 hours to determine the concentrations of L-valine, L-leucine, L-isoleucine, bacterial cell concentration, and residual sugar in the fermentation broth.

[0045] In the above fermentation process, defoaming agent can be added according to actual needs to carry out defoaming.

[0046] Comparative Example 1

[0047] L-valine was prepared by fermentation using the method in Example 1, except that the strain used was VAL1.

[0048] Comparative Example 2

[0049] L-valine was prepared by fermentation using the method in Example 1, except that the strain used was E. coli MG1655.

[0050] After the fermentation of the above examples and comparative examples was completed, the yield and purity of L-valine were determined by the same separation and purification method.

[0051] Experimental results:

[0052]

[0053] Val-valine, Leu-leucine, Ile-isoleucine

[0054] According to the experimental structure, the yield of L-valine in Example 1 is the highest.

[0055] It can be verified that the present invention obtains a genetically engineered strain VAL2 for producing high-purity L-valine by systematically metabolically engineering the key enzymes of the L-leucine and L-isoleucine synthesis pathways, thereby obtaining high-purity L-valine. The CRISPR-Cas9 technology is used to knock out the leucine synthesis pathway gene leuA and the isoleucine synthesis pathway gene ilvA, thereby achieving high-purity production of L-valine, with an output of 48 g / L and a purity of more than 99%. The method has the advantages of high efficiency, environmental protection, and low cost, and is suitable for industrial production.

Claims

1. A genetically engineered bacterium based on Escherichia coli modification, characterized in that: The genetically engineered bacteria uses Escherichia coli as a host, and the genetically engineered bacteria is genetically modified, and one or all of the leuA gene and the ilvA gene are knocked out or replaced by gene editing technology to obtain the genetically modified strain VAL2.

2. The genetically engineered bacterium according to claim 1, characterized in that The genetically modified strain is constructed by the following steps: (a) Designing specific gRNA sequences for the leucine biosynthesis pathway gene leuA and the isoleucine biosynthesis pathway gene ilvA; (b) The CRISPR-Cas9 plasmid and the gRNA sequence were introduced into Escherichia coli MG1655 by electroporation.

3. The genetically engineered bacterium according to claim 1, characterized in that: The host is one of E. coli MG1655, EcoliW3110, E. coli BL21 or E. coli BW25113.

4. The genetically engineered bacterium according to claim 2, characterized in that: The host is E coli MG1655.

5. The genetically engineered bacterium according to any one of claims 1 to 4, characterized in that: include: The introduction of foreign gene sequences or other base sequences into the host Escherichia coli results in the inhibition or inactivation of the leucine synthesis pathway gene leuA and the isoleucine synthesis pathway gene ilvA.

6. A method for producing L-valine using the genetically engineered bacteria described in claim 1, characterized in that: The L-valine is obtained by culturing on a culture medium, the culture medium is placed in a fermentation tank, the culture medium includes the strain VAL2 described in claim 1, and the production of the L-valine includes three steps: strain activation, seed culture, and fermentation culture.

7. The method for producing L-valine as claimed in claim 6, characterized in that The seed culture medium in the seed culture step is composed of: glucose 30 g / L, KH2PO4 1.5 g / L, MgSO4·7H2O 0.4 g / L, MnSO4·H2O 10 mg / L, V B1 0.3 mg / L, V H 0.2mg / L, yeast powder 5g / L, methionine 1g / L, glutamic acid 1g / L, corn steep liquor powder 20g / L (121℃, sterilized separately for 20min), soybean meal hydrolyzate 10mL / L, the rest is water, sterilized at 115℃ for 15min.

8. The method for producing L-valine according to claim 6, wherein the fermentation medium in the fermentation culture step comprises: 80 g / L glucose, 3 g / L (NH4)2SO4, 2.5 g / L KH2PO4, 2 g / L MgSO4·7H2O, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4·H2O, 10 mg / L V B1 0.2mg / L,V H 0.08mg / L, yeast powder 5g / L, methionine 0.7g / L, glutamic acid 5g / L, choline chloride 1g / L, corn powder 16g / L (121℃, 20min sterilized separately), soybean meal hydrolyzate 20mL / L, the rest is water, sterilized at 115℃ for 15min.

9. The method for producing Valine according to claim 6, wherein The fermentation conditions in the production of L-valine are: temperature 32-38°C, pH value 6.5-7.5, ventilation volume 0.5-0.8vvm, pressure: 0.01-0.1MPa, stirring speed 200-800rpm, and fermentation time 12-36 hours.

10. The method for producing L-valine according to claim 6, wherein The fermentation tank comprises equipment for controlling fermentation conditions, including a temperature control system, a pH value adjustment system, a ventilation system and a stirring system.

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