Method for producing ergothioneine by fermentation of mixed strains

Abstract

The invention discloses a method for producing ergothioneine by fermentation of a mixed strain. The method comprises a step of performing mixed fermentation on a high-yield histidine EGT strain and a histidine defect type EGT strain to obtain ergothioneine, the high-yield histidine EGT strain and the histidine defect type EGT strain both comprise fusion proteins with EgtD, Egt1 and EgtE catalytic functions. According to the present invention, the fermentation is performed by using the method, such that the yield of the ergothioneine is high, the histidine does not need to be added during the fermentation process so as to save the cost, and the method is suitable for the industrial production of the ergothioneine.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology, and specifically relates to a method for fermenting and producing ergothioneine by using a mixed strain. Background Art

[0002] Ergothioneine (EGT), also known as 2-thio-L-histidine trimethyl inner salt, is a rare natural chiral histidine-derived thiol compound, first isolated from Claviceps purpurea by Tamcet in 1909. Ergothioneine (L-ergothioneine, EGT, ERGO) is a natural and rare chiral amino acid-based strong antioxidant and an important physiologically active substance in the body. It participates in regulating the redox reaction in cells, scavenging free radicals, preventing ultraviolet radiation damage, regulating intracellular energy metabolism, anti-fatigue, protecting the cornea and other functions. Due to the biological and pharmacological properties of ergothioneine and its advantages such as safety and stability, it shows good application prospects in the fields of cosmetics, functional foods, pharmaceuticals and biomedicine.

[0003] In 2010, Seeback et al. first analyzed the biosynthetic pathway of L-EGT in the bacterium Mycobacterium smegmatis and identified the enzymes involved in the biosynthesis reaction of L-EGT. This pathway involves an enzymatic reaction composed of five enzymes, including EgtA (γ-glutamylcysteine ligase), EgtB (Fe2+-dependent enzyme), EgtC (glutaminyl aminotransferase), EgtD (histidine methyltransferase) and EgtE (PLP-dependent C-S lyase). First, in bacterial synthesis, EgtD transfers three methyl groups from S-adenosylmethionine (SAM) to histidine to form histidine trimethyl inner salt; at the same time, EgtA converts cysteine into γ-glutamyl-cysteine. Secondly, using divalent ions and oxygen as cofactors, histidine trimethyl inner salt and γ-glutamyl-cysteine are catalyzed by EgtB to generate γ-glutamyl-hexynylcysteine sulfoxide. Subsequently, γ-glutamyl-hexynylcysteine sulfoxide is catalyzed by EgtC to generate hexenylcysteine sulfoxide. Finally, hexenylcysteine sulfoxide is catalyzed by EgtE to produce L-EGT, while releasing pyruvate and NH3. In addition, the latest research found that the biosynthesis of L-EGT in Methylobacterium strains only requires three steps, namely EgtD → MsEgtB ( Figure 1The part marked with "#" in → EgtE; among them, MsEgtB can use cysteine as a sulfur donor to convert histidine betaine into hercynine cysteine sulfoxide, reducing the bacterial L-EGT biosynthesis pathway from 5 steps to 3 steps. Therefore, this shorter bacterial synthesis pathway with special EgtB is more attractive for the biological preparation of L-EGT. In cyanobacteria and anaerobic green sulfur bacteria, the L-EGT synthesis pathway consists of two enzymes, EanA and EanB. Histidine is converted into histidine betaine under the action of the EanA enzyme and directly catalyzed to generate L-EGT by the EanB enzyme. In some fungi, such as four small fungi, Neurospora crassa, Schizosaccharomyces pombe, Aspergillus fumigatus, and Rhodotorula mucilaginosa, the L-EGT synthesis pathway is different from that of Mycobacterium smegmatis, cyanobacteria, and anaerobic green sulfur bacteria. This pathway consists of two synthases, Egt1 and Egt2. In these four fungi, Egt1 transfers three methyl groups from s-adenosylmethionine (SAM) to histidine to form histidine betaine, and further catalyzes histidine betaine to generate hercynine cysteine sulfoxide using oxygen and cystine. Finally, it is catalyzed by Egt2 to generate L-EGT. In addition, the fungal L-EGT biosynthesis pathway also eliminates the participation of γ-glutamyl-cysteine, thus eliminating the biosynthetic competition between L-EGT and glutathione and greatly improving the biosynthetic efficiency of L-EGT. These studies indicate that the L-EGT biosynthesis pathways of natural microorganisms are diverse, and there may be some unknown L-EGT biosynthesis methods.

[0004] At present, researchers have improved the production of ergothioneine by screening related enzymes in ergothioneine synthesis, gene editing, or constructing engineered bacteria. For example, CN119101636A screened several EgtD gene sequences from a database for in vitro enzyme activity characterization and finally successfully screened an EgtD strain with a 71% increase in activity. This highly active EgtD is a methyltransferase gene from Gammaproteobacteria bacterium, and its enzyme activity is 71% higher than that of the control. The production of ergothioneine by the engineered strain containing this enzyme increased by 30%; CN118703410A discloses a genetically engineered strain for synthesizing ergothioneine. The genetically engineered strain is Escherichia coli, and the Escherichia coli contains a plasmid for synthesizing ergothioneine. The plasmid clones the coding gene of methyltransferase NNEgtD from Nocardia nova, the coding gene of sulfoxide synthase Ntegt1 from Neurospora tetrasperma FGSC 2508, and the coding gene of C-S bond lyase SmEgt2 from Sordariamacrospora k-hell, and a high-yield strain of ergothioneine was obtained through the above method; CN118480457A can achieve the efficient synthesis of ergothioneine by expressing the ergothioneine synthesis pathway genes Tregt1 and Tregt2 in Pichia pastoris and combining fed-batch supply, providing a new way for the efficient synthesis of ergothioneine.

[0005] Although the production of ergothioneine has been continuously improved through the above methods, histidine is directly or indirectly used as a raw material in the enzymatic synthesis or fermentation process, which greatly limits the industrial production of ergothioneine. Summary of the Invention

[0006] In view of the above technical problems, the inventors first proposed using a histidine-producing strain in the fermentation process of ergothioneine, using the histidine produced by this strain as a fermentation substrate, and no additional histidine needs to be added during the fermentation process to achieve the purpose of cost savings. Further, the inventors adopted the technology of co-fermentation of mixed strains, and on the basis of not adding histidine, the production of ergothioneine was also increased.

[0007] Specifically, the present invention includes the following technical solutions.

[0008] A method for fermenting and producing ergothioneine using a mixed strain, the method comprising the step of co-fermenting a high-histidine-producing EGT strain and a histidine-deficient EGT strain to obtain ergothioneine; both the high-histidine-producing EGT strain and the histidine-deficient EGT strain contain a fusion protein having the catalytic functions of EgtD, Egt1, and EgtE.

[0009] In one or more embodiments, the fusion protein comprises a first domain having the catalytic function of EgtD, a second domain having the catalytic function of Egt1, and a third domain having the catalytic function of EgtE; wherein,

[0010] The amino acid sequence of the first domain having the catalytic function of EgtD is shown in SEQ ID NO.1;

[0011] The amino acid sequence of the second domain having the catalytic function of Egt1 is shown in SEQ ID NO.2;

[0012] The amino acid sequence of the third domain having the catalytic function of EgtE is shown in SEQ ID NO.3.

[0013] In one or more embodiments, the first domain and the second domain of the fusion protein are connected by a linker peptide, and the linker peptide is RGGG.

[0014] In one or more embodiments, the construction of the high-histidine EGT strain comprises the following steps:

[0015] (1) Using Escherichia coli MG1655 as the starting strain, knocking out the hisGL gene and integrating hisD-I at this site in the Escherichia coli genome, and then integrating the hisG* mutant gene on the Escherichia coli genome, the amino acid sequence of the hisG* mutant is shown in SEQ ID NO.4, to obtain a high-histidine strain;

[0016] (2) Constructing a fusion protein plasmid having the catalytic functions of EgtD, Egt1, and EgtE, and transferring it into the above high-histidine strain to obtain a high-histidine EGT strain.

[0017] Preferably, in the above step (1), the hisG* mutant gene is integrated into the following sites respectively: yidJ (NCBI sequence number: Gene ID:948188), yjiV (NCBI sequence number: Gene ID:2847669), and ycgH (NCBI sequence number: Gene ID:2847703).

[0018] In one or more embodiments, the construction of the histidine-deficient EGT strain comprises the following steps:

[0019] (1) Using Escherichia coli MG1655 as the starting strain, knocking out the SpeD gene and the hisG gene to obtain a histidine-deficient strain;

[0020] (2) Constructing a fusion protein plasmid having the catalytic functions of EgtD, Egt1, and EgtE, and transferring it into the above histidine-deficient strain to obtain a histidine-deficient EGT strain.

[0021] In one or more embodiments, before mixing and fermenting the two strains, the high-yield histidine EGT strain and the histidine-deficient EGT strain are first cultured at 37°C for 12 h. When the OD 600 reaches 3-5, the fermentation broth of the two strains is mixed and fermented in a fermentation tank, and the total addition amount of the broth of the two strains is 4%-5% of the volume of the tank body.

[0022] Preferably, when mixing and fermenting, the initial addition volume ratio of the high-yield histidine EGT strain to the histidine-deficient EGT strain is 1:1 - 4:1.

[0023] In one or more embodiments, the mixed strains are fermented at 37°C in the early stage. When the OD 600 reaches 50, the temperature is lowered to 30°C, and amino acids are added continuously; the feeding material is 80% glucose monohydrate; the pH is controlled at 7.0.

[0024] Preferably, the continuously added amino acids are a mixture of cysteine and methionine, and the addition concentration ratio of the two is 1:1 - 1:3. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the map of plasmid ETTE-pDonor in Example 1.

[0026] Figure 2 is the electrophoresis detection diagram of the ETTE band in Example 1.

[0027] Figure 3 is the verification diagram of the histidine-deficient strain in Example 1.

[0028] Figure 4 is the map of plasmid ETTE-pSC101 in Example 2.

[0029] Figure 5 is the detection diagram of ergothioneine after the strains are mixed and fermented at a ratio of 3 for 70 h in Example 3.

[0030] Figure 6 is the detection diagram of histidine after the strains are mixed and fermented at a ratio of 3 for 70 h in Example 3.

[0031] Figure 7 is the detection diagram of the contents of the two strains after the strains are mixed and fermented at a ratio of 2 for 70 h in Example 4.

[0032] Figure 8 is the detection diagram of the contents of the two strains after the strains in Example 4 are mixed and fermented at a ratio of 3 for 70 h.

[0033] Figure 9 is the detection diagram of the contents of the two strains after the strains in Example 4 are mixed and fermented at a ratio of 4 for 70 h.

[0034] Figure 10 It is a detection chart of the contents of the two strains after the strain of Example 4 is fermented in a mixture at a ratio of 5 for 70 h. Detailed implementation mode

[0035] According to the prior art in this field, EgtD can transfer three methyl groups from S-adenosylmethionine (SAM) to histidine to form histidine betaine; then Egt1 uses cysteine as a sulfur donor to convert histidine betaine into hercynine sulfoxide; finally, hercynine sulfoxide is catalyzed by EgtE to produce L-EGT, while pyruvate and NH3 are released. Therefore, the inventors believe that a fusion protein containing domains with the catalytic functions of EgtD, Egt1, and EgtE can be used to produce ergothioneine. Preferably, the sources of EgtD, Egt1, and EgtE are as follows.

[0036] In the present invention, the EgtD is from Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155), and the amino acid sequence of the first domain having the catalytic function of EgtD is as shown in SEQ ID NO.1:

[0037] MNAPLPHLAPSASLDDTADKGFLRDVLAGLGAPHKHLSAKYFYDRRGSELFEAITRLPEYYPTRTELAILDRYGPEIAAGLPPGAALVEFGSGSTTKVRRLLPHLGDLSAYVPVDVSEEFLRSEAEELCRDFPRLRVEPVAADFTKPFPLPDDLAEAPKAGFFPGSTIGNFEPDAAAGLLGSFARTLGAGATLIVGIDLVKEKAVLDAAYDDEAQVTAAFNLNLLTRINRELGADFDLDAFAHHAFFDENLGRIEMHLVSRRSQLVTVAGVPFHFASGETIHTENSYKYTVPGFRALARRAGWEHTRVWTDEDGLFSVHALTASTIRRH*

[0038] In the present invention, the Egt1 is from Trichoderma reesei (strain QM6a), specifically TtrEgt1, and the amino acid sequence of the second domain having the catalytic function of Egt1 is as shown in SEQ ID NO.2:

[0039] MPFPRLRELYASDTLPTWADWENLWAAWDTVTRKMLPDAELNEKPIKLRNACIFYLGHIPAFLDIQLKKTTKAGGTEPLYFHTIFERGIDPDVDNPEKCHDHSEVPDEWPPLEDILKYQDRVRERLRKLYASPDELVGDVRRAVWIGFEHEALHLETLLYMLLQSDKTLPPPHTVVPDFPKMAQKAYAARVPNQWFDVPEQTITIGMDDPEDEHESNRHFGWDNEKPARQETVRAFQAKARPITNEEYAKYLYSSHIENLPASWSVIPPNYHHNTNATTPGKPILSELPESFLHDAVRTVYGLVPLRYALDWPVFASYDELAGCAAWMGGRIPTMEEAKSIYAYVERQKDIAKQSKLSNKVPAVNGHLVNDGVEETPPSKPSPASLFVDLSTTNTGFLHWHPVPVTPNGGSLAGQAELGGVWEWTSSVLRPHQGFRPMSLYPGYTADFVDDKHNVVLGGSWATHPRIAGRKSFVNWYQRNYLYAWVGARLVRDV*

[0040] In the present invention, the EgtE is from Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155), specifically MSegtE, and the amino acid sequence of the third domain with catalytic function is as shown in SEQ ID NO.3:

[0041] MLAQQWRDARPKVAGLHLDSGACSRQSFAVIDATTAHARHEAEVGGYVAAEAATPALDAGRAAVASLIGFAASDVVYTSGSNHAIDLLLSSWPGKRTLACLPGEYGPNLSAMAANGFQVRALPVDDDGRVLVDEASHELSAHPVALVHLTALASHRGIAQPAAELVEACHNAGIPVVIDAAQALGHLDCNVGADAVYSSSRKWLAGPRGVGVLAVRPELAERLQPRIPPSDWPIPMSVLEKLELGEHNAAARVGFSVAVGEHLAAGPTAVRERLAEVGRLSRQVLAEVDGWRVVEPVDQPTAITTLESTDGADPASVRSWLIAERGIVTTACELARAPFEMRTPVLRISPHVDVTVDELEQFAAALREAP*

[0042] In the present invention, during the construction of the high - histidine - producing EGT strain, the hisG* mutant mutates N to K at the 215th position of the hisG protein, and its mutated amino acid sequence is as shown in SEQ ID NO.4:

[0043] MLKIAVPNKGSLSERAMEILAEAGYAGRGDSKSLNVFDEANNVEFFFLRPKDIAIYVAGGQLDLGITGRDLARDSQADVHEVLSLGFGSSTFRYAAPADEEWSIEKLDGKRIATSYPNLVRDDLAARGLSAEVLRLDGAVEVSIKLGVADAIADVVSTGRTLRQQGLAPFGEVLCTSEAVIVGRKDEKVTPEQQILLRRIQGILHAQNFLMLDYKVDRDNLDAATAVTPGLSGPTVSPLARDNWVAVRAMVPRRSANAIMDKLAGLGAEAILASEIRIARI

[0044] The amino acid sequences of the fusion protein and the hisG* mutant in the present invention are clear. Therefore, those skilled in the art can easily obtain their encoding genes, expression cassettes and plasmids containing these genes, as well as transformants containing the plasmid.

[0045] In the present invention, the construction of the high - histidine - producing EGT strain comprises the following steps:

[0046] (1) Using Escherichia coli MG1655 as the starting strain, the hisGL gene was knocked out and hisD-I was integrated at this locus in the Escherichia coli genome; then the hisG* mutant gene was integrated into the Escherichia coli genome at the following loci: yidJ (Gene ID: 948188), yjiV (Gene ID: 2847669), and ycgH (Gene ID: 2847703). The amino acid sequence of the hisG* mutant is shown in SEQ ID NO.4, and a strain with high histidine production was obtained;

[0047] (2) A fusion protein plasmid with the catalytic functions of EgtD, Egt1, and EgtE was constructed and transferred into the above-mentioned strain with high histidine production to obtain a high histidine-producing EGT strain.

[0048] In the present invention, the construction of the histidine-deficient EGT strain includes the following steps:

[0049] (1) Using Escherichia coli MG1655 as the starting strain, the SpeD gene and the hisG gene were knocked out to obtain a histidine-deficient strain;

[0050] (2) A fusion protein plasmid with the catalytic functions of EgtD, Egt1, and EgtE was constructed and transferred into the above-mentioned histidine-deficient strain to obtain a histidine-deficient EGT strain.

[0051] In the present invention, the high histidine-producing EGT strain and the histidine-deficient EGT strain constructed according to the above method were co-fermented. During the fermentation process, histidine can be not added, and the production of ergothioneine has also been greatly improved. Therefore, it can be used for industrial production of ergothioneine.

[0052] The following specific examples illustrate the embodiments of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0053] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments and not for limiting the protection scope of the present invention.

[0054] When numerical ranges are given in the examples, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any value therebetween can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the present invention pertains.

[0055] For those technical or conditions not specified in the examples, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through regular channels.

[0056] Regarding the addition amounts, contents and concentrations of various substances in the examples, unless otherwise specified, the percentage contents all refer to mass percentage contents.

[0057] Embodiment

[0058] Materials and Methods

[0059] The full gene synthesis, primer synthesis and sequencing in the examples were entrusted to Sangon Biotech (Shanghai) Co., Ltd.

[0060] The molecular biology experiments in the examples, including plasmid construction, restriction digestion, ligation, preparation of competent cells, transformation, medium preparation, etc., were mainly carried out with reference to the third edition of "Molecular Cloning: A Laboratory Manual" (edited by J. Sambrook, D.W. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). Specific experimental conditions can be determined through simple experiments when necessary.

[0061] The pTargetF and pCassacB plasmids were stored in the laboratory of Huarui Biotechnology (Chuzhou) Co., Ltd.

[0062] The PCR amplification experiments were carried out according to the reaction conditions provided by the plasmid or DNA template suppliers or the kit instructions. Adjustments can be made through simple experiments when necessary.

[0063] LB solid medium: 10 g / L peptone, 10 g / L sodium chloride, 5 g / L yeast extract, 15 g / L agar powder.

[0064] LB liquid medium: 10 g / L sodium chloride, 5 g / L yeast extract, 10 g / L peptone.

[0065] Metal ions: 20 mg / L ferrous sulfate heptahydrate, 20 mg / L manganese sulfate monohydrate, pH adjusted to <2 with hydrochloric acid.

[0066] Vitamins: 2 mg / L thiamine, 2 mg / L hydrochloric acid, 2 mg / L cobalamin, 2 mg / L pantothenic acid.

[0067] Trace elements: 2.5 g / L sodium molybdate dihydrate, 1.6 g / L nickel chloride hexahydrate, 10 g / L calcium chloride, 0.4 g / L copper sulfate pentahydrate, 2.25 g / L aluminum sulfate, 1.8 g / L cobalt chloride hexahydrate, 0.5 g / L nickel chloride hexahydrate, 0.14 g / L zinc sulfate heptahydrate, 0.14 g / L boric acid.

[0068] Antibiotic concentrations: 50 mg / L kanamycin, 50 mg / L spectinomycin, 35 mg / L chloramphenicol, 50 mg / L bleomycin.

[0069] Inducer concentrations: 10 mM rhamnose, 10 mM arabinose, 0.001 mM anhydrotetracycline.

[0070] In the following examples, the conditions for culturing the strain seeds were: 37 °C, 220 rpm, 500 ml shake flask, 80 ml culture medium (LB + antibiotic*), cultured for 12 h, OD > 3.

[0071] In the following examples, the components of the medium for fed-batch fermentation are shown in Table 1 and Table 2:

[0072] Table 1 Fed-batch fermentation medium (1.6 L per tank, made up to 1400 ml)

[0073]

[0074]

[0075] Table 2 Configuration of T019-1 and T019-1

[0076]

[0077] In the above T019-1 and V019-2, the trace elements were sterilized by high-temperature sterilization (except for iron, the others were sterilized together).

[0078] In the following examples, amino acids were fed during the fed-batch fermentation (cysteine concentration: methionine concentration = 35 g / L: 70 g / L), histidine was not added, and the feed was 80% glucose monohydrate. The pH was controlled at 7.0 during the fermentation process. Tank pressure: initially 0.005 mpa, up to 0.02 mpa at the highest. Aeration rate: initially 1.5, up to 3 at the highest.

[0079] In the following examples, the steps for fed-batch fermentation were:

[0080] (1), 80 ml of the seed solution was inoculated into 1600 ml of the fermentation culture medium (final volume), cultured at 37 °C, and after OD = 50, the temperature was lowered and amino acids were supplemented, with an initial rate of approximately 12 ml H -1 L -1 .

[0081] (2) After the initial sugar is consumed, supplement with glucose solution (80%) (before supplementing amino acids, the dissolved oxygen > 25%, after supplementing amino acids, the dissolved oxygen is maintained at 20%, if the dissolved oxygen is relatively low in the later stage, maintain the dissolved oxygen above 5% by reducing the feeding rate, and control the pH = 7.0). The glucose feeding rate is approximately around 16 - 18 ml / h.

[0082] (3) During the fermentation process, detect the amino acid concentration. If there is no residue, appropriately increase the supplementing rate.

[0083] In the following examples, when using a medium containing kanamycin (kan), the final concentration of kanamycin in the medium is 50 μg / ml; when using a medium containing chloramphenicol (cm), the final concentration of chloramphenicol in the medium is 30 μg / ml; when using a medium containing apramycin resistance (Apr), the final concentration of apramycin in the medium is 50 μg / ml; when using IPTG for induction, the final concentration of IPTG in the medium is 0.2 mM.

[0084] In the following examples, the EgtD is from Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155), Egt1 is from Trichoderma reesei (strain QM6a, specifically TtrEgt1), and EgtE is from Mycobacterium smegmatis (strain ATCC700084 / mc(2)155, specifically MSegtE).

[0085] In the following examples, the detection method for ergothioneine is as follows:

[0086] Use an Agilent 1260II instrument for high - performance liquid chromatography detection of ergothioneine. The chromatographic column model is Agilent SB - Aq (5um 4.6x250mm), the detection wavelength of the ultraviolet detector is 258 nm, the detection mobile phase is 0.1% perchloric acid:acetonitrile = 95:5 (v / v), the flow rate is 1 mL / min, the injection volume is 5 μL, and the column temperature is 40°C. The ergothioneine standard is dissolved in ddH20, and the concentrations of the ergothioneine standard are 0.05 g / L - 1 g / L respectively. Perform HPLC determination on the prepared standard solution and sample solution respectively, record the peak area, and calculate the ergothioneine content by the external standard method. Diluent: ultrapure water; blank solution: ultrapure water.

[0087] The calculation formula is as follows:

[0088]

[0089] In the following examples, the detection method for histidine refers to Patent CN 118147236 A.

[0090] In the following examples, the primers used are shown in Table 3:

[0091] Table 3 Primer Information

[0092]

[0093]

[0094]

[0095] It should be noted that for the sake of convenience in description, in the examples, the strain numbers, plasmid numbers, enzyme numbers, and enzyme-encoding gene numbers can share the same number, which is easily understood by those skilled in the art, that is, the same number can refer to different biological forms in different environments.

[0096] Example 1: Construction of Histidine-Deficient EGT Strain

[0097] Using Escherichia coli MG1655 as the starting strain, the SpeD gene and hisG gene were knocked out to obtain a histidine-deficient strain; a fusion protein plasmid with the catalytic functions of EgtD, Egt1, and EgtE was constructed and transferred into the above histidine-deficient strain. The specific steps are as follows:

[0098] (1) Knockout of the SpeD gene

[0099] The gene speD (WP_000734287.1) was knocked out using CRISPR-Cas9.

[0100] The constructed plasmid pCassac containing the Cas9 protein was transferred into E. coli Mg1655 competent cells by electroporation, plated on a plate containing Kana resistance, and cultured overnight at 37°C for 18 h; single colonies were selected and inoculated into liquid LB containing Kana, cultured at 37°C and 220 r / min for 18 h; transferred to fresh LB at an inoculation amount of 1% by volume, and at the same time, 10 g / L arabinose was added; cultured at 37°C for 1.5 - 2 h until OD 600 rose to 0.6 - 0.8, and the cells were harvested to prepare electrocompetent cells. The electrocompetent cells need to be washed twice with 10% sterile glycerol, then resuspended with 10% glycerol, and stored at -80°C.

[0101] According to the primers sg-speD-F and sg-speD-R, they were integrated into the sgRNA expression plasmid pTarget by seamless splicing to obtain the speD-sgRNA plasmid. The homologous fragment speD-donor was amplified by PCR using the primers speD-F and speD-R. The homologous fragment speD-donor and the speD-sgRNA plasmid were added to the competent cells, gently pipetted and mixed well, and left standing on ice for 20 min. The mixture was then added to a 1 mm electroporation cuvette (pre-cooled on ice), electroporated at 1.8 kV for 5 ms, and 800 μL of pre-cooled LB liquid medium was added. After recovery in a shaker at 37 °C and 220 r / min for 1 h, it was spread on an LB plate supplemented with the corresponding antibiotic and cultured at 37 °C for 24 h.

[0102] Colony PCR verification was performed using the primers speD-CF / speD-CR to screen for positive transformants, and the strain Mg1655-ΔspeD was obtained.

[0103] (2) Knockout of the hisG gene

[0104] First, starting from the Mg1655-ΔspeD strain, self-annealing was performed using the primer pair hisG-N20-F / R to obtain sgRNA. The pTarget plasmid was digested with BsaI enzyme, and then these two fragments were ligated with T4 ligase to obtain the pTarget-hisG plasmid for knocking out hisG. The donor fragments for knocking out hisG were amplified by PCR using the primer pairs up-KOHisG-F / R and dn-KOHisG-F / R respectively. The pCassac plasmid was transferred into the starting strain Mg1655-ΔspeD by electroporation, and 10 mM arabinose was added to induce the expression of the λRed recombinase system; the pTarget-hisG and the donor fragments were electroporated into the above-mentioned Escherichia coli containing pCassac, and then spread on an LB plate containing spectinomycin and kanamycin and cultured at 37 °C for 15 - 24 h. Colony PCR verification was performed on the single colonies on the plate. After successful colony PCR verification, samples were sent for sequencing to verify whether the integrated genes in the relevant genome were complete. The relevant sequencing work was responsible for by Shanghai Sangon Biotech Co., Ltd. The successfully verified single colonies were transferred to 5 mL of LB liquid test tubes, and 10 mM rhamnose was added to induce the removal of the pTarget plasmid. After shaking overnight, a part of the bacterial liquid was streaked and cultured overnight, and then spotted on a plate to verify whether the pTarget plasmid was removed. The single colonies with successful removal of the pTarget plasmid were inoculated into antibiotic-free LB liquid test tubes and cultured overnight at 37 °C and 200 rpm. A part of the bacterial liquid was streaked onto an LB solid plate containing sucrose, and then single colonies were picked to verify whether the pCassac plasmid was removed. After successful verification, the recombinant Escherichia coli Mg1655-ΔspeD-ΔhisG strain was obtained.

[0105] (3) Transfer of a fusion protein with EgtD, Egt1, and EgtE catalytic functions

[0106] Fragments with EgtD, Egt1, and EgtE catalytic functions and the pDonor component of the expression plasmid were amplified by PCR respectively, and then recovered by gel cutting. Among them, EgtD is from Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155), Egt1 is from Trichoderma reesei (strain QM6a, TtrEgt1), and EgtE is from Mycobacterium smegmatis (strain ATCC700084 / mc(2)155, MSegtE). The primers for amplifying the EgtD fragment are EgtD-F and Linker-EgtD-R, the primers for amplifying the Egt1 fragment are linker-TtrEgt1-F and TtrEgt1-R, the primers for amplifying the EgtE fragment are EgtE-F and EgtE-R, and the primers for amplifying the pDonor component of the expression plasmid are V-F and V-R.

[0107] The vector was constructed by the pEASY-Basic Seamless Cloning and Assembly Kit homologous recombinase. Multiple gene fragments with homologous arms were homologously recombined to construct a circular plasmid ETTE-pDonor (as shown in Figure 1 ). The specific reaction system and reaction conditions are shown in Table 4:

[0108] Table 4 Homologous recombination reaction system and reaction conditions

[0109]

[0110] The constructed plasmid ETTE-pDonor was verified by PCR with primers EgtD(wt)-seqF1 and pDonor-V-R2. After the PCR products were identified by 1% agarose gel electrophoresis, the colonies with correct band sizes (such as the ETTE band shown in Figure 2 ) were preserved, the plasmids were extracted, and sent for sequencing.

[0111] The plasmid with correct sequenced was introduced into the strain Mg1655-ΔspeD-ΔhisG, and identified by PCR with primers EgtD(wt)-seqF1 and pDonor-V-R2 to confirm that the plasmid had been transferred into the strain. The histidine-deficient EGT strain Mg1655-ΔspeD-ΔhisG+ETTE-pDonor was obtained, named CI70ΔhisG, and stored in a glycerol tube.

[0112] (4) Verify whether CI70ΔhisG is histidine-deficient

[0113] According to the above method, but without knocking out the hisG gene described in step (2), the obtained strain is named CI70.

[0114] As Figure 3 shown, prepare two LB solid plates, and add histidine and no histidine respectively when preparing the culture medium. Divide the plate with added histidine and the plate without added histidine into two parts, and inoculate CI70 and CI70ΔhisG on each plate respectively. It is found that on the plate without added histidine, the strain CI70ΔhisG does not grow, indicating that this strain is a histidine auxotrophic strain.

[0115] Example 2: Construction of a high-histidine EGT strain

[0116] In this example, Escherichia coli MG1655 was used as the starting strain. The hisGL gene was knocked out and hisD-I was integrated at this site in the Escherichia coli genome. The hisG* mutant (at position 215 of the hisG protein, N was mutated to K) gene was integrated at the Escherichia coli genome sites yidJ (NCBI accession number: Gene ID: 948188), yjiV (NCBI accession number: Gene ID: 2847669) and ycgH (NCBI accession number: Gene ID: 2847703) respectively to obtain a high-histidine-producing strain. The amino acid sequence of the hisG* mutant is as shown in SEQ ID NO.4;; A fusion protein plasmid with the catalytic functions of EgtD, Egt1 and EgtE was constructed and transferred into the above high-histidine-producing strain.

[0117] (1) The construction steps of the high-histidine-producing strain are as follows:

[0118] Using E. coli Mg1655 as the starting strain, self-annealing was carried out using primers hisGL-N20-F / R (the reaction system and procedure are shown in Table 5) to obtain sgRNA. The pTarget plasmid was digested with BsaI enzyme, and then these two fragments were ligated with T4 ligase to obtain the pTarget-hisGL plasmid for knocking out hisGL. The hisGL and hisD-I genes, namely their upstream homologous arms and donor fragments, were amplified by PCR using primers up-KOHisGL-F / R and HisD-I-F / R respectively; the two amplified fragments were fused by overlap PCR to obtain the donor fragment integrating hisD-I at the hisGL locus. The pCassacB plasmid was transformed into the starting strain MG1655 by electroporation, and 10 mM arabinose was added to induce the expression of the λRed recombinase system; the pTarget-hisGL and donor fragments were electrotransformed into the above-mentioned E. coli containing pCassacB, and then spread on an LB plate containing spectinomycin and kanamycin, and cultured at 37 °C for 15 - 24 h. Colony PCR was used to verify the single colonies on the plate; after successful colony PCR verification, the samples were sent for sequencing to verify whether the integrated genes in the relevant genome were complete. The relevant sequencing work was responsible by Sangon Biotech (Shanghai) Co., Ltd. The successfully verified single colonies were transferred to 5 mL of LB liquid test tubes, and 10 mM rhamnose was added to induce the removal of the pTarget plasmid. After shaking overnight, part of the bacterial liquid was streaked and cultured overnight, and then spotted on the plate to verify whether the pTarget plasmid was removed; the single colonies with successful removal of the pTarget plasmid were inoculated into antibiotic-free LB liquid test tubes and cultured overnight at 37 °C and 200 rpm. Part of the bacterial liquid was streaked onto an LB solid plate containing sucrose, and then single colonies were picked to verify whether the pCassacB plasmid was removed. The successful verification was the recombinant E. coli MG1655ΔhisGL::hisD-I.

[0119] The pDonor plasmid was specifically amplified using the primer pair pdonor-GJ-F / R to obtain the plasmid vector fragment 1; the Escherichia coli genome was amplified using the primer pair hisG-F / hisG-N215K-R to obtain the target gene fragment 2, and the Escherichia coli genome was amplified using the primer pair hisG-N215K-F / hisG-R to obtain the target gene fragment 3. Fragments 1, 2, and 3 were assembled using a gibson assembly kit (the gibson reaction system and assembly conditions are shown in Table 6) to construct the plasmid pDonor-hisG* for multi-copy gene integration. The recombinant plasmid pDonor-hisG* and pQCas7 were electrotransformed into the chassis strain MG1655ΔhisGL::hisD-I, respectively, and spread on an LB solid plate containing kanamycin and chloramphenicol, and cultured at 30 °C for 12 - 14 h. The positive transformants obtained in the above steps were resuspended with sterile LB liquid and then spread on an LB solid plate with a final concentration of 0.001 mM anhydrous tetracycline, kanamycin, and chloramphenicol. Anhydrous tetracycline was used to induce the expression of the transposase, and the culture conditions were 30 °C for 16 h. The positive transformants on the above plate were diluted by a certain multiple with sterile LB, and the diluted bacterial solution was spread on an LB solid plate with a final concentration of 0.01 mM anhydrous tetracycline, kanamycin, and chloramphenicol. Anhydrous tetracycline was used to induce the expression of the transposase, and the culture conditions were 30 °C for 16 h.

[0120] To increase the gene copy number of the genome, the transposition time can be extended by repeated streaking. The positive transformants on the above plate were appropriately diluted and resuspended with sterile LB liquid, and then streaked on a new LB solid plate with a final concentration of 0.01 mM anhydrous tetracycline, kanamycin, and chloramphenicol to isolate single colonies. The gene copy number of the positive transformants on the plate was verified by colony PCR to obtain engineered Escherichia coli with different copy numbers of the hisG* gene: The same positive transformant was verified by colony PCR using the primer pairs pQcas-1-F / R, pQcas-2-F / R, pQcas-3-F / R, pQcas-4-F / R, pQcas-4-F / R, pQcas-5-F / R, pQcas-6-F / R, pQcas-7-F / R. According to the nucleic acid electrophoresis band conditions of different primer pairs, the specific integration site situation of the hisG* gene was judged. Finally, recombinant Escherichia coli with the hisG* gene integrated at sites 3, 4, and 5 (i.e., sites yidJ (NCBI accession number: Gene ID: 948188), yjiV (NCBI accession number: Gene ID: 2847669), and ycgH (NCBI accession number: Gene ID: 2847703)) was obtained, and the copy number was 3.

[0121] After successful verification of a single colony, the integrated genes in the genome were sequenced, and the relevant sequencing work was responsible by Sangon Biotech (Shanghai) Co., Ltd. Removal of the two plasmids pDonor and pQCas7 of the above system required plasmid removal through plasmid pfree to obtain a plasmid-free Escherichia coli strain; the integrated host obtained in the above step was prepared into electrocompetent cells, electrotransformed with plasmid pfree, and after activation, it was spread on an LB solid medium containing bleomycin, and the culture condition was overnight at 30 °C. Several positive transformants on the plate were selected, 1.0 mM rhamnose was added, and the culture was continued overnight at 30 °C. Part of the bacterial liquid was streaked on an LB solid plate, and then it was verified on an LB solid plate containing a single antibiotic of kanamycin, chloramphenicol, or bleomycin to screen out single colonies without pDonor-hisG* and pQCas7, and at the same time remove plasmid pfree, that is, a plasmid-free engineered Escherichia coli MG1655ΔhisGL::hisD-I 3hisG* was obtained, named SH2428.

[0122] Table 5 Self-annealing reaction system and reaction conditions

[0123]

[0124] Table 6 Gibson reaction system and assembly conditions

[0125]

[0126] (2) Fermentation ability detection of the strain constructed above

[0127] The above strain SH2428 was inoculated into 50 mL of LB culture medium in a shake flask, and the culture conditions were 37 °C and 220 rpm. After culturing for 10 - 12 h, the seed liquid in the shake flask was transferred to a 1.5 L fermenter at an inoculation amount of 35% (v / v), and fermentation was carried out at 30 °C. Samples of the fermentation broth were taken at fixed intervals, diluted by an appropriate multiple to prepare samples for OD and product detection, and the results are shown in Table 7.

[0128] Table 7 Fermentation results of SH2428 in a 1.5 L fermenter

[0129]

[0130] From the above data, it can be seen that for the strain constructed according to this example, the histidine yield can reach 18.66 g / L after 77 hours of fermentation, and the product concentration is very high, indicating that this strain is a high-histidine-producing strain.

[0131] (3) Transfer of a fusion protein with the catalytic functions of EgtD, Egt1, and EgtE

[0132] Using the Psc101 - plasmid as a template, amplify it with the primer pair Vpsc101 - F / R; using the plasmid ETTE - pDonor constructed in step (3) of Example 1 as a template, amplify the ETTE gene part with the primer pair IETTE - F / R, and recover the two amplified fragments. Construct a vector through the pEASY - Basic Seamless Cloning and Assembly Kit homologous recombinase, and perform homologous recombination on the two gene fragments with homologous arms to construct a plasmid ETTE - pSC101 with a circular structure, as Figure 4 shown. The specific reaction system and reaction conditions are shown in Table 8:

[0133] Table 8 Reaction system and reaction conditions for homologous recombination

[0134]

[0135] Verify the constructed plasmid ETTE - pSC101 by PCR and sequencing according to the method in step (3) of Example 1, and transfer the plasmid ETTE - pSC101 with correct sequencing into the high - histidine - producing strain SH2428. After PCR verification again, obtain the high - histidine - producing EGT strain, named CI50, and store it in a glycerol tube.

[0136] Example 3: Separate and mixed fermentation of histidine - defective EGT strain and high - histidine - producing EGT strain

[0137] Take 50 μL of the CI50 and CI70ΔhisG bacterial solutions from the glycerol tubes respectively, spread them on plates (7 cm × 7 cm, kanamycin resistance), and culture them overnight in a 37 - degree incubator; take 1 / 5000 of the bacteria on the plate and inoculate them into a seed LB shake flask (80 / 500 ml), culture them at 37 degrees for 12 h, OD 600 = 3 - 5, and transfer to the fermenter. The fermentation volume is 1.6 L, and inoculate according to the volume ratio of different seeds, with a total inoculation amount of 5%. Ferment at 37 degrees, when OD 600 = 50, cool down to 30 degrees, and feed amino acids (cysteine concentration: methionine concentration = 35 g / L: 70 g / L), without adding histidine, and the feed is 80% glucose monohydrate. Control the pH at 7.0 and perform zero - sugar fermentation.

[0138] The HPLC detection results of ergothioneine and histidine in the fermentation broth after the strains are mixed and fermented for 70 h according to the ratio 4 in the following table are shown in Figure 5 and Figure 6 shown; the ergothioneine product concentration results after the strains are mixed and fermented for 70 h in different ratios are shown in Table 9.

[0139] Table 9 Fermentation results of the two strains mixed in different ratios

[0140]

[0141]

[0142] As can be seen from the above data, when the two strains are mixed and fermented for 70 h in the ratio of 1:1 - 1:4, the product concentration of ergothioneine can reach more than 6 g / L. Among them, when the strains are mixed and fermented for 70 h in the ratio of 4:6, the product concentration of ergothioneine can reach 7.72 g / L. In addition, as Figure 6 shown, there is still enough histidine available in the fermentation broth after 70 h of fermentation. Therefore, in the fermentation process of the present invention, it is not necessary to add histidine additionally, which can save costs and is very suitable for industrial production.

[0143] Example 4: Growth situation of histidine - deficient EGT strain and high - histidine - producing EGT strain during mixed fermentation

[0144] Mix the two strains according to the ratios 2 - 5 in Table 7 and ferment them separately, and detect the content of the two strains after 70 h of fermentation to verify whether there is competition between the two strains during the mixed fermentation process.

[0145] Method for detecting the content of the two strains: Select the speD locus for verification. One primer is on the genome and the other primer is in the knockout part. The specific primers are SpeD - CF2 and SpeD - CR2. Using the fermented bacterial liquid as a template, perform PCR amplification. If there is a band with a size of more than 1 kb, it is the CI50 strain; if there is no band, it is the CI70 strain.

[0146] The detection results are as Figures 7 - 10 shown, Figure 7 is the detection result of the bacterial cells after mixed fermentation in the ratio of 2. The proportion of the CI50 strain is 33.3%; Figure 8 is the detection result of the bacterial cells after mixed fermentation in the ratio of 3. The proportion of the CI50 strain is 25%; Figure 9 is the detection result of the bacterial cells after mixed fermentation in the ratio of 4. The proportion of the CI50 strain is 8.3%; Figure 10 is the detection result of the bacterial cells after mixed fermentation in the ratio of 5. The proportion of the CI50 strain is 8.3%. Combining the strain mixing ratios 2 - 4 (the specific ratios of the strains are 2:8, 3:7, and 4:6) and Figures 7 - 9 it can be known that when the initial addition amount of the CI50 strain in the mixed strains continuously decreases, the content of the CI50 strain after 70 h of mixed fermentation also decreases accordingly, indicating that there is no competition relationship between the two mixed strains during the mixed fermentation process; in addition, when the strains are mixed and fermented for 70 h in the ratio of 5 (the specific ratio of the strains is 5:5), as Figure 10It can be seen that the content of CI50 strain in the fermentation broth is the same as the result of mixed fermentation according to ratio 4, indicating that the CI50 strain will maintain a certain amount of bacteria and produce a relatively stable amount of histidine in the later stage of fermentation( Figure 6 ), further indicating that the histidine produced by the CI50 strain during the mixed fermentation process is sufficient for the production of ergothioneine.

[0147] As described above, it is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for fermenting and producing ergothioneine using a mixed strain, characterized in that, The method includes the step of mixing and fermenting a high-histidine EGT strain and a histidine-deficient EGT strain to obtain ergothioneine; both the high-histidine EGT strain and the histidine-deficient EGT strain contain a fusion protein having the catalytic functions of EgtD, Egt1, and EgtE.

2. The method according to claim 1, characterized in that, The fusion protein includes a first domain having the catalytic function of EgtD, a second domain having the catalytic function of Egt1, and a third domain having the catalytic function of EgtE; wherein, the amino acid sequence of the first domain having the catalytic function of EgtD is as shown in SEQ ID NO.1; the amino acid sequence of the second domain having the catalytic function of Egt1 is as shown in SEQ ID NO.2; the amino acid sequence of the third domain having the catalytic function of EgtE is as shown in SEQ ID NO.

3.

3. The method according to claim 2, characterized in that, The first domain and the second domain of the fusion protein are connected by a linker peptide, and the linker peptide is RGGG.

4. The method according to claim 1, wherein The construction of the high-histidine EGT strain includes the following steps: (1) Using Escherichia coli MG1655 as the starting strain, knocking out the hisGL gene and integrating hisD-I at this site in the Escherichia coli genome, and then integrating the hisG* mutant gene on the Escherichia coli genome, the amino acid sequence of the hisG* mutant is as shown in SEQ ID NO.4, to obtain a high-histidine strain; (2) Constructing a fusion protein plasmid having the catalytic functions of EgtD, Egt1, and EgtE, and transferring it into the above high-histidine strain to obtain a high-histidine EGT strain.

5. The obtaining method according to claim 4, characterized in that, In step (1), the hisG* mutant gene is integrated into the following sites respectively: yidJ (Gene ID: 948188), yjiV (Gene ID: 2847669), and ycgH (Gene ID: 2847703) sites.

6. The method according to claim 1, wherein The construction of the histidine-deficient EGT strain includes the following steps: (1) Using Escherichia coli MG1655 as the starting strain, knocking out the SpeD gene and the hisG gene to obtain a histidine-deficient strain; (2) Constructing a fusion protein plasmid having the catalytic functions of EgtD, Egt1, and EgtE, and transferring it into the above histidine-deficient strain to obtain a histidine-deficient EGT strain.

7. The method according to claim 1, wherein Before mixing the two strains for fermentation, first culture the high - histidine EGT strain and the histidine - deficient EGT strain at 37 °C for 12 h. When the OD 600 is 3 - 5, mix the fermentation broth of the two strains and transfer it to the fermentation tank for fermentation. The total addition amount of the broth of the two strains is 4% - 5% of the volume of the fermentation tank.

8. The method according to claim 7, wherein When the fermentation broths of the two strains are mixed and fermented in a fermenter, the initial added broth volume ratio of the high-histidine EGT strain to the histidine-deficient EGT strain is 1:1 - 4:

1.

9. The method according to claim 1, wherein The mixed strain is fermented at 37°C in the early stage. When OD 600 reaches 50, the temperature is lowered to 30°C, and amino acids are fed continuously; the feeding medium is 80% glucose monohydrate; the pH is controlled at 7.

0.

10. The method according to claim 9, wherein The amino acids added by feeding are a mixture of cysteine and methionine, and the added concentration ratio of the two is 1:1 - 1:3.

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