CN116904380A - Construction and application of genetic engineering bacteria for producing ergothioneine - Google Patents
Construction and application of genetic engineering bacteria for producing ergothioneine Download PDFInfo
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- CN116904380A CN116904380A CN202310930735.3A CN202310930735A CN116904380A CN 116904380 A CN116904380 A CN 116904380A CN 202310930735 A CN202310930735 A CN 202310930735A CN 116904380 A CN116904380 A CN 116904380A
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- ergothioneine
- gene
- escherichia coli
- cfegt2
- cfegt1
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- SSISHJJTAXXQAX-ZETCQYMHSA-N L-ergothioneine Chemical compound C[N+](C)(C)[C@H](C([O-])=O)CC1=CNC(=S)N1 SSISHJJTAXXQAX-ZETCQYMHSA-N 0.000 title claims abstract description 61
- 229940093497 ergothioneine Drugs 0.000 title claims abstract description 60
- 241000894006 Bacteria Species 0.000 title claims abstract description 30
- 238000010353 genetic engineering Methods 0.000 title claims abstract description 13
- 238000010276 construction Methods 0.000 title abstract description 10
- 238000000855 fermentation Methods 0.000 claims abstract description 29
- 230000004151 fermentation Effects 0.000 claims abstract description 29
- 101150002764 purA gene Proteins 0.000 claims abstract description 13
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- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 20
- 241000588724 Escherichia coli Species 0.000 claims description 18
- 150000001413 amino acids Chemical group 0.000 claims description 13
- 239000002609 medium Substances 0.000 claims description 12
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- 239000008103 glucose Substances 0.000 claims description 10
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- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 7
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- 229910052708 sodium Inorganic materials 0.000 claims description 4
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- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 3
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- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
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- GPPYTCRVKHULJH-QMMMGPOBSA-N N(alpha),N(alpha),N(alpha)-trimethyl-L-histidine Chemical class C[N+](C)(C)[C@H](C([O-])=O)CC1=CNC=N1 GPPYTCRVKHULJH-QMMMGPOBSA-N 0.000 description 1
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Abstract
The invention discloses construction and application of a genetic engineering bacterium for producing ergothioneine, and belongs to the technical field of genetic engineering. According to the invention, genes purH, purA and purB are integrated on a ppnn site of E coli.BW25113, genes ndk and adk are integrated on a mazG site, cfegt1 and cfegt2 are over-expressed, and cfegt1 and cfegt2 are connected through a flexible joint, so that the genetic engineering bacterium E2-cfegt1-linker-cfegt2 for producing ergothioneine is constructed. Fermenting the obtained genetically engineered bacterium E2-cfegt1-linker-cfegt2 in a 20ml shake flask for 48 hours, and producing 112mg/L of ergothioneine; fermenting for 96 hours in a 2L system fermentation tank, and producing the ergothioneine with the yield of 8g/L, thereby being beneficial to the industrial production of the ergothioneine.
Description
Technical Field
The invention relates to construction and application of a genetic engineering bacterium for producing ergothioneine, and belongs to the technical field of genetic engineering.
Background
Ergothioneine (ergothioneine) is a naturally occurring amino acid that was first isolated from ergot fungi in 1909 and named after this fungus. The structure was determined in 1911 and ergothioneine was found to be a histidine betaine derivative. Ergothioneine is a natural and precious sulfur-containing amino acid, colorless and odorless, and is very soluble in water, methanol and ethanol. Ergothioneine has the functions of resisting oxidation and aging, inhibiting cardiovascular diseases, maintaining DNA biosynthesis, scavenging free radicals, promoting neurogenesis and the like, has better protection effect on cell and tissue injury in various disease models, and therefore, can be often used as an antioxidant capable of being transported between cells. In 2014, ergothioneine was formally listed by the national food and drug administration as a catalog of cosmetic raw materials for use. The new resource food list is listed in the european union in 2018. The ergothioneine in nature is mainly synthesized by biological pathways such as fungi, bacteria and the like, but the ergothioneine cannot be directly synthesized by human or animals and can only be indirectly taken up by eating, and then is absorbed and accumulated in tissues or cells.
Ergothioneine can currently be prepared by chemical synthesis and biological fermentation. For such optically active components, the synthetic and purification processes of chemical synthesis are highly demanding. Compared with chemical synthesis and natural biological extraction processes, the biological fermentation method for producing ergothioneine has the advantages of low cost, easily obtained raw materials, easily enlarged yield, high product safety and the like. The fermentation technology of ergothioneine comprises solid fermentation and liquid fermentation, wherein the solid fermentation is to inoculate fungus strain into a solid culture medium to obtain metabolite produced by mycelium. The solid fermentation microorganism is metabolized, grown and formed on the surface of a solid medium, and is easy to be contaminated by miscellaneous bacteria, long in fermentation period and low in product content. Liquid fermentation is a common means for synthesizing ergothioneine, and the current literature reports that escherichia coli is fermented for 94 hours in a 5L fermentation tank through fed-batch fermentation, and the yield of the ergothioneine is 5.4g/L.
Protein linker refers to short amino acid sequence with 2 or more protein molecules or domains connected, has important function on protein function and stability, and is an important means for improving space and time contact efficiency of enzyme and substrate. Wherein, the flexible joint is mainly composed of neutral amino acids (such as Gly and Ser) with small molecular weight, which can lead the connecting structural domains to have certain swinging degree and interaction, and the flexible joint is most commonly used in the design of fusion proteins. However, there is no protein linker suitable for engineering strains producing ergothioneine.
Disclosure of Invention
According to the invention, E coli.BW25113 is taken as an initial strain, genes purH, purA, purB, ndk and adk are integrated on a genome, and chassis cells expressing ergothioneine are constructed; further, cfegt1 and cfegt2 are over-expressed in the obtained chassis cells, the cfegt1 and cfegt2 are connected through a flexible joint with an amino acid sequence of GGGGSGGGGS, so that the genetic engineering bacteria for producing the ergothioneine are constructed, and the yield of producing the ergothioneine by the strain can be remarkably improved.
The invention provides an escherichia coli genetic engineering bacterium for producing ergothioneine, which overexpresses an inosine cyclohydrolase coding gene (purH), an adenylyl-glass-peruvic acid synthase coding gene (purA), an adenylyl-succinic acid lyase coding gene (purB), a nucleoside diphosphate kinase coding gene (ndk) and an adenylyl kinase coding gene (adk), and expresses an ergothioneine synthesis gene 1 (cfegt 1) and an ergothioneine synthesis gene 2 (cfegt 2) from anthrax (Colletotrichum fioriniae).
In one embodiment, the nucleotide sequence of purH is shown as SEQ ID NO.10, the nucleotide sequence of purA is shown as SEQ ID NO.11, the nucleotide sequence of purB is shown as SEQ ID NO.12, the nucleotide sequence of adk is shown as SEQ ID NO.13, the nucleotide sequence of ndk is shown as SEQ ID NO.14, the nucleotide sequence of cfegt1 is shown as SEQ ID NO.6, and the nucleotide sequence of cfegt2 is shown as SEQ ID NO. 8.
In one embodiment, the escherichia coli genetically engineered bacteria integrate genes purH, purA and purB at the ppnn site, wherein the nucleotide sequence of the gene purH is shown as SEQ ID NO.10, the nucleotide sequence of the gene purA is shown as SEQ ID NO.11, and the nucleotide sequence of the gene purB is shown as SEQ ID NO. 12.
In one embodiment, the escherichia coli genetically engineered bacterium integrates genes ndk and adk at a mazG site, the nucleotide sequence of the gene adk is shown in SEQ ID NO.13, and the nucleotide sequence of the gene ndk is shown in SEQ ID NO. 14.
In one embodiment, the cfegt1 and cfegt2 are linked by the amino acid sequence shown in SEQ ID No. 9.
The invention also provides a method for producing ergothioneine by fermentation, which comprises the step of fermenting the escherichia coli genetic engineering bacteria in a culture medium.
In one embodiment, the medium comprises peptone, yeast powder, na 2 HPO 4 ·12H 2 O、KH 2 PO 4 、NH 4 Cl、Na 2 SO 4 Glycerol, glucose, mgSO 4 ·7H 2 O, trace salt solution, arabinose, ampicillin and methionine.
In one embodiment, the feed medium is fed when glucose is depleted, and the residual sugar content in the fermentation broth is controlled to be no more than 5g/L.
In one embodiment, the feed medium comprises glucose: 500-600g/L, histidine: 20-30g/L, methionine: 20-30g/L, cysteine: 20-30g/L.
The invention also provides application of the escherichia coli genetic engineering bacteria in preparation of products containing ergothioneine.
The beneficial effects are that:
the invention integrates genes purH, purA and purB on the ppnn locus of E coli.BW25113, integrates genes ndk and adk on the mazG locus, and constructs chassis cells for expressing ergothioneine; and further over-expressing cfegt1 and cfegt2 in the obtained chassis cells, wherein the cfegt1 and the cfegt2 are connected through a flexible joint with an amino acid sequence of GGGGSGGGGS, and the genetic engineering bacterium E2-cfegt1-linker-cfegt2 for producing ergothioneine is constructed. The obtained genetically engineered bacterium E2-cfegt1-linker-cfegt2 is fermented for 48 hours in a 20mL shake flask, the yield of the produced ergothioneine is 112mg/L, the yield is improved by 0.8 times compared with the yield of the genetically engineered bacterium without a flexible joint, and the yield is improved by 0.96 times compared with the yield of the genetically engineered bacterium over-expressing the ncegt1 and the ncegt2; fermenting in a 5L fermentation tank of a 2L system for 96 hours, wherein the yield of the produced ergothioneine is 8g/L, and the method is favorable for industrial production of the ergothioneine.
Drawings
Fig. 1: shake flask fermentation of ergothioneine yield; wherein 1 is E2-ncegt1-ncegt2;2 is E2-cfegt1-cfegt2;3 is E2-ncegt1-linker-ncegt2;4 is E2-cfegt1-linker-cfegt2;5 is BW-ncegt1-ncegt2;6 is BW-cfegt1-cfegt2;7 is BW-ncegt1-linker-ncegt2;8 is BW-cfegt1-linker-cfegt2;
fig. 2: the method comprises the steps of (1) feeding and fermenting an engineering bacterium 5L fermentation tank, wherein the yield of ergothioneine at different time points;
fig. 3: HPLC detection result diagram of engineering bacteria E2-cfegt1-linker-cfegt2 for synthesizing ergothioneine by fermentation, wherein figure A is 15mg/L ergothioneine standard, and figure B is sample with 160 times dilution of supernatant of bacterial liquid after fermentation.
Detailed Description
According to the research of the literature, the ncegt1 and the ncegt2 derived from the fungus MAICHO (Neurospora crassa) are commonly used for biosynthesis of ergothioneine, so that two genes derived from the species are used as a reference, and are screened in a database through enzyme activity comparison by intelligent calculation software to obtain cfegt1 and cfegt2 with the same function from Colletotrichum fioriniae, and the four genes are subjected to codon optimization to synthesize the genes. The amino acid sequence of NCegt1 protein is shown as SEQ ID NO.1, the nucleotide sequence of the codon-optimized NCegt1 gene is shown as SEQ ID NO.2, the amino acid sequence of the NCegt2 protein is shown as SEQ ID NO.3, the nucleotide sequence of the codon-optimized NCegt2 gene is shown as SEQ ID NO.4, the amino acid sequence of the CFegt1 protein is shown as SEQ ID NO.5, the nucleotide sequence of the codon-optimized CFegt1 gene is shown as SEQ ID NO.6, the amino acid sequence of the CFegt2 protein is shown as SEQ ID NO.7, and the nucleotide sequence of the codon-optimized CFegt2 gene is shown as SEQ ID NO. 8.
EXAMPLE 1 E.coli expression vector construction
The amplification of the vector fragment was performed using pBAD/HisA (available from Semer Feiche technologies Co., ltd.) plasmids as templates and pBAD-F and pBAD-R as primers. 2 kinds of Egt1 and Egt2 fragments from different sources are combined to construct plasmid, and a flexible linker (the amino acid sequence is shown as SEQ ID NO. 9) is respectively introduced to connect Egt1 and Egt2 into fusion protein. The synthesized ancegt 1 gene is used as a template, and F1 and R1 are used as primers to amplify to obtain a fragment ancegt 1. The synthesized ancegt 2 gene is used as a template, and F2 and R2 are used as primers to amplify to obtain a fragment ancegt 2. The synthesized ncegt1 gene is used as a template, and F1 and R6 are used as primers to amplify to obtain a fragment ncegt1-linker. The synthesized ancegt 2 gene is used as a template, F6 and R2 are used as primers to amplify to obtain a fragment linker-ancegt 2. Synthesizing cfegt1 gene as a template, and amplifying F3 and R3 as primers to obtain fragment cfegt1. Synthesizing cfegt2 gene as a template, and amplifying F4 and R4 as primers to obtain fragment cfegt2. And synthesizing cfegt1 genes as templates, and amplifying F3 and R5 serving as primers to obtain fragments cfegt1-linker. And synthesizing cfegt2 genes as templates, and amplifying with F5 and R4 as primers to obtain fragment linker-cfegt2. The amplified vector fragment, the gene fragment with/without flexible joint, and the gene fragment ancegt 1 and ancegt 2 are respectively connected by adopting a Norwegian recombination cloning kit (Clone MultiS One Step Cloning Kit, (C113-02)) to construct and obtain plasmids pBAD-ancet 1-ancet 2 and pBAD-ancet 1-linker-ancet 2 for producing ergothioneine; the amplified vector fragment, the gene fragment cfegt1 with/without the flexible linker and the gene fragment cfegt2 are respectively connected to construct plasmids pBAD-cfegt1-cfegt2 and pBAD-cfegt1-linker-cfegt2 for producing ergothioneine. And respectively converting the obtained recombinant plasmids into competent cells Trans1-T1 (Phage Chemically Comptent Cell) by a chemical conversion method, picking positive clones, and extracting plasmids for sequencing.
TABLE 1 primer sequences
EXAMPLE 2 construction of genetically engineered bacteria producing ergothioneine
1. Construction of Chassis cells
The synthesis of ergothioneine requires the participation of ATP and increases the ability of ATP synthesis in E.coli by overexpression of purH, purA, purB and adk, ndk in the chassis strain.
Modification of the metabolic pathway of Chassis E coli.BW25113, integration of the genes purH, purA and purB at the ppnn site. Gene knock-in experiments were performed using CRISPR-CAS9 technology. Firstly constructing pTargetF plasmid of PPNN, inputting target gene nucleotide sequence on sgRNA design website (http:// crispor.tefor.net /), selecting sgRNA sequence with low off-target rate. PCR was performed using pTargetF plasmid as a template and primers PPNN-N20-F and PPNN-N20-R, DH 5. Alpha. Competent cells were transformed after purification and recovery, plates containing 50mg/L spectinomycin were incubated overnight at 37℃on LB plates, and single colony plasmids were picked up for sequencing. The construction of Donor DNA is carried out by using overlap extension PCR, taking the genome of escherichia coli BW25113 as a template, respectively amplifying upstream and downstream homologous arm fragments PPNN-UP and PPNN-DOWN by using primers PPNN-UP/R and PPNN-DOWN, amplifying purH gene by using primers PURH-F/R, amplifying purA by using primers PURA-F/R, amplifying purB by using primers PURB-F/R, purifying and recovering fragments by using purified purH, purA and purB fragments as templates, carrying out overlap extension PCR by using primers PURH-F and PURB-R, and cutting the PCR product into gel and recovering target fragment PUR. And (3) performing overlap extension PCR by using the PUR, the PPNN-UP and the PPNN-DOWN as templates and using the primers PPNN-UP-F and the PPNN-DOWN-R, and cutting the PCR product into gel to recover a target fragment PPNN Donor.
Preparation of electrotransformation competent cells, 50 mu L E of coll.BW25113 glycerol bacteria were taken and inoculated in 5mL of antibiotic-free LB medium for overnight culture, and 100mL of antibiotic-free LB medium was inoculated in an inoculum size of 1%In which culture was carried out at 37℃to OD 600 =0.6, pre-chilled on ice for 10min, then the supernatant was decanted by centrifugation at 4000rpm for 10min, the cells were resuspended in pre-chilled 10% glycerol, centrifuged at 4000rpm for 10min, and the cells were washed twice with 10% glycerol. Cells were resuspended in 1mL of pre-chilled 10% glycerol and dispensed into sterile EP tubes at 100. Mu.L per tube. One tube of competent cells was taken and added to 2. Mu.L of pCas9 plasmid, the above system was added to a 2mm electric beaker, 2.5kV shocked, 900. Mu.L of antibiotic-free LB medium was rapidly added, and after resuspension, transferred to a sterile EP tube, resuscitated at 30℃for 60min at 200 rpm. Centrifugation at 4000rpm for 5min, pouring out a part of supernatant, re-suspending, coating a kanamycin LB plate containing 50mg/L, culturing overnight at 30 ℃, and gathering single colonies in LB culture medium to prepare BW25113, wherein pCas9 electrotransformation competence. The pTargetF plasmid of PPNN and the Donor DNA of PPNN were co-transformed into BW25113: pCas9 electrotransformation competent cells to obtain E1 strain. E1 strain was added to 900. Mu.L of antibiotic-free LB medium, resuscitated at 30℃and 200rpm for 60min, and then plated with kanamycin and spectinomycin double-resistant plates, and cultured overnight at 30 ℃. And selecting a single colony for colony PCR, selecting a positive result for measurement, and selecting an E1 strain with correct sequencing for subsequent experiments.
Continuing to integrate genes ndk and adk on the mazG locus of the E1 strain, carrying out PCR (polymerase chain reaction) by using a pTargetF plasmid as a template and using primers MAZG-N20-F and MAZG-N20-R, purifying and recovering, converting the obtained pTargetF fragment of MAZG into competent cells, coating a spectinomycin LB plate containing 50mg/L, culturing overnight at 37 ℃, and picking a single colony plasmid for sequencing. The construction of Donor DNA is carried out by overlapping extension PCR, wherein the genome of escherichia coli BW25113 is used as a template, primers MAZG-UP-F/R and MAZG-DOWN-F/R are respectively used for amplifying upstream and downstream homologous arm fragments MAZG-UP and MAZG-DOWN, primer ADK-F/R is used for amplifying ADK genes, primer NDK-F/R is used for amplifying NDK, gel running is used for verifying fragment size, ADK and NDK fragments are used as templates for overlapping extension PCR after purification and recovery, and primers ADK-F and NDK-R are used for carrying out gel cutting on PCR products to recover target fragments ADNK. And performing overlap extension PCR by using ADNK, MAZG-UP and MAZG-DOWN fragments as templates and using primers MAZG-UP-F and MAZG-DOWN-R, and cutting a PCR product to recover a target fragment MAZG Donor. Competent cells of E1 strain were prepared in the same manner as described above, and pTargetF plasmid of MAZG and Donor DNA of MAZG were electrotransformed into competent cells of E1 strain to obtain E2 strain. The E2 strain was added to 900. Mu.L of the antibiotic-free LB medium, resuscitated at 30℃and 200rpm for 60min, and then plated with kanamycin and spectinomycin double-resistant plates, and cultured overnight at 30 ℃. And selecting a single colony for colony PCR, selecting a positive result for measurement, and selecting an E2 strain with correct sequencing for subsequent experiments.
2. Construction of genetically engineered bacteria for producing ergothioneine
The plasmids pBAD-ncegt1-ncegt2, pBAD-cfegt1-cfegt2, pBAD-ncegt1-linker-ncegt2 and pBAD-cfegt1-linker-cfegt2 which are sequenced correctly in example 1 are electrically transferred into the strain E2 of the chassis fungus, the prepared strain E2 is placed on ice for melting, 1 mu L of plasmid is respectively added into the strain E2 competence, the mixture is gently blown and mixed, competent cells containing the plasmids are added into a 2mm electrorotating cup, the electric shock conversion condition of the electrorotating instrument is that the voltage is 2.5kV, the resistance is 200 omega, 1mL of LB culture medium is immediately added, and then the culture is carried out for 1h at 37 ℃ and 200rpm in an oscillating way. The transformed strains are respectively coated on a screening culture medium containing 100mg/L ampicillin, and after inversion culture is carried out at 37 ℃ for overnight, monoclonal is selected for PCR identification, and correct strains E2-ncegt1-ncegt2, E2-cfegt1-cfegt2, E2-ncegt1-linker-ncegt2 and E2-cfegt1-linker-cfegt2 are obtained for subsequent verification of the yield of ergothioneine.
The same experimental procedure was followed to transform four plasmids into E.coli BW25113, obtaining the strains BW-ncegt1-ncegt2, BW-cfegt1-cfegt2, BW-ncegt1-linker-ncegt2 and BW-cfegt1-linker-cfegt2.
EXAMPLE 3 shaking flask culture of engineering bacteria
8 engineering bacteria obtained in example 2 were subjected to shake flask culture, respectively.
Shake flask 1L medium was peptone: 10g, yeast powder: 5g, na 2 HPO 4 ·12H 2 O：9g，KH 2 PO 4 ：3.4g，NH 4 Cl：2.7g，Na 2 SO 4 :0.7g, glycerol: 5g, glucose: 0.5g of MgSO 4 ·7H 2 O:1.2g, 1mL of trace salt solution (1L of trace salt solution contains FeCl) 3 ·6H 2 O：13.51g，CaCl 2 ：2.22g，MnCl 2 ·4H 2 O：1.98g，ZnSO 4 ·7H 2 O：2.88g，CoCl 2 ·6H 2 O：0.48g，NiCl 2 ·6H 2 O：0.48g，Na 2 MoO 4 ·2H 2 O：0.48g，Na 2 SeO 3 ：0.35g，H 3 BO 3 :0.12 g) arabinose: 2g, methionine: 1g, volume was set to 1L with water. The resistance component used in the medium was ampicillin at a final concentration of 100. Mu.g/mL.
The shake flask culture conditions were: the strain cultured for 8-10 hours was added to a 20mL fermentation system in an amount of 1mL/100mL and the initial OD was 0.1, and cultured at 30 ℃. After 24h of incubation, histidine, methionine and cysteine were added at a final concentration of 1g/L, 2g/L glucose, 20mg/L FeSO 4 ·7H 2 O, culturing at 30 ℃ at 200rpm, sampling, carrying out protein electrophoresis to observe protein expression, centrifuging to remove bacterial cells, and reserving supernatant for ergothioneine content detection, wherein the result is shown in figure 1. Wherein, 1 is E2-ncegt1-ncegt2 strain with the yield of the ergothioneine of 57mg/L at 48 hours, 2 is E2-cfegt1-cfegt2 strain with the yield of the ergothioneine of 62mg/L at 48 hours, 3 is E2-ncegt1-linker-ncegt2 strain with the yield of the ergothioneine of 42mg/L at 48 hours, and 4 is E2-cfegt1-linker-cfegt2 strain with the yield of the ergothioneine of 112mg/L at 48 hours. 5 is BW-ncegt1-ncegt2 strain with a yield of 25mg/L in 48 hours, 6 is BW-cfegt1-cfegt2 strain with a yield of 35mg/L in 48 hours, 7 is BW-ncegt1-linker-ncegt2 strain with a yield of 20mg/L in 48 hours, and 8 is BW-cfegt1-linker-cfegt2 strain with a yield of 70mg/L in 48 hours.
From the results of the synthesis of ergothioneine, the yield of each plasmid in the engineering strain E2 is higher, which indicates that the transformation of the chassis strain has positive significance for improving the synthesis capacity of ergothioneine. Meanwhile, after CFEgt1 and CFEgt2 are over-expressed, the ability of the strain to produce ergothioneine is stronger, and the yield is obviously improved by flexible protein joints, which is probably that two proteins of CFEgt1 and CFEgt2 are closer through the joint space distance, and the reaction speed is faster. However, the synthesis yield of ergothioneine decreases after NCEgt1 and NCEgt2 are linked by a protein linker, which may affect the spatial structure of the two proteins.
Example 4 feed fermentation of engineering bacteria 5L fermenters
Seed culture medium formulation (1L): yeast extract: 5g, peptone: 10g, sodium chloride: 10g.
Fermentation medium formula (1L): glucose: 5g, (NH) 4 ) 2 HPO 4 ：4g，KH 2 PO 4 :13.3g, citric acid: 1.7g, trace salt solution: 10mL (1L trace salt solution contains FeCl) 3 ·6H 2 O：13.51g，CaCl 2 ：2.22g，MnCl 2 ·4H 2 O：1.98g，ZnSO 4 ·7H 2 O：2.88g，CoCl 2 ·6H 2 O：0.48g，NiCl 2 ·6H 2 O：0.48g，Na 2 MoO 4 ·2H 2 O：0.48g，Na 2 SeO 3 ：0.35g，H 3 BO 3 ：0.12g)。
Feed medium (1L): glucose: 500g, histidine: 20g, methionine: 20g, cysteine: 20g.
Inoculating single colonies of 4 engineering bacteria into a seed culture medium respectively, culturing overnight at 37 ℃, inoculating seed solution into a 5L fermentation tank at a volume ratio of 10%, culturing at 30 ℃ until glucose is exhausted, starting feeding a feed supplement culture medium, controlling dissolved oxygen in a fermentation process to be about 20%, and adjusting the flow acceleration of the feed supplement culture medium during the fermentation process to ensure that residual sugar in the tank is not higher than 5g/L, and maintaining pH at 7.0 in the fermentation process. The detection results of ergothioneine by taking fermentation liquor at different time points are shown in figure 2, and the yield of the ergothioneine obtained by fermenting E2-cfegt1-linker-cfegt2 strain for 96 hours can reach 8g/L.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. An escherichia coli genetically engineered bacterium producing ergothioneine, characterized by overexpressing an inosine cyclohydrolase gene purH, an adenylyl-glass-perc-acid synthase gene purA, an adenylyl-succinic-acid lyase gene purB, a nucleoside diphosphate kinase gene ndk, and an adenylyl-kinase gene adk, and expressing a histidine methyltransferase/betaine-cysteine thiooxygenase gene cfegt1 and a betaine-cysteine thiooxygenase gene cfegt2, which are derived from anthrax (Colletotrichum fioriniae).
2. The genetically engineered escherichia coli of claim 1, wherein the genetically engineered escherichia coli integrates genes purH, purA, and purB at the ppnn site.
3. The genetically engineered escherichia coli of claim 1 or 2, wherein the genetically engineered escherichia coli integrates genes ndk and adk at the mazG locus.
4. The escherichia coli genetically engineered bacterium of any one of claims 1 to 3, wherein the histidine methyltransferase/betaine-cysteine thiooxygenase and betaine-cysteine thiooxygenase are linked by an amino acid sequence shown in SEQ ID No. 9.
5. The application of the gene cfegt1 encoding histidine methyltransferase/betaine-cysteine thiooxygenase and the gene cfegt2 encoding betaine-cysteine thiooxygenase in improving the yield of the ergothioneine of escherichia coli.
6. A method for producing ergothioneine by fermentation, which is characterized in that the escherichia coli genetic engineering bacteria of any one of claims 1 to 4 are fermented in a culture medium.
7. The method of claim 6, wherein the medium comprises peptone, yeast powder, na 2 HPO 4 ·12H 2 O、KH 2 PO 4 、NH 4 Cl、Na 2 SO 4 Glycerin and grapeSugar, mgSO 4 ·7H 2 O, trace salt solution, arabinose and methionine; the trace salt solution comprises: feCl 3 ·6H 2 O，CaCl 2 ，MnCl 2 ·4H 2 O，ZnSO 4 ·7H 2 O，CoCl 2 ·6H 2 O，NiCl 2 ·6H 2 O，Na 2 MoO 4 ·2H 2 O，Na 2 SeO 3 ，H 3 BO 3 。
8. The method of claim 7, wherein the feeding is performed with a feeding medium when glucose is depleted, and wherein the residual sugar content in the fermentation broth is controlled to be not higher than 5g/L.
9. The method of claim 8, wherein the feed medium comprises 500-600g/L glucose, 20-30g/L histidine, 20-30g/L methionine, and 20-30g/L cysteine.
10. Use of the genetically engineered escherichia coli of any one of claims 1-4 in the preparation of a product containing ergothioneine.
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