CN112251392A - Genetic engineering strain for producing ergothioneine and application thereof - Google Patents

Genetic engineering strain for producing ergothioneine and application thereof Download PDF

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CN112251392A
CN112251392A CN202011154568.0A CN202011154568A CN112251392A CN 112251392 A CN112251392 A CN 112251392A CN 202011154568 A CN202011154568 A CN 202011154568A CN 112251392 A CN112251392 A CN 112251392A
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马倩
田道光
谢希贤
吴鹤云
李镠
蒋帅
秦臻
朱彦凯
王加初
朱永铎
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Abstract

The invention provides a genetic engineering bacterium for high-yield ergothioneine and application thereof, wherein the strain takes escherichia coli as a host, and a coding gene hisG of a corynebacterium glutamicum ATP transphosphoribosylase HisG mutant is integrated on the genome of the host; and also at itThe copy number of histidine operon gene hisDBCHAFI is increased on the genome; also integrates the ergothioneine operator gene egtCDE of Mycobacterium smegmatis on the genome; the gene gshA of the escherichia coli glutamylcysteine ligase coding gene is also integrated on the genome of the strain, so that the synthesis of ergothioneine is promoted; also integrates a gene egtE encoding C-S lyase of the Neurospora crassa on the genomencrFurther promoting the synthesis of ergothioneine; also integrated in its genome is the gene egtB of a sulfoxide synthase mutantmsmFurther promote the synthesis of ergothioneine.

Description

Genetic engineering strain for producing ergothioneine and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a genetic engineering bacterium for producing ergothioneine and application thereof.
Background
Ergothioneine (EGT), a sulfhydryl histidine trimethyl inner salt, is a natural amino acid derivative found in ergot in 1909. Ergothioneine has multiple functions of resisting oxidation, eliminating free radicals, regulating oxidation-reduction reaction, participating in intracellular energy regulation and the like. Because of its excellent cell physiological protective function, ergothioneine can be applied to various fields including medicine, food, feed and cosmetic industries. In 2017, ergothioneine is approved by the European food safety agency as a safe new resource food for diets of infants, pregnant women and lactating women. In 2019, ergothioneine received GRAS certification by the U.S. food and drug administration.
At present, the main production mode of ergothioneine is submerged fermentation of fungal mycelium, but the fermentation period of the fungal mycelium is long, the yield of the ergothioneine is low, and the ergothioneine does not have the potential of industrial production. The microbial fermentation method for producing the ergothioneine has the advantages of low raw material cost, environmental friendliness, simplicity in operation and short period, is suitable for industrial production, and becomes a hotspot of the current ergothioneine production research. The bottleneck in microbial fermentation processes for ergothioneine production is the lack of strains that produce ergothioneine efficiently. Although the biosynthetic pathway of ergothioneine has been resolved, the heterologous synthesis of ergothioneine using microbial cell factories remains a formidable challenge. Firstly, the construction of a microbial cell factory needs to introduce a heterologous ergothioneine anabolic module, also relates to the modification of a central metabolic module and a plurality of precursor anabolic modules, and is a difficult task of efficiently combining and optimizing the metabolic modules; secondly, the supply of histidine, adenosylmethionine and cysteine directly influences the synthesis of ergothioneine, the anabolic flux of the ergothioneine in cells is low, and the difficulty of synergistically improving the synthesis flux of the precursors is high; finally, the three main precursors are related to different metabolic modules, the expression levels of genes related to central metabolism and different branch metabolism need to be coordinated, the intracellular contents of intermediate metabolites and synthetic precursors are strictly controlled, and otherwise, the cellular metabolism imbalance is easily caused. Researchers have attempted to synthesize ergothioneine using various host microorganisms (e.g., Methylobacterium, Saccharomyces cerevisiae, Escherichia coli, Aspergillus oryzae, etc.), but the yield is still low, the fermentation time is still long, and the cost is still high.
The highest yield of ergothioneine produced by fermentation of Escherichia coli is reported in the literature (Naoyuki tanaka, Yusuke Kawano, Yasuharu satoh, tohru Dairi & Iwao ohtsu (2019). "Gram-scale selective production of ergothioneine drive by genetic expression of mycobacterial in Escherichia coli," Scientific reports 9(1): 1895-) and 1.3g/L of ergothioneine can be obtained by heterogeneously expressing the synthetic pathway of ergothioneine derived from mycobacterial in Escherichia coli, studying the fermentation tank, culturing, continuously adding precursors such as histidine, and fermenting for 216 h. Under the condition of supplementing various precursors, the escherichia coli realizes the synthesis of ergothioneine, but the yield and the production intensity are still low.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for improving the fermentation yield of ergothioneine and provide a novel ergothioneine fermentation production strain.
The technical scheme of the invention is summarized as follows:
the invention provides a genetically engineered bacterium E.coli EGT12 for high-yield ergothioneine, which takes escherichia coli as a host, and integrates a coding gene hisG of a corynebacterium glutamicum ATP transphosphoribosyl enzyme HisG mutant with a sequence shown as SEQ ID NO. 1 on the genome of the host so as to relieve the feedback regulation effect of the HisG; the copy number of histidine operon gene hisDBCHAFI is also increased on the genome, thereby enhancing the terminal synthetic pathway of histidine; also integrates the ergothioneine operator gene egtCDE of Mycobacterium smegmatis on the genome; the gene gshA of the escherichia coli glutamylcysteine ligase coding gene is integrated on the genome of the gene, so that more sulfuration substrates glutamylcysteine are provided for the synthesis of ergothioneine, and the synthesis of the ergothioneine is promoted; also integrates a gene egtE of the C-S lyase coding gene of the Neurospora crassa with the sequence shown as SEQ ID NO. 2 on the genomencrFurther promoting the synthesis of ergothioneine; also integrated into its genome is the gene egtB which codes for a sulfoxide synthase mutant with the amino acid sequence shown in SEQ ID NO. 4msmFurther promote the synthesis of ergothioneine.
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 histidine operon on the E.coli genome in double copies.
Further, the above-mentioned gene integrated into the genome of E.coli is transcriptionally expressed from a strong promoter.
In one embodiment of the invention, the strong promoter is promoter Ptrc。
The sulfoxide synthase mutant is obtained by replacing amino acid R (arginine) at the 374 th site of an amino acid sequence (NCBI-protein ID: WP-011731158.1) of original Mycolibacillosis sulfoxide synthase with Q (glutamine), wherein the mutated amino acid sequence is shown as SEQ ID NO:4, respectively.
In a specific embodiment of the invention, the nucleotide sequence of the gene encoding the mutant sulfoxide synthase is shown as SEQ ID NO: 3, respectively.
In a specific embodiment of the invention, the E.coli glutamyl cysteine ligase encoding gene gshA is replaced by the Mycobacterium smegmatis glutamyl cysteine ligase encoding gene egtA.
In one embodiment of the invention, the C-S lyase-derived gene egtE of Neurospora crassancrReplacement by Mycobacterium smegmatis C-S lyase-encoding gene egtEmsm。
In one embodiment of the invention, the sulfoxide synthase mutant encodes the gene egtBmsmSubstitution to the sulfoxide synthase encoding Gene egtE derived from Mycobacterium smegmatismsm。
As a preferred embodiment of the invention, the genetically engineered bacterium e.coli EGT12 is obtained by directionally modifying a host e.coli MG1655 by using a CRISPR/Cas 9-mediated gene editing technology, and specifically comprises the following steps:
(1) construction of the promoter PtrcAnd nucleotide sequence shown as SEQ ID NO:1 of Corynebacterium glutamicumtrc-hisG and integrating it at the tdcD gene site on the host genome;
(2) the histidine operon gene hisDBCHAF was integrated at the yghX gene site in the host genome and expressed from the promoter PtrcStarting;
(3) the ergothioneine operator egtCDE is integrated in the host genome at the ilvG gene site and is obtained from the promoter PtrcStarting;
(4) construction of the promoter PtrcConnecting fragment P of gene gshA encoding glutamylcysteine ligase of escherichia colitrc-gshA, and integrating it at the mbhA gene site on the host genome;
(5) construction of the promoter PtrcAnd nucleotide sequence shown as SEQ ID NO:2 of the gene egtE for the C-S lyase of Venus crassancrConnecting fragment P oftrc-egtEncrAnd integrating it at the site of the tehB gene on the host genome;
(6) construction of the promoter PtrcAnd nucleotide sequence shown as SEQ ID NO: 3, and coding gene egtB of sulfoxide synthase lyase mutantmsmConnecting fragment P oftrc-egtB*msmAnd integrated at the locus of the yeeP gene on the genome.
The invention also provides a method for preparing ergothioneine, which comprises the steps of culturing the genetically engineered bacterium E.coli EGT12 under appropriate conditions and collecting the ergothioneine from the culture.
Has the advantages that:
at present, the construction of ergothioneine synthesis strains is basically a direct exogenous introduction way, and the yield is very low, so that the requirement of industrial production cannot be met. The invention combines a high-efficiency ergothioneine synthesis way by sequentially expressing and comparing glutamylcysteine ligase, C-S lyase and sulfoxide synthase from different sources in escherichia coli. Double copy of self glutamylcysteine ligase encoding gene gshA in E.coli, introduction of mutant Mycobacterium smegmatis-derived sulfoxide synthetase encoding gene egtBmsmCodon-optimized C-S lyase-derived gene egtE from Neurospora crassancrAnd a high-efficiency non-natural ergothioneine synthesis path is reconstructed in the escherichia coli. The genetic engineering bacterium E.coli EGT12 for stably and efficiently producing the ergothioneine has the advantages that the yield of the ergothioneine can reach 105.7mg/L after 30-hour shake flask fermentation; the ergothioneine can be produced for 52h in a 5L fermentation tank, and the ergothioneine can be produced for 2.9g/L, and the histidine can be produced for 14.77 g/L. Compared with the reported strains (216 h fermentation in a 3L fermentation tank, 1.31g/L accumulation of L-ergothioneine) of Tohru Dairii and the like in 2019, the strain has the advantages of no mutagenesis treatment, no plasmid vector, short fermentation period, clear genetic background, stable metabolism, strong production and the likeHigh degree and good industrial application prospect. The existing ergothioneine producing strains need exogenously added synthetic precursors of histidine, methionine and cysteine. The bacterial strain has good histidine synthesis capacity, no exogenous histidine is required to be added in the ergothioneine synthesis process, and the highest yield of histidine in the fermentation process can reach about 20 g/L.
Drawings
Fig. 1 (a): (iv) pREDCas9 plasmid map.
Fig. 1 (b): pGRB plasmid map.
FIG. 2: construction and validation of electropherograms for hisG integration fragments. Wherein: m: 1kb DNA marker; 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 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. 4: construction and electrophoretogram verification of the hisC-hisB integrated fragment. Wherein: m: 1kb DNAmarker; 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. 5: construction and electrophoretogram validation of the hisH-hisA-hisF-hisI integrated fragment. Wherein: m: 1kb DNA marker; 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. 6: construction and confirmation of the egtBCE-1 integration fragment. M: 1kb DNA marker; 1: an upstream homology arm; 2: a downstream homology arm; 3: an egtBCDE-1 gene fragment; 4: overlapping segments; 5: original bacteria control; 6: identification fragments of positive bacteria.
FIG. 7: construction and validation of the egtBCE-2 integration fragment. M: 1kb DNA marker; 1: an egtBCDE-2 gene fragment; 2: a downstream homology arm; 3: overlapping segments; 4: original bacteria control; 5: identification fragments of positive bacteria.
FIG. 8: construction of the egtA integration fragment and validation of the electropherogram. M: 1kb DNA marker; 1: an upstream homology arm; 2: a downstream homology arm; 3: an egtA gene fragment; 4: overlapping segments; 5: original bacteria control; 6: identification fragments of positive bacteria.
FIG. 9: gshA integrated fragment construction and validation of electropherograms. Wherein: m: 1kb DNA marker; 1: an upstream homology arm; 2: a downstream homology arm; 3: gshA gene fragment 4: overlapping segments; 5: original bacteria control; 6: identification fragments of positive bacteria.
FIG. 10: egtEmsmAnd (5) integrating fragment construction and verifying an electrophoretogram. Wherein: m: 1kb DNA marker; 1: upstream homology arm 2: a downstream homology arm; 3: egtEmsmGene fragment 4: overlapping segments; 5: original bacteria control; 6: identification fragments of positive bacteria.
FIG. 11: egtEncrAnd (5) integrating fragment construction and verifying an electrophoretogram. Wherein: m: 1kb DNA marker; 1: upstream homology arm 2: a downstream homology arm; 3: egtEncrGene fragment 4: overlapping segments; 5: original bacteria control; 6: identification fragments of positive bacteria.
FIG. 12: egtBmsmAnd (5) integrating fragment construction and verifying an electrophoretogram. Wherein: m: 1kb DNA marker; 1: an upstream homology arm; 2: a downstream homology arm; 3: egtBmsmA gene fragment; 4: overlapping segments; 5: control of original bacteria
FIG. 13: egtBmsmAnd (5) integrating fragment construction and verifying an electrophoretogram. Wherein: m: 1kb DNA marker; 1: upstream homology arm 2: a downstream homology arm; 3: egtBmsmGene fragment 4: overlapping segments; 5: original bacteria control; 6: identification fragment of positive bacterium
FIG. 14: coli EGT5 and e.coli EGT6 shake flask fermentation results.
FIG. 15: coli EGT9 and e.coli EGT10 shake flask fermentation results.
FIG. 16: coli EGT11 and e.coli EGT12 shake flask fermentation results.
FIG. 17: coli EGT12 fermentation process profile on a 5L fermentor.
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.
Reference to percent in the embodiments is to volume percent unless otherwise specified; percent of solution "% (m/v)" refers to the grams of solute contained in 100mL of solution.
Example 1:
constructing a genetic engineering bacterium for producing ergothioneine.
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. metabolic engineering of Escherichia coli 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
Figure BDA0002742354100000061
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
Figure BDA0002742354100000062
II One Step Cloning Kit series of enzymes, recombination conditions: 30min at 37 ℃.
Recombination system
Figure BDA0002742354100000063
Figure BDA0002742354100000071
1.1.5 transformation of plasmids
Adding 10 μ L of reaction solution into 100 μ L of DH5 alpha transformation competent cells, mixing gently, ice-cooling for 20min, heat-shocking at 42 deg.C for 45-90s, immediately ice-cooling for 2-3min, adding 900 μ L of SOC, and resuscitating at 37 deg.C for 1 h. The cells were centrifuged at 8000rpm for 2min, a portion of the supernatant was discarded, about 200. mu.L of the supernatant was retained, and the cells were resuspended and spread on a plate containing 100mg/L ampicillin, and the plate was inverted and cultured overnight at 37 ℃. And (4) after the single bacterium grows out from the plate, carrying out colony PCR identification, and selecting a positive recon.
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). Designing an upstream and downstream homologous arm primer (amplification length is about 400-500bp) by using a primer design software primer 5 and taking an upstream and downstream sequence of a gene to be knocked out or a site to be integrated as a template; 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
Figure BDA0002742354100000072
The system of overlapping PCR is as follows:
overlapping PCR amplification system
Figure BDA0002742354100000081
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 electrotransferred to MG1655 in an electrotransferred state by an electrotransfer method, and the cells were applied to LB plates containing spectinomycin after recovery culture 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 OD6000.1M IPTG (final concentration: 0.1mM) was added to the medium at 0.1 to 0.15, and the medium was further cultured until OD reached600And when the ratio is 0.2-0.3, performing competent preparation. 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:
Figure BDA0002742354100000091
Figure BDA0002742354100000101
Figure BDA0002742354100000111
3 specific Process for Strain construction
3.1 mixing PtrcIntegration of hisG at the site of the tdcD gene
Taking an E.coli MG1655 genome as ｃA template, designing an upstream homology arm primer (tdcD-UP-S, tdcD-UP-A) and ｃA downstream homology arm primer (tdcD-DN-S, tdcD-DN-A) according to an upstream sequence and ｃA downstream sequence of ｃA tdcD gene, and carrying out PCR amplification on upstream and downstream homology arm fragments; and designing a primer (hisG-S, hisG-A) according to the hisG gene (the nucleotide sequence is shown as SEQ ID NO: 1), and then amplifying the hisG gene fragment. 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), DNA fragment containing target sequence used for construction of pGRB-tdcD was prepared by annealing primers tdcD-gRNA-S and tdcD-gRNA-a. Coli MG1655 competent cells were prepared and manipulated according to the methods shown in 1.3 and 1.4 to finally obtain strain e. 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 484bp, the length of the amplified hisG gene fragment should be 627bp, the length of the downstream homology arm should be 642bp, the total length of the integrated fragment should be 1864bp, the length of the PCR amplified fragment of the positive bacterium should be 1864bp, and the length of the PCR amplified fragment of the original bacterium should be 2269 bp.
3.2 double-copy of E.coli MG 1655's own histidine operon gene at the yghX Gene site
In the invention, histidine operator genes (hisDBCHAFI, comprising seven genes of hisD, hisB, hisC, hisH, hisA, hisF and hisI) in E.coli MG1655 are sequentially integrated at a pseudo-gene yghX site on the E.coli EGT1 genome, and a promoter PtrcTranscription expression of the operon is initiated, and a strain E.coli EGT2-3 is constructed.
The integration of the histidine operon genes was accomplished in three segments.
3.2.1PtrcIntegration of hisD
Taking E.coli MG1655 genome as template, designing upstream homology arm primer (yghX-UP-S, yghX-UP-A) and downstream homology arm primer (yghX-DN-S1, yghX-DN-A) according to upstream and downstream sequences of yghX gene, and PCR amplifying 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 yghX-gRNA-S and yghX-gRNA-a. Competent cells of e.coli.egt1 were prepared and operated according to the methods shown in 1.3 and 1.4 to finally obtain the strain e.coli EGT 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. 3. 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 MG1655 genome as ｃA template, designing an upstream homology arm primer (hisCB-UP-S, hisCB-UP-A) according to hisB-hisC and an upstream sequence thereof, and carrying out PCR amplification on an upstream homology arm fragment; coli MG1655 genome as template, based on the downstream sequence of yghX gene to design downstream homology arm primer (yghX-DN-S2, yghX-DN-A), and PCR amplify its downstream homology arm segment. 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 used was prepared by annealing primers his1-gRNA-S and his 1-gRNA-S. Competent cells of e.coli EGT2-1 were prepared and manipulated according to the methods shown in 1.3 and 1.4 to finally obtain strain e.coli EGT 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
Taking E.coli MG1655 genome as ｃA template, designing an upstream homology arm primer (hisHAFI-UP-S, hisHAFI-UP-A) according to hisH-hisA-hisF-hisI and an upstream sequence thereof, and carrying out PCR amplification on an upstream homology arm fragment; coli MG1655 genome as template, based on the downstream sequence of yghX gene to design downstream homology arm primer (yghX-DN-S3, yghX-DN-A), and PCR amplify its downstream homology arm segment. 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. Preparing competent cells of E.coli EGT2-2, and operating according to the methods shown in 1.3 and 1.4 to finally obtain the strain E.coli EGT 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. 5. 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.
3.3 integration of the ergothioneine operon gene of Mycolibacillosis MC2155 at the ilvG Gene site
The ergothioneine operon gene (egtBCDE, including egtB, egtC, egtD and egtE four genes) in Mycolibacillosis MC2155 is sequentially integrated at the ilvG locus of a pseudogene on the E.coli EGT2-3 genome, and the promoter PtrcTranscription expression of the operon is initiated, and a strain E.coli EGT4-2 is constructed.
The integration of the ergothioneine operon genes was accomplished in two stages.
3.3.1PtrcIntegration of egtBCDE-1
Taking an E.coli MG1655 genome as ｃA template, designing an upstream homology arm primer (ilvG-UP-S, ilvG-UP-A) and ｃA downstream homology arm primer (FZ-ilvG-DN-S, ilvG-DN-A) according to the upstream and downstream sequences of an ilvG gene, and carrying out PCR amplification on upstream and downstream homology arm fragments; primers (msm-S, FZ-msm-A) are designed according to the sequence of the egtBCDE gene in Mycolibacillosis MC2155, and then the egtBCDE-1 gene fragment is amplified. Promoter PtrcThen designed into the downstream primer of the upstream homology arm and the upstream primer of the egtBCDE-1 gene. The integrated fragment of the egtBCDE-1 gene (upstream homology arm-P) is obtained by the overlapping PCR method of the above fragmentstrc-egtBCDE-1-downstream homology arm), the DNA fragment containing the target sequence used for the construction of pGRB-ilvG was prepared by annealing the primers ilvG-gRNA-S and ilvG-gRNA-A. Preparing competent cells of E.coli EGT2-3, and operating according to the methods shown in 1.3 and 1.4 to finally obtain the strain E.coli EGT 4-1. PtrcConstruction of the-egtBCDE-1 integration fragment and PCR-validation of the positive strain the electrophoretogram is shown in FIG. 6. Wherein, the length of the upstream homologous arm should be 412bp, the length of the amplified egtBCDE-1 gene fragment should be 2122bp, the length of the downstream homologous arm should be 461bp, the total length of the integrated fragment should be 2930bp, during PCR verification, an identifying primer is designed and PCR verification is carried out, the length of the amplified fragment of the positive recon is 749bp, and the original bacterium has no band.
3.3.2PtrcIntegration of egtBCDE-2
Taking E.coli MG1655 genome as template, designing downstream homology arm primer (ilvG-DN-S, ilvG-DN-A) according to the downstream sequence of ilvG gene, and PCR amplifying the downstream homology arm segment; a primer (FZ-msm-S, msm-A) is designed according to the sequence of the egtBCDE gene in Mycolibacillosis MC2155, and then an egtBCDE-2 gene fragment is amplified. The integrated fragment of the egtBCDE-2 gene (egtBCDE-2-downstream homologous arm) is obtained by the overlapping PCR method of the above fragments, and a DNA fragment containing a target sequence used for constructing pGRB-egt2 is prepared by annealing primers egt2-gRNA-S and egt 2-gRNA-A. Competent cells of e.coli EGT4-1 were prepared and manipulated according to the methods shown in 1.3 and 1.4 to finally obtain strain e.coli EGT 4-2. The electrophoresis pattern of the construction of the integrated fragment and the PCR validation of the positive strain during the integration of egtBCDE-2 is shown in FIG. 7. The length of the amplified egtBCDE-2 gene fragment is 2696bp, the length of the downstream homologous arm is 524bp, the total length of the overlapped fragment is 3181bp, an identifying primer is designed and PCR verification is carried out, the length of the amplified fragment of the positive recon is 1403bp, and the original bacterium has no band.
3.4PtrcIntegration of egtA
Using E.coli MG1655 genome as template, designing upstream homology arm primer (mbｃA-UP-S, mbhA-UP-A) and downstream homology arm primer (mbｃA-DN-S, mbhA-DN-A) according to upstream and downstream sequences of the mbｃA gene, and PCR amplifying upstream and downstream homology arm fragments; takes Mycolibacillosis MC2155 genome as a template, designs a primer (egtA-S, egtA-A) according to the egtA gene sequence (NCBI-GeneID: 4532453) and amplifies an egtA fragment by PCR, and a promoter PtrcA downstream primer of the upstream homology arm and an upstream primer of the egtA gene are designed. The fragments are fused by an overlapping PCR method to obtain PtrcIntegration fragment of-egtA (upstream homology arm-P)trc-egtA downstream homology arm), a DNA fragment containing a target sequence used for construction of pGRB-mbhA was prepared by annealing primers mbhA-gRNA-S and mbhA-gRNA-A. Preparing competent cells of E.coli EGT4-2, and performing the operations according to the methods shown in 1.3 and 1.4 to finally obtain the strain E.coli EGT 5. PtrcFIG. 8 shows the electrophoresis chart of the construction of the integrated fragment and the PCR verification of the positive strain in the process of integrating the egtA fragment. Wherein the length of the upstream homology arm is 690bp, the length of the egtA gene fragment is 1272bp, the length of the downstream homology arm is 749bp, the length of the overlapped fragment is 2632bp, an identification primer is designed and PCR verification is carried out, the length of the fragment amplified by the positive recombinant is 1330bp, and the original bacterium has no band.
3.5PtrcIntegration of-gshA
Using E.coli MG1655 genome as template, designing upstream homology arm primer (mbｃA-UP-S, mbhA-UP-A) and downstream homology arm primer (mbｃA-DN-S, mbhA-DN-A) according to upstream and downstream sequences of the mbｃA gene, and PCR amplifying upstream and downstream homology arm fragments; coli MG1655 genome as template, based on gshA gene sequence (NCBI-GeneID: BAA16555), designing primer (gshA-S, gshA-A) and PCR amplifying gshA fragment, promoter PtrcThe downstream primer of the upstream homology arm and the upstream primer of the gshA gene are designed. The fragments are fused by an overlapping PCR method to obtain Ptrc-integrated fragment of gshA (upstream homology arm-P)trcA gshA downstream homology arm), a DNA fragment containing the target sequence used for the construction of pGRB-mbhA was prepared by annealing the primers mbhA-gRNA-S and mbhA-gRNA-A. Preparing competent cells of E.coli EGT4-2, and performing the operations according to the methods shown in 1.3 and 1.4 to finally obtain the strain E.coli EGT 6. PtrcThe electrophoretogram of the construction of the integrated fragment and the PCR verification of the positive strain during integration of the gshA fragment is shown in fig. 9. The length of the upstream homology arm is 690bp, the length of the gshA gene fragment is 1680bp, the length of the downstream homology arm is 749bp, the length of the overlapped fragment is 3040bp, an identification primer is designed and PCR verification is carried out, the length of the fragment amplified by the positive recon is 3040bp, and the length of the fragment amplified by the PCR of the original bacterium is 1831 bp.
3.6Ptrc-egtEmsmIntegration of
Taking E.coli MG1655 genome as template, designing upstream homology arm primer (tehB-UP-S, tehB-UP-A) and downstream homology arm primer (tehB-DN-S, tehB-DN-A) according to the upstream and downstream sequences of its tehB gene, and PCR amplifying its upstream and downstream homology arm fragments; takes Mycolibacillosis MC2155 genome as template, according to egr Emsm(NCBI-GeneID: 4531386) Gene sequence design primer (egtE)msm-S、egtEmsm-A) and PCR amplification of egtEmsmPromoter PtrcDesign the downstream primer and egtE of the upstream homology armncrThe upstream primer of the gene. The fragments are fused by an overlapping PCR method to obtain Ptrc-egtEmsmOf (2)Syntagm fragment (upstream homology arm-P)trc-egtEmsmDownstream homology arm), a DNA fragment containing the target sequence used for the construction of pGRB-tehB was prepared by annealing the primers tehB-gRNA-S and tehB-gRNA-a. Preparing competent cells of E.coli EGT6, and performing the operations according to the methods shown in 1.3 and 1.4 to finally obtain the strain E.coli EGT 9. Ptrc-egtEmsmThe electrophoresis chart of the construction of the integrated fragment and the PCR verification of the positive strain in the fragment integration process is shown in FIG. 10. Wherein the upstream homology arm is 538bp, egtEmsmThe length of the gene fragment is 1116bp, the length of the downstream homology arm is 493bp, the length of the overlapped fragment is 2066bp, an identifying primer is designed and PCR verification is carried out, the length of the amplified fragment of the positive recon is 2066bp, and the length of the amplified fragment of the original bacterium PCR is 1437 bp.
3.7Ptrc-egtEncrIntegration of
Taking E.coli MG1655 genome as template, designing upstream homology arm primer (tehB-UP-S, tehB-UP-A) and downstream homology arm primer (tehB-DN-S, tehB-DN-A) according to the upstream and downstream sequences of its tehB gene, and PCR amplifying its upstream and downstream homology arm fragments; based on the Neurospora crassa genome, egtE of the Neurospora crassa genome is used as a basisncrThe gene is obtained by a chemical synthesis method after codon optimization, and egtE is obtained after codon optimizationncrGene (nucleotide sequence is shown as SEQ ID NO: 2) design primer (egtE)ncr-S，egtEncr-A) and PCR amplification of egtEncrFragment, promoter PtrcDesign the downstream primer and egtE of the upstream homology armncrThe upstream primer of the gene. The fragments are fused by an overlapping PCR method to obtain Ptrc-egtEncrIntegration fragment of (upstream homology arm-P)trc-egtEncrDownstream homology arm), a DNA fragment containing the target sequence used for the construction of pGRB-tehB was prepared by annealing the primers tehB-gRNA-S and tehB-gRNA-a. Preparing competent cells of E.coli EGT6, and performing the operations according to the methods shown in 1.3 and 1.4 to finally obtain the strain E.coli EGT 10. Ptrc-egtEncrThe electrophoretogram of the construction of the integrated fragment and the PCR verification of the positive strain during the integration of the fragment is shown in FIG. 11. Wherein the upstream homology arm is 538bp, egtEncrGeneThe length of the fragment is 1545bp, the length of the downstream homology arm is 493bp, the length of the overlapped fragment is 2495bp, an identifying primer is designed and PCR verification is carried out, the length of the fragment amplified by the positive recon is 2495bp, and the length of the fragment amplified by the PCR of the original bacterium is 1437 bp.
3.8Ptrc-egtBmsmIntegration of
Taking an E.coli MG1655 genome as ｃA template, designing an upstream homology arm primer (yeeP-UP-S, yeeP-UP-A) and ｃA downstream homology arm primer (yeeP-DN-S, yeeP-DN-A) according to the upstream and downstream sequences of ｃA yeeP gene, and carrying out PCR amplification on upstream and downstream homology arm fragments; takes Mycolibacillosis MC2155 genome as template, according to egtBmsm(NCBI-GeneID: 4533015) Gene sequence design primer (egtB)msm-S、egtBmsm-A) and PCR amplification of egtBmsmPromoter PtrcDesign the downstream primer and egtB of the upstream homology armmsmThe upstream primer of the gene. The fragments are fused by an overlapping PCR method to obtain Ptrc-egtBmsmIntegration fragment of (upstream homology arm-P)trc-egtBmsmDownstream homology arm), a DNA fragment containing a target sequence used for constructing pGRB-yeeP was prepared by annealing primers yeeP-gRNA-S and yeeP-gRNA-a. Preparing competent cells of E.coli EGT10, and performing the operations according to the methods shown in 1.3 and 1.4 to finally obtain the strain E.coli EGT 11. Ptrc-egtBmsmThe electrophoresis chart of the construction of the integrated fragment and the PCR verification of the positive strain in the fragment integration process is shown in FIG. 12. Wherein the length of the upstream homology arm is 572bp, egtBmsmThe length of the gene fragment is 1287bp, the length of the downstream homology arm is 580bp, the length of the overlapped fragment is 2473bp, an identifying primer is designed and PCR verification is carried out, the length of the fragment amplified by the positive recon is 1121bp, and the original bacterium has no band.
3.9Ptrc-egtB*msmIntegration of
Taking an E.coli MG1655 genome as ｃA template, designing an upstream homology arm primer (yeeP-UP-S, yeeP-UP-A) and ｃA downstream homology arm primer (yeeP-DN-S, yeeP-DN-A) according to the upstream and downstream sequences of yeeP genes, and carrying out PCR amplification on upstream and downstream homology arm fragments; homology based on 10 mycobacterial sulfoxide synthases, paired survivalCarrying out phylogenetic mutation on the sexual site, the vicinity of the active site and the conserved region to obtain the sulfoxide synthase mutant. Based on the Mycolibacillosis MC2155 genome, mutation of egtBmsmGene (nucleotide sequence is shown as SEQ ID NO: 3) design primer (egtB)msm-S，egtB*msm-A，egtB*msm-S，egtBmsm-A) and PCR amplification of egtBmsm-1 and egtBmsm-2 fragment, promoter PtrcDesign the downstream primer and egtB of the upstream homology arm msm1 fragment upstream primer. The fragments are fused by an overlapping PCR method to obtain Ptrc-egtB*msmIntegration fragment of (upstream homology arm-P)trc-egtB*msmDownstream homology arm), a DNA fragment containing a target sequence used for constructing pGRB-yeeP was prepared by annealing primers yeeP-gRNA-S and yeeP-gRNA-a. Preparing competent cells of E.coli EGT10, and performing the operations according to the methods shown in 1.3 and 1.4 to finally obtain the strain E.coli EGT 12. Ptrc-egtB*msmThe electrophoretogram of the construction of the integrated fragment and the PCR verification of the positive strain during the integration of the fragment is shown in FIG. 13. Wherein the upstream homology arm is 572bp, egtBmsmThe length of the gene fragment is 1396bp, the length of the downstream homology arm is 580bp, the length of the overlapped fragment is 2473bp, an identifying primer is designed and PCR verification is carried out, the length of the amplified fragment of the positive recon is 1121bp, and the original bacterium has no band.
Example 2:
the method for producing ergothioneine by using the genetically engineered bacteria E.coli EGT5 and E.coli EGT6, E.coli EGT9, E.coli EGT10, E.coli EGT11 and E.coli EGT12 constructed in the embodiment 1 through shake flask fermentation is as follows:
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 8-10 h;
and (3) shake flask fermentation culture: inoculating the seed solution 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 fermentation period is 26-30 h;
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, and 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 5-20mg/L，MnSO4·H2O 5-20mg/L，VB1 1-3mg/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 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，MnSO4·7H2O5-20 mg/L, methionine 1-2g/L, cysteine 0.5-1g/L VB1、VB3、VB5、VB12、VHEach 1-3mg/L, VB65-10mg/L, the balance of water and pH 7.0-7.2.
Results of shake flask fermentations of e.coli EGT5 and e.coli EGT6 are shown in fig. 14.
Results of shake flask fermentations of e.coli EGT9 and e.coli EGT10 are shown in fig. 15.
Results of shake flask fermentations of e.coli EGT11 and e.coli EGT12 are shown in fig. 16.
The expression comparison of glutamylcysteine ligase, C-S lyase and sulfoxide synthase from different sources is carried out in sequence in escherichia coli. The shaking flask fermentation of 26h proves that E.coli EGT5 can generate 4.2mg/L of ergothioneine, and E.coli EGT6 can generate 6.2mg/L of ergothioneine, which indicates that the self-glutamylcysteine ligase of escherichia coli can synthesize more glutamylcysteine; coli EGT9 can produce 23.5mg/L of ergothioneine, E.coli EGT10 can produce 34.4mg/L of ergothioneine, and the C-S lyase from the Neurospora crassa has higher activity; coli EGT11 produced 80mg/L of ergothioneine and E. coli EGT12 produced 84.7mg/L of ergothioneine, indicating that the mutated sulfoxide synthase has higher ergothioneine synthesis capacity.
The fermentation of a shake flask verifies that the glutamyl cysteine ligase (coding gene is gshA) derived from escherichia coli; neurospora crassa-derived sulfoxide synthase (coding gene egtE)ncr) (ii) a C-S lyase derived from Mycobacterium smegmatis (coding gene egtB)msm) The combination mode of (A) reconstructs a high-efficiency ergothioneine synthesis way in the escherichia coli. Carrying out shake flask fermentation for 26h, wherein the yield of ergothioneine in E.coli EGT12 strain fermentation liquor is 84.7 mg/L; the fermentation is continued for 30h, and the ergothioneine can be accumulated to 105.7 mg/L.
Example 3:
coli EGT12 fermentation experiments on 5L tanks.
Coli EGT12, which was constructed in example 1, was used as a production strain to produce ergothioneine as follows:
bevel activation: taking glycerol preservation strains, streaking and inoculating the glycerol preservation strains to a test tube slant culture medium, and culturing for 12h at 37 ℃; then the slant-preserved strain is streaked and inoculated in a slant culture medium of a eggplant-shaped bottle, and the culture is carried out for 14h at 37 ℃. Seed culture: taking one inclined plane of an activated fresh eggplant-shaped bottle, washing the inclined plane with 150mL of sterile water, inoculating the bottle 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 20-30% in the culture process, and culturing seeds until the OD is obtained600Is about 15.
Culturing in a fermentation tank: inoculating seed liquid (discharged to 450mL, and poured into a sterilized fermentation culture medium under flame protection) into seeds of a fermentation tank in an inoculation amount of 15%, controlling the temperature at 35 ℃, automatically feeding ammonia water (or 20% sulfuric acid) to control the pH value at 7.0, controlling the initial aeration rate at 2L/min, the aeration ratio at 0.667vvm, controlling the initial stirring speed at 400rpm, controlling the dissolved oxygen at 20-30% by adjusting the rotation speed and the air quantity, manually dropping foam killer for defoaming, feeding 80% glucose solution in the fermentation process, and feeding 5g/L methionine and 2g/L cysteine along with the sugar flow.
The slant culture medium comprises: 1g/L glucose, 10g/L peptone, 10g/L beef extract, 5g/L yeast powder, 2.5g/L NaCl, 25g/L agar and the balance 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 KH2PO4 5g/L，MgSO4·7H2O 2g/L，FeSO4·7H2O 10mg/L，MnSO4·H2O 10mg/L，VB1 2mg/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, K2HPO4 3g/L，MgSO4·7H2O 1.5g/L，FeSO4·7H2O 20mg/L，MnSO4·H2O20 mg/L, methionine 5-10g/L, cysteine 2-5g/L, VB6 10-20mg/L，VB1、VB3、VB5、VB12、VHEach 2mg/L, the rest is water, and the pH value is 7.0-7.2.
Coli EGT12 fermentation profile on 5L fermentor as shown in fig. 17.
As can be seen from the fermentation curve, the thallus begins to enter the logarithmic growth phase after fermenting for 8h, and the OD is 44h600Entering a stable period after reaching the maximum value; starting to enter a rapid accumulation stage of ergothioneine after fermenting for 12 hours, and fermenting for 52 hours until the yield of the ergothioneine is 2.9 g/L; histidine is continuously accumulated in the first 32h of the thalli, the maximum yield is about 20g/L in 32h, then the yield of histidine is reduced, and the final yield of histidine is 14.77g/L after fermentation is finished; since the methionine concentration increased 8h before the methionine addition with the sugar stream and then decreased, the fermentation broth could not detect methionine after 24 h.
SEQUENCE LISTING
<110> Tianjin science and technology university
<120> genetic engineering strain for producing ergothioneine and application thereof
<160> 4
<170> PatentIn version 3.5
<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
<210> 2
<211> 1422
<212> DNA
<213> Artificial sequence
<400> 2
atggttgcga ccaccgttga gctgccgctc cagcagaaag cggatgcggc ccaaaccgtt 60
acgggcccac tgccgttcgg caacagtctg ctgaaggagt tcgttctgga cccagcctat 120
cgtaatctga atcacggcag cttcggcacc atcccaagcg ccatccagca gaaactccgc 180
agttatcaga ccgccgccga agcgcgtcca tgcccgtttc tgcgttacca gaccccagtt 240
ctgctcgacg aaagccgtgc cgcggttgcg aatctgctga aagtgccggt tgaaaccgtt 300
gtgttcgtgg ccaatgccac gatgggcgtg aataccgttc tgcgcaacat cgtgtggagc 360
gccgatggca aagacgagat cctctacttc gacaccattt acggtgcgtg cggcaaaacg 420
atcgactacg tgatcgaaga caagcgcggt atcgtgagca gccgctgcat tccgctcatc 480
tacccggccg aagacgacga tgttgttgcc gcgtttcgcg atgcgatcaa gaagagccgc 540
gaggaaggta aacgcccacg tctggcggtg attgatgtgg tgagcagcat gccgggcgtg 600
cgtttcccgt tcgaggacat cgtgaagatc tgtaaagaag aggaaatcat tagctgcgtg 660
gatggcgcgc aaggcatcgg catggtggac ctcaaaatca ccgagaccga cccggacttc 720
ctcatcagca actgccataa gtggctgttc accccgcgtg gttgcgccgt gttctacgtt 780
ccggtgcgta accagcatct gattcgtagt acgctgccga cgagccatgg tttcgtgccg 840
caagttggta atcgtttcaa cccgctggtg ccagccggca acaagagtgc cttcgtgagc 900
aacttcgagt tcgtgggtac ggtggacaac agcccgttct tctgcgtgaa agacgccatc 960
aagtggcgtg aggaagttct cggtggcgag gagcgcatta tggagtacat gacgaagctg 1020
gcccgcgaag gcggccaaaa ggttgcggaa attctgggta cccgcgttct ggagaacagt 1080
acgggtaccc tcattcgctg cgcgatggtt aacatcgcgc tgccgtttgt ggttggcgaa 1140
gatccgaaag cgccggtgaa gctgaccgag aaagaggaga aagacgtgga aggtctgtac 1200
gaaatcccac acgaggaagc gaacatggcc ttcaagtgga tgtacaacgt gctgcaagat 1260
gagttcaata cgtttgtgcc aatgaccttc catcgtcgcc gtttttgggc ccgtctgagt 1320
gcccaagttt atctggaaat gagcgacttc gagtgggccg gcaaaacgct caaagagctg 1380
tgcgagcgcg tggccaaagg cgaatacaaa gagagcgcct aa 1422
<210> 3
<211> 1287
<212> DNA
<213> Artificial sequence
<400> 3
atgatcgcac gcgagacact ggccgacgag ctggccctgg cccgcgaacg cacgttgcgg 60
ctcgtggagt tcgacgacgc ggaactgcat cgccagtaca acccgctgat gagcccgctc 120
gtgtgggacc tcgcgcacat cgggcagcag gaagaactgt ggctgctgcg cgacggcaac 180
cccgaccgcc ccggcatgct cgcacccgag gtggaccggc tttacgacgc gttcgagcac 240
tcacgcgcca gccgggtcaa cctcccgttg ctgccgcctt cggatgcgcg cgcctactgc 300
gcgacggtgc gggccaaggc gctcgacacc ctcgacacgc tgcccgagga cgatccgggc 360
ttccggttcg cgctggtgat cagccacgag aaccagcacg acgagaccat gctgcaggca 420
ctcaacctgc gcgagggccc acccctgctc gacaccggaa ttcccctgcc cgcgggcagg 480
ccaggcgtgg caggcacgtc ggtgctggtg ccgggcggcc cgttcgtgct cggggtcgac 540
gcgctgaccg aaccgcactc actggacaac gaacggcccg cccacgtcgt ggacatcccg 600
tcgttccgga tcggccgcgt gccggtcacc aacgccgaat ggcgcgagtt catcgacgac 660
ggtggctacg accaaccgcg ctggtggtcg ccacgcggct gggcgcaccg ccaggaggcg 720
ggcctggtgg ccccgcagtt ctggaacccc gacggcaccc gcacccggtt cgggcacatc 780
gaggagatcc cgggtgacga acccgtgcag cacgtgacgt tcttcgaagc cgaggcctac 840
gcggcgtggg ccggtgctcg gttgcccacc gagatcgaat gggagaaggc ctgcgcgtgg 900
gatccggtcg ccggtgctcg gcgccggttc ccctggggct cagcacaacc cagcgcggcg 960
ctggccaacc tcggcggtga cgcacgccgc ccggcgccgg tcggggccta cccggcgggg 1020
gcgtcggcct atggcgccga gcagatgctg ggcgacgtgt gggagtggac ctcctcgccg 1080
ctgcggccgt ggcccggttt cacgccgatg atctacgagc agtacagcac gccgttcttc 1140
gagggcacca catccggtga ctaccgcgtg ctgcgcggcg ggtcatgggc cgttgcaccg 1200
ggaatcctgc ggcccagctt ccgcaactgg gaccacccga tccggcggca gatcttctcg 1260
ggtgtccgcc tggcctggga cgtctga 1287
<210> 4
<211> 428
<212> PRT
<213> Artificial sequence
<400> 4
Met Ile Ala Arg Glu Thr Leu Ala Asp Glu Leu Ala Leu Ala Arg Glu
1 5 10 15
Arg Thr Leu Arg Leu Val Glu Phe Asp Asp Ala Glu Leu His Arg Gln
20 25 30
Tyr Asn Pro Leu Met Ser Pro Leu Val Trp Asp Leu Ala His Ile Gly
35 40 45
Gln Gln Glu Glu Leu Trp Leu Leu Arg Asp Gly Asn Pro Asp Arg Pro
50 55 60
Gly Met Leu Ala Pro Glu Val Asp Arg Leu Tyr Asp Ala Phe Glu His
65 70 75 80
Ser Arg Ala Ser Arg Val Asn Leu Pro Leu Leu Pro Pro Ser Asp Ala
85 90 95
Arg Ala Tyr Cys Ala Thr Val Arg Ala Lys Ala Leu Asp Thr Leu Asp
100 105 110
Thr Leu Pro Glu Asp Asp Pro Gly Phe Arg Phe Ala Leu Val Ile Ser
115 120 125
His Glu Asn Gln His Asp Glu Thr Met Leu Gln Ala Leu Asn Leu Arg
130 135 140
Glu Gly Pro Pro Leu Leu Asp Thr Gly Ile Pro Leu Pro Ala Gly Arg
145 150 155 160
Pro Gly Val Ala Gly Thr Ser Val Leu Val Pro Gly Gly Pro Phe Val
165 170 175
Leu Gly Val Asp Ala Leu Thr Glu Pro His Ser Leu Asp Asn Glu Arg
180 185 190
Pro Ala His Val Val Asp Ile Pro Ser Phe Arg Ile Gly Arg Val Pro
195 200 205
Val Thr Asn Ala Glu Trp Arg Glu Phe Ile Asp Asp Gly Gly Tyr Asp
210 215 220
Gln Pro Arg Trp Trp Ser Pro Arg Gly Trp Ala His Arg Gln Glu Ala
225 230 235 240
Gly Leu Val Ala Pro Gln Phe Trp Asn Pro Asp Gly Thr Arg Thr Arg
245 250 255
Phe Gly His Ile Glu Glu Ile Pro Gly Asp Glu Pro Val Gln His Val
260 265 270
Thr Phe Phe Glu Ala Glu Ala Tyr Ala Ala Trp Ala Gly Ala Arg Leu
275 280 285
Pro Thr Glu Ile Glu Trp Glu Lys Ala Cys Ala Trp Asp Pro Val Ala
290 295 300
Gly Ala Arg Arg Arg Phe Pro Trp Gly Ser Ala Gln Pro Ser Ala Ala
305 310 315 320
Leu Ala Asn Leu Gly Gly Asp Ala Arg Arg Pro Ala Pro Val Gly Ala
325 330 335
Tyr Pro Ala Gly Ala Ser Ala Tyr Gly Ala Glu Gln Met Leu Gly Asp
340 345 350
Val Trp Glu Trp Thr Ser Ser Pro Leu Arg Pro Trp Pro Gly Phe Thr
355 360 365
Pro Met Ile Tyr Glu Gln Tyr Ser Thr Pro Phe Phe Glu Gly Thr Thr
370 375 380
Ser Gly Asp Tyr Arg Val Leu Arg Gly Gly Ser Trp Ala Val Ala Pro
385 390 395 400
Gly Ile Leu Arg Pro Ser Phe Arg Asn Trp Asp His Pro Ile Arg Arg
405 410 415
Gln Ile Phe Ser Gly Val Arg Leu Ala Trp Asp Val
420 425

Claims (10)

1. A genetically engineered bacterium for producing ergothioneine is characterized in that the genetically engineered bacterium takes escherichia coli as a host, and a glutamic acid stick with a sequence shown as SEQ ID NO. 1 is integrated on the genome of the hostThe coding gene hisG of bacillus ATP transphosphoribosyl enzyme HisG mutant; the copy number of histidine operon gene hisDBCHAFI is also increased on the genome; also integrates the ergothioneine operator gene egtCDE of Mycobacterium smegmatis on the genome; the gene gshA for encoding the glutamylcysteine ligase of the escherichia coli is also integrated on the genome of the gene gshA; also integrates a gene egtE of the C-S lyase coding gene of the Neurospora crassa with the sequence shown as SEQ ID NO. 2 on the genomencr(ii) a Also integrated into its genome is the gene egtB which codes for a sulfoxide synthase mutant with the amino acid sequence shown in SEQ ID NO. 4msm。
2. The genetically engineered bacterium of claim 1, wherein said host is e.
3. The genetically engineered bacterium of claim 1, wherein the histidine operon gene hisDBCHAFI comprises seven genes hisD, hisB, hisC, hisH, hisA, hisF and hisI, and wherein the histidine operon is increased as two copies in the host genome.
4. The genetically engineered bacterium of claim 1, wherein the gene integrated into the host genome is transcriptionally expressed from a strong promoter.
5. The genetically engineered bacterium of claim 4, wherein the strong promoter is promoter Ptrc。
6. The genetically engineered bacterium of claim 1, wherein the nucleotide sequence of the gene encoding the sulfoxide synthase mutant is as set forth in SEQ ID NO: 3, respectively.
7. The genetically engineered bacterium of claim 1, wherein said E.coli glutamylcysteine ligase encoding gene gshA is replaced with Mycobacterium smegmatis glutamylcysteine ligase encoding gene egtA, and/or said E.coli glutamylcysteine ligase encoding gene egtAC-S lyase coding gene egtE from Neurospora crassancrReplacement by Mycobacterium smegmatis C-S lyase-encoding gene egtEmsmAnd/or the gene egtB coding for the sulfoxide synthase mutantmsmSubstitution to the sulfoxide synthase encoding Gene egtE derived from Mycobacterium smegmatismsm。
8. A construction method of a genetic engineering bacterium for producing ergothioneine is characterized in that the genetic engineering bacterium is obtained by directionally transforming a host E.coli MG1655 by using a CRISPR/Cas9 mediated gene editing technology, and specifically comprises the following steps:
(1) construction of the promoter PtrcAnd nucleotide sequence shown as SEQ ID NO:1 of Corynebacterium glutamicumtrc-hisG and integrating it at the tdcD gene site on the host genome;
(2) the histidine operon gene hisDBCHAF was integrated at the yghX gene site in the host genome and expressed from the promoter PtrcStarting;
(3) the ergothioneine operator egtCDE is integrated in the host genome at the ilvG gene site and is obtained from the promoter PtrcStarting;
(4) construction of the promoter PtrcConnecting fragment P of gene gshA encoding glutamylcysteine ligase of escherichia colitrc-gshA, and integrating it at the mbhA gene site on the host genome;
(5) construction of the promoter PtrcAnd nucleotide sequence shown as SEQ ID NO:2 of the gene egtE for the C-S lyase of Venus crassancrConnecting fragment P oftrc-egtEncrAnd integrating it at the site of the tehB gene on the host genome;
(6) construction of the promoter PtrcAnd nucleotide sequence shown as SEQ ID NO: 3, and coding gene egtB of sulfoxide synthase lyase mutantmsmConnecting fragment P oftrc-egtB*msmAnd integrated at the locus of the yeeP gene on the genome.
9. A process for producing ergothioneine, comprising culturing the genetically engineered bacterium according to any one of claims 1 to 7 under suitable conditions, and collecting ergothioneine from the culture.
10. The method of claim 9, wherein the genetically engineered bacteria are activated to prepare a seed solution, the seed solution is inoculated to a fermentation medium in an inoculation amount of 15%, the temperature is controlled at 35 ℃, ammonia water or 20% sulfuric acid is automatically fed, the pH is controlled at 7.0, the aeration ratio is 0.667vvm, the initial stirring speed is 400rpm, the dissolved oxygen is controlled at 20-30%, an 80% glucose solution is fed during fermentation, and 5g/L methionine and 2g/L cysteine are fed with sugar; the fermentation medium comprises the following components: 10g/L glucose, 5g/L yeast extract, 4g/L tryptone, K2HPO4 3g/L，MgSO4·7H2O 1.5g/L，FeSO4·7H2O 20mg/L，MnSO4·H2O20 mg/L, methionine 5-10g/L, cysteine 2-5g/L, VB6 10-20mg/L，VB1、VB3、VB5、VB12、VHEach 2mg/L, the rest is water, and the pH value is 7.0-7.2.
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CN114032229A (en) * 2021-11-09 2022-02-11 南开大学 Single enzyme mutant prepared by replacing ACS double-enzyme activity key structural domain of higher plant and application thereof
CN114085782A (en) * 2021-11-23 2022-02-25 深圳中科欣扬生物科技有限公司 Ergothioneine synthetic gene derived from natural hot spring of Quzhuomu and development and application thereof
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Publication number Priority date Publication date Assignee Title
CN112980760A (en) * 2021-03-08 2021-06-18 华东理工大学 Genetic engineering strain for producing ergothioneine and construction method and application thereof
CN112980760B (en) * 2021-03-08 2023-09-29 百开盛(上海)生物科技有限公司 Genetic engineering strain for producing ergothioneine, construction method and application thereof
CN114032229A (en) * 2021-11-09 2022-02-11 南开大学 Single enzyme mutant prepared by replacing ACS double-enzyme activity key structural domain of higher plant and application thereof
CN114032229B (en) * 2021-11-09 2023-11-17 南开大学 Single enzyme mutant prepared by replacing ACS (ACS) double-enzyme activity key structural domain of higher plant
CN114085782A (en) * 2021-11-23 2022-02-25 深圳中科欣扬生物科技有限公司 Ergothioneine synthetic gene derived from natural hot spring of Quzhuomu and development and application thereof
CN114085782B (en) * 2021-11-23 2023-10-24 深圳中科欣扬生物科技有限公司 Ergothioneine synthetic gene from Qu Zhuomu natural hot spring and development and application thereof
CN114262702A (en) * 2021-12-31 2022-04-01 西南大学 Application of ergothioneine synthesis gene in reconstruction of ergothioneine metabolic pathway in corynebacterium glutamicum and method thereof
CN114262702B (en) * 2021-12-31 2023-12-08 西南大学 Application of ergothioneine synthesis gene in reconstructing ergothioneine metabolic pathway in corynebacterium glutamicum and method thereof
JP7450959B2 (en) 2022-01-07 2024-03-18 シャメン オアミック バイオテック カンパニー リミテッド Yeast and its use in ergothioneine production
CN114606170A (en) * 2022-03-07 2022-06-10 深圳中科欣扬生物科技有限公司 Ergothioneine biosynthesis method based on CRISPR-Cas9 and application
CN114606170B (en) * 2022-03-07 2023-07-18 深圳中科欣扬生物科技有限公司 CRISPR-Cas 9-based ergothioneine biosynthesis method and application

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