WO2021109467A1 - 生产l-精氨酸的基因工程菌及其构建方法与应用 - Google Patents

生产l-精氨酸的基因工程菌及其构建方法与应用 Download PDF

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WO2021109467A1
WO2021109467A1 PCT/CN2020/090626 CN2020090626W WO2021109467A1 WO 2021109467 A1 WO2021109467 A1 WO 2021109467A1 CN 2020090626 W CN2020090626 W CN 2020090626W WO 2021109467 A1 WO2021109467 A1 WO 2021109467A1
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gene
arginine
fragment
encoding
coli
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French (fr)
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谢希贤
蒋帅
文晨辉
吴鹤云
刘益宁
李旋
田道光
熊博
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天津科技大学
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Priority to JP2022525726A priority Critical patent/JP7373661B2/ja
Priority to BR112022009979A priority patent/BR112022009979A2/pt
Priority to EP20896807.3A priority patent/EP4056676A4/en
Priority to US17/781,124 priority patent/US20220411833A1/en
Priority to CA3161765A priority patent/CA3161765A1/en
Priority to KR1020227016823A priority patent/KR20220088451A/ko
Publication of WO2021109467A1 publication Critical patent/WO2021109467A1/zh
Priority to ZA2022/05587A priority patent/ZA202205587B/en

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Definitions

  • the invention belongs to the technical field of genetic engineering, and specifically relates to a genetically engineered bacterium for stably and efficiently producing L-arginine, and a construction method and application thereof.
  • L-arginine is a semi-essential basic amino acid or conditionally essential amino acid in humans and animals, and has important biochemical and physiological functions. At present, L-arginine has been widely used in medicine, industry, food, cosmetics, animal husbandry and other fields, and has important economic and social value.
  • L-arginine mainly include protein hydrolysis and microbial fermentation. Compared with protein hydrolysis extraction, microbial fermentation has the advantages of relatively simple production process, relatively small impact on the environment, and high product purity. It is suitable for large-scale production. Industrial production.
  • the main strain of arginine production is Corynebacterium glutamicum.
  • the existing fermentation process of Corynebacterium glutamicum is subject to auxiliary materials such as The quality of corn steep liquor has a greater impact and production fluctuates easily.
  • the gene editing of Corynebacterium glutamicum is difficult, so that in the existing arginine strains, plasmid expression vectors are often used to enhance the key genes related to arginine synthesis.
  • multiple copies of the plasmid cause certain effects on the growth of the bacteria during the fermentation process.
  • arginine synthesis metabolic pathway Since there are many feedback controls in the arginine synthesis metabolic pathway, and there are many arginine metabolic pathways, the metabolic network involved in the precursors required for the synthesis of arginine is complex, so the initial research and development of arginine industrial production strains mainly used traditional Mutagenesis combined with arginine structural analogue resistance screening method.
  • the selected starting strains are mainly Brevibacterium flavum, Corynebacterium crenatum, and Corynebacterium glutamicum.
  • the research strategy focuses on screening arginine structural analogue mutants to relieve feedback regulation during arginine synthesis and improve intracellular The accumulation of L-arginine.
  • Li Shaoping was screened by NTG stepwise mutagenesis and found a strain with histidine deficiency, sulfaguanidine resistance, D-arginine resistance, homoarginine resistance, and S-methylcysteine
  • the resistant Corynebacterium crenatum (CN201010610917.5) was verified by fermentation experiments, and the accumulation of L-arginine was 32.8g/L when it was cultured in a 5L fermentor for 96h.
  • the production strains obtained through mutagenesis and structural analog screening are difficult to be put into mass industrial production due to the disadvantages of poor genetic stability and easy back mutation.
  • Corynebacterium glutamicum has no genes involved in the degradation of arginine; and glutamic acid is one of the main precursors of arginine synthesis, and the intracellular glucose taken up by Corynebacterium glutamicum undergoes the glycolytic pathway
  • the metabolic flux for producing glutamic acid is relatively strong, so Corynebacterium glutamicum is the main choice for constructing L-arginine producing strains.
  • the above-mentioned production strains generally have the problems of long production cycle and low production intensity, and the key arginine synthesis gene is connected to the expression vector during the strain construction process to increase the transcription amount of key enzymes, thereby enhancing the metabolism of arginine synthesis pathway
  • the expression vector is easy to lose or need to add a certain selective pressure, and it is difficult to put into industrial production.
  • the gene argA214 (H15Y) is used to relieve the feedback inhibition of arginine to ArgA, knock out the arginine degradation related gene adiA, knock out the ornithine degradation related genes speC and speF, so that the carbon flux of the intermediate metabolites flows more to L -Arginine.
  • the accumulation of L-arginine reached 11.64g/L.
  • the conversion rate is 0.44g arginine/g glucose, and the production intensity is 0.29g arginine/L/h.
  • the purpose of the present invention is to provide a genetically engineered bacterium for the stable and efficient production of L-arginine and its construction method and application.
  • the engineered bacterium has a good industrial application prospect.
  • the present invention provides a genetically engineered strain for producing L-arginine, which contains the genes pyrAA and pyrAB encoding carbamoyl phosphate synthase.
  • the genetically engineered strain uses Escherichia coli or Corynebacterium glutamicum as the starting strain; for example, E. coli W3110 or E. coli MG1655 as the starting strain.
  • the pyrAA and pyrAB genes are integrated into the yjiT gene locus of Escherichia coli.
  • the pyrAA and pyrAB are derived from Bacillus subtilis, specifically, the pyrAA and pyrAB are derived from the gene encoding carbamoyl phosphate synthase in B. subtilis A260.
  • the genetically engineered strain further contains genes encoding L-arginine biosynthetic pathway enzymes, and the -arginine biosynthetic pathway enzymes are selected from one or more of the following enzymes: argC, argJ , ArgB, argD, argF, argG, argH; the gene encoding the L-arginine biosynthetic pathway enzyme is derived from Corynebacterium glutamicum (Corynebacterium glutamicum ATCC13032); in one embodiment, the encoding L-arginine The gene for the enzyme of the amino acid biosynthetic pathway is initiated by the P trc promoter; in one embodiment, the gene encoding the enzyme of the L-arginine biosynthetic pathway is integrated into the yghX gene locus of Escherichia coli.
  • the genetically engineered strain further contains a gene lysE (NCBI Reference Sequence: WP_143758438.1) encoding an arginine transporter, and the transporter gene is derived from Corynebacterium efficiency; in one implementation In the scheme, the lysE gene is integrated into the ilvG gene locus of Escherichia coli.
  • lysE NCBI Reference Sequence: WP_143758438.1
  • the genetically engineered strain does not contain a gene encoding L-arginine, such as knocking out one or more of the following genes: a gene encoding arginine decarboxylase, a gene encoding arginine succinyl transfer Enzyme gene, gene encoding acetylornithine deacetylase.
  • the gene encoding arginine decarboxylase includes at least one of speA (NCBI-GeneID: 12933352) and adiA (NCBI-GeneID: 12934085); the gene encoding arginine succinyltransferase is astA (NCBI -GeneID: 12933241); the gene encoding acetylornithine deacetylase is argE (NCBI-GeneID: 12930574).
  • the genetically engineered strain is Escherichia coli with speA, adiA, and astA genes knocked out at the same time.
  • the genetically engineered strain contains pyrAA, pyrAB, argC, argJ, argB, argD, argF, argG, argH, and lysE genes. In one embodiment, the genetically engineered strain does not contain speA, adiA, astA and argE genes.
  • the pyrAA, pyrAB, argC, argJ, argB, argD, argF, argG, argH, lysE, speA, adiA, astA and argE genes are not limited to wild-type genes, and may also be mutants or mutants encoding corresponding proteins.
  • the artificially modified gene includes substitution, deletion or insertion of one or more amino acid residues at one or more sites, as long as the protein encoded by the mutant or the artificially modified gene has the corresponding activity and has no functional defects. These genes have been registered in GenBank, and those skilled in the art can obtain these genes by PCR.
  • the pyrAA gene is NCBI-GeneID: 937368
  • the pyrAB gene is NCBI-GeneID: 936608
  • the argC gene is NCBI-GeneID: 1019370
  • the argJ gene is NCBI-GeneID: 1019371
  • the argB gene is NCBI-GeneID: 1019372
  • the argD gene is NCBI-GeneID: 1019372.
  • the argF gene is NCBI-GeneID: 1019374
  • the argG gene is NCBI-GeneID: 1019376
  • the argH gene is NCBI-GeneID: 1019377
  • the lysE gene is the nucleotide sequence shown in SEQ ID NO: 68 ( NCBI Sequence ID: WP_143758438.1);
  • speA gene is NCBI-GeneID: 12933352
  • adiA gene is NCBI-GeneID: 12934085
  • astA gene is NCBI-GeneID: 12933241;
  • argE gene is NCBI-GeneID: 12930574.
  • the present invention provides a method for constructing the above genetically engineered strain, which includes the following steps: (1) pyrAA and pyrAB genes are integrated into the genome of the starting strain.
  • the starting strain is Escherichia coli, such as E. coli W3110 (ATCC27325).
  • the construction method further includes optionally one or more of the following steps:
  • Integrating arginine biosynthetic pathway enzyme genes including one or more of argC, argJ, argB, argD, argF, argG, and argH; and/or integrating the gene lysE encoding arginine transporter;
  • the gene includes at least one of speA and adiA; the gene encoding arginine succinyltransferase is astA; the gene encoding acetylornithine deacetylase is argE.
  • the construction method includes the following steps:
  • steps (1) to (5) of the above construction method of the present invention is not limited, and can be carried out in any order that can be implemented by a person skilled in the art.
  • steps (1) to (5) are carried out in sequence.
  • gene knockout or gene silencing method known in the art can be used to achieve the above-mentioned gene knockout, and any method known in the art can also be used to achieve gene integration, such as homologous recombination, overlap PCR, mutagenesis screening or Gene editing and other technologies.
  • gene knockout can be done by removing a specific region in the gene so that it does not have the function of expressing the target protein, or by introducing site-specific mutations in the coding region or promoter region of one or more nucleotide sequence substitutions, Deletions, insertions, etc., chemical reagents can also be used to decrease or disappear the transcription of the specific gene.
  • the construction method includes using CRISPR/Cas9-mediated gene editing technology for gene integration and knockout.
  • the construction method includes constructing a recombinant fragment and a pGRB plasmid.
  • the construction of the pGRB plasmid includes: designing a target sequence, preparing a DNA fragment containing the target sequence, and recombining the DNA fragment containing the target sequence with a linearized vector fragment; in a specific embodiment, the The target sequence is 5'-NGG-3'.
  • constructing a recombinant fragment in the construction method includes constructing a gene-integrated recombinant fragment or constructing a gene knock-out recombinant fragment.
  • the steps of constructing a recombinant fragment of gene integration include: using the genome of the starting strain as a template, designing upstream and downstream homology arm primers according to the upstream and downstream sequences of the target gene to be inserted into the site, and designing primers according to the target genome to amplify the target gene Fragments, and then recombined fragments are obtained by PCR overlap technology.
  • the steps of constructing a knock-out recombinant fragment include: using the upstream and downstream sequences of the gene to be knocked out as a template, designing primers for the upstream and downstream homology arms; respectively amplifying the upstream and downstream homology arms by PCR, and then preparing the recombination by overlapping PCR Fragment.
  • the construction method includes the steps of simultaneously transforming the pGRB plasmid and the above-mentioned recombinant fragment into electrocompetent cells containing pREDCas9, and removing the plasmid to obtain a recombinant genetically engineered strain.
  • the present invention provides the application of the above genetically engineered bacteria in the preparation of L-arginine.
  • the present invention also provides a method for fermentation and production of L-arginine by using the above-mentioned genetically engineered bacteria, which includes: contacting the above-mentioned genetically engineered strain of Escherichia coli with a fermentation medium, performing fermentation and cultivation, and preparing L-arginine.
  • the fermentation culture includes shake flask fermentation or fermentor fermentation.
  • the inoculation amount of shake flask fermentation is 10-15%
  • the fermentation conditions are 37° C., 200 r/min shaking culture
  • the pH is maintained at 7.0-7.2 during the fermentation process
  • the pH can be adjusted by adding ammonia water.
  • a glucose solution can also be added to maintain the fermentation.
  • the mass volume concentration of the glucose solution is 60% (m/v).
  • the fermentation time of the shake flask fermentation is 26-30h.
  • the supplementary amount of the glucose solution is not particularly limited, and the glucose concentration in the fermentation broth can be maintained at 5 g/L or less, for example, 1-5 g/L.
  • the shake flask fermentation uses a 500m L Erlenmeyer flask for fermentation.
  • the concentration of L-arginine in the fermentation broth can reach 30-32g/L.
  • the inoculum during fermentation in the fermentor is 15-20%, the fermentation temperature is 35°C, and the dissolved oxygen is between 25-35%.
  • the pH is controlled to stabilize at 7.0-7.2, and the pH can be adjusted by adding ammonia; when the glucose in the medium is consumed, 80% (m/v) glucose solution is added to maintain the glucose concentration in the fermentation medium In 0.1-5g/L.
  • the fermentor fermentation adopts a 5L fermentor for fermentation. After culturing in a 5L fermentor for 50-55h, the accumulation of L-arginine reached 130-135g/L. The conversion rate reaches 0.48g arginine/g glucose, and the production intensity reaches 2.5g arginine/L/h.
  • the fermentation medium of Escherichia coli known in the art can be used for fermentation.
  • the fermentation medium composition of shake flask fermentation is: glucose 20-40g/L, yeast extract 1-3g/L, peptone 2-3g/L, K 2 HPO 4 3-6g/L, MgSO 4 ⁇ 7H 2 O 1-2g/L, FeSO 4 ⁇ 7H 2 O 15-20mg/L, MnSO 4 ⁇ 7H 2 O 15-20mg/L, V B1 , V B3 , V B5 , V B12 , V H each 1 -3mg/L, the balance is water, pH 7.0-7.2.
  • the composition of the fermentation medium for fermentation in the fermentor is: glucose 10-25g/L, yeast extract 1-5g/L, peptone 1-5g/L, K 2 HPO 4 1-5g/L, MgSO 4 ⁇ 7H 2 O 1-3g/L, FeSO 4 ⁇ 7H 2 O 10-30mg/L, MnSO 4 ⁇ H 2 O 10-30mg/L, V B1 , V B3 , V B5 , V B12 , V H each 1-3mg/L, the balance is water, pH 7.0-7.2.
  • the present invention selects Escherichia coli with short growth cycle, clear metabolic pathway and convenient molecular operation as the starting strain.
  • the arginine in E. coli The synthesis pathway and the metabolic flow related to arginine in the entire amino acid metabolism network were analyzed and reconstructed to obtain a genetically engineered strain with a clear genetic background, no plasmid, no mutagenesis, and stable and efficient production of L-arginine .
  • the Escherichia coli obtained by the present invention constructs the circulation path of L-arginine, improves the flux of L-arginine and the supply of precursors, reduces the degradation of L-arginine, and promotes the accumulation and accumulation of L-arginine. Transport, thereby effectively increasing the production of L-arginine.
  • the genetically engineered bacteria for producing L-arginine of the present invention can accumulate 130-135 g/L of L-arginine after being cultured in a 5L fermentor for 50-55 hours.
  • the conversion rate can reach 0.48g arginine/g glucose, and the production intensity can reach 2.5g arginine/L/h.
  • the accumulation of L-arginine is 92.5g/L, the conversion rate is 0.35g arginine/g glucose, the maximum production intensity is 0.9g arginine/L/h)
  • the L-arginine production capacity of this strain is stronger, and it has not undergone mutagenesis. It has the advantages of processing, no plasmid carrier, short fermentation cycle, clear genetic background, stable metabolism, high production intensity, etc., and has good industrial application prospects.
  • Figure 1 (a) pREDCas9 plasmid map, (b) pGRB plasmid map.
  • Figure 2 Construction and verification electrophoresis of speA gene knockout fragment. Among them: M: 1kb DNA marker; 1: upstream homology arm; 2: downstream homology arm; 3: overlapping fragments; 4: original bacteria control; 5: positive bacteria identification fragments.
  • Figure 3 Construction and verification electropherogram of adiA gene knockout fragment. Among them: M: 1kb DNA marker; 1: upstream homology arm; 2: downstream homology arm; 3: overlapping fragments; 4: original bacteria control; 5: positive bacteria identification fragments.
  • Figure 4 Construction and verification electropherogram of astA gene knockout fragment. Among them: M: 1kb DNA marker; 1: upstream homology arm; 2: downstream homology arm; 3: overlapping fragments; 4: original bacteria control; 5: positive bacteria identification fragments.
  • Figure 5 Construction and verification electropherogram of argJ gene integration fragment. Among them: M: 1kb DNA marker; 1: upstream homology arm; 2: argJ fragment; 3: downstream homology arm; 4: overlapping fragment; 5: original bacteria control; 6: positive bacteria identification fragment.
  • Figure 6 Construction and verification electropherogram of argC-argJ integration fragment.
  • M 1kb DNA marker
  • 1 upstream homology arm
  • 2 argC-argJ fragment
  • 3 downstream homology arm
  • 4 overlapping fragment
  • 5 original bacteria control
  • 6 positive bacteria identification fragment.
  • Figure 7 Construction and verification electropherogram of argB-argD-argF integration fragment.
  • M 1kb DNA marker
  • 1 argB-argD-argF upstream fragment-argB-argD-argF gene fragment
  • 2 downstream homology arm
  • 3 overlapping fragment
  • 4 original bacteria control
  • 5 positive bacteria identification fragment .
  • Figure 8 Construction and verification electropherogram of argG-argH integration fragment.
  • M 1kb DNA marker
  • 1 upstream homology arm
  • 2 argG-argH fragment
  • 3 downstream homology arm
  • 4 overlapping fragment
  • 5 original bacteria control
  • 6 positive bacteria identification fragment.
  • Figure 9 Construction and verification electropherogram of the first integrated fragment of pyrAA-pyrAB.
  • M 1kb DNA marker
  • 1 upstream homology arm
  • 2 1-pyrAA-pyrAB fragment
  • 3 downstream homology arm
  • 4 overlapping fragment
  • 5 original bacteria control
  • 6 positive bacteria identification fragment.
  • Figure 10 Construction and verification electropherogram of the second integration fragment of pyrAA-pyrAB.
  • M 1kb DNA marker
  • 1 upstream fragment of pyrAA-pyrAA-pyrAB-downstream homology arm
  • 2 downstream homology arm
  • 3 overlapping fragment
  • 4 original bacteria control
  • 5 positive bacteria identification fragment.
  • Figure 11 lysE integration fragment construction and verification electropherogram.
  • M 1kb DNA marker
  • 1 upstream homology arm
  • 2 lysE fragment
  • 3 downstream homology arm
  • 4 overlapping fragment
  • 5 original bacteria control
  • 6 positive bacteria identification fragment.
  • Figure 12 Fed-batch fermentation process curve of strain E.coli W3110 ARG10 in a 5L fermentor.
  • the gene editing method used in 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-Cas9 meditated gene editing. Metabolic engineering, 2015, 31: 13-21.), The two plasmid maps used in this method are shown in Figure 1.
  • pREDCas9 carries gRNA expression plasmid pGRB elimination system, lambda phage Red recombination system and Cas9 protein expression system, spectinomycin resistance (working concentration: 100mg/L), cultured at 32°C;
  • pGRB uses pUC18 as the backbone, including the promoter J23100, gRNA-Cas9 binding region sequence and terminator sequence, ampicillin resistance (working concentration: 100mg/L), cultured at 37°C.
  • the purpose of constructing the plasmid pGRB is to transcribe the corresponding gRNA to form a complex with the Cas9 protein, and to recognize the target gene target site through base pairing and PAM to achieve the target DNA double-strand break.
  • the pGRB plasmid was constructed by recombining the DNA fragment containing the target sequence with the linearized vector fragment.
  • Design primers 5'-linearized vector end sequence (15bp)-restriction site-target sequence (not including PAM sequence)-linearized vector end sequence (15bp)-3' and its reverse complementary primer, through a single
  • the annealing of stranded DNA produces a DNA fragment containing the target sequence.
  • Reaction conditions pre-denaturation 95°C, 5min; annealing 30-50°C, 1min.
  • the annealing system is as follows:
  • the linearization of the vector adopts the method of inverse PCR amplification.
  • the reorganization system is shown in Table 2. All recombinases used are One Step Cloning Kit series of enzymes, recombination conditions: 37°C, 30min.
  • the PCR-positive colonies were inoculated into LB medium containing 100mg/L ampicillin and cultured overnight to preserve the bacteria.
  • the plasmids were extracted and identified by restriction enzyme digestion.
  • the recombination fragment used for knockout consists of the upstream and downstream homology arms of the gene to be knocked out (upstream homology arm-downstream homology arm); the recombination fragment used for integration is the upstream and downstream homology arms of the integration site and the homology arms to be integrated
  • the composition of the gene fragments (upstream homology arm-target gene-downstream homology arm).
  • primer design software primer5 use the upstream and downstream sequences of the gene to be knocked out or the site to be integrated as a template to design the upstream and downstream homology arm primers (amplification length is about 400-500bp); use the gene to be integrated as a template to design the integrated gene
  • the amplification primers After amplifying the upstream and downstream homology arms and the target gene fragments by PCR, the recombinant fragments are prepared by overlapping PCR.
  • Table 3 The PCR system and method are shown in Table 3:
  • PCR reaction conditions (PrimeSTAR HS enzyme): pre-denaturation (95°C) for 5min; then 30 cycles of denaturation (98°C) for 10s, annealing ((Tm-3/5)°C) for 15s, 72°C for extension; 72 Continue to extend for 10 min at °C; maintain (4 °C).
  • the pREDCas9 plasmid was electrotransformed into the electrotransformation competence of W3110 by the method of electroporation, the bacteria were resuscitated and cultured and spread on the LB plate containing spectinomycin, and cultured overnight at 32°C. A single colony grown on the resistant plate was subjected to colony PCR with identification primers to screen positive recombinants.
  • IPTG IPTG
  • a final concentration of 0.1 mM 0.1 mM
  • the pGRB and recombinant DNA fragments were simultaneously electrotransformed into electrocompetent cells containing pREDCas9. After electrotransformation, the resuscitated cells were spread on LB plates containing ampicillin and spectinomycin, and cultured overnight at 32°C.
  • the positive recombinants were cultured overnight in an LB medium containing 0.2% arabinose, diluted appropriately and spread on an LB plate containing spectinomycin resistance, and cultured overnight at 32°C. Transfer single colonies to the LB plates containing ampicillin and spectinomycin resistance respectively, and select the single colonies that did not grow on the ampicillin plate and the spectinomycin resistant plates to protect the bacteria.
  • the positive recombinants were transferred to non-resistant LB liquid medium and cultured at 42°C overnight, diluted appropriately and spread on a non-resistant LB plate, and cultured overnight at 37°C.
  • upstream homology arm primers UP-speA-S, UP-speA-A
  • downstream homology based on the upstream and downstream sequences of its speA gene (NCBI-GeneID: 12933352) Arm primers (DN-speA-S, DN-speA-A), and PCR amplification of its upstream and downstream homology arm fragments.
  • the above fragments were fused by overlapping PCR to obtain knock-out fragments of the speA gene (upstream homology arm-downstream homology arm).
  • the DNA fragments obtained by annealing the primers gRNA-speA-S and gRNA-speA-A were connected to the plasmid pGRB to construct the recombinant plasmid pGRB-speA.
  • Prepare competent cells of E. coli W3110 operate according to the methods shown in 1.3 and 1.4, and simultaneously electrotransform plasmid pGRB-speA and speA knock-out fragments into competent cells, and finally obtain the strain E. coli W3110 ARG1. See Figure 2 for the construction of the speA knockout fragment and the PCR verification of the positive strain.
  • the length of the upstream homology arm should be 397 bp
  • the length of the downstream homology arm should be 468 bp
  • the total length of overlapping fragments should be 865 bp.
  • the length of the PCR amplified fragment of the positive bacteria should be 2752 bp
  • the original bacteria PCR amplification The fragment length should be 865bp.
  • upstream homology arm primers UP-adiA-S, UP-adiA-A
  • downstream homology based on the upstream and downstream sequences of its adiA gene (NCBI-GeneID: 12934085) Arm primers (DN-adiA-S, DN-adiA-A), and PCR amplification of its upstream and downstream homology arm fragments.
  • the above fragments were fused by overlapping PCR to obtain knock-out fragments of the adiA gene (upstream homology arm-downstream homology arm).
  • the DNA fragments obtained by annealing the primers gRNA-adiA-S and gRNA-adiA-A are connected to the plasmid pGRB to construct pGRB-adiA.
  • Figure 3 shows the electropherogram of the construction of the adiA knockout fragment and the PCR verification of the positive strain.
  • the length of the upstream homology arm should be 806bp
  • the length of the downstream homology arm should be 402bp
  • the total length of overlapping fragments should be 1208bp.
  • the length of the PCR amplified fragment of positive bacteria should be 2124bp
  • the original bacteria should be amplified by PCR.
  • the fragment length should be 1208bp.
  • upstream homology arm primers UP-astA-S, UP-astA-A
  • downstream homology based on the upstream and downstream sequences of its adiA gene (NCBI-GeneID: 12933241)
  • Arm primers DN-astA-S, DN-astA-A
  • PCR amplification of its upstream and downstream homology arm fragments were fused by overlapping PCR to obtain knock-out fragments of the astA gene (upstream homology arm-downstream homology arm).
  • the DNA fragments prepared by annealing the primers gRNA-astA-S and gRNA-astA-A were connected to the plasmid pGRB to construct pGRB-astA.
  • Figure 4 shows the construction of the astA knockout fragment and the electrophoresis diagram of the PCR verification of the positive strain.
  • the length of the upstream homology arm should be 443bp
  • the length of the downstream homology arm should be 523bp
  • the total length of overlapping fragments should be 965bp.
  • the length of the PCR amplified fragment of the positive bacteria should be 1869bp
  • the original bacteria PCR amplification The fragment length should be 965bp.
  • E.coli W3110 (ATCC27325) genome as template, design the upstream homology arm primers (UP-argE-S, UP-argE-A) and downstream homology according to the upstream and downstream sequences of its argE gene (NCBI-GeneID: 12930574) Arm primers (DN-argE-S, DN-argE-A), PCR amplification of its upstream and downstream homology arm fragments; using the Corynebacterium glutamicum (ATCC13032) genome as a template, according to its argJ gene sequence (NCBI-GeneID: 1019371) Design primers (argJ-S, argJ-A), PCR amplify the argJ fragment; the promoter P trc is designed in the downstream primer of the upstream homology arm and the upstream primer of the argJ gene.
  • the above fragments were fused by overlapping PCR to obtain an integrated fragment (upstream homology arm-P trc- argJ-downstream homology arm) that knocked out the argE gene and integrated the argJ gene.
  • the primers gRNA-argE-S and gRNA-argE- A DNA fragment prepared by annealing was ligated with plasmid pGRB to construct pGRB-argE.
  • Prepare competent cells of E. coli W3110 ARG3 operate according to the methods shown in 1.3 and 1.4, transform plasmid pGRB-argE and knock-out argE gene and integrate the integrated fragment of argJ gene at the same time into competent cells, and finally obtain strain E .coli W3110 ARG4.
  • the construction of the integrated fragment and the electrophoresis diagram of the PCR verification of the positive strains are shown in Figure 5.
  • the length of the upstream homology arm is 510bp
  • the length of the argJ gene fragment is 1206bp
  • the length of the downstream homology arm is 668bp
  • the length of the overlapping fragment is 2458bp.
  • the length of the fragment amplified by the positive recombinant should be 2458bp.
  • the length of the amplified fragment from the original bacteria should be 2154bp.
  • arginine synthesis operating genes including argC, argJ, argB, argD, argF, argG, and argH
  • the transcriptional expression of the foreign operon was initiated, and the strain E.coli W3110 ARG7 was constructed.
  • E.coli W3110 (ATCC27325) genome as template, design the upstream homology arm primers (UP-yghX-S, UP-yghX-A) and downstream homology arm primers (DN-yghX- S1, DN-yghX-A), PCR amplification of its upstream and downstream homology arm fragments; using the Corynebacterium glutamicum (ATCC 13032) genome as a template, according to its argC-argJ gene sequence (NCBI-GeneID: 1019370, 1019371) The primers (argC-argJ-S, argC-argJ-A) were designed to amplify the argC-argJ fragment by PCR; the promoter P trc was designed in the downstream primer of the upstream homology arm and the upstream primer of the argC-argJ gene.
  • UP-yghX-S, UP-yghX-A downstream homology arm primers
  • the above fragments were fused by overlapping PCR to obtain an integrated fragment of the argC-argJ gene (upstream homology arm-P trc- argC-argJ-downstream homology arm), and the primers gRNA-yghX-S and gRNA-yghX-A were annealed
  • the DNA sequence containing the target sequence is prepared, and the plasmid pGRB is connected to construct pGRB-yghX.
  • the upstream homology arm primers (UP-argB-argD-argF-S) were designed according to argB-argD-argF (NCBI-GeneID: 1019372, 1019373, 1019374) and its upstream sequence.
  • UP-argB-argD-argF-A PCR amplifies the upstream homology arm fragment; using E. coli W3110 (ATCC27325) genome as a template, the downstream homology arm primer (DN- yghX-S2, DN-yghX-A), PCR amplification of its downstream homology arm fragments.
  • the above fragments were fused by overlapping PCR to obtain an integrated fragment of argB-argD-argF (upstream fragment of argB-argB-argD-argF-downstream homology arm).
  • the primers gRNA-argBDF-S and gRNA-argBDF-A were annealed to prepare a DNA fragment containing the target sequence, which was ligated with plasmid pGRB to construct pGRB-argBDF.
  • Prepare E. coli W3110 ARG5 competent cells operate according to the methods shown in 1.3 and 1.4, and simultaneously electrotransform the integrated fragments of plasmid pGRB-argBDF and argB-argD-argF into competent cells, and finally obtain the strain E.
  • the upstream homology arm primers (UP-argG-argH-S, UP-argG-argH) were designed according to argG-argH (NCBI-GeneID: 1019376, 1019377) and its upstream sequence.
  • -A) and argG-argH fragment primers (argG-argH-S, argG-argH-A), PCR amplification of the upstream homology arm fragment and argG-argH fragment; using E.
  • the downstream homology arm primers (DN-yghX-S3, DN-yghX-A) were designed according to the downstream sequence of the yghX gene, and the downstream homology arm fragments were amplified by PCR. The above fragments were fused by overlapping PCR to obtain an integrated fragment of argG-argH (upstream fragment of argG-argG-argH-downstream homology arm).
  • the primers gRNA-argG-argH-S and gRNA-argG-argH-A were degraded to prepare a DNA sequence containing the target sequence, which was connected with the plasmid pGRB to construct pGRB-argG-argH. Prepare E.
  • coli W3110 ARG6 competent cells operate according to the methods shown in 1.3 and 1.4, and simultaneously electrotransform the integrated fragments of plasmid pGRB-argG-argH and argG-argH into competent cells, and finally obtain the strain E. coli W3110 ARG7.
  • the construction of the integrated fragment and the electrophoresis diagram of the PCR verification of the positive strains are shown in Figure 8.
  • the total length of the upstream fragment of argG is 405bp
  • the total length of the argG-argH fragment is 2826bp
  • the length of the downstream homology arm is 561bp
  • the total length of overlapping fragments should be 3875bp
  • the length of the fragment amplified by the identification primer should be 1521bp.
  • the bacteria should have no bands.
  • B.subtilis A260 is the starting strain of Bacillus subtilis 168 strain, which is selected by the method of combining ARTP mutagenesis and high-throughput screening (this strain was deposited in the Chinese Microbial Culture Collection on December 2, 2015 General Microbiology Center of the Management Committee, Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Zip Code: 100101, Culture Collection Number: CGMCC No.11775).
  • the pyrAA-pyrAB gene of Bacillus subtilis is 4292bp integrated into E. coli in two segments, the first segment is 2651bp in length and the second segment is 1641bp in length.
  • upstream homology arm primers UP-yjiT-S, UP-yjiT-A
  • downstream homology arm primers DN-yjiT-A
  • 11775 genome as a template, according to the gene pyrAA (NCBI-GeneID: 937368), pyrAB (NCBI- GeneID: 936608) design primers (1-pyrAA-pyrAB-S, 1-pyrAA-pyrAB-A) to amplify the first pyrAA-pyrAB gene fragment.
  • the promoter P trc is designed in the downstream primer of the upstream homology arm and the upstream primer of the pyrAA-pyrAB gene.
  • the above fragments were fused by overlapping PCR to obtain the integrated fragment of the first pyrAA-pyrAB gene (upstream homology arm-P trc- pyrAA-pyrAB-downstream homology arm), and the primers gRNA-yjiT-S and gRNA-yjiT- A is annealed to prepare a DNA fragment containing the target sequence, which is ligated with plasmid pGRB to construct pGRB-yjiT.
  • Prepare competent cells of E.coli W3110 ARG7 operate according to the methods shown in 1.3 and 1.4, and simultaneously electrotransform the plasmid pGRB-yjiT and the integrated fragment of the first pyrAA-pyrAB gene into competent cells, and finally obtain strain E.
  • Figure 9 shows the construction of the first P trc -pyrAA-pyrAB integration fragment and the electrophoresis diagram of the PCR verification of the positive strains.
  • the length of the upstream homology arm should be 316bp
  • the length of the first pyrAA-pyrAB gene fragment should be 2651bp
  • the length of the downstream homology arm should be 667bp
  • the total length of the integrated fragment should be 3634bp.
  • the length of the fragment amplified by the identification primer It should be 1100bp, and the original bacteria should have no bands.
  • PCR amplification of the upstream homology arm fragments including the first pyrAA-pyrAB downstream sequence 266bp and the integrated second pyrAA-pyrAB sequence 1641bp total 1907bp; using the E.coli W3110 (ATCC27325) genome as a template, according to its The downstream sequence of yjiT gene was designed with downstream homology arm primers (DN-yjiT-S1, DN-yjiT-A), and the downstream homology arm fragment was amplified by PCR. The above fragments were fused by overlapping PCR to obtain the second pyrAA-pyrAB integrated fragment (the second pyrAA-pyrAB-downstream homology arm).
  • the primers gRNA-pyrAA-pyrAB-S and gRNA-pyrAA-pyrAB-A are annealed to prepare a DNA fragment containing the target sequence, which is connected with the plasmid pGRB to construct pGRB-pyrAA-pyrAB.
  • E.coli W3110 (ATCC27325) genome as template, design the upstream homology arm primers (UP-ilvG-S, UP-ilvG-A) and downstream homology arm primers (DN-ilvG- S, DN-ilvG-A), PCR amplification of its upstream and downstream homology arm fragments; according to the lysE gene (NCBI Reference Sequence: WP_143758438.1) sequence (SEQ ID NO: 68) design primers (lysE-S, lysE-A) ), amplify the lysE gene fragment.
  • the promoter P trc is designed in the downstream primer of the upstream homology arm and the upstream primer of the lysE gene.
  • the above fragments were fused by overlapping PCR to obtain the integrated fragment of the lysE gene (upstream homology arm-P trc- lysE-downstream homology arm), and the ilvG primers gRNA-ilvG-S and gRNA-ilvG-A were annealed to obtain the target containing
  • the DNA fragment of the sequence was ligated with plasmid pGRB to construct pGRB-ilvG.
  • Prepare competent cells of E.coli W3110 ARG9 operate according to the methods shown in 1.3 and 1.4, and simultaneously electrotransform the integrated fragments of plasmid pGRB-ilvG and lysE genes into competent cells, and finally obtain the strain E.coli W3110 ARG10.
  • the length of the upstream homology arm should be 412 bp
  • the length of the P trc -lysE gene fragment should be 806 bp
  • the length of the downstream homology arm should be 481 bp
  • the total length of the integrated fragment should be 1699 bp.
  • the positive bacteria PCR amplification The fragment length should be 1699bp
  • the original bacteria PCR amplified fragment length should be 1426bp.
  • Slant culture streak inoculate the preserved strains on the activated slant at -80°C, culture for 12h at 37°C, and pass them once;
  • Shake flask seed culture Use an inoculating loop to scrape a loop of slant seeds and inoculate them in a 500mL Erlenmeyer flask containing 30mL seed culture medium, seal with nine layers of gauze, cultivate for 7-10h at 37°C, 200rpm;
  • Shake flask fermentation culture inoculate 15% of the seed culture solution volume into a 500mL Erlenmeyer flask containing fermentation medium (final volume 30mL), seal with nine layers of gauze, 37°C, 200r/min shaking culture, fermentation process
  • the pH is maintained at 7.0-7.2 by adding ammonia water; the fermentation is maintained by adding 60% (m/v) glucose solution; the fermentation period is 26-30h;
  • composition of the slant medium is: glucose 1g/L, peptone 10g/L, beef extract 10g/L, yeast powder 5g/L, NaCl 2.5g/L, agar 20g/L, the rest is water, pH 7.0-7.2;
  • the composition of the seed culture medium is: glucose 25g/L, yeast extract 5g/L, peptone 3g/L, K 2 HPO 4 1g/L, MgSO 4 ⁇ 7H 2 O 1g/L, FeSO 4 ⁇ 7H 2 O 10mg/L , MnSO 4 ⁇ 7H 2 O 10mg/L, V B1 , V B3 , V B5 , V B12 , V H each 1 mg/L, the rest is water, pH 7.0-7.2.
  • the composition of the fermentation medium is: glucose 25g/L, yeast extract 3g/L, peptone 2g/L, K 2 HPO 4 3g/L, MgSO 4 ⁇ 7H 2 O 2g/L, FeSO 4 ⁇ 7H 2 O 10mg/L , MnSO 4 ⁇ 7H 2 O 10mg/L, V B1 , V B3 , V B5 , V B12 , V H each 1 mg/L, the rest is water, pH 7.0-7.2.
  • the yield of L-arginine in the fermentation broth of E.coli W3110 ARG10 strain is 30-32g/L.
  • Slope activation culture scrape a ring of bacteria from the -80°C refrigerator preservation tube, spread it evenly on the activation slope, incubate at 37°C for 12-16h, transfer to eggplant-shaped flask and continue culturing for 12-16h;
  • Seed culture Take an appropriate amount of sterile water in an eggplant-shaped bottle, and connect the bacterial suspension to the seed culture medium.
  • the pH is stabilized at about 7.0, the temperature is constant at 37°C, and the dissolved oxygen is between 25-35%, and cultivate until the cells Dry weight reaches 5-6g/L;
  • Fermentation culture Connect fresh fermentation medium according to 15% inoculum to start fermentation. During the fermentation process, control pH to stabilize at about 7.0, maintain temperature at 35°C, and dissolved oxygen between 25-35%; After the glucose is consumed, add 80% (m/v) glucose solution to maintain the glucose concentration in the fermentation medium at 0.1-5g/L;
  • Slant medium, seed medium and fermentation medium are the same as shake flask fermentation.

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Abstract

公开了一种生产L-精氨酸的基因工程菌及其构建方法与应用。该方法包括在大肠杆菌中通过整合编码氨甲酰磷酸合成酶的基因和编码L-精氨酸生物合成途径酶的基因，对大肠杆菌中精氨酸合成途径和整个氨基酸代谢网络中与精氨酸相关的代谢流进行分析重构，得到遗传背景清晰，不携带质粒，不经诱变且能稳定高效生产L-精氨酸的基因工程菌。

Description

生产L-精氨酸的基因工程菌及其构建方法与应用
本申请要求2019年12月02日向中国国家知识产权局提交的专利申请号为201911211097.X，发明名称为“生产L-精氨酸的基因工程菌及其构建方法与应用”的在先申请的优先权，该在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明属于基因工程技术领域，具体涉及一种稳定高效生产L-精氨酸的基因工程菌及其构建方法与应用。
背景技术
L-精氨酸是人体和动物体内的半必需碱性氨基酸或条件性必需氨基酸，具有重要的生化和生理学功能。目前，L-精氨酸已在医药、工业、食品、化妆品、畜牧业等领域有广泛的应用，具有重要的经济和社会价值。
L-精氨酸的生产方法主要包括蛋白质水解法和微生物发酵法，相比蛋白质水解提取法，微生物发酵法具有生产工艺相对简单、对环境影响相对较小、产品纯度高的优势，适合大规模工业生产。
目前精氨酸生产菌种主要为谷氨酸棒状杆菌，在生产过程中存在发酵周期长(90h-120h)，生产强度低的问题，同时现有的谷氨酸棒杆菌的发酵工艺受辅料如玉米浆的质量影响较大，生产易波动。并且谷氨酸棒状杆菌的基因编辑难度较大，致使在现有精氨酸菌株多采用质粒表达载体强化精氨酸合成相关的关键基因，但是在发酵过程中质粒多拷贝对菌体生长造成一定的负担，造成发酵后期产量下降，而且在生产过程中质粒表达载体容易丢失或者需要添加一定的选择性压力，造成工业生产过程中成本过高的问题。诸多因素造成目前的精氨酸菌株很难投入到工业化生产中。
由于精氨酸合成代谢途径中存在诸多反馈调控，且精氨酸代谢途径多，合成精氨酸所需前体物涉及到的代谢网络复杂，因此最初精氨酸工业生产菌株的研发主要采用传统诱变结合精氨酸结构类似物抗性筛选的方法。选用的出发菌株主要为黄色短杆菌、钝齿棒杆菌、谷氨酸棒杆菌，研究策略集中在筛选精氨酸结构类似物突变体，以解除精氨酸合成过程中的反馈 调控，提高胞内L-精氨酸的积累量。其中，李少平等经过NTG逐级诱变筛选到一株具有组氨酸缺陷型、磺胺胍抗性、D-精氨酸抗性、高精氨酸抗性、S-甲基半胱氨酸抗性的钝齿棒杆菌(CN201010610917.5)，经过发酵实验验证，在5L发酵罐中培养96h，L-精氨酸的积累量为32.8g/L。但是经过诱变和结构类似物筛选得到的生产菌株，由于遗传稳定性差，容易产生回复突变等缺点很难投入到大批量工业生产中。
随着基因工程技术的飞速发展，运用代谢工程技术构建L-精氨酸生产菌株的方法逐渐替代了传统诱变育种方法。谷氨酸棒状杆菌中没有涉及到精氨酸降解的相关基因；而且谷氨酸作为精氨酸合成的主要的前体物之一，谷氨酸棒状杆菌胞内摄取的葡萄糖经过糖酵解途径生成谷氨酸的代谢流量较强，因此谷氨酸棒状杆菌作为构建L-精氨酸生产菌株的主要选择。徐美娟等(Xu M,Rao Z,Yang J,et al.J Ind Microbiol Biotechnol,2012,39(3):495-502.)将合成L-精氨酸的基因簇argCJBDFRGH连接到pJCtac穿梭表达载体上并将其导入钝齿棒状杆菌，该菌株经过96h发酵后，L-精氨酸产量提高到45.6g/L。Park等(Park S H,Kim H U,Kim T Y,et al.Nature Communications,2014,5:4618---)以谷氨酸棒状杆菌为出发菌株，通过随机诱变提高谷氨酰胺对L-精氨酸结构类似物的耐受性，运用系统代谢工程技术解除精氨酸合成过程中反馈抑制，加强合成过程中NADPH的供应，增强前体物的供应，最终经过5L发酵罐发酵96h，L-精氨酸的积累量为92.5g/L，转化率为0.35g精氨酸/g葡萄糖，最大生产强度0.9g精氨酸/L/h。上述生产菌株普遍存在生产周期长，生产强度低的问题，而且在菌株构建过程中采用将精氨酸合成关键基因连接到表达载体上以提高关键酶的转录量，进而增强精氨酸合成途径代谢流量，但是在生产过程中表达载体容易丢失或者需要加入一定的选择性压力，难以投入工业化生产。
大肠杆菌由于发酵周期短，遗传背景清晰，分子操作便捷和发酵工艺稳定等优势，成为构建L-精氨酸工业生产菌株的更好选择。Ginesy等(Ginesy M,Belotserkovsky J,Enman J,et al.Microbial Cell Factories,2015,14(1):29.)以大肠杆菌为出发菌株，敲除argR以解除精氨酸的反馈阻遏，整合突变基因argA214(H15Y)以解除精氨酸对ArgA的反馈抑制，敲除精氨酸降解相关基因adiA，敲除鸟氨酸降解相关基因speC和speF，使中间代谢产物的碳流量更多的流向L-精氨酸。在1L发酵罐中培养42h后，L-精氨酸的积累量达到11.64g/L。转化率为0.44g精氨酸/g葡萄糖，生产强度为0.29g精氨酸/L/h。虽然该菌株发酵周期明显缩短，但其精氨酸积累量和生产强度还不能满足工业生产的要求。
发明内容
针对上述存在问题，本发明目的是提供一株稳定高效生产L-精氨酸的基因工程菌及其构建方法与应用，该工程菌具有很好的工业应用前景。
本发明提供如下技术方案：
第一方面，本发明提供一种生产L-精氨酸的基因工程菌株，含有编码氨甲酰磷酸合成酶的基因pyrAA、和pyrAB。
在一个实施方案中，所述基因工程菌株以大肠杆菌或谷氨酸棒杆菌为出发菌株；例如以E.coli W3110或者E.coli MG1655为出发菌株。
在一个实施方案中，所述pyrAA、和pyrAB基因整合于大肠杆菌的yjiT基因位点。
在一个实施方案中，所述pyrAA和pyrAB来源于枯草芽孢杆菌，具体地，所述pyrAA和pyrAB来源于B.subtilis A260中编码氨甲酰磷酸合成酶的基因。
在一个实施方案中，所述基因工程菌株还含有编码L-精氨酸生物合成途径酶的基因，所述-精氨酸生物合成途径酶选自以下酶的一种或多种：argC、argJ、argB、argD、argF、argG、argH；所述编码L-精氨酸生物合成途径酶的基因来源于谷氨酸棒杆菌(Corynebacterium glutamicum ATCC13032)；在一个实施方案中，所述编码L-精氨酸生物合成途径酶的基因由P trc启动子启动；在一个实施方案中，所述编码L-精氨酸生物合成途径酶的基因整合于大肠杆菌的yghX基因位点。
在一个实施方案中，所述基因工程菌株进一步含有编码精氨酸转运蛋白的基因lysE(NCBI Reference Sequence:WP_143758438.1)，所述转运蛋白基因来源于有效棒杆菌(Corynebacterium efficiens)；在一个实施方案中，所述lysE基因整合于大肠杆菌的ilvG基因位点。
在一个实施方案中，所述基因工程菌株不含有分解L-精氨酸的编码基因，例如敲除如下一种或多种基因：编码精氨酸脱羧酶的基因、编码精氨酸琥珀酰转移酶的基因、编码乙酰鸟氨酸脱乙酰基酶的基因。所述编码精氨酸脱羧酶的基因包括speA(NCBI-GeneID:12933352)、adiA(NCBI-GeneID:12934085)中的至少一种；所述编码精氨酸琥珀酰转移酶的基因为astA(NCBI-GeneID:12933241)；所述编码乙酰鸟氨酸脱乙酰基酶的基因为argE(NCBI-GeneID:12930574)。在一个实施方案中，所述基因工程菌株是同时敲除speA、adiA、和astA基因的大肠杆菌。
在一个实施方案中，所述基因工程菌株含有pyrAA、pyrAB、argC、argJ、argB、argD、argF、argG、argH、和lysE基因。在一个实施方案中，所述基因工程菌株不含有speA、adiA、 astA和argE基因。
在本发明中，pyrAA、pyrAB、argC、argJ、argB、argD、argF、argG、argH、lysE、speA、adiA、astA和argE基因并不限于野生型基因，也可以是编码相应蛋白的突变体或者经人工修饰的基因，包括一个或多个位点的取代、缺失或者插入一个或多个氨基酸残基，只要该突变体或经人工修饰的基因所编码的蛋白具有相应的活性，没有功能缺陷。这些基因均已在GenBank有登记，本领域技术人员可通过PCR获得这些基因。作为示例，pyrAA基因为NCBI-GeneID:937368，pyrAB基因为NCBI-GeneID:936608，argC基因为NCBI-GeneID:1019370，argJ基因为NCBI-GeneID:1019371，argB基因为NCBI-GeneID:1019372，argD基因为NCBI-GeneID:1019373，argF基因为NCBI-GeneID:1019374，argG基因为NCBI-GeneID:1019376，argH基因为NCBI-GeneID:1019377，lysE基因为SEQ ID NO:68所示的核苷酸序列(NCBI Sequence ID:WP_143758438.1)；speA基因为NCBI-GeneID:12933352，adiA基因为NCBI-GeneID:12934085，astA基因为NCBI-GeneID:12933241；argE基因为NCBI-GeneID:12930574。
第二方面，本发明提供上述基因工程菌株的构建方法，包括如下步骤：(1)出发菌株的基因组中整合pyrAA、pyrAB基因。
示例性地，所述出发菌株为大肠杆菌，例如E.coli W3110(ATCC27325)。
在一个实施方案中，所述构建方法还包括任选地如下一个或多个步骤：
(2)整合精氨酸生物合成途径酶基因，包括argC、argJ、argB、argD、argF、argG、argH中的一种或多种；和/或整合编码精氨酸转运蛋白的基因lysE；
(3)敲除编码精氨酸脱羧酶的基因、编码精氨酸琥珀酰转移酶的基因，和/或编码乙酰鸟氨酸脱乙酰基酶的基因；例如，所述编码精氨酸脱羧酶的基因包括speA、adiA中的至少一种；所述编码精氨酸琥珀酰转移酶的基因为astA；所述编码乙酰鸟氨酸脱乙酰基酶的基因为argE。
在一个实施方案中，所述构建方法包括如下步骤：
(1)将大肠杆菌上编码精氨酸脱羧酶的基因speA、编码精氨酸脱羧酶的基因adiA和编码精氨酸琥珀酰转移酶的基因astA这三个基因敲除；
(2)敲除大肠杆菌中编码乙酰鸟氨酸脱乙酰基酶的基因argE，任选地将编码谷氨酸乙酰基转移酶的基因argJ整合到大肠杆菌中；
(3)整合如下精氨酸生物合成相关基因簇：argC、argJ、argB、argD、argF、argG和argH，并由P trc启动子启动；
(4)整合编码氨甲酰磷酸合成酶的基因pyrAA和pyrAB；
(5)将编码精氨酸转运蛋白的基因lysE整合到大肠杆菌基因组上。
本领域技术人员可以理解，本发明的上述构建方法的步骤(1)至(5)的顺序并不限制，可以本领域技术人员可实施的任何顺序进行。优选地采用如步骤(1)至(5)依次进行的方式。
可以采用本领域已知的任何基因敲除或基因沉默的方法实现上述基因的敲除，也可以采用本领域已知的任何方法实现基因的整合，例如同源重组，重叠PCR，诱变筛选或者基因编辑等技术。例如，基因敲除可以通过去除基因中某一特定区域使其不具有表达目的蛋白的功能，或者通过位点特异性突变在编码区或启动子区引入一个或多个核苷酸序列的取代、缺失、插入等等，还可以用化学试剂是的该特定基因编码的转录下降或消失。
在一个实施方案中，所述构建方法包括采用CRISPR/Cas9介导的基因编辑技术进行基因整合和敲除。
在一个实施方案中，所述构建方法包括构建重组片段和pGRB质粒。
在一个实施方案中，所述pGRB质粒的构建包括：设计靶序列，制备包含靶序列的DNA片段，将包含靶序列的DNA片段与线性化的载体片段重组；在一个具体实施方案中，所述靶序列为5’-NGG-3’。
在一个实施方案中，所述构建方法中构建重组片段包括构建基因整合的重组片段或者构建基因敲除的重组片段。其中，构建基因整合的重组片段的步骤包括：以出发菌株的基因组为模板，根据目的基因拟插入位点的上下游序列设计上下游同源臂引物，并根据目的基因组设计引物，扩增目的基因片段，再通过PCR重叠技术获得重组片段。构建基因敲除的重组片段的步骤包括：以待敲除基因的上下游序列为模板，设计上下游同源臂引物；通过PCR的方法分别扩增上下游同源臂，再经过重叠PCR制备重组片段。
在一个实施方案中，所述构建方法包括：将pGRB质粒和上述重组片段同时转化至含有pREDCas9的电转感受态细胞中，以及质粒消除的步骤，获得重组的基因工程菌株。
本发明提供上述基因工程菌在制备L-精氨酸中的应用。
本发明还提供了利用上述基因工程菌发酵生产L-精氨酸的方法，包括：将上述大肠杆菌基因工程菌株与发酵培养基接触，进行发酵培养，制备得到L-精氨酸。
根据本发明，所述发酵培养包括摇瓶发酵或者发酵罐发酵。
在一个实施方案中，摇瓶发酵接种量为10-15％，发酵条件为，37℃，200r/min振荡培养，发酵过程中维持pH在7.0-7.2，可通过补加氨水调节pH。在发酵过程中还可以补加葡萄糖溶液维持发酵进行，优选所述葡萄糖溶液的质量体积浓度为60％(m/v)。优选地，所述摇瓶发酵的发酵时间为26-30h。本发明中对于葡萄糖溶液的补加量没有特别的限定，可维持发酵液中葡萄糖浓度为5g/L以下，例如1-5g/L。
在一个实施方案中，所述摇瓶发酵用500m L三角瓶进行发酵。当摇瓶发酵26-30h，发酵液中L-精氨酸的浓度可以达到30-32g/L。
在一个实施方案中，发酵罐发酵时接种量为15-20％，发酵温度为35℃，溶氧在25-35％之间。发酵过程中控制pH稳定在7.0-7.2，可通过补加氨水调节pH；当培养基中的葡萄糖消耗完之后，流加80％(m/v)的葡萄糖溶液，维持发酵培养基中的葡萄糖浓度在0.1-5g/L。
在一个实施方案中，所述发酵罐发酵采用5L发酵罐进行发酵。在5L发酵罐中培养50-55h后，L-精氨酸的积累量达到130-135g/L。转化率达到0.48g精氨酸/g葡萄糖，生产强度达到2.5g精氨酸/L/h。
本发明可以采用本领域已知的大肠杆菌发酵培养基进行发酵。
在一个实施方案中，摇瓶发酵的发酵培养基组成为：葡萄糖20-40g/L，酵母提取物1-3g/L，蛋白胨2-3g/L，K 2HPO 4 3-6g/L，MgSO 4·7H 2O 1-2g/L，FeSO 4·7H 2O 15-20mg/L，MnSO 4·7H 2O15-20mg/L，V B1、V B3、V B5、V B12、V H各1-3mg/L，余量为水，pH 7.0-7.2。
在一个实施方案中，发酵罐发酵的发酵培养基组成为：葡萄糖10-25g/L，酵母提取物1-5g/L，蛋白胨1-5g/L，K 2HPO 4 1-5g/L，MgSO 4·7H 2O 1-3g/L，FeSO 4·7H 2O 10-30mg/L，MnSO 4·H 2O 10-30mg/L，V B1、V B3、V B5、V B12、V H各1-3mg/L，余量为水，pH 7.0-7.2。
有益效果：
本发明选用生长周期短、代谢途径清晰、分子操作便捷的大肠杆菌为出发菌株，从L-精氨酸合成代谢途径的基因工程改造和整个代谢网络的工程改造出发，对大肠杆菌中精氨酸合成途径和整个氨基酸代谢网络中与精氨酸相关的代谢流进行分析重构，得到一株遗传背景清晰，不携带质粒，不经诱变且能稳定高效生产L-精氨酸的基因工程菌株。
本发明获得的大肠杆菌构建了L-精氨酸的循环路径，提高L-精氨酸通量和前体物供应，降低L-精氨酸的降解，并促进L-精氨酸的积累和转运，从而有效提高了L-精氨酸的产量。
本发明的生产L-精氨酸的基因工程菌，在5L发酵罐中培养50-55h后，L-精氨酸的积累量可达到130-135g/L。转化率可达0.48g精氨酸/g葡萄糖，生产强度可达2.5g精氨酸/L/h，与2014年Park等报道菌株(5L发酵罐发酵96h，L-精氨酸的积累量为92.5g/L，转化率为0.35g精氨酸/g葡萄糖，最大生产强度0.9g精氨酸/L/h)相比，该菌株产L-精氨酸能力更强，并且不经过诱变处理、无质粒载体、发酵周期短，具有遗传背景清晰、代谢稳定、生产强度高等优势，具有很好的工业应用前景。
附图说明
图1：(a)pREDCas9质粒图谱，(b)pGRB质粒图谱。
图2：speA基因敲除片段的构建及验证电泳图。其中：M：1kb DNA marker；1：上游同源臂；2：下游同源臂；3：重叠片段；4：原菌对照；5：阳性菌鉴定片段。
图3：adiA基因敲除片段的构建及验证电泳图。其中：M：1kb DNA marker；1：上游同源臂；2：下游同源臂；3：重叠片段；4：原菌对照；5：阳性菌鉴定片段。
图4：astA基因敲除片段的构建及验证电泳图。其中：M：1kb DNA marker；1：上游同源臂；2：下游同源臂；3：重叠片段；4：原菌对照；5：阳性菌鉴定片段。
图5：argJ基因整合片段构建及验证电泳图。其中：M：1kb DNA marker；1：上游同源臂；2：argJ片段；3：下游同源臂；4：重叠片段；5：原菌对照；6：阳性菌鉴定片段。
图6：argC-argJ整合片段构建及验证电泳图。其中：M：1kb DNA marker；1：上游同源臂；2：argC-argJ片段；3：下游同源臂；4：重叠片段；5：原菌对照；6：阳性菌鉴定片段。
图7：argB-argD-argF整合片段的构建及验证电泳图。其中：M：1kb DNA marker；1：argB-argD-argF上游片段-argB-argD-argF基因片段；2：下游同源臂；3：重叠片段；4:原菌对照；5：阳性菌鉴定片段。
图8：argG-argH整合片段构建及验证电泳图。其中：M：1kb DNA marker；1：上游同源臂；2：argG-argH片段；3：下游同源臂；4：重叠片段；5：原菌对照；6：阳性菌鉴定片段。
图9：pyrAA-pyrAB第一段整合片段构建及验证电泳图。其中：M：1kb DNA marker；1：上游同源臂；2：1-pyrAA-pyrAB片段；3：下游同源臂；4：重叠片段；5：原菌对照；6：阳性菌鉴定片段。
图10：pyrAA-pyrAB第二段整合片段构建及验证电泳图。其中：M：1kb DNA marker；1：pyrAA的上游片段-pyrAA-pyrAB-下游同源臂；2：下游同源臂；3：重叠片段；4:原菌对照； 5：阳性菌鉴定片段。
图11：lysE整合片段构建及验证电泳图。其中：M：1kb DNA marker；1：上游同源臂；2：lysE片段；3：下游同源臂；4：重叠片段；5：原菌对照；6：阳性菌鉴定片段。
图12：菌株E.coli W3110 ARG10在5L发酵罐中分批补料发酵过程曲线。
具体实施方式
下文通过对本发明实施例的描述，更加详细地对本发明的上述及其他特性和优势进行解释和说明。应当理解，下列实施例旨在对本发明的技术方案进行示例性的说明，而并非旨在对由权利要求及其等价方案所限定的本发明保护范围进行任何限制。
除非另有说明，本文中的材料和试剂均为市售商品，或可由本领域技术人员根据现有技术制备。
实施例1：
基因工程菌E.coli W3110 ARG10的构建：
1基因编辑的方法
本发明中采用的基因编辑方法参照文献(Li Y,Lin Z,Huang C,et al.Metabolic engineering of Escherichia coli using CRISPR–Cas9 meditated genome editing.Metabolic engineering,2015,31:13-21.)进行，该方法所用的两个质粒图谱见图1。其中pREDCas9携带gRNA表达质粒pGRB的消除系统，λ噬菌体的Red重组系统及Cas9蛋白表达系统，奇霉素抗性(工作浓度：100mg/L)，32℃培养；pGRB以pUC18为骨架，包括启动子J23100，gRNA-Cas9结合区域序列和终止子序列，氨苄青霉素抗性(工作浓度：100mg/L)，37℃培养。
该方法的具体步骤如下：
1.1 pGRB质粒构建
构建质粒pGRB的目的是为了转录相应的gRNA，从而与Cas9蛋白形成的复合体，并通过碱基配对和PAM识别目的基因靶位点，实现目的DNA双链断裂。采用包含靶序列的DNA片段与线性化的载体片段重组的方法构建pGRB质粒。
1.1.1靶序列设计
使用CRISPR RGEN Tools设计靶序列(PAM:5’-NGG-3’)
1.1.2包含靶序列的DNA片段的制备
设计引物：5’-线性化载体末端序列(15bp)-酶切位点-靶序列(不包括PAM序列)-线性化载体末端序列(15bp)-3’及其反向互补的引物，通过单链DNA的退火制备包含靶序列的DNA片段。反应条件：预变性95℃，5min；退火30-50℃，1min。退火体系如下表1：
表1退火体系
Figure PCTCN2020090626-appb-000001
1.1.3线性载体的制备
载体的线性化采用反向PCR扩增的方法。
1.1.4重组反应
重组体系如下表2。所用重组酶均为
Figure PCTCN2020090626-appb-000002
One Step Cloning Kit系列的酶，重组条件：37℃，30min。
表2重组体系
Figure PCTCN2020090626-appb-000003
1.1.5质粒的转化
取10μL反应液，加入到100mL DH5α化转感受态细胞中，轻轻混匀后冰浴20min，42℃热激45-90s，立即冰浴2-3min，加入900μL SOC，于37℃复苏1h。8000rpm离心2min，弃部分上清，留200μL左右将菌体重悬后涂布到含有100mg/L氨苄青霉素的平板，将平板倒置，于37℃过夜培养。待平板长出单菌落后通过菌落PCR鉴定，挑选阳性重组子。
1.1.6克隆鉴定
将PCR阳性菌落接种至含有100mg/L氨苄青霉素的LB培养基中过夜培养后保菌，提取质粒，酶切鉴定。
1.2重组DNA片段的制备
用于敲除的重组片段由需敲除基因的上下游同源臂组成(上游同源臂-下游同源臂)；用于整合的重组片段以整合位点的上下游同源臂及待整合的基因片段组成(上游同源臂-目的基因-下游同源臂)。利用引物设计软件primer5，以待敲除基因或待整合位点的上下游序列为模板，设计上下游同源臂引物(扩增长度约400-500bp)；以待整合基因为模板，设计整合基 因的扩增引物。通过PCR的方法分别扩增上下游同源臂和目的基因片段后，再经过重叠PCR制备重组片段。PCR的体系和方法如下表3：
表3 PCR扩增体系
Figure PCTCN2020090626-appb-000004
重叠PCR的体系如下表4：
表4重叠PCR扩增体系
Figure PCTCN2020090626-appb-000005
PCR反应条件(宝生物PrimeSTAR HS酶)：预变性(95℃)5min；然后进行30轮循环：变性(98℃)10s，退火((Tm-3/5)℃)15s，72℃延伸；72℃继续延伸10min；维持(4℃)。
1.3质粒和重组DNA片段的转化
1.3.1 pREDCas9的转化
利用电转的方法将pREDCas9质粒电转至W3110的电转感受态中，将菌体复苏培养后涂布于含奇霉素的LB平板上，32℃过夜培养。抗性平板上生长单菌落用鉴定引物进行菌落PCR,筛选阳性重组子。
1.3.2含pREDCas9的目的菌株电转化感受态制备
32℃培养至OD 600＝0.1～0.2时，添加终浓度为0.1mM的IPTG，继续培养至OD 600＝0.6～0.7时进行感受态制备。添加IPTG的目的是使pREDCas9质粒上的重组酶诱导表达。感受态制备所需培养基及制备过程参照常规标准操作。
1.3.3 pGRB和重组DNA片段的转化
将pGRB和重组DNA片段同时电转化至含有pREDCas9的电转感受态细胞中。将电转化后复苏培养的菌体涂布于含氨苄青霉素和奇霉素的LB平板上，32℃过夜培养。用上游同源臂上游引物和下游同源臂的下游引物，或设计专门的鉴定引物，进行菌落PCR验证，筛选阳性重组子并保菌。
1.4质粒的消除
1.4.1 pGRB的消除
将阳性重组子置于含有0.2％阿拉伯糖的LB培养基中过夜培养，适量稀释后涂布于含有奇霉素抗性的LB平板上，32℃过夜培养。分别转接单菌落到含有氨苄青霉素和奇霉素抗性的LB平板，挑选氨苄青霉素平板不生长，奇霉素抗性平板生长的单菌落保菌。
1.4.2 pREDCas9质粒的消除
将阳性重组子转接到无抗性的LB液体培养基中，42℃过夜培养，适量稀释后涂布于无抗性的LB平板上，37℃过夜培养。对点含有奇霉素抗性和无抗性的LB平板，挑选奇霉素抗性平板不生长，无抗性平板生长的单菌落保菌。
2.菌株构建过程中用到的引物见表5
表5菌株构建过程中所涉及的引物：
Figure PCTCN2020090626-appb-000006
Figure PCTCN2020090626-appb-000007
Figure PCTCN2020090626-appb-000008
Figure PCTCN2020090626-appb-000009
Figure PCTCN2020090626-appb-000010
Figure PCTCN2020090626-appb-000011
3菌株构建的具体过程
3.1 speA、adiA和astA三个基因的敲除。
3.1.1 speA基因的敲除
以E.coli W3110(ATCC27325)基因组为模板，根据其speA基因(NCBI-GeneID:12933352)的上下游序列设计上游同源臂引物(UP-speA-S、UP-speA-A)和下游同源臂引物(DN-speA-S、DN-speA-A)，并PCR扩增其上下游同源臂片段。上述片段通过重叠PCR的方法融合获得speA基因的敲除片段(上游同源臂-下游同源臂)。将引物gRNA-speA-S和gRNA-speA-A退火后得到的DNA片段与质粒pGRB连接，构建重组质粒pGRB-speA。制备E.coli W3110的感受态细胞，按照1.3和1.4所示的方法操作，将质粒pGRB-speA和speA敲除片段同时电转化入感受态细胞，最终获得菌株E.coli W3110 ARG1。speA敲除片段的构建和阳性菌株的PCR验证的电泳图见图2。其中，上游同源臂的长度应为397bp，下游同源臂的长度应为468bp，重叠片段的总长应为865bp，PCR验证时，阳性菌PCR扩增片段长度应为2752bp，原菌PCR扩增片段长度应为865bp。
3.1.2 adiA基因的敲除
以E.coli W3110(ATCC27325)基因组为模板，根据其adiA基因(NCBI-GeneID:12934085) 的上下游序列设计上游同源臂引物(UP-adiA-S、UP-adiA-A)和下游同源臂引物(DN-adiA-S、DN-adiA-A)，并PCR扩增其上下游同源臂片段。上述片段通过重叠PCR的方法融合获得adiA基因的敲除片段(上游同源臂-下游同源臂)。将引物gRNA-adiA-S和gRNA-adiA-A退火得到的DNA片段与质粒pGRB连接，构建pGRB-adiA。制备E.coli W3110 ARG1的感受态细胞，按照1.3和1.4所示的方法操作，将质粒pGRB-adiA和adiA敲除片段同时电转化入感受态细胞，最终获得菌株E.coli W3110 ARG2。adiA敲除片段的构建和阳性菌株的PCR验证的电泳图见图3。其中，上游同源臂的长度应为806bp，下游同源臂的长度应为402bp，重叠片段的总长应为1208bp，PCR验证时，阳性菌PCR扩增片段长度应为2124bp，原菌PCR扩增片段长度应为1208bp。
3.1.3 astA基因的敲除
以E.coli W3110(ATCC27325)基因组为模板，根据其adiA基因(NCBI-GeneID:12933241)的上下游序列设计上游同源臂引物(UP-astA-S、UP-astA-A)和下游同源臂引物(DN-astA-S、DN-astA-A)，并PCR扩增其上下游同源臂片段。上述片段通过重叠PCR的方法融合获得astA基因的敲除片段(上游同源臂-下游同源臂)。将引物gRNA-astA-S和gRNA-astA-A的退火制得的DNA片段与质粒pGRB连接，构建pGRB-astA。制备E.coli W3110 ARG2的感受态细胞，按照1.3和1.4所示的方法操作，将质粒pGRB-astA和astA敲除片段同时电转化入感受态细胞，最终获得菌株E.coli W3110 ARG3。astA敲除片段的构建和阳性菌株的PCR验证的电泳图见图4。其中，上游同源臂的长度应为443bp，下游同源臂的长度应为523bp，重叠片段的总长应为965bp，PCR验证时，阳性菌PCR扩增片段长度应为1869bp，原菌PCR扩增片段长度应为965bp。
3.2敲除大肠杆菌中argE基因，并在该位点整合来源于谷氨酸棒状杆菌的argJ基因
以E.coli W3110(ATCC27325)基因组为模板，根据其argE基因(NCBI-GeneID:12930574)的上下游序列设计上游同源臂引物(UP-argE-S、UP-argE-A)和下游同源臂引物(DN-argE-S、DN-argE-A)，PCR扩增其上下游同源臂片段；以谷氨酸棒状杆菌(ATCC13032)基因组为模板，根据其argJ基因序列(NCBI-GeneID:1019371)设计引物(argJ-S、argJ-A)，PCR扩增argJ片段；启动子P trc则设计在上游同源臂的下游引物和argJ基因的上游引物中。上述片段通过重叠PCR的方法融合，获得敲除argE基因同时整合argJ基因的整合片段(上游同源臂-P trc-argJ-下游同源臂)，将引物gRNA-argE-S和gRNA-argE-A退火制得的DNA片段与质粒pGRB连接，构建pGRB-argE。制备E.coli W3110 ARG3的感受态细胞，按照1.3和1.4所示的方法操 作，将质粒pGRB-argE和敲除argE基因同时整合argJ基因的整合片段同时电转化入感受态细胞，最终获得菌株E.coli W3110 ARG4。P trc-argJ片段整合过程中，整合片段的构建和阳性菌株的PCR验证的电泳图见图5。其中上游同源臂长度为510bp，argJ基因片段长度为1206bp，下游同源臂长度为668bp，重叠片段的长度为2458bp，PCR验证重组子时，阳性重组子所扩增的片段长度应为2458bp，原菌扩增出来的片段长度应为2154bp。
3.2将谷氨酸棒状杆菌的精氨酸合成操纵子整合在大肠杆菌中yghX基因位点。
将谷氨酸棒状杆菌中精氨酸合成操纵基因(包含argC、argJ、argB、argD、argF、argG、argH七个基因)按顺序依次整合至大肠杆菌yjhX基因位点，并由启动子P trc启动该外源操纵子的转录表达，构建了菌株E.coli W3110 ARG7。
谷氨酸棒状杆菌中的精氨酸合成操纵基因的整合共分了三段。
3.2.1 P trc-argC-argJ的整合
以E.coli W3110(ATCC27325)基因组为模板，根据其yghX基因的上下游序列设计上游同源臂引物(UP-yghX-S、UP-yghX-A)和下游同源臂引物(DN-yghX-S1、DN-yghX-A)，PCR扩增其上下游同源臂片段；以谷氨酸棒状杆菌(ATCC 13032)基因组为模板，根据其argC-argJ基因序列(NCBI-GeneID:1019370、1019371)设计引物(argC-argJ-S、argC-argJ-A)，PCR扩增argC-argJ片段；启动子P trc则设计在上游同源臂的下游引物和argC-argJ基因的上游引物中。上述片段通过重叠PCR的方法融合，获得argC-argJ基因的整合片段(上游同源臂-P trc-argC-argJ-下游同源臂)，将引物gRNA-yghX-S和gRNA-yghX-A退火制得含靶序列的DNA序列，与质粒pGRB连接，构建pGRB-yghX。制备E.coli W3110 ARG4的感受态细胞，按照1.3和1.4所示的方法操作，将质粒pGRB-yghX和argC-argJ基因的整合片段同时电转化入感受态细胞，最终获得菌株E.coli W3110 ARG5。P trc-argC-argJ片段整合过程中，整合片段的构建和阳性菌株的PCR验证的电泳图见图6。其中上游同源臂长度为602bp，argC-argJ基因片段长度为2324bp，下游同源臂长度为561bp，重叠片段的长度为3650bp，鉴定引物所扩增片段长度应为1068bp，原菌应无条带。
3.2.2 argB-argD-argF的整合
以谷氨酸棒状杆菌(ATCC13032)基因组为模板，根据argB-argD-argF(NCBI-GeneID:1019372、1019373、1019374)及其上游序列设计上游同源臂引物(UP-argB-argD-argF-S、UP-argB-argD-argF-A)，PCR扩增其上游同源臂片段；以E.coli W3110(ATCC27325)基因组为模板，根据其yghX基因的下游序列设计下游同源臂引物(DN-yghX-S2、DN-yghX-A)，PCR 扩增其下游同源臂片段。上述片段通过重叠PCR的方法融合，获得argB-argD-argF的整合片段(argB的上游片段-argB-argD-argF-下游同源臂)。将引物gRNA-argBDF-S和gRNA-argBDF-A退火制得含靶序列的DNA片段，与质粒pGRB连接，构建pGRB-argBDF。制备E.coli W3110 ARG5的感受态细胞，按照1.3和1.4所示的方法操作，将质粒pGRB-argBDF和argB-argD-argF的整合片段同时电转化入感受态细胞，最终获得菌株E.coli W3110 ARG6。argB-argD-argF片段整合过程中，整合片段的构建和阳性菌株的PCR验证的电泳图见图7。其中argB的上游片段-argB-argD-argF的总长度为3575bp，下游同源臂长度为561bp，重叠片段的长度为4219bp，鉴定引物所扩增片段长度应为1034bp，原菌应无条带。
3.2.3 argG-argH的整合
以谷氨酸棒状杆菌(ATCC13032)基因组为模板，根据argG-argH(NCBI-GeneID:1019376、1019377)及其上游序列设计上游同源臂引物(UP-argG-argH-S、UP-argG-argH-A)及argG-argH片段引物(argG-argH-S、argG-argH-A)，PCR扩增其上游同源臂片段和argG-argH片段；以E.coli W3110(ATCC27325)基因组为模板，根据其yghX基因的下游序列设计下游同源臂引物(DN-yghX-S3、DN-yghX-A)，PCR扩增其下游同源臂片段。上述片段通过重叠PCR的方法融合，获得argG-argH的整合片段(argG的上游片段-argG-argH-下游同源臂)。将引物gRNA-argG-argH-S和gRNA-argG-argH-A退化制得制得含靶序列的DNA序列，与质粒pGRB连接，构建pGRB-argG-argH。制备E.coli W3110 ARG6的感受态细胞，按照1.3和1.4所示的方法操作，将质粒pGRB-argG-argH和argG-argH的整合片段同时电转化入感受态细胞，最终获得菌株E.coli W3110 ARG7。argG-argH的整合过程中，整合片段的构建和阳性菌株的PCR验证的电泳图见图8。其中argG的上游片段的总长度为405bp，argG-argH片段的总长度为2826bp，下游同源臂长度为561bp，重叠片段的总长度应为3875bp，鉴定引物所扩增片段长度应为1521bp，原菌应无条带。
3.3将来源于枯草芽孢杆菌的pyrAA-pyrAB基因整合在大肠杆菌的yjiT基因位点。
B.subtilis A260是以枯草芽孢杆菌168菌株为出发菌株，采用ARTP诱变和高通量筛选相结合的方法选育而来(该菌株已于2015年12月2日保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路l号院3号，中国科学院微生物研究所，邮编：100101，菌种保藏编号：CGMCC No.11775)。该菌株解除了尿苷酸及精氨酸对氨甲酰磷酸合成酶的反馈调节作用，通过对其嘧啶核苷操纵子基因进行测序发现其氨甲酰磷酸大亚基(pyrAB编码)第949位的谷氨酸缺失(申请公布号CN105671007A)。将B.subtilis A260中不 受精氨酸反馈抑制的氨甲酰磷酸合成酶(pyrAA、pyrAB)整合至大肠杆菌中，以提高精氨酸合成过程中前体物氨甲酰磷酸的供应量。
枯草芽孢杆菌中pyrAA-pyrAB基因共4292bp分两段整合至大肠杆菌中，其中第一段长度为2651bp，第二段长度为1641bp。
3.3.1第一段P trc-pyrAA-pyrAB的整合
以E.coli W3110(ATCC27325)基因组为模板，根据其yjiT基因的上下游序列设计上游同源臂引物(UP-yjiT-S、UP-yjiT-A)和下游同源臂引物(DN-yjiT-S、DN-yjiT-A)，PCR扩增其上下游同源臂片段；以B.subtilis A260(CGMCC No.11775)基因组为模板，根据基因pyrAA(NCBI-GeneID:937368)、pyrAB(NCBI-GeneID:936608)设计引物(1-pyrAA-pyrAB-S、1-pyrAA-pyrAB-A)，扩增第一段pyrAA-pyrAB基因片段。启动子P trc则设计在上游同源臂的下游引物和pyrAA-pyrAB基因的上游引物中。上述片段通过重叠PCR的方法融合获得第一段pyrAA-pyrAB基因的整合片段(上游同源臂-P trc-pyrAA-pyrAB-下游同源臂)，将引物gRNA-yjiT-S和gRNA-yjiT-A退火制得含靶序列的DNA片段，与质粒pGRB连接，构建pGRB-yjiT。制备E.coli W3110 ARG7的感受态细胞，按照1.3和1.4所示的方法操作，将质粒pGRB-yjiT和第一段pyrAA-pyrAB基因的整合片段同时电转化入感受态细胞，最终获得菌株E.coli W3110 ARG8。第一段P trc-pyrAA-pyrAB整合片段的构建和阳性菌株的PCR验证的电泳图见图9。其中，上游同源臂的长度应为316bp，第一段pyrAA-pyrAB基因片段长度应为2651bp，下游同源臂的长度应为667bp，整合片段的总长应为3634bp，鉴定引物所扩增片段长度应为1100bp，原菌应无条带。
3.3.2第二段pyrAA-pyrAB的整合
以B.subtilis A260(CGMCC No.11775)基因组为模板，根据第二段pyrAA-pyrAB基因序列及其上游序列设计上游同源臂引物(2-pyrAA-pyrAB-S、2-pyrAA-pyrAB-A)，PCR扩增其上游同源臂片段(包括第一段pyrAA-pyrAB下游序列266bp和整合第二段pyrAA-pyrAB序列1641bp共1907bp)；以E.coli W3110(ATCC27325)基因组为模板，根据其yjiT基因的下游序列设计下游同源臂引物(DN-yjiT-S1、DN-yjiT-A)，PCR扩增其下游同源臂片段。上述片段通过重叠PCR的方法融合，获得第二段pyrAA-pyrAB的整合片段(第二段pyrAA-pyrAB-下游同源臂)。将引物gRNA-pyrAA-pyrAB-S和gRNA-pyrAA-pyrAB-A退火制得含靶序列的DNA片段，与质粒pGRB连接，构建pGRB-pyrAA-pyrAB。制备E.coli W3110 ARG8的感受态细胞，按照1.3和1.4所示的方法操作，将质粒pGRB-pyrAA-pyrAB和第二段pyrAA-pyrAB基因的整合片段 同时电转化入感受态细胞，最终获得菌株E.coli W3110 ARG9。第二段pyrAA-pyrAB的整合过程中，整合片段的构建和阳性菌株的PCR验证的电泳图见图10。其中第二段pyrAA-pyrAB的上游片段的总长度为1907bp，下游同源臂长度为667bp，重叠片段的总长度应为2574bp，鉴定引物所扩增片段长度应为1135bp，原菌应无条带。
3.4将有效棒杆菌中lysE基因整合至大肠杆菌ilvG基因位点。
以E.coli W3110(ATCC27325)基因组为模板，根据其ilvG基因的上下游序列设计上游同源臂引物(UP-ilvG-S、UP-ilvG-A)和下游同源臂引物(DN-ilvG-S、DN-ilvG-A)，PCR扩增其上下游同源臂片段；根据lysE基因(NCBI Reference Sequence:WP_143758438.1)序列(SEQ ID NO:68)设计引物(lysE-S、lysE-A)，扩增lysE基因片段。启动子P trc则设计在上游同源臂的下游引物和lysE基因的上游引物中。上述片段通过重叠PCR的方法融合获得lysE基因的整合片段(上游同源臂-P trc-lysE-下游同源臂)，将ilvG引物gRNA-ilvG-S和gRNA-ilvG-A退火制得含靶序列的DNA片段，与质粒pGRB连接，构建pGRB-ilvG。制备E.coli W3110 ARG9的感受态细胞，按照1.3和1.4所示的方法操作，将质粒pGRB-ilvG和lysE基因的整合片段同时电转化入感受态细胞，最终获得菌株E.coli W3110 ARG10。P trc-lysE整合片段的构建和阳性菌株的PCR验证的电泳图见图11。其中，上游同源臂的长度应为412bp，P trc-lysE基因片段长度应为806bp，下游同源臂的长度应为481bp，整合片段的总长应为1699bp，PCR验证时，阳性菌PCR扩增片段长度应为1699bp，原菌PCR扩增片段长度应为1426bp。
实施例2：
利用基因工程菌E.coli W3110 ARG10发酵生产精氨酸的方法如下：
(1)摇瓶发酵：
斜面培养：取-80℃保藏菌种划线接种于活化斜面，37℃培养12h，并传代一次；
摇瓶种子培养：用接种环刮取一环斜面种子接种于装有30mL种子培养基的500mL三角瓶中，九层纱布封口，37℃，200rpm培养7-10h；
摇瓶发酵培养：按种子培养液体积15％的接种量接种到装有发酵培养基的500mL三角瓶中(终体积为30mL)，九层纱布封口，37℃，200r/min振荡培养，发酵过程中通过补加氨水维持pH在7.0-7.2；补加60％(m/v)葡萄糖溶液维持发酵进行；发酵周期26-30h；
斜面培养基组成为：葡萄糖1g/L，蛋白胨10g/L，牛肉膏10g/L，酵母粉5g/L，NaCl 2.5g/L，琼脂20g/L，其余为水，pH 7.0-7.2；
种子培养基组成为：葡萄糖25g/L，酵母提取物5g/L，蛋白胨3g/L，K 2HPO 4 1g/L， MgSO 4·7H 2O 1g/L，FeSO 4·7H 2O 10mg/L，MnSO 4·7H 2O 10mg/L，V B1、V B3、V B5、V B12、V H各1mg/L其余为水，pH 7.0-7.2。
发酵培养基组成为：葡萄糖25g/L，酵母提取物3g/L，蛋白胨2g/L，K 2HPO 4 3g/L，MgSO 4·7H 2O 2g/L，FeSO 4·7H 2O 10mg/L，MnSO 4·7H 2O 10mg/L，V B1、V B3、V B5、V B12、V H各1mg/L其余为水，pH 7.0-7.2。
经过26-30h摇瓶发酵，E.coli W3110 ARG10菌株发酵液中L-精氨酸的产量为30-32g/L。
(2)发酵罐发酵：
斜面活化培养：从-80℃冰箱保菌管中刮一环菌种，均匀涂布于活化斜面，37℃培养12-16h，转接茄形瓶继续培养12-16h；
种子培养：取适量无菌水于茄形瓶中，将菌悬液接入种子培养基中，pH稳定在7.0左右，温度恒定在37℃，溶氧在25-35％之间，培养至细胞干重达到5-6g/L；
发酵培养：按照15％接种量接入新鲜的发酵培养基，开始发酵，发酵过程中控制pH稳定在7.0左右，温度维持在35℃，溶氧在25-35％之间；当培养基中的葡萄糖消耗完之后，流加80％(m/v)的葡萄糖溶液，维持发酵培养基中的葡萄糖浓度在0.1-5g/L；
斜面培养基、种子培养基和发酵培养基与摇瓶发酵相同。
在5L发酵罐中培养50-55h后，L-精氨酸的积累量达到130-135g/L。转化率为0.48g精氨酸/g葡萄糖，生产强度为2.5g精氨酸/L/h。发酵过程曲线见图12。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种生产L-精氨酸的基因工程菌株，含有编码氨甲酰磷酸合成酶的基因pyrAA和pyrAB。
  2. 根据权利要求1所述的基因工程菌株，以大肠杆菌或谷氨酸棒杆菌为出发菌株；例如以E.coli W3110或者E.coli MG1655为出发菌株；
    优选地，所述pyrAA和pyrAB基因整合于大肠杆菌的yjiT基因位点；
    优选地，所述pyrAA和pyrAB来源于枯草芽孢杆菌。
  3. 根据权利要求1或2所述的基因工程菌株，所述基因工程菌株还含有编码L-精氨酸生物合成途径酶的基因，所述精氨酸生物合成途径酶选自以下的一种或多种：argC、argJ、argB、argD、argF、argG、argH；
    优选地，所述编码L-精氨酸生物合成途径酶的基因由P trc启动子启动；
    优选地，所述编码L-精氨酸生物合成途径酶的基因整合于大肠杆菌的yghX基因位点。
  4. 根据权利要求1-3任一项所述的基因工程菌株，所述基因工程菌株进一步含有编码精氨酸转运蛋白的基因lysE；优选地，所述lysE基因整合于大肠杆菌的ilvG基因位点；优选地，所述lysE基因为SEQ ID NO：68所示的核苷酸序列。
  5. 根据权利要求1-4任一项所述的基因工程菌株，所述基因工程菌株不含有分解L-精氨酸的编码基因，例如敲除如下一种或多种基因：编码精氨酸脱羧酶的基因、编码精氨酸琥珀酰转移酶的基因、编码乙酰鸟氨酸脱乙酰基酶的基因；优选地，所述编码精氨酸脱羧酶的基因包括speA、adiA中的至少一种；所述编码精氨酸琥珀酰转移酶的基因为astA；所述编码乙酰鸟氨酸脱乙酰基酶的基因为argE；优选地，所述基因工程菌株是同时敲除speA、adiA、astA和argE基因的大肠杆菌。
  6. 一种基因工程菌株的构建方法，包括如下步骤：(1)出发菌株的基因组中整合pyrAA和pyrAB基因；
    优选地，所述构建方法还包括任选地如下一个或多个步骤：
    (2)整合精氨酸生物合成途径酶基因，包括argC、argJ、argB、argD、argF、argG、argH中的一种或多种；和/或整合编码精氨酸转运蛋白的基因lysE；
    (3)敲除编码精氨酸脱羧酶的基因、编码精氨酸琥珀酰转移酶的基因、编码乙酰鸟氨酸脱乙酰基酶的基因；例如，所述编码乙酰鸟氨酸脱乙酰基酶的基因包括speA、adiA中的至少一种；所述编码精氨酸琥珀酰转移酶的基因为astA；所述编码乙酰鸟氨酸脱乙 酰基酶的基因为argE。
  7. 根据权利要求6所述的构建方法，包括如下步骤：
    (1)将大肠杆菌上编码精氨酸脱羧酶的基因speA、编码精氨酸脱羧酶的基因adiA和编码精氨酸琥珀酰转移酶的基因astA三个基因敲除；
    (2)敲除大肠杆菌中编码乙酰鸟氨酸脱乙酰基酶的基因argE，任选地将编码谷氨酸乙酰基转移酶的基因argJ整合到大肠杆菌中；
    (3)整合精氨酸生物合成相关基因簇，argC、argJ、argB、argD、argF、argG和argH；
    (4)整合编码氨甲酰磷酸合成酶的基因pyrAA和pyrAB；
    (5)将编码精氨酸转运蛋白的基因lysE整合到大肠杆菌基因组上。
  8. 根据权利要求6或7所述的构建方法，包括采用CRISPR/Cas9介导的基因编辑技术进行基因整合和敲除。
  9. 根据权利要求6-8任一项所述的构建方法，包括构建重组片段和pGRB质粒；
    优选地，所述pGRB质粒的构建包括：设计靶序列，制备包含靶序列的DNA片段，将包含靶序列的DNA片段与线性化的载体片段重组；
    优选地，所述构建方法中构建重组片段包括构建基因整合的重组片段或者构建基因敲除的重组片段；优选地，构建基因整合的重组片段的步骤包括：以出发菌株的基因组为模板，根据目的基因拟插入位点的上下游序列设计上下游同源臂引物，并根据目的基因组设计引物，扩增目的基因片段，再通过PCR重叠技术获得重组片段；优选地，构建基因敲除的重组片段的步骤包括：以待敲除基因的上下游序列为模板，设计上下游同源臂引物；通过PCR的方法分别扩增上下游同源臂，再经过重叠PCR制备重组片段；
    优选地，所述构建方法包括将pGRB质粒和上述重组片段同时转化至含有pREDCas9的电转感受态细胞中，还包括质粒消除的步骤，获得重组的基因工程菌株。
  10. 权利要求1-5任一项所述的基因工程菌发酵生产L-精氨酸的方法，包括：将上述大肠杆菌基因工程菌株与发酵培养基接触，进行发酵培养，制备得到L-精氨酸；
    优选地，所述发酵培养包括摇瓶发酵或者发酵罐发酵；
    优选地，L-精氨酸的积累量达到130-135g/L。转化率达到0.48g精氨酸/g葡萄糖，生产强度达到2.5g精氨酸/L/h。
PCT/CN2020/090626 2019-12-02 2020-05-15 生产l-精氨酸的基因工程菌及其构建方法与应用 WO2021109467A1 (zh)

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US17/781,124 US20220411833A1 (en) 2019-12-02 2020-05-15 Gene engineering bacteria for producing l-arginine and construction method and application of gene engineering bacteria
CA3161765A CA3161765A1 (en) 2019-12-02 2020-05-15 Gene engineering bacteria for producing l-arginine and construction method and application of gene engineering bacteria
KR1020227016823A KR20220088451A (ko) 2019-12-02 2020-05-15 L-아르기닌을 생산하는 유전자 조작 박테리아 및 이의 구축 방법과 응용
ZA2022/05587A ZA202205587B (en) 2019-12-02 2022-05-20 Gene engineering bacteria for producing l-arginine and construction method and application of gene engineering bacteria

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