CN111321102A - Genetically engineered bacterium for producing L-histidine and application thereof - Google Patents
Genetically engineered bacterium for producing L-histidine and application thereof Download PDFInfo
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Abstract
The invention provides a genetically engineered bacterium for producing L-histidine and application thereof, wherein the bacterium integrates a corynebacterium glutamicum ATP transphosphoribosylase HisG mutant coding gene hisG with a nucleotide sequence shown as SEQ ID NO. 1 on a genome of escherichia coli*And the expression is strong, so that the activity of HisG which is a key enzyme for synthesizing histidine is enhanced; the copy number of the escherichia coli self histidine operon gene hisDBCHAFI is also increased on the genome, so that the terminal synthetic pathway of histidine is enhanced; a gene lysE encoding an arginine/lysine transporter from Corynebacterium glutamicum was also integrated into the genome and strongly expressed to promote extracellular secretion of intracellular histidine; the glutamic acid dehydrogenase-encoding gene rocG of bacillus subtilis is also integrated into the genome and strongly expressed to promote the production of histidine.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a genetic engineering bacterium for producing L-histidine, and a construction method and application thereof.
Background
L-histidine (hereinafter referred to as "histidine") is an important functional amino acid and is involved in various physiological and biochemical processes such as body development, oxidation resistance, immune regulation and the like. The compound amino acid nutrient supplement is mainly applied to the food, feed and medicine industries, can be used as a nutrient supplement and a feed additive, can be used for producing amino acid infusion and comprehensive amino acid preparations, can be used for assisting in treating diseases such as heart diseases, anemia and gastrointestinal ulcers, and has high economic and social values.
Currently, histidine is mainly produced by a method of extracting from protein hydrolysate, but the method has high loss rate, serious equipment corrosion and high separation cost, and is not an optimal histidine production method. The microbial fermentation method for producing histidine has the advantages of low raw material cost, environmental friendliness, simple operation and short period, is suitable for industrial production, and becomes a hotspot of the current histidine production research. The bottleneck of producing histidine by a microbial fermentation method is the lack of a strain for efficiently producing histidine, and the main reason is that the biosynthesis path of histidine is long and is strictly and complexly controlled by feedback; the coordination supply of various preconditioners is needed, particularly a large amount of energy is supplied, so that the thalli are difficult to accumulate histidine in large amount. In order to obtain a production strain with high histidine yield, researchers apply a plurality of technical means including traditional mutagenesis screening, genetic engineering, metabolic engineering and the like to transform strains of different species and obtain a series of achievements. Although these studies differ in technical means, the strategy is generally as follows: relieving feedback inhibition of histidine synthesis pathway; enhancing the synthetic pathway of histidine; increasing the supply of precursors; blocking the histidine metabolic pathway; and promoting secretion of histidine to the outside of cells.
The currently reported strain with the highest histidine fermentation level is E.coli WHY3(CN 110184230A), the strain is constructed by taking E.coli W3110 as an original strain and starting from three aspects of releasing feedback control of histidine synthesis, enhancing a histidine synthesis pathway and promoting extracellular secretion of histidine, 40-55g/L of histidine can be produced by fermenting in a 5L fermentation tank for 40-50h, the average production intensity is 1.0-1.5g/(L × h), and the conversion rate is 0.18-0.22g of histidine/g of glucose, but the histidine yield and the conversion rate still have great promotion space.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for improving the fermentation yield of histidine and provide a novel histidine fermentation production strain.
The technical scheme of the invention is summarized as follows:
the invention provides a genetically engineered bacterium E.coli WHY3-1 for high yield of L-histidine, which integrates a Corynebacterium glutamicum ATP transphosphoribosyl enzyme HisG mutant coding gene hisG with a nucleotide sequence shown as SEQ ID NO. 1 on the genome of Escherichia coli, and makes the gene hisG strongly express so as to enhance the activity of histidine to synthesize a key enzyme HisG; the expression of the histidine operon gene hisDBCHAFI of the Escherichia coli is enhanced on the genome, so that the terminal synthetic pathway of histidine is enhanced; a gene lysE encoding an arginine/lysine transporter from Corynebacterium glutamicum was also integrated into the genome and strongly expressed to promote extracellular secretion of intracellular histidine; the glutamic acid dehydrogenase-encoding gene rocG of bacillus subtilis is also integrated into the genome and strongly expressed to promote the production of histidine.
Further, the escherichia coli is e.
Further, the histidine operon gene hisDBCHAFI, which comprises seven genes of hisD, hisB, hisC, hisH, hisA, hisF and hisI, increases the copy number of the seven genes on the genome of Escherichia coli.
Further, the c.glutamicum ATP transphosphoribosylase HisG mutant encoding gene HisG is integrated in the genome of e.coli at least two gene sites.
Further, strong expression of the foreign gene can be achieved by constructing a strong promoter.
In a preferred embodiment of the invention, the gene hisG encoding the mutant ATP transphosphoribosylase HisG of Corynebacterium glutamicum is integrated into the gene sites tdcD and ybE of Escherichia coli genome respectively and is derived from promoter PtrcAnd (5) starting.
In a preferred embodiment of the present invention, the glutamate dehydrogenase encoding gene rocG is integrated into the yjhE gene site on the E.coli genome from the promoter PtrcControlling its transcriptional expression.
As a preferred embodiment of the invention, the genetically engineered bacterium e.coli WHY3-1 is obtained by directionally modifying e.coli W3110 by using a CRISPR/Cas 9-mediated gene editing technology, and specifically comprises the following steps:
(1) construction of the promoter PtrcThe junction fragment P of the gene hisG with the nucleotide sequence shown as SEQ ID NO. 1trc-hisG and integrating it into the genome at the tdcD and ybe gene sites, respectively;
(2) construction of the promoter PtrcConnecting fragment P with Escherichia coli histidine operon genetrc-hisD-hisC-hisB-hisH-hisA-hisF-hisI and integration into the genome at the yghX locus by means of segmental integration;
(3) construction of the promoter PtrcLigation fragment P to lysE Gene derived from Corynebacterium glutamicumtrclysE and its integration into the genome at the yjiT locus;
(4) construction of the promoter PtrcConnecting fragment P with glutamic acid dehydrogenase encoding gene rocG from bacillus subtilistrc-rocG and its integration at the yjhE gene locus on the genome.
The invention also provides a method for preparing L-histidine, which comprises the steps of culturing the genetically engineered bacterium E.coli WHY3-1 under proper conditions and collecting histidine from the culture.
Has the advantages that:
the method for improving the yield of histidine at present focuses on relieving feedback inhibition borne by a histidine synthesis pathway, enhancing the histidine synthesis pathway, increasing the supply of precursors, blocking the histidine metabolic pathway, promoting the extracellular secretion of histidine and the like+And glutamic acid and NAD+Respectively an ammonia donor and a coenzyme in the synthesis process of histidine, thereby further improving the production efficiency of histidine. WHY3-1 is used for producing histidine by fermentation method, and can produce histidine 50-65g/L, average production intensity 1.5-2.0g/L/h, maximum value up to 2.5g/L/h, and conversion rate 0.2-0.24g histidine/g glucose by fermenting for 36-48 h in 5L fermentation tank.
Drawings
FIG. 1: (a) (ii) pREDCas9 plasmid map, (b) pGRB plasmid map.
FIG. 2: the electropherograms were constructed and validated by integration of the fragment at the site of tdcD gene, hisG. Wherein: m: 1kb DNAmarker; 1: an upstream homology arm; 3: hisG gene fragment; 3: a downstream homology arm; 4: overlapping segments; 5: original bacteria control; 6: identification fragments of positive bacteria.
FIG. 3: construction and validation of the electropherograms of the integrated fragments upon integration of hisG at the ybE gene site. Wherein: m: 1kb DNAmarker; 1: an upstream homology arm; 3: hisG gene fragment; 3: a downstream homology arm; 4: overlapping segments; 5: original bacteria control; 6: identification fragments of positive bacteria.
FIG. 4: construction and validation of the hisD integration fragment electropherograms. Wherein: m: 1kb DNA marker; 1: an upstream homology arm; 3: a hisD gene fragment; 3: a downstream homology arm; 4: overlapping segments; 5: original bacteria control; 6: identification fragments of positive bacteria.
FIG. 5: construction and electrophoretogram verification of the hisC-hisB integrated fragment. Wherein: m: 1kb DNA marker; 1: hisC upstream sequence-hisC-hisB fragment; 2: a downstream homology arm; 3: overlapping segments; 4: original bacteria control; 5: identification fragments of positive bacteria.
FIG. 6: construction and electrophoretogram validation of the hisH-hisA-hisF-hisI integrated fragment. Wherein: m: 1kb DNAmarker; 1: the hisH upstream sequence-hisH-hisA-hisF-hisI fragment; 2: a downstream homology arm; 3: overlapping segments; 4: original bacteria control; 5: identification fragments of positive bacteria.
FIG. 7: ptrcConstruction and validation of the lysE integration fragment electrophorogram. Wherein: m: 1kb DNA marker; 1: an upstream homology arm; 2: a lysE gene fragment; 3: a downstream homology arm; 4: overlapping segments; 5: original bacteria control; 6: and (4) identifying fragments of positive bacteria.
FIG. 8: construction and validation of the electropherogram of the integrated fragment when rocG is integrated at the yjhE gene locus. Wherein: m: 1kb DNAmarker; 1: an upstream homology arm; 3: a downstream homology arm; 3: a segment of the rocG gene; 4: overlapping segments; 5: original bacteria control; 6: identification fragments of positive bacteria.
FIG. 9: results of fermentation of coli WHY3-1 on a 5L tank.
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.
The percent symbols "%" referred to in the following examples refer to volume percent unless otherwise specified without the definition well known in the art; percent of solution "% (m/v)" refers to the grams of solute contained in 100mL of solution.
Example 1:
coli WHY 3-1.
1 Gene editing method
The gene editing method employed in the present invention is performed with reference to the literature (Li Y, Lin Z, Huang C, et al. methylation using CRISPR-Cas 9 programmed genetic engineering,2015,31:13-21.) and the two plasmid maps used in the method are shown in FIG. 1. Wherein pREDCas9 carries an elimination system of gRNA expression plasmid pGRB, a Red recombination system of lambda phage and a Cas9 protein expression system, spectinomycin resistance (working concentration: 100mg/L) and is cultured at 32 ℃; pGRB comprises a pUC 18-based skeleton, a promoter J23100, a gRNA-Cas 9-binding region sequence and a terminator sequence, ampicillin resistance (working concentration: 100mg/L), and culture at 37 ℃.
The method comprises the following specific steps:
1.1pGRB plasmid construction
The plasmid pGRB is constructed for the purpose of transcribing the corresponding gRNA to form a complex with the Cas9 protein, and recognizing a target site of a target gene through base pairing and PAM, thereby realizing double strand break of the target DNA. pGRB plasmids are constructed by recombination of DNA fragments containing the target sequence with linearized vector fragments.
1.1.1 target sequence design
Design of target sequence using CRISPR RGEN Tools (PAM:5 '-NGG-3')
1.1.2 preparation of DNA fragments containing the target sequence
Designing a primer: 5 '-linearized vector terminal sequence (15bp) -enzyme cutting site-target sequence (excluding PAM sequence) -linearized vector terminal sequence (15bp) -3' and reverse complementary primer thereof, and preparing DNA fragment containing target sequence by annealing single-stranded DNA. Reaction conditions are as follows: pre-denaturation at 95 deg.C for 5 min; annealing at 30-50 deg.C for 1 min. The annealing system is as follows:
annealing system
1.1.3 preparation of Linear vectors
The linearization of the vector adopts a reverse PCR amplification method.
1.1.4 recombination reactions
The recombination system is shown in the following table. All recombinant enzymes used are
II One Step Cloning Kit series of enzymes, recombination conditions: 30min at 37 ℃.
Recombination system
1.1.5 transformation of plasmids
Taking 10 mu L of reaction liquid, adding the reaction liquid into 100 mu L of DH5 α transformation competent cells, gently mixing the reaction liquid, carrying out ice bath for 20min, carrying out heat shock for 45-90s at 42 ℃, immediately carrying out ice bath for 2-3min, adding 900 mu L of SOC, recovering at 37 ℃ for 1h, carrying out centrifugation at 8000rpm for 2min, abandoning part of supernatant, leaving about 200 mu L of supernatant, coating the suspended thallus on a plate containing 100mg/L ampicillin, inverting the plate, carrying out overnight culture at 37 ℃, identifying the single thallus after the plate grows out, and selecting positive recombinants by colony PCR.
1.1.6 cloning identification
Inoculating the PCR positive colony to LB culture medium containing 100mg/L ampicillin for overnight culture, preserving the bacteria, extracting plasmid, and performing enzyme digestion identification.
1.2 preparation of recombinant DNA fragments
The recombination segment for knockout consists of an upstream homology arm and a downstream homology arm of a gene to be knocked out (upstream homology arm-downstream homology arm); the recombinant fragment used for integration consists of the upstream and downstream homology arms of the integration site and the gene fragment to be integrated (upstream homology arm-target gene-downstream homology arm). Using primer design software primer5, using the upstream and downstream sequences of the gene to be knocked out or the site to be integrated as templates, designing upstream and downstream homologous arm primers (amplification length is about 400-; the gene to be integrated is used as a template, and an amplification primer of the integrated gene is designed. Respectively amplifying upstream and downstream homologous arms and target gene fragments by a PCR method, and preparing recombinant fragments by overlapping PCR. The PCR system and method is as follows:
PCR amplification system
The system of overlapping PCR is as follows:
overlapping PCR amplification system
Note: the template consists of amplified fragments of upstream and downstream homology arms and target genes in equimolar amount, and the total amount is not more than 10 ng.
PCR reaction conditions (precious organism PrimeSTAR HS enzyme): pre-denaturation (95 ℃) for 5 min; then 30 cycles of circulation were performed: denaturation (98 ℃) for 10s, annealing ((Tm-3/5) ° C) for 15s, and extension at 72 ℃ (the enzyme activity extends about 1kb in 1 min); continuing to extend for 10min at 72 ℃; maintained at (4 ℃).
1.3 transformation of plasmids and recombinant DNA fragments
1.3.1 transformation of pREDCas9
The pREDCas9 plasmid was electroporated into W3110 by electroporation, and after recovery culture, the cells were plated on LB plates containing spectinomycin and cultured overnight at 32 ℃. And (3) growing a single colony on the resistant plate, carrying out colony PCR by using an identification primer, and screening positive recombinants.
1.3.2 electrotransformation-competent preparation of the Strain of interest containing pREDCas9
Culturing at 32 deg.C to OD600When the concentration is 0.1 to 0.2, 0.1M IPTG (final concentration: 0.1mM) is added and the culture is continued until OD is reached600When the ratio is 0.6-0.7, the competent preparation is carried out. The purpose of the addition of IPTG was to induce expression of the recombinase on the pREDCas9 plasmid. The culture medium required by the competent preparation and the preparation process refer to the conventional standard operation.
1.3.3 transformation of pGRB and recombinant DNA fragments
pGRB and donor DNA fragments were simultaneously electroporated into electroporation competent cells containing pREDCas 9. The thalli which are recovered and cultured after the electrotransformation are coated on an LB plate containing ampicillin and spectinomycin, and cultured overnight at 32 ℃. And (3) carrying out colony PCR verification by using an upstream primer of the upstream homology arm and a downstream primer of the downstream homology arm or designing a special identification primer, screening positive recombinants and preserving bacteria.
1.4 Elimination of plasmids
1.4.1 Elimination of pGRB
The positive recombinants are placed in an LB culture medium containing 0.2% of arabinose for overnight culture, and are coated on an LB plate containing spectinomycin resistance after being diluted by a proper amount, and are cultured at 32 ℃ overnight. And (3) selecting a single colony which does not grow on the ampicillin plate and grows on the spectinomycin resistant plate to preserve bacteria on the LB plate containing ampicillin and spectinomycin resistance.
1.4.2 Elimination of pREDCas9 plasmid
Transferring the positive recombinants into a nonresistant LB liquid culture medium, culturing at 42 ℃ overnight, diluting the positive recombinants in a proper amount, coating the diluted positive recombinants on a nonresistant LB plate, and culturing at 37 ℃ overnight. And (3) selecting a single colony which does not grow on the spectinomycin resistant plate and does not grow on the non-resistant plate to preserve the bacteria on the LB plate containing spectinomycin resistance and non-resistance.
2. All primers involved in the strain construction process are shown in the following table:
3 specific Process for Strain construction
3.1 Release of feedback inhibition by HisG and Strong expression thereof
3.1.1 treatment of PtrcIntegration of hisG at the site of the tdcD gene
Using E.coli W3110(ATCC27325) genome as a template, designing an upstream homology arm primer (UP-tdcD-S, UP-tdcD-A) and a downstream homology arm primer (DN-tdcD-S, DN-tdcD-A) according to upstream and downstream sequences of tdcD gene, and carrying out PCR amplification on upstream and downstream homology arm fragments; primers (hisG-S, hisG-A) were designed based on hisG gene (nucleotide sequence shown in SEQ ID No. 2), and then fragments of hisG gene were amplified. Promoter PtrcThe downstream primer of the upstream homology arm and the upstream primer of the hisG gene were designed. The above fragments were subjected to overlap PCR to obtain an integrated fragment of hisG gene (upstream homology arm-P)trc-hisG-downstream homology arm), the DNA fragment containing the target sequence used for the construction of pGRB-tdcD was prepared by annealing the primers gRNA-tdcD-S and gRNA-tdcD-a. Coli W3110, and the procedure was performed according to the methods shown in 1.3 and 1.4, to finally obtain the strain E.coli WHY 1-1. PtrcConstruction of-hisG integration and PCR-verified electropherograms of positive strains are shown in FIG. 2. Wherein, the length of the upstream homology arm should be 496bp, the length of the amplified hisG gene fragment should be 627bp, the length of the downstream homology arm should be 1902bp, the total length of the integrated fragment should be 3024bp, when PCR verification is carried out by using the identifying primer, the length of the PCR amplified fragment of the positive bacterium should be 627bp, and the original bacterium has no band.
3.1.2 treatment of PtrcIntegration of hisG at the ybE gene site
Taking E.coli W3110(ATCC27325) genome as a template, designing an upstream homology arm primer (UP-ybE-S, UP-ybE-A) and a downstream homology arm primer (DN-ybE-S, DN-ybE-A) according to the upstream and downstream sequences of the ybE gene, and carrying out PCR amplification on upstream and downstream homology arm fragments; the hisG gene fragment was amplified using primers (hisG-S, hisG-A). The above fragments were used to obtain an integrated fragment of hisG gene (upstream) by overlap PCRHomologous arm-Ptrc-hisG-downstream homology arm), a DNA fragment containing the target sequence used for the construction of pGRB-ybe was prepared by annealing the primers gRNA-ybe-S and gRNA-ybe-a. Competent cells of E.coli WHY1-1 were prepared and manipulated according to the methods shown in 1.3 and 1.4 to finally obtain the strain E.coli WHY1-2. PtrcConstruction of-hisG integration and PCR-verified electropherograms of positive strains are shown in FIG. 3. Wherein, the length of the upstream homology arm should be 601bp, the length of the amplified hisG gene fragment should be 627bp, the length of the downstream homology arm should be 547bp, the total length of the integrated fragment should be 1815bp, when PCR verification is carried out by using the identifying primer, the length of the PCR amplified fragment of the positive bacterium should be 903bp, and the original bacterium has no band.
3.2 integration of the histidine operon gene of E.coli W3110 at the yghX gene site
In the present invention, histidine operator (hisDBCHAFI, comprising seven genes of hisD, hisB, hisC, hisH, hisA, hisF and hisI) in E.coli W3110 was sequentially integrated at the yghX site of pseudogene on the E.coli WHY1-2 genome in order from promoter PtrcTranscription expression of the operon is initiated, and a strain E.coli HIS3-3 is constructed.
The integration of the histidine operon genes is divided into three segments.
3.2.1PtrcIntegration of hisD
Taking E.coli W3110(ATCC27325) genome as a template, designing upstream homology arm primers (UP-yghX-S, UP-yghX-A) and downstream homology arm primers (DN-yghX-S1 and DN-yghX-A) according to upstream and downstream sequences of yghX gene, and carrying out PCR amplification on upstream and downstream homology arm fragments; designing a primer (hisD-S, hisD-A) according to the hisD gene sequence, and carrying out PCR amplification on the hisD fragment; promoter PtrcA downstream primer of the upstream homology arm and an upstream primer of the hisD gene are designed. The fragments are fused by an overlapping PCR method to obtain PtrcIntegration fragment of the hisD Gene (upstream homology arm-P)trc-hisD-downstream homology arm), a DNA fragment containing the target sequence used for the construction of pGRB-yghX was prepared by annealing the primers gRNA-yghX-S and gRNA-yghX-a. Competent cells of E.coli WHY1-2 were prepared and manipulated according to the methods shown in 1.3 and 1.4 to finally obtain strain E.coli WHY 2-1. PtrcThe electrophoretogram of the construction of the integrated fragment and the PCR verification of the positive strain during integration of the hisD fragment is shown in FIG. 4. Wherein the length of the upstream homologous arm is 602bp, the length of the hisD gene fragment is 1305bp, the length of the downstream homologous arm is 561bp, the length of the overlapped fragment is 2542bp, an identifying primer is designed and PCR verification is carried out, the length of the amplified fragment of the positive recon is 1208bp, and the original bacterium is free from a band.
3.2.2 integration of hisB-hisC
Taking an E.coli W3110(ATCC27325) genome as a template, designing an upstream homology arm primer (UP-hisBC-S, UP-hisBC-A) according to hisB-hisC and an upstream sequence thereof, and carrying out PCR amplification on an upstream homology arm fragment; coli HIS3-1 genome as template, downstream homology arm primers (DN-yghX-S2, DN-yghX-A) are designed according to the downstream sequence of the yghX gene, and the downstream homology arm fragment is amplified by PCR. The above fragments were fused by the overlap PCR method to obtain an integrated fragment of hisB-hisC (the upstream fragment of hisB-hisC-downstream homology arm). Construction of pGRB-his 1A DNA fragment containing the target sequence was prepared by annealing the primers gRNA-his1-S and gRNA-his 1-A. Competent cells of E.coli WHY2-1 were prepared and manipulated according to the methods shown in 1.3 and 1.4 to finally obtain strain E.coli WHY 2-2. The electrophoresis chart of the construction of the integration fragment and the PCR verification of the positive strain during integration of the hisB-hisC fragment is shown in FIG. 4. Wherein the total length of an upstream fragment-hisB-hisC of the hisB is 2696bp, the length of a downstream homologous arm is 561bp, the length of an overlapped fragment is 3317bp, an identifying primer is designed and PCR verification is carried out, the length of an amplified fragment of the positive recon is 1118bp, and the original bacterium has no band.
3.2.3 integration of hisH-hisA-hisF-hisI
Using E.coli W3110(ATCC27325) genome as template, designing upstream homology arm primer (UP-hisHAFI-S, UP-hisHAFI-A) according to hisH-hisA-hisF-hisI and upstream sequence thereof, and PCR amplifying upstream homology arm fragment; coli HIS3-2 genome as template, downstream homology arm primers (DN-yghX-S3, DN-yghX-A) are designed according to the downstream sequence of yghX gene, and the downstream homology arm fragment is amplified by PCR. The above fragments were fused by the overlap PCR method to obtain an integrated fragment of hisH-hisA-hisF-hisI (the upstream fragment of hisH-hisA-hisF-hisI-downstream homology arm). Construction of pGRB-his 2A DNA fragment containing the target sequence was prepared by annealing the primers gRNA-his2-S and gRNA-his 2-A. Competent cells of E.coli WHY2-2 were prepared and manipulated according to the methods shown in 1.3 and 1.4 to finally obtain strain E.coli WHY 2-3. The electrophoresis pattern of the construction of the integration fragment and the PCR verification of the positive strain during integration of hisH-hisA-hisF-hisI is shown in FIG. 6. Wherein the total length of an upstream fragment-hisH-hisA-hisF-hisI of the hisH is 3265bp, the length of a downstream homologous arm is 561bp, the total length of an overlapped fragment is 3317bp, an identifying primer is designed and PCR verification is carried out, the length of an amplified fragment of the positive recon is 1136bp, and the original bacterium has no band.
3.3PtrcIntegration of lysE
Taking E.coli W3110(ATCC27325) genome as a template, designing upstream homology arm primers (UP-yjiT-S, UP-yjiT-A) and downstream homology arm primers (DN-yjiT-S1 and DN-yjiT-A) according to upstream and downstream sequences of yjiT gene, and carrying out PCR amplification on upstream and downstream homology arm fragments; designing a primer (lysE-S, lysE-A) based on the gene sequence of lysE (NCBI-GeneID:1019244) using the genome of Corynebacterium glutamicum (ATCC 13032) as a template, and PCR-amplifying a lysE fragment; promoter PtrcA downstream primer of the upstream homology arm and an upstream primer of the lysE gene were designed. The fragments are fused by an overlapping PCR method to obtain PtrcIntegration fragment of lysE (upstream homology arm-P)trclysE-downstream homology arm), a DNA fragment containing the target sequence used for the construction of pGRB-yjiT was prepared by annealing the primers gRNA-yjiT-S and gRNA-yjiT-A. Competent cells of e.coli why2-3 were prepared and operated according to the methods shown in 1.3 and 1.4 to finally obtain strain e.coli WHY 3. PtrcThe electrophoretogram of the construction of the integrated fragment and the PCR verification of the positive strain during the integration of the lysE fragment is shown in FIG. 7. Wherein the length of the upstream homologous arm is 372bp, the length of the lysE gene fragment is 834bp, the length of the downstream homologous arm is 530bp, the length of the overlapped fragment is 1655bp, an identifying primer is designed and PCR verification is carried out, the length of the amplified fragment of the positive recon is 1429bp, and the original bacterium has no band.
3.4PtrcIntegration of the RocG
Coli W3110 genome as template, based on itDesigning an upstream homology arm primer (UP-yjhE-S, UP-yjhE-A) and a downstream homology arm primer (DN-yjhE-S, DN-yjhE-A) from the upstream and downstream sequences of the yjhE gene, and carrying out PCR amplification on upstream and downstream homology arm fragments; designing a primer (rocG-S, rocG-A) according to a rocG gene sequence (NCBI-GeneID:937066) by taking a B.subtilis 168 genome as a template, and carrying out PCR amplification on a rocG gene fragment; promoter PtrcThe downstream primer of the upstream homology arm and the upstream primer of the rocG gene are designed. The fragments are fused by an overlapping PCR method to obtain PtrcIntegration fragment of the rocG gene (upstream homology arm-P)trcrocG-downstream homology arm), the DNA fragment containing the target sequence used for the construction of pGRB-yjhE was prepared by annealing the primers gRNA-yjhE-S and g RNA-yjhE-a. Competent cells of e.coli WHY3 were prepared and operated according to the methods shown in 1.3 and 1.4 to finally obtain strain e.coli WHY 3-1. PtrcThe electrophoretogram of the construction of the integrated fragment and the PCR verification of the positive strain during integration of the rocG fragment is shown in FIG. 8. The length of the upstream homology arm is 602bp, the length of the rocG gene fragment is 503bp, the length of the downstream homology arm is 491bp, the length of the overlapped fragment is 2419bp, primers UP-yjhE-S and DN-yjhE-A are used for carrying out PCR verification, the length of the amplified fragment of the positive recon is 2419bp, and the length of the original bacterium is 1547 bp.
Example 2:
the genetically engineered bacterium E.coli WHY3-1 is utilized to produce histidine by fermentation as follows:
(1) by shaking flask culture
Slant culture: taking a preserved strain at the temperature of minus 80 ℃, streaking and inoculating the strain on an activated inclined plane, culturing for 12h at the temperature of 37 ℃, and carrying out passage once;
and (3) seed culture in a shaking flask: scraping a ring of inclined plane seeds by using an inoculating ring, inoculating the seeds into a 500mL triangular flask filled with 30mL seed culture medium, sealing by nine layers of gauze, and culturing at 37 ℃ and 200rpm for 6-8 h;
and (3) shake flask fermentation culture: inoculating the strain into a 500mL triangular flask (the final volume is 30mL) filled with a fermentation culture medium according to the inoculation amount of 10-15%, sealing with nine layers of gauze, performing shaking culture at 37 ℃ at 200r/min, and maintaining the pH value at 7.0-7.2 by adding ammonia water in the fermentation process; adding 60% (m/v) glucose solution to maintain fermentation;
the slant culture medium comprises: 1-5g/L glucose, 5-10g/L peptone, 5-10g/L beef extract, 1-5g/L yeast powder, 1-2.5g/L NaCl, 20-25g/L agar, and the balance of water, wherein the pH value is 7.0-7.2;
the seed culture medium comprises the following components: 15-30g/L of glucose, 5-10g/L of yeast extract, 5-10g/L of peptone, 5-15g/L of KH2PO 45-15 g/L, MgSO4 & 7H2O 2-5g/L, FeSO4 & 7H2O 5-20mg/L, MnSO4 & H2O 5-20mg/L, VB 11-3 mg/L, VH 0.1-1mg/L, 2 drops of antifoaming agent and the balance of water, wherein the pH value is 7.0-7.2;
the fermentation medium comprises the following components: 20-30g/L of glucose, 2-5g/L of yeast extract, 2-4g/L of peptone, KH2PO41-3g/L, MgSO4 & 7H2O 1-2g/L, FeSO4 & 7H2O 5-20mg/L, MnSO4 & 7H2O 5-20mg/L, VB1, VB3, VB5, VB12 and VH are respectively 1-3mg/L, and the balance is water, and the pH value is 7.0-7.2.
(2) Or culturing in a fermenter
Slant activation culture: scraping a ring of strains from a refrigerator bacteria-protecting tube at the temperature of-80 ℃, uniformly coating the strains on an activated inclined plane, culturing for 12-16h at the temperature of 37 ℃, and transferring to an eggplant-shaped bottle for further culturing for 12-16 h;
seed culture: placing appropriate amount of sterile water in eggplant-shaped bottle, inoculating the bacterial suspension into seed culture medium, stabilizing pH at about 7.0, maintaining temperature at 37 deg.C, and culturing until fermentation liquid OD is 25-35%600The value reaches 10-15;
inoculating into fresh fermentation culture medium according to 15-20% inoculum size, starting fermentation, controlling pH to be stabilized at about 7.0, maintaining temperature at 37 deg.C, and dissolving oxygen at 25-35%; when the glucose in the culture medium is completely consumed, feeding 80% (m/v) of glucose solution to maintain the glucose concentration in the fermentation culture medium at 0.1-5 g/L;
the slant culture medium comprises: 1-5g/L glucose, 5-10g/L peptone, 5-10g/L beef extract, 1-5g/L yeast powder, 1-2.5g/L NaCl, 20-25g/L agar, and the balance of water, wherein the pH value is 7.0-7.2;
the seed culture medium comprises the following components: 15-30g/L glucose, 5-10g/L yeast extract, 5-10g/L peptone and KH2PO45-15g/L，MgSO4·7H2O 2-5g/L，FeSO4·7H2O 5-15mg/L，MnSO4·H2O 5-15mg/L，VB11-3mg/L，VH0.1-1mg/L, 2 drops of defoaming agent and the balance of water, and the pH value is 7.0-7.2;
the fermentation medium comprises the following components: glucose 10-25g/L, yeast extract 1-5g/L, peptone 1-5g/L, K2HPO41-5g/L，MgSO4·7H2O 1-3g/L，FeSO4·7H2O 10-30mg/L，MnSO4·H2O 10-30mg/L，VB1、VB3、VB5、VB12、VH1-3mg/L of each, the balance of water, and the pH value of 7.0-7.2.
Example 3:
fermentation experiments on a 5L tank with coli WHY 3-1.
Histidine was produced using the strain e.coli WHY3-1 constructed in example 1 as a production strain:
bevel activation: taking glycerol preservation strains, streaking and inoculating the glycerol preservation strains to a test tube slant culture medium, and culturing at 37 ℃ for 12; then, the slant-preserved strain is streaked and inoculated in a slant culture medium of a eggplant-shaped bottle, and is cultured for 14 hours at 37 ℃;
seed culture: taking 1 inclined plane of an activated fresh eggplant-shaped bottle, washing the inclined plane with 150mL of sterile water, inoculating the inclined plane into a fermentation tank under the protection of flame, controlling the temperature at 37 ℃, automatically feeding ammonia water in a flowing manner to control the pH value to be 7.0, controlling the initial aeration rate to be 2L/min, controlling the initial stirring rotation speed to be 200rpm, maintaining the DO value to be between 20 and 30 percent in the culture process, and culturing seeds until the OD600 is about 15;
culturing in a fermentation tank: inoculating 15% of seed solution (discharging to 450mL, pouring sterilized fermentation culture medium under flame protection) into seeds of a fermentation tank, controlling the temperature to 35 ℃, automatically feeding ammonia water (or 20% sulfuric acid) to control the pH to be 7.0, controlling the initial aeration rate to be 2L/min, controlling the aeration ratio to be 0.667vvm, controlling the initial stirring rotating speed to be 400rpm, controlling the dissolved oxygen to be 20-30% by adjusting the rotating speed and the air quantity, manually dropping foam killer for defoaming, feeding 80% of glucose solution in the fermentation process, ensuring sufficient sugar supply and ensuring that the sugar concentration is not higher than 5 g/L;
the slant culture medium comprises: 1g/L glucose, 10g/L peptone, 10g/L beef extract, 5g/L yeast powder, NaCl2.5g/L agar, 25g/L the rest water, and the pH value is 7.0-7.2;
the seed culture medium comprises the following components: 10g/L glucose, 5g/L yeast extract, 5g/L peptone and KH2PO45g/L，MgSO4·7H2O 2g/L，FeSO4·7H2O 10mg/L，MnSO4·H2O 10mg/L，VB12mg/L，VH1mg/L, 2 drops of defoaming agent and the balance of water, and the pH value is 7.0-7.2;
the fermentation medium comprises the following components: 10g/L glucose, 5g/L yeast extract, 4g/L tryptone, K2HPO43g/L，MgSO4·7H2O 1.5g/L，FeSO4·7H2O 20mg/L，MnSO4·H2O 20mg/L，VB1、VB3、VB5、VB12、VHEach 2mg/L, the rest is water, and the pH value is 7.0-7.2.
The fermentation profile of coli WHY3-1 on a 5L fermentor is shown in FIG. 9.
As can be seen from the fermentation curve, the thallus begins to enter the logarithmic growth phase after 4 hours of fermentation, and the OD is 32 hours600Enter a stationary phase after reaching the maximum value. The fermentation starts to enter a histidine rapid accumulation stage after 8 hours, and the histidine yield is up to 65g/L after 44 hours of fermentation. The final conversion was 0.223g histidine/g glucose. Compared with WHY3 under the same fermentation conditions, WHY3-1 is reduced in biomass, but the yield and the production intensity of histidine are both improved by 18.2%, and the conversion rate is improved by 11.5%. It can be seen that E.coli WHY3-1 has very significant advantages as a histidine-producing bacterium.
Sequence listing
<110> Zhejiang Shayuan pharmacy Co., Ltd., Tianjin science and technology university
<120> genetic engineering bacterium for producing L-histidine and application thereof
<130>2020
<141>2020-03-06
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>627
<212>DNA
<213> Artificial sequence ()
<400>1
atgttgaaaa tcgctgtccc aaacaaaggc tcgctgtccg agcgcgccat ggaaatcctc 60
gccgaagcag gctacgcagg ccgtggagat tccaaatccc tcaacgtttt tgatgaagca 120
aacaacgttg aattcttctt ccttcgccct aaagatatcg ccatctacgt tgctggtggc 180
cagctcgatt tgggtatcac cggccgcgac cttgctcgcg attcccaggc tgatgtccac 240
gaagttcttt ccctcggctt cggttcctcc actttccgtt acgcagcacc agctgatgaa 300
gagtggagca tcgaaaagct cgacggcaag cgcatcgcta cctcttaccc caaccttgtt 360
cgcgatgacc tcgcagcacg tgggctttcc gctgaggtgc tccgcctcga cggtgcagta 420
gaggtattca tcaagcttgg tgtcgcagat gccatcgccg atgttgtatc caccggccgc 480
acgctgcgtc agcaaggtct tgcacctttc ggcgaggttc tgtgcacctc tgaggctgtc 540
attgttggcc gcaaggatga aaaggtcacc ccagagcagc agatcctgct tcgccgcatc 600
cagggaattt tgcacgcgca gaactag 627
Claims (9)
1. A genetically engineered bacterium for producing L-histidine is characterized in that the genetically engineered bacterium integrates a Corynebacterium glutamicum ATP transphosphoribosylase HisG mutant coding gene hisG with a nucleotide sequence shown as SEQ ID NO. 1 on a genome of Escherichia coli and enables the gene hisG to be strongly expressed; the expression of the E.coli histidine operon genes hisD, hisB, hisC, hisH, hisA, hisF and hisI was also enhanced on the genome; a gene lysE encoding an arginine/lysine transporter from Corynebacterium glutamicum was also integrated in the genome and strongly expressed; the glutamic acid dehydrogenase-encoding gene rocG of bacillus subtilis is also integrated into the genome and strongly expressed.
2. The genetically engineered bacterium of claim 1, wherein said E.coli is E.
3. The genetically engineered bacterium of claim 1 or 2, wherein the c.glutamicum ATP transphosphoribosylase HisG mutant-encoding gene HisG is integrated in the genome at least two gene sites and is driven by a strong promoter.
4. The genetically engineered bacterium of claim 1 or 2, wherein the c.glutamicum ATP transphosphoribosylase HisG mutant encoding gene HisG is integrated in the genome at the tdcD and ybe gene sites, respectively, and is derived from promoter PtrcStarting; the Escherichia coli histidine operon genes hisD, hisB, hisC, hisH, hisA, hisF and hisI are sequentially integrated on the yghX gene locus on the genome in sequence and are controlled by a promoter PtrcStarting; the coding gene lysE of the arginine/lysine transporter is integrated on the yjiT gene site on the genome and is obtained by a promoter PtrcStarting; the glutamate dehydrogenase encoding gene rocG is integrated to yjhE gene site on the genome of escherichia coli and is expressed by a promoter PtrcAnd (5) starting.
5. Use of the genetically engineered bacterium of any one of claims 1 to 4 for the fermentative production of L-histidine.
6. A construction method of a gene engineering bacterium for producing L-histidine is characterized in that the gene engineering bacterium is obtained by directionally transforming E.coli W3110 by using a CRISPR/Cas9 mediated gene editing technology, and comprises the following steps:
(1) construction of the promoter PtrcThe junction fragment P of the gene hisG with the nucleotide sequence shown as SEQ ID NO. 1trc-hisG and integrating it into the genome at the tdcD and ybe gene sites, respectively;
(2) construction of the promoter PtrcAnd Escherichia coli groupJunction fragment P of the amino acid operon genetrc-hisD-hisC-hisB-hisH-hisA-hisF-hisI and integration into the genome at the yghX locus by means of segmental integration;
(3) construction of the promoter PtrcLigation fragment P to lysE Gene derived from Corynebacterium glutamicumtrclysE and its integration into the genome at the yjiT locus;
(4) construction of the promoter PtrcConnecting fragment P with glutamic acid dehydrogenase encoding gene rocG from bacillus subtilistrc-rocG and its integration at the yjhE gene locus on the genome.
7. A method for producing L-histidine, characterized in that the method comprises culturing the genetically engineered bacterium according to any one of claims 1 to 4 under suitable conditions, and collecting histidine from the culture.
8. The method of claim 7, wherein the culture is performed in a shake flask: activating the genetically engineered bacteria, preparing a seed solution, inoculating the seed solution into a 500mL triangular flask filled with a fermentation culture medium according to the inoculation amount of 10-15%, sealing by nine layers of gauze, carrying out shaking culture at 37 ℃ at 200r/min, and maintaining the pH value at 7.0-7.2 by adding ammonia water in the fermentation process; adding 60% (m/v) glucose solution to maintain fermentation; the composition of the fermentation medium is as follows: glucose 20-30g/L, yeast extract 2-5g/L, peptone 2-4g/L, KH2PO41-3g/L，MgSO4·7H2O 1-2g/L，FeSO4·7H2O 5-20mg/L，MnSO4·7H2O 5-20mg/L，VB1、VB3、VB5、VB12、VH1-3mg/L of each, the balance of water, and the pH value of 7.0-7.2.
9. The method of claim 7, wherein the fermenter culture is used: activating the genetically engineered bacteria to prepare seed liquid, inoculating 15-20% of the inoculum size into fresh fermentation culture medium, fermenting, controlling pH to be stabilized at about 7.0, maintaining temperature at 37 deg.C, and dissolving oxygen at 25-35%To (c) to (d); when the glucose in the culture medium is completely consumed, feeding 80% (m/v) of glucose solution to maintain the glucose concentration in the fermentation culture medium at 0.1-5 g/L; the composition of the fermentation medium is as follows: glucose 10-25g/L, yeast extract 1-5g/L, peptone 1-5g/L, K2HPO41-5g/L，MgSO4·7H2O1-3g/L，FeSO4·7H2O 10-30mg/L，MnSO4·H2O 10-30mg/L，VB1、VB3、VB5、VB12、VH1-3mg/L of each, the balance of water, and the pH value of 7.0-7.2.
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