CN116555362A - 使用解脂耶氏酵母生产脂质的方法 - Google Patents
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- CN116555362A CN116555362A CN202210110527.4A CN202210110527A CN116555362A CN 116555362 A CN116555362 A CN 116555362A CN 202210110527 A CN202210110527 A CN 202210110527A CN 116555362 A CN116555362 A CN 116555362A
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- oleaginous yeast
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- yarrowia lipolytica
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- C—CHEMISTRY; METALLURGY
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- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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Abstract
本发明涉及生产脂质的方法,包括培养产油酵母,其中所述产油酵母已被转化以表达脂滴相关蛋白和/或二酰甘油酰基转移酶。
Description
技术领域
本发明涉及生产脂质的方法,其包括培养产油酵母,其中所述产 油酵母已被转化以表达脂滴相关蛋白。
背景技术
本发明涉及通过培养产油酵母生产脂质(如二十二碳六烯酸(或 DHA)、富含DHA的三酰基甘油或磷脂)的方法。
脂质与蛋白质和碳水化合物一起构成大量营养素的三个主要家 族之一。在脂质中,甘油三酯和磷脂是特别感兴趣的。
甘油三酯(也称为三酰基甘油、三酰基甘油酯或TAG)是其中甘 油的三个羟基被脂肪酸酯化的甘油酯。它们是植物油和动物脂肪的主 要成分。
甘油三酯占人类摄取的膳食脂质的约95%。在机体内,它们主要 存在于脂肪组织中并构成能量储存的主要形式。
磷脂是两亲性脂质,即由极性(亲水性)“头”和两个脂族(疏 水性)“尾”组成的脂质。磷脂是结构脂质,因为它们是细胞膜的成 分,它们尤其提供流动性。
大多数磷脂是磷酸甘油酯,其头围绕用极性分子酯化的甘油-3- 磷酸残基组织,并且其两个尾是两个脂肪酸的脂族链。
其它磷脂是鞘磷脂,其在结构上衍生自鞘氨醇而不是衍生自甘 油,鞘氨醇构成两个脂族尾之一。
从活组织中分离的第一磷脂从蛋黄卵磷脂中表征;它们更特别地 是磷脂酰胆碱。此外,这就是为什么磷脂酰胆碱也被称为卵磷脂。
磷脂酰胆碱是由肝脏天然产生的。它们是胆汁的重要成分,其中 它们乳化十二指肠中存在的脂肪。除了胆汁盐之外,它们对于防止脂 滴再凝集也是必需的。
作为磷脂,磷脂酰胆碱参与细胞膜并用于保持其粘弹性。它们是 神经***的必要组分并且构成接近30%的脑干重和15%的神经。
甘油三酯和磷脂主要由脂肪酸组成,它们都由饮食提供,并且对 于它们中的一些,由生物体合成。
生物化学分类(基于脂肪酸分子中所含的双键数)区分饱和脂肪 酸(SFA)、单不饱和脂肪酸(MUFA)和多不饱和脂肪酸(PUFA)。
从生理观点来看,区别如下:
-必需脂肪酸,是人体发育和正确功能所需的,但机体不能产生;
-“条件性”必需肪酸,其对于细胞的正常生长和生理功能是必 需的,但如果通过饮食提供,其可由其前体产生,并且如果其必需前 体不存在,则其因此是严格所需的;以及
-非必需脂肪酸。
必需和“条件性”必需脂肪酸的组构成必需脂肪酸。
其它脂肪酸被称为非必需的。
特别地,非必需脂肪酸包含ω3脂肪酸家族的二十碳五烯酸 (EPA)、油酸、我们饮食中主要的单不饱和脂肪酸和饱和脂肪酸(如 月桂酸、肉豆蔻酸或棕榈酸)。
多不饱和脂肪酸根据从最终甲基官能团开始的第一个双键的位 置分类。因此,在术语中,对于ω“x”或“nx”,“x”对应于第一不饱和 的位置。
必需脂肪酸的两个主要家族被区分为:ω6脂肪酸(或n-6PUFA), 其中前体和主要代表是亚油酸(LA),和ω3脂肪酸(或n-3PUFA), 例如α-亚麻酸(ALA)及其衍生物(如二十碳五烯酸(EPA)和二十 二碳六烯酸(DHA))。
大多数生物学感兴趣的多不饱和脂肪酸属于ω6家族(花生四烯 酸或ARA)或ω3家族(如EPA或DHA)。
此外,在术语中,构成链的碳原子数也定义为:因此,EPA被描 述为C20:5并且DHA被描述为C22:6。
因此,“5”和“6”对应于分别由EPA和DHA表示的碳链的不饱和 数。
ω3脂肪酸家族的DHA是生物体可从α-亚麻酸合成的脂肪酸,或 其通过食用油性鱼(金枪鱼、鲑鱼、鲱鱼等)提供。
DHA在膜的结构以及脑和视网膜的发育和功能中起重要作用。
鱼油主要用作ω3脂肪酸(如DHA和EPA)的来源,但它们也 存在于微藻的油中,其中它们作为混合物或单独提取,例如来自某些 选择的菌株的油,如裂殖壶菌属(Schizochytrium genus)的那些,其 仅含有痕量EPA但具有高DHA含量。
富含DHA的微藻的生物质的商业制剂是可获得的。因此,可以 提及例如由Aquafauna Bio-Marine Inc.销售的Algamac系列产品,建 议用于轮虫水产养殖中的营养,或由DSM以商品名DHA GoldTM销售 的产品。
申请人还开发了产生DHA的红树林裂殖壶菌(Schizochytrium mangrovei)菌株,其具有产生非常少的高胆固醇血的饱和脂肪酸(小 于6%的月桂酸和肉豆蔻酸,本领域技术人员已知是已知的最多的高 胆固醇血的)和大于40%的棕榈酸(在此理解为按总脂肪酸的重量计 的%)的特性。所述菌株及其用于生产脂质的用途描述于例如国际公 布WO2014/122158中。
产油酵母(如解脂耶氏酵母(Yarrowia lipolytica或Y.lipolytica)) 也已被工程化并用作宿主以产生高水平的n-3PUFA(如DHA、EPA 和ALA...)和脂溶性类胡萝卜素(番茄红素、β-胡萝卜素、虾青素…)。 因此,具有增加的脂质含量的工程化解脂耶氏酵母菌株可用于增强功 能性脂质和类胡萝卜素的生产。
例如,Xue等人(Nature Biotechnology,2013,vol 31:8,p 734-740) 描述了产油酵母解脂耶氏酵母的代谢工程,以增加通过所述酵母生产 ω-3二十碳五烯酸。
类似地,Xie等人(Appl Microbiol Biotechnol 2015,99:1599-1610 描述了用于在细胞脂质谱的混合物中产生定制的ω-3(EPA、DHA) 和/或ω-6(ARA、GLA)脂肪酸的耶氏酵母平台。
Zhu等人(Current Opinion in Biotechnology,2015,36:65-72)综述 了用于生产生物柴油燃料、功能性脂肪酸和类胡萝卜素的解脂耶氏酵 母代谢工程的最新进展。
除了引入在生产脂质(如EPA)的代谢途径中有用的特定酶之外, 还需要增加脂质的总产量。
本领域仍然需要增加通过解脂耶氏酵母生产所需脂质的其它方 法。
因此,申请人的功劳在于开发了这样的方法,其将在下文更详细 地公开。
发明概述
本发明涉及生产脂质的方法,包括培养产油酵母,其中所述产油 酵母已被转化以表达具有如SEQ ID NO:2所示氨基酸序列的蛋白质 和/或具有如SEQ ID NO:4所示氨基酸序列的蛋白质和/或具有如SEQ ID NO:6所示氨基酸序列的蛋白质。
本发明还涉及用编码具有如SEQ ID NO:2所示氨基酸序列的蛋 白质和/或具有如SEQ ID NO:4所示氨基酸序列的蛋白质和/或具有如 SEQ ID NO:6所示氨基酸序列的蛋白质的表达载体转化的宿主细胞, 其中所述宿主细胞是产油酵母。
本发明还涉及于2021年12月9日在布达佩斯条约下以CCTCC M 20211578保藏于中国典型培养物保藏中心(中国湖北省武汉市武昌区八 一路299号,武汉大学)的解脂耶氏酵母(Yarrowia lipolytica)菌株。
最后,本发明还涉及包含如SEQ ID NO:1、SEQ ID NO:3和/或 SEQ ID NO:5所示序列的核酸用于转化产油酵母细胞的用途。
发明详述
本发明的第一个目的是生产脂质的方法,包括培养产油酵母,其 中所述产油酵母已被转化以表达具有如SEQ ID NO:2所示氨基酸序 列的蛋白质和/或具有如SEQ ID NO:4所示氨基酸序列的蛋白质和/或 具有如SEQ ID NO:6所示氨基酸序列的蛋白质。
实际上,本发明人惊奇地发现来自微藻红树林裂殖壶菌 (Schizochytriummangrovei)的某些基因能够显著增加转化的产油酵 母(如解脂耶氏酵母)中脂肪酸的产量。
特别地,本发明人已经分离并发现了编码具有SEQ ID NO:2的蛋 白质的基因,它们被称为“脂滴相关蛋白(LDAP)”。
当该基因在产油酵母(如解脂耶氏酵母)中表达时,脂肪酸的总 产量增加。
ldap的mRNA序列如SEQ ID NO:1所示:
ATGCCTGCCGCCAAGACCATGCAAGAGTCCAAAGTCCCGG ACGATGACTATGTGCTCCACGAGCCGCACAATGGCCCCGTTGGT AAGCTCATCGACATGAGCTACGAACAGTACTCGGGTGCCAAGG ACATGCTCGCCAAGAACCAGGTGATCGGCAAGCGCCTCACTCC ATTCGTGGACTGGACTGAGAACACCACCAAGGCGATTCTCAGA AAGTCCCCGGTCGCTGTCGACAAGGTCGTCTCGGCCGTCGACAC CCGCGCAGAGCGTGTTGTTAACTTCACCTCTGACAAGGTACAGA CCATTCGTGACACTCCCAAAAATACTCTTGGATATGTTCATGGC AAGCTGAAATCCTTGCGTACTGAGCCCCAGGAAGCCGAGAATG AGGAACCTGGTGTCCGCACCATCATCATCACCGGCAAGTCTGCC GCTGCTGAGCGCCTCAATGTCCTCCTCGATTCTAGCGAGGGGTA CCTCAAGCAGTACCTCCCCATCTCTGATGATGAGAAGGCGCTCA TGGTCAATGGCTCCAAGTCTGAGATCCGCACCGTCGCTACTCGC ACGGTCAACCTTTCCAAGGTGGCCATCTCCAAGGCCATCGAGC GCGCTCTTAACCGCGCCGGTGAAATCAAGGAGCGCACCAAGGA GACCATTCATGTGGATCTCATTCGCTACAACGAGTGGCTTGACA TGAACGTTAAGCAGCCCGTTTCGCGTCGCCTCAAGATTGTCGAC GACAAGCTTGCCATCTCTGAGAAGGTTGACAAGGTCGACCAGA AGGTTGTCAAGCCCATTAAAAACTCCATCAACCGTCGTGTCGAG CTCATTGATGCCAAGGTTGTGACCCCAGTTAAGGAAAAGTTTGT ACTCGTTGTCACTCGTGTCAGCGACACTTACCAGCACAAGGTTG TTGAGCCTCGTGACCAGATCATCCAAATGTTCCGCGAGGAACTT AGCCTCCAGCAAGAAATCGCCAAGAGCAAGTCCGGTGAGGAGG AGCTTACCATTAGTGCTGGCCTCTCTGCCGTCATTGCCGCCGCT AAGTCTCGCCTCGAGAAGGAGTACGAGGTTCGCGTCTCCCCGG CCCTCCACCGCTTCATGGGCCGCGAAAAGGAAGAGGAGATTGA GGACCAGTCTTTCGGTGATGATGTTGAGGAGGACGAGGATGTCGATGAGACCTACTAA
LDAP的蛋白质序列如SEQ ID NO:2所示:
MPAAKTMQESKVPDDDYVLHEPHNGPVGKLIDMSYEQYSGA KDMLAKNQVIGKRLTPFVDWTENTTKAILRKSPVAVDKVVSAVD TRAERVVNFTSDKVQTIRDTPKNTLGYVHGKLKSLRTEPQEAENE EPGVRTIIITGKSAAAERLNVLLDSSEGYLKQYLPISDDEKALMVN GSKSEIRTVATRTVNLSKVAISKAIERALNRAGEIKERTKETIHVDLI RYNEWLDMNVKQPVSRRLKIVDDKLAISEKVDKVDQKVVKPIKN SINRRVELIDAKVVTPVKEKFVLVVTRVSDTYQHKVVEPRDQIIQM FREELSLQQEIAKSKSGEEELTISAGLSAVIAAAKSRLEKEYEVRVS PALHRFMGREKEEEIEDQSFGDDVEEDEDVDETY
另外,本发明人发现了两个二酰基甘油酰基转移酶(dgat2)基因的 序列,其编码称为DGAT2-1和DGAT2-2的蛋白质。
DGAT2-1mRNA的序列如SEQ ID NO:3所示:
ATGTCTTCAAGCCCCGCCAAAGCTCCTCCTGCTCGCCAGCA AACTGGCAATGGGGTGTCTAGTCGGAGCAGCGTGTCCCAGCGT CGTCGCGTAGAGAAGCTCAAGCAAGGCTATGGCGAGGAGGAGG ATGGAAGCAACACTCCTACAGGCCTGAACACGCCTTACAGCAC ATCCATGGGCTCTATTTCAAGTTATTCGTCTTCTGGAGACTACG CTCAGTCCGAAGGCGAAGGATCTGAGCCTGCTCTGGACCCTGC GGACAGTGTGGATGGCAGTCCAGGCAAGCGGGATTCTGCGTAC AACAAATCGTCCAAAAGACAACTCACGCAAGAAGAGCGCGAAC TCTTCTTGCGTCTTGAGAAGGAATGGCGCGAGGAGGACTCTTGG GCAGAACAACCTGGGTCCTGGTACTCAATGCTTGCGTGGATGCC TGTTCTTATTGCACTTCGCGTGTTTAACGTCTTCCTCTCGATCCT CTTTTGGCCTGTATCATTTGTGGCGCGGGTCTTCTTTGGAAAGG AGATCCACTCGGTTAGTTTTTGGGACGTTCCGCTAGCTCGACGC AAGCAAACTGCAGTGGTCTTGCTTTTCGTTATGCTTTTGCCGAT GATTATGGTAGTCTACTCTTGGACCCTGATCCTCCTCATTTTCCC ACTCACGACCCTTCCCACACTTTCATACCTTATCTGGATTATGTA TATTGACAAGTCGCATGAGACTGGTAAGCGCAAGCCGTTCATG CGTTATTGGAAAATGTGGCGCCACTTTGCAAACTACTTCCCTTT GCGCCTCATCCGTACAACCCCCCTTGATCCGCGCAGGAAATATG TATTTTGCTACCACCCGCACGGGATCATTTCGCTTGGTGCCTTTG GTAACTTCGCAACTGATTCCACTGGTTTCTCGCGCAAGTTTCCA GGCATTGACTTGCGCCTTCTTACTTTGCAGATCAACTTTTATTGC CCAATCATTCGGGAGCTTCTGCTGTATATGGGTCTCTGCTCTGC CGCCAAGAAGTCATGCAATCAAATTCTCCAGCGCGGACCTGGG TCAGCTATCATGCTTGTCGTAGGTGGTGCTGCAGAATCCCTTGA CTCGCAACCTGGTACATATCGTCTCACGCTTGGTCGCAAGGGCT TCGTTCGTGTTGCACTTGACAACGGTGCCGATCTTGTTCCTGTTCTAGGATTTGGTGAGAATGATGTCTTCGATACCGTCTACTTACCA CCCAACTCCTGGGCACGAAATGTTCAAGAGTTCGTTCGAAAGA AGCTTGGCTTTGCGACTCCAATTTTTAGTGGACGAGGTATCTTC CAATACAACATGGGCCTCATGCCGCACCGCAAGCCTATCATTAT TCCCAAGATCCCGGATGAATTAAAGGGCCGTGCTCTCTCTACAA CCGCAGAAGGTGTCGCCTTAGTTGATAAGTACCACGAGAAATA TGTCCGGGCTCTGCGTGAGCTATGGAACTTGTACAAGGAGCGCT GGGCTGTTCACCGCCAAGGTTCTCTTTTAATTCAGAAGTAA
DGAT2-1蛋白的序列如SEQ ID NO:4所示:
MSSSPAKAPPARQQTGNGVSSRSSVSQRRRVEKLKQGYGEEE DGSNTPTGLNTPYSTSMGSISSYSSSGDYAQSEGEGSEPALDPADS VDGSPGKRDSAYNKSSKRQLTQEERELFLRLEKEWREEDSWAEQP GSWYSMLAWMPVLIALRVFNVFLSILFWPVSFVARVFFGKEIHSVS FWDVPLARRKQTAVVLLFVMLLPMIMVVYSWTLILLIFPLTTLPTL SYLIWIMYIDKSHETGKRKPFMRYWKMWRHFANYFPLRLIRTTPL DPRRKYVFCYHPHGIISLGAFGNFATDSTGFSRKFPGIDLRLLTLQI NFYCPIIRELLLYMGLCSAAKKSCNQILQRGPGSAIMLVVGGAAES LDSQPGTYRLTLGRKGFVRVALDNGADLVPVLGFGENDVFDTVY LPPNSWARNVQEFVRKKLGFATPIFSGRGIFQYNMGLMPHRKPIIIP KIPDELKGRALSTTAEGVALVDKYHEKYVRALRELWNLYKERWA VHRQGSLLIQK
DGAT2-2mRNA的序列如SEQ ID NO:5所示:
ATGGATCGCACAGCAGAATGCTCTGCGAAGGAACCTTATT ACTCACTTGACGTATGGCGAATCCCAACCGCTTTGCACGCGTCA AAAGTGGTGCCACAAGATGCAGACGAGGAGACTCGCACAAGGT TAGAAGATGAGGCAAACGAGGTGCTTCTCCGGGAGTTCGCCGT GAAGCATGATGGTCCATTTCCTGAAGACTTTCTAACAGCAAAG GCTGACATCACATTTTCAGAAGAAATCATGGTGCTTTCTGCTTT GCTCGTGACTCTCGGTGGCCCACTCTTTTGGTTTTCGGCTGGCCT CCTCACGTTCATCGCTGCATCATGGACATCAGTTATGTTGTACA TAATCCTGACAGCCTTCCTTGCTTTCCACCCACTCCCTAACTCTA TTCCTGCTTTATGGACTTCGCCTTTGATCATCGCAATGTACAAGT ACTTCTCGTACAGGTTTGTGTGGAAAGGCAACGCACGAGAAAT GCTACGATCGAACAAGCAATTTCTTGGTTCAGGCGTGCCTCACG GTGTGATGCCGTTTGCTAATCTTCTTTGCATCCCTGCGACGAAC TCTGTTCTGTACAGAGGCTGCAATTTTTGGGGTGCCCCAGCGTC AGTTGTGTTTCATACTCCTTTCCTGCGATACTTGTCAATTCTGCA GTGTTGCCATGTTGGCCGCGAAGCAATAATGCGCGAACTTGAA GTTGGGCACTCTGTAGGTTTAGTTGGTGATGGAATTGCTGGAAT CTTTCAATCAAACCATGACGACGAGGTTGTTGCTCTGAAGCACC GCAAGGGCTTGGCGAAGCTTGCGTTACGGACAGGTACTCCTGT GCTTCCTTGCTACTCACTGGGAAATACCGCTGCCTTTTCTGCAT GGTTTGACAGTTATGGAGTCATGGAGTGGCTTTCTCGCAAAGCT CAGGCCTCTATTTTCTTTTATTGGGGTCGGTTTGGCCTACCTATC CCACATCGTGTAAATATTACTATGATTGTAGGCAACATGGTTCT TGTAGAAAAAGTAGACGAGCCATCAGAAGATCAGATCCAAGTC CTTCATGATAAGATTTTGGATGGGTTCCGGGATGCGTTTGACTC GCATAAAGCATCGCTAGGATGGGCGCACGGAAGTTGCGCTTTG TGTAACAAAGCGTTGCTACTGCAGGGCGGAATCTAG
DGAT2-2蛋白的序列如SEQ ID NO:6所示:
MDRTAECSAKEPYYSLDVWRIPTALHASKVVPQDADEETRTR LEDEANEVLLREFAVKHDGPFPEDFLTAKADITFSEEIMVLSALLV TLGGPLFWFSAGLLTFIAASWTSVMLYIILTAFLAFHPLPNSIPALW TSPLIIAMYKYFSYRFVWKGNAREMLRSNKQFLGSGVPHGVMPFA NLLCIPATNSVLYRGCNFWGAPASVVFHTPFLRYLSILQCCHVGRE AIMRELEVGHSVGLVGDGIAGIFQSNHDDEVVALKHRKGLAKLAL RTGTPVLPCYSLGNTAAFSAWFDSYGVMEWLSRKAQASIFFYWG RFGLPIPHRVNITMIVGNMVLVEKVDEPSEDQIQVLHDKILDGFRDAFDSHKASLGWAHGSCALCNKALLLQGGI
本领域技术人员可以容易地选择合适的产油酵母。合适的酵母包 括通常用于产生高产量脂质的任何酵母种类。
在一个实施方案中,产油酵母选自下组:耶氏酵母属(Yarrowia)、 假丝酵母属(Candida)、红酵母属(Rhodotorula)、红冬孢酵母属 (Rhodosporidium)、隐球酵母属(Cryptococcus)、丝孢酵母属 (Trichosporon)和油脂酵母属(Lipomyces)。
在优选实施方案中,产油酵母选自下组:斯达氏油脂酵母 (Lipomycesstarkeyi)、圆红冬孢酵母(Rhodosporidium toruloides)、 粘红酵母(Rhodotorulaglutinis)和解脂耶氏酵母(Yarrowia lipolytica)。
在优选实施方案中,所述产油酵母是解脂耶氏酵母。
可以使用任何合适的表达给定核酸的方法。
典型地,本领域技术人员知道在Lv Y,Edwards H,Zhou J,Xu P. Combining 26srDNA and the Cre-loxP System for Iterative Gene Integration and EfficientMarker Curation in Yarrowia lipolytica.ACS Synthetic Biology.2019Mar;8(3):568-576.DOI: 10.1021/acssynbio.8b00535.PMID:30695641的出版物中描述的方法。
在优选实施方案中,可以进一步转化产油酵母以表达已知增加脂 质产量的其它基因,如Zhu等人(Current Opinion in Biotechnology, 2015,36:65-72)描述的那些。
本发明还涉及用编码具有如SEQ ID NO:2所示氨基酸序列的蛋 白质和/或具有如SEQ ID NO:4所示氨基酸序列的蛋白质和/或具有如 SEQ ID NO:6所示氨基酸序列的蛋白质的表达载体转化的宿主细胞, 其中所述宿主细胞是产油酵母。
本发明还涉及于2021年12月9日在布达佩斯条约下以CCTCC M 20211578保藏于中国典型培养物保藏中心(中国湖北省武汉市武昌区八 一路299号,武汉大学)的解脂耶氏酵母(Yarrowia lipolytica)菌株。
最后,本发明还涉及包含如SEQ ID NO:1、SEQ ID NO:3和/或 SEQ ID NO:5所示序列的核酸用于转化产油酵母细胞的用途。
通过阅读下文的实施例将更清楚地理解本发明,这些实施例仅仅 是说明性的,并不以任何方式限制本发明的保护范围。
具体实施方式
实施例1:分离来自“脂滴相关蛋白”的红树林裂殖壶菌株D5
以5000g收集细胞10分钟,并用30mL磷酸盐缓冲盐水(PBS) 洗涤两次。在冰上在30ml缓冲液A(25mM tricine、250mM蔗糖, pH 7.8)中孵育20分钟后,将细胞均质化。将细胞匀浆在50mL管中 以6000g离心10分钟以除去细胞碎片和未破碎的细胞。将用2mL缓 冲液B(20mM HEPES、100mM KCl、2mM MgCl2,pH7.4)覆盖 的上清液部分(10mL)在4℃以38000rpm离心1h(BeckmanSW40)。 使用200μL移液管尖端收集在梯度顶部含有脂滴(LD)的白色条带,并转移至1.5mL Eppendorf管中。将LD用200μL缓冲液B洗涤三次。 通过观察染色确认LD的纯度。然后,从LD中提取总蛋白并通过蛋 白质组学分析。
通过质谱法分离一种特定的蛋白质,称为LDAP,并对相应的 mRNA测序。
ldap mRNA的序列如SEQ ID NO:1所示。
LDAP蛋白的序列如SEQ ID NO:2所示。
实施例2:生产表达ldap的工程化解脂耶氏酵母
本发明人使用载体pYLXP’2在解脂耶氏酵母中过表达ldap。它 是生产如Lv Y、Edwards H、Zhou J、Xu P所述的工程化解脂耶氏酵 母的标准程序。将26srDNA与Cre-loxP***结合用于解脂耶氏酵母 中的反复基因整合和有效的标记固化。ACS SyntheticBiology.2019 Mar;8(3):568-576.DOI:10.1021/acssynbio.8b00535.PMID:30695641。
分离所得转化菌株并进一步表征脂质生产。
实施例3:生物质和总脂质产量的增加
解脂耶氏酵母不天然产生DHA,它只能合成通常的脂肪酸,如 C18:0、C18:1和C18:2。油质解脂耶氏酵母是一种宿主,因为它能产 生丰富的乙酰CoA,其是生物合成功能性脂质和类胡萝卜素的前体。 此外,只有油质微生物可以形成明显的LD,并且已经证明TAG主要沉积在解脂耶氏酵母中的LD中。LDAP可能参与LD中脂质的积聚。 结果表明,当ldap过表达时,LD增大或稳定,并且在解脂耶氏酵母 中沉积并产生更多的TAG。通过在解脂耶氏酵母中过表达单独的 ldap,总脂质滴度和产量都极大增强(下表1)。LDAP-5和LDAP-12 代表两种选择的工程化菌株。本文显示的数据重复三次。
表1:原始Polf菌株和菌株LDAP-5和LDAP-12的生物质和TFA产量。结果以平均值±
标准差表示
此外,本发明人注意到单不饱和脂肪酸C18:1在LDAP-5和 LDAP-12中均显著增加(参见表2),表明过表达ldap可能影响解脂 耶氏酵母中脂肪酸的生物合成。
表2:原始Polf菌株和菌株5和12中的脂肪酸谱。结果以平均值±标准差表示
菌株LDAP-12(也称为pLDAP)于2021年12月9日按照布达 佩斯条约以保藏号CCTCCM 20211578保藏于CCTCC。
实施例4:表达ldap和/或dgat的工程化解脂耶氏酵母
使用与实施例2中相同的方法来产生过表达二酰基甘油酰基转移 酶(DGAT2)的解脂耶氏酵母菌株。DGAT2是参与TAG生物合成的 限制酶。
实际上,本发明人从D5的基因组中发现了两个二酰基甘油酰基 转移酶(DGAT2)基因。对这两个编码DGAT2的基因进行密码子优 化并合成。过表达dgat2-1或dgat2-2导致总脂肪酸(TFA)的增加。
生物质略微降低。
表3:在原始Polf菌株和过表达根据本发明的各种基因的菌株中的生物质和TFA生
产。结果以平均值±标准差表示
Po1f,Y.lipolytica,MATa,leu2-270,ura3-302,xpr2-322,axp-2, (Leu2-,Ura3-);LDAP,Po1f harboring pYLXP’::ldap;DGAT2-1,Po1f harboring dgat2-;DGAT2-2,Po1f harboring pYLXP’::dgat2-2; LDAP-DGAT2-1,pYLXP’::ldap::dgat2-1;LDAP-DGAT2-2, pYLXP’::ldap::dgat2-2。
本发明人已经表明,表达来自红树林裂殖壶菌株的ldap或dgat2-1 或dgat2-2基因的解脂耶氏酵母可用于生产脂肪酸,特别是DHA。
Claims (7)
1.生产脂质的方法,包括培养产油酵母,其中所述产油酵母已被转化以表达具有如SEQID NO:2所示氨基酸序列的蛋白质和/或具有如SEQ ID NO:4所示氨基酸序列的蛋白质和/或具有如SEQ ID NO:6所示氨基酸序列的蛋白质。
2.宿主细胞,其用编码具有如SEQ ID NO:2所示氨基酸序列的蛋白质和/或具有如SEQID NO:4所示氨基酸序列的蛋白质和/或具有如SEQ ID NO:6所示氨基酸序列的蛋白质的表达载体转化,其中所述宿主细胞是产油酵母。
3.根据权利要求1所述的方法或根据权利要求2所述的宿主细胞,其中所述产油酵母选自下组:耶氏酵母属、假丝酵母属、红酵母属、红冬孢酵母属、隐球酵母属、丝孢酵母属和油脂酵母属。
4.根据权利要求3所述的方法或宿主细胞,其中所述产油酵母选自下组:斯达氏油脂酵母、圆红冬孢酵母、粘红酵母和解脂耶氏酵母。
5.根据权利要求4所述的方法或宿主细胞,其中所述产油酵母是解脂耶氏酵母菌株。
6.解脂耶氏酵母,其于2021年12月9日在布达佩斯条约下以CCTCC M 20211578保藏。
7.包含如SEQ ID NO:1、SEQ ID NO:3和/或SEQ ID NO:5所示序列的核酸用于转化产油酵母细胞的用途。
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