CN117487679A - Method for improving fermentation yield of ergothioneine in process of enhancing product excretion by over-expression transporter encoding gene - Google Patents

Method for improving fermentation yield of ergothioneine in process of enhancing product excretion by over-expression transporter encoding gene Download PDF

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CN117487679A
CN117487679A CN202311361676.9A CN202311361676A CN117487679A CN 117487679 A CN117487679 A CN 117487679A CN 202311361676 A CN202311361676 A CN 202311361676A CN 117487679 A CN117487679 A CN 117487679A
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ergothioneine
saccharomyces cerevisiae
scw10
mrh1
phm7
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王亮
程婧妍
郭越
陈喜媚
李倩
聂钒宇
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Dalian Polytechnic University
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Abstract

The invention discloses a method for improving fermentation yield of ergothioneine in the process of enhancing product excretion by over-expression transporter coding gene. The invention obtains transporter coding genes MRH1, PHM7, HNM1, TPO5 and SCW10 possibly involved in ergothioneine efflux in saccharomyces cerevisiae based on transcriptome analysis and screening. The saccharomyces cerevisiae engineering strain is constructed by constructing a recombinant vector which over-expresses the key genes and introducing the recombinant vector into a saccharomyces cerevisiae original strain for producing ergothioneine. Research results show that after the key genes are over-expressed, the growth of yeast engineering strains is obviously improved, the fermentation yield of ergothioneine is obviously improved, and the extracellular ergothioneine duty ratio is obviously increased. The yield of the ergothioneine obtained by fermenting the engineering strain M02-SCW10 for 144 hours reaches 105.50 +/-0.81 mg/L, which is improved by 415.39 percent compared with the control group. The invention provides a method for constructing an engineering strain for high-yield ergothioneine by modifying saccharomyces cerevisiae based on efflux transport engineering, which provides a basis for high-efficiency production of ergothioneine.

Description

Method for improving fermentation yield of ergothioneine in process of enhancing product excretion by over-expression transporter encoding gene
Technical Field
The invention relates to a method for improving the yield of ergothioneine by modifying saccharomyces cerevisiae engineering bacteria by using a transport engineering strategy, belonging to the technical field of bioengineering.
Background
Ergothioneine (EGT), a 2-mercapto-L-histidine trimethylinner salt, is a unique sulfur-containing compound isolated by Tanret et al in 1909 when studying the fungus Claviceppurpore, and is a rare natural amino acid derivative. Under physiological pH conditions, ergothioneine exists mainly in the form of thioketone, and has higher stability and stronger antioxidant activity than other thiol antioxidants (such as glutathione) because of higher oxidation-reduction potential and difficult self-oxidation. The ergothioneine has the functions of scavenging free radicals, maintaining normal growth of cells, maintaining DMA biosynthesis, preventing injury caused by ultraviolet radiation, whitening skin, resisting aging, resisting inflammation and the like, plays an important role in resisting oxidation and regulating energy, and is a multifunctional cell physiological protective agent. The unique physiological function and physicochemical properties of ergothioneine determine that the ergothioneine has wide application prospect in the fields of cosmetics, foods, biomedicine and the like.
At present, three methods for obtaining ergothioneine at home and abroad are respectively an extraction method, a chemical synthesis method and a microbial fermentation method. The method for extracting the ergothioneine from the edible fungi has the problems of time consumption, low extraction efficiency, more impurities and the like, so that the industrialization of the ergothioneine is limited. The chemical synthesis method cannot ensure the safety of the catalyst, and the raw material cost is high. The microbial fermentation method has the advantages of easily available raw materials, low production cost, easily enlarged productivity and the like, and is the development direction of ergothioneine industrialization. However, at present, chemical synthesis occupies most markets, so that the price of the chemical synthesis is high, and the future price of ergothioneine is also high, which severely limits the multi-aspect application of ergothioneine. Therefore, the production of ergothioneine by a more economical and efficient method is an important research direction for synthesis and development of ergothioneine.
Products made by microorganisms, ranging from natural products, polymers to biofuels, new chemicals, have been used in the pharmaceutical, chemical, energy and agricultural fields. However, conventional engineering strategies including enzyme engineering, metabolic engineering are not sufficient to obtain efficient microbial production systems. In the synthesis of target compounds, the target compounds usually accumulate in microbial cells, and when the intracellular product concentration exceeds a certain level, strong feedback inhibition is induced, thereby affecting the yield of target metabolites. Another strategy is provided based on the transport engineering of the efflux transport protein, so that the transmembrane transfer of the metabolite in the microbial cells is enhanced, and the intracellular target metabolite concentration is timely reduced, thereby weakening feedback inhibition and cytotoxicity caused by the metabolite to improve the product yield.
The transfer engineering strategy is utilized in the research to reform the ergothioneine producing strain so as to achieve the purpose of improving the yield of the ergothioneine, thereby promoting the application of the ergothioneine in more fields and meeting various market demands.
Disclosure of Invention
When natural compounds are produced by a microbial cell factory, excessive intracellular product concentration can cause feedback inhibition and generate certain cytotoxicity, reduce the catalytic activity of upstream pathway metabolic enzymes, and further limit the fermentation yield of target metabolites. In order to solve the problems, the invention promotes the excretion of intracellular products based on a transport engineering strategy and improves the flux of a biosynthesis pathway so as to realize the efficient production of target metabolites.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the primary purpose of the invention is to request protection of related protein coding genes involved in ergothioneine efflux transport and application thereof in improving the yield of ergothioneine, and related genes MRH1, PHM7, HNM1, TPO5 and SCW10 sequentially have sequences shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.
In one embodiment of the present invention, the nucleotide sequences of MRH1, PHM7, HNM1, TPO5 and SCW10 are GeneBank numbers in order: 851597, 854070, 852803, 853680, 855352.
The invention provides a method for improving the fermentation yield of ergothioneine based on a transport engineering strategy, which is to construct a saccharomyces cerevisiae engineering strain over-expressing related genes by a genetic engineering means; the related genes are MRH1, PHM7, HNM1, TPO5 and SCW10.
Specifically, the saccharomyces cerevisiae engineering bacteria of the overexpression genes MRH1, PHM7, HNM1, TPO5 or SCW10 are constructed according to the following method:
(1) The gene sequence of MRH1, PHM7, HNM1, TPO5 or SCW10 is amplified by taking Saccharomyces cerevisiae Saccharomyces cerevisiae S288C genome as a template, or the gene coding protein is referred to for codon optimization to obtain the gene sequence.
(2) And (3) after the free expression vector is subjected to enzyme digestion, the gel is recovered to obtain the linearized vector fragment.
(3) The linearized free expression vector is respectively subjected to seamless cloning connection with target genes MRH1, PHM7, HNM1, TPO5 or SCW10, and then transferred into escherichia coli Escherichia coli DH5 alpha competent cells, and after screening verification, recombinant vectors respectively containing over-expression genes are obtained;
(4) Transferring the recombinant expression vectors constructed in the above way into S.cerevisiae BY4741 strain respectively, and carrying out S C-URA solid culture screening to obtain Saccharomyces cerevisiae transformants respectively containing the recombinant vectors of the over-expressed target genes;
(5) After bacterial liquid PCR and sequencing verification are carried out on the Saccharomyces cerevisiae transformant obtained by the preliminary screening, the Saccharomyces cerevisiae engineering bacteria M02-MRH1, M02-PHM7, M02-HNM1, M02-TPO5 or M02-SCW10 which are successfully transformed are obtained;
(6) And (3) carrying out liquid fermentation by using engineering bacteria, sampling, detecting the content of the ergothioneine in the fermentation liquor by HPLC, and carrying out subculture for multiple times and liquid fermentation verification to finally obtain the saccharomyces cerevisiae recombinant strain with high yield of the ergothioneine and stable inheritance.
Further, in the step (1), the gene MRH1, PHM7, HNM1, TPO5 or SCW10 may be obtained by ordinary PCR or nested PCR cloning.
Further, the linearized episomal expression vector of step (2) is pYES2-URA3-EGT1-EGT2 comprising an autonomously replicating sequence, an ampicillin resistance gene (Amp r ) Expression cassette, URA3 gene expression cassette, EGT1&2 (ergothioneine synthesis key genes EGT1 and EGT 2), a promoter sequence and a terminator sequence.
Further, the SC-URA screening medium in the step (4) comprises the following components: 8g of SC-URA powder, 20g of glucose, adding deionized water to a volume of 1L, sterilizing at 121 ℃ under high temperature and high pressure for 20min, and preserving at room temperature. 2-3% of agar powder is added into the solid culture medium.
Further, the recombinant expression vector in the step (5) is pYES2-URA3-EGT1-EGT2-MRH1, pYES2-URA3-EGT1-EGT2-PHM7, pYES2-URA3-EGT1-EGT2-HNM1, pYES2-URA3-EGT1-EGT2-TPO5 or pYES2-URA3-EGT1-EGT2-SCW10, respectively.
Further, the liquid fermentation medium in the step (6) comprises the following components (1L): 6.7g of YNB (ammonium sulfate) -containing glucose, 20-30 g of D-glucose, 0.2-0.5g of L-arginine, 0.05-0.07g of L-cysteine, 0.05-0.1g of L-tryptophan, 0.05-0.1g of L-threonine, 0.1-0.3g of L-aspartic acid, 0.2-0.4g of L-isoleucine, 0.02-0.05g of L-phenylalanine, 0.05-0.1g of L-proline, 0.2-0.4g of L-serine, 0.01-0.05g of L-tyrosine, 0.02-0.05g of L-valine, 0.2-0.5g of L-methionine, 0.2-0.5g of L-tryptophan, 0.2-0.5g of L-histidine, 0.1-0.3g of L-aspartic acid, 0.05-0.1g of adenine, 0.02-0.04g of L-phenylalanine, 0.02-0.04g of L-glutamine, 0.05-0.05 g of L-glutamic acid, 0.5mg of L-glutamic acid, 0.05-5 mg of L-glutamic acid, 0.05-1 mg of L-glutamic acid, and 0.5mg of hydrochloric acid.
Compared with the prior art, the invention has the beneficial effects that: based on metabolic engineering and efflux transport engineering strategies, a recombinant strain capable of stably inheriting and producing ergothioneine at high yield is constructed, compared with a control strain, the yield of the ergothioneine through liquid fermentation is obviously improved in an experimental group, and the fermentation yields of the ergothioneine through fermentation of 144h M02-MRH1, M02-PH M7, M02-HNM1, M02-TPO5 and M02-SCW10 reach 78.44+/-6.35 mg/L, 86.08 +/-3.20 mg/L, 104.77 +/-7.63 mg/L, 103.38 +/-0.35 mg/L and 105.50 +/-0.81 mg/L respectively, and are respectively improved by 283.19%, 320.52%, 411.82%, 405.03% and 415.39% compared with the control group. Provides an important target for the high-efficiency production of ergothioneine based on genetically engineered Saccharomyces cerevisiae, and has an industrial application prospect.
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FIG. 1 shows the result of PCR amplification of the target gene and enzyme digestion of the expression vector. Wherein, (a) M: D L2000DNA Marker,1-2: MRH1; (b) M: DL5000 DNA Marker,1-2: PHM7; (c) M: DL2000DNA Marker,1-2: HNM1; (d) M: DL2000DNA Marker,1-2: T PO5; (e) M: DL2000DNA Marker,1-2: SCW10; (f) DL15000DNA Marker,1-2 pYES2-URA3-EGT1-EGT2 single cleavage (Sal I-HF).
FIG. 2 shows the PCR results of the E.coli transformant after the enzyme digestion verification of the over-expression recombinant vector constructed in the present invention. Note that (a) M: DL15000DNA Marker,1-3: pYES2-URA3-EGT1-EGT2-MRH1 original plasmid and enzyme cutting band (Bgl II enzyme cutting band size is 14010bp, bam HI-HF enzyme cutting band size is 2631bp and 11367 bp); (b) M: DL15000DNA Marker,1-3: pYES2-URA3-E GT1-EGT2-PHM7 original plasmid and enzyme cutting band (Bgl II enzyme cutting band size is 16023bp, sal I-HF enzyme cutting band size is 2982bp and 13041 bp); (c) M: DL15000DNA Marker,1-3: pY ES2-URA3-EGT1-EGT2-HNM1 original plasmid and enzyme cutting band (XbaI enzyme cutting band size is 14739 bp, salI-HF enzyme cutting band size is 1692bp and 13041 bp); (d) M: DL15000DNA Marker,1-3: pYES2-URA3-EGT1-EGT2-TPO5 original plasmid and enzyme cutting band (Bgl II enzyme cutting band size is 1490 bp, sal I-HF enzyme cutting band size is 1863bp and 13041 bp); (e) M: DL15000DNA Marker,1-3: pYES2-URA3-EGT1-EGT2-SCW10 original plasmid and enzyme cutting band (Bgl II enzyme cutting band size is 14217bp, sal I-HF enzyme cutting band size is 1176bp and 13041 bp); (f) M: DL2000DNA Marker,1-5: PCR verification (847 bp) of pYES2-URA3-EGT1-EGT2-MRH1/PHM7/HNM1/TPO5/SCW10 bacterial liquid.
FIG. 3 shows the result of PCR verification of the Saccharomyces cerevisiae engineering strain constructed in accordance with the present invention. And (a) verifying bacterial liquid PCR genes of the yeast engineering bacteria of which the M is DL2000DNA Marker and the genes are respectively expressed MRH1, PHM7, HNM1, TPO5 and SCW10.
FIG. 4 shows the construction flow of the recombinant vectors of the overexpression genes MRH1 (a), PHM7 (b), HNM1 (c), TPO5 (d) and SCW10 (e) constructed by the invention.
FIG. 5 shows the liquid fermentation phenotype parameters of the overexpression genes MRH1, PHM7, HNM1, TPO5 and SCW10 Saccharomyces cerevisiae engineering strains M02-MRH1, M02-PHM7, M02-HNM1, M02-TPO5 and M02-S CW10 constructed by the invention. Note (a) 144h biomass, ergothioneine yield; (b) 144h unit OD 600 Ergothioneine yield; (c) intracellular/extracellular ergothioneine yield analysis.
Detailed Description
The invention is further illustrated below in connection with specific examples, but the scope of the invention is not limited thereto, as set forth in the following claims. The Saccharomyces cerevisiae of the present invention is a species known in the art and is commercially available.
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless clearly defined otherwise herein in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
In the present invention, the expression "recombinant vector overexpressing the gene MRH1/PHM7/HNM1/TPO5/SCW10" refers specifically to an overexpressing recombinant vector containing the gene MRH1 or PHM7 or HNM1 or TPO5 or SCW10. The term "target gene MRH1/PHM7/HNM1/TPO5/SCW10" specifically means a target gene MRH1 or PHM7 or HNM1 or TPO5 or SCW10. The recombinant expression vector (or recombinant vector) "pYES2-URA3-EGT1-EGT2-MRH1/PHM7/HNM1/TPO5/SCW10" specifically means that the recombinant expression vector pYES2-URA3-EGT1-EGT2-MRH1 or pYES2-URA3-EGT1-EGT2-PHM7 or pYES2-URA3-EGT1-EGT2-HNM1 or pYES2-URA3-EGT1-EGT2-TPO5 or pYES2-URA3-EGT1-EGT2-SCW10.
In the present invention, the above-mentioned gene may be naturally occurring, for example, it may be cloned, isolated or purified from an automatic plant or microorganism. In addition, the gene may be artificially prepared, for example, the gene may be obtained by synthesis using a gene synthesis apparatus based on a known gene sequence.
The invention also relates to a variant of the gene, the coded polypeptide is similar to the corresponding wild type polypeptide in amino acid sequence and is a fragment, analogue or derivative of the wild type polypeptide. Variants of the polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. Such nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded polypeptide.
It should be understood that while each gene of the present invention is preferably obtained from Saccharomyces cerevisiae, other genes obtained from other microorganisms that are highly homologous (e.g., have more than 70%, such as 80%, 90%, 95%, or even 98% sequence identity) to the corresponding genes in Saccharomyces cerevisiae are also within the contemplation of the present invention. Methods and means for aligning sequence identity are also well known in the art.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used are not specific to the manufacturer and are commercially available conventional products
Example 1: liquid culture of Saccharomyces cerevisiae
This example examined the effect of overexpression of MRH1, PHM7, HNM1, TPO5 and SCW10 on ergothioneine production in a Saccharomyces cerevisiae liquid fermentation system. The culture medium used for liquid fermentation is synthetic culture medium (including non-amino yeast nitrogen source, glucose, inorganic salt, amino acid and vitamin B) 6 ) The liquid fermentation medium comprises the following components (1L): 6.7g of YNB (ammonium sulfate) -containing solution, 20g of D-glucose anhydrous, 0.5g of L-arginine, 0.07g of L-cysteine, 0.1g of L-lysine, 0.1g of L-threonine, 0.3g of L-aspartic acid, 0.4g of L-isoleucine, 0.05g of L-phenylalanine, 0.1g of L-proline, 0.4g of L-serine, 0.05g of L-tyrosine, 0.05g of L-valine, 0.5g of L-methionine, 0.5g of L-tryptophan, 0.5g of L-histidine, 0.3g of L-leucine, 0.1g of adenine, 0.04g of L-glutamic acid, 0.04g of L-glutamine, 0.2g of L-glycine, 0.05g of L-alanine, 0.08g of L-asparagine, 10g of ammonium sulfate, 0.16mg of copper sulfate, 1.6mg of pyridoxine hydrochloride (vitamin B), 0.5mg of biotin and 0.05mg of ferrous sulfate. The preparation process comprises the following steps: preparing 20 times of concentrated solution independently from an amino-free yeast nitrogen source, and filtering and sterilizing by a 0.22 mu m filter membrane; preparing 10 times of concentrated solution of anhydrous glucose, and sterilizing at 121deg.C for 20min; the amino acid mixture is prepared separately, and 800mL of deionized water is added; inorganic salt is singly prepared into 20 times of concentrated solution, and a 0.22 mu m filter membrane is used for filtering and sterilizing; mixing under aseptic condition, regulating pH with 2mol/L NaOH to 5.4+ -0.2,2-8deg.C, and preserving.
Liquid fermentation experiments were performed using 250mL Erlenmeyer flasks (50 mL liquid loading) to control the initial OD 600 Shaking culture at 0.2 and 30deg.C at 180r/min for 6d, sampling during the period to determine OD 600 And ergothioneine yield.
Example 2: screening of key genes involved in ergothioneine efflux transport using transcriptome analysis data
In this example, transcriptomics analysis was performed using a liquid fermentation system of an engineering strain S.cerevisiae E01 (S.cerevisiae BY 4741-introduced plasmid pYES2-URA3-EGT1-EGT 2) in which a starting strain S.cerevisiae BY4741 strain and a recombinant expression plasmid were transferred to produce ergothioneine as a subject. The original strain S.cerevisiae BY4741 strain is selected as a control group because the strain lacks the key genes EGT1 and EGT2 for synthesizing the ergothioneine and can not synthesize the ergothioneine, and a sample at a time point of 30 hours is selected for transcriptome analysis; the experimental group s.cerevisiae E01 strain started to rapidly accumulate ergothioneine at 20h, and samples at three time points of 20h, 30h, and 60h were selected for transcriptome analysis. Transcriptome data at four time points were analyzed in comparison to each other and 5 proteins likely to promote ergothioneine efflux were initially screened (Table 1). As can be seen from Table 1, the expression levels of the genes MRH1, PHM7, HNM1, TPO5 and SCW10 were all significantly up-regulated in the comparison of the different groups.
TABLE 1 analysis of expression of genes encoding transporters potentially involved in ergothioneine efflux
Example 3: construction of recombinant expression vector pYES2-URA3-EGT1-EGT2-MRH1/PHM7/HNM1/TPO5/SCW10
The genome of Saccharomyces cerevisiae S288C was extracted and the genes MRH1, PHM7, HNM1, TPO5 and SCW10 were amplified by nested PCR using the genome as a template (FIGS. 1 a-e). The PCR product was subjected to agarose gel electrophoresis, and the target fragment was recovered using a DNA gel recovery kit (stored at-20℃for use). And (3) carrying out gene sequencing on the gel-collected products by the large gene technology Co-Ltd, and selecting PCR gel-collected products with correct sequencing for subsequent experiments.
The pYES2-URA3-EGT1-EGT2 expression vector is subjected to single cleavage (SalI-HF) (figure 1 f), the genes MRH1, PHM7, HNM1, TPO5 and SCW10 are respectively connected to the linearized expression vector by using a seamless cloning technology (figure 4), and finally the MRH1 overexpression vector pYES2-URA3-EGT1-EGT2-MRH1, PHM7 overexpression vector pYES2-URA3-EGT1-EGT2-PHM7, HNM1 overexpression vector pYES2-URA3-EGT 1-HNM 1, TPO5 overexpression vector pYES2-URA3-EGT 2-5 and SCW10 overexpression vector pYES2-URA3-EGT1-EGT2-SCW10 are constructed through enzyme cleavage, PCR and sequencing verification (figure 2 a-f).
TABLE 2 seamless cloning primers for use in this experiment
Example 4: saccharomyces cerevisiae LiAc high-efficiency conversion method
The recombinant expression vectors pYES2-URA3-EGT1-EGT2-MRH1, pYES2-URA3-EGT1-EGT2-PHM7, pYES2-URA3-EGT1-EGT2-HNM1, pYES2-URA3-EGT1-EGT2-TPO5 and pYES2-URA3-EGT1-EGT2-SCW10 are respectively transferred into escherichia coli DH5 alpha by a chemical transformation method, and plasmids are extracted after liquid culture of escherichia coli to obtain a large number of recombinant expression plasmids. Further, the recombinant expression vectors pYES2-URA3-EGT1-EGT2-MRH1, pYES2-URA3-EGT1-E GT2-PHM7, pYES2-URA3-EGT1-EGT2-HNM1, pYES2-URA3-EGT1-EGT2-TPO5, pYES2-URA3-EGT1-EGT2-SCW10 were transferred into S.cerevisiae BY4741 strain, respectively, to obtain strain containing pYES2-URA3-EGT1-EGT2-MRH1, pYES2-URA3-EGT1-EGT2-PHM7, pYES 2-EGT 2-URA3-EGT 1-HNM 1, pYES2-URA3-EGT1-EGT 2-5, pYES2-URA3-EGT 1-SCT 2-SCW10, and strain containing pYES2-URA3-EGT1-EGT2-SCW10 (FIG. 5/MRH 1/PHM 10).
SC-URA culture medium used for culturing saccharomyces cerevisiae: 8g of SC-URA powder, 20g of anhydrous glucose, adding deionized water to a volume of 1L, sterilizing at 121 ℃ under high temperature and high pressure and humidity for 20min, and preserving at room temperature. If a solid culture medium is prepared, 2-3% of agar powder is added for sterilization. LB liquid medium: 10g/L tryptone, 5g/L yeast extract powder, 10g/L NaCl, and distilled water to dissolve, 3mol/L NaOH to adjust the pH to 7.0. The solid culture medium is added with 20g/L of agar powder.
Example 5: liquid fermentation of saccharomyces cerevisiae engineering bacteria and intracellular/extracellular ergothioneine content analysis
Liquid fermentation of yeast engineering bacteria M02-MRH1, M02-PHM7, M02-HNM1, M02-TPO5 and M02-SCW10 was carried out using a Saccharomyces cerevisiae strain S.cerevisiae BY4741 (M02) containing the recombinant vector pYES2-URA3-EGT1-EGT2 as a control group (FIG. 5). The content of ergothioneine in the fermentation broth is detected by high performance liquid chromatography, and the conditions of the high performance liquid chromatography are as follows: waters e2695, waters 2998, 260nm wavelength ultraviolet detector, XCHARE C18 (4.6X250 mm,5 μm) column temperature setting 25 ℃, mobile phase condition methanol: 0.1% formic acid (5:95), flow rate 0.8mL/min, and sample injection amount 10 μl.
As shown in the results of fig. 5, cell growth of the engineering strain over-expressing the efflux transporter was significantly improved over the control strain (fig. 5 a), fermentation yield of ergothioneine (fig. 5 a) and yield of ergothioneine per unit OD (fig. 5 b) were also significantly improved, and extracellular ergothioneine content and occupancy rate were significantly improved (fig. 5 c). The intracellular ergothioneine fermentation yields of the strains M02-MRH1, M02-PHM7, M02-HNM1, M02-TPO5 and M02-SCW10 reach 37.56+/-1.83 mg/L, 37.61 +/-3.28 mg/L, 18.45+/-4.38 mg/L, 32.37+/-3.00 mg/L and 39.71+/-3.76 mg/L respectively, which are respectively improved by 268.96%, 269.45%, 81.24%, 217.98% and 290.08% compared with the control strain, the ergothioneine fermentation yields of the strains M02-MRH1, M02-PHM7, M02-HNM1, M02-TPO5 and M02-SCW10 reach 78.44+/-6.35 mg/L, 86.08 +/-3.20 mg/L, 104.77 +/-7.63 mg/L, 5235+/-0.35 mg/L and 105.50 +/-0.81 mg/L respectively, which are respectively improved by 38395%, 320.52%, 3286%, 142.86%, 103.38%, 196.60% and 196.60% respectively compared with the starting strain. Wherein, the yield of the ergothioneine of the engineering bacterium M02-SCW10 is highest and is 105.50 +/-0.81 mg/L, which is 415.39 percent higher than that of the control group. The above results indicate that overexpression of the transporter plays an important role in relieving feedback inhibition and enhancing ergothioneine biosynthesis flux.

Claims (6)

1. The application of the overexpression of the efflux transporter coding gene in improving the fermentation production of ergothioneine is characterized in that the expression of the gene or the efflux transporter with high protein sequence similarity in other organisms is used for producing the ergothioneine;
the genes MRH1, PHM7, HNM1, TPO5 or SCW10 sequentially have sequences shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5; the gene is derived from Saccharomyces cerevisiae.
2. A method for enhancing the output of ergothioneine fermentation by enhancing the output transfer of ergothioneine through over-expression key genes is characterized in that target genes MRH1, PHM7, HNM1, TPO5 or SCW10 are respectively introduced into saccharomyces cerevisiae engineering bacteria producing ergothioneine through a genetic engineering technology, and saccharomyces cerevisiae engineering strains M02-MRH1, M02-PHM7, M02-HNM1, M02-TPO5 or M02-SCW10 are constructed.
3. The method according to claim 2, wherein the recombinant vector of the overexpressed gene MRH1/PHM7/HNM1/TPO5/SCW10 is introduced into Saccharomyces cerevisiae by using LiAc/ssDNA/PEG high-efficiency transformation or electrotransformation to construct Saccharomyces cerevisiae engineering bacteria.
4. The method according to claim 2, wherein the construction flow is as follows: connecting a target gene MRH1/PHM7/HNM1/TPO5/SCW10 with a linearization vector pYES2-URA3-EGT1-EGT2 to obtain a recombinant vector pYES2-URA3-EGT1-EGT2-MRH1/PHM7/HNM1/TPO5/SCW10; the recombinant vector is used for transforming the saccharomyces cerevisiae for producing the ergothioneine, and the saccharomyces cerevisiae engineering strain containing the recombinant vector pYES2-URA3-EGT1-EGT2-MRH1/PHM7/HNM1/TPO5/SCW10 is obtained through SC-URA solid culture screening.
5. The method of claim 2, wherein the linearization vector is an episomal expression vector pYES2-URA3-EGT1-EGT2 comprising an ampicillin resistance gene expression cassette, a URA3 gene expression cassette, an EGT1&2 gene expression cassette, a yeast promoter, a yeast terminator.
6. The method according to claim 2, wherein the saccharomyces cerevisiae engineering strain is subjected to liquid fermentation in a synthetic medium to accumulate ergothioneine, the liquid fermentation medium composition being (1L): 6.7g of YNB, 20-30 g of D-glucose, 0.2-0.5g of L-arginine, 0.05-0.07g of L-cysteine, 0.05-0.1g of L-lysine, 0.05-0.1g of L-threonine, 0.1-0.3g of L-aspartic acid, 0.2-0.4g of L-isoleucine, 0.02-0.05g of L-phenylalanine, 0.05-0.1g of L-proline, 0.2-0.4g of L-serine, 0.01-0.05g of L-tyrosine, 0.02-0.05g of L-valine, 0.2-0.5g of L-methionine, 0.2-0.5g of L-tryptophan, 0.2-0.5g of L-histidine, 0.1-0.3g of L-leucine, 0.05-0.1g of adenine, 0.02-0.04g of L-glutamic acid, 0.02-0.04g of L-glutamine, 0.02-0.05-0.5 mg of L-glycine, 0.05-1 mg of aspartic acid, 0.5-1.05 mg of aspartic acid, 0.1-1.08 mg of L-1, 0.5mg of L-alanine, and 1.5 mg of biological sulfuric acid.
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