Recombinant strain, construction method and application thereof, and method for producing ergothioneine

Abstract

The invention relates to the technical field of biology, and discloses a recombinant strain, a construction method and application of the recombinant strain and a method for producing ergothioneine. The recombinant strain can be used for simultaneously expressing the ergothioneine synthetase 1, the ergothioneine synthetase 2, the glucan endonuclease II, the exonuclease II and the beta-glucosidase. The recombinant strain disclosed by the invention can effectively utilize the carboxymethyl cellulose, breaks through the limitation that the carboxymethyl cellulose is very difficult to convert by saccharomyces cerevisiae, realizes the production of ergothioneine, effectively reduces the production cost of biological fermentation, is safer and more efficient, and has a better industrial prospect.

Description

Technical Field

[0001] The present invention relates to biotechnology, and in particular to a recombinant strain, a construction method and application thereof, and a method for producing ergothioneine. Background Art

[0002] Ergothioneine ( Ergothioneine As a natural amino acid derivative, ERG plays a variety of important physiological roles in the body. Its potent antioxidant capacity effectively scavenges free radicals, protecting cells from oxidative damage and playing a crucial role in maintaining normal cellular function and metabolic balance. This property holds great promise for its application in a wide range of fields, including medicine, food, and cosmetics.

[0003] In the medical field, ergothioneine has potential therapeutic and preventive effects on a variety of diseases. Studies have found that it can reduce inflammatory responses and has certain auxiliary therapeutic value for inflammation-related diseases such as arthritis. At the same time, ergothioneine can also protect neurons and has important research significance in the prevention and treatment of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In the food field, due to its antioxidant properties, ergothioneine can be used as a natural food preservative and nutritional enhancer. Adding ergothioneine can extend the shelf life of food, maintain the quality and nutritional content of food, and at the same time enhance the antioxidant properties of food, providing consumers with healthier food choices. In the cosmetics field, ergothioneine can resist the damage of ultraviolet rays to the skin and reduce the production of free radicals, thereby delaying skin aging and improving the appearance and texture of the skin. Therefore, it has attracted much attention in the research and development of skin care products and cosmetics.

[0004] At present, the production methods of ergothioneine mainly include extraction, chemical synthesis and microbial synthesis. The extraction method is limited by natural resources, the chemical synthesis method has high production costs, and the microbial synthesis method has received widespread attention in recent years as a green and sustainable production method. Saccharomyces cerevisiae ) has become an ideal host strain for microbial synthesis of ergothioneine due to its clear genetic background, simple genetic manipulation, and rapid growth. However, the current method of using Saccharomyces cerevisiae to synthesize ergothioneine faces two major challenges. First, the traditional Saccharomyces cerevisiae's ability to synthesize ergothioneine itself is weak, making it difficult to meet the requirements of industrial production. Second, in the process of using Saccharomyces cerevisiae to produce ergothioneine, traditional carbon sources such as glucose are relatively expensive, which would significantly increase production costs in large-scale production.

[0005] Therefore, developing a recombinant bacterial strain that can utilize a low-cost carbon source and efficiently produce thioneine has important practical significance for reducing production costs, improving production efficiency, and realizing large-scale industrial production of thioneine. Summary of the Invention

[0006] The present invention aims to overcome the problem that the ability of traditional saccharomyces cerevisiae self-synthesizing thioneine in the prior art is weak and the traditional carbon source costs such as glucose are higher, and a recombinant strain and its construction method and application and a method for producing thioneine are provided, the recombinant strain has the ability to efficiently utilize carboxymethyl cellulose to produce thioneine, and realizes low-cost, large-scale industrial production of thioneine.

[0007] In order to achieve the above-mentioned object, the first aspect of the present invention provides a recombinant strain, which is obtained by genetically modifying the starting strain. Compared with the starting strain, the recombinant strain simultaneously expresses ergothioneine synthase 1, ergothioneine synthase 2, endoglucanase II, exocellulase II and β-glucosidase.

[0008] Preferably, the amino acid sequence of the ergothioneine synthase 1 is shown as SEQ ID NO. 1, the amino acid sequence of the ergothioneine synthase 2 is shown as SEQ ID NO. 2, the amino acid sequence of the endoglucanase II is shown as SEQ ID NO. 3, the amino acid sequence of the exocellulase II is shown as SEQ ID NO. 4, and the amino acid sequence of the β-glucosidase is shown as SEQ ID NO. 5.

[0009] Preferably, the nucleotide sequence of the gene encoding ergothioneine synthase 1, Tregt1, is shown in SEQ ID NO. 6, the nucleotide sequence of the gene encoding ergothioneine synthase 2, Tregt2, is shown in SEQ ID NO. 7, the nucleotide sequence of the gene encoding EGⅡ of the endoglucanase II, is shown in SEQ ID NO. 8, the nucleotide sequence of the gene encoding CBHⅡ of the exocellulase II, is shown in SEQ ID NO. 9, and the nucleotide sequence of the gene encoding BGL1 of the β-glucosidase, is shown in SEQ ID NO. 10.

[0010] Preferably, the starting strain is Saccharomyces cerevisiae BY4741.

[0011] The second aspect of the present invention provides a method for constructing a recombinant strain, which comprises heterologously expressing ergothioneine synthase 1, ergothioneine synthase 2, endoglucanase II, exocellulase II and β-glucosidase in a starting strain.

[0012] Preferably, the starting strain is Saccharomyces cerevisiae BY4741.

[0013] Preferably, the amino acid sequence of the ergothioneine synthase 1 is shown as SEQ ID NO. 1, the amino acid sequence of the ergothioneine synthase 2 is shown as SEQ ID NO. 2, the amino acid sequence of the endoglucanase II is shown as SEQ ID NO. 3, the amino acid sequence of the exocellulase II is shown as SEQ ID NO. 4, and the amino acid sequence of the β-glucosidase is shown as SEQ ID NO. 5.

[0014] Preferably, the nucleotide sequence of the gene encoding ergothioneine synthase 1, Tregt1, is shown in SEQ ID NO. 6, the nucleotide sequence of the gene encoding ergothioneine synthase 2, Tregt2, is shown in SEQ ID NO. 7, the nucleotide sequence of the gene encoding EGⅡ of the endoglucanase II, is shown in SEQ ID NO. 8, the nucleotide sequence of the gene encoding CBHⅡ of the exocellulase II, is shown in SEQ ID NO. 9, and the nucleotide sequence of the gene encoding BGL1 of the β-glucosidase, is shown in SEQ ID NO. 10.

[0015] Preferably, the process of exogenous introduction comprises: transforming the starting strain with a transformation mixture containing a recombinant vector I expressing ergothioneine synthase 1, ergothioneine synthase 2 and endoglucanase II and a recombinant vector II expressing exocellulase II and β-glucosidase to produce exogenous genes.

[0016] Preferably, the transformation mixture further contains: 65-80 volume % polyethylene glycol 4000 solution, 8-14 volume % lithium acetate solution and 12-18 volume % carrier DNA solution, wherein the concentration of the polyethylene glycol 4000 solution is 40-60 weight %, and the concentration of the lithium acetate solution is 0.8-1.2 mol / L.

[0017] Preferably, relative to 100 μL of the transformation mixture, the content of the recombinant vector I is 15-40 ng, the content of the recombinant vector II is 15-40 ng, and the amount of the starting strain is 1×10 11 -2×10 11 CFU.

[0018] Preferably, the expression vector of the recombinant vector I is pYES2-URA3, and the expression vector of the recombinant vector II is pESC-LEU.

[0019] Preferably, the conversion conditions include: temperature of 35-48° C. and time of 20-30 min.

[0020] The third aspect of the present invention provides the use of the recombinant strain as described above or the recombinant strain constructed by the method as described above in the fermentation production of ergothioneine.

[0021] A fourth aspect of the present invention provides a method for producing ergothioneine by fermentation, comprising: inoculating the recombinant strain as described above or the recombinant strain prepared by the method as described above into a fermentation medium containing carboxymethyl cellulose for fermentation and culturing.

[0022] Preferably, the fermentation medium comprises: 8-10 g / L yeast powder, 18-22 g / L peptone, 0.1-0.3 g / L glucose, 18-22 g / L carboxymethyl cellulose, 35-45 mg / L methionine and 1-2 g / L anhydrous magnesium sulfate.

[0023] Preferably, the fermentation culture conditions include: temperature of 25-35° C., time of 1-8 days, and rotation speed of 200-300 rpm.

[0024] Through the above technical scheme, the present invention adopts an efficient cerevisiae expression system, and finally obtains a recombinant strain capable of utilizing carboxymethyl cellulose as a carbon source to efficiently synthesize ergothioneine by expressing highly active ergothioneine synthase 1 (Tregt1 enzyme) and endoglucanase II (EGII enzyme) derived from Trichoderma reesei, ergothioneine synthase 2 (Tregt2 enzyme) and exocellulase II (CBHⅡ enzyme) derived from Aspergillus niger, and β-glucosidase (BGL1 enzyme) derived from Aspergillus aculeatus, thereby realizing low-cost, large-scale industrial production of ergothioneine (ERG).

[0025] The recombinant strain provided by the present invention has broad application prospects in the fields of medicine, food, cosmetics and health products. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the ERG biomass monitoring graph in Example 3; Figure 2 This is a graph showing the determination of ERG production in Example 3; Figure 3 is a standard curve diagram for determining the activity of cellulose exoglucanase in Example 4; Figure 4 is a standard curve diagram for determining the activity of cellulose endoglucanase in Example 4; Figure 5 This is a standard curve diagram for determining β-glucosidase activity in Example 4. DETAILED DESCRIPTION

[0027] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0028] The first aspect of the present invention provides a recombinant strain, which is obtained by genetically modifying a starting strain. Compared with the starting strain, the recombinant strain simultaneously expresses ergothioneine synthase 1, ergothioneine synthase 2, endoglucanase II, exocellulase II and β-glucosidase.

[0029] The inventors discovered that cellulose, one of the most abundant biomass resources on Earth, is widely available and inexpensive, making it a highly promising alternative carbon source. Carboxymethyl cellulose (CMC), in particular, has significant industrial applications due to its unique chemical structure and properties. However, cerevisiae yeast lacks the ability to efficiently utilize CMC. Its high viscosity limits the diffusion of the hydrolyzed product, hindering its uptake and utilization, making it difficult to effectively convert CMC into ergothioneine.

[0030] Ergothioneine synthase 1 plays an important role in the synthesis of ergothioneine. It catalyzes the methylation of histidine, forms a CS bond with cysteine, and then generates histidine betaine cysteine ​​sulfoxide; ergothioneine synthase 2 is mainly responsible for cleaving the CS bond of the precursor substance generated by ergothioneine synthase 1, and finally forms ergothioneine; EGⅡ enzyme breaks down carboxymethyl cellulose into oligosaccharide fragments of different lengths by cutting the β-1,4-glycosidic bond, thereby increasing the availability of the substrate; CBHⅡ enzyme can specifically recognize the structure of carboxymethyl cellulose, starting from its non-reducing end to cut the β-1,4-glycosidic bond to produce cellobiose; BGL1 enzyme can specifically recognize and bind to cellobiose, cut its glycosidic bond through a hydrolysis reaction, and decompose cellobiose into two glucose molecules. The recombinant strain provided by the present invention not only enables the recombinant strain to utilize carboxymethyl cellulose through the coordinated expression of ergothioneine synthase 1, ergothioneine synthase 2, endoglucanase II, exocellulase II and β-glucosidase, but also further improves the efficiency and ability of the recombinant strain to synthesize ergothioneine, thereby realizing low-cost, large-scale industrial production of ergothioneine.

[0031] According to the present invention, preferably, the amino acid sequence of the ergothioneine synthase 1 is shown as SEQ ID NO. 1, the amino acid sequence of the ergothioneine synthase 2 is shown as SEQ ID NO. 2, the amino acid sequence of the endoglucanase II is shown as SEQ ID NO. 3, the amino acid sequence of the exocellulase II is shown as SEQ ID NO. 4, and the amino acid sequence of the β-glucosidase is shown as SEQ ID NO. 5.

[0032] According to the present invention, preferably, the nucleotide sequence of the gene encoding ergothioneine synthase 1, Tregt1, is as shown in SEQ ID NO. 6, the nucleotide sequence of the gene encoding ergothioneine synthase 2, Tregt2, is as shown in SEQ ID NO. 7, the nucleotide sequence of the gene encoding EGⅡ of the endoglucanase II is as shown in SEQ ID NO. 8, the nucleotide sequence of the gene encoding CBHⅡ of the exocellulase II is as shown in SEQ ID NO. 9, and the nucleotide sequence of the gene encoding BGL1 of the β-glucosidase is as shown in SEQ ID NO. 10. The nucleotide sequence provided by the present invention can generally be obtained by polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis.

[0033] The starting strain can be a prokaryotic cell or a eukaryotic cell. According to the present invention, preferably, the starting strain is Saccharomyces cerevisiae BY4741.

[0034] The second aspect of the present invention provides a method for constructing a recombinant strain, which comprises heterologously expressing ergothioneine synthase 1, ergothioneine synthase 2, endoglucanase II, exocellulase II and β-glucosidase in a starting strain.

[0035] In the present invention, the recombinant vector can be transformed, transduced, or transfected into the starting strain by conventional methods in the art, such as chemical transformation or high-voltage electroporation. Chemical transformation involves treating cells with chemical reagents to alter the permeability of the cell membrane, allowing exogenous DNA to enter the cell.

[0036] The starting strain can be a prokaryotic cell or a eukaryotic cell. According to the present invention, preferably, the starting strain is Saccharomyces cerevisiae BY4741.

[0037] According to the present invention, preferably, the amino acid sequence of the ergothioneine synthase 1 is shown as SEQ ID NO. 1, the amino acid sequence of the ergothioneine synthase 2 is shown as SEQ ID NO. 2, the amino acid sequence of the endoglucanase II is shown as SEQ ID NO. 3, the amino acid sequence of the exocellulase II is shown as SEQ ID NO. 4, and the amino acid sequence of the β-glucosidase is shown as SEQ ID NO. 5.

[0038] According to the present invention, preferably, the nucleotide sequence of the gene encoding ergothioneine synthase 1, Tregt1, is shown as SEQ ID NO. 6, the nucleotide sequence of the gene encoding ergothioneine synthase 2, Tregt2, is shown as SEQ ID NO. 7, the nucleotide sequence of the gene encoding EGⅡ of the endoglucanase II is shown as SEQ ID NO. 8, the nucleotide sequence of the gene encoding CBHⅡ of the exocellulase II is shown as SEQ ID NO. 9, and the nucleotide sequence of the gene encoding β-glucosidase, BGL1, is shown as SEQ ID NO. 10.

[0039] According to the present invention, preferably, the process of exogenous introduction comprises: transforming the starting strain with a transformation mixture containing a recombinant vector I expressing ergothioneine synthase 1, ergothioneine synthase 2 and endoglucanase II and a recombinant vector II expressing exocellulase II and β-glucosidase to produce exogenous genes.

[0040] According to the present invention, preferably, the transformation mixture further contains 65-80 volume% of polyethylene glycol 4000, 8-14 volume% of lithium acetate and 12-18 volume% of carrier DNA solution, wherein the concentration of the polyethylene glycol 4000 solution is 40-60 weight%, and the concentration of the lithium acetate solution is 0.8-1.2 mol / L.

[0041] According to the present invention, preferably, relative to 100 μL of the transformation mixture, the content of the recombinant vector I is 15-40 ng, the content of the recombinant vector II is 15-40 ng, and the amount of the starting strain is 1×10 11 -2×10 11 CFU.

[0042] According to the present invention, preferably, the expression vector of the recombinant vector I is pYES2-URA3, and the expression vector of the recombinant vector II is pESC-LEU.

[0043] In the present invention, the "vector" used in the recombinant vector can be selected from various vectors known in the art, such as various commercially available plasmids, cosmids, phages and retroviruses. The expression vector of the recombinant vector I expressing the genes of ergothioneine synthase 1, ergothioneine synthase 2 and EGⅡ enzyme of the present invention is pYES2-URA3, and the expression vector of the recombinant vector II expressing the genes of CBHⅡ enzyme and BGL1 enzyme is pESC-LEU.

[0044] According to the present invention, preferably, the conversion conditions include: temperature of 35-48° C. and time of 20-30 min.

[0045] The present invention utilizes a transformation mixture consisting of PEG 4000 (polyethylene glycol 4000), LiAc (lithium acetate), and carrier DNA to achieve transformation. LiAc (lithium acetate) is a key chemical reagent, neutralizing the negative charge on the yeast cell surface, weakening the cell membrane's repulsion of DNA. It also alters the cell membrane's structure, increasing its permeability and allowing the recombinant vector to enter the cell more easily. PEG 4000 (polyethylene glycol 4000) improves transformation efficiency by promoting DNA binding to the cell surface and aiding DNA aggregation. While carrier DNA is not a chemical that traditionally alters cell membrane permeability, it protects the plasmid DNA being transformed from degradation by intracellular nucleases, providing a supporting and synergistic effect throughout the transformation process.

[0046] The inventors have found that by adopting the above-mentioned preferred embodiment, the recombinant strain can efficiently express ergothioneine synthase 1 (Tregt1 enzyme), ergothioneine synthase 2 (Tregt2 enzyme), EGⅡ enzyme (endoglucanase II), CBHⅡ enzyme (exocellulase II) and BGL1 enzyme (β-glucosidase), thereby further improving the ability of the recombinant strain to produce ergothioneine using carboxymethyl cellulose.

[0047] The third aspect of the present invention provides the use of the recombinant strain as described above or the recombinant strain constructed by the method as described above in the fermentation production of ergothioneine.

[0048] A fourth aspect of the present invention provides a method for producing ergothioneine by fermentation, the method comprising: inoculating the recombinant strain as described above or the recombinant strain prepared by the method as described above into a fermentation medium containing carboxymethyl cellulose for fermentation and culturing.

[0049] In the present invention, carboxymethyl cellulose (CMC) can be derived from plant fiber raw material processing. Cotton linters are a byproduct of cotton processing and are abundantly available. To prepare CMC from cotton linters, the linters are first pretreated to remove impurities before reacting with chloroacetic acid under alkaline conditions. Because cotton linters are produced in large quantities and have a relatively low market price, the cost of producing CMC from them is also low. my country, a major cotton producer and consumer, has abundant cotton linters resources, with large quantities used in industrial production annually. This ensures a stable supply of raw materials for CMC production and effectively reduces costs. According to the present invention, preferably, the fermentation medium comprises: 8-10 g / L yeast powder, 18-22 g / L peptone, 0.1-0.3 g / L glucose, 18-22 g / L carboxymethyl cellulose, 35-45 mg / L methionine and 1-2 g / L anhydrous magnesium sulfate.

[0050] In the present invention, the fermentation conditions of the recombinant strain are not particularly limited, as long as the recombinant strain can be proliferated in large quantities through the fermentation process. Exemplarily, the fermentation process includes: first, randomly picking a single colony of the recombinant strain and inoculating it into a seed culture medium (the seed culture medium preferably contains: 15-25 g / L tryptone, 5-15 g / L yeast powder, and 15-25 g / L glucose) for seed culture, shaking and culturing at room temperature at a speed of 200-300 rpm for 20-30 hours, and then inoculating it into a fermentation medium (the fermentation medium preferably contains: 15-25 g / L peptone, 8-10 g / L yeast powder, 0.1-0.3 g / L glucose, 18-22 g / L carboxymethyl cellulose, 35-45 mg / L methionine, and 1-2 g / L MgSO4) for expression for 1-8 days, and then centrifuging the resulting fermentation broth at 10,000-13,000 rpm for 10-15 minutes to collect the bacterial cells and discard the supernatant. The collected bacteria were resuspended in pure water, incubated at 90-95°C for 8-12 min, then mixed using a vortex instrument, and finally filtered through a 0.22 μm water filter membrane.

[0051] According to the present invention, preferably, the conditions of the fermentation culture include: a temperature of 25-35 ° C, a time of 1-8 days, more preferably 5-8 days, and a rotation speed of 200-300 rpm. The inventors have found that by adopting the above preferred embodiment, the ability of the recombinant strain to produce ergothioneine can be significantly improved.

[0052] The present invention is described in detail below through examples. In the following examples, carboxymethyl cellulose was purchased from Yuanye; a DNA rapid extraction kit was purchased from Shanghai Biotech; PEG 4000 was purchased from Shanghai Biotech; LiAc was purchased from Maclean; carrier DNA was purchased from Solebol; and Do supplement was purchased from Shanghai Weidi Biotechnology. All other reagents and raw materials were conventional commercially available products.

[0053] YPD solid medium composition: peptone 20 g / L, glucose 20 g / L, yeast powder 10 g / L, agar 20 g / L.

[0054] YPD liquid medium composition: peptone 20 g / L, glucose 20 g / L, yeast powder 10 g / L.

[0055] Fermentation medium: yeast powder 10 g / L, peptone 20 g / L, glucose 0.2 g / L, carboxymethyl cellulose 20 g / L, methionine 40 mg / L and MgSO4 1.5 g / L.

[0056] The yield of ergothioneine was determined using HPLC analysis. The following are the parameters for the HPLC analysis: The liquid chromatography column for detection was a HILIC column (Poroshell 120, HILIC, 250 × 4.6 mm, 4 μm). The column temperature was maintained at 30°C, and the UV detector (detection wavelength was 254 nm) was used. The mobile phase consisted of 80% acetonitrile and 20% ultrapure water. The injection volume was 10 μL, the injection flow rate was 1 mL / min, and the single sample detection time was 20 min.

[0057] Example 1 The amino acid sequences of ergothioneine synthase 1 (Tregt1), ergothioneine synthase 2 (Tregt2), endoglucanase II (EGII), exocellulase II (CBHII), and β-glucosidase (BGL1), and the nucleotide sequences of the genes encoding Tregt1, Tregt2, EGII, CBHII, and BGL1 are shown in SEQ ID NOs. 1-10. Blunt-ended primer pairs designed using Snap Gene software were used as templates to obtain gene fragments. The PCR system was as follows:

[0058] 1. The process of constructing the recombinant vector pYES2-URA3-EG-Tregt1-Tregt2 (a recombinant vector capable of expressing EG, Tregt1, and Tregt2 genes) is as follows: S1, Tregt1, and Tregt2 genes are derived from Trichoderma reesei QM9414. For details, see reference 1: Chen Z, He Y, Wu X, Wang L, Dong Z, Chen X. Toward more efficient ergothioneine production using the fungal ergothioneine biosynthetic pathway. Microb Cell Fact. 2022 May 7;21(1):76. doi: 10.1186 / s12934-022-01807-3. S2 and promoter pTPI1 were obtained by PCR from the genome of Saccharomyces cerevisiae CH1. Saccharomyces cerevisiae CH1 was obtained from Professor Huang He's laboratory at Nanjing Normal University. This CH1 was prepared and preserved in the laboratory and deposited with the Guangdong Provincial Microbial Culture Collection on October 26, 2024, with the strain number: GDMCC No. 63756. The genome was extracted using the Shanghai Sangon Yeast Genomic DNA Rapid Extraction Kit. S3, using primers α-factor-EG-tAOX1 R / F to obtain the EG gene and tAOX1 terminator as fragment 1 on the vector pPicz-gap-eg-pARS (for details, please refer to reference 2: Qiao, J., Sheng, Y, Wang, M., Li, A., Li, X., Huang, H., Evolving Robust and Interpretable Enzymes for the Bioethanol Industry. Angewandte Chemie Novit. 2023 Mar. 62 (12). doi: 10.1002 / anie.202300320. PMID: 36701239. PMCID: Free PMCarticle.); using primers pTPI1 F / R to obtain the promoter fragment as fragment 2 on the Saccharomyces cerevisiae genome; using primers G1 F / R was expressed in the vector pYES2-TEF-CYC1-BamHICAN1.Y-ZeocinR (for details, please refer to the literature: Zhang, ZX., Wang, YZ., Xu, YS. et al. Developing GDi-CRISPR System forMulti-copy Integration in Saccharomyces cerevisiae . Appl Biochem Biotechnol193,2379–2388 (2021).) to obtain backbone G1; finally, fragments 1, 2 and backbone G1 were connected to obtain the recombinant vector pYES2-EG.

[0059] S4. Use primers G2 R / F to obtain backbone 2 on the vector pETDuet-1-Tregt1- tregt2 (for details, see the patent literature, patent publication number: CN118291417A). Use primers tADH1 R / F to obtain the tADH1 terminator. After connecting backbone 2 and the terminator, the recombinant vector pETDuet-Tregt1-tADH1-Tregt2 was obtained.

[0060] S5. Based on the recombinant vector pETDuet-Tregt1-tADH1-Tregt2 prepared in step S4, backbone 3 was obtained using primers G3 F / R, and promoter pTPTI1 fragment 3 was obtained using primers pTPTI1 F / R. Backbone 3 and pTPTI1 fragment 3 were ligated to obtain the recombinant vector pETDuet-Tregt1-tADH1- pTPTI1-Tregt2. Fragment 4 was obtained on the recombinant vector pETDuet-Tregt1-tADH1- pTPTI1-Tregt2 using primers Tregt1 F / Tregt2 R. Backbone 4 was obtained on the recombinant vector pYES2-EG prepared in step S3 using primers pTEF1 R / tCYC1 F. Backbone 4 and fragment 4 were ligated to obtain the recombinant vector pYES2-LEU-EG-Tregt1-tregt2.

[0061] The S6, CBHⅡ, and BGL1 genes and the vector pESC-LEU-CBHⅡ-BGL1 were synthesized by GenScript. The nucleotide sequence of the gene encoding the CBHⅡ enzyme is shown in SEQ ID NO. 9, and the nucleotide sequence of the gene encoding the BGL1 enzyme is shown in SEQ ID NO. 10.

[0062] The above PCR products were detected by agarose gel electrophoresis and then recovered. The fragments and backbones in the recovered products were connected and 20 μL was transferred to 100 μL of E. coliInitial screening was performed in DH5α competent bacteria using the antibiotic Amp. Because the recombinant vector contains an Amp-resistance gene, only bacteria that successfully ingested the recombinant vector were resistant to the antibiotic and able to grow on plates containing the antibiotic. DNA sequencing was then performed to confirm the successful construction of the recombinant vector. The resulting recombinant vector, pYES2-URA3-EG-Tregt1-Tregt2, expressing the EG, Tregt1, and Tregt2 genes (sequencing results shown as SEQ ID NO. 11), and the recombinant vector pESC-LEU-CBHⅡ-BGL1, expressing the CBHⅡ and BGL1 genes (sequencing results shown as SEQ ID NO. 12), were optimized and synthesized by GenScript.

[0063] The Tregt1 and Tregt2 genes, vector pPicz-gap-eg-pARS, Saccharomyces cerevisiae CH1, vector pYES2-TEF-CYC1-BamHICAN1.Y-ZeocinR, and recombinant vector pETDuet-1-Tregt1-Tregt2 were obtained from the laboratory of Academician Huang He, School of Food and Pharmaceutical Engineering, Nanjing Normal University. The recombinant vector pETDuet-1-Tregt1-Tregt2 was synthesized by GenScript and then stored in the laboratory of Academician Huang He.

[0064] Example 2 Transformation of recombinant vector Saccharomyces cerevisiae BY4741 was cultured on a streaked YPD medium plate at 30°C until a single colony of about 1-3 mm was grown; a single colony was picked and inoculated into 5 mL of YPD liquid medium and cultured at 30°C and 150 rpm for 12 h; 50 μL of the above culture solution was inoculated into 5 mL of YPD liquid medium and cultured at 30°C and 150 rpm until the OD 600 0.5-1; Collect 1 mL of bacterial culture in a 1.5 mL centrifuge tube by centrifugation at 3800 rpm for 30 seconds and discard the supernatant. Wash the cells with 1 mL of sterile water and centrifuge to remove the residual liquid. Repeat three times. Add 360 µL of the transformation mixture to the centrifuge tube and vortex vigorously to mix using a Kylin-Bell VORTEX-5 cell shaker at setting 5 for 10 seconds.

[0065] The composition of the transformation mixture is as follows: 1. Polyethylene glycol 4000, 50% (w / v), 240 µL; 2. Lithium acetate, concentration 1.0 mol / L, 36 µL; 3. Carrier DNA, purchased from Solebro, product number: H1060, 50 µL (Carrier DNA can prevent the recombinant vector to be transformed from nuclease degradation during the transformation process, ensuring that a sufficient amount of recombinant vector enters the cells and is successfully transformed, thereby improving the success rate of the experiment); 4. 34 µL of the recombinant vectors (pESC-LEU-CBH-BGL1 and pYES2-URA3-EG-Tregt1-Tregt2) to be transformed (100 ng of each recombinant vector, the rest is ultrapure water).

[0066] Strictly control the total volume to 360 µL and oscillate for several minutes to mix thoroughly. Place the mixed transformation system in a 42°C water bath for 25 minutes, then centrifuge at 5000 rpm for 30 seconds to collect the cells and discard the supernatant. The collected bacteria were resuspended in 400 μL of sterile water, spread on plates containing a medium omitting specific leucine and uracil components (Do supplement: 0.6 g / L, YNB: 6.7 g / L, glucose: 20 g / L, agar powder: 20 g / L, methionine: 20 mg / L, and histidine: 100 mg / L), and cultured at 30°C for 2 days. Single colonies that grew were inoculated into liquid medium omitting leucine and uracil (Do supplement: 0.6 g / L, YNB: 6.7 g / L, glucose: 20 g / L, agar powder: 20 g / L, methionine: 20 mg / L, and histidine: 100 mg / L) to further examine and confirm their nutritional deficiency markers.

[0067] After identification, these single colonies were able to grow in liquid culture medium without leucine and uracil, indicating that the successfully transformed Saccharomyces cerevisiae cells had obtained the corresponding nutritional deficiency markers, proving that the recombinant vector had been successfully introduced into Saccharomyces cerevisiae cells to obtain recombinant strain T1, laying the foundation for the subsequent use of this recombinant bacteria for the production of ergothioneine and related research.

[0068] Example 3 Fermentation of recombinant Saccharomyces cerevisiae A single colony of the recombinant strain T1 prepared in Example 2 was inoculated into 5 mL of YPD liquid medium and cultured at 30°C and 250 rpm for 24 h. The cells were centrifuged and resuspended twice in sterile water to wash away the residual YPD liquid medium. The initial OD 600 =0.1 was inoculated into the fermentation medium. The strain was fermented at 30℃ and 250 rpm. The bacterial growth was as follows Figure 1 As shown, Figure 1 The graph shows the biomass monitoring of ergothioneine produced when carboxymethyl cellulose is used as the substrate. As can be seen from the graph, as the shake flask fermentation time is extended from 0 days to 8 days, the biomass of the bacteria (measured in OD 600At the beginning of fermentation, OD 600 The value is low, indicating that the initial number of bacteria is small; it then gradually increases, indicating that in the culture system with carboxymethyl cellulose as the substrate, the bacteria can use the substrate to grow and reproduce, and the growth process continues to advance. 1-5 days is the optimal fermentation time.

[0069] Ergothioneine is produced intracellularly and is essentially absent extracellularly because Saccharomyces cerevisiae has a limited ability to transport ergothioneine to the extracellular space. Take 2 mL of fermentation broth each day, centrifuge at 12,000 rpm for 10 min to collect the bacteria, resuspend the bacteria in 2 mL of ultrapure water, incubate at 94°C for 10 minutes, and then mix using a vortexer. After dilution 30 times, filter through a 0.22 μm water filter membrane and transfer to a liquid phase vial. The ergothioneine content was detected by HPLC, and the test results are as follows: Figure 2 As shown, ergothioneine was well expressed from 5 to 8 days.

[0070] Example 4 Determination of cellulose-utilizing enzyme activity 1. Determination of Cellulose Exoglucanase Activity 1.1 Principle of determination: p-Nitrophenol cellobiose (pNPC) is an analog of cellobiose. After degradation by cellulose exoglucanase (CBH), p-nitrophenol (pNP) is released. pNP is an acid-base indicator. It is a colorless to pale yellow crystal that is easily soluble in water. Its color in aqueous solution changes with pH. In alkaline conditions, the color changes from colorless to yellow. The absorbance is measured at 405 nm and the standard curve (such as Figure 3 ) can be used to calculate its concentration and thus the enzyme activity.

[0071] 1.2 Determination method: 0.2 mL of 1 mg / mL pNPC, 0.4 mL of 50 mmol / L sodium acetate buffer (pH 5.0), and a bacterial suspension (2 mL of the fermentation broth prepared in Example 3 on the third day of fermentation, purged evenly, and 0.12 mL) were added. The reaction was allowed to proceed at 50°C for 4 h, followed by the addition of 0.48 mL of 1 mol / L Na2CO3 solution to terminate the reaction.

[0072] 1.3 Definition of enzyme activity unit: Under the assay conditions, 1 unit (U / mL) of enzyme was required to hydrolyze 1 µmol of pNP per hour.

[0073] Draw a standard curve such as Figure 3As shown, the concentration c1 can be calculated according to the standard curve, and thus the enzyme activity can be calculated. The calculation formula is (n1*c1*1.2 / 4)*(1 / 0.12), where n1 is the absorbance value at 405 nm. The enzyme activity measured after 3 days of fermentation (72 hours) is calculated to be: 75.91 U / mL 2. Determination of cellulose endoglucanase activity 2.1 Determination of enzyme activity using sodium carboxymethyl cellulose as a substrate: 0.3 mL of a 5-fold diluted crude enzyme solution (the fermentation broth prepared in Example 3 on the third day of fermentation was homogenized using a cell homogenizer; homogenization parameters: 60 Hz for 15 s, 5 s interval, total time 20 min) was added to 0.4 mL of carboxymethyl cellulose (1 wt %, prepared with 50 mmol / L citric acid buffer at pH 5). The solution was incubated at 50°C for 1 h, followed by the addition of 0.5 mL of DNS reagent (3,5-dinitrosalicylic acid reagent). The solution was incubated in a boiling water bath for 10 min, immediately cooled with an ice-water mixture, and the absorbance of the reaction solution at 540 nm was measured after cooling for 10 min.

[0074] 2.2 The crude enzyme solution in step 2.1 added to the blank control tube was first boiled in a boiling water bath for 10 minutes to inactivate the enzyme. The remaining reagents were the same as those in step 2.1. Figure 4 ) to calculate enzyme activity. 2.3 Definition of enzyme activity unit: The amount of enzyme required to produce 1 μmol of reducing sugar per hour under experimental conditions is 1 enzyme activity unit (U / mL).

[0075] 2.4 Draw the standard curve as Figure 4 As shown, the concentration c2 can be calculated based on the standard curve using the formula: enzyme activity = (n2*c2)*(1 / 0.3), where c2 is the absorbance at 540 nm. The enzyme activity measured after 3 days of fermentation (72 h) is 10.25 U / mL. 3. β-glucosidase activity assay 3.1 Determination principle: p-Nitrophenyl-glucoside (pNPG) is an analogue of cellobiose. Its structure is a glucose residue connected to one p-nitrobenzene through a glycosidic bond. After being degraded by β-glucosidase, p-nitrophenol (pNP) is released. The concentration of pNP can be calculated from the absorbance at 405 nm according to the standard curve, and thus the enzyme activity can be calculated.

[0076] 3.2 Determination method: The assay volume (1 mL) consisted of 0.1 mL of 50 mmol / L sodium acetate buffer, 0.25 mL of 10 mmol / L pNPG, and 0.3 mL of a 5-fold diluted crude enzyme solution (the fermentation broth prepared in Example 3, obtained on day 3, was homogenized using a cell homogenizer with homogenization parameters: 60 Hz for 15 s, 5 s intervals, for a total of 20 minutes). Ultrapure water was added to bring the volume up to 0.5 mL. After incubation at 50°C for 10 minutes, the reaction was immediately terminated by the addition of 0.5 mL of 1 mol / L NaCO reaction stop solution.

[0077] 3.3 Definition of enzyme activity unit: The amount of enzyme required to hydrolyze 1 µmol of pNP per minute under the assay conditions is 1 unit (U).

[0078] 3.4 Draw the standard curve as Figure 5 As shown, the concentration c3 can be calculated according to the standard curve. The calculation formula is: enzyme activity = (n3*c3) / 10, where n3 is the absorbance value at 405 nm. After 72 hours of fermentation, the enzyme activity is measured to be: 75.91 U / mL In this example, the enzymatic activities of cellulose exoglucanase, cellulose endoglucanase and β-glucosidase were determined to prove that the enzymes were expressed and active.

[0079] Example 6 The five enzymes were respectively constructed into recombinant vectors and introduced into the starting strain to obtain the recombinant strain T2. ​​The specific process is as follows: Backbones G1 and G2 were obtained by PCR on plasmids pYES2-URA3-EG-Tregt1-Tregt2 and pESC-LEU-CBHⅡ-BGL1 using primers G1-F / R and G2-F / R, respectively. Backbones G3 and G4 were obtained by PCR on plasmid pESC-HIS (this plasmid was donated by Suzhou Silicon-Based Biotechnology Co., Ltd. and is publicly available at https: / / www.snapgene.com / plasmids / yeast_plasmids / pESC-HIS) using primers G3-F / R and G4-F / R, respectively. Primers P1-F / R, P2-F / R, P3-F / R, P4-F / R, and P5-F / R were used to obtain fragments P1, P2, P3, P4, and a linearized hygromycin fragment P5 from the plasmid (the plasmid was donated by Suzhou Silicon Biotechnology Co., Ltd., and the nucleotide sequence of the plasmid is shown in SEQ ID NO. 13). Then, G1 / P1, G2 / P2, G3 / P3, and G4 / P4 / P5 were ligated to obtain recombinant vectors pYES2-URA3-Tregt1-Tregt2, pESC-LEU-CBHII, pESC-HIS-BGL1, and pESC-HPH-EG. The transformation steps were the same as in Example 2, and the transformed strains were plated on LB solid plate medium (containing hygromycin supplement: 0.6 dl) without leucine, uracil, and histidine. g / L, YNB: 6.7 g / L, glucose: 20 g / L, agar powder: 20 g / L, methionine: 20 mg / L, and hygromycin: 400 μg / ml) to further examine and confirm their nutritional deficiency markers, and successfully obtained the recombinant strain T2 containing Tregt1-Tregt2, the recombinant strain T3 containing CBH II, the recombinant strain T4 containing BGL1, and the recombinant strain T5 containing EG.

[0080] Recombinant strain T2-T5 is fermented and detected according to the method of Example 3, the inventor finds that the thioneine output of the recombinant strain T1 provided in Example 2 is significantly higher than that provided in Example 5 Recombinant strain T2-T5, illustrating that the present invention provides a recombinant strain T1 containing a recombinant vector I expressing thioneine synthase 1, thioneine synthase 2 and endoglucanase II and a recombinant vector II expressing exocellulase II and beta-glucosidase, can form better synergy, can more efficiently utilize carboxymethyl cellulose to synthesize thioneine.

[0081] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A recombinant strain, characterized in that The recombinant strain is obtained by genetically modifying the starting strain. Compared with the starting strain, the recombinant strain simultaneously expresses ergothioneine synthase 1, ergothioneine synthase 2, endoglucanase II, exocellulase II and beta-glucosidase.

2. The recombinant strain according to claim 1, characterized in that The amino acid sequence of the ergothioneine synthase 1 is shown in SEQ ID NO. 1, the amino acid sequence of the ergothioneine synthase 2 is shown in SEQ ID NO. 2, the amino acid sequence of the endoglucanase II is shown in SEQ ID NO. 3, the amino acid sequence of the exocellulase II is shown in SEQ ID NO. 4, and the amino acid sequence of the β-glucosidase is shown in SEQ ID NO.

5.

3. The recombinant strain according to claim 2, characterized in that The nucleotide sequence of the gene encoding ergothioneine synthase 1, Tregt1, is shown in SEQ ID NO. 6, the nucleotide sequence of the gene encoding ergothioneine synthase 2, Tregt2, is shown in SEQ ID NO. 7, the nucleotide sequence of the gene encoding EGⅡ of the endoglucanase II, is shown in SEQ ID NO. 8, the nucleotide sequence of the gene encoding CBHⅡ of the exocellulase II, is shown in SEQ ID NO. 9, and the nucleotide sequence of the gene encoding BGL1 of the β-glucosidase, is shown in SEQ ID NO.

10.

4. The recombinant strain according to any one of claims 1 to 3, characterized in that The starting strain is Saccharomyces cerevisiae BY4741.

5. A method for constructing a recombinant strain, characterized in that: The method comprises the following steps: heterologously expressing ergothioneine synthase 1, ergothioneine synthase 2, endoglucanase II, exocellulase II and beta-glucosidase in a starting strain.

6. The method according to claim 5, characterized in that The starting strain is Saccharomyces cerevisiae BY4741; Preferably, the amino acid sequence of the ergothioneine synthase 1 is shown in SEQ ID NO. 1, the amino acid sequence of the ergothioneine synthase 2 is shown in SEQ ID NO. 2, the amino acid sequence of the endoglucanase II is shown in SEQ ID NO. 3, the amino acid sequence of the exocellulase II is shown in SEQ ID NO. 4, and the amino acid sequence of the β-glucosidase is shown in SEQ ID NO. 5; Preferably, the nucleotide sequence of the gene encoding ergothioneine synthase 1, Tregt1, is shown in SEQ ID NO. 6, the nucleotide sequence of the gene encoding ergothioneine synthase 2, Tregt2, is shown in SEQ ID NO. 7, the nucleotide sequence of the gene encoding EGⅡ of the endoglucanase II, is shown in SEQ ID NO. 8, the nucleotide sequence of the gene encoding CBHⅡ of the exocellulase II, is shown in SEQ ID NO. 9, and the nucleotide sequence of the gene encoding BGL1 of the β-glucosidase, is shown in SEQ ID NO.

10.

7. The method according to claim 5 or 6, characterized in that The process of exogenous introduction comprises: transforming the starting strain with a transformation mixture containing a recombinant vector I expressing ergothioneine synthase 1, ergothioneine synthase 2 and endoglucanase II and a recombinant vector II expressing exocellulase II and β-glucosidase to perform exogenous gene transformation; Preferably, the transformation mixture further contains: 65-80% by volume of polyethylene glycol 4000 solution, 8-14% by volume of lithium acetate solution, and 12-18% by volume of carrier DNA solution, wherein the concentration of the polyethylene glycol 4000 solution is 40-60% by weight, and the concentration of the lithium acetate solution is 0.8-1.2 mol / L; Preferably, relative to 100 μL of the transformation mixture, the content of the recombinant vector I is 15-40 ng, the content of the recombinant vector II is 15-40 ng, and the amount of the starting strain is 1×10 11 -2×10 11 CFU; Preferably, the expression vector of the recombinant vector I is pYES2-URA3, and the expression vector of the recombinant vector II is pESC-LEU; Preferably, the conversion conditions include: temperature of 35-48° C. and time of 20-30 min.

8. the application of the recombinant strain ... described in any one in claim 1 to 4 or the recombinant strain of the method described in any one in claim 5 to 7 in fermentative production of thioneine.

9. A method for producing thioneine by fermentation, characterized in that, The method comprises: inoculating the recombinant strain according to any one of claims 1 to 4 or the recombinant strain prepared by the method according to any one of claims 5 to 7 into a fermentation medium containing carboxymethyl cellulose for fermentation culture.

10. The method according to claim 9, characterized in that: The fermentation medium comprises: 8-10 g / L yeast powder, 18-22 g / L peptone, 0.1-0.3 g / L glucose, 18-22 g / L carboxymethyl cellulose, 35-45 mg / L methionine and 1-2 g / L anhydrous magnesium sulfate; Preferably, the fermentation culture conditions include: a temperature of 25-35° C., a time of 1-8 days, and a rotation speed of 200-300 rpm.

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