WO2017000366A1 - 一种利用酿酒酵母酶法制备瑞鲍迪甙m的方法 - Google Patents

一种利用酿酒酵母酶法制备瑞鲍迪甙m的方法 Download PDF

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WO2017000366A1
WO2017000366A1 PCT/CN2015/087751 CN2015087751W WO2017000366A1 WO 2017000366 A1 WO2017000366 A1 WO 2017000366A1 CN 2015087751 W CN2015087751 W CN 2015087751W WO 2017000366 A1 WO2017000366 A1 WO 2017000366A1
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rebaudioside
ugt
saccharomyces cerevisiae
udp
plasmid
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French (fr)
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杜好勉
魏喜换
谢新开
陶军华
李托马斯
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苏州汉酶生物技术有限公司
百事可乐公司
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Priority to US15/740,572 priority Critical patent/US20180320211A1/en
Publication of WO2017000366A1 publication Critical patent/WO2017000366A1/zh

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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
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    • C12N9/10—Transferases (2.)
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    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C12N1/185—Saccharomyces isolates
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae

Definitions

  • the invention belongs to the field of bioengineering enzymes, and particularly relates to a method for preparing rebaudioside M by using Saccharomyces cerevisiae enzymatic method.
  • Sweeteners are a class of food additives that are widely used in the production of food, beverages and confectionery. They can be added either during the production of foods or as appropriate substitutes for sucrose in household baking. Sweeteners include natural sweeteners and artificial sweeteners, such as sucrose, high fructose corn syrup, honey, etc., such as aspartame, saccharin, and the like.
  • Stevia is a natural sweetener extracted from plant stevia and is currently widely used in foods and beverages.
  • the extract of Stevia contains a variety of steviosides including rebaudioside, and the different batch components of naturally extracted stevioside vary greatly, requiring subsequent purification.
  • the current commercial product, Rebaudio A contains some other stevia such as Rebaudioside C, D and F.
  • the stevioside prepared by the extraction method is usually mixed with some impurities, which may have a certain influence on the range of its use.
  • Rebaudioside M has an advantage over Rebaudioside A, but its content in stevia leaves is extremely low and is only detected in stevia Morita plants (2010, J. Appl. Glycosci., 57, 199-209). ). There is currently no commercial production of Rebaudio M.
  • Chinese patent document CN103397064A discloses a method for preparing rebaudioside M by enzymatic method, which utilizes UDP-glucosyltransferase prepared by Escherichia coli or Escherichia coli containing UDP-glucosyltransferase in the presence of a glucose-based donor.
  • Rebaudioside A or Rebaudioside D produces Rebaudioside M.
  • the genetically engineered bacterium producing UDP-glucosyltransferase which is a genetically-loving strain (GRAS), which produces toxins during the cultivation process, produces rebaudio ⁇ M cannot be directly applied, and E. coli is a prokaryote.
  • GRAS genetically-loving strain
  • the gene of UDP-glucosyltransferase is derived from eukaryotes. Because eukaryotes and prokaryotes have different levels of protein expression, prokaryotic bacteria express eukaryotic origin. The enzyme affects the activity of the enzyme. Saccharomyces cerevisiae is a well-recognized strain, but the expression level of a foreign gene expressed in yeast is low.
  • the technical problem to be solved by the present invention is to overcome the safety problem of the produced rebaudioside M produced by the Escherichia coli containing UDP-glucosyltransferase in the prior art in the process of preparing Rebaudioside M due to the production of toxin by the engineering bacteria itself.
  • a method for preparing rebaudioside M by enzymatic method by a recognized safe strain which can produce a high-purity Rebaudioside M product at a relatively low cost and in a short cycle.
  • the present invention adopts the following technical solutions:
  • a method for preparing rebaudioside M by using Saccharomyces cerevisiae enzymatic method using recombinant Saccharomyces cerevisiae containing UDP-glucosyltransferase or UDP-glucosyltransferase prepared thereby, in the presence of glucose-based donor, catalyzing Rebaudioside A or Rebaudioside D produces Rebaudioside M, and the recombinant Saccharomyces cerevisiae is constructed by introducing a strong promoter into a plasmid to obtain a vector plasmid, and UDP-glucosyltransferase gene is digested. A site is inserted into the vector plasmid under the control of the strong promoter to obtain an expression vector, and then transformed into Saccharomyces cerevisiae to obtain recombinant Saccharomyces cerevisiae.
  • the UDP-glucosyltransferase is UGT-A from Stevia rebaudiana and/or UGT-B from rice (Oryza sativa).
  • the amino acid sequence of UGT-A has at least 60% identity with the amino acid sequence shown by SEQ ID NO. 1 in the sequence listing; the amino acid of UGT-B derived from rice The sequence has at least 60% identity to the amino acid sequence set forth in SEQ ID NO. 3 of the Sequence Listing.
  • the amino acid sequence of the UGT-A is as shown in SEQ ID NO. 1 in the Sequence Listing
  • the amino acid sequence of the UGT-B is as shown in SEQ ID NO. 3 in the Sequence Listing. .
  • the cleavage sites are HindIII and XbaI.
  • the strong promoter is ADH2 or TEF1.
  • the plasmid is pYES2.
  • the vector plasmid is constructed by introducing an AgeI restriction site into the plasmid and introducing a strong promoter through the AgeI/HindIII site.
  • Saccharomyces cerevisiae is Saccharomyces cerevisiae BY4742.
  • the glucose-based donor is UDP-glucose or a UDP-glucose regeneration system consisting of sucrose, sucrose synthase and UDP.
  • recombinant Saccharomyces cerevisiae is formed into a recombinant Saccharomyces cerevisiae permeable cell for catalysis by a cell permeabilizing agent.
  • the recombinant Saccharomyces cerevisiae is cultured, ultrasonically disrupted in an ice bath, the crushed liquid is centrifuged, and the supernatant is collected and lyophilized to obtain a freeze-dried powder of UGT-A or UGT-B. For catalysis.
  • the catalytic reaction is carried out in an aqueous phase system having a temperature of 4 ° C to 50 ° C and a pH of 5.0 to 9.0.
  • the present invention has the following advantages compared with the prior art:
  • the method for preparing rebaudioside M by using Saccharomyces cerevisiae enzymatic method provided by the invention, preparing UDP-glucosyltransferase by preparing high-safety recombinant Saccharomyces cerevisiae for catalytic production, and producing rebaudioside M safety High, can be used directly as a food additive.
  • the method for preparing rebaudioside M by using the Saccharomyces cerevisiae enzymatic method provided by the present invention using Saccharomyces cerevisiae as an engineering strain, introducing a strong promoter ADH2 or TEF1 through the AgeI/HindIII cleavage site on the plasmid pYES2, replacing the pYES2 plasmid.
  • the original Gal promoter was inserted into the expression vector of UDP-glucosyltransferase gene derived from eukaryotes through HindIII and XbaI sites.
  • the UDP-glucosyltransferase gene was under the control of a strong promoter and cultured in Saccharomyces cerevisiae BY4742.
  • the method for preparing rebaudioside M by using the Saccharomyces cerevisiae enzymatic method provided by the invention the produced rebaudioside M has good safety and high purity, and is directly used for food additives without subsequent treatment, thereby significantly shortening the production cycle. Increased production and reduced costs.
  • Figure 1 is a 1 H NMR chart of the prepared rebaudioside M.
  • Reb A The following rebaudio A, Rebaudi D, and Rebaudi M are referred to as Reb A, Reb D and Reb M, respectively.
  • the structural formulas of the three are shown in Formulas I, II and III, respectively.
  • the invention mainly provides four routes for synthesizing Reb M:
  • the UGT-A or UGT-B used may be present in the form of an enzyme lyophilized powder or present in recombinant yeast cells.
  • UGT-A or UGT-B can be obtained as follows:
  • the recombinant Saccharomyces cerevisiae expression strain of UGT-A or UGT-B was obtained by molecular cloning technology and genetic engineering technology, and then the recombinant Saccharomyces cerevisiae was fermented to prepare recombinant cells containing UGT-A or UGT-B, or UGT-A was prepared. Or lyophilized powder of UGT-B.
  • the expression vector was transformed into S. cerevisiae BY4742 to obtain a recombinant S. cerevisiae expression strain of UGT-A or UGT-B or SUS.
  • Recombinant cells containing UGT-A or UGT-B, or lyophilized powder of UGT-A or UGT-B, were prepared using recombinant S. cerevisiae expression strains containing UGT-A or UGT-B.
  • Primers pYES2-AgeI-F (GATGATCCACTAGTAACCGGTAGAAGCCGCCG) and pYES2-AgeI-R (CGGCGGCTTCTACCGGTTACTAGTGGATCATC) were designed based on the pYES2 plasmid sequence, and the AgeI cut-point was introduced into the pYES2 plasmid by expand-loop PCR to obtain plasmid pYES2-AgeI.
  • the primers ADH2-F CACTAGTAACCGGTGCAAAACGTAGGGGC
  • ADH2-R GTCCAGCCCAAGCTTGTATTACGATATAG
  • AgeI/HindIII was digested, and the purified fragment was recovered, and the fragment was ligated into the pYES2-AgeI corresponding restriction site by adding T4 ligase to obtain a pEZADH2 plasmid.
  • the gene was synthesized into a UGT-A gene fragment, and ligated into the pUC57 vector to obtain PUC57-UGT-A (Suzhou Jinweizhi Biotechnology Co., Ltd.).
  • UGT-A gene was obtained by PCR using pUC57-UGT-A as template, and HindIII and XbaI restriction sites were added to both ends.
  • the UGT-A gene fragment was digested with restriction endonucleases HindIII and XbaI to recover the purified fragment.
  • the TEZ ligase was added to ligate the fragment into the pEZADH2 corresponding cleavage site to obtain the pEZADH2-UGT-A plasmid, and the plasmid was transformed into the yeast BY4742 strain to obtain the recombinant EZ-A.
  • the recombinant cells of UGT-A prepared in Example 1 were ultrasonically disrupted in an ice bath, and the disrupted solution was centrifuged (8,000 rpm, 10 min), and the supernatant was collected and lyophilized for 24 hours to obtain a lyophilized powder of UGT-A.
  • Method 2 Pick the plate monoclonal to SC-Ura+2% glucose medium, incubate at 30 °C, shaking at 200 rpm for 48 h, inoculate EZ-A strain to 50 ml YPD+1% glucose medium at 2%, 30 °C After incubation for 48 h at 200 rpm, add 20% Triton X-100 (V/V) 500 ul, continue to shake for 2 h, collect the cells by centrifugation (4,000 rpm, 10 min), and use 5 ml of 0.1 mol/L phosphate buffer (pH 7.0). The cells were resuspended and recombinant S. cerevisiae cells containing UGT-A were obtained for catalysis.
  • V/V Triton X-100
  • Primer design primers pYES2-AgeI-F (GATGATCCACTAGTAACCGGTAGAAGCCGCCG) and pYES2-AgeI-R (CGGCGGCTTCTACCGGTTACTAGTGGATCATC) were designed according to the pYES2 plasmid sequence, and the AgeI cleavage point was introduced into the pYES2 plasmid by expand-loop PCR to obtain plasmid pYES2-AgeI.
  • TEF1-F CCACTAGTAACCGGTCACACACCATAGCTTC
  • TEF1-R GTCCAGCCCAAGCTTTGTAATTAAAACTTAG
  • AgeI/HindIII was digested, and the purified fragment was recovered, and the fragment was ligated into the pYES2-AgeI corresponding cleavage site by adding T4 ligase to obtain a pEZTEF1 plasmid.
  • the gene was synthesized into a UGT-B gene fragment, and ligated into the pUC57 vector to obtain PUC57-UGT-B (Suzhou Jinweizhi Biotechnology Co., Ltd.).
  • PUC57-UGT-B Suzhou Jinweizhi Biotechnology Co., Ltd.
  • the UGT-B gene was obtained by PCR, and HindIII and XbaI restriction sites were added to both ends, and the UGT-B gene fragment was digested with restriction endonucleases HindIII and XbaI to recover the purified fragment.
  • the T4 ligase was added to ligate the fragment into the pEZTEF1 corresponding cleavage site to obtain the pEZTEF1-UGT-B plasmid, and the plasmid was transformed into the yeast BY4742 strain to obtain the recombinant EZ-B.
  • the cells were resuspended (pH 7.0) to obtain recombinant cells containing UGT-B.
  • the recombinant cells of UGT-B prepared in Example 4 were ultrasonically disrupted in an ice bath, and the disrupted solution was centrifuged (8,000 rpm, 10 min), and the supernatant was collected and lyophilized for 24 hours to obtain a lyophilized powder of UGT-B.
  • Recombinant S. cerevisiae permeable cells are used for catalysis
  • Method 2 Pick the plate monoclonal to SC-Ura+2% glucose medium, incubate at 30 °C, shaking at 200 rpm for 48 h, inoculate EZ-B strain to 50 ml YPD+1% glucose medium at 2%, 30 °C After incubation for 48 h at 200 rpm, add 20% Triton X-100 (V/V) 500 ul, continue to shake for 2 h, collect the cells by centrifugation (4,000 rpm, 10 min), and use 5 ml of 0.1 mol/L phosphate buffer (pH 7.0). The cells were resuspended and recombinant S. cerevisiae cells containing UGT-B were obtained for catalysis.
  • V/V Triton X-100
  • Primers pYES2-AgeI-F (GATGATCCACTAGTAACCGGTAGAAGCCGCCG) and pYES2-AgeI-R (CGGCGGCTTCTACCGGTTACTAGTGGATCATC) were designed based on the pYES2 plasmid sequence, and the AgeI cut-point was introduced into the pYES2 plasmid by expand-loop PCR to obtain plasmid pYES2-AgeI.
  • primers ADH1-F (TCCACTAGTAACCGGTCTCCCTAACATGTAGG) and ADH1-R (GTCCAGcccAAGCTTAGTTGATTGTATGC) were designed, and the ADH1 promoter gene fragment was amplified by PCR and recovered by gelatinization.
  • the obtained gene fragment AgeI/HindIII was digested, and the purified fragment was recovered, and the fragment was ligated into the pYES2-AgeI corresponding restriction site by adding T4 ligase to obtain a pEZADH1 plasmid.
  • the fragment was ligated into the corresponding pEZADH1 restriction site to obtain a pEZADH1-SUS plasmid, and the plasmid was transformed into yeast BY4742 strain to obtain a recombinant strain EZ-S.
  • the recombinant cells of SUS prepared in Example 7 were ultrasonically disrupted in an ice bath, and the disrupted solution was centrifuged (8,000 rpm, 10 min), and the supernatant was collected and lyophilized for 24 hours to obtain a lyophilized powder of SUS.
  • Method 2 Pick the plate monoclonal to SC-Ura+2% glucose medium, incubate at 30 °C, shaking at 200 rpm for 48 h, inoculate EZ-S strain to 50 ml YPD+1% glucose medium at 2%, 30 °C After incubation for 48 h at 200 rpm, add 20% Triton X-100 (V/V) 500 ul, continue to shake for 2 h, collect the cells by centrifugation (4,000 rpm, 10 min), and use 5 ml of 0.1 mol/L phosphate buffer (pH 7.0). The cells were resuspended to obtain SUS-containing recombinant Saccharomyces cerevisiae permeable cells for catalysis.
  • V/V Triton X-100
  • Example 13 Whole cell synthesis of Reb M using Reb A as a substrate
  • the conversion rate of Reb A is 40% or more.
  • Reb M 0.04 g was obtained, and the purity was more than 99%.
  • the above crude solution was distilled under reduced pressure (40-50 ° C) to about 50 mL of the solution, centrifuged at 9900 rpm for 10 minutes, and the supernatant was discarded.
  • the pellet was washed with 20 mL of water, centrifuged at 9900 rpm for 10 minutes, and the supernatant was discarded.
  • the precipitate was suspended in a 50% aqueous solution of ethanol, heated to 65 ° C to dissolve, and an equal volume of water was added until the ethanol concentration was 25%. After gradually cooling to room temperature, the solid was precipitated, suction filtered and dried in vacuo to give a Reb M sample having a purity of more than 99%.
  • the conversion rate of the substrate is shown in Table 1-2, and the glucose-based donor is UDP-G.
  • Table 1 shows the conversion rate of the substrate during the catalytic preparation of Reb M by UDP-glucosyltransferase gene under the action of pYES2 and Gal promoters and different host bacteria.
  • Table 2 shows the conversion of the substrate during the catalytic preparation of Reb M by the UDP-glucosyltransferase gene under the pYES2 plasmid containing different promoters in CEN.PK2-1C or BY4742.

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Abstract

本发明提供了一种酶法制备瑞鲍迪甙M的方法，其利用含有UDP-葡萄糖基转移酶的重组酿酒酵母或其制备的UDP-葡萄糖基转移酶，催化生成瑞鲍迪甙M。所述重组酿酒酵母是通过转化含有在强启动子控制下的UDP-葡萄糖基转移酶基因的表达载体得到的。

Description

一种利用酿酒酵母酶法制备瑞鲍迪甙M的方法 技术领域
本发明属于生物工程制酶领域，具体涉及一种利用酿酒酵母酶法制备瑞鲍迪甙M的方法。
背景技术
甜味剂是一类广泛应用于食品、饮料及糖果生产的食品添加剂，其既可以在食品的生产过程中添加，也可以在家庭烘焙时经过适当稀释作为蔗糖的替代品使用。甜味剂包括天然甜味剂和人工甜味剂，前者如蔗糖、高果糖玉米糖浆、蜜糖等，后者如阿斯巴甜、糖精等。甜菊糖是一类从植物甜菊中提取出来的天然甜味剂，目前已被广泛用在食品及饮料中。甜菊的提取物中含有包含瑞鲍迪甙在内的多种甜菊糖，天然提取的甜菊糖不同的批次成分差异较大，需要后续的提纯。目前商业化的产品瑞鲍迪甙A包含一些其它的甜菊糖如瑞鲍迪甙C，D及F等。提取的方法制备的甜菊糖通常还有混有一些杂质，有可能对其使用范围造成一定的影响。瑞鲍迪甙M相较于瑞鲍迪甙A具有优势，但其在甜菊叶中的含量极少，且只在甜菊Morita植株中被检测到(2010，J.Appl.Glycosci.,57,199-209)。目前尚未有瑞鲍迪甙M的商业化生产。
中国专利文献CN103397064A公开了一种酶法制备瑞鲍迪甙M的方法，该方法利用大肠杆菌制备的UDP-葡萄糖基转移酶或含有UDP-葡萄糖基转移酶的大肠杆菌，在葡萄糖基供体存在的情况下，催化瑞鲍迪甙A或瑞鲍迪甙D生成瑞鲍迪甙M。上述方法中，所利用的生产UDP-葡萄糖基转移酶的基因工程菌--大肠杆菌并不是安全的菌株(GRAS，general regarded as safe)，其在培养过程中会产生毒素，生产的瑞鲍迪甙M不能直接应用，并且大肠杆菌为原核生物，UDP-葡萄糖基转移酶的基因来自于真核生物，因为真核生物和原核生物的蛋白表达过程的复杂程度不同，用原核细菌表达真核来源的酶，会影响酶的活性。酿酒酵母是公认安全的菌株，但是在酵母菌中表达外源基因的表达量较低。
发明内容
本发明所要解决的技术问题是克服现有技术中含有UDP-葡萄糖基转移酶的大肠杆菌在制备瑞鲍迪甙M的过程中因工程菌自身产生毒素使生产的瑞鲍迪甙M存在安全问题，提供一种通过公认安全菌株酶法制备瑞鲍迪甙M的方法，该方法可以较低的成本，较短的周期生产出高纯度的瑞鲍迪甙M产品。
为解决以上技术问题，本发明采取如下技术方案：
一种利用酿酒酵母酶法制备瑞鲍迪甙M的方法，利用含有UDP-葡萄糖基转移酶的重组酿酒酵母或其制备的UDP-葡萄糖基转移酶，在葡萄糖基供体存在的情况下，催化瑞鲍迪甙A或瑞鲍迪甙D生成瑞鲍迪甙M，所述重组酿酒酵母通过以下方法构建：在质粒中引入强启动子得到载体质粒，将UDP-葡萄糖基转移酶基因通过酶切位点插入到所述载体质粒上置于所述强启动子控制之下得到表达载体，然后转化酿酒酵母，得到重组酿酒酵母。
上述制备瑞鲍迪甙M的方法中，所述UDP-葡萄糖基转移酶为来自甜菊(Stevia rebaudiana)的UGT-A和/或来自水稻(Oryza sativa)的UGT-B。
上述制备瑞鲍迪甙M的方法中，所述UGT-A的氨基酸序列与序列表中SEQ ID NO.1所示氨基酸序列具有至少60％的一致性；所述来自水稻的UGT-B的氨基酸序列与序列表中SEQ ID NO.3所示氨基酸序列具有至少60％的一致性。
上述制备瑞鲍迪甙M的方法中，所述UGT-A的氨基酸序列如序列表中SEQ ID NO.1所示，所述UGT-B的氨基酸序列如序列表中SEQ ID NO.3所示。
上述制备瑞鲍迪甙M的方法中，所述酶切位点为HindIII和XbaI。
上述制备瑞鲍迪甙M的方法中，所述强启动子为ADH2或TEF1。
上述制备瑞鲍迪甙M的方法中，所述质粒为pYES2。
上述制备瑞鲍迪甙M的方法中，所述载体质粒的构建方法为：在质粒内部引入AgeI酶切位点，通过AgeI/HindIII位点引入强启动子。
上述制备瑞鲍迪甙M的方法中，所述酿酒酵母为酿酒酵母BY4742。
上述制备瑞鲍迪甙M的方法中，所述葡萄糖基供体为UDP-葡萄糖或由蔗糖、蔗糖合成酶和UDP组成的UDP-葡萄糖再生体系。
上述制备瑞鲍迪甙M的方法中，将重组酿酒酵母在细胞通透剂作用下形成重组酿酒酵母透性细胞用于催化。
上述制备瑞鲍迪甙M的方法中，将重组酿酒酵母进行培养，于冰浴中超声波破碎，将破碎液离心，收集上清液冻干，获得UGT-A或UGT-B的冻干粉用于催化。
上述制备瑞鲍迪甙M的方法中，所述催化的反应在温度4℃～50℃以及pH5.0～9.0的水相体系中进行。
由于以上技术方案的实施，本发明与已有技术相比具有如下优势：
1、本发明提供的利用酿酒酵母酶法制备瑞鲍迪甙M的方法，通过制备高安全性的重组酿酒酵母制备UDP-葡萄糖基转移酶的进行催化生产，生产的瑞鲍迪甙M安全性高，可以直接作为食品添加剂使用。
2、本发明提供的利用酿酒酵母酶法制备瑞鲍迪甙M的方法，采用酿酒酵母作为工程菌，在质粒pYES2上通过AgeI/HindIII酶切位点引入强启动子ADH2或TEF1，替换pYES2质粒上原有的Gal启动子，通过HindIII和XbaI位点插入来源于真核生物的UDP-葡萄糖基转移酶基因构建表达载体，UDP-葡萄糖基转移酶基因位于强启动子控制下，在酿酒酵母BY4742培养过程中不需要诱导可以直接表达，节省了发酵时间和步骤，并且相对于原核工程菌生产的UDP-葡萄糖基转移酶活性更高，UDP-葡萄糖基转移酶催化底物的转化率最高为90％，经纯化，生产的瑞鲍迪甙M纯度大于99％。
3、本发明提供的利用酿酒酵母酶法制备瑞鲍迪甙M的方法，生产的瑞鲍迪甙M安全性好、纯度高，不用后续处理，直接用于食品添加剂，从而显著缩短了生产周期，提高了产量，降低了成本。
附图说明
下面将通过附图和具体实施方式进行一步说明本发明。
图1为制备的瑞鲍迪甙M的1HNMR图。
具体实施方式
以下瑞鲍迪甙A、瑞鲍迪甙D、瑞鲍迪甙M分别简称Reb A、Reb D和Reb M， 三者的结构式分别参见式I、II和III。
Figure PCTCN2015087751-appb-000001
本发明主要提供四条合成Reb M的路线：
路线一：
Figure PCTCN2015087751-appb-000002
路线二：
Figure PCTCN2015087751-appb-000003
路线三：
Figure PCTCN2015087751-appb-000004
路线四：
Figure PCTCN2015087751-appb-000005
根据本发明，所用的UGT-A或UGT-B可以酶冻干粉形式存在或存在于重组酵母细胞中。
UGT-A或UGT-B的获得方法如下：
利用分子克隆技术、基因工程技术获得UGT-A或UGT-B的重组酿酒酵母表达菌株，然后将重组酿酒酵母发酵，制备得到含有UGT-A或UGT-B的重组细胞，或者制备得到UGT-A或UGT-B的冻干粉。
本发明所述的分子克隆技术和基因工程技术如无特殊说明均是已知的。分子克隆技术可参见《分子克隆实验指南》第三版(J.沙姆布鲁克著,2005)。
采用基因工程技术构建本发明重组菌株的表达步骤如下：
(1)根据UGT-A、UGT-B或SUS的氨基酸序列或者UGT-A、UGT-B或SUS基因的核苷酸序列合成UGT-A、UGT-B或SUS基因片段，连入pUC57载体；
(2)通过PCR、双酶切、连接，将各基因片段插入重组质粒pEZADH2、pEZTEF1、pEZADH1相应的酶切位点中，使各基因置于不同启动子的控制之下；
(3)将表达载体转化进入酿酒酵母BY4742中，得到UGT-A或UGT-B或SUS的重组酿酒酵母表达菌株。
利用含有UGT-A或UGT-B的重组酿酒酵母表达菌株制备含有UGT-A或UGT-B的重组细胞，或者UGT-A或UGT-B的冻干粉。
上述方法的具体步骤见下述实施例。
实施例1制备含UGT-A的重组酿酒酵母细胞
根据pYES2质粒序列，设计引物pYES2-AgeI-F(GATGATCCACTAGTAACCGGTAGAAGCCGCCG)和pYES2-AgeI-R(CGGCGGCTTCTACCGGTTACTAGTGGATCATC)，通过扩环PCR，在pYES2质粒内部引入AgeI切点，得到质粒pYES2-AgeI。
以INVSc2基因组为模板，设计引物ADH2-F(CACTAGTAACCGGTGCAAAACGTAGGGGC)和ADH2-R(GTCCAGCCCAAGCTTGTATTACGATATAG)，PCR扩增得到ADH2启动子基因片段，切胶回收。将得到的基因片段AgeI/HindIII酶切，回收纯化片段，加入T4连接酶将片段连入pYES2-AgeI对应酶切位点，得到pEZADH2质粒。
根据序列表中SEQ ID NO.2所示的核苷酸序列，基因合成UGT-A基因片段，连入pUC57载体获得PUC57-UGT-A(苏州金唯智生物技术有限公司)。以pUC57-UGT-A为模板，PCR获得UGT-A基因，两端分别加上HindIII和XbaI酶切位点，将UGT-A基因片段用限制性内切酶HindIII和XbaI酶切，回收纯化片段，加入T4连接酶将片段连入pEZADH2对应酶切位点，得到pEZADH2-UGT-A质粒，将质粒转化酵母BY4742菌株，得到重组菌EZ-A。
挑取平板单克隆至SC-Ura+2％葡萄糖培养基，30℃，200rpm震荡培养48h，以2％比例将EZ-A菌种接种到50ml YPD+1％葡萄糖培养基，30℃，200rpm震荡培养48h。离心收集细胞(4,000rpm，10min)，用5ml 0.1mol/L磷酸缓冲液(pH7.0)重悬细胞，获得含UGT-A的重组细胞。
实施例2制备UGT-A冻干粉
将实施例1中制得的UGT-A的重组细胞于冰浴中超声波破碎细胞，将破碎液离心(8,000rpm，10min)，收集上清液冻干24h，获得UGT-A的冻干粉。
实施例3制备含UGT-A的重组酿酒酵母透性细胞
方法一：称取实施例1中制得的UGT-A的重组细胞1g湿菌体，重悬至20ml75mmol咪唑-0.1mol/L KCl-10mmol/L MgCl2+1ml甲苯：无水乙醇(v/v)＝1：4，25℃，160rpm摇床震荡15min，离心收集细胞(4,000rpm，10min)，浓缩10 倍重悬到0.1mol/L磷酸缓冲液(pH7.0)，获得UGT-A重组酿酒酵母透性细胞用于催化。
方法二：挑取平板单克隆至SC-Ura+2％葡萄糖培养基，30℃，200rpm震荡培养48h，以2％比例将EZ-A菌种接种到50ml YPD+1％葡萄糖培养基，30℃，200rpm震荡培养48h后，添加20％Triton X-100(V/V)500ul，继续震荡培养2h，离心收集细胞(4,000rpm，10min)，用5ml 0.1mol/L磷酸缓冲液(pH7.0)重悬细胞，获得含UGT-A的重组酿酒酵母透性细胞用于催化。
实施例4制备含UGT-B的重组酿酒酵母细胞
根据pYES2质粒序列，设计引物设计引物pYES2-AgeI-F(GATGATCCACTAGTAACCGGTAGAAGCCGCCG)和pYES2-AgeI-R(CGGCGGCTTCTACCGGTTACTAGTGGATCATC)，通过扩环PCR，在pYES2质粒内部引入AgeI切点，得到质粒pYES2-AgeI。
以INVSc2基因组为模板，设计引物TEF1-F(CCACTAGTAACCGGTCACACACCATAGCTTC)和TEF1-R(GTCCAGCCCAAGCTTTGTAATTAAAACTTAG)，PCR扩增得到TEF1启动子基因片段，切胶回收。将得到的基因片段AgeI/HindIII酶切，回收纯化片段，加入T4连接酶将片段连入pYES2-AgeI对应酶切位点，得到pEZTEF1质粒。
根据序列表中SEQ ID NO.4所示的核苷酸序列，基因合成UGT-B基因片段，连入pUC57载体获得PUC57-UGT-B(苏州金唯智生物技术有限公司)。以pUC57-UGT-B为模板，PCR获得UGT-B基因，两端分别加上HindIII和XbaI酶切位点，将UGT-B基因片段用限制性内切酶HindIII和XbaI酶切，回收纯化片段，加入T4连接酶将片段连入pEZTEF1对应酶切位点，得到pEZTEF1-UGT-B质粒，将质粒转化酵母BY4742菌株，得到重组菌EZ-B。
挑取平板单克隆至SC-Ura+2％葡萄糖培养基，30℃，200rpm震荡培养48h，以2％比例将EZ-B菌种接种到50mlYPD+2％葡萄糖培养基，30℃，200rpm震荡培养48h。离心收集细胞(4,000rpm，10min)，用5ml 0.1mol/L磷酸缓冲液
(pH7.0)重悬细胞，获得含UGT-B的重组细胞。
实施例5制备UGT-B冻干粉
将实施例4中制得的UGT-B的重组细胞于冰浴中超声波破碎细胞，将破碎液离心(8,000rpm，10min)，收集上清液冻干24h，获得UGT-B的冻干粉。
实施例6制备含UGT-B的重组酿酒酵母透性细胞
方法一：称取实施例4中制得的UGT-B的重组细胞将1g湿菌体，重悬至20ml 75mmol咪唑-0.1mol/L KCl-10mmol/L MgCl2+1ml甲苯：无水乙醇(v/v)＝1：4，25℃，160rpm摇床震荡15min，离心收集细胞(4,000rpm，10min)，浓缩10倍重悬到0.1mol/L磷酸缓冲液(pH7.0)，获得UGT-B重组酿酒酵母透性细胞用于催化。
方法二：挑取平板单克隆至SC-Ura+2％葡萄糖培养基，30℃，200rpm震荡培养48h，以2％比例将EZ-B菌种接种到50ml YPD+1％葡萄糖培养基，30℃，200rpm震荡培养48h后，添加20％Triton X-100(V/V)500ul，继续震荡培养2h，离心收集细胞(4,000rpm，10min)，用5ml 0.1mol/L磷酸缓冲液(pH7.0)重悬细胞，获得含UGT-B的重组酿酒酵母透性细胞用于催化。
实施例7制备含SUS的重组酿酒酵母细胞
根据pYES2质粒序列，设计引物pYES2-AgeI-F(GATGATCCACTAGTAACCGGTAGAAGCCGCCG)和pYES2-AgeI-R(CGGCGGCTTCTACCGGTTACTAGTGGATCATC)，通过扩环PCR，在pYES2质粒内部引入AgeI切点，得到质粒pYES2-AgeI。
以INVSc2基因组为模板，设计引物ADH1-F(TCCACTAGTAACCGGTCTCCCTAACATGTAGG)和ADH1-R(GTCCAGcccAAGCTTAGTTGATTGTATGC)，PCR扩增得到ADH1启动子基因片段，切胶回收。将得到的基因片段AgeI/HindIII酶切，回收纯化片段，加入T4连接酶将片段连入pYES2-AgeI对应酶切位点，得到pEZADH1质粒。
根据序列表中SEQ ID NO.5所示的氨基酸序列或SEQ ID NO.6所示的核苷 酸序列，基因合成SUS基因片段，连入pUC57载体获得PUC57-SUS(苏州金唯智生物技术有限公司)。以pUC57-SUS为模板，PCR获得SUS基因，两端分别加上HindIII和XbaI酶切位点，将SUS基因片段用限制性内切酶HindIII和XbaI酶切，回收纯化片段，加入T4连接酶将片段连入pEZADH1对应酶切位点，得到pEZADH1-SUS质粒，将质粒转化酵母BY4742菌株，得到重组菌EZ-S。
挑取平板单克隆至SC-Ura+2％葡萄糖培养基，30℃，200rpm震荡培养48h，以2％比例将EZ-S菌种接种到50mlYPD+2％葡萄糖培养基，30℃，200rpm震荡培养48h。离心收集细胞(4,000rpm，10min)，用5ml 0.1mol/L磷酸缓冲液(pH7.0)重悬细胞，获得含SUS的重组细胞用于催化。
实施例8制备SUS冻干粉
将实施例7中制得的SUS的重组细胞于冰浴中超声波破碎细胞，将破碎液离心(8,000rpm，10min)，收集上清液冻干24h，获得SUS的冻干粉。
实施例9制备含SUS的重组酿酒酵母透性细胞
方法一：称取实施例7中制得的SUS的重组细胞将1g湿菌体，重悬至20ml75mmol咪唑-0.1mol/L KCl-10m mol/L MgCl2+1ml甲苯：无水乙醇(v/v)＝1：4，25℃，160rpm摇床震荡15min，离心收集细胞(4,000rpm，10min)，浓缩10倍重悬到0.1mol/L磷酸缓冲液(pH7.0)，获得SUS重组酿酒酵母透性细胞用于催化。
方法二：挑取平板单克隆至SC-Ura+2％葡萄糖培养基，30℃，200rpm震荡培养48h，以2％比例将EZ-S菌种接种到50ml YPD+1％葡萄糖培养基，30℃，200rpm震荡培养48h后，添加20％Triton X-100(V/V)500ul，继续震荡培养2h，离心收集细胞(4,000rpm，10min)，用5ml 0.1mol/L磷酸缓冲液(pH7.0)重悬细胞，获得含SUS的重组酿酒酵母透性细胞用于催化。
实施例10以Reb D为底物酶法合成Reb M
在反应体系中依次加入100mL 0.1mol/L磷酸缓冲液(pH7.0)，0.224g UDP，34.2g蔗糖，0.2g Reb D，UGT-A冻干粉1g以及SUS冻干粉0.4g，混合均匀 后置于37℃水浴，200rpm搅拌反应18h。反应结束后，取300μl反应液加入600μl 3％甲酸水混匀，10,000rpm离心5min取上清液过滤膜后用高效液相色谱检测(色谱条件：色谱柱：Agilent eclipse sb-C18 4.6X150mm；检测波长：210nm；流动相：甲醇：水＝68％﹕32％；流速：1.0mL/min；柱温：30℃)。Reb D的转化率为90％以上。经树脂分离、结晶等后处理纯化后得到Reb M 0.1g，纯度大于99％，其1HNMR图如图1所示。
实施例11以Reb A为底物酶法合成Reb M
在反应体系中依次加入100mL 0.1mol/L磷酸缓冲液(pH7.0)，0.18g UDP，41.04g蔗糖，0.2g Reb A，UGT-A和UGT-B以及SUS冻干粉分别2g、1g和0.5g，混合均匀后置于37℃水浴，200rpm搅拌反应18h。反应结束后，取300μl反应液加入600μl 3％甲酸水混匀，10,000rpm离心5min取上清液过滤膜后用高效液相色谱检测(色谱条件：色谱柱：Agilent eclipse sb-C18 4.6X150mm；检测波长：210nm；流动相：甲醇：水＝68％﹕32％；流速：1.0mL/min；柱温：30℃)。Reb A的转化率为50％以上。经树脂分离、结晶等后处理纯化后得到Reb M 0.09g，纯度大于99％。
实施例12以Reb D为底物全细胞合成Reb M
在反应体系中依次加入100mL 0.1mol/L磷酸缓冲液(pH7.0)，0.54g UDP-G，0.1g Reb D，UGT-A全细胞1g，混合均匀后置于37℃水浴，200rpm搅拌反应18h。反应结束后，取300μl反应液加入600μl 3％甲酸水混匀，10,000rpm离心5min取上清液过滤膜后用高效液相色谱检测(色谱条件：色谱柱：Agilent eclipse sb-C18 4.6X150mm；检测波长：210nm；流动相：甲醇：水＝68％﹕32％；流速：1.0mL/min；柱温：30℃)。Reb D的转化率为90％以上。经树脂分离、结晶等后处理纯化后得到Reb M 0.09g，纯度大于99％。
实施例13以Reb A为底物全细胞合成Reb M
在反应体系中依次加入100mL 0.1mol/L磷酸缓冲液(pH7.0)，1.08g UDP-G，0.1g Reb A，UGT-A和UGT-B全细胞分别2g和1g，混合均匀后置于37℃水浴，200rpm搅拌反应18h。反应结束后，取300μl反应液加入600μl 3％ 甲酸水混匀，10,000rpm离心5min取上清液过滤膜后用高效液相色谱检测(色谱条件：色谱柱：Agilent eclipse sb-C18 4.6X150mm；检测波长：210nm；流动相：甲醇：水＝68％﹕32％；流速：1.0mL/min；柱温：30℃)。Reb A的转化率为40％以上。经树脂分离、结晶等后处理纯化后得到Reb M 0.04g，纯度大于99％。
实施例14Reb M的分离纯化方法
500ml反应液加1.5L去离子水，55℃加热1小时并超声处理，6700转/分钟离心30分钟，上清液为样品A。离心后沉淀加500ml水，55℃加热0.5小时并超声处理，6700转/分钟离心30分钟，上清液为样品B。将样品A及样品B混合后得到样品C，将样品C用大孔吸附树脂(AB-8)分离。先用水冲洗4个柱体积，再用70％乙醇洗脱3.5个柱体积，得到Reb M粗品溶液。将上述粗品溶液减压蒸馏(40-50℃)至溶液剩余约50mL，9900rpm离心10分钟，弃上清液。沉淀加20mL水洗涤，9900rpm离心10分钟，弃上清液。沉淀用50％乙醇水溶液悬浮，加热至65℃溶解，加入等体积的水至乙醇浓度为25％。逐渐冷却至室温，析出固体后，抽滤并真空干燥，得到纯度大于99％的Reb M样品。
实施例15
UDP-葡萄糖基转移酶基因在不同的启动子的质粒、宿主菌中催化制备RebM过程中，底物的转化率如表1-2所示，葡萄糖基供体为UDP-G。
表1为UDP-葡萄糖基转移酶基因在pYES2、Gal启动子作用下、不同宿主菌中催化制备Reb M过程中底物的转化率
Figure PCTCN2015087751-appb-000006
表2为UDP-葡萄糖基转移酶基因在含有不同启动子的pYES2质粒下、在CEN.PK2-1C或BY4742中催化制备Reb M过程中底物的转化率
Figure PCTCN2015087751-appb-000007
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (13)

  1. 一种利用酿酒酵母酶法制备瑞鲍迪甙M的方法，利用含有UDP-葡萄糖基转移酶的重组酿酒酵母或其制备的UDP-葡萄糖基转移酶，在葡萄糖基供体存在的情况下，催化瑞鲍迪甙A或瑞鲍迪甙D生成瑞鲍迪甙M，其特征在于，所述重组酿酒酵母通过以下方法构建：在质粒中引入强启动子得到载体质粒，将UDP-葡萄糖基转移酶基因通过酶切位点插入到所述载体质粒上置于所述强启动子控制之下得到表达载体，然后转化酿酒酵母，得到重组酿酒酵母。
  2. 根据权利要求1所述的制备瑞鲍迪甙M的方法，其特征在于，所述UDP-葡萄糖基转移酶为来自甜菊的UGT-A和/或来自水稻的UGT-B。
  3. 根据权利要求2所述的制备瑞鲍迪甙M的方法，其特征在于，所述UGT-A的氨基酸序列与序列表中SEQ ID NO.1所示氨基酸序列具有至少60％的一致性；所述来自水稻的UGT-B的氨基酸序列与序列表中SEQ ID NO.3所示氨基酸序列具有至少60％的一致性。
  4. 根据权利要求3所述的制备瑞鲍迪甙M的方法，其特征在于，所述UGT-A的氨基酸序列如序列表中SEQ ID NO.1所示，所述UGT-B的氨基酸序列如序列表中SEQ ID NO.3所示。
  5. 根据权利要求1-4任一项所述的制备瑞鲍迪甙M的方法，其特征在于，所述酶切位点为HindIII和XbaI。
  6. 根据权利要求1-5任一项所述的制备瑞鲍迪甙M的方法，其特征在于，所述强启动子为ADH2或TEF1。
  7. 根据权利要求1-6任一项所述的制备瑞鲍迪甙M的方法，其特征在于，所述质粒为pYES2。
  8. 根据权利要求7所述的制备瑞鲍迪甙M的方法，其特征在于，所述载体质粒的构建方法为：在质粒内部引入AgeI酶切位点，通过AgeI/HindIII位点引入强启动子。
  9. 根据权利要求1-8任一项所述的制备瑞鲍迪甙M的方法，其特征在于，所述酿酒酵母为酿酒酵母BY4742。
  10. 根据权利要求1-9任一项所述的制备瑞鲍迪甙M的方法，其特征在于，所述葡萄糖基供体为UDP-葡萄糖或由蔗糖、蔗糖合成酶和UDP组成的UDP-葡萄糖再生体系。
  11. 根据权利要求1-10任一项所述的制备瑞鲍迪甙M的方法，其特征在于，将重组酿酒酵母在细胞通透剂作用下形成重组酿酒酵母透性细胞用于催化。
  12. 根据权利要求1-11任一项所述的制备瑞鲍迪甙M的方法，其特征在于，将重组酿酒酵母进行培养，于冰浴中超声波破碎，将破碎液离心，收集上清液冻干，获得UGT-A或UGT-B的冻干粉用于催化。
  13. 根据权利要求1-12任一项所述的制备瑞鲍迪甙M的方法，其特征在于，所述催化的反应在温度4℃～50℃以及pH 5.0～9.0的水相体系中进行。
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