CN113444701B - 酿酒酵母内源角鲨烯单加氧酶突变体及应用 - Google Patents
酿酒酵母内源角鲨烯单加氧酶突变体及应用 Download PDFInfo
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- CN113444701B CN113444701B CN202110732545.1A CN202110732545A CN113444701B CN 113444701 B CN113444701 B CN 113444701B CN 202110732545 A CN202110732545 A CN 202110732545A CN 113444701 B CN113444701 B CN 113444701B
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Abstract
本发明公开了酿酒酵母内源角鲨烯单加氧酶突变体及应用,属于基因工程和酶工程技术领域。本发明提供的酿酒酵母角鲨烯单加氧酶突变体G27S、G30S、G25S、Y90F、Y90A、Q63N、C416A、I483A、G27S/Y90A、G27S/I483A、Y90A/I483A、G27S/Y90A/I483A的酶活性比野生型均有不同程度的降低,表达上述突变体的酿酒酵母胞内角鲨烯产量可提高了40%以上,角鲨烯积累量可高达6.82g/L,有利于扩大其工业化应用范围,实现大规模的安全生产。
Description
技术领域
本发明涉及酿酒酵母内源角鲨烯单加氧酶突变体及应用,属于基因工程和酶工程技术领域。
背景技术
角鲨烯(squalene)是一种重要的萜类化合物,它是许多生物活性化合物的前体,如类固醇和藿烯等。角鲨烯本身具有较高的应用价值,可用于化妆品行业作为润肤霜。角鲨烯对人体健康也有益,具有包括抗肿瘤、抗真菌/真菌,增强机体免疫能力,降低胆固醇等作用。另外,角鲨烯还有一种重要的用途是制作稳定的乳化液,例如疫苗、药物及其他的药用底物。目前,角鲨烯传统的生产方法主要通过非法手段从鲨鱼肝提取或低效的从植物中提取,但鉴于其来源有限,价格昂贵,利用微生物生产角鲨烯具有可持续性,环境友好等的优点,故与传统角鲨烯生产相比采用微生物发酵法生产角鲨烯具有很好的前景。
角鲨烯的合成从乙酰辅酶A开始,首先通过HMG-CoA合成酶催化生成3-羟基-3-甲基戊二酸单酰辅酶A(3-hydroxy-3-methyl glutaryl coenzyme A,HMG-CoA),接下来HMG-CoA还原酶(HMGR)催化HMG-CoA生成甲羟戊酸(Mevalonate,MVA),是MVA途径中第一个限速酶,IDI1编码的异戊烯焦磷酸酯异构酶催化异戊烯二磷酸(IPP)形成烯丙基二磷酸(DMAPP),其与HMG1是细胞质萜类化合物的代谢中的重要调控点。接下来通过一系列Erg蛋白酶催化,角鲨烯合成酶(Erg9p)将法尼烯基焦磷酸(FPP)催化形成角鲨烯。角鲨烯单加氧酶Erg1催化2,3-环氧角鲨烯的形成。天然酿酒酵母MVA途径中角鲨烯几乎不积累。现有技术中有文献记载了对酿酒酵母进行基因工程改造提高角鲨烯产量的方案,但仍存在发酵周期长、产量有限等问题。
目前,角鲨烯主要以微生物发酵的方式生产。酿酒酵母菌Saccharomycescerevisiae是最主要的生产菌株,针对酿酒酵母菌生产角鲨烯的研究主要集中在提高角鲨烯产量和产率等方面。角鲨烯单加氧酶是催化角鲨烯生成环氧角鲨烯的关键酶,研究人员发现特比萘芬能有效抑制角鲨烯单加氧酶的催化活性,而特比萘芬作为抗生素添加为角鲨烯的工业化生产应用带来困难。如何选择合适的方式对角鲨烯单加氧酶进行改性,获得理想的角鲨烯单加氧酶活性,在保持低活性的基础上不影响酵母细胞生长,从而扩大其工业应用是目前亟待解决的问题。
已有研究对人源的角鲨烯单加氧酶进行蛋白质结构的解析,并对不同来源的角鲨烯单加氧酶进行蛋白质序列的比对,但目前还没有报道对酿酒酵母来源的ERG1的蛋白质结构及功能研究。因此,筛选具有低角鲨烯催化活性的角鲨烯单加氧酶对于改造高产角鲨烯的酿酒酵母菌株具有重要的意义。
发明内容
针对目前存在的问题,本发明利用基因工程和酶工程的手段,降低角鲨烯单加氧酶的活性,提高角鲨烯的产量,为其工业化生产创造了条件。
本发明提供了如下(a)或(b)的角鲨烯单加氧酶突变体:
(a)在SEQ ID NO.1的氨基酸基础上,第25位、27位、30位、63位、90位、416位或483位进行了一个或多个突变的角鲨烯单加氧酶突变体,
(b)在(a)中的氨基酸序列经过取代、缺失或添加一个或几个氨基酸且具有角鲨烯单加氧酶活性的由(a)衍生的蛋白质。
在一种实施方式中,所述亲本的氨基酸序列如SEQ ID NO.1所示。
在一种实施方式中,所述S.cerevisiae S288C来源的角鲨烯单加氧酶的编码基因的核苷酸序列如SEQ ID NO.2所示。
在一种实施方式中,所述突变体是将亲本第25位甘氨酸(Gly)突变为丝氨酸(Ser),获得的突变体命名为G25S。
在一种实施方式中,所述突变体是将亲本第27位甘氨酸(Gly)突变为丝氨酸(Ser),获得的突变体命名为G27S。
在一种实施方式中,所述突变体是将亲本第30位甘氨酸(Gly)突变为丝氨酸(Ser),获得的突变体命名为G30S。
在一种实施方式中,所述突变体是将亲本第90位酪氨酸(Tyr)突变为苯丙氨酸(Phe),获得的突变体命名为Y90F。
在一种实施方式中,所述突变体是将亲本第90位酪氨酸(Tyr)突变为丙氨酸(Ala),获得的突变体命名为Y90A。
在一种实施方式中,所述突变体是将亲本第63位谷氨酰胺(Gln)突变为天冬酰胺(Asn),获得的突变体命名为Q63N。
在一种实施方式中,所述突变体是将亲本第416位半胱氨酸(Cys)突变为丙氨酸(Ala),获得的突变体命名为C416A。
在一种实施方式中,所述突变体是将亲本第483位异亮氨酸(Ile)突变为丙氨酸(Ala),获得的突变体命名为I483A。
在一种实施方式中,所述突变体是将亲本第27位甘氨酸(Gly)突变为丝氨酸(Ser),并将第90位酪氨酸(Tyr)突变为丙氨酸(Ala),获得的突变体命名为G27S/Y90A。
在一种实施方式中,所述突变体是将亲本第27位甘氨酸(Gly)突变为丝氨酸(Ser),并将第483位异亮氨酸(Ile)突变为丙氨酸(Ala),获得的突变体命名为G27S/I483A。
在一种实施方式中,所述突变体是将亲本第90位酪氨酸(Tyr)突变为丙氨酸(Ala),并将第483位异亮氨酸(Ile)突变为丙氨酸(Ala),获得的突变体命名为Y90A/I483A。
在一种实施方式中,所述突变体是将亲本第27位甘氨酸(Gly)突变为丝氨酸(Ser),并将亲本第90位酪氨酸(Tyr)突变为丙氨酸(Ala),并将第483位异亮氨酸(Ile)突变为丙氨酸(Ala),获得的突变体命名为G27S/Y90A/I483A。
本发明提供了编码所述角鲨烯单加氧酶突变体的基因。
在一种实施方式中,所述基因是在SEQ ID NO.2所示核苷酸序列的基础上,引入对突变位点编码突变后的氨基酸的密码子。
本发明提供了携带所述基因的表达载体。
在一种实施方式中,所述表达载体包括但不限于pET系列、Duet系列、pGEX系列、pHY300、pHY300PLK、pPIC3K或pPIC9K系列。
在一种实施方式中,所述表达载体包括pET-28a。
本发明提供了表达所述酿酒酵母角鲨烯单加氧酶突变体,或含有编码所述酿酒酵母角鲨烯单加氧酶的基因的微生物细胞。
在一种实施方式中,所述微生物细胞以大肠杆菌为宿主。
在一种实施方式中,所述大肠杆菌包括但不限于E.coli BL21(DE3)系列。
在一种实施方式中,所述微生物细胞以酿酒酵母为宿主。
在一种实施方式中,所述宿主包括但不限于CENPK系列的细胞,例如酿酒酵母CENPK2-1C、酿酒酵母CENPK2-1D,酿酒酵母BY4741、酿酒酵母BY4742,酿酒酵母C800,酿酒酵母CP08。
在一种实施方式中,所述酿酒酵母基因组上整合了所述基因。
本发明提供了一种体外生产酿酒酵母角鲨烯单加氧酶的方法,所述方法是将所述微生物细胞在含有卡纳抗生素的LB培养基中培养8-10h,获得种子液;以1%的转接量转接到TB培养基中37℃培养2-4h至OD600=0.6~0.8,加入终浓度为0.1~0.5mM的IPTG诱导20h,将细胞破碎后离心取上清,即得到角鲨烯单加氧酶液。
在一种实施方式中,将破碎细胞得到的上清纯化后加入含有100μM Tris-HCl,100μMFAD;3mM NADPH;1mM EDTA;1mM AMO-1618的反应体系中。
在一种实施方式中,所述角鲨烯单加氧酶在反应体系中的浓度为6g/L。
在一种实施方式中,反应体系pH为7.5,在30℃水浴摇床中,搅拌转速50~100r/min,预孵化10min,添加角鲨烯开始反应。
在一种实施方式中,添加0.6ml含15%KOH和0.2%焦棓酸90%的乙醇终止催化反应。
在一种实施方式中,通过酿酒酵母基因组中角鲨烯单加氧酶点突变测定突变体对胞内角鲨烯积累的影响。
本发明提供了所述角鲨烯单加氧酶,或所述基因,或所述微生物细胞在生产角鲨烯中的应用。
在一种实施方式中,所述应用是将表达所述突变体的酿酒酵母用于发酵生产角鲨烯。
在一种实施方式中,所述应用是将所述酿酒酵母接种至培养基中,于25~35℃发酵至少48h。
在一种实施方式中,所述应用是将酿酒酵母在YPD培养基中于28~32℃培养至少48h,并在第12h后补料。
在一种实施方式中,用于补料的培养基含有:葡萄糖、(NH4)2SO4、KH2PO4、MgSO4·7H2O、K2SO4、Na2SO4和微量元素。
在一种实施方式中,所述微量元素包括金属离子和维生素。
在一种实施方式中,所述微量元素包括金属盐溶液和维生素溶液;所述金属盐溶液含有Zn2+、Mn2+、Co2+、Ca2+和Fe2+;所述维生素溶液包括生物素、泛酸钙、烟酸、肌醇、硫胺素和吡哆醛。
在一种实施方式中,所述补料具体为:第12~24h以4mL/h的流速补料,第25~48h以12mL/h的流速补料,第49~84h以4mL/h的流速补料。
在一种实施方式中,所述方法还包括破碎细胞,收集细胞内的角鲨烯。
有益效果:
本发明提供了酿酒酵母角鲨烯单加氧酶酶活降低的突变体,以角鲨烯为底物,突变体G25S、G27S、G30S、Q63N、Y90F、Y90A、C416A、I483A、G27S/Y90A、G27S/I483A、Y90A/I483A、G27S/Y90A/I483A的角鲨烯单加氧酶活性比野生型分别降低了22%、16%、5%、10%、42%、9%、4%、33%、58%、43%、61%、68%,在具有角鲨烯合成能力的酿酒酵母中表达上述突变体,可使酿酒酵母体内角鲨烯的产量提高146mg/L、109g/L、17mg/L、32mg/L、333mg/L、58mg/L、24mg/L、211mg/L,383mg/L、311mg/L、407mg/L、457mg/L,提高比例分别为45.3%、33.9%、5.3%、9.9%、103.4%、18.0%、7.5%、65.5%、118.9%、96.6%、126.4%、141.9%。
本发明提供的5株表达角鲨烯单加氧酶突变体Y90A、G27S/Y90A、G27S/I483A、Y90A/I483A、G27S/Y90A/I483A的酿酒酵母菌株相比野生型菌株,在摇瓶水平的角鲨烯产量分别提高了648mg/L、1004mg/L、596mg/L、1184mg/L、1289mg/L,提高比例分别为44.8%、67.5%、40.1%、79.6%、86.6%。
本发明提供的表达突变体G27S/Y90A/I483A的酿酒酵母在5L发酵罐上发酵120h后,胞内角鲨烯的产量达6.82g/L,相比突变前野生型角鲨烯单加氧酶菌株,产量提高了2.31g/L,提高比例为51.2%,且生产过程不涉及危害食品安全的物质,有助于实现大规模的安全生产。
附图说明
图1为不同角鲨烯突变体的相对酶活及表达该突变体的酿酒酵母角鲨烯产量。
具体实施方式
LB培养基(每L):酵母粉5g,蛋白胨10g,氯化钠10g,1L去离子水灭菌,121℃,20min。
TB培养基(每L):酵母粉24g,蛋白胨12g,KH2PO4 2.31g,K2HPO4 12.54g,甘油4mL。
角鲨烯单加氧酶突变体的体外酶活测定:
(1)500μL的反应体系中包含有300μL的缓冲液,100μL纯化酶液(6g/L),100μMTris-HCl,100μM FAD;3mM NADPH;1mM EDTA;1mM AMO-1618;
(2)将反应体系在水浴锅中30℃水浴预孵化10min,添加100μL纯度为98%的角鲨烯开始反应,反应30min,反应每组三个平行。
(3)添加0.6ml含15%KOH、0.2%焦棓酸和90%的乙醇终止反应。
(4)向反应体系中加入600μL乙酸乙酯,震荡混匀,12000r/min,离心5min,使乙酸乙酯相和水相分层。
(5)取适量上层清液用针式过滤器过滤,并转移至液相小瓶中,待进行液相色谱检测。
(6)在30℃的条件下进行酶催化反应,本实施例中纯化后角鲨烯单加氧酶酶活为32pmol/mg/min。
角鲨烯的测定方法:
使用岛津液相检测,使用C18柱(30cm×0.25mm,0.25μm),紫外检测波长195nm,流动相纯乙腈,进样量3μL,等度洗脱,流速1.6mL/min,分析时间15min,出峰时间13.8min。
实施例1:酿酒酵母来源的角鲨烯单加氧酶的表达
以pET-28a为模版,用引物pET28a-F/pET28a-R扩增得到载体片段;以酿酒酵母S288C基因组为模板,用引物ERG1-F/ERG1-R进行PCR扩增得到基因片段(核苷酸序列如SEQID NO.2所示),经过琼脂糖凝胶检测后,进行回收纯化,纯化之后的片段通过同源重组克隆到载体片段中,将重组表达载体转化至E.coli JM109,通过DNA测序(Sangon,China)证实表达质粒pET-28a-ERG1正确构建,将成功构建的质粒pET-28a-ERG1转化到E.coli BL21(DE3)。
表1本实施例所需的引物序列
PCR反应体系均为:正向引物(10μM)1μL,反向引物(10μM)1μL,模板DNA 1μL,2×Phanta Max Master Mix 25μL,加入双蒸水至50μL。
PCR扩增条件为:95℃预变性3min;随后25个循环(95℃15s,55℃5s,72℃15s);72℃继续延伸10min。
将测序正确的重组质粒转入大肠杆菌E.coli BL21(DE3)感受态中37℃培养12h.挑取单菌落接种于5mL的LB培养基中,加入终浓度为50μg/mL的kana抗生素,37℃、220r/min下恒温摇床振荡培养8-10h,将种子接种于50mL的终浓度为50μg/mL的kana抗生素的TB培养基中,37℃、220r/min下恒温摇床振荡培养到OD600至0.6-0.8之间,加入终浓度为0.5mM的IPTG(异丙基硫代半乳糖苷,Isopropylβ-D-Thiogalactoside),20℃、220r/min诱导20h,离心收集细胞(7000r/min,5min),用PBS缓冲液洗涤菌体2次,高压均质机破碎细胞,得到利用蛋白纯化仪和镍柱纯化蛋白,90%A液,10%B液洗脱,测定得到蛋白浓度3mg/mL,测定纯酶液酶活(0.12U/g),4℃下保存备用。
实施例2:体外角鲨烯单加氧酶突变体的制备及表达
(1)体外角鲨烯单加氧酶突变体的构建
利用快速PCR技术,根据实施例1所扩增得到的基因序列(核苷酸序列如SEQ IDNO.1所示),分别设计并合成引入G27S、G30S、G25S、Y90F、Y90A、Q63N、C416A、I483A的突变的引物对角鲨烯单加氧酶基因进行定点突变(突变核苷酸用下划线标注)。
引入序列G27S突变的定点突变引物为:
正向引物:5’-ATGCGATTGTCATCGGTGCTTCTGTTATCGGTCCATGTGTTGC-3’,
反向引物:5’-AGCACCGATGACAATCGCAT-3’;
引入序列如G30S突变的定点突变引物为:
正向引物:5’-ATCGGTGCTGGTGTTATCTCTCCATGTGTTGCT-3’,
反向引物:5’-GATAACACCAGCACCGATGACAATCG-3’;
引入序列如G25S突变的定点突变引物为:
正向引物:5’-ACCTACGATGCGATTGTCATCTCTGCTGGTGTTATCG-3’,
反向引物:5’-GATGACAATCGCATCGTAGGTAATTGTGT-3’;
引入序列如Y90F突变的定点突变引物为:
正向引物:5’-CGAAGCATATCCTGTTACCGGTTTTACCGTCTTTTTCAAC-3’,
反向引物:5’-ACCGGTAACAGGATATGCTTCGAT-3’;
引入序列如Y90A突变的定点突变引物为:
正向引物:5’-CGAAGCATATCCTGTTACCGGTGCTACCGTCTTTTTCAAC-3’,
反向引物:5’-ACCGGTAACAGGATATGCTTCGAT-3’。
引入序列如Q63N突变的定点突变引物为:
正向引物:5’-AGAATTGTTGGTGAATTGATGAATCCAGGTGGTGTTAG-3’,
反向引物:5’-CATCAATTCACCAACAATTCTATCAGGCATAGC-3’。
引入序列如C416A突变的定点突变引物为:
正向引物:5’-CTTGAAGGCATTACAAAAAGGTGCTTTCAAATATTTCC-3’,
反向引物:5’-ACCTTTTTGTAATGCCTTCAAGTTATCGCT-3’。
引入序列如I483A突变的定点突变引物为:
正向引物:5’-TTATGATTTTGATCACAGCTGCTAGAGTATTCACCCCA-3’,
反向引物:5’-AGCTGTGATCAAAATCATAATACCTTCCAATAAAGC-3’。
PCR反应体系均为:正向引物(10μM)1μL,反向引物(10μM)1μL,模板DNA 1μL,2×Phanta Max Master Mix 25μL,加入双蒸水至50μL。
PCR扩增条件为:95℃预变性3min;随后25个循环(95℃15s,55℃5s,72℃15s);72℃继续延伸10min。
PCR产物经验证正确后,用DpnⅠ消化后转化至大肠杆菌JM109感受态,将感受态细胞在LB固体培养基(含50mg/L kana)培养过夜后,挑克隆于含50mg/L kana的LB液体培养基培养后提取质粒,将突变质粒转化表达宿主大肠杆菌BL21(DE3)感受态细胞,所有突变质粒均测序正确。获得重组菌株,分别命名为G27S、G30S、G25S、Y90F、Y90A、Q63N、C416A、I483A。
(2)突变体酶的表达与纯化
将步骤(1)筛选获得的阳性克隆挑取单菌落接种于装有5mL LB培养基的试管中,37℃、220r/min培养8-10h,获得种子液;以2%(1mL/50mL)接种量将种子液转接至50mL的终浓度为50μg/mL的kana抗生素的TB培养基中,37℃、220r/min下恒温摇床振荡培养到OD600至0.6-0.8之间,加入终浓度为0.5mM的IPTG(异丙基硫代半乳糖苷,Isopropylβ-D-Thiogalactoside),20℃,220r/min诱导20h。离心收集细胞(7000r/min,5min),用PBS缓冲液洗涤菌体2次,高压均质机破碎细胞,利用蛋白纯化仪和镍柱纯化蛋白,90%A液,10%B液洗脱,4℃下保存备用。
(3)酶活测定:检测突变体的酶活,表2的结果表明各个突变体的酶活均低于野生型,表达G27S、G30S、G25S、Y90F、Y90A、Q63N、C416A、I483A的重组大肠杆菌的体外酶活分别降低了22%、16%、5%、10%、42%、9%、4%、33%,显示表达上述突变体的酿酒酵母对角鲨烯的积累的能力增加。
表2野生型酿酒酵母角鲨烯单加氧酶和突变体酶的相对酶活
实施例3:多突变体的制备和表达及酶的性能分析
分别以实施例2构建的携带突变体G27S、Y90A、I483A基因的质粒作为模板,并根据实施例2设计的定点突变的引物,使用快速PCR技术,对携带编码突变体G27S、Y90A、I483A的基因的质粒进行定点突变,构建多突变体G27S/Y90A、G27S/I483A、Y90A/I483A、G27S/Y90A/I483A。
分别测序确认酿酒酵母角鲨烯单加氧酶多突变体的编码基因是否正确,并将测序结果正确的质粒导入大肠杆菌中进行表达,得到表达角鲨烯单加氧酶多突变的大肠杆菌。按照实施例2和3中的方法,检测突变体的酶活及角鲨烯的产量。
野生型酿酒酵母角鲨烯单加氧酶(WT)和突变体体外酶活检测结果于表2,结果表明,各个突变体的酶活均低于野生型,分别降低了58%、43%、61%、68%。
实施例4:表达突变体的重组酿酒酵母的构建
分别以实施例2和实施例3构建的携带突变体G27S、G30S、G25S、Y90F、Y90A、Q63N、C416A、I483A、G27S/Y90A、G27S/I483A、Y90A/I483A、G27S/Y90A/I483A基因的质粒作为模板,并利用实施例1中的引物ERG1-F/ERG1-R使用快速PCR技术,对ERG1突变体的基因片段进行扩增,得到突变体片段G27S、G30S、G25S、Y90F、Y90A、Q63N、C416A、I483A、G27S/Y90A、G27S/I483A、Y90A/I483A、G27S/Y90A/I483A,分别用设计带有整合同源臂的引物mutant-F/mutant-R对获得的突变片段进行扩增,得到整合用片段arm-G27S、arm-G30S、arm-G25S、arm-Y90F、arm-Y90A、arm-Q63N、arm-C416A、arm-I483A、arm-G27S/Y90A、arm-G27S/I483A、arm-Y90A/I483A、arm-G27S/Y90A/I483A用于酵母基因组整合。
mutant-F:CAATACAGGTTATTTCGAACAATTGAAAAAAAAAAATCACAGAAAAACATATCGAGAAAAGGGTCATGTCTGCTGTTAACGTTGCACCT;
mutant-R:AAAAAAAAAAGGTGCAGCTTAATGTTTGACGGTTCCTATCCTCTCTCCCTTATAAGCTGTAGCTACATAAGAACACCTTTGGTGGAGGG
用于基因组整合的片段1μg、筛选标记片段1μg、sgRNA500ng一同利用酵母转化试剂盒Frozen-EZ Yeast Transformation II转化至具有合成角鲨烯能力的酿酒酵母中,包括但不限于酿酒酵母CENPK2-1C、酿酒酵母CENPK2-1D、酿酒酵母BY4741、酿酒酵母BY4742。以本研究室保藏的一株生产角鲨烯的酿酒酵母CP08(菌株基因型列于表3)为例。将转化至CP08细胞后的重组酿酒酵母细胞涂布于SD-LEU-TRP筛选固体培养基上,30℃培养72h,分别得到酿酒酵母突变体菌株G27S、G30S、G25S、Y90F、Y90A、Q63N、C416A、I483A、G27S/Y90A、G27S/I483A、Y90A/I483A、G27S/Y90A/I483A挑取突变体菌株单菌落于含有YPD培养基的24孔板中,30℃培养96h,进行胞内角鲨烯含量检测。
胞内角鲨烯含量检测方法:取500μL菌液,离心去上清,加入破碎珠和1mL丙酮利用fastprep破碎后使用高效液相色谱法检测。
表3本发明中涉及的菌株
注:C800菌株公开于论文《Promoter-Library-Based Pathway Optimization forEfficient(2S)-Naringenin Production from p-Coumaric Acid in Saccharomycescerevisiae》;CP02是在C800的基础上在基因组上ARO10处整合了tHMG1和IDI1;CP08是在CP02的基础上在基因组上EXG1处整合了tHMG1和IDI1;tHMG1的核苷酸序列如SEQ ID NO.3所示;IDI1的核苷酸序列如SEQ ID NO.4所示。
检测结果显示,在酵母基因组上整合角鲨烯单加氧酶突变基因可以促进角鲨烯的积累能力增加,角鲨烯的产量提高,体内角鲨烯生产能力列于表4。相比于野生型角鲨烯单加氧酶菌株,突变体菌株G27S、G30S、G25S、Y90F、Y90A、Q63N、C416A、I483A、G27S/Y90A、G27S/I483A、Y90A/I483A、G27S/Y90A/I483A中角鲨烯的产量分别提高了146mg/L、109mg/L、17mg/L、32mg/L、333mg/L、58mg/L、24mg/L、211mg/L、383mg/L、311mg/L、407mg/L、457mg/L,提高比例分别为45.3%、33.9%、5.3%、9.9%、103.4%、18.0%、7.5%、65.5%、118.9%、96.6%、126.4%、141.9%。
表4分别表达野生酶和突变酶的酿酒酵母胞内角鲨烯产量
实施例5:重组酿酒酵母摇瓶发酵生产角鲨烯
选取实施例4中在孔板发酵结果较优的前5株酿酒酵母角鲨烯单加氧酶突变体菌株Y90A、G27S/Y90A、G27S/I483A、Y90A/I483A、G27S/Y90A/I483A进行摇瓶发酵验证,野生型角鲨烯单加氧酶菌株为对照。挑取各菌株单菌落于含有25mL YPD培养基的250mL摇瓶中30℃培养96h。
角鲨烯单加氧酶突变体菌株Y90A、G27S/Y90A、G27S/I483A、Y90A/I483A、G27S/Y90A/I483A胞内角鲨烯产量分别达2136mg/L、2492mg/L、2084mg/L、2672mg/L、2777mg/L,相比野生型角鲨烯单加氧酶菌株(1488mg/L),角鲨烯产量分别提高了648mg/L、1004mg/L、596mg/L、1184mg/L、1289mg/L,提高比例分别为44.8%、67.5%、40.1%、79.6%、86.6%。
实施例6:重组酿酒酵母在5L发酵罐放大培养
选取实施例5摇瓶发酵结果最优的角鲨烯单加氧酶突变体菌株G27S/Y90A/I483A进行5L发酵罐的放大培养。挑取大而圆润的菌落在25mL YPD装液量的250ml摇瓶中,30℃、220r/min培养至OD600 4-5(14~16个小时),以1%接种量接种至25mL YPD装液量的250mL摇瓶中,30℃、220rpm培养至OD600 25(14~16小时),将25mL种子液全部接种至装有2.5LYPD、10g/L CaCO3培养基的5L发酵罐中。
控制初始转速300rpm,通气量1.5vvm,氨水控制pH 5.5。溶氧关联搅拌,前48h控制溶氧60%,48h后控制溶氧30%。第12~24h补料培养基流速为4mL/h,第25~48h补料培养基流速为12mL/h,第49~84h补料培养基流速为4mL/h。
补料培养基:葡萄糖400g/L,(NH4)2SO4 50g/L,KH2PO4 20g/L,MgSO4·7H2O 12g/L,K2SO4 8g/L,Na2SO4 2g/L,微量元素A(金属离子)20mL/L,微量元素B(维生素)20mL/L。
微量元素A(金属离子,每L):ZnSO4·7H2O 5g,MnCl2·4H2O 0.5g,CoCl2·6H2O0.8g,CaCl2·2H2O 4g,FeSO4·7H2O 4g,pH 8.0。
微量元素B(维生素,每L):生物素0.1g,泛酸钙2g,烟酸2g,肌醇20g,硫胺素2g,吡哆醛1.5g。
表达G27S/Y90A/I483A的重组酿酒酵母发酵120小时即可在细胞内检测获得角鲨烯,发酵120h,OD600可达32,每L发酵液可检测到胞内角鲨烯的产量达6.82g/L,相比突变前野生型角鲨烯单加氧酶菌株,胞内角鲨烯产量提高了2.31g/L,提高比例为51.2%。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 江南大学
<120> 酿酒酵母内源角鲨烯单加氧酶突变体及应用
<130> BAA210912A
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 496
<212> PRT
<213> Saccharomyces cerevisiae
<400> 1
Met Ser Ala Val Asn Val Ala Pro Glu Leu Ile Asn Ala Asp Asn Thr
1 5 10 15
Ile Thr Tyr Asp Ala Ile Val Ile Gly Ala Gly Val Ile Gly Pro Cys
20 25 30
Val Ala Thr Gly Leu Ala Arg Lys Gly Lys Lys Val Leu Ile Val Glu
35 40 45
Arg Asp Trp Ala Met Pro Asp Arg Ile Val Gly Glu Leu Met Gln Pro
50 55 60
Gly Gly Val Arg Ala Leu Arg Ser Leu Gly Met Ile Gln Ser Ile Asn
65 70 75 80
Asn Ile Glu Ala Tyr Pro Val Thr Gly Tyr Thr Val Phe Phe Asn Gly
85 90 95
Glu Gln Val Asp Ile Pro Tyr Pro Tyr Lys Ala Asp Ile Pro Lys Val
100 105 110
Glu Lys Leu Lys Asp Leu Val Lys Asp Gly Asn Asp Lys Val Leu Glu
115 120 125
Asp Ser Thr Ile His Ile Lys Asp Tyr Glu Asp Asp Glu Arg Glu Arg
130 135 140
Gly Val Ala Phe Val His Gly Arg Phe Leu Asn Asn Leu Arg Asn Ile
145 150 155 160
Thr Ala Gln Glu Pro Asn Val Thr Arg Val Gln Gly Asn Cys Ile Glu
165 170 175
Ile Leu Lys Asp Glu Lys Asn Glu Val Val Gly Ala Lys Val Asp Ile
180 185 190
Asp Gly Arg Gly Lys Val Glu Phe Lys Ala His Leu Thr Phe Ile Cys
195 200 205
Asp Gly Ile Phe Ser Arg Phe Arg Lys Glu Leu His Pro Asp His Val
210 215 220
Pro Thr Val Gly Ser Ser Phe Val Gly Met Ser Leu Phe Asn Ala Lys
225 230 235 240
Asn Pro Ala Pro Met His Gly His Val Ile Leu Gly Ser Asp His Met
245 250 255
Pro Ile Leu Val Tyr Gln Ile Ser Pro Glu Glu Thr Arg Ile Leu Cys
260 265 270
Ala Tyr Asn Ser Pro Lys Val Pro Ala Asp Ile Lys Ser Trp Met Ile
275 280 285
Lys Asp Val Gln Pro Phe Ile Pro Lys Ser Leu Arg Pro Ser Phe Asp
290 295 300
Glu Ala Val Ser Gln Gly Lys Phe Arg Ala Met Pro Asn Ser Tyr Leu
305 310 315 320
Pro Ala Arg Gln Asn Asp Val Thr Gly Met Cys Val Ile Gly Asp Ala
325 330 335
Leu Asn Met Arg His Pro Leu Thr Gly Gly Gly Met Thr Val Gly Leu
340 345 350
His Asp Val Val Leu Leu Ile Lys Lys Ile Gly Asp Leu Asp Phe Ser
355 360 365
Asp Arg Glu Lys Val Leu Asp Glu Leu Leu Asp Tyr His Phe Glu Arg
370 375 380
Lys Ser Tyr Asp Ser Val Ile Asn Val Leu Ser Val Ala Leu Tyr Ser
385 390 395 400
Leu Phe Ala Ala Asp Ser Asp Asn Leu Lys Ala Leu Gln Lys Gly Cys
405 410 415
Phe Lys Tyr Phe Gln Arg Gly Gly Asp Cys Val Asn Lys Pro Val Glu
420 425 430
Phe Leu Ser Gly Val Leu Pro Lys Pro Leu Gln Leu Thr Arg Val Phe
435 440 445
Phe Ala Val Ala Phe Tyr Thr Ile Tyr Leu Asn Met Glu Glu Arg Gly
450 455 460
Phe Leu Gly Leu Pro Met Ala Leu Leu Glu Gly Ile Met Ile Leu Ile
465 470 475 480
Thr Ala Ile Arg Val Phe Thr Pro Phe Leu Phe Gly Glu Leu Ile Gly
485 490 495
<210> 2
<211> 1491
<212> DNA
<213> Saccharomyces cerevisiae
<400> 2
atgtctgctg ttaacgttgc acctgaattg attaatgccg acaacacaat tacctacgat 60
gcgattgtca tcggtgctgg tgttatcggt ccatgtgttg ctactggtct agcaagaaag 120
ggtaagaaag ttcttatcgt agaacgtgac tgggctatgc ctgatagaat tgttggtgaa 180
ttgatgcaac caggtggtgt tagagcattg agaagtctgg gtatgattca atctatcaac 240
aacatcgaag catatcctgt taccggttat accgtctttt tcaacggcga acaagttgat 300
attccatacc cttacaaggc cgatatccct aaagttgaaa aattgaagga cttggtcaaa 360
gatggtaatg acaaggtctt ggaagacagc actattcaca tcaaggatta cgaagatgat 420
gaaagagaaa ggggtgttgc ttttgttcat ggtagattct tgaacaactt gagaaacatt 480
actgctcaag agccaaatgt tactagagtg caaggtaact gtattgagat attgaaggat 540
gaaaagaatg aggttgttgg tgccaaggtt gacattgatg gccgtggcaa ggtggaattc 600
aaagcccact tgacatttat ctgtgacggt atcttttcac gtttcagaaa ggaattgcac 660
ccagaccatg ttccaactgt cggttcttcg tttgtcggta tgtctttgtt caatgctaag 720
aatcctgctc ctatgcacgg tcacgttatt cttggtagtg atcatatgcc aatcttggtt 780
taccaaatca gtccagaaga aacaagaatc ctttgtgctt acaactctcc aaaggtccca 840
gctgatatca agagttggat gattaaggat gtccaacctt tcattccaaa gagtctacgt 900
ccttcatttg atgaagccgt cagccaaggt aaatttagag ctatgccaaa ctcctacttg 960
ccagctagac aaaacgacgt cactggtatg tgtgttatcg gtgacgctct aaatatgaga 1020
catccattga ctggtggtgg tatgactgtc ggtttgcatg atgttgtctt gttgattaag 1080
aaaataggtg acctagactt cagcgaccgt gaaaaggttt tggatgaatt actagactac 1140
catttcgaaa gaaagagtta cgattccgtt attaacgttt tgtcagtggc tttgtattct 1200
ttgttcgctg ctgacagcga taacttgaag gcattacaaa aaggttgttt caaatatttc 1260
caaagaggtg gcgattgtgt caacaaaccc gttgaatttc tgtctggtgt cttgccaaag 1320
cctttgcaat tgaccagggt tttcttcgct gtcgcttttt acaccattta cttgaacatg 1380
gaagaacgtg gtttcttggg attaccaatg gctttattgg aaggtattat gattttgatc 1440
acagctatta gagtattcac cccatttttg tttggtgagt tgattggtta a 1491
<210> 3
<211> 1575
<212> DNA
<213> 人工序列
<400> 3
gaccaattgg tgaaaactga agtcaccaag aagtctttta ctgctcctgt acaaaaggct 60
tctacaccag ttttaaccaa taaaacagtc atttctggat cgaaagtcaa aagtttatca 120
tctgcgcaat cgagctcatc aggaccttca tcatctagtg aggaagatga ttcccgcgat 180
attgaaagct tggataagaa aatacgtcct ttagaagaat tagaagcatt attaagtagt 240
ggaaatacaa aacaattgaa gaacaaagag gtcgctgcct tggttattca cggtaagtta 300
cctttgtacg ctttggagaa aaaattaggt gatactacga gagcggttgc ggtacgtagg 360
aaggctcttt caattttggc agaagctcct gtattagcat ctgatcgttt accatataaa 420
aattatgact acgaccgcgt atttggcgct tgttgtgaaa atgttatagg ttacatgcct 480
ttgcccgttg gtgttatagg ccccttggtt atcgatggta catcttatca tataccaatg 540
gcaactacag agggttgttt ggtagcttct gccatgcgtg gctgtaaggc aatcaatgct 600
ggcggtggtg caacaactgt tttaactaag gatggtatga caagaggccc agtagtccgt 660
ttcccaactt tgaaaagatc tggtgcctgt aagatatggt tagactcaga agagggacaa 720
aacgcaatta aaaaagcttt taactctaca tcaagatttg cacgtctgca acatattcaa 780
acttgtctag caggagattt actcttcatg agatttagaa caactactgg tgacgcaatg 840
ggtatgaata tgatttctaa aggtgtcgaa tactcattaa agcaaatggt agaagagtat 900
ggctgggaag atatggaggt tgtctccgtt tctggtaact actgtaccga caaaaaacca 960
gctgccatca actggatcga aggtcgtggt aagagtgtcg tcgcagaagc tactattcct 1020
ggtgatgttg tcagaaaagt gttaaaaagt gatgtttccg cattggttga gttgaacatt 1080
gctaagaatt tggttggatc tgcaatggct gggtctgttg gtggatttaa cgcacatgca 1140
gctaatttag tgacagctgt tttcttggca ttaggacaag atcctgcaca aaatgttgaa 1200
agttccaact gtataacatt gatgaaagaa gtggacggtg atttgagaat ttccgtatcc 1260
atgccatcca tcgaagtagg taccatcggt ggtggtactg ttctagaacc acaaggtgcc 1320
atgttggact tattaggtgt aagaggcccg catgctaccg ctcctggtac caacgcacgt 1380
caattagcaa gaatagttgc ctgtgccgtc ttggcaggtg aattatcctt atgtgctgcc 1440
ctagcagccg gccatttggt tcaaagtcat atgacccaca acaggaaacc tgctgaacca 1500
acaaaaccta acaatttgga cgccactgat ataaatcgtt tgaaagatgg gtccgtcacc 1560
tgcattaaat cctaa 1575
<210> 4
<211> 867
<212> DNA
<213> 人工序列
<400> 4
atgactgccg acaacaatag tatgccccat ggtgcagtat ctagttacgc caaattagtg 60
caaaaccaaa cacctgaaga cattttggaa gagtttcctg aaattattcc attacaacaa 120
agacctaata cccgatctag tgagacgtca aatgacgaaa gcggagaaac atgtttttct 180
ggtcatgatg aggagcaaat taagttaatg aatgaaaatt gtattgtttt ggattgggac 240
gataatgcta ttggtgccgg taccaagaaa gtttgtcatt taatggaaaa tattgaaaag 300
ggtttactac atcgtgcatt ctccgtcttt attttcaatg aacaaggtga attactttta 360
caacaaagag ccactgaaaa aataactttc cctgatcttt ggactaacac atgctgctct 420
catccactat gtattgatga cgaattaggt ttgaagggta agctagacga taagattaag 480
ggcgctatta ctgcggcggt gagaaaacta gatcatgaat taggtattcc agaagatgaa 540
actaagacaa ggggtaagtt tcacttttta aacagaatcc attacatggc accaagcaat 600
gaaccatggg gtgaacatga aattgattac atcctatttt ataagatcaa cgctaaagaa 660
aacttgactg tcaacccaaa cgtcaatgaa gttagagact tcaaatgggt ttcaccaaat 720
gatttgaaaa ctatgtttgc tgacccaagt tacaagttta cgccttggtt taagattatt 780
tgcgagaatt acttattcaa ctggtgggag caattagatg acctttctga agtggaaaat 840
gacaggcaaa ttcatagaat gctataa 867
Claims (9)
1.角鲨烯单加氧酶突变体,其特征在于,相比于SEQ ID NO.1所示的酶,第27位甘氨酸突变为丝氨酸,并将第90位酪氨酸突变为丙氨酸,并将第483位异亮氨酸突变为丙氨酸。
2.编码权利要求1所述角鲨烯单加氧酶突变体的基因。
3.携带权利要求2所述基因的表达载体。
4.表达权利要求1所述角鲨烯单加氧酶突变体,或含有权利要求2所述基因的酿酒酵母,所述酿酒酵母自身角鲨烯单加氧酶被替代或敲除。
5.一种重组酿酒酵母,其特征在于,为(a)或(b):
(a)基因组上整合有权利要求2所述的基因;
(b)含有携带权利要求2所述基因的表达载体;
酿酒酵母自身角鲨烯单加氧酶被替代或敲除。
6.权利要求1所述的角鲨烯单加氧酶突变体,或权利要求2所述的基因,或权利要求4所述的酿酒酵母,或权利要求5所述的重组酿酒酵母在生产角鲨烯中的应用。
7.一种生产角鲨烯的方法,其特征在于,将权利要求5所述的重组酿酒酵母接种至培养基中,于25~35℃发酵至少48 h。
8.根据权利要求7所述的方法,其特征在于,自发酵第12 h后补料。
9.根据权利要求7或8所述的方法,其特征在于,用于补料的培养基含有:葡萄糖、(NH4)2SO4 、KH2PO4、MgSO4·7H2O 、K2SO4、Na2SO4和微量元素。
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