CN116590165A - 一株利用木糖生产香叶醇的酿酒酵母菌株及其应用 - Google Patents
一株利用木糖生产香叶醇的酿酒酵母菌株及其应用 Download PDFInfo
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- CN116590165A CN116590165A CN202310836102.6A CN202310836102A CN116590165A CN 116590165 A CN116590165 A CN 116590165A CN 202310836102 A CN202310836102 A CN 202310836102A CN 116590165 A CN116590165 A CN 116590165A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/165—Yeast isolates
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/85—Saccharomyces
- C12R2001/865—Saccharomyces cerevisiae
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Abstract
本发明涉及一株利用木糖生产香叶醇的酿酒酵母菌株及其应用,属于生物工程领域。利用酿酒酵母合成香叶醇,存在GES表达活性较低、前体供应不足以及单萜类化合物对细胞毒性等问题制约香叶醇的高效合成。本发明提供一株利用木糖生产香叶醇的重组酿酒酵母菌株,所述菌株为酿酒酵母MH1,分类命名为酿酒酵母Saccharomyces cerevisiae,已于2023年06月08日保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏地址:北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC No.27584;该菌株在发酵制备香叶醇中有很好的应用。通过发酵测试分析重组酿酒酵母菌株MH1香叶醇产量的影响,结果显示本发明所述利用木糖生产香叶醇的重组酿酒酵母菌株产量为156.65 mg/L。
Description
技术领域
本发明涉及一株利用木糖生产香叶醇的酿酒酵母菌株及其应用,属于生物工程领域。
背景技术
萜类化合物种类繁多,结构复杂,化学合成困难,目前主要从天然植物中直接提取,这不仅造成植物资源的浪费,而且分离纯化产率很低。随着生物合成技术的不断发展,通过改造微生物细胞,使之成为能利用廉价的底物高效合成萜类化合物的细胞工厂,可以大大降低生产成本,具有广泛的应用前景。
单萜类香叶醇,因其具有玫瑰般的香气、有效的药理特性以及高热值、低吸湿性和低挥发性等特点,在化妆品、制药和生物燃料领域中都有着较为广泛的应用。另外,香叶醇作为一些临床抗癌药物的合成前体,在喜树碱、长春新碱等单萜吲哚生物碱的生物合成过程中发挥关键作用。正因为香叶醇的上述用途,使其具有较大的市场应用潜力。因此可利用酿酒酵母内源性的甲羟戊酸(Mevalonate, MVA)途径,以乙酰辅酶A为关键前体合成单萜类前体香叶基焦磷酸(Geranyldiphosphate, GPP),然后引入香叶醇合成酶(Geraniolsynthase, GES)转化GPP合成香叶醇,是一种高效、绿色的生产方式。但是在酿酒酵母中存在GES表达活性较低、前体供应不足以及单萜类化合物对细胞毒性等问题制约香叶醇的高效合成。另外,单萜对细胞的毒性也是限制其产量的重要因素之一。
酿酒酵母(Saccharomyces cerevisiae)作为真核模式生物,具有生长速度快、抑制物耐受性高,优良的工业生产特性以及成熟的遗传操作体系使其成为最具潜力的微生物细胞工厂之一。天然的酿酒酵母存在木糖代谢途径基因,如编码醛糖还原酶(Aldosereductase, GRE3)、山梨醇脱氢酶(Sorbitoldehydrogenase, SOR1)和木酮糖激酶(Xylulosekinase, XKS1)基因。然而,由于这些基因的表达不足并且缺乏把木糖转化为木酮糖的木糖代谢上游途径,使菌株不能有效地利用木糖。这无疑大大限制了酿酒酵母在生物基产品合成领域的应用。在天然木糖利用微生物中共有三种木糖分解代谢途径,包括Dahms或Weimberg途径、木糖-1-磷酸酯(Xylulose-1-phosphate, X-1-P)或核糖-1-磷酸酯(Ribulose-1-phosphate, R-1-P)途径和木糖还原酶XR/木糖醇脱氢酶XDH(XR-XDH)或木糖异构酶(Xyloseisomerase, XI)途径。其中,Dahms或Weimberg途径和X-1-P或R-1-P途径都因途径中酶基因活性低导致过度积累中间代谢物等一些限制因素使得木糖代谢效率低,无法在酿酒酵母中得到良好的运用。XR-XDH或XI途径是在酿酒酵母中研究最广泛的木糖代谢途径,这两条途径虽然路径不同,但目的都是将木糖转化为木酮糖。其中XR-XDH途径大多使用了来源于树干毕赤酵母的XYL1和XYL2基因分别编码的NAD(P)H依赖的XR和NADP+依赖的XDH,能够将木糖还原为木糖醇,然后再被氧化为木酮糖,在这一途径中,XR更倾向于NADPH,而XDH则严格依赖于NAD+,该途径对辅因子依赖性不同,会造成NAD+缺乏或NADPH过量,导致氧化还原代谢失衡,造成木糖醇等中间代谢物的积累,难以提高糖醇转化率。
基于木质纤维素生物质的生物炼制技术的发展,且随着酿酒酵母工程菌株木糖代谢能力的显著提升,以木糖为碳源合成化学品受到越来越多的关注,拓展木糖作为碳源高效合成产物对实现木质纤维素生物转化高效经济生产至关重要。结合上述单萜合成的限制性因素,经检索利用木糖生产香叶醇的酿酒酵母重组菌株的专利及文献还未见报道。
发明内容
针对现有技术单萜合成的限制性因素,基于木质纤维素生物质的生物炼制技术的发展,本发明提供一株利用木糖生产香叶醇的重组酿酒酵母菌株,该菌株在发酵制备香叶醇中有很好的应用。
本发明的技术方案如下:
一株利用木糖生产香叶醇的酿酒酵母菌株,所述菌株为酿酒酵母MH1,分类命名为酿酒酵母Saccharomyces cerevisiae,已于2023年06月08日保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏地址:北京市朝阳区北辰西路1号院3号,保藏编号为CGMCCNo. 27584。
本发明的另一个目的,保护上述利用木糖生产香叶醇的酿酒酵母菌株在发酵生产香叶醇中的应用。
本发明的另一个目的,保护上述利用木糖生产香叶醇的酿酒酵母菌株在发酵生产香叶醇衍生产物中的应用。
本发明所述利用木糖生产香叶醇重组酿酒酵母菌株构建方法概述:将含有香叶醇合成酶和法呢基焦磷酸合成酶突变体融合蛋白基因tVoGES-ERG20 WW 和含有木糖异构酶基因Ru-xylA的表达载体pJX7GE与含有异戊烯基二磷酸异构酶基因IDI1、HMG-CoA还原酶基因tHMG1和甾醇调节转录因子基因UPC2-1表达载体pZMVA4共转化酿酒酵母单倍体出发菌株BSPX051(XK,gre3::PPP,Cox4Δ,AE)中,得到含有两个质粒的重组酿酒酵母菌株MH1,即为利用木糖生产香叶醇的重组酿酒酵母菌株。
上述技术方案较具体的步骤是:构建辅助质粒pJX7-XI,通过Gibson组装的方法将来源于牛瘤胃宏基因组的木糖异构酶基因Ru-xylA克隆到表达载体pJFE3的SbfI/BamHI位点处得到表达载体pJX7-XI,以尿嘧啶为筛选标记;构建辅助质粒pJX7G,通过融合PCR的方法将香叶醇合成酶基因tVoGES与启动子P PGK1 和终止子T CYC1 融合,并通过Gibson组装的方法将融合片段克隆到表达载体pJX7-XI的EcoRI位点处得到表达载体pJX7G,以尿嘧啶为筛选标记;构建质粒pJX7GE,通过融合PCR的方法将香叶醇合成酶和法呢基焦磷酸合成酶突变体融合蛋白基因tVoGES-ERG20 WW 与启动子P PGK1 和终止子T CYC1 融合并克隆到表达载体pJX7-XI的EcoRI位点处得到表达载体pJX7GE,以尿嘧啶为筛选标记;质粒pZMVA4:用于表达香叶醇合成上游途径(即酿酒酵母内源的MVA途径)的关键基因IDI、tHMG1、UPC2-1,以组氨酸为筛选标记;将质粒pJX7GE和质粒pZMVA4共转化酿酒酵母单倍体BSPX051(XK,gre3::PPP,Cox4Δ,AE)菌株(该菌株为申请人实验室保存的酿酒酵母实验室菌株)中,通过SX-Ura-His培养基(酵母基础氮源1.7 g/L,木糖40 g/L,硫酸铵5 g/L,缺尿嘧啶和组氨酸的氨基酸混合物CSM-Ura-His 0.65g/L)筛选得到转化成功的转化子,即为重组酿酒酵母菌株MH1,其基因型为:pJX7GE,pZMVA4。
通过发酵测试分析重组酿酒酵母菌株MH1香叶醇产量的影响,结果显示本发明所述利用木糖生产香叶醇的重组酿酒酵母菌株产量为156.65 mg/L。具体的,好氧摇瓶发酵:20 mL SX-Ura-His培养基中添加4 mL十二烷作为萃取剂,初始接种OD值为0.2,培养108 h,香叶醇产量达到最大值156.65 mg/L。
本发明的有益效果
本发明的重组酿酒酵母菌株能够以木糖为唯一碳源生产香叶醇,对以木质纤维素生物质为原料进行生物精炼,生产单萜类化学品具有指导意义。通过发酵测试分析重组酿酒酵母菌株MH1香叶醇产量的影响,结果显示本发明所述利用木糖生产香叶醇的重组酿酒酵母菌株产量为156.65 mg/L。本发明的技术策略为构建酿酒酵母利用木糖生产合成其它高值单萜类化合物并实现工业化生产打下了坚实的基础。
保藏信息
保藏菌株:酿酒酵母MH1;
保藏时间:2023年06月08日;
分类命名:酿酒酵母Saccharomyces cerevisiae;
保藏单位:中国微生物菌种保藏管理委员会普通微生物中心;
保藏地址:北京市朝阳区北辰西路1号院3号;
保藏编号:CGMCC No. 27584。
附图说明
图1 辅助质粒pJX7-XI的构建流程图;
图2 辅助质粒pJX7G质粒构建流程图;
图3 质粒pJX7GE质粒构建流程图;
图4 香叶醇标准品与对照菌株生产香叶醇GC-MS图谱,其中A为香叶醇标准品GC图谱,B为对照菌株的GC图谱;
图5重组酿酒酵母菌株MH0与菌株MH1生产香叶醇GC-MS图谱,其中A为菌株MH0的GC图谱,B为菌株MH1的GC图谱;
图6菌株MH1的MS图谱;
图7 重组酿酒酵母菌株MH0好氧摇瓶发酵过程中OD值和香叶醇产量的变化;其中A为MH0好氧摇瓶发酵过程中OD值的变化;B为MH0好氧摇瓶发酵过程中香叶醇产量的变化;
图8 重组酿酒酵母菌株MH1好氧摇瓶发酵过程中OD值和香叶醇产量的变化;其中A为MH1好氧摇瓶发酵过程中OD值的变化;B为MH1好氧摇瓶发酵过程中香叶醇产量的变化。
具体实施方式
实施例1微生物材料来源及培养和分子生物学技术方法
(1)培养基
大肠杆菌(Escherichia coli)培养使用的LB培养基:10 g/L蛋白胨,5 g/L酵母提取物,10 g/LNaCl;固体培养基需添加20 g/L琼脂粉;灭菌条件:115 °C,30 min;使用时,添加氨苄青霉素(Amp)至终浓度200 μg/mL用于筛选E. coli转化子。
酿酒酵母培养使用的培养基包括:①YEPD培养基:20 g/L葡萄糖,20 g/L蛋白胨,10 g/L酵母粉;② SX-Ura-His合成培养基:40 g/L木糖,1.7 g/L酵母基础氮源,5 g/L硫酸铵,0.65 g/L缺尿嘧啶和组氨酸的氨基酸混合物;③SX-Ura合成培养基:20 g/L木糖,1.7g/L酵母基础氮源,5 g/L硫酸铵,0.77 g/L缺尿嘧啶的氨基酸混合物。其中,木糖需单独灭菌后添加;固体培养基需添加20 g/L琼脂粉;灭菌条件:115℃,30 min。摇瓶培养需添加20%培养基体积十二烷用于萃取香叶醇(过滤除菌)。
(2)酶与试剂
2×MultiF Seamless Assembly Mix连接酶(ABclonal Technology Co.,Ltd.);Restriction Enzymes, FastDigest enzymes, GeneRuler 1 kb DNA Ladder(ThermoFisher Scientific Inc.);蛋白胨、酵母提取物(购自OXOID公司)和琼脂粉(购自Solarbio公司);木糖(国药化学试剂有限公司);其它化学试剂均为国产分析纯。
E.Z.N.A.® Plasmid Mini Kit I,E.Z.N.A.® Cycle-Pure Kit,E.Z.N.A.®Gel Extraction kit (Omega Bio-Tek Inc.);酵母质粒小提试剂盒(天根生化科技(北京)有限公司);相应的实验操作按产品说明书进行;苯酚氯仿法(25:24:1)提取基因组DNA。
Trans5α Chemically Competent Cell为实验室制备。
PCR使用的2×Rapid Taq Master Mix和Phanta Max Super-Fidelity DNAPolymerase购于南京诺唯赞生物科技股份有限公司。反应体系按说明书进行添加,PCR反应程序分别设置如下:①95°C 3 min,1个循环;95°C 15 s,Tm-5°C 15 s,72°C 1 kb/15s,32个循环;72°C 5 min,1个循环;4°C保温。②95°C 5 min,1个循环;95°C 15 s,55-61°C 15s,72°C 1 kb/30s,30-35个循环;72°C 5 min,1个循环;4°C保温。
(3)引物及测序
引物合成、质粒及DNA片段测序由金唯智生物科技有限公司、青岛蔚来生物科技有限公司、北京擎科生物科技有限公司等公司完成。
(4)酵母转化
酿酒酵母转化通过醋酸锂/聚乙二醇/单链DNA共转化方法(Daniel Gietz&Woods,2002)进行转化。
实施例2质粒pJX7-XI的构建
(1)PCR扩增DNA片段
以下片段通过PCR进行扩增:片段Ru-xylA。
以质粒pJFE-XI为模板,用引物1和引物2扩增片段Ru-xylA。
(3)质粒构建
如图1所示,用BamHI和SbfI双酶切质粒pJFE3(本实验室构建)得到线性化的载体片段,然后采用Gibson组装的方法将片段连接到线性化载体pJFE3上得到重组质粒pJX7-XI;
质粒pJX7-XI中片段Ru-xylA用引物3和引物4通过PCR进行扩增跑电泳验证条带大小,并测序验证序列无碱基突变。
表1 PCR所用引物列表
实施例3质粒pJX7G的构建
(1)PCR扩增DNA片段
通过普通PCR扩增得到:tVoGES,P PGK1 ,T CYC1 片段
以质粒pUC57-tVoGES为模板,用引物5和引物6扩增片段tVoGES;以质粒pIYC04为模板,用引物7和引物8扩增启动子P PGK1 ,用引物9和引物10扩增终止子T CYC1 ;所述引物的序列见表1。
(2)PCR扩增P PGK1 -tVoGES-T CYC1 融合片段
通过融合PCR扩增片段P PGK1 -tVoGES-T CYC1 ;将步骤(1)中得到的DNA片段tVoGES,P PGK1 和T CYC1 同时作为模板,用引物7和引物10进行PCR扩增;上述步骤中所述引物的序列见表1。
(3)质粒的构建
如图2所示,用EcoRI单酶切质粒pJFE-XI得到线性化的载体片段,然后采用Gibson组装的方法将片段连接到线性化载体pJFE-XI上得到重组质粒pJX7G;质粒pJX7G中片段P PGK1 -tVoGES-T CYC1 用引物11和引物12通过PCR进行扩增跑电泳验证条带大小,并测序验证序列无碱基突变。
实施例4质粒pJX7GE的构建
(1)PCR扩增DNA片段
通过普通PCR扩增得到:tVoGES-GGGS-ERG20 WW ,P PGK1 ,T CYC1 片段
以质粒pJFE-tVoGES-ERG20WW为模板,用引物13和引物14扩增片段tVoGES-GGGS-ERG20 WW ;以质粒pIYC04为模板,用引物7和引物8扩增启动子P PGK1 ,用引物9和引物10扩增终止子T CYC1 ;所述引物的序列见表1。
(2)PCR扩增P PGK1 -tVoGES-GGGS-ERG20 WW -T CYC1 融合片段
通过融合PCR扩增片段P PGK1 -tVoGES-T CYC1 ;将步骤(1)中得到的DNA片段tVoGES- GGGS-ERG20 WW ,P PGK1 和T CYC1 同时作为模板,用引物7和引物10进行PCR扩增;上述步骤中所述引物的序列见表1。
(3)质粒的构建
如图3所示,用EcoRI单酶切质粒pJFE-XI得到线性化的载体片段,然后采用Gibson组装的方法将片段连接到线性化载体pJFE-XI上得到重组质粒pJX7GE;质粒pJX7GE中片段P PGK1 -tVoGES-GGGS-ERG20 WW -T CYC1 用引物11和引物12通过PCR进行扩增跑电泳验证条带大小,并测序验证序列无碱基突变。
以上质粒转化大肠杆菌Trans 5α感受态细胞,含有氨苄青霉素钠LB固体平板上筛选得到正确的转化子,然后提取质粒对基因片段进行测序验证。
实施例5菌株MH0的构建
BSPX051(XK,gre3::PPP,Cox4Δ,AE)菌株的制备:采用Journal of Bioscienceand Bioengineering, VOL. 121 No. 2, 160-165, 2016公开的BSPX042,将其敲除KanMX基因即可得到BSPX051。
将实施例2中所得质粒pJX7G(Ura缺陷型)转化进BSPX051(XK,gre3::PPP,Cox4Δ,AE)菌株中,通过SX-Ura固体培养基筛选得到正确的转化子,然后挑取2个单菌落于5 mLSX-Ura液体培养基中,30℃,200 rpm,培养24 h,提取酵母质粒通过PCR验证质粒pJX7G存在,将正确的转化子划线于SX-Ura固体培养基平板,30℃培养两天,待形成明显单菌落放4℃冰箱备用。
实施例6重组酿酒酵母菌株MH1的构建
BSPX051(XK,gre3::PPP,Cox4Δ,AE)菌株的制备同实施例5。
将实施例4中所得质粒pJX7GE(Ura缺陷型)和pZMVA4(His缺陷型)共转化BSPX051(XK,gre3::PPP,Cox4Δ,AE),得到酿酒酵母MH1,通过SX-Ura-His固体培养基筛选得到正确的转化子,然后挑取2个单菌落于5 mL SX-Ura-His液体培养基中,30℃,200 rpm,培养24h,提取酵母质粒通过PCR验证两个质粒存在,将正确的转化子划线于SX-Ura-His固体培养基平板,30℃培养两天,待形成明显单菌落放4℃冰箱备用。
实施例7香叶醇产量检测
待测菌株单菌落接种到5 mLSX-Ura-His/SX-Ura液体培养基,30℃,200 rpm培养24 h,再转接至10 mL SX-Ura-His/SX-Ura液体培养基二次活化12 h。将活化菌种接种至盛有20 mL SX-Ura-His/SX-Ura液体培养基的100 mL的三角瓶中,加入20%体积(4mL)十二烷,初始接种OD值为0.2,棉塞封口,30℃,200 rpm 培养60 h/72 h/84 h/96 h,取十二烷层13000 rpm离心10 min,转移至气相小瓶,-20℃保存。气相色谱质谱联用仪(GC-MS)检测香叶醇,根据标准曲线计算样品的香叶醇含量。
1、标准曲线的制备:5 mL容量瓶加入4 mL十二烷,再精确称取6 mg香叶醇溶于十二烷,十二烷定容至5 mL,充分混匀后制成1.20 mg/mL香叶醇标准品母液;然后,5倍梯度稀释得到浓度分别为0.24、0.048、0.0096、0.00192 mg/mL的香叶醇标准品,进行GC-MS或GC/FID检测,根据不同浓度标准品的峰面积与对应浓度绘制标准曲线,R2≥0.99;
2、GC-MS检测方法:以安捷伦GC-MS系统定性检测香叶醇标准品和样品,及定量检测实验样品;GC-MS色谱条件:使用DB-FFAP色谱柱(规格为30 m×0.25 mm×0.25 µm);样品进样体积为1 µL,流速为0.78 mL/min,分流比设定为10:1;柱温箱初始温度为60℃,进样口温度设置为260℃,检测器温度设置为280℃;程序升温条件:仪器在60℃的初始温度维持2min,然后以10 ℃/min的速率升温至150℃,保持5 min,分析时间为29 min,载气为氦气。
结果如图4、5、6所示,图4中A为香叶醇标准品GC图谱,图4中B为对照菌株的GC图谱,图5中A为菌株MH0的GC图谱,图5中B为菌株MH1的GC图谱;图6为菌株MH1的MS图谱,通过与香叶醇标准品的GC图谱对比,以及对菌株MH1进行MS质谱检测,据此确定了检测样品在12.841 min为香叶醇产物的出峰时间。通过绘制标准曲线,对峰面积进行积分,进一步进行定量分析,测定菌株的香叶醇产量。
实施例8重组酿酒酵母菌株MH0好氧摇瓶发酵
使用SX-Ura培养基(酵母基础氮源1.7 g/L,木糖20 g/L,硫酸铵5 g/L,缺尿嘧啶的氨基酸混合物CSM-Ura 0.77g/L)在好氧摇瓶发酵条件下检测重组酿酒酵母菌株MH0生产香叶醇的能力,并以转入pJFE3质粒的菌株作为对照。菌株活化如实施例5中所述,发酵条件如下所述:培养温度为30°C,100 mL摇瓶中装有20 mL SX-Ura培养基,摇床转速为200 rpm,棉塞封口,初始接种OD值为0.2,加入20%体积十二烷萃取香叶醇,培养84 h。发酵过程中分别在60 h、72 h和84 h取十二烷层200 μL,按照实施例7中描述的GC-MS方法定性香叶醇的存在并定量香叶醇的产量。发酵实验重复三次,数据取平均值进行计算。菌株MH0发酵过程中香叶醇的积累量变化(图7中A)和菌株生长情况(图7中B)如图7所示,以20g/L木糖为碳源进行发酵培养时,对照菌株发酵代谢物没有检测到香叶醇的存在,而菌株MH0检测到香叶醇的存在,并且产量随着培养时间的延长而增加。菌株MH0在72 h达到最大生长点,并且香叶醇产量也在72 h达到最大值,达到12.72 mg/L,84 h以后香叶醇的产量开始下降。证明了在出发菌株BSPX051(XK,gre3::PPP,Cox4Δ,AE)中超表达香叶醇合成酶基因tVoGES和木糖异构酶基因Ru-xylA获得MH0菌株有较好的木糖代谢能力,以及能够以木糖为唯一碳源合成香叶醇。
实施例9重组酿酒酵母菌株MH1好氧摇瓶发酵
使用SX-Ura-His培养基(酵母基础氮源1.7 g/L,木糖40 g/L,硫酸铵5 g/L,缺尿嘧啶和组氨酸的氨基酸混合物CSM-Ura-His 0.65g/L)在好氧摇瓶发酵条件下检测重组酿酒酵母菌株MH1生产香叶醇的能力,并以含有pJFE3和pIYC04质粒的菌株作为对照。菌株活化如实施例6中所述,发酵条件如下所述:培养温度为30 °C,100 mL摇瓶中装有20 mL SX-Ura-His/SX-Ura培养基,摇床转速为200 rpm,棉塞封口,初始接种OD值为0.2,加入20%体积十二烷萃取香叶醇,培养96 h。发酵过程中分别在60 h、72 h、84 h和96 h取十二烷层200 μL,按照实施例7中描述的GC-MS方法定性香叶醇的存在并定量香叶醇的产量。发酵实验重复三次,数据取平均值进行计算。菌株MH1发酵过程中香叶醇的积累量变化(图8中B)和菌株生长情况(图8中A)如图8所示,以40 g/L木糖为碳源进行发酵培养时,对照菌株发酵代谢物没有检测到香叶醇的存在,而菌株MH1检测到香叶醇的存在,并且产量随着培养时间的延长而增加,在84 h达到最大值,达到156.65 mg/L,96 h以后香叶醇的产量开始下降。本发明的菌株MH1摇瓶发酵的香叶醇产量较实施例8中重组酿酒酵母菌株MH0提高了11倍,说明菌株MH1在木糖为唯一碳源下具有较强的香叶醇生产能力。
Claims (3)
1.一株利用木糖生产香叶醇的酿酒酵母菌株,其特征在于,所述菌株为酿酒酵母MH1,分类命名为酿酒酵母 Saccharomyces cerevisiae,已于2023年06月08日保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏地址:北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC No. 27584。
2.权利要求1所述的利用木糖生产香叶醇的酿酒酵母菌株在发酵生产香叶醇中的应用。
3.权利要求1所述的利用木糖生产香叶醇的酿酒酵母菌株在发酵生产香叶醇衍生产物中的应用。
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