WO2021185039A1 - 一种构建麦角硫因生产菌的方法 - Google Patents

一种构建麦角硫因生产菌的方法 Download PDF

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WO2021185039A1
WO2021185039A1 PCT/CN2021/077815 CN2021077815W WO2021185039A1 WO 2021185039 A1 WO2021185039 A1 WO 2021185039A1 CN 2021077815 W CN2021077815 W CN 2021077815W WO 2021185039 A1 WO2021185039 A1 WO 2021185039A1
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thioneine
pgpd
gene expression
sccyc1t
plasmid
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范文超
高书良
王金刚
任亮
俞想
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浙江华睿生物技术有限公司
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  • the present invention belongs to the field of genetic engineering, and relates to a method for constructing a thioneine-producing bacterium, in particular to a method for constructing a rhododendron torus for the production of thiothioine.
  • L-ergothionine (EGT), whose chemical name is 2-mercaptohistidine trimethyl inner salt, has the following structural formula:
  • Ergothioneine is the only natural 2-thioimidazole amino acid known so far. Ergothioneine has many physiological effects such as anti-oxidation, anti-inflammatory, prolonging cell life cycle or anti-cell senescence, and improving nerve cell production. At the same time, in a variety of disease models including Alzheimer's, diabetes and other complications, it has a better effect of protecting cells and fighting damage, and has a broad market application prospect.
  • thioneine can be obtained by chemical synthesis, edible fungus extraction and microbial fermentation production.
  • a variety of microorganisms have been confirmed to have the ability to synthesize thioneine, including mycobacteria, streptomyces, molds and yeasts.
  • CN102978121B discloses that the edible fungus White Mushroom catalyzes the production of thioneine from the histidine substrate, the substrate conversion rate can reach 70%, and the product yield is not reported;
  • CN103184246A discloses that the large filamentous fungus S.
  • sibiricum has a shake flask fermentation for 10 days and the ergothione output is 51mg/L;
  • WO2017150304A1 discloses that Streptomyces lividans has been fermented for 7 days, and the ergothione output is 900mg/L;
  • WO2015180492A1 discloses Pleurotus ostreatus was fermented in a 75L fermentor for 14 days, and the ergothione yield was 352 mg/L;
  • CN103734022 disclosed that the edible fungus Pleurotus ostreatus was fermented for 7-15 days, and the highest yield of ergothioine reached 143.7 mg/L;
  • CN107250347 disclosed the gene Engineering Aspergillus includes Aspergillus oryzae, Aspergillus sojae and Aspergillus niger.
  • the fermentation yield can reach 438mg/L; Escherichia coli has been genetically engineered to express the thioneine gene synthesis cluster of mycobacterium heterologously, thioneine Yield 640mg/L; CN106661585 discloses that Escherichia coli has been genetically engineered to heterologously express the thioneine gene synthesis cluster of mycobacteria, and the engineered bacteria fermented thioneine yield 12mg/L; CN105296559A discloses that Pleurotus ostreatus edible fungus undergoes multiple Fermentation formula composition adjustment, fermentation for at least 6 days, the highest ergothioine output is 315.7mg/L; CN201910664772.8A discloses that Bacillus subtilis 168 is used as a host to express foreign genes, and the constructed genetically engineered bacteria ergothioine yield reaches 568.4mg /L.
  • the present invention uses genetic engineering technology to genetically modify natural Rhodotorula toruloides (or Rhodotorula toruloides) to construct A high-yielding strain of ergothioneine.
  • the present invention includes the following technical solutions:
  • An EGT1 gene expression cassette which is selected from SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • SEQ ID NO: 1 contains the promoter pGPD, the encoding gene NcEGT1 of the enzyme egt1 from Neurospora crassa, and the CYC1t terminator from Saccharomyces cerevisiae, and the XbaI and PmeI restriction sites are added at the beginning and the end, respectively, for Subsequent plasmid construction, named Pgpd-NcEGT1-ScCYC1t in this article;
  • SEQ ID NO: 2 contains the promoter pGPD, the coding gene CpEGT1 of the enzyme egt1 derived from Claviceps purpurea and the CYC1t terminator from Saccharomyces cerevisiae, and add XbaI and PmeI restriction sites at the beginning and the end, respectively, for subsequent use
  • the plasmid construction named Pgpd-CpEGT1-ScCYC1t in this article;
  • SEQ ID NO: 3 contains the promoter pGPD, the encoding gene RmEGT1 of the enzyme egt1 derived from Rhodotorula mucilaginosa, and the CYC1t terminator derived from Saccharomyces cerevisiae.
  • XbaI and PmeI restriction sites are added at the beginning and the end, respectively, for subsequent use Plasmid construction, named Pgpd-RmEGT1-ScCYC1t herein.
  • an EGT1 gene expression plasmid which is obtained by cloning the above-mentioned gene expression cassette on a Puc57 plasmid.
  • Puc57-Pgpd-NcEGT1-ScCYC1t Puc57-Pgpd-CpEGT1-ScCYC1t
  • Puc57-Pgpd-RmEGT1-ScCYC1t respectively.
  • a thioneine functional gene expression plasmid (or EGT1 gene transformation plasmid), which is constructed by a method including the following steps:
  • step 3 Connect the EGT1 gene expression fragment obtained in step 1) with the plasmid backbone obtained in step 2) to obtain an expression plasmid for the thioneine functional gene.
  • the ergothione functional gene expression plasmids are respectively pZPK-NcEGT1, pZPK-NcEGT1, pZPK1PK-CpEG-CpEG .
  • a method for constructing thiolgoine producing bacteria which includes the following steps: the above-mentioned thiolgoine functional gene expression plasmids (pZPK-NcEGT1, pZPK-CpEGT1, pZPK-RmEGT1) Transform into a strain capable of producing ergothioneine to obtain a positive clone.
  • the above-mentioned strain may be Rhodotorula toruloides (or Rhodotorula toruloides).
  • the above method is to transfect Rhodotorula vulgaris with thioneine functional gene expression plasmids (pZPK-NcEGT1, pZPK-CpEGT1, pZPK-RmEGT1) mediated by Agrobacterium to obtain positive transformants.
  • the above method may specifically include the following steps:
  • Rhodotorula vulgaris Mix the Agrobacterium engineering bacteria liquid with the Rhodotorula vulgaris, perform resistance screening, and obtain positive transformants after phenotypic and PCR verification, so as to realize Agrobacterium-mediated transfection of Rhodotorula vulgaris.
  • a thiolgoine-producing bacterium which is obtained by a method of constructing a thiolgoine-producing bacterium.
  • the above-mentioned thionin-producing bacterium is Rhodotorula vulgaris, and preferably the original Rhodotorula vulgaris is Rhodotorula toruloides 2.1389 deposited by the China General Microorganism Collection Management Center.
  • the above-mentioned thioneine-producing bacteria can produce thioneine by a fermentation method.
  • the present invention genetically transforms the natural Rhodotorula vulgaris by means of genetic engineering, and obtains a high-yielding ergothione strain with food safety. After fermentation verification, the yield of ergothioneine can reach about 1.5g/L, and the constructed engineering strain has been passaged many times, and the genetic traits are relatively stable, and it has good industrial application potential.
  • Figure 1 is a schematic diagram of the map structure of the plasmid pZPK-PGPD-Hyg-Tnos constructed in the present invention.
  • egt1 is a multifunctional enzyme that can catalyze the production of thioneine precursor S-(histidine betaine)-2-)yl-L-cysteine S-oxide (S-(hercyn- 2-yl)-L-cysteine S-oxide). Studies have found that by expressing the egt1 gene in a strain that can produce ergothioneine, the production of ergothioneine can be promoted.
  • egt1 and its coding gene (DNA) name are mixed, and those skilled in the art should understand that they represent different substances in different description occasions.
  • DNA coding gene
  • the coding genes are NcEGT1, CpEGT1 and RmEGT1, respectively.
  • NcEGT1 gene according to NCBI database, Genbank accession: XP_956324.3 sequence, after codon optimization, the following base sequence can be obtained:
  • RmEGT1 For RmEGT1, use the NcEGT1 protein sequence and use the blastp tool in the NCBI database to define the Rhodotorula mucilaginosa genome and perform sequence alignment to obtain the following egt1 sequence:
  • the egt1 gene exists in the form of a gene expression cassette.
  • the terms "egt1 gene expression cassette”, “gene expression cassette” and “expression cassette” have the same meaning and can be used interchangeably.
  • three gene expression cassettes for egt1 can be constructed, namely Pgpd-NcEGT1-ScCYC1t (i.e. SEQ ID NO:1) and Pgpd-CpEGT1-ScCYC1t (i.e. SEQ ID NO: 2) And Pgpd-RmEGT1-ScCYC1t (ie SEQ ID NO: 3).
  • the three expression cassettes are designed to add XbaI and PmeI restriction sites at the head and tail, respectively, for subsequent plasmid construction.
  • Rhodotorula vulgaris By expressing the egt1 gene in a suitable host, such as some Rhodotorula vulgaris, the ability of Rhodotorula vulgaris to produce thioneine is improved.
  • a suitable host such as some Rhodotorula vulgaris
  • the constructed Rhodotorula vulgaris engineered bacteria produces thioneine through fermentation, it can produce a large amount of thioneine without adding histidine betaine, histidine or cysteine to the culture solution.
  • the molecular biology experiments in this article include plasmid construction, restriction enzyme digestion, competent cell preparation, transformation, etc. Mainly refer to "Molecular Cloning Experiment Guide” (third edition), edited by J. Sambrook, DW Russell (US), Translated by Huang Peitang and others, Science Press, Beijing, 2002).
  • the competent cell transformation method and the competent preparation method are carried out in accordance with the "Molecular Cloning Experiment Guide” (Third Edition) Chapter 1, page 96. If necessary, specific experimental conditions can be determined through simple experiments.
  • LB medium 5g/L yeast extract, 10g/L tryptone, 10g/L sodium chloride. (LB solid medium plus 20g/L agar powder.)
  • YEB medium beef extract 5g/L, yeast extract 1g/L, peptone 5g/L, sucrose 5g/L, MgSO 4 ⁇ 7H 2 O 0.5g/L, pH 7.4. (Add 15g/L agar powder to YEB solid medium.)
  • YPD medium 1wt% yeast extract, 2wt% peptone, 2wt% glucose.
  • MM solution 3.625g KH 2 PO 4 , 5.125g K 2 HPO 4 , 1.250g MgSO 4 ⁇ 7H 2 O, 0.375g NaCl, 0.165g CaCl 2 ⁇ 2H 2 O, 0.0062g FeSO 4 ⁇ 7H 2 O, 1.250g (NH 4 ) 2 SO 4 , dissolved in ddH 2 O and dilute to 1L;
  • M-100 trace element solution 30mg H 3 BO 3 , 70mg MnCl 2 ⁇ 4H 2 O, 200mg ZnCl 2 , 20mg Na 2 MoO 4 ⁇ 2H 2 O, 50mg FeCl 3 ⁇ 6H 2 O, 200mg CuSO 4 ⁇ 5H 2 O , Dissolved in ddH 2 O and dilute to 500ml.
  • M-100 salt solution 16g KH 2 PO 4 , 4g Na 2 SO 4 , 8g KCl, 2g MgSO 4 ⁇ 7H 2 O, 1g CaCl 2 , 8ml M-100 trace element solution, dissolved in ddH 2 O to a constant volume 1L.
  • IM liquid medium 80ml MM solution, 0.36g glucose, 1ml glycerol, dissolved in ddH 2 O and dilute to 200ml. After sterilizing and cooling to about 50°C, add the following ingredients: 8ml 1M MES, 4ml 10mM AS, the final concentrations of MES and AS are 40mM and 200 ⁇ M, respectively.
  • IM solid medium 160ml MM salt solution, 0.36g glucose, 2ml glycerol, dissolved in ddH 2 O to make the volume 400ml, and finally add 6g agar powder. After sterilizing and cooling to about 50°C, add the following ingredients: 16ml 1M MES, 8ml 10mM AS, the final concentrations of MES and AS are 40mM and 200 ⁇ M, respectively.
  • Ergothioneine standards were purchased from China National Pharmaceutical (Group) Shanghai Chemical Reagent Company.
  • Extracellular product detection the fermentation broth was centrifuged at 10000rpm for 4min, the supernatant was taken, filtered with a 2 ⁇ m filter, and the sample was tested by HPLC.
  • Intracellular product detection Centrifuge the fermentation broth at 10000rpm for 4min, collect the bacteria, weigh 100mg wet bacteria, transfer to a clean 1.5ml centrifuge tube, add 1ml sterile water, vortex, resuspend the bacteria, then Warm-bath at 90°C for 30 minutes, end the warm-bath, centrifuge at 12000 rpm for 10 minutes, take the supernatant, filter it with a 2 ⁇ m filter, and perform HPLC detection on the sample.
  • yeast seed culture medium composition glucose 20g/L, yeast powder 10g/L, peptone 20g/L.
  • Yeast shake flask fermentation medium ammonium sulfate 0.5g/L, yeast powder 10g/L, peptone 10g/L, glutamic acid 3g/L, biotin 0.6mg/L, thiamine hydrochloride 0.1g/L, magnesium sulfate 5g/L, ferrous sulfate heptahydrate 0.278g/L, manganese sulfate monohydrate 0.085g/L, glucose 25g/L, potassium dihydrogen phosphate 2.5g/L.
  • Shake flask fermentation use a 250ml Erlenmeyer flask with a liquid volume of 50ml, an inoculation ratio of 2%, and a shaker at 30°C and 250rpm for 72h.
  • Rhodotorula vulgaris Total output mg/L Rhodotorula touloides 2.2424 120 Rhodotorula toruloides 2.1003 230 Rhodotorula toruloides 2.1609 187 Rhodotorula toruloides 2.1389 385 Rhodotorula toruloides 2.499 281 Rhodotorula toruloides 2.278 80 Rhodotorula toruloides 2.107 34
  • Rhodotorula vulgaris subspecies have different ergothioine production abilities, and Rhodotorula toruloides 2.1389 is the best.
  • this strain is used as the starting strain for genetic modification.
  • Rhodosporidium toruloides For the genetic manipulation method of Rhodosporidium toruloides, please refer to the following documents: Lin,X.,et al. (2014). “Functional integration of multiple genes into the gene of the oleaginous yeast Rhodosporidium toruloides. "FEMS Yeast Research 14(4): 547-555.
  • pGPD is designed as a promoter to control the expression of the functional gene; the CYC1t terminator derived from Saccharomyces cerevisiae is used to control the expression termination of the functional gene; Design and add XbaI restriction site (tctaga) and PmeI restriction site (gtttaaac) respectively for subsequent plasmid construction.
  • the designed gene expression cassettes are Pgpd-NcEGT1-ScCYC1t (i.e. SEQ ID NO: 1), Pgpd-CpEGT1-ScCYC1t (i.e. SEQ ID NO: 2) and Pgpd-RmEGT1-ScCYC1t (i.e. SEQ ID NO: 3)
  • the designed gene expression cassette sequence was sent to Suzhou Jinweizhi Biotechnology Company for synthesis, and the above gene expression cassette was cloned on the Puc57 plasmid to obtain the EGT1 gene expression plasmid, which was used for subsequent enzyme digestion ligation cloning construction.
  • the names of gene expression cassettes named Puc57-Pgpd-NcEGT1-ScCYC1t, Puc57-Pgpd-CpEGT1-ScCYC1t, Puc57-Pgpd-RmEGT1-ScCYC1t, respectively.
  • Pgpd-NcEGT1-ScCYC1t ie SEQ ID NO: 1
  • Pgpd-CpEGT1-ScCYC1t ie SEQ ID NO: 2
  • Pgpd-RmEGT1-ScCYC1t ie SEQ ID NO: 3
  • Puc57-Pgpd-NcEGT1-ScCYC1t After receiving the above three plasmids, Puc57-Pgpd-NcEGT1-ScCYC1t, Puc57-Pgpd-CpEGT1-ScCYC1t and Puc57-Pgpd-RmEGT1-ScCYC1t, they were digested with XbaI/PmeI restriction enzymes and gelled separately. The 3.7kb, 3.6kb and 4.9kb fragments were recovered.
  • the backbone plasmid pZPK-PGPD-Hyg-Tnos was digested with XbaI/PmeI, and then an 8.3kb fragment was recovered. Connect this fragment with the digested fragments of the above three gene expression cassettes to obtain three ergothione functional gene expression plasmids (or EGT1 gene transformation plasmids) pZPK-NcEGT1, pZPK-CpEGT1 and pZPK-RmEGT1, Used to transform the host.
  • the ligation method is: use the T4 ligase system purchased from NEB, refer to the instructions for use, add the vector and gene expression cassette to the T4 ligase system at a molar ratio of 1:5, 16°C, ligate for 2 hours, and transform the ligation product into DH5a Escherichia coli , Using kanamycin for cloning screening, after colony PCR, plasmid digestion and plasmid sequencing verification, the above three ergothione functional gene expression plasmids (or EGT1 gene transformation plasmids) pZPK-NcEGT1, p ZPK-CpEGT1 were obtained And p ZPK-RmEGT1.
  • Plasmid transformation to Agrobacterium tumefaciens AGL-1 Place the Agrobacterium competently on ice for 10 minutes. Add 1-2 ⁇ g of the EGT1 gene transformation plasmids pZPK-NcEGT1, pZPK-CpEGT1 and pZPK-RmEGT1 constructed in Example 4, gently pipette and mix with a pipette, transfer to a 0.2cm sterile electro-rotor cup, and place on ice for 5 minutes , 2.5kv electric shock, add 1ml liquid YEB, incubate at 28°C, 150rpm for 3h. The cells were collected by centrifugation at 8000 rpm for 2 min.
  • transformants were verified by PCR, and the bacterial solution was stored at -80°C (containing 15% glycerol) to obtain Agrobacterium engineering bacteria AGL-1/pZPK-NcEGT1, AGL-1/pZPK-CpEGT1 and AGL-1/pZPK- RmEGT1.
  • Example 2 According to the method of Example 1, the positive transformant obtained in Example 6 was subjected to shake flask fermentation investigation. After shake flask fermentation, the intracellular and extracellular thioneine production of each engineered bacteria after fermentation was evaluated. The results are shown in Table 2.
  • EGT1 genes from three different microorganisms are expressed in the same Rhodotorula toruloides 2.1389 to promote the production of ergothione.
  • NcEGT1 is the best, and EGT1 genes from different microorganisms are different.
  • the expression of thioneine can increase the production of ergothioneine.
  • an inoculating loop to take a single colony inoculum culture medium (glucose 20g/L, peptone 10g/L, yeast extract 5g/L, pH 7.0) on a streak plate of Rhodotorula Cultivate overnight at 230 rpm; inoculate the seed medium shake flask with an inoculum of 5% v/v, fill 50 ml of liquid in a 250ml Erlenmeyer flask, cultivate overnight at 30°C and 230 rpm shaker to prepare seeds.
  • a single colony inoculum culture medium (glucose 20g/L, peptone 10g/L, yeast extract 5g/L, pH 7.0) on a streak plate of Rhodotorula Cultivate overnight at 230 rpm
  • inoculate the seed medium shake flask with an inoculum of 5% v/v fill 50 ml of liquid in a 250ml Erlenmeyer flask, cultiv
  • the seed solution was transferred to the fermentation tank medium (glucose 60g/L, peptone 10g/L, corn steep liquor 10g/L, ferrous sulfate 10mg/L, pH 6.5) at an inoculation ratio of 10% v/v.
  • the stirring speed of the fermenter is 400 ⁇ 800rpm, the dissolved oxygen is coupled, the dissolved oxygen is controlled at 40%, the temperature is 30°C, the air flow is 3L/min, ammonia and histidine are added, and the pH is controlled at 6.5, the fermentation can continue until 120h .
  • 500g/L glucose solution was added at a rate of 20ml/h; for 24h-72h, 500g/L glucose solution was added at a rate of 10ml/h; 72h-96h, at a rate of 3-5ml/ Add 500g/L glucose solution at a rate of h; add 100g/L corn steep liquor solution at a rate of 5-8ml/h for 24-96h.
  • the production of intracellular and extracellular ergothioine after fermentation of each engineered bacteria was evaluated. The results are shown in Table 3.
  • Rhodotorula toruloides 2.1389, Rhodotorula toruloides 2.1389-NcEGT1, Rhodotorula toruloides 2.1389-CpEGT1, Rhodotorula toruloides 2.1389-RmEGT1 were subcultured on non-resistant seeds in plate streaking, and after passage, they were subjected to shake-flask engineering verification, and the yield was evaluated. The genetic stability of bacteria.

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Abstract

提供了一种圆红酵母工程菌及其构建方法，该圆红酵母工程菌能够表达外源的egt1酶，从而提高生产麦角硫因的能力，发酵后麦角硫因产量接近1.5g/L，且工程菌遗传性状稳定。

Description

一种构建麦角硫因生产菌的方法 技术领域
本发明属于基因工程领域，涉及一种构建麦角硫因生产菌的方法，尤其涉及一种构建用于生产麦角硫因的圆红酵母菌的方法。
背景技术
麦角硫因(L-ergothionine，EGT)，化学名为2-巯基组氨酸三甲基内盐，结构式如下：
Figure PCTCN2021077815-appb-000001
麦角硫因是至今唯一为人们所知的天然2-硫代咪唑氨基酸。麦角硫因具有抗氧化、抗炎症、延长细胞生存周期或抗细胞衰老活性，改善神经细胞生成等多种生理功效。同时在多种疾病模型中包括阿兹海默、糖尿病等的并发症中，具有较好的保护细胞和抗击损伤的功效，市场应用前景广阔。
目前，麦角硫因可以经化学合成、食用真菌提取和微生物发酵生产获得。有多种微生物被证实具有麦角硫因的合成能力，包括分枝杆菌、链霉菌、霉菌和酵母等多类微生物。有许多专利技术涉及利用微生物发酵生产麦角硫因，比如CN102978121B公开了食用菌白香蘑菌催化组氨酸底物可生成麦角硫因，底物转化率可达70％，未报道产物产率；CN103184246A公开了大型丝状真菌花脸香蘑摇瓶发酵10天，麦角硫因产量51mg/L；WO2017150304A1公开了青紫链霉菌Streptomyces lividans经7天发酵，麦角硫因产量900mg/L；WO2015180492A1公开了糙皮侧耳在75L发酵罐中经14天发酵，麦角硫因产量352mg/L；CN103734022公开了食用菌糙皮侧耳经7～15天发酵，麦角硫因产量最高产量达143.7mg/L；CN107250347公开了基因工程曲霉包括米曲霉、酱油曲霉和黑曲霉，经基因工程改造后，发酵产量可达438mg/L；大肠杆菌经基因工程改造，异源表达分枝杆菌的麦角硫因基因合成簇，麦角硫因产量640mg/L；CN106661585公开了大肠杆菌经基因工程改造，异源表达分枝杆菌的麦角硫因基因合成簇，工程菌发酵麦角硫因产量12mg/L； CN105296559A公开了糙皮侧耳食用菌经过多重发酵配方成分的调整，发酵至少6天，麦角硫因产量最高315.7mg/L；CN201910664772.8A公开了以枯草芽孢杆菌168为宿主表达外源基因，构建的基因工程菌麦角硫因产量达到568.4mg/L。
文献(Takusagawa,S.,Y.Satoh,I.Ohtsu and T.Dairi(2018)."Ergothioneine production with Aspergillus oryzae." Bioscience,Biotechnology,and Biochemistry:1-4.)公开了米曲霉Aspergillus oryzae经基因工程改造，麦角硫因产量231mg/L。此外，目前微生物发酵麦角硫因的最高产量为文献(Tanaka,N.,Y.Kawano,Y.Satoh,T.Dairi and I.Ohtsu(2019)."Gram-scale fermentative production of ergothioneine driven by overproduction of cysteine in Escherichia coli." Scientific reports9(1):1895-1895.)报道，该研究利用基因工程手段，在大肠杆菌中异源表达分枝杆菌来源的麦角硫因的合成，研究获得工程菌，进行发酵罐培养，通过添加IPTG诱导打开合成开关，并持续性补加前体物质组氨酸，进行长达216h即9天的发酵，最终可获得1.3g/L麦角硫因产量。但作为一种条件致病菌以及转基因菌株，其产品应用前景令人担忧。寻找天然来源且具有食源安全属性的微生物，并开发合适的生产工艺，对解决麦角硫因高效生产尤为重要。文献(van der Hoek,S.A.,B.Darbani,K.E.Zugaj,B.K.Prabhala,M.B.Biron,M.Randelovic,J.B.Medina,D.B.Kell and I.Borodina(2019)."Engineering the yeast&lt；em&gt；Saccharomyces cerevisiae&lt；/em&gt；for the production of L-(+)-ergothioneine." bioRxiv:667592.)公开了酿酒酵母Saccharomyces cerevisiae经基因工程改造，对外源基因进行表达，可合成麦角硫因，产量可达到630mg/L。
发明内容
为了探索利用食源安全性的微生物通过发酵法生产麦角硫因的工业化途径，本发明利用基因工程技术来对天然圆红酵母(或称圆红冬孢酵母，Rhodotorula toruloides)进行遗传改造，构建出了高产麦角硫因的菌株。具体而言，本发明包括如下技术方案：
一种EGT1基因表达盒，其选自SEQ ID NO:1、SEQ ID NO:2或者SEQ ID NO:3。
其中SEQ ID NO:1包含启动子pGPD、来源于粗糙链孢霉Neurospora crassa的酶egt1的编码基因NcEGT1和酿酒酵母来源的CYC1t终止子，并在首尾分别添加XbaI和PmeI酶切位点，用于后续的质粒构建，本文中命名为Pgpd-NcEGT1-ScCYC1t；
SEQ ID NO:2包含启动子pGPD、来源于黑麦麦角菌Claviceps purpurea的酶egt1的编码基因CpEGT1和酿酒酵母来源的CYC1t终止子，并在首尾分别添加XbaI和PmeI酶切位点，用于后续的质粒构建，本文中命名为Pgpd-CpEGT1-ScCYC1t；
SEQ ID NO:3包含启动子pGPD、来源于胶红酵母Rhodotorula mucilaginosa的酶egt1的编码基因RmEGT1和酿酒酵母来源的CYC1t终止子，并在首尾分别添加XbaI和PmeI酶切位点，用于后续的质粒构建，本文中命名为Pgpd-RmEGT1-ScCYC1t。
根据本发明的第二个方面，提供了一种EGT1基因表达质粒，其通过将上述的基因表达盒克隆在Puc57质粒上而获得。对应于基因表达盒名称，分别命名为Puc57-Pgpd-NcEGT1-ScCYC1t、Puc57-Pgpd-CpEGT1-ScCYC1t、Puc57-Pgpd-RmEGT1-ScCYC1t。
根据本发明的第三个方面，提供了一种麦角硫因功能基因表达质粒(或称EGT1基因转化质粒)，其通过包括下述步骤的方法构建：
1)使用限制性内切酶XbaI/PmeI对如权利要求2所述EGT1基因表达质粒Puc57-Pgpd-NcEGT1-ScCYC1t、Puc57-Pgpd-CpEGT1-ScCYC1t、Puc57-Pgpd-RmEGT1-ScCYC1t进行酶切，分别凝胶回收3.7kb、3.6kb、4.9kb片段，得到EGT1基因表达片段；
2)使用限制性内切酶XbaI/PmeI对核苷酸序列为SEQ ID NO:4的质粒pZPK-PGPD-Hyg-Tnos进行酶切，凝胶回收8.3kb片段，得到质粒骨架；
3)将步骤1)中得到的EGT1基因表达片段与步骤2)中得到的质粒骨架相连接，得到麦角硫因功能基因表达质粒。与质粒Puc57-Pgpd-NcEGT1-ScCYC1t、Puc57-Pgpd-CpEGT1-ScCYC1t、Puc57-Pgpd-RmEGT1-ScCYC1t相对应地，麦角硫因功能基因表达质粒分别为pZPK-NcEGT1、pZPK-CpEGT1、pZPK-RmEGT1质粒。
文献(Lin,X.,et al.(2014)."Functional integration of multiple genes into the genome of the oleaginous yeast Rhodosporidium toruloides."FEMS Yeast Research 14(4):547-555.)中描述了上述质粒pZPK-PGPD-Hyg-Tnos。
根据本发明的第四个方面，提供了一种构建麦角硫因生产菌的方法，包括下述步骤：将上述的麦角硫因功能基因表达质粒(pZPK-NcEGT1、pZPK-CpEGT1、pZPK-RmEGT1)转化入能够产生麦角硫因的菌株中，得到阳性克隆株。
上述菌株可以为圆红酵母(或称圆红冬孢酵母，Rhodotorula toruloides)。
在一种实施方式中，上述方法是，将麦角硫因功能基因表达质粒(pZPK-NcEGT1、pZPK-CpEGT1、pZPK-RmEGT1)通过农杆菌介导转染圆红酵母，得到阳性转化子。
例如，上述方法可以具体包括如下步骤：
A.将上述的麦角硫因功能基因表达质粒(pZPK-NcEGT1、pZPK-CpEGT1、pZPK-RmEGT1)转化入农杆菌感受态细胞，得到农杆菌工程菌；优选农杆菌是根癌农杆菌；
B.将农杆菌工程菌菌液与圆红酵母菌液相混合，进行抗性筛选，经表型及PCR验证，获得阳性转化子，从而实现农杆菌介导转染圆红酵母。
根据本发明的第五个方面，提供了一种麦角硫因生产菌，其通过构建麦角硫因生产菌的方法得到。
在一种实施方式中，上述麦角硫因生产菌为圆红酵母，优选原始圆红酵母菌为中国普通微生物保藏管理中心保藏的Rhodotorula toruloides 2.1389。
上述麦角硫因生产菌可以通过发酵法生产麦角硫因。
本发明通过基因工程手段，对天然圆红酵母进行遗传改造，获得具有食源安全性的麦角硫因高产菌种。经发酵验证，麦角硫因产量可达到约1.5g/L，构建的工程菌株经多次传代，遗传性状相对稳定，具有较好的工业应用潜力。
附图说明
图1是本发明构建的质粒pZPK-PGPD-Hyg-Tnos的图谱结构示意图。
具体实施方式
egt1是个多功能酶，可以催化底物组氨酸生成麦角硫因前体S-(组氨酸甜菜碱)-2-)基-L-半胱氨酸S-氧化物(S-(hercyn-2-yl)-L-cysteine S-oxide)。研究发现，通过在可产生麦角硫因的菌株中表达egt1基因，能够促进麦角硫因的产生。
在本文中，为了描述简便，有时会将某种蛋白比如egt1与其编码基因(DNA)名称混用，本领域技术人员应能理解它们在不同描述场合表示不同的物质。本领域技术人员根据语境和上下文容易理解它们的含义。例如，对于egt1，用于描述酶功能或类别时，指的是蛋白质；在作为一种基因描述时，指的是编码该酶的基因。
我们筛选出三种微生物来源的egt1酶，分别来源于粗糙链孢霉Neurospora crassa、黑麦麦角菌Claviceps purpurea和胶红酵母Rhodotorula mucilaginosa，其编码基因分别为NcEGT1、CpEGT1和RmEGT1。
NcEGT1基因，根据NCBI数据库，Genbank accession:XP_956324.3序列，经密码子优化，可以获得如下碱基序列：
Figure PCTCN2021077815-appb-000002
Figure PCTCN2021077815-appb-000003
CpEGT1序列参考文献van der Hoek,S.A.,et al.(2019)."Engineering the yeast Saccharomyces cerevisiae for the production of L-(+)-ergothioneine."bioRxiv:667592.其为
Figure PCTCN2021077815-appb-000004
Figure PCTCN2021077815-appb-000005
RmEGT1，使用NcEGT1蛋白序列，在NCBI数据库blastp工具，限定胶红酵母Rhodotorula mucilaginosa基因组，进行序列比对，获得如下egt1序列：
Figure PCTCN2021077815-appb-000006
Figure PCTCN2021077815-appb-000007
Figure PCTCN2021077815-appb-000008
在本发明构建的工程菌中，egt1基因以基因表达盒的形式存在。在本文中，术语“egt1基因表达盒”、“基因表达盒”和“表达盒”表示相同的意义，可以互换使用。
以上述NcEGT1、CpEGT1和RmEGT1为目的基因，可以构建出三种egt1基因表达盒，分别为Pgpd-NcEGT1-ScCYC1t(即SEQ ID NO:1)、Pgpd-CpEGT1-ScCYC1t(即SEQ ID NO:2)和Pgpd-RmEGT1-ScCYC1t(即SEQ ID NO:3)。三个表达盒均在首尾分别设计添加XbaI和PmeI酶切位点，用于后续的质粒构建。
将上述基因表达盒元件序列送苏州金唯智生物技术公司，进行合成，分别获得Puc57-Pgpd-NcEGT1-ScCYC1t，Puc57-Pgpd-CpEGT1-ScCYC1t，Puc57-Pgpd-RmEGT1-ScCYC1t质粒，用于后续的酶切连接克隆构建。
通过在合适的宿主比如某些圆红酵母中表达egt1基因，提高了圆红酵母生产麦角硫因的能力。构建的圆红酵母工程菌在通过发酵生产麦角硫因时，不需要在培养液中添加组氨酸甜菜碱、组氨酸或者半胱氨酸等就能够产生大量的麦角硫因。
以下结合具体实施例对本发明做进一步详细说明。应理解，以下实施例仅用于说明本发明而非用于限定本发明的范围。
本文中涉及到多种物质的添加量、含量及浓度，其中所述的百分含量，除特别说明外，皆指质量百分含量。
实施例
材料和方法
本文中的全基因合成、引物合成及测序皆由苏州金唯智生物技术公司完成。
本文中的分子生物学实验包括质粒构建、酶切、感受态细胞制备、转化等主要参照《分子克隆实验指南》(第三版)，J.萨姆布鲁克，D.W.拉塞尔(美)编著，黄培堂等译，科学出版社，北京，2002)进行。比如感受态细胞转化方法及感受态制备方法均参照《分子克隆实验指南》(第三版)第1章96页进行。必要时可以通过简单试验确定具体实验条件。
主要培养基及溶液：
LB培养基：5g/L酵母提取物，10g/L胰蛋白胨，10g/L氯化钠。(LB固体培养基另加20g/L琼脂粉。)
YEB培养基：牛肉浸膏5g/L，酵母膏1g/L，蛋白胨5g/L，蔗糖5g/L，MgSO 4·7H 2O0.5g/L，pH 7.4。(YEB固体培养基另加15g/L琼脂粉。)
YPD培养基：1wt％酵母膏，2wt％蛋白胨，2wt％葡萄糖。
农杆菌介导酵母转化溶液及培养基：
MM溶液：3.625g KH 2PO 4，5.125g K 2HPO 4，1.250g MgSO 4·7H 2O，0.375g NaCl，0.165g CaCl 2·2H 2O，0.0062g FeSO 4·7H 2O，1.250g(NH 4) 2SO 4，溶解于ddH 2O中定容至1L；
M-100微量元素溶液：30mg H 3BO 3，70mg MnCl 2·4H 2O，200mg ZnCl 2，20mg Na 2MoO 4·2H 2O，50mg FeCl 3·6H 2O，200mg CuSO 4·5H 2O，溶解于ddH 2O中定容至500ml。
M-100盐溶液：16g KH 2PO 4，4g Na 2SO 4，8g KCl，2g MgSO 4·7H 2O，1g CaCl 2，8ml M-100微量元素溶液，溶解于ddH 2O中定容至1L。
IM液体培养基：80ml MM溶液，0.36g葡萄糖，1ml甘油，溶解于ddH 2O中定容至200ml。灭菌冷却到50℃左右后添加下列成分：8ml 1M MES，4ml 10mM AS，MES和AS的终浓度分别为40mM和200μM。
IM固体培养基：160ml MM盐溶液，0.36g葡萄糖，2ml甘油，溶解于ddH 2O中定容至400ml，最后加入6g琼脂粉。灭菌冷却到50℃左右后添加下列成分：16ml 1M MES，8ml 10mM AS，MES和AS的终浓度分别为40mM和200μM。
麦角硫因检测材料和方法：
麦角硫因标准品，购自中国医药(集团)上海化学试剂公司。
HPLC检测条件为：安捷伦高效液相色谱仪1260infinity II，依利特ODS-BP色谱柱，柱温40℃，流动相：A，磷酸二氢铵(配置方法，称取1.1503g磷酸二氢铵+400mL纯化水，氨水调pH至5.0，再加100mL纯化水)；B：乙腈。A:B＝99:1，流速1mg/min，进样量10μl，检测波长258nm。
胞外产物检测：发酵菌液进行10000rpm，4min离心，取上清，用2μm过滤器进行过滤处理后，样品进行HPLC检测。
胞内产物检测：发酵菌液进行10000rpm，4min离心，收集菌体，称取100mg湿菌体，转移至干净的1.5ml离心管中，加1ml无菌水，涡旋，重悬菌体，然后于90℃进行温浴30min，温浴结束，12000rpm，10min离心，取上清，用2μm过滤器进行过滤处理后，样品进行HPLC检测。
实施例1：原始菌种筛选
根据检索到的文献报道，筛选能够产生麦角硫因的食源安全性微生物。自中国普通微生物保藏管理中心购买多株圆红酵母进行初步筛选对比，评估各菌株麦角硫因产量。
将购买的菌种接种在酵母培养基中，进行摇瓶发酵。酵母种子培养基组成：葡萄糖20g/L，酵母粉10g/L，蛋白胨20g/L。
将甘油管种子，接种种子培养基，30℃，250rpm，17h培养，OD600>16时可转接。
酵母摇瓶发酵培养基：硫酸铵0.5g/L，酵母粉10g/L，蛋白胨10g/L，谷氨酸3g/L，生物素0.6mg/L，盐酸硫胺素0.1g/L，硫酸镁5g/L，七水硫酸亚铁0.278g/L，一水硫酸锰0.085g/L，葡萄糖25g/L，磷酸二氢钾2.5g/L。
摇瓶发酵：使用250ml三角瓶，装液量50ml，接种比例2％，于摇床30℃，250rpm，培养72h。
摇瓶发酵后，评估各菌发酵后的胞内和胞外麦角硫因产量。结果见表1。
表1、不同圆红酵母发酵生产麦角硫因能力的对比
圆红酵母菌种 总产量mg/L
Rhodotorula toruloides 2.2424 120
Rhodotorula toruloides 2.1003 230
Rhodotorula toruloides 2.1609 187
Rhodotorula toruloides 2.1389 385
Rhodotorula toruloides 2.499 281
Rhodotorula toruloides 2.278 80
Rhodotorula toruloides 2.107 34
由表1可见，不同的圆红酵母亚种生产麦角硫因的能力是有差别的，其中Rhodotorula toruloides 2.1389最优。下述实施例中将该菌株作为遗传改造的出发菌。
实施例2：构建质粒pZPK-PGPD-Hyg-Tnos
圆红酵母的遗传操作方法可参考下述文献：Lin,X.,et al.(2014)."Functional integration of multiple genes into the genome of the oleaginous yeast Rhodosporidium toruloides."FEMS Yeast Research 14(4):547-555.
参考该文献，根据公开序列，确定质粒pZPK-PGPD-Hyg-Tnos完整序列，并送苏州金唯智生物科技有限公司进行合成，获得质粒pZPK-PGPD-Hyg-Tnos(SH205)，其结构如图1所示，其碱基序列为SEQ ID NO:4，8378bp。
以此为质粒骨架，进行后续的质粒构建。
实施例3：构建EGT1基因表达盒和表达质粒
对于三种微生物来源的目的基因NcEGT1、CpEGT1和RmEGT1，设计以pGPD为启动子来控制该功能基因的表达；以酿酒酵母来源的CYC1t终止子来控制该功能基因的表达终止；并且在首、尾分别设计添加XbaI酶切位点(tctaga)和PmeI酶切位点(gtttaaac)，以便用于后续的质粒构建。设计的基因表达盒分别为Pgpd-NcEGT1-ScCYC1t(即SEQ ID NO:1)、Pgpd-CpEGT1-ScCYC1t(即SEQ ID NO:2)和Pgpd-RmEGT1-ScCYC1t(即SEQ ID NO:3)
将设计的基因表达盒序列送苏州金唯智生物技术公司进行合成，并将上述的基因表达盒克隆在Puc57质粒上，获得EGT1基因表达质粒，用于后续的酶切连接克隆构建。对应于基因表达盒名称，分别命名为Puc57-Pgpd-NcEGT1-ScCYC1t、Puc57-Pgpd-CpEGT1-ScCYC1t、Puc57-Pgpd-RmEGT1-ScCYC1t。
分别获得Pgpd-NcEGT1-ScCYC1t(即SEQ ID NO:1)、Pgpd-CpEGT1-ScCYC1t(即SEQ ID NO:2)和Pgpd-RmEGT1-ScCYC1t(即SEQ ID NO:3)质粒。
实施例4：构建麦角硫因功能基因表达质粒
4.1在收到上述Puc57-Pgpd-NcEGT1-ScCYC1t、Puc57-Pgpd-CpEGT1-ScCYC1t和Puc57-Pgpd-RmEGT1-ScCYC1t三个质粒后，分别使用XbaI/PmeI限制性内切酶进行酶切，分别凝胶回收3.7kb，3.6kb和4.9kb片段。
4.2骨架质粒pZPK-PGPD-Hyg-Tnos经XbaI/PmeI酶切后，回收8.3kb片段。将该片段与上述3种基因表达盒的酶切片段分别进行连接，获得三种麦角硫因功能基因表达质粒(或称EGT1基因转化质粒)pZPK-NcEGT1、p ZPK-CpEGT1和p ZPK-RmEGT1，用于转化宿主。连接方法为：使用购买自NEB公司的T4连接酶体系，参照使用说明，按载体和基因表达盒摩尔比例1:5添加到T4连接酶体系，16℃，连接2h，将连接产物转化DH5a大肠杆菌，使用卡那霉素进行克隆筛选，经过菌落PCR、质粒酶切和质粒测序验证后，获得上述三种麦角硫因功能基因表达质粒(或称EGT1基因转化质粒)pZPK-NcEGT1、p ZPK-CpEGT1和p ZPK-RmEGT1。
实施例5：制备根癌农杆菌工程菌
5.1根癌农杆菌菌株AGL-1感受态的制备：从含50μg/ml羧苄青霉素(Carb)的YEB 平板上挑取菌株AGL-1(上海康朗生物科技有限公司)的单菌落到5ml YEB液体培养基中(含50μg/ml Carb)，于28℃、200rpm摇床培养过夜。取2ml菌液到50ml YEB(含50μg/ml Carb)中，于28℃、200rpm摇床培养至菌液在600nm处的OD吸收值为0.5左右。取出菌液，冰上冰浴30min后，4℃，8000rpm离心5min收集菌体。用10ml ddH 2O洗一次。4℃，8000rpm离心5min再次收集菌体。弃上清液，加入2ml终浓度为10％无菌甘油，每份100μl分装，即为根癌农杆菌菌株AGL-1感受态。
5.2质粒转化至根癌农杆菌AGL-1：将农杆菌感受态置于冰上10min。加1～2μg实施例4中构建的EGT1基因转化质粒pZPK-NcEGT1、pZPK-CpEGT1和pZPK-RmEGT1，用移液器轻轻吹打混匀，转移至0.2cm无菌电转杯中，冰上放置5min，2.5kv电击，加入1ml液体YEB，于28℃，150rpm培养3h。8000rpm离心2min收集菌体。去掉上清后用100μl液体YEB重悬菌体，并均匀涂布在YEB平板上(含50μg/ml Carb和50μg/ml Kan)，于28℃培养2～3天。挑取转化子，用3ml液体YEB(含50μg/ml Carb和50μg/ml Kan)于28℃、220rpm摇菌过夜。PCR验证转化子，并将其菌液保存于-80℃(含15％的甘油)，得到农杆菌工程菌AGL-1/pZPK-NcEGT1、AGL-1/pZPK-CpEGT1和AGL-1/pZPK-RmEGT1。
实施例6：农杆菌介导转染圆红酵母
6.1农杆菌工程菌培养：将实施例5中构建的农杆菌工程菌AGL-1/pZPK-NcEGT1、AGL-1/pZPK-CpEGT1、AGL-1/pZPK-RmEGT1单菌落接种于4-5ml的LB培养基(含抗生素Amp和Kan)中，30℃过夜培养。测定OD值，一般需要达到OD 600＝4.2以上，计算稀释倍数，应稀释(据实计算)倍，可达到OD＝0.6。取一定量的菌液于1.5ml离心管中，12000rpm离心30s，将菌体沉底，加入灭菌蒸馏水悬浮，再次离心沉底。最后用计算好的水体积将菌悬浮至OD＝0.6。备用。
6.2圆红酵母宿主培养：将圆红酵母Rhodotorula toruloides 2.1389单菌落接种于4-5ml的YPD培养基中过夜培养，约15-16h。测定OD 600值，计算稀释倍数，应稀释(据实计算)倍，稀释OD至0.6。按需要，取一定量的菌液于1.5ml离心管中，13000rpm离心30s，将菌体沉底，加入灭菌蒸馏水悬浮，再次离心沉底。最后用计算好的水体积将菌悬浮至OD＝0.6。备用。
6.3平板培养转染：将上述稀释好的农杆菌工程菌液和酵母菌液混合后，涂布平板：分别取100ul混合后，涂在IM平板上，24℃培养2-3天。
6.4抗性筛选：IM平板上获得的菌落，使用YPD液体培养基洗脱刮下，将菌悬液涂布到YPD+潮霉素抗性平板上，30℃培养4天。待有转化子出现后，挑选克隆子，进行YPD+潮霉素抗性平板上进行划线传代培养，需连续传代3代，对连续传代抗性表型稳定的克隆，进行基因组抽提，进行特定的靶点序列PCR验证，并经表型验证，获得阳性转化子。
实施例7：工程菌摇瓶发酵考察
按照实施例1的方法，对实施例6中获得的阳性转化子进行摇瓶发酵考察。摇瓶发酵后，评估各工程菌发酵后的胞内和胞外麦角硫因产量。结果见表2。
表2、不同圆红酵母工程菌摇瓶发酵生产麦角硫因能力的对比
Figure PCTCN2021077815-appb-000009
由表2可见，三种不同微生物来源的EGT1基因在同一个圆红酵母亚种Rhodotorula toruloides 2.1389中表达来促进麦角硫因生产的能力是有差别的，其中NcEGT1最优，且不同微生物来源EGT1基因的表达都能提高麦角硫因产量。
实施例8：工程菌发酵罐发酵考察
圆红酵母划线平板上，使用接种环取单菌落接种子培养基(葡萄糖20g/L，蛋白胨10g/L，酵母提取物5g/L，pH7.0)试管，4ml装液量，30℃，230rpm培养过夜；按5％v/v的接种量，接种到种子培养基摇瓶，250ml三角瓶装液50ml，30℃，230rpm摇床培养过夜，准备种子。将种子液按10％v/v的接种比例，转接发酵罐培养基(葡萄糖60g/L，蛋白胨 10g/L，玉米浆粉10g/L，硫酸亚铁10mg/L，pH6.5)。
发酵罐搅拌转速为400～800rpm，溶氧偶联，溶氧控制40％，温度为30℃，空气流量3L/min，补加氨水和组氨酸，控制pH6.5，发酵可以一直持续到120h。其间，在12-24h，以20ml/h的速率流加500g/L的葡萄糖溶液；24h-72h，以10ml/h的速率流加500g/L的葡萄糖溶液；72h-96h，以3-5ml/h的速率流加500g/L的葡萄糖溶液；24-96h，以5-8ml/h的速率流加100g/L的玉米浆溶液。发酵结束后，评估各工程菌发酵后的胞内和胞外麦角硫因产量。结果见表3。
表3、不同圆红酵母工程菌发酵罐发酵生产麦角硫因能力的对比
Figure PCTCN2021077815-appb-000010
由表3可见，与摇瓶发酵类似，三种不同微生物来源的EGT1基因在同一个圆红酵母亚种Rhodotorula toruloides 2.1389中表达来促进麦角硫因生产的能力是有差别的，不同微生物来源EGT1基因的表达都能提高麦角硫因产量，其中NcEGT1仍是最优，发酵后麦角硫因产量接近1.5g/L，具有工业化应用前景。
实施例9：工程菌遗传稳定性评估
将上述菌株Rhodotorula toruloides 2.1389，Rhodotorula toruloides 2.1389-NcEGT1，Rhodotorula toruloides 2.1389-CpEGT1，Rhodotorula toruloides 2.1389-RmEGT1，进行无抗种子平板划线传代培养，传代后对其进行摇瓶发酵验证，评估产量，判断工程菌的遗传稳定性。
Figure PCTCN2021077815-appb-000011
上述实验结果表明，本发明方法构建的基因工程菌株经过6次传代，产物产量仍高于出发菌株Rhodotorula toruloides 2.1389，表明工程菌的遗传性状相对稳定。

Claims (10)

  1. 一种EGT1基因表达盒，其选自SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3。
  2. 一种EGT1基因表达质粒，其通过将如权利要求1所述的基因表达盒克隆在pUC57质粒上而获得，分别命名为Puc57-Pgpd-NcEGT1-ScCYC1t、Puc57-Pgpd-CpEGT1-ScCYC1t、Puc57-Pgpd-RmEGT1-ScCYC1t。
  3. 一种麦角硫因功能基因表达质粒，其通过包括下述步骤的方法构建：
    1)使用限制性内切酶XbaI/PmeI对如权利要求2所述EGT1基因表达质粒Puc57-Pgpd-NcEGT1-ScCYC1t、Puc57-Pgpd-CpEGT1-ScCYC1t、Puc57-Pgpd-RmEGT1-ScCYC1t进行酶切，分别凝胶回收3.7kb、3.6kb、4.9kb片段，得到EGT1基因表达片段；
    2)使用限制性内切酶XbaI/PmeI对核苷酸序列为SEQ ID NO:4的质粒pZPK-PGPD-Hyg-Tnos进行酶切，凝胶回收8.3kb片段，得到质粒骨架；
    3)将步骤1)中得到的EGT1基因表达片段与步骤2)中得到的质粒骨架相连接，得到麦角硫因功能基因表达质粒。
  4. 一种构建麦角硫因生产菌的方法，其特征在于，将如权利要求3所述的麦角硫因功能基因表达质粒转化入能够产生麦角硫因的菌株中，得到阳性克隆株。
  5. 如权利要求4所述的方法，其特征在于，所述菌株为圆红酵母Rhodotorula toruloides。
  6. 如权利要求5所述的方法，其特征在于，将如权利要求3所述的麦角硫因功能基因表达质粒通过农杆菌介导转染圆红酵母，得到阳性转化子。
  7. 如权利要求6所述的方法，其特征在于，包括如下步骤：
    A.将如权利要求3所述的麦角硫因功能基因表达质粒转化入农杆菌感受态细胞，得到农杆菌工程菌；
    B.将农杆菌工程菌菌液与圆红酵母菌液相混合，进行抗性筛选，经表型及PCR验证，获得阳性转化子。
  8. 一种麦角硫因生产菌，其通过如权利要求4-7中任一项所述的方法构建得到。
  9. 如权利要求8所述的麦角硫因生产菌，其特征在于，为圆红酵母。
  10. 如权利要求8或9所述麦角硫因生产菌在发酵法生产麦角硫因中的用途。
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