CN101563356A - 高山被孢霉c16/18脂肪酸延伸酶 - Google Patents
高山被孢霉c16/18脂肪酸延伸酶 Download PDFInfo
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- CN101563356A CN101563356A CNA2005800518880A CN200580051888A CN101563356A CN 101563356 A CN101563356 A CN 101563356A CN A2005800518880 A CNA2005800518880 A CN A2005800518880A CN 200580051888 A CN200580051888 A CN 200580051888A CN 101563356 A CN101563356 A CN 101563356A
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Abstract
本发明涉及能够催化棕榈酸(16:0)向硬脂酸(18:0)的转化的真菌C16/18脂肪酸延伸酶。特别地,提供了高山被孢霉(Mortierella alpina)C16/18脂肪酸延伸酶的核苷酸序列(称为“ELO3”)。在此描述了通过过量表达C16/18脂肪酸延伸酶来提高微生物油产量、提高进入多不饱和脂肪酸生物合成途径的碳通量以及提高多不饱和脂肪酸的含量的方法。最理想地,当前的ELO3的底物特异性将对于允许长链多不饱和脂肪酸在产油酵母例如解脂耶罗维亚酵母(Yarrowia lipolytica)中的积累是特别有用的。
Description
发明领域
本发明属于生物技术领域。更具体地,本发明涉及编码对于提高产油微生物中硬脂酸产量有用的C16/18脂肪酸延伸酶的核酸片段的分离和表征。
发明背景
细胞中的油生物合成一般是指甘油三酯(TAG)的合成,其中TAG被定义为由酯化到甘油分子的三个脂肪酰基残基构成的中性脂质。这样的油可以含有光谱的脂肪酸,包括饱和的和不饱和的脂肪酸以及短链和长链脂肪酸。并且,不意外地,大量的因素影响了特定微生物中这样产生的油的数量和它最终的脂肪酸成分。
虽然传统方法(即,育种)和遗传工程方法已经成功地应用于产生具有改善的油含量的含油种子植物[通过例如高月桂酸油菜、高硬脂酸油菜、高油酸大豆和高油酸玉米的商业可获得性展现],在产油微生物中油含量的类似操纵在过去没有显著地进行。然而,将具有在它们的油成分内商业上产生长链ω-3和/或ω-6多不饱和脂肪酸(“PUFA”,例如,18 3、18 4、20 3、20 4、20 5、22 6脂肪酸)能力的微生物工程化的新近努力已经产生了对提高流入脂质代谢的碳的方法的需求。
在大多数有机体中的脂质代谢由多酶脂肪酸合成酶复合物(“FAX”)催化,首先通过八个双碳片段(来自乙酰CoA的乙酰基)的缩合来形成棕榈酸,一种16碳饱和脂肪酸(Smith,S FASEB J,8(15)1248-59(1994))。一旦游离棕榈酸(160)从FAS上释放,该分子经历延伸(即,经由C16/18脂肪酸延伸酶来产生硬脂酸(180)或不饱和现象(即,经由A9脱饱和酶来产生棕榈油酸(161))。从这两种代谢前体合成所有其他脂肪酸分子。由于棕榈酸的主要结局是延伸,然而(去饱和仅是大多数有机体中的很小的反应),断定的是,C16/18脂肪酸延伸酶在确定进入脂肪酸生物合成途径的碳通量方面起到重要作用,从而在这样产生的油的数量和组成方面起到决定性作用。
近年来已经分离和表征了多种脂肪酸延伸酶。例如,讨论酵母、哺乳动物、植物和低等真核生物中的长链脂肪酸的延伸的有用的综述是Leonard,A E,等人(Prog Lipid Res 43 36-54(2004))。Leonard等人的表1提供了脂肪酸延伸酶基因的概述、GenBank Accession No.和每种酶催化的反应(即,来自酿酒酵母(S.cerevisiae)[ELO1、ELO2、ELO3],人类,小鼠[Elov1、Elov2、Elov3、Elov4、Lee],大鼠[rELO1、rELO2],秀丽小杆线虫(Caenorhabditis elegans)[CEELO1],高山被孢霉(Mortierella alpina)[GEELO,MAELO],球等鞭金藻(Isochrysisgalbana)[IgASE1]和小立碗藓(Physcomitrella patens)[PSE1])。已经描述和功能上表征的其他脂肪酸延伸酶包括来自Ostreococcus tauri[OtELO1、OtELO2]、Thalassiosira pseudomana[TpELO1、TpELO2]、光滑爪蟾(Xenopus laevis)[XIELO]和虹鳟(Oncorhynchus mykiss)[OmELO](Meyer,A,等人,J.Lipid Res 45(10)1899-1909(2004))金黄色破囊壶菌(Thraustochytrium aureum)(U S 6,677,145)、P.patens[pavELO](Pereira,S等人,Biochem J 384357-366(2004))和秀丽小杆线虫(Caenorhabditis elegans)CeELO2(Kniazeva,M等人,Genetics163 159-169(2003))的那些。然而,仅大鼠rELO2和秀丽小杆线虫(C.elegans)CeELO2延伸酶被分类为具有适合的底物特异性以允许棕榈酸向硬脂酸的转化的C16/18脂肪酸延伸酶。因而,在此希望的是利用遗传工程的技术鉴定和表征新的C16/18脂肪酸延伸酶,作为允许产油微生物中流入脂质代谢的碳的增量调节的手段。
申请人通过从高山被孢霉(Mortierella alpina)分离编码C16/18脂肪酸延伸酶的基因、展现在产油酵母解脂耶罗维亚酵母(Yarrowialipolytica)中过量表达该基因时提高了160向180的转化解决了声明的难题。这允许提高微生物油中PUFA含量和提高油生物合成。
发明概述
本发明涉及分离自被孢霉(Mortierella)的编码C16/18脂肪酸延伸酶的基因,对于操作引起微生物油的产生、特别是在产油酵母中微生物油的产生的生物化学途径是有用。
因而,本发明提供了编码C16/18脂肪酸延伸酶的分离的核酸分子,所述分离的核酸分子选自:
(a)编码SEQ ID NO:2中所示氨基酸序列的分离的核酸分子,
(b)在以下杂交条件下与(a)杂交的分离的核酸分子:0.1×SSC,0.1%SDS,65℃,以及用2×SSC、0.1%SDS随后用0.1×SSC、0.1%SDS洗涤,
与(a)或(b)完全互补的分离的核酸分子。
此外,本发明提供了由本发明的分离的核酸分子编码的多肽、遗传嵌合体和表达所述多肽的宿主细胞。
在类似的实施方式中,本发明提供了分离的核酸分子,其包含编码至少275个氨基酸的C16/18脂肪酸延伸酶的第一核苷酸序列,当根据BLAST比对方法与具有SEQ ID NO:2中所示序列的多肽相比较时,所述C16/18脂肪酸延伸酶具有至少90%的同一性,
或包含所述第一核苷酸序列的互补物的第二核苷酸序列。
此外提供的是产生硬脂酸的方法,包括
a)提供产油酵母,所述产油酵母包含处在适合的调节序列的控制下的、编码如SEQ ID NO:2中所示具有C16/18脂肪酸延伸酶活性的多肽的分离的核酸片段,
b)提供包含棕榈酸的延伸酶底物源,
c)在一定条件下与(b)的延伸酶底物一起生长步骤(a)的产油酵母,在所述条件中步骤(a)(i)或步骤(a)(ii)的核酸分子被表达并且产生硬脂酸,和
d)任选地回收步骤(c)的硬脂酸。
类似地,本发明提供了生产多不饱和脂肪酸的方法,包括
a)提供产油酵母,其包含
(i)编码如SEQ ID NO:2中所示具有C16/18脂肪酸延伸酶活性的多肽的分离的核酸片段,
(ii)编码功能性多不饱和脂肪酸生物合成途径的基因,
b)提供包含棕榈酸的延伸酶底物源,
c)在一定条件下与(b)的延伸酶底物一起生长步骤(a)的产油酵母,在所述条件下产生多不饱和脂肪酸,和
d)任选地回收步骤(c)的多不饱和脂肪酸。
在另一个实施方式中,本发明提供了通过本发明的方法产生的微生物油。
附图的简要描述和序列描述
附图1图形地表示了SEQ ID NO:1、2、7、14、17、18、19和29之间的关系,它们每一个都涉及高山被孢霉(Mortierella alpina)中的C16/18脂肪酸延伸酶酶(ELO3)。
附图2提供了以下质粒的质粒图:(A)pZF5T-PPC、(B)pZUF6S和(C)pZUF6S-E3WT。
附图3说明了PUFA生物合成途径和前体。根据以下的详细说明和构成本申请的一部分的附随的序列描述可以更完整地理解本发明。
以下序列符合37C.F.R.§1.821-1.825(“含有核苷酸序列和/或氨基酸序列公开内容的专利申请的要求-序列规则”),并且符合世界知识产权组织(WIPO)标准ST 25(1998)以及EPO和PCT(Rules 5 2和49 5(a-bis),以及实施细则的小节208和附录C)的序列表要求。核苷酸和氨基酸序列数据的符号和格式复合37C.F.R.§1.822的规则。
SEQ ID NO:1、2、7、14、17-22和25-28是ORF编码基因或蛋白质(或其部分)或质粒,如表1中所标识的。
表1基因和蛋白质SEQ ID编号的概述
描述 | ORF核酸SEQ ID NO. | 蛋白质SEQ ID NO. |
高山被孢霉(Mortierella alpina)C16/18脂肪酸延伸酶(ELO3)-编码区 | 1(828bp) | 2(275AA) |
高山被孢霉(Mortierella alpina)ELO3-部分cDNA序列 | 7(607bp) | - |
通过基因组步移(genome walking)获得的高山被孢霉(Mortierella alpina)ELO3-3′序列 | 14(1,042bp) | - |
通过基因组步移获得的高山被孢霉(Mortierella alpina)ELO3-5′序列 | 17(2,223bp) | - |
高山被孢霉(Mortierella alpina)ELO3-cDNA重叠群 | 18(3,557bp) | - |
高山被孢霉(Mortierella alpina)ELO3-内含子 | 19(542bp) | - |
高山被孢霉(Mortierella alpina)ELO3-基因组重叠群 | 29(4,099bp) | - |
质粒pKUNT2 | 20(6,457bp) | - |
解脂耶罗维亚酵母(Yarrowia lipolytica)A12脱饱和酶 | 21(1,936bp) | 22(419AA) |
质粒pZF5T-PPC | 25(5,553bp) | - |
质粒pZF5T-PPC-E3 | 26(5,031bp) | - |
质粒pZUF6S | 27(8462bp) | - |
质粒pZUF6S-E3WT | 28(11,046bp) | - |
SEQ ID NO:6相应于用于cDNA库测序的M13正向引物
SEQ ID NO:8和9相应于来自ClonTech的UniversalGenomeWalkerTM试剂盒的Genome Walker接头,用于基因组步移来分离高山被孢霉(M.alpina)ELO3的3′末端区域。
SEQ ID NO:10-13分别相应于引物MA Elong 3′1、AP1、MA Elong3′2和AP2,用于基因组步移来分离高山被孢霉(M.alpina)ELO3的3′末端区域。
SEQ ID NO:15和16分别相应于引物MA Elong 5′1和MA elong5′2,用于基因组步移来分离高山被孢霉(M.alpina)ELO3的5′末端区域。
SEQ ID NO:23和24相应于引物MA ELONG 5′Ncol 3和MAELONG 3′Notl 1,用于从高山被孢霉(M.alpina)cDNA扩增完整的ELO3。
发明详述
在此引用的所有专利、专利申请和出版物通过以它们的整体引用而合并在此。这特别地包括,但不限于,以下的申请人的受让人的共同待决申请美国专利申请No.10/840478(2004年5月6日提交)、美国专利申请No.10/840579(2004年5月6日提交,美国专利申请No.10/869630(2004年6月16日提交)、美国专利申请No.10/987548(2004年12月12日提交),美国专利申请No.11/225354(2005年9月12日提交)、美国专利申请No.60/624812(2004年11月4日提交)、美国专利申请No.11/183664(2005年7月18日提交)和美国专利申请No.11/185301(2005年7月20日提交)。
申请人已经分离了编码C16/18脂肪酸延伸酶的高山被孢霉(Mortierella alpina)基因,其对棕榈酸向硬脂酸的转化负责。这个基因(在此标识为“ELO3”)对于改变在产油酵母例如解脂耶罗维亚酵母(Yarrowia lipolytica)中产生的长链多不饱和脂肪酸(PUFA)的数量是有用的。
PUFA的重要性是毫无疑义的。例如,某些PUFA是健康细胞的重要生物成分,并被认为是“必需”脂肪酸,其在哺乳动物中不能从头合成而必需在膳食中获得或通过亚油酸(LA)或α-亚麻酸(ALA)的脱饱和延伸来衍生。此外,长链ω-3PUFA的高摄入产生心血管保护效果(Dyerberg,J等人,Amer.J Clin Nutr 28958-966(1975)、Dyerberg,J等人,Lancet 2(8081)117-119(July 15,1978)、Shimokawa,H,World Rev Nutr Diet,88100-108(2001)、von Schacky,C,and Dyerberg,J.,World Rev Nutr Diet,8890-99(2001))。大量其他研究记载了通过施用ω-3和/或ω-6脂肪酸所赋予的针对多种症状和疾病(例如,哮喘、银屑病、湿疹、糖尿病、癌症)的广泛健康效益。
因而,本发明存在许多应用。通过在此公开的方法积累的PUFA或其衍生物可以用作饮食代替物或增补剂,特别是婴儿配制食品,用于经历静脉给养的患者或用于预防或治疗营养不良。做为选择,纯化的PUFA(或其衍生物)可以被掺入烹调油、脂肪或人造黄油配方,从而在正常的使用中接受者将接受期望数量的饮食补充。PUFA也可以掺入婴儿配制食品、营养增补剂或其他食物产品,可以用作抗炎试剂或胆固醇降低试剂。任选地,组合物可以用于药物用途(人类或兽医学)。在这种情况下,PUFA一般口服地施用,但可以通过可以成功地吸收它们的任何途径来施用,例如,胃肠外地(例如,皮下地、肌内注射地或静脉内地)、直肠地、阴道地或体表地(例如,作为皮肤软膏剂或洗液)。
具有通过重组方法产生的PUFA对人类或动物的增补可以产生提高水平的所添加PUFA,以及它们的代谢产物。例如,用ARA处理可以不仅引起提高水平的ARA,还产生ARA的下游产品例如前列腺素。复杂的调节机制使得希望组合各种PUFA或添加不同的PUFA的轭合物,以防止、控制或克服这些机制来实现个体中期望水平的特定PUFA。
定义
在本公开中,使用了许多术语和缩写。提供了以下定义。“开放阅读框”缩写为ORF。
“聚合酶链式反应”缩写为PCR。
“美国典型培养物保藏所”缩写为ATCC。
“多不饱和脂肪酸”缩写为PUFA。
“微生物油”或“单细胞油”是由微生物(例如,藻类、产油酵母和丝状真菌)在它们生命期期间天然产生的那些油。这些油一般作为“中性脂质”保持在细胞中。术语“甘油三酯”、“油”和“TAG”是指由酯化到甘油分子的三个脂肪酰基残基构成的中性脂质(这些术语将在此处的当前公开中可互换地使用)。这些油可以含有短链和长链饱和的和不饱和的脂肪酸。因而,“油生物合成”一般是指TAG在细胞中的合成。
术语“脂肪酸”是指各种链长度的长链脂肪族酸(链烷酸),从约C12到C22(虽然更长和更短链长度的酸是已知的)。主要链长度是在C16和C22之间。脂肪酸的结构由简单的符号系统“X Y”表示,其中X是特定脂肪酸中碳(C)原子的总数,Y是双键的数目。涉及“饱和脂肪酸”对比“不饱和脂肪酸”、“单不饱和脂肪酸”对比“多不饱和脂肪酸”(或PUFA)、以及“ω-6脂肪酸”(ω-6或n-6)对比“ω-3脂肪酸”(ω-3或n-3)的其他细节在WO2004/101757中提供了。
在本公开中用于描述PUFA的命名以下在表2中示出。在标为“简化符号”的列中,ω-参考系统被用于标明碳的数目、双键的数目和最靠近Ω碳的双键的位置,从Ω碳(为此被编号为1)从开始数起。表格的其余部分概述了ω-3和ω-6脂肪酸及其前体的常见名称,将在整个说明书中和每个化合物的化学名称上使用缩写。
表2某些脂肪酸的命名
常用名 | 缩写 | 化学名称 | 简化符号 |
肉豆蔻酸 | - | 十四烷酸 | 14 0 |
棕榈酸 | 棕榈酸 | 十六烷酸 | 16 0 |
棕榈油酸 | - | 9-十六烯酸 | 16 1 |
硬脂酸 | - | 十八烷酸 | 18 0 |
油酸 | - | 顺式-9-十八烯酸 | 18 1 |
亚油酸 | LA | 顺式-9,12-十八碳二烯酸 | 18 2ω-6 |
γ-亚麻酸 | GLA | 顺式-6,9,12-十八碳三烯酸 | 18 3ω-6 |
二十碳二烯酸 | EDA | 顺式-11,14-二十碳二烯酸 | 20 2ω6 |
二高-γ-亚麻酸 | DGLA | 顺式-8,11,14-二十碳三烯酸 | 20 3ω-6 |
花生四烯酸 | ARA | 顺式-5,8,11,14-二十碳四烯酸 | 20 4ω-6 |
α-亚麻酸 | ALA | 顺式-9,12,15-十八碳三烯酸 | 18 3ω-3 |
stearidonic acid | STA | 顺式-6,9,12,15-十八碳四烯酸 | 18 4ω-3 |
二十碳三烯酸 | ETrA | 顺式-11,14,17-二十碳三烯酸 | 20 3ω-3 |
二十碳四烯酸 | ETA | 顺式-8,11,14,17-二十碳四烯酸 | 20 4ω-3 |
二十碳五烯酸 | EPA | 顺式-5,8,11,14,17-二十碳五烯酸 | 20 5ω-3 |
二十二碳五烯酸 | DPA | 顺式-7,10,13,16,19-二十二碳五烯酸 | 22 5ω-3 |
二十二碳六烯酸 | DHA | 顺式-4,7,10,13,16,19-二十二碳六烯酸 | 22 6ω-3 |
“延伸酶系统”是指四种酶的套系,其对脂肪酸碳链的延伸负责,来产生比延伸酶系统作用的脂肪酸底物更长2个碳的酸。更具体地,延伸的过程与脂肪酸合成酶相关地发生,其中CoA是酰基载体(Lassner等人,The Plant Cell 8281-292(1996))。在第一步中,已经发现这一步是底物特异性和速度限制性的,丙二酰基CoA与长链酰基CoA缩合来产生CO2和β-酮酰基-CoA(其中酰基部分被延长两个碳原子)。为此,催化这个第一缩合反应的酶将在下文中称为“延伸酶”或“脂肪酸延伸酶”。随后的反应包括还原成β-羟基酰基-CoA、脱水成烯酰基-CoA,并二次还原来产生延长的酰基CoA。
本领域公知的是,脂肪酸延伸酶可以具有不同的底物特异性和选择性,其中“底物特异性”是指当面对单个底物时酶的活性,“底物选择性”描述了从底物混合物中特定底物的选择。例如,C16/18脂肪酸延伸酶优先C16底物,C18/20脂肪酸延伸酶优先C18底物,C20/22脂肪酸延伸酶优先C20底物。同样地,Δ9脂肪酸延伸酶能够分别催化LA和ALA向EDA和ETrA的转化。重要的是注意到,某些脂肪酸延伸酶具有广泛的特异性,因而单个酶可能能够催化几种脂肪酸延伸酶反应(例如,从而作为C16/18脂肪酸延伸酶和C18/20脂肪酸延伸酶)。在优选的实施方式中,最希望的是能够通过用脂肪酸延伸酶的基因转化适合的宿主并确定它对宿主的脂肪酸分布的影响来经验性地确定脂肪酸延伸酶的特异性。
在本公开的上下文中,术语“ELO3”是指高山被孢霉(Mortierellaalpina)C16/18脂肪酸延伸酶酶(在此作为SEQ ID NO:2提供),由elo3基因(SEQ ID NO:1)编码。根据在此报告的数据,ELO3优先地催化棕榈酸(160)向硬脂酸(180)的转化。
术语“脱饱和酶”是指一种多肽,其可以在一种或多种脂肪酸中脱饱和,即,引入双键来产生单或多不饱和脂肪酸。尽管在整个说明书中对于特定脂肪酸使用ω参考系统,更方便的是使用Δ系统从底物的羧基末端开始计数来表明脱饱和酶的活性。在此特别感兴趣的是Δ9脱饱和酶,其催化棕榈酸向棕榈油酸(16 1)和/或硬脂酸向油酸(18 1)的转化。与本公开相关的其他脱饱和酶包括Δ12脱饱和酶,其在分子从羧基末端数起第12和13个碳原子之间脱饱和脂肪酸并催化油酸向LA的转化;Δ15脱饱和酶,其催化LA向ALA的转化;Δ17脱饱和酶,其催化DGLA向ETA和/或ARA向EPA的转化;Δ6脱饱和酶,其催化LA向GLA和/或ALA向STA的转化;Δ5脱饱和酶,其催化DGLA向ARA和/或ETA向EPA的转化,Δ4脱饱和酶,其催化DPA向DHA的转化;以及,Δ脱饱和酶,其催化EDA向DGLA和/或ETrA向ETA的转化。
术语“转化效率”和“底物转化百分比”是指特定的酶(例如脱饱和酶或延伸酶)将底物转化成产物的效力。转化效率根据以下公式测量([产物]/[底物+产物]*100,其中“产物”包括即时产物和来自它的途径中的所有产物。
术语“PUFA生物合成途径酶”是指与PUFA的生物合成相关的任何以下的酶(和编码所述酶的基因),包括Δ4脱饱和酶、Δ5脱饱和酶、Δ6脱饱和酶、Δ12脱饱和酶、Δ15脱饱和酶、Δ17脱饱和酶、Δ9脱饱和酶、Δ8脱饱和酶、Δ9脂肪酸延伸酶、C14/16脂肪酸延伸酶、C16/18脂肪酸延伸酶、C18/20脂肪酸延伸酶和/或C20/22脂肪酸延伸酶。类似地,术语“ω-3/ω-6脂肪酸生物合成途径”是指一组基因,当在适合的条件下表达时它们编码酶,所述酶催化ω-3或ω-6脂肪酸的直接或间接产生。在附图3中说明了代表性的途径,提供了肉豆蔻酸通过各种中间物向DHA的转化,这展现了ω-3和ω-6脂肪酸如何从共同的来源产生。该途径天然地分成两个部分,其中一个部分将产生ω-3脂肪酸,另一个部分仅产生ω-6脂肪酸。仅产生ω-3脂肪酸的部分在此将称为ω-3脂肪酸生物合成途径,而仅产生ω-6脂肪酸的部分在此将称为ω-6脂肪酸生物合成途径。
在此关于PUFA生物合成途径的上下文中使用的术语“功能性”是指在途径中的某些(或所有)基因表达活性的酶。要理解的是,“PUFA生物合成途径”或“功能性PUFA生物合成途径”不意味着需要上文段落中列出的所有基因,因为许多脂肪酸产物将仅需要这个途径的基因的子集的表达。
术语“产油的”是指倾向于以脂质形式保存它们的能源的那些有机体(Weete,In Fungal Lipid Biochemistry,2nd ed,Plenum,1980)。术语“产油酵母”是指可以产生油的、分类为酵母的那些微生物。一般地,产油微生物的细胞油或TAG含量跟随S形曲线,其中脂质的浓度提高直到它在晚对数生长期或早期稳定生长期达到最大值,然后在晚期稳定期和死亡期期间逐渐地降低(Yongmanitchai and Ward,Appl.Environ.Microbiol 57 419-25(1991))。对于产油微生物罕有的是将超过它们细胞干重的约25%积累成油产油酵母的实例包括但不限制于以下的属:耶罗维亚酵母属(Yarrowia)、假丝酵母属(Candida)、红酵母属(Rhodotorula)、红冬孢酵母属(Rhodosporidium)、隐球菌属(Cryptococcus)、丝孢酵母属(Trichosporon)和油脂酵母属(Lipomyces)。
如在此使用的,术语“分离的核酸片段”或“分离的核酸分子”将可互换地使用,是指单链或双链的、任选地含有合成的、非天然的或改变的核苷酸碱基的RNA或DNA的聚合物。DNA聚合物形式的分离的核酸片段可以由cDNA、基因组DNA或合成DNA的一种或多种片段组成。
当核酸分子的单链形式可以在适合的温度和溶液离子强度条件下与另一个核酸分子退火时,核酸分子是与另一个核酸分子,例如cDNA、基因组DNA或RNA分子“可杂交的”。杂交和洗涤条件是公知的,在Sambrook,J,Fntsch,E F and Maniatis,T Molecular Cloning A Laboratory Manual,2nd ed,Cold Spring Harbor Laboratory Cold Spring Harbor,NY(1989)、特别是其中的第11章和其中的表111中(通过引用完全合并在此)举例说明了。温度和离子强度条件决定了杂交的“严格度”。可以调节严格条件来筛选中度相似的片段(例如,来自远缘相关有机体的同源序列)、到高度相似的片段(例如,来自近相关有机体的同功能酶)。杂交后洗涤决定了严格条件一组优选的条件利用一系列的洗涤,从用6×SSC、0.5%SDS在室温下15分钟开始,然后用2×SSC、0.5%SDS在45℃30分钟重复,然后用0.2×SSC、0.5%SDS在50℃30分钟重复两次。一组更优选的严格条件利用更高的温度,其中洗涤与上述的相同,只是在0.2×SSC、0.5%SDS中最后两次30分钟洗涤的温度提高到60℃。另一组优选的高严格条件利用在65℃下在0.1×SSC、0.1%SDS中的两次最后的洗涤。其他的严格条件组包括例如在0.1×SSC、0.1%SDS、65℃杂交,和用2×SSC、0.1%SDS最后通过0.1×SSC、0.1%SDS洗涤。
杂交需要两个核酸含有互补序列,然而取决于杂交的严格度,碱基之间的错配是可能性。杂交核酸的合适的严格度取决于本领域公知的核酸的长度和互补的程度、变量。两个核苷酸序列之间的相似度或同源性越高,具有这些序列的核酸的杂交的Tm的值越大。核酸杂交的相对稳定性(与更高的Tm相应)按照以下顺序降低:RNA RNA、DNARNA、DNA DNA。对于长度超过100个核苷酸的杂交,已经得出了计算Tm的方程式(参见Sambrook等人,supra,950-951)。对于更短的核酸即寡核苷酸的杂交,错配的位置变得更为重要,寡核苷酸的长度决定了它的特异性(参见Sambrook等人,supra,117-118)。在一个实施方式中,可杂交核酸的长度是至少约10个核苷酸。优选的,可杂交核酸的最小长度是至少约15个核苷酸,更优选的至少约20个核苷酸;最优选的,该长度是至少约30个核苷酸。此外,熟练的技术人员将认识到,温度和洗涤溶液盐浓度可以按需要根据一些因素如探针的长度来调节。
氨基酸或核苷酸序列的“基本部分”是包含足够的多肽氨基酸序列或基因核苷酸序列以推定性地鉴定该多肽或基因的部分,所述鉴定通过由本领域技术人员人工评估序列,或通过使用例如BLAST(BasicLocal Alignment Search Tool;Altschul,S F,等人,J Mol.Biol 215403-410(1993))的算法计算机自动化的序列比较和鉴定。一般地,为了推定性地鉴定多肽或核酸序列同源于已知蛋白或基因,十个或更多连续氨基酸或者三十个或更多核苷酸的序列是必需的。此外,对于核苷酸序列,包含20-30个连续核苷酸的基因特异性寡核苷酸探针可以用于序列依赖性的基因鉴定方法(例如,Southern杂交)和分离(例如,细节菌落或噬菌斑的原位杂交)。此外,12-15个碱基的短寡核苷酸可以用作PCR中的扩增引物以获得包含该引物的特定核酸片段。因而,核苷酸序列的“基本部分”包含足够的序列以特异性地鉴别和/或分离包含该序列的核酸片段。本说明书教导了编码一种或多种特定真菌蛋白质的部分或完整氨基酸和核苷酸序列。熟练的技术人员得益于在此报道的序列,现在可以使用所公开序列的所有或基本部分,用于本领域技术人员已知的目的。因而,本发明包含如在附随的序列表中报道的完整序列,以及以上定义的那些序列的基本部分。
术语“互补”被用于描述能够相互杂交的核苷酸碱基之间的关系。例如,对于DNA,腺苷酸互补于胸腺嘧啶,胞嘧啶互补于鸟嘌呤。因而,本发明还包括与附随的序列表中报道的完整序列互补的分离的核酸片段,以及那些基本上相似的核酸序列。
术语“同一性百分比”,如本领域已知的,是通过比较序列确定的、两种或多种多肽序列或两种或多种多核苷酸序列之间的相互关系。在本领域中,“同一性”还意指多肽或多核苷酸序列之间的序列相关性的程度,视情况而定,如通过这些序列的串列之间的匹配所确定的。“同一性”和“相似性”可以通过已知的方法容易地计算,包括但不限于在1)Computational Molecular Biology(Lesk,A M,Ed)OxfordUniversity NY(1988),2)Biocomputinq Informatics and Genome Projects(Smith,D W,Ed)Academic NY(1993),3)Computer Analysis of Sequence Data,Part I(Griffin,A M,and Griffin,H G.,Eds)HumaniaNJ(1994),4)Sequence Analysis in Molecular Biology(von Heinje,G,Ed)Academic(1987),和5)Sequence Analysis Primer(Gribskov,M and Devereux,J,Eds)Stockton NY(1991)中描述的那些。确定同一性的优选的方法被设计以给出测试的序列之间的最佳匹配。确定同一性和相似性的方法被编写成公众可获得的计算机程序。序列比对和同一性百分比计算可以使用LASERGENE生物信息学计算套件(DNASTAR Inc,Madison,Wl)的Megalign程序来进行。使用Clustal比对方法(Higgins and Sharp,cabios.5151-153(1989)))用默认参数(GAP MENALTY=10,GAP LENGTH PENALTY=10)进行多个序列比对。使用CLUSTAL方法的配对式比对的默认参数是KTUPLE 1、GAPPENALTY=3、WINDOW=5和DIAGONALS SAVED=5。
适合的核酸片段(本发明的分离的多核苷酸)编码多肽,所述多肽与在此报道的氨基酸序列至少约70%相同、优选的至少约75%相同、更优选的至少约80%相同。优选的核酸片段编码约85%相同于在此报道的氨基酸序列的氨基酸序列。更优选的核酸片段编码至少约90%相同于在此报道的氨基酸序列的氨基酸序列。最优选的是编码氨基酸序列的核酸片段,所述氨基酸序列至少约95%相同于在此报道的氨基酸序列。适合的核酸片段不仅具有上述同源性,而且一般地编码具有至少50个氨基酸、优选的至少100个氨基酸、更优选的至少150个氨基酸、再更优选的至少200个氨基酸、最优选的至少250个氨基酸的多肽。
术语“序列分析软件”是指对于分析核苷酸或氨基酸序列有用的任何计算机算法或软件程序。“序列分析软件”可以是商业上可获得的或独立地开发的。典型的序列分析软件包括但不限于:1)GCG程序套件(Wisconsin Package Version 90,Genetics Computer Group(GCG),Madison,Wl),2)BLASTP,BLASTN,BLASTX(Altschul等人,JMol Biol.215.403-410(1990)),3)DNASTAR(DNASTAR,IncMadison,Wl),4)Sequencher(Gene Codes Corporation,Ann Arbor,Ml)和5)包括Smith-Waterman算法的FASTA程序(W R Pearson,Comput Methods Genome Res.,[Proc Int Symp](1994),Meeting Date1992,111-20 Suhai,Sandor,Ed Plenum New York,NY)。在本申请的上下文中,要理解的是,当序列分析软件用于分析时,分析的结果将基于所涉及程序的“默认值”,除非另作说明。如在此使用的“默认值”将意指当首次初始化时软件最初加载的任何数值或参数集合。
“密码子简并性”是指在遗传密码中允许核苷酸序列的变异而不影响编码的多肽的氨基酸序列的性质。熟练的技术人员熟知在指定给定氨基酸的核苷酸密码子的利用率方面特定宿主细胞所展现的“密码子偏差”。因而,当为了改善宿主细胞中的表达合成基因时,希望的是设计基因使得它的密码子利用的频率接近宿主细胞的优选的密码子利用频率。
术语“优化的密码子”当涉及基因或核酸片段的编码区时,是指在基因或核酸分子的编码区中改变密码子以反映宿主有机体的典型密码子利用率而不改变DNA编码的多肽。
“基因”是指表达特定蛋白质的核酸片段,并且可以指单独的编码区,或可以包括在编码序列之前(5′非编码序列)和之后(3′非编码序列)的调节序列。“天然基因”是指在自然中存在具有其自身的调节序列的基因。“嵌合基因”是指任何基因,其不是天然基因,包含在自然中不在一起存在的调节和编码序列。因而,嵌合基因可以包含来自不同来源的调节序列和编码序列,或来自相同来源但以不同于自然中存在的方式排列的调节序列和编码序列。“内源基因”是指在有机体的基因组中处在其自然位置的天然基因。“外源的”基因是指通过基因转移导入宿主有机体的基因。外源基因可以包含插入非天然有机体中的天然基因、导入天然宿主内的新位置的天然基因、或嵌合基因。“密码子优化的基因”是一种基因,它的密码子利用的频率被设计以模拟宿主细胞的优选的密码子利用的频率。
“编码序列”是指编码特定氨基酸序列的DNA序列。“适合的调节序列”是指位于编码序列的上游(5′非编码序列)、之内、或下游(3′非编码序列)的核苷酸序列,其影响相连的编码序列的转录、RNA加工或稳定性、或翻译。调节序列可以包括启动子、翻译前导序列、内含子、多聚腺苷酸识别序列、RNA加工位点、效应物结合位点和茎-环结构。
“启动子”是指能够控制编码序列或功能性RNA的表达的DNA序列。一般地,编码序列位于启动子序列的3′。启动子可以整体地来自天然的基因,或由来自自然中发现的不同启动子的不同元件组成,或甚至包含合成的DNA片段。本领域技术人员理解的是,不同的启动子可以指导在不同组织或细胞类型中、或在不同的发育阶段、或响应于不同的环境或生理条件的基因表达。使得基因在大多数时间在大多数细胞类型中表达的启动子通常称为“组成型启动子”。进一步公认的是,大多数情况下,调节序列的确切边界没有被完全地划定,不同长度的DNA片段可能具有相同的启动子活性。
术语“3′非编码序列”或“转录终止子”是指位于编码序列下游的DNA序列。这包括多聚腺苷酸识别序列和编码能够影响mRNA加工或基因表达的调节信号的其他序列。多腺苷酸化信号通常以影响多聚腺苷酸段向mRNA前体的3′末端的添加为特征。3′区域可以影响转录、RNA加工或稳定性,或相连的编码序列的翻译。
“RNA转录产物”是指产自DNA序列的RNA聚合酶催化转录的产物。当RNA转录产物是DNA序列的理想互补拷贝时,它被称为原始转录产物,或者它可以是来自原始转录产物的翻译后加工的RNA序列,被称为成熟RNA。“信使RNA”或“mRNA”是指没有内含子并且可以被细胞翻译成蛋白质的RNA。“cDNA”是指互补于并来自mRNA的双链DNA。“正义”RNA是指包括mRNA并因此可以被细胞翻译成蛋白质的RNA转录产物。“反义RNA”是指一种RNA转录产物,其与目标原始转录产物或mRNA的全部或部分互补并且阻断目标基因的表达(US 5,107,065、WO 99/28508)。反义RNA的互补性可以是对于特定基因转录产物的任何部分,即,在5′非编码序列、3′非编码序列、或编码序列中。“功能性RNA”是指反义RNA、核酶RNA、或不被翻译但仍影响细胞过程的其他RNA。
术语“可操纵地连接的”是指核酸序列在单个核酸片段上的相连,从而一个核酸序列的功能受到另一个的影响。例如,当启动子能够影响编码序列的表达时(即,编码序列处在启动子的转录控制之下),启动子是与编码序列可操纵地连接的。编码序列可以以正义或反义的取向与调节序列可操纵地连接。
如在此使用的,术语“表达”是指来自本发明的核酸片段的正义(mRNA)或反义RNA的转录和稳定积累。表达也可以指mRNA翻译成多肽。
“转化”是指核酸分子进入宿主有机体的转移,引起遗传稳定的遗传。例如,核酸分子可以是自主复制的质粒;或,它可以整合到宿主有机体的基因组中。含有转化的核酸片段的宿主有机体被称为“转基因的”或“重组的”或“转化的”有机体。
术语“质粒”、“载体”和“盒”是指通常携带基因的染色体外元件,通常处于环形双链DNA片段的形式,所述基因不是细胞的中心新陈代谢的部分。这样的元件可以是自主复制的序列、基因组整合序列、噬菌体或核苷酸序列、线性或环形的、单链或双链的DNA或RNA,来自任何来源,其中许多核苷酸序列已经连接或重组成独特的结构,所述结构能够将启动子片段和选定基因产物的DNA序列与合适的3′非翻译序列一起导入细胞。“表达盒”是指含有外源基因并具有除了外源基因之外的元件的特定载体,其容许该基因在外源宿主中增强的表达。
术语“同源重组”是指在两个DNA分子之间DNA片段的交换(在交叉期间)。交换的片段的侧翼是这两个DNA分子之间相同核苷酸序列的位点(即,“同源性区域”)。
术语“同源性区域”是指在参与同源重组、具有相互的同源性的核酸片段上核苷酸序列的延伸。有效的同源重组一般将在这些同源性区域长度至少约10bp的地方发生,长度至少约50bp的地方是优选的。一般地,意图重组的片段含有至少两个同源性区域,在那里目标基因破坏或替换是期望的。
在此使用的标准重组DNA和分子扩增技术是本领域公知的,由Sambrook,J,Fntsch,E F and Maniatis,T,Molecular Cloning A Laboratory Manual,2nd ed,Cold Spring Harbor Laboratory Cold SpringHarbor,NY(1989)(hereinafter″Maniatis″),by Silhavy,T J,Bennan,M L and Enquist,L W,Experiments with Gene Fusions,Cold SpringHarbor Laboratory Cold Spring Harbor,NY(1984),以及由Ausubel,FM等人,Current Protocols in Molecular Biology,published by GreenePublishing Assoc and Wiley-lnterscience(1987)所描述。
高山被孢霉(Mortierella alpina)C 16/18 脂肪酸延伸酶的序列鉴定
在本发明中,编码C16/18脂肪酸延伸酶(在此称为“ELO3”)的基因已经从高山被孢霉(Mortierella alpina)的专有DuPont cDNA库分离。高山被孢霉(M.alpina)是在它的TAG级分中天然地积累具有等于或大于C20链长度的脂肪酸的有机体,因而表明C16/18脂肪酸延伸酶可能以高效率起作用来确保硬脂酸进入PUFA生物合成途径的大的流量。
使用BLAST算法(Altschul,S F,等人,Nucleic Acids Res 253389-3402(1997))与公众数据库比较C16/18脂肪酸延伸酶核苷酸碱基和推断的氨基酸序列揭示了,大多数相似的已知序列在275个氨基酸的长度上约37%相同于在此报道的C16/18脂肪酸延伸酶的氨基酸序列。优选的氨基酸片段至少约70%-80%相同于此处的序列,其中85%-90%相同的那些序列是特别适合的,约95%相同的那些序列是最优选的。类似地,编码相应于当前ORF的优选的C16/18脂肪酸延伸酶编码核酸序列是编码活性蛋白质的那些,其至少约70%-80%相同于编码在此报道的C16/18脂肪酸延伸酶的核酸序列,其中85%-90%相同的那些序列是特别合适的,约95%相同的那些序列是最优选的。
同源物的鉴定和分离
本发明的C16/18脂肪酸延伸酶核酸片段可以用于从相同的或其他的细菌、藻类、真菌或植物物种鉴定和分离编码同源蛋白质的基因。
鉴定技术。例如,在此描述的高山被孢霉(Mortierella alpina)C16/18脂肪酸延伸酶氨基酸或核苷酸序列的基本部分可以用于推定地鉴定相关的多肽或基因,通过本领域技术人员的人工评估,或通过利用算法例如BLAST(Basic Local Alignment Search Tool,Altschul,S F,等人,J.Mol Biol 215403-410(1993))和ClustalW(DNASTAR软件的Megahgn程序)的计算机自动化序列比较和鉴定。
做为选择,当前的脂肪酸延伸酶序列可以用作杂交试剂用于同源物的鉴定。核酸杂交测试的基本成分包括探针、怀疑含有感兴趣的基因或基因片段的样品和特定的杂交方法。本发明的探针一般是互补于要检测的核酸序列的单链核酸序列。探针是与要检测的核酸序列“可杂交的”。探针长度可以从5个碱基变化到数万碱基,将取决于所要进行的特定测试。一般地,约15个碱基到约30个碱基的探针长度是适合的。探针分子的仅一部分需要与要检测的核酸序列互补。此外,探针和目标序列之间的互补性不必是完美的。在不完全互补的分子之间确实发生杂交,结果是杂交区域中的碱基的某些部分不与适当的互补碱基配对。
杂交方法是明确定义的。一般地,探针和样品必须在允许核酸杂交的条件下混合。这涉及在适当的浓度和温度条件下在存在无机盐或有机盐的情况下接触探针和样品。探针和样品核酸必需接触足够长时间,从而可以发生在探针和样品核酸之间任何可能的杂交。混合物中探针或目标的浓度将决定发生杂交需要的时间。探针或目标浓度越高,所需的杂交孵育时间越短。任选的,可以添加离液剂。离液剂通过抑制核酸酶活性稳定了核酸。此外,离液剂容许短寡核苷酸探针在室温下的敏感和严格的杂交(Van Ness and Chen,Nucl.Acids Res 195143-5151(1991))。适合的离液剂包括盐酸胍、硫氰酸胍、硫氰酸钠、四氯乙酸锂、高氯酸钠、四氯乙酸铷、碘化钾和三氟乙酸铯,等等。一般地,离液剂将以约3M的终浓度存在。如果希望,人们可以向杂交混合物添加甲酰胺,一般30-50%(v/v)。
可以采用各种杂交溶液。一般地,这些包括约20到60%体积、优选的30%的极性有机溶剂。常见的杂交溶液采用约30-50%v/v甲酰胺、约0.15到1M氯化钠、约0.05到0.1M缓冲液(例如,柠檬酸钠、Tris-HCl、PIPES或HEPES(pH范围约6-9)、约0.05-0.2%洗涤剂(例如,十二烷基磺酸钠),或0.5-20mM EDTA、FICOLL(Pharmacia Inc)(约300-500kdal)、聚乙烯吡咯烷酮(约250-500kdal)和血清白蛋白。还包括在典型的杂交溶液中的是约0.1到5mg/mL的未标记的载体核酸、片段化的核DNA(例如,小牛胸腺或鲑鱼精子DNA,或酵母RNA),和任选的约0.5到2%wt/vol甘氨酸。其他添加剂还可以包括,例如体积排阻试剂,其包括各种极性水可溶的或可膨胀试剂(例如,聚乙二醇)、阴离子聚合物(例如,聚丙烯酸酯或聚甲基丙烯酸甲酯)和阴离子糖聚合物(例如,硫酸葡聚糖)。
核酸杂交可适应各种分析形式。最适合的一种是夹心分析形式。夹心分析特别适合于在非变性条件下的杂交。夹心型分析的基本元件是固相支持物。固相支持物具有吸附在其上或共价联结到其上的固定的核酸探针,所述核酸探针是未标记的并与序列的一部分互补。
分离方法 在此鉴定的高山被孢霉(Mortierella alpina)C16/18脂肪酸延伸酶可以用于从相同的或其他的细菌、藻类、真菌或植物物种鉴定和分离编码同源蛋白质的基因,基于序列依赖性的方案。例如,这些方案包括但不限于1)核酸杂交的方法,2)DNA和RNA扩增的方法,例如核酸扩增技术的各种应用[例如,聚合酶链式反应(PCR)Mullis等人,美国专利4,683,202,连接酶链式反应(LCR)Tabor,S.等人,Proc.Natl Acad Sci USA.,89392(1992)],和3)库构建和通过互补筛选的方法。
例如,编码与在此描述的脂肪酸延伸酶相似的蛋白质或多肽的基因可以利用本领域技术人员公知的方法通过使用本核酸片段的全部或部分作为DNA杂交探针筛选来自任何期望的酵母或真菌(其中产生PUFA的那些酵母或真菌将是优选的)的库来直接分离。基于当前核酸序列的特定寡核苷酸探针可以通过本领域已知的方法来设计和合成(Maniatis,同上)。此外,整个序列可以通过熟练的技术人员已知的方法直接使用来合成DNA探针(例如,随机引物DNA标记、切口转译或末端标记技术),或合成体外转录系统中可用的RNA探针。此外,可以涉及特定引物并用于控制当前序列的一部分(或全长)。产生的扩增产物可以在扩增反应期间直接标记或在扩增反应之后标记,并用作探针来在合适严格度的条件下分离全长DNA片段。
一般地,在PCR型扩增技术中,引物具有不同的序列并且相互不互补。取决于期望的试验条件,引物的序列将被设计以提供目标核酸的有效和可靠的复制。PCR引物设计的方法是本领域常见和公知的(Them and Wallace,″The use of oligonucleotides as specifichybridization probes in the Diagnosis of Genetic Disorders″,in HumanGenetic Diseases.A Practical Approach,K E Davis Ed,(1986)pp 33-50,IRL Herndon,VA,and Rychhk,W,In Methods in Molecular Biology,White,B A Ed,(1993)Vol 15,pp 31-39,PCR Protocols Current Methodsand Applications Humania Totowa,NJ)。
一般地,当前ELO3序列的两个短片段可以用于PCR方案中来从DNA或RNA扩增编码同源基因的更长的核酸片段。PCR也可以在克隆的核酸片段的库上进行,其中一个引物的序列来自当前核酸片段,另一个引物的序列利用了mRNA前体编码微生物基因的3′末端的多聚腺苷酸段的存在。或者,第二引物序列可以基于来自克隆载体的序列,例如,使用RACE方案(Frohman等人,PNAS USA 85 8998(1988))通过使用PCR来扩增转录产物中的单个点和3′或5′末端之间的区域的拷贝来产生cDNA。在3′和5′方向上定向的引物可以从当前序列设计。利用商业上可获得的3′RACE或5′RACE系统(Gibco/BRL,Gaithersburg,MD),可以分离特定的3′或5′cDNA片段(Ohara等人,PNAS USA 86 5673(1989),Loh等人,Science 243 217(1989))。
当前核苷酸和推断的氨基酸C16/18脂肪酸延伸酶序列的可用性便于DNA表达库的免疫学筛选。可以合成代表当前氨基酸序列的部分的合成肽。这些肽可以用于免疫动物来产生具有对包含该氨基酸序列的肽或蛋白质的特异性的多克隆或单克隆抗体。这些抗体然后可以用于筛选DNA表达库来分离感兴趣的全长DNA克隆(Lerner,R A AdvImmunol.361(1984);Maniatis,同上)。
改善异源表达的基因优化
在鉴定和分离特定的C16/18脂肪酸延伸酶(例如,SEQ ID NO:1和2)之后,可以利用各种技术来改善感兴趣的C16/18脂肪酸延伸酶在可选择的宿主中的表达。两种这种技术包括密码子优化和基因的诱变。
本领域技术人员理解的是,修改要在特定外源宿主中表达的、编码特定多肽的一部分密码子通常是有用的(从而修饰的多肽利用该可选择寄主优选的密码子),因为这可以充分地提高编码多肽的外源基因的表达。本领域技术人员将熟悉确定特定宿主物种中宿主优选的密码子并计算和合成可选择的编码序列的技术(例如,参见WO2004/101753),因而,例如,在本发明的一个实施方式中,可能希望的是修改编码ELO3多肽的一部分密码子来提高基因在解脂耶罗维亚酵母(Yarrowia lipolytica)中的表达。
在可选择的实施方式中,诱变技术,例如体外诱变和选择、定点诱变、error prone PCR(Melnikov等人,Nucleic Acids Research,27(4)1056-1062(February 15,1999))、“基因混洗”(US 5,605,793、US5,811,238、US 5,830,721和US 5,837,458)或其他手段可以被采用来获得天然发生的延伸酶基因例如在此描述的C16/18脂肪酸延伸酶的突变体(其中这种突变体可以包括删除、插入、点突变或其组合)。这将允许产生具有体内脂肪酸延伸酶活性、对于在特定宿主细胞中起作用具有更期望的物理和动力学参数(例如,更长的半衰期或更高的期望脂肪酸的生产率)的多肽。或者,如果希望,对于酶活性重要的目标多肽的区域可以通过常规诱变、表达产生的突变多肽并测定它们的活性来确定。这些技术的综述在WO 2004/101757中描述了。
来自在此描述的C16/18脂肪酸延伸酶基因的所有这些突变的蛋白质和编码它们的核苷酸序列处于本发明的范围之内。并且,本发明的方法包括使用如附随的序列表中报道的C16/18脂肪酸延伸酶的完整序列、这种完整序列的互补物、这个序列的基本部分、由此衍生的密码子优化的延伸酶和与之基本上同源的那些序列。
脂肪酸的微生物生物合成
脂质代谢是几乎所有的动物、植物和微生物具有的基础代谢过程,因而,几乎所有这些有机体具有合成棕榈酸(16 0)、硬脂酸(18 0)和油酸(18 1)的能力(因为这些脂肪酸对于磷脂生物合成是必需的)。如附图3中所说明的,这些脂肪酸的每一个都来自肉豆蔻酸(14 0)。特别地,游离棕榈酸经由延伸反应从肉豆蔻酸产生(即,通过C14/15脂肪酸延伸酶催化),随后,棕榈酸可以通过C16/18脂肪酸延伸酶延伸来产生硬脂酸,或脱饱和来产生棕榈油酸(16 1)。由于棕榈酸的原始结局是延伸,然而,断定的是C16/18脂肪酸延伸酶在确定进入脂肪酸生物合成途径的总体碳通量方面起到重要作用,因为这些酶控制了微生物细胞中产生的硬脂酸的数量。
对于操作生物化学途径有用的方法是本领域技术人员公知的。预计的是,在合适的启动子的控制下导入编码在此描述的C16/18脂肪酸延伸酶的嵌合基因将引起转化的宿主有机体中硬脂酸的提高的产生。因而,本发明的一个目的是提供在产油酵母中产生硬脂酸的方法,其中向所述产油酵母提供(a)编码SEQ ID NO:2所示C16/18脂肪酸延伸酶的分离的核酸片段,和(b)包含棕榈酸的延伸酶底物的来源,其中所述酵母在一定条件下生长从而所述嵌合延伸酶基因被表达并且棕榈酸被转化成硬脂酸,其中任选的回收所述硬脂酸。
此外,在此描述的C16/18脂肪酸延伸酶基因和它相应的酶产物可以间接地地用于ω-3和/或ω-6 PUFA的产生。特别地,预期的是在此描述的C16/18脂肪酸延伸酶可以与编码其他酶的一种或多种基因一同表达,从而发生一系列的反应来产生期望的PUFA产物。
虽然许多微生物(包括藻类、细菌、霉菌、真菌和酵母)可以在普通的细胞代谢过程中合成PUFA和Ω脂肪酸,还可能的是将这种能力导入不天然地产生PUFA的有机体中(或产生期望的PUFA和/或脂质分布型)。本领域技术人员将熟悉将编码用于PUFA生物合成的合适的酶的表达盒导入所选宿主有机体所必需的考虑和技术。为了这些目的,已经通过遗传学方法鉴定了涉及PUFA产生的多种脱饱和酶和延伸酶基因,这些基因的某一些的DNA序列是公众可获得的(例如,对于在GenBank中可获得基因和/或关于选择具有脱饱和酶或者延伸酶活性的特定多肽的专利文献和考虑的综述,参见WO2004/101757)。并且,虽然在此没有详细描述,在文献中提供了大量教导,其中各种有机体被工程化来产生特定PUFA,以下提供了某些说明性的参考文献,而这些不应被看作是限制性的:WO 98/46763,WO 98/46764,WO98/46765,WO 99/64616,WO 2002/077213,WO 2003/093482,WO2004/057001,WO 2004/090123,WO 2004/087902,U S 6,140,486,U.S6,459,018,U S 6,136,574,U S.2003/0172399,U S 2004/0172682,U S.2004/098762;U S 2004/0111763,U S 2004/0053379,U S 2004/0049805,U S 2004/0237139;U.S 04/0172682,Beaudoin F等人,PNAS USA,97(12).6421-6426(2000),Dyer,J M等人,Appl.Envi Microbiol,59224-230(2002),Domergue,F.等人Eur.J.Biochem.269 4105-4113(2002),Qi,B等人,Nature Biotech.22 739-745(2004)和Abbadi等人,The Plant Cell,162734-2748(2004))。
因此,例如,在此描述的C16/18脂肪酸延伸酶可以与编码PUFA酶的一种或多种PUFA生物合成途径基因一同表达,从而发生一系列的反应来产生期望的PUFA产物。在优选的实施方式中,例如,宿主有机体可以用1)编码本发明的高山被孢霉(M.alpina)C16/18脂肪酸延伸酶的嵌合基因,和2)包含编码PUFA生物合成途径的酶(例如,Δ9脱饱和酶、Δ12脱饱和酶、Δ6脱饱和酶、C18/20脂肪酸延伸酶、Δ5脱饱和酶、Δ17脱饱和酶、C20/22脂肪酸延伸酶、Δ4脱饱和酶、Δ15脱饱和酶、Δ9脂肪酸延伸酶和/或Δ8脱饱和酶)的其他基因的载体转化。与在未表达本发明的编码高山被孢霉(M.alpina)C16/18脂肪酸延伸酶的嵌合基因的相同转化宿主中发生的相比,这种转化宿主预计将产生提高数量的ω-3和/或ω-6脂肪酸(例如,LA、ALA、EDA、GLA、STA、ETrA、DGLA、ETA、ARA、EPA、DPA和DHA,参见附图3)。包括在特定表达盒内的特定基因将取决于宿主细胞(和它的PUFA分布型和/或脱饱和酶/延伸酶分布型)、底物的可用性、以及期望的终产物。
在可选择的实施方式中,可能有用的是破坏宿主有机体的天然C16/18脂肪酸延伸酶,根据在此描述的完整序列、那些完整序列的互补物、那些序列的基本部分、由此衍生的密码子优化的脱饱和酶和那些与之基本上同源的序列。例如,宿主有机体中C16/18脂肪酸延伸酶的定向破坏产生了不能合成硬脂酸的突变菌株,因而为了存活需要C18脂肪酸的补充。
在可选择的实施方式中,包含其天然C16/18脂肪酸延伸酶的破坏或钝化的转化宿主有机体然后可以被有益地转化来表达异源C16/18脂肪酸延伸酶(例如,如果异源C16/18脂肪酸延伸酶具有与天然C16/18脂肪酸延伸酶相比不同的底物特异性)。这种操作可以降低天然和异源C16/18脂肪酸延伸酶之间的底物竞争。因而,与相应的天然解脂耶罗维亚酵母(Yarrowia lipolytica)C16/18脂肪酸延伸酶的敲除一同、异源C16/18脂肪酸延伸酶(即,SEQ ID NO:2)的表达可以显著地提高在为PUFA生物合成工程化的转化体解脂耶罗维亚酵母(Y.lipolytica)宿主细胞中产生的总体长链ω-3/ω-6PUFA。
对于基因破坏,外源DNA片段(一般是可选择标记基因)被插入要破坏的结构基因中来打断它的编码序列从而功能上钝化该基因。破坏盒对宿主细胞中的转化引起功能性天然基因通过同源重组被无功能的破坏的基因替换(参见,例如Hamilton等人,J.Bactenol 1714617-4622(1989),Balbas等人,Gene 136 211-213(1993),Gueldener等人,Nucleic Acids Res 24 2519-2524(1996),和Smith等人,MethodsMol.Cell Biol 5 270-277(1996))。
反义技术是当目标基因的序列已知时减量调节基因的另一种方法。为了实现这一点,来自期望基因的核酸片段被克隆并可操纵地连接到启动子,从而将转录RNA的反义链。这个构建体然后被导入宿主细胞,产生RNA的反义链。反义RNA通过防止编码目标蛋白质的mRNA的积累来抑制基因表达。本领域的技术人员将知道,与使用反义技术以降低特定基因的表达相关的专门的考虑。例如,反义基因的适当表达水平可能需要使用利用熟练的技术人员已知的不同调节元件的不同嵌合基因。
虽然当序列已知时目标基因破坏和反义技术提供了减量调节基因的有效手段,已经开发了不基于序列的其他较低特异性的方法(例如,经由UV照射/化学试剂的诱变,或使用可转座元件/转座子;参见USSN10/840579)。
表达系统、盒和载体
在此描述的本序列的基因和基因产物可以在异源微生物宿主细胞中、特别是在产油酵母(例如,解脂耶罗维亚酵母(Yarrowia lipolytica))的细胞中产生。
含有指导外源蛋白的高水平表达的调节序列的微生物表达系统和表达载体是本领域技术人员公知的。任何这些可以用于构建嵌合基因,用于产生当前脂肪酸延伸酶序列的基因产物。这些嵌合基因然后可以经由转化被导入合适的微生物中以提供所编码的酶的高水平表达。
对于转化适合的宿主细胞有用的载体或DNA盒是本领域公知的。在构建体中存在的序列的特定选择取决于期望的表达产物(上文)、宿主细胞的性质以及分离转化的细胞与未转化的细胞的预计手段。然而,一般地,载体或盒含有指导相关基因的转录和翻译的序列、可选择标记以及容许自主复制或染色体整合的序列。适合的载体包含基因5′的控制转录起始的区域,和DNA片段的3′的控制转录终止的区域。当两个控制区域都来源于来自被转化宿主细胞的基因时是最优选的,然而要理解的是这种控制区域不必来源于对于选择作为生产宿主的特定物种是天然的基因(对于有用的起始控制区域和终止子,参见,例如,USSN 10/840579)。然而,在某些优选的实施方式中,转录起始调节区域获自甘油醛-3-磷酸-脱氢酶(USSN 10/869630,共同待决美国专利申请No.11/183664)、磷酸甘油酸变位酶(USSN 10/869630)、果糖-二磷酸醛缩酶(USSN 10/987548)、甘油-3-磷酸O-酰基转移酶(美国专利申请No.60/610060)、铵转运蛋白(共同待决美国专利申请No.11/185301)和翻译延伸因子EF1-α(TEF)蛋白(US 6,265,185)。
技术人员了解,仅仅将基因插入克隆载体中不能确保它将以所需的水平成功地表达。响应于高表达速度的需求,许多专业化表达载体已经通过操作许多不同的遗传元件来创建,所述遗传元件控制转录、翻译、蛋白质稳定性、氧显著、从宿主细胞的分泌的方面。更具体地,被操作来控制基因表达的一些分子特征包括1)相关转录启动子和终止子序列的性质,2)克隆的基因的拷贝数,无论该基因是质粒携带的还是整合到宿主细胞的基因组中的,3)合成的外源蛋白质的最终的细胞位置,4)在宿主有机体中的翻译效力,5)克隆的基因蛋白质在宿主细胞内的固有稳定性;和6)克隆的基因内的密码子利用率,从而它的频率接近宿主细胞的优选的密码子利用的频率。这些修饰类型的每一种都包括在本发明中,作为进一步优化在此描述的C16/18脂肪酸延伸酶的表达的手段。
C
16/18
脂肪酸延伸酶的重组表达的优选的微生物宿主
用于表达当前C16/18脂肪酸延伸酶基因和核酸片段的宿主细胞可以包括微生物宿主,其在广泛的温度和pH值下在各种进料(feedstock)上生长,包括简单的或复杂的碳水化物、有机酸和醇类、和/或烃类。虽然本发明中描述的基因被分离用于在产油酵母、特别是解脂耶罗维亚酵母(Yarrowia lipolytica)中表达,预期的是,由于转录、翻译和蛋白质生物合成机构是高度保守的,任何细菌、酵母、藻类和/或丝状真菌将是表达当前核酸片段的适合的宿主。
优选的微生物宿主是产油有机体,例如产油酵母。这些产油有机体天然能够进行油合成和积累,其中总油含量可以包括大于约25%的细胞干重,更优选的大于约30%的细胞干重,以及最优选的大于约40%的细胞干重。另外,存在着使用这些有机体用于产生PUFA的基础,如在USSN 10/840579和共同待决美国专利申请No.60/624812中所见。
一般地鉴定为产油酵母的属包括但不限于耶罗维亚酵母属(Yarrowia)、假丝酵母属(Candida)、红酵母属(Rhodotorula)、红冬孢酵母属(Rhodosporidium)、隐球菌属(Cryptococcus)、丝孢酵母属(Trichosporon)和油脂酵母属(Lipomyces)。更具体地,说明性的油合成酵母包括圆红冬孢酵母属(Rhodosporidium toruloides)、斯达氏油脂酵母(Lipomyces starkeyii)、L hpoferus,Candida revkaufi、C.pulchernma、C.tropicalis、C.utilis、Trichosporon pullans、T.cutaneum、Rhodotorulaglutmus、R.graminis和解脂耶罗维亚酵母(Yarrowia lipolytica)(之前分类为Candida lipolytica)。
最优选的是产油酵母解脂耶罗维亚酵母(Yarrowia lipolytica);以及,在进一步的实施方式中,最优选的是指定为ATCC#20362、ATCC#8862、ATCC#18944、ATCC#76982、ATCC#90812和/或LGAM S(7)1(Papanikolaou S,and Aggelis G.,Bioresour.Technol 82(1)43-9(2002))的解脂耶罗维亚酵母(Y.lipolytica)菌株。
微生物宿主的转化
一旦获得了编码适合于在产油酵母中表达的多肽的DNA,它被置于能够在宿主细胞中自主复制的质粒载体中,或它被直接整合到宿主细胞的基因组中。表达盒的整合可以在宿主基因组内随机地发生,或可以通过使用含有与宿主基因组同源的区域的构建体,其足以将重组物靶向宿主基因座。当构建体被靶向内源基因座时,全部或某些转录和翻译调节区域可以由内源基因座提供。
当从独立复制的载体表达两种或多种基因时,期望的是每种载体具有不同的选择手段并缺少与另一个构建体的同源性,以维持稳定表达和防止构建体之间元件的重新分配。导入的构建体的调节区、选择手段和增殖方法的明智选择可以实验上地确定,从而所有导入的基因以必要的水平表达以提供期望产物的合成。
包含感兴趣的C16/18脂肪酸延伸酶基因的构建体可以通过任何标准技术导入宿主细胞。这些技术包括转化(例如,乙酸锂转化[Methods inEnzymology,194 186-187(1991)])、原生质体融合、基因枪(biohsticimpact)、电穿孔、显微注射或将感兴趣的基因导入宿主细胞的任何其他方法。适合于产油酵母(即,解脂耶罗维亚酵母(Yarrowia lipolytica))的更具体的教导包括美国专利NO.4,880,741和NO.5,071,764以及Chen,D C et al(Appl Microbiol Biotechnol 48(2)232-235(1997))
为了方便起见,已经通过任何方法操作来接受DNA序列(例如,表达盒)的宿主细胞在此将称为“转化的”或“重组的”。转化的宿主将具有表达构建体的至少一个拷贝,可以具有两个或多个,取决于基因是整合到基因组中、扩增还是存在于具有多个拷贝数的染色体外元件上。转化的宿主细胞可以通过各种选择技术来鉴别,如USSN10/840579和USSN 10/869630中所描述的。在此使用的一些优选的选择方法是对卡那霉素、匀霉素、氨基糖苷G418的抗性,以及在缺乏尿嘧啶、亮氨酸、赖氨酸、色氨酸或组氨酸的培养基上生长的能力。
在转化之后,适合于当前C16/18脂肪酸延伸酶(以及,在宿主细胞内共表达的任选的其他PUFA酶)的底物可以通过宿主天然地或转基因地生产,或者可以外源地提供它们。
硬脂酸生产的发酵过程
转化的微生物宿主细胞在一定条件下生长,所述条件优化脂肪酸生物合成基因的活性并产生最大和最经济的脂肪酸产量(例如,硬脂酸,其可以随后提高各种ω-3和/或ω-6脂肪酸的产量)。一般地,可以被优化的培养基条件包括碳源的类型和数量、氮源的类型和数量、碳氮比例、含氧量、生长温度、pH值、生物质生产阶段的长度、油积累阶段的长度以及细胞收获的时间。感兴趣的微生物,例如产油酵母在复合培养基(例如,酵母提取物-蛋白胨-葡萄糖肉汤(YPD))中生长,或在缺乏生长必需成分的限定的基本培养基中生长从而促使选择期望的表达盒(例如Yeast Nitrogen Base(DIFCO Laboratories,Detroit,M1))。
本发明中的发酵培养基必需含有适合的碳源。适合的碳源可以包括,但不限于单糖(例如,葡萄糖、果糖)、二糖(例如,乳糖或蔗糖)、低聚糖、多糖(例如,淀粉、纤维素或其混合物)、糖醇(例如,甘油)或来自可更新进料的混合物(例如,干酪乳清渗透物、玉米浸泡液、甜菜糖蜜、大麦芽)。另外,碳源可以包括烷烃、脂肪酸、脂肪酸的酯、甘油单酯、甘油二酯、甘油三酯、磷脂和各种脂肪酸的商业来源,包括植物油(例如,大豆油)和动物脂肪。此外,碳底物可以包括一碳底物(例如,二氧化碳或甲醇),已经展现了它们代谢转变成关键的生物化学中间物。因此预期的是,在本发明中利用的碳源可以包括各种各样的含碳底物,将仅由宿主有机体的选择来限制。虽然所有以上提及的碳底物和其混合物预计在本发明中是适合的,优选的碳底物是糖类和/或脂肪酸。最优选的是葡萄糖和/或含有10-22个碳之间的脂肪酸。
氮可以从无机的(例如,(NH4)2SO4)或有机的(例如,尿素或谷氨酸)来源提供。除了合适的碳和氮源之外,发酵培养基还必需含有适合的矿物质、盐、辅助因子、缓冲液、维生素和本领域技术人员已知的其他成分,适合于微生物的生长并促进PUFA生产所必需的酶途径。
特别注意的是给予促进脂质和PUFA合成的几种金属离子(例如,Mn+2、Co+2、Zn+2、Mg+2)(Nakahara,T等人,Ind Appl.Single Cell Oils,D J Kyle and R Colin,eds pp 61-97(1992))。
本发明中优选的生长培养基是常见的商业上制备的培养基,例如Yeast Nitrogen Base(DIFCO Laboratories,Detroit,Ml)。其他限定的或合成的生长培养基也可以使用,对于特定微生物的生长适合的培养基将是微生物学或发酵科学领域的技术人员已知的。发酵的适合的pH值范围一般在约pH 4.0到pH 8.0之间,其中pH5.5到pH 7.0是初始生长条件优选的范围。发酵可以在有氧或缺氧条件下进行,其中微有氧条件是优选的。
一般地,产油酵母细胞中高水平的PUFA的积累需要两个阶段的过程,因为代谢状态必需在生长和脂肪的合成/储存之间“平衡”。因而,最优选地,二阶段发酵过程是产油酵母中PUFA的生产所必需的。在WO 2004/101757中描述了这种方法,以及各种适合的发酵过程设计(即,分批、进料分批和连续)和生长期间的考虑。
PUFA的纯化
脂肪酸、包括PUFA可以在宿主微生物中作为游离脂肪酸或以酯化的形式存在,例如酰基甘油、磷脂、硫脂或糖脂,可以通过各种本领域公知的方式从宿主细胞提取。酵母脂质的提取技术、质量分析和接受标准的一篇综述是Z Jacobs的综述(Critical Reviews inBiotechnology 12(5/6)463-491(1992))。下游加工的简要综述也是可获得的,A Singh和O Ward(Adv.Appl.Microbiol 45271-312(1997))。
一般地,纯化PUFA的方法可以包括用有机溶剂提取、超声处理、超临界流体提取(例如,使用二氧化碳)、皂化、和物理方式,例如压榨,或其组合。对于其他细节参考WO 2004/101757的教导。
优选实施方式的说明
在此描述的工作的最终目标是开发积累在ω-3和/或ω-6PUFA中富集的油的产油酵母。为此,必需鉴定在产油酵母中有效地起作用的脂肪酸延伸酶,以允许在这些宿主中优选的PUFA的合成和高积累。在早先的工作中,申请人过量表达了大鼠C16/18脂肪酸延伸酶蛋白质(来自Rattus norvegicus的rELO2,GenBank Accession No.AB071986),虽然rELO2在解脂耶罗维亚酵母(Yarrowia lipolytica)中被成功地表达作为提高下游PUFA的产量的手段(美国专利申请No.60/624812),在转化的宿主中这个基因的存在无助于管理批号的获得。结果,进行了当前的工作来鉴定更合适的C16/18脂肪酸延伸酶。
根据BLAST分析,在专有的高山被孢霉(Mortierella alpina)cDNA库中鉴定了cDNA片段。该基因的编码区(暂定名称“elo3”)在强天然启动子的控制下在解脂耶罗维亚酵母(Yarrowia lipolytica)中过量表达,从而产生与对照菌株相比产生多35%的C18脂肪酸(18 0,C 18 1,C18 2和GLA)和少31%的C16脂肪酸。因此,这些数据表明高山被孢霉(M.alpina)ELO3使用C16脂肪酸作为底物来产生C18脂肪酸。
预计的是,高山被孢霉(M.alpina)ELO3将适合于提高进入ω-3/ω-6脂肪酸生物合成途径的碳通量,从而提高工程化的耶罗维亚酵母属(Yarrowia)菌株中更长链的脂肪酸油含量。因而,本发明的一个实施方式是改变产油酵母中脂肪酸分布型的方法,其中高山被孢霉(M.alpina)ELO3被单独表达或与其他PUFA生物合成途径基因(例如,Δ9脱饱和酶、Δ12脱饱和酶、Δ6脱饱和酶、C18/20脂肪酸延伸酶、Δ5脱饱和酶、Δ17脱饱和酶、C20/22脂肪酸延伸酶、Δ4脱饱和酶、Δ15脱饱和酶、Δ9脂肪酸延伸酶和/或Δ8脱饱和酶)一同表达以允许长链PUFA(例如LA、ALA、EDA、GLA、STA、ETrA、DGLA、ETA、ARA、EPA、DPA和/或DHA)的提高的产生。
实施例
在以下实施例中进一步定义本发明。要理解的是,代表本发明的优选的实施方式的这些实施例仅通过例示的方式给出。根据以上的说明和这些实施例,本领域技术人员可以容易地确定本发明的基本特征而不背离本发明的精神和范围,可以进行本发明的各种变化和修改来使其适应各种用途和状况。
一般方法
在实施例中使用的标准重组DNA和分子扩增技术是本领域公知的,由1)Sambrook,J,Fntsch,E F and Maniatis,T.Molecular Cloning.A Laboratory Manual,Cold Spring Harbor Laboratory Cold SpringHarbor,NY(1989)(Maniatis);2)T J Silhavy,M L Bennan,and LW Enquist,Experiments with Gene Fusions,Cold Spring HarborLaboratory Cold Spring Harbor,NY(1984)和3)Ausubel,F M等人,Current Protocols in Molecular Biology,published by Greene PublishingAssoc and Wiley-lnterscience(1987)所描述。
适合于细菌培养物的维持和生长的材料和方法是本领域公知的。适用于以下实施例的技术可以在Manual of Methods for General Bacteriology(Philhpp Gerhardt,R.G E Murray,Ralph N.Costilow,Eugene W Nester,Willis A Wood,Noel R Kneg and G Bnggs Phillips,Eds)、American Society for Microbiology Washington,D C(1994))或Thomas D Brock in Biotechnology A Textbook of Industrial Microbiology,2nd ed,Sinauer Associates Sunderland,MA(1989)中找到。对于细菌细胞的生长与维持使用的所有试剂、限制性内切酶和材料是从Aldnch Chemicals(Milwaukee,Wl)、DIFCO Laboratories(Detroit,Ml)、GIBCO/BRL(Gaithersburg,MD)或Sigma ChemicalCompany(St Louis,MO)获得的,除非另作说明。
E.coli TOP10细胞和E.coll Electromax DH10B细胞获自Invitrogen(Carlsbad,CA)。E.coli DH5α的最大效力感受态细胞获自GIBCO/BRL(Gaithersburg,MD)。Ecoli(XL1-Blue)感受态细胞购自StratageneCompany(San Diego,CA)。所有E.coli菌株一般在37℃在Luna Bertani(LB)平板上生长。
一般的,根据标准方法(Sambrook等人,上文)进行分子克隆。通过Sigma-Genosy(Spring,TX)合成寡核苷酸。PCR产物克隆到Promega的pGEM-T-easy载体(Madison,WI)中。
利用载体和间插特异性引物的组合使用染料终止子技术(US5,366,860、EP 272,007)在ABI自动测序仪上产生DNA序列。序列编辑在Sequencher(Gene Codes Corporation,Ann Arbor,MI)中进行。所有序列在两个方向上呈现覆盖至少两次。遗传序列的比较使用DNASTAR软件(DNASTAR Inc,Madison,WI)进行。
缩写的含义如下:“sec”是指秒,“min”是指分钟,“h”是指小时,“d”是指天,“μl”是指微升,“ml”是指毫升,“L”是指升,“μM”是指微摩,“mM”是指毫摩,“M”是指摩,“mmol”是指毫摩尔,“μmole”是指微摩尔,“g”是指克,“μg”是指微克,“ng”是指纳克,“U”是指单位,“bp”是指碱基对,“kB”是指千碱基
解脂耶罗维亚酵母(Yarrowia lipolytica)的培养
解脂耶罗维亚酵母(Yarrowia lipolytica)菌株ATCC#76982和ATCC#90812购自美国典型培养物保藏所(Rockville,MD)。解脂耶罗维亚酵母(Y.lipolytica)菌株通常在28℃在YPD琼脂(1%酵母提取物、2%bactopeptone、2%葡萄糖、2%琼脂)上生长。
解脂耶罗维亚酵母(Yarrowia lipolytica)的转化根据Chen,D C等人(Appl.Microbiol Biotechnol 48(2)232-235(1997))的方法进行,除非另作说明。简要地,Yarrowia在YPD平板上划线,在30℃生长大约18小时。从平板上刮下几个大菌环的细胞,重悬浮在1mL转化缓冲液中,所述转化缓冲液含有2.25ml 50%PEG、平均MW 3350,0.125mL 2M醋酸锂,pH 6.0,0.125mL 2M DTT,和50μg剪切的鲑鱼精子DNA。然后,约500ng线性化的质粒DNA在100μl重悬浮细胞中孵育,以15分钟间隔的涡旋混合维持在39℃1小时。细胞平铺到选择培养基平板上,在30℃维持2到3天。
对于转化体的选择,一般使用基本培养基(“MM”),其中MM的组成如下:0.17%yeast nitrogen base(DIFCO Laboratories,Detroit,M1),没有硫酸铵或氨基酸,2%葡萄糖,0.1%脯氨酸,pH 6.1)。视情况添加尿嘧啶的补充到0.01%的终浓度(从而产生“MMU”选择培养基,用20g/L琼脂制备)。
做为选择,在5-氟乳清酸(“FOA”,也称5-氟尿嘧啶-6-羧酸一水合物)选择培养基上选择转化体,其包含0.17%酵母氮碱(DIFCOLaboratories,Detroit,Ml),没有硫酸铵或氨基酸、2%葡萄糖、0.1%脯氨酸、75mg/L尿嘧啶、75mg/L尿嘧啶核苷、900mg/L FOA(ZymoResearch Corp,Orange,CA)和20g/L琼脂。
最后,如下制备高葡萄糖培养基(“HGM”):14g/L KH2PO4、4g/LK2HPO4、2g/L MgSO47H20、80g/L葡萄糖(pH 6.5)。设计这种培养基以促进油的条件。
解脂耶罗维亚酵母(Yarrowia lipolytica)的脂肪酸分析
对于脂肪酸分析,通过离心收集细胞,如Bligh,E G & Dyer,WJ.(Can.J.Biochem.Physiol.37.911-917(1959))中描述的提取脂质。通过用甲醇钠转酯化脂质提取物制备脂肪酸甲酯(Roughan,G,andNishida I Arch Biochem Biophys 276(1)38-46(1990)),随后用装备有30-m X 0 25mm(id)HP-INNO.WAX(Hewlett-Packard)柱的Hewlett-Packard 6890GC分析。烘箱温度以3.5℃/min从170℃(保持25分钟)到185℃。
对于直接碱转酯化,收获耶罗维亚酵母属(Yarrowia)培养物(3mL),在蒸馏水中洗涤一次,在在Speed-Vac中真空度中干燥5-10分钟。甲醇钠(1%的100μl)添加到样品,然后涡旋和摇摆样品20min。
在添加3滴1M NaCl和400μl己烷之后,涡旋和旋转样品。除去上层,如上所述通过GC分析。
实施例1
高山被孢霉(Mortierella alpina)cDNA库的构建
本实施例描述了利用BD-Clontech CreatorcDNA库试剂盒(Mississauga,ON,Canada)根据厂家的方案构建高山被孢霉(Mortierella alpina)的cDNA库。
特别地,高山被孢霉(M.alpina)菌株ATCC#16266在60mL YPD培养基(2%Bacto酵母提取物,3%Bactor蛋白胨,2%葡萄糖)中在23℃生长3天。通过在Beckman GH38转子中在3750rpm离心10min来团化细胞,重悬浮在6×0.6mL Trizole试剂(Invitrogen)中。重悬浮的细胞转移到六个2mL螺旋盖管中,各含有0.6mL的0.5mm玻璃珠。在Biospec(Bartlesville,OK)迷你珠子搅拌器上以HOMOGENIZE设置匀化细胞2min。试管短暂旋转来沉淀珠子。液体转移到4个新鲜的1.5mL微离心试管中,向每个试管添加0.2mL氯仿/异戊醇(24∶1)。用手摇动试管1min,放置3min。试管然后在4℃在14,000rpm旋转10min。上层转移到4个新试管中。向每个试管添加异丙醇(0.5mL)。在室温下孵育试管15min,随后在4℃以14,000rpm离心10min。团粒各用1mL的75%乙醇(用无RNase的水制成)洗涤并风干。总RNA样品然后重新溶解在500μl水中,使用1∶50稀释的RNA样品通过A260nm测量RNA的数量。获得总共3.14mg RNA。
这个总RNA样品进一步用Qiagen RNeasy总RNA Midi试剂盒根据厂家的方案纯化。这样,总RNA样品稀释到2mL,与具有80μl β-巯基乙醇和5.6mL 100%乙醇的8mL缓冲液RLT混合。样品分成4个部分,加载到4个RNeasy midid柱上。柱然后在4500×g离心5min。为了洗涤柱,加载2ml缓冲液RPE,柱在4500×g离心2min。洗涤步骤重复一次,只是离心时间延长到5min。通过向每个柱施加250μl无RNase的水,等待1min并在4500×g离心3min来洗脱总RNA。
然后根据Amersham Biosciences的mRNA纯化试剂盒的方案,从上述总RNA样品分离PolyA(+)RNA。简要地,使用2 oligo-dT-纤维素柱。每次用1mL高盐缓冲液洗涤柱两次。来自前面步骤的总RNA样品稀释到2mL总体积,调节到10mM Tris/HCl、pH 8.0、1mM EDTA。样品在65℃加热5min,然后置于冰上。添加样品缓冲液(0.4mL),然后将样品自动供料地加载到两个oligo-dt-纤维素柱上。柱在350×g离心2min,每次用0.25mL高盐缓冲液洗涤2×,每次随后是350×g离心2min。用低盐缓冲液进一步洗涤柱3次,随后是相同的常规离心。Poly(A)+RNA通过每次用0.25mL预热到65℃的洗脱缓冲液洗涤柱4次、随后是相同的离心操作来洗脱。整个纯化过程重复一次。纯化的poly(A)+RNA以30.4ng/μl的浓度获得。
产生cDNA,使用BD-Clontech说明的LD-PCR方法和0.1μg polyA(+)RNA样品。具体地,对于第一链cDNA合成,3μl的poly(A)+RNA样品与1μl的SMART IV寡核苷酸(SEQ ID NO:3)和1μl的CDSIII/3′PCR引物(SEQ ID NO:4)混合。混合物在72℃加热2min,在冰上冷却2min。向试管中添加以下2μl第一链缓冲液、1μl 20mMDTT、1μl 10mM dNTP混合物和1μl Powerscnpt逆转录酶。混合物在42℃孵育1hr并在冰上冷却。
第一链cDNA合成混合物用作PCR反应的模板。具体地,反应混合物含有以下2μl第一链cDNA混合物、2μl 5′PCR引物(SEQ ID NO:5)、2μl CDSIII/3′PCR引物(SEQ ID NO:4)、80μl水、10μl 10×Advantage 2PCR缓冲液、2μl 50×dNTP混合物和2μl 50×Advantage2聚合酶混合物。在GenAmp 9600仪器上,热循环仪条件设置为95℃20秒,随后是95℃5秒和68℃6分钟的14个循环。通过琼脂糖凝胶电泳和溴化乙锭染色来测定PCR产物。
75μl上述PCR产物(cDNA)与随试剂盒提供的3μl 20ng/μl蛋白酶K混合。混合物在45℃孵育20min,然后添加75μl水,用150μl苯酚∶氯仿∶异戊醇混合物(25∶24∶1)提取混合物。水相进一步用150μl氯仿∶异戊醇(25∶1)提取。水相然后与15μl 3M醋酸钠、2μl 20ng/μl糖原和400μl的100%乙醇混合。混合物立即在微离心管中以14000rpm在室温下离心20min。团粒用150μl 80%乙醇洗涤一次,风干,溶于79μl水中。
溶解的cDNA随后用Sfil消化(79μl的cDNA与10μl的10×Sfil缓冲液、10μl Sfi酶和1μl 100×BSA混合,混合物在50℃孵育2小时)。添加二甲苯蓝染料(2μl 1%)。完全根据厂家的操作,混合物然后在试剂盒提供的Chroma Spin-400柱上分级。通过琼脂糖凝胶电泳分析从柱收集的级分。集中含有cDNA的前三个级分,用乙醇沉淀cDNA。沉淀的cDNA重新溶解在7μl水中,连接到试剂盒提供的pDNR-LIB中。
库测序
使用连接产物来转化E.coli XL-1 Blue电穿孔感受态细胞(Stratagene)。获得估计总共2×106个菌落。cDNA库的测序由Agencourt Bioscience Corporation(Beverly,MA),使用M13正向引物(SEQ ID NO:6)进行。
实施例2
来自高山被孢霉(Mortierella alpina)的部分脂肪酸延伸酶序列的鉴定
本实施例描述了从9,984个cDNA序列中编码高山被孢霉(M.alpina)脂肪酸延伸酶的cDNA片段(SEQ ID NO:7)的鉴定。
对于与BLAST“nr”数据库(包括所有非冗余GenBank CDS翻译、来自3维结构Brookhaven蛋白质数据库的序列、SWISS-PROT蛋白质序列数据库、EMBL和DDBJ数据库)中含有的序列的相似性通过进行高山被孢霉(M.alpina)cDNA序列的BLAST(Basic Local AlignmentSearch Tool,Altschul,S F,等人,J Mol Biol 215 403-410(1993))检索来测定序列的同一性。在所有阅读框中翻译cDNA序列,使用NCBI提供的BLAST算法(Gish,W and States,D J Nature Genetics 3 266-272(1993))与“nr”数据库中含有的所有公众可获得的蛋白质序列比较相似性。一个cDNA片段(SEQ ID NO:7)带有与许多脂肪酸延伸酶的显著同源性,因而被暂时鉴定为延伸酶。
概况了与SEQ ID NO:7具有最大相似性的序列的BLAST比较的结果是根据同一性%、相似性%和期望值报告的。“同一性%”被定义为两个蛋白质之间相同的氨基酸的百分比。“相似性%”被定义为在两个蛋白质之间相同或保守的氨基酸的百分比。“期望值”使用给定的分值估计了匹配的统计显著性,指出了在这个绝对大小的数据库检索中偶然预期的匹配数量。因而,SEQ ID NO:7的翻译的氨基酸序列与来自Candida albicans SC5314的潜在脂肪酸延伸酶的蛋白质序列(GenBank Accession No.EAL04510 1,其中注释为与酿酒酵母(S.cerevisiae)EUR4、FEN1和ELO1类似的三种潜在脂肪酸延伸酶基因之一)具有32%同一性和46%相似性,期望值4e-13。另外,SEQ ID NO:7与来自酿酒酵母(Saccharomyces cerevisiae)的ELO1(GenBankAccession No.NC_001142,染色体X的碱基67849-68781)具有35%同一性和53%相似性。S.cerevisiae ELO1被描述为中链酰基延伸酶,其催化不饱和C12-C16脂肪酰基-CoA向C16-18脂肪酸的羧基末端延伸。
在以上报道的同源性的基础上,SEQ ID NO:7的解脂耶罗维亚酵母(Yarrowia lipolytica)基因产物在此被称为“延伸酶3”或“ELO3”。
实施例3
完整高山被孢霉(M.alpina)ELO3的测序
部分脂肪酸延伸酶cDNA序列(SEQ ID NO:7)的分析表明5′和3′末端都是不完整的。为了获得缺失的区域,利用了基因组步移技术。
基因组步移来分离高山被孢霉(M.alpina)ELO3的3′末端区域
Clontech Universal GenomeWalkerTM试剂盒被用于获得与高山被孢霉(M.alpina)ELO3的3′末端区域相应的基因组DNA的片段。简要地,25μg每种高山被孢霉(M.alpina)基因组DNA用DraI、EcoRV、PvuII或StuI分别消化,消化的DNA样品使用Qiagen Qiaquick PCR纯化试剂盒来纯化,各用30μg试剂盒缓冲液EB洗脱,纯化的样品然后与Genome Walker接头(SEQ ID NO:8[顶部链]和9[底部链])连接,如下所示:
5’-GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT-3’
3’-H2N-CCCGACCA-5’
每种连接反应混合物含有1.9μl的25μM Genome Walker接头,1.6μl 10×连接缓冲液、0.5μl T4 DNA连接酶以及4μl纯化的消化基因组DNA样品之一。反应混合物在16℃孵育过夜。通过在70℃孵育5min终止反应。然后,向每个连接反应混合物添加72μl 10mM TrisHCl、1mM EDTA、pH 7.4缓冲液。
进行四个独立的PCR反应,每个使用四种连接混合物之一作为模板。PCR反应混合物含有1μl连接混合物、0.5μl 20μM引物MA Elong3′1(SEQ ID NO:10)、1μl 10μM试剂盒引物AP1(SEQ ID NO:11)、22.5μl水和25μl ExTaq premix Taq 2×PCR溶液(TaKaRa Bio Inc,Otsu,Shiga,520-2193,Japan)。PCR反应使用以下条件进行32个循环,在94℃30秒变性、在55℃30秒退火以及在72℃3分钟延伸。最后的延伸循环在72℃进行7分钟,随后在4℃反应终止。
每个PCR反应的产物分别1∶50稀释,用作第二轮PCR的模板。每个反应混合物含有1μl稀释的PCR产物之一作为模板,0.5μl 20μM引物MA elong 3′2(SEQ ID NO:12)、1μl 10μM试剂盒引物AP2(SEQID NO:13)、22.5μl水和25μl ExTaq premix Taq 2X PCR溶液(TaKaRa)。PCR反应使用如上所述相同的热循环仪条件进行32个循环。
从第二轮PCR获得1041bp DNA片段(SEQ ID NO:14)。这个片段被纯化并克隆到pCR2.1-TOPO中并测序。序列分析显示该片段含有ELO3的3′末端,包括基因的“TAA”终止密码子的~640bp下游。
基因组步移来分离高山被孢霉(M.alpina)ELO3的5′末端区域
在Clontech 3′末端RACE(上文)中使用的相同的四个连接混合物集合也被用于获得高山被孢霉(M.alpina)ELO3的5′末端区域。具体地,使用如上所述相同的成分和条件进行第一轮PCR,只是MA Elong 5′1(SEQ ID NO:15,在5′末端嵌套)和AP1作为引物(即,代替引物MA Elong 3′1和AP1)。第二轮PCR使用MA Elong 5′2(SEQ ID NO:16,在5′末端嵌套)和AP2作为引物。获得了2223bp DNA片段(SEQID NO:17)。它被纯化并克隆到pCR2.1-TOPO中,序列的了分析显示它含有ELO3基因的5′区域。
因而,通过组合由基因组步移获得的(分别为SEQ ID NO:14和17,在附图1中图示说明)、具有重叠的5′和3′序列的原始部分cDNA序列(SEQ ID NO:7)获得了高山被孢霉(M.alpina)ELO3的完整cDNA序列(SEQ ID NO:1)。这产生了3557bp的序列,在此标为SEQ ID NO:18,包括ELO3的推定的“ATG”翻译起始密码子的2091bp上游、828bp ELO3ORF(即,SEQ ID NO:1,相应于SEQ ID NO:18的碱基2092-2919)和ELO3终止密码子的638bp下游(相应于SEQ ID NO:18的碱基2920-3557)。
高山被孢霉(M.alpina)ELO3的相应基因组序列比SEQ ID NO:18所提供的cDNA片段更长。具体地,在含有ELO3基因的基因组DNA的ORF的318bp处存在542bp内含子(SEQ ID NO:19);因而,在此由SEQ ID NO:29所提供的基因组DNA片段是4,099bp(附图1)。
根据BLAST程序分析(上文,实施例2)翻译的ELO3蛋白质序列(SEQ ID NO:2)具有与已知脂肪酸延伸酶的以下同源性:与来自白假丝酵母(Candida albicans)SC5314的可能的脂肪酸延伸酶(GenBank Accession No.EAL04510 1)的37%同一性和51%相似性,4e-43的期望值。此外,翻译的ELO3与来自Neurospora crassa的XP_331368(其中注释为“假想的蛋白”)享有33%的同一性和44%的相似性,3e-44的期望值。
实施例4
解脂耶罗维亚酵母(Yarrowia lipolytica)ATCC#20362菌株Y2031
的产生
本实施例描述了来自解脂耶罗维亚酵母(Yarrowia lipolytica)ATCC#20362的菌株Y2031的构建。通过将来自质粒pKUNT2的TEF YΔ12Pex20嵌合基因到ATCC#20362的Ura3基因座,从而产生Ura基因型,来产生Y2031菌株。质粒pKUNT2含有以下组件:
表3 质粒pKUNT2的描述(SEQ ID NO:20)
SEQ ID NO:20内的RE位点和核苷酸 | 片段和嵌合基因组件的描述 |
Ascl/Bsi WI(3225-3015) | Yarrowia Ura3基因(GenBank Accession No.AJ306421)的784bp 5′部分 |
Sphl/PacI(5933-13) | Yarrowia Ura3基因(GenBank Accession No.AJ306421)的516bp 3′部分 |
EcoRI/BsiWI(6380-8629) | TEF YA12 Pex20,包括:·TEF:TEF启动子(GenBank Accession No.AF054508)·YA12:YarrowiaΔ12脱饱和酶基因(SEQ IDNO.21)·Pex20:来自YarrowiaPex20基因(GenBank Accession No.AF054613)的Pex20终止子序列 |
用Ascl/Sphl消化pKUNT2质粒,然后用于根据一般方法的野生型Y.lipolyticaATCC#20362的转化。转化体细胞平铺在FOA选择培养基平板上,在30℃维持2到3天。挑选FOA抗性菌落并在MM和MMU选择平板上划线。选择可以在MMU平板上生长但不能在M平板上生长的菌落作为Ura菌株。然后在30℃将Ura菌株的单个菌落接种到液体MMU中,在250rpm/min摇动2天。通过离心收集细胞,提取脂质,通过转酯化制备脂肪酸甲酯,随后用Hewlett-Packard 6890GC分析。
GC分析显示,与野生型ATCC#20362中的约20%LA相比,在两种Ura菌株(菌株#2和#3)中有约45%LA。转化体菌株#2被称为菌株“Y2031”。
实施例5
高山被孢霉(Mortierella alpina)脂肪酸ELO3在解脂耶罗维亚酵母
(Yarrowia lipolytica)菌株Y2031中的异源表达
本实施例描述了在解脂耶罗维亚酵母(Y.lipolytica)菌株Y2031中在解脂耶罗维亚酵母(Y.lipolytica)启动子的控制下高山被孢霉(M.alpina)ELO3ORF在嵌合基因中的过量表达,以及通过TAG含量分析确定的过量表达的效果。
包含A FBAIN ELO3 PEX16-3′嵌合基因的质粒pZUF6S-E3WT的
构建
高山被孢霉(M.alpina)脂肪酸ELO3 ORF如下克隆:引物MA Elong5′Ncol 3和MA Elong 3′Notl(SEQ ID NO:23和24)被用于从高山被孢霉(M.alpina)的cDNA(实施例1)通过PCR扩增ELO3 ORF。反应混合物含有1μl cDNA、1μl每种引物、22μl水和25μl ExTaq premix 2×Taq PCR溶液(TaKaRa)。扩增如下进行:在94℃30秒首先变性,随后是94℃30秒变性、55℃30秒退火和72℃120秒延伸的32个循环。最后的延伸循环在72℃进行7min,随后在4℃终止反应。从PCR反应获得830bp DNA片段。使用Qiagen(Valencia,CA)PCR纯化试剂盒根据厂家的方案来纯化。纯化的PCR产物分成两个等份,其中一个用Ncol和Nspl消化,而另一个用Nspl和Notl消化。270bp Ncol-Nspl和560bp Nspl-Notl片段通过三片段连接克隆到Nco l-No.t l酶切的pZF5T-PPC载体中(附图2A;SEQ ID NO:25),从而基因处于用于在解脂耶罗维亚酵母(Y.lipolytica)中表达的自我复制载体中解脂耶罗维亚酵母(Y.lipolytica)FBAIN启动子和PEX16-3′终止子区域(GenBankAccession No.U75433)的控制下。“FBAIN启动子”是指在fbal基因编码的果糖-二磷酸醛缩酶(EC 4 12 13)的“ATG”翻译起始密码子之前的5′上游非翻译区域,并且它是表达所必需的,加上具有fbal基因的内含子的一部分5′编码区(进一步细节参见WO 2005/049805)。通过miniprep分析确认了正确的转化体,产生的质粒称为“pZF5T-PPC-E3”(SEQ ID NO:26)。
用Clal和Pad消化质粒pZF5T-PPC-E3,使用Qiagen凝胶提取试剂盒从琼脂糖凝胶纯化~2.2kB条带(即,FBAIN ELO 3 PEX16-3′片段)。将该片段克隆到Clal-Pacl酶切的pZUF6S(附图2B,SEQ ID NO:27)中,这是一种含有处在FBAIN启动子的控制下的高山被孢霉(Mortierella alpina)ΔA6脱饱和酶ORF(“D6S”,GenBank Accession No.AF465281)的自我复制质粒,具有Pex20-3′终止子(即,FBAIN D6S.Pex20嵌合基因)和Ura3基因。通过miniprep分析确认了正确的转化体,产生的质粒称为“pZUF6S-E3WT”(SEQ ID NO:28,附图2C)。
过量表达高山被孢霉(M.alpina)ELO3的转化体解脂耶罗维亚酵母(Y.
lipolytica)中脂质含量的分析
用质粒pZUF6S(对照,包含FBAIN D6S Pex20嵌合基因)和质粒pZUF6S-E3WT(包含FBAIN D6S Pex20嵌合基因和FBAIN ELO 3PEX16嵌合基因)根据一般方法转化解脂耶罗维亚酵母(Y.lipolytica)菌株Y2031。转化体在补充有氨基酸的合成MM上生长2天,随后在HGM中4天。含有pZUF6S的六个克隆(克隆#1-6,来自单个的转化体)和含有pZUF6S-E3WT的22个克隆(来自四种不同的转化体[即,#3、5、6和7]的脂肪酸分布型以下在表4中显示,根据GC分析(如一般方法中所描述的)。脂肪酸被标示为16 0(棕榈酸)、16 1(棕榈油酸)、18 0、18 1(油酸)、18 2(LA)和GLA,每一种的组成表示为总脂肪酸的%。
表4被工程化以过量表达高山被孢霉(M.alpina)脂肪酸ELO3的
Yarrowia菌株Y2031中的脂质含量
一些样品(以粗体和斜体字标记)偏离了预期读数。特别地,Y2031+pZUF6S-E3WT#3-3和Y2031+pZUF6S-E3WT#5-6都不产生GLA。类似地,Y2031+pZUF6S-E3WT#7-1、#7-3和#7-4具有GC错误,其中160和161峰被GC读取为单个的峰。作为这些变体结果的结果,表5报道了在对照和表达ELO3的转化体菌株中的平均脂质。特别地,表5显示了来自表4中脂肪酸分布型的平均值,而在计算这平均值时在表4中通过粗体和斜体标示为不正确的株系没有被包括。“总C16”代表160和161的平均面积,而“总C 18”反映了180、181、182和GLA的平均面积。
表5
被工程化以过量表达高山被孢霉(M.alpina)脂肪酸ELO3的耶罗维
亚酵母(Yarrowia)菌株Y2031中的平均脂质含量
根据以上报道的数据,相对于仅过量表达高山被孢霉(M.alpina)Δ6脱饱和酶的对照菌株Y2031,当在解脂耶罗维亚酵母(Yarrowialipolytica)菌株Y2031中与高山被孢霉(M.alpina)Δ6脱饱和酶共表达时,高山被孢霉(M.alpina)ELO3的过量表达产生C18的提高的百分比和C 16的降低的百分比。这表明高山被孢霉(M.alpina)ELO3实际上是C16/18脂肪酸延伸酶。
对本领域技术人员明显的是,其他嵌合基因可以在工程化的Yarrowia中与高山被孢霉(M.alpina)ELO3基因共表达来提高各种其他脂肪酸的产生。例如,除了高山被孢霉(M.alpina)ELO3(其任选的可以是为提高表达密码子优化的)之外,人们可以容易地表达Cum脂肪酸延伸酶和/或Δ9脱饱和酶作为允许流入脂质代谢的碳的增量调节的手段。在可选择的实施方式中,高山被孢霉(M.alpina)ELO3可以容易地与任何以下的PUFA生物合成途径基因共表达,包括,例如,Δ12脱饱和酶、Δ15脱饱和酶、Δ6脱饱和酶、Δ5脱饱和酶、Δ17脱饱和酶、Δ9脂肪酸延伸酶、Δ8脱饱和酶、C18/20脂肪酸延伸酶、C20/22脂肪酸延伸酶和/或Δ4脱饱和酶,以允许各种PUFA的提高的生产(附图3)。
序列表
<110>E.I.duPont de Nemours and Company
<120>高山被孢霉C16/18脂肪酸延伸酶
<130>CL3027PCT
<160>29
<170>PatentIn version 3.3
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cccaaggttg gcaaagtctc gcgcgtggta gccaagtcgg ctaacgccaa gccggcagag 180
cgcacgcagt ccggcgccgc catgaccgcc tttgtctttg tccacaacct tatcctctgc 240
gtgtactctg gaatcacctt ctactacatg ttcccagcca tggtcaagaa ctttagaaca 300
cataccctcc atgaggccta ctgcgatacg gatcagagcc tgtggaacaa cgcccttggc 360
tactggggct acctcttcta cctttcaaag ttttacgagg tcattgacac catcatcatc 420
atcttgaagg ggcgccgctc gtccctgctc cagacctacc accacgccgg cgctatgatc 480
accatgtggt ccggcatcaa ctaccaggca acgcccattt ggatttttgt cgtcttcaac 540
tcgttcatcc acaccatcat gtactgttac tatgccttca cctcaatcgg cttccacccc 600
ccaggcaaga agtacctcac ctccatgcag atcacccagt ttttggtcgg catcactatc 660
gccgtctctt atctcttcgt ccctggatgt atccgcacac ccggtgctca gatggctgtc 720
tggatcaacg tcggatacct ctttcccctc acttatctct ttgtggattt tgccaagcgt 780
acttactcca agcgtagtgc catcgccgct cagaagaagg cccagtaa 828
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Met Glu Ser Gly Pro Met Pro Ala Gly Ile Pro Phe Pro Glu Tyr Tyr
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Asp Phe Phe Met Asp Trp Lys Thr Pro Leu Ala Ile Ala Ala Thr Tyr
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Thr Ala Ala Val Gly Leu Phe Asn Pro Lys Val Gly Lys Val Ser Arg
35 40 45
Val Val Ala Lys Ser Ala Asn Ala Lys Pro Ala Glu Arg Thr Gln Ser
50 55 60
Gly Ala Ala Met Thr Ala Phe Val Phe Val His Asn Leu Ile Leu Cys
65 70 75 80
Val Tyr Ser Gly Ile Thr Phe Tyr Tyr Met Phe Pro Ala Met Val Lys
85 90 95
Asn Phe Arg Thr His Thr Leu His Glu Ala Tyr Cys Asp Thr Asp Gln
100 105 110
Ser Leu Trp Asn Asn Ala Leu Gly Tyr Trp Gly Tyr Leu Phe Tyr Leu
115 120 125
Ser Lys Phe Tyr Glu Val Ile Asp Thr Ile Ile Ile Ile Leu Lys Gly
130 135 140
Arg Arg Ser Ser Leu Leu Gln Thr Tyr His His Ala Gly Ala Met Ile
145 150 155 160
Thr Met Trp Ser Gly Ile Asn Tyr Gln Ala Thr Pro Ile Trp Ile Phe
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Val Val Phe Asn Ser Phe Ile His Thr Ile Met Tyr Cys Tyr Tyr Ala
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Phe Thr Ser Ile Gly Phe His Pro Pro Gly Lys Lys Tyr Leu Thr Ser
195 200 205
Met Gln Ile Thr Gln Phe Leu Val Gly Ile Thr Ile Ala Val Ser Tyr
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Leu Phe Val Pro Gly Cys Ile Arg Thr Pro Gly Ala Gln Met Ala Val
225 230 235 240
Trp Ile Asn Val Gly Tyr Leu Phe Pro Leu Thr Tyr Leu Phe Val Asp
245 250 255
Phe Ala Lys Arg Thr Tyr Ser Lys Arg Ser Ala Ile Ala Ala Gln Lys
260 265 270
Lys Ala Gln
275
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技术
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tgtaaaacga cggccagt 18
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cccgtcttca ccctccctct tccccccgtt cctacgtcta cagccgttgg ctcatcttgc 60
agttgcttgt ctactatttg gtgccgacct atattcctct gtcacccaac ctaccgcact 120
cacactcgca taatggagtc tggacctatg cctgccggga tccccttccc tgaatactat 180
gactttttca tggactggaa gacacccctg gcaattgctg ccacctacac cgccgctgtt 240
gggctcttca accccaaggt tggcaaagtc tcgcgcgtgg tagccaagtc ggctaacgcc 300
aagccggcag agcgcacgca gtccggcgcc gccatgaccg cctttgtctt tgtccacaac 360
cttatcctct gcgtgtactc tggaatcacc ttctactaca tgttcccagc catggtcaag 420
aactttagaa cacataccct ccatgaggcc tactgcgata cggatcagag cctgtggaac 480
aacgcccttg gctactgggg ctacctcttc tacctttcaa agttttacga ggtcattgac 540
accatcatca tcatcttgaa ggggcgccgc tcgtccctgc tccagaccta ccaccacgcc 600
ggcgcta 607
<210>8
<211>44
<212>DNA
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<223>基因组步移者衔接子-1
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accagccc 8
<210>10
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tgaaggggcg ccgctcgtcc ctgctccaga cctaccacca cgccggcgct atgatcacca 60
tgtggtccgg catcaactac caggcaacgc ccatttggat ttttgtcgtc ttcaactcgt 120
tcatccacac catcatgtac tgttactatg ccttcacctc aatcggcttc caccccccag 180
gcaagaagta cctcacctcc atgcagatca cccagttttt ggtcggcatc actatcgccg 240
tctcttatct cttcgtccct ggatgtatcc gcacacccgg tgctcagatg gctgtctgga 300
tcaacgtcgg atacctcttt cccctcactt atctctttgt ggattttgcc aagcgtactt 360
actccaagcg tagtgccatc gccgctcaga agaaggccca gtaaagaggc cacttcttgt 420
cgtttactct actgtttcca acatttacat ctctcaatgg cagcctcctc cctctatcct 480
tcgcactttc attttagttc cttgtattca taaaargtca aaattcattg taactccaaa 540
agtgccaatg gctttatcat ggatgagtgc catgtttatg gcccattgca tcaatacagg 600
aagatatgag cggtcagcga tagctattcc tctgagttgt cctggccttg tatgcactct 660
cgaaggagga aatggatcgg agcatcagca ctatttcagg actaggagag atacatccaa 720
aattttaaag gcatctctgg tcagtacaag agcgacttgg caacaagtct ttagtcaaac 780
ttcctcacct ttgaacttcc tcagaggtcg ccgtggcact gtggacggag acccgtgaaa 840
agaactcatg cagcaggtta agcaaccttg gaaatccatc gatgaatgcc tgatgtaaaa 900
attgagaagc tgcaagagcc aaaagttcag ttcagcacat ataagagact ttggccaaga 960
gcgatgaagt cacatgggaa ccacgtaaac aaacctcgtg cggagsacca gcccgggccg 1020
tcgaccacgc gtgccctata gt 1042
<210>15
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gcataggtcc agactccatt atgcg 25
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<211>25
<212>DNA
<213>人工序列
<220>
<223>引物MA Elong 5’2
<400>16
cagaggaata taggtcggca ccaaa 25
<210>17
<211>2223
<212>DNA
<213>高山被孢霉
<220>
<221>misc_feature
<222>(2092)..(2094)
<223>ATG翻译起始密码子
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actatagggc acgcgtggtc gacggcccgg gctggtccta cctctcaatc ttgttgccct 60
gtagtcctga aatgagctcg tcatgggtct ctgtcctctc ctcgttatgg tcaaacgcat 120
tggacccggt tttctttaga ggagcattct tcttgtttaa gccgactcct cttcccgtcc 180
cgaacgacaa cttgataggt ccaccatttc catttggtgg gtgaagcggg ctgtcctcag 240
cggacatgac tctgagctag aaggaagggt gtagaggacg aaggagtaag tgctaccgtt 300
cactggcagg tgaggagcgt cgggtctaag ccacatgcct gccaattgtc ctcgccaagt 360
gcttggaagg tgtattgatg ctcatgcagc tccactctgg acctggaaac gccaccgcat 420
cgcattctta atgagctctg ctacaaacga caatctgcga accagtttgc acagggagct 480
agtcgagtct ggaaagcggg accagtgagt gactcggcgg ggcttgcatc gctgaacatc 540
gccattggga tttcttagaa agcagctctt gctcttatac aaacaccttc ctgcgtgcct 600
caccttactt acgaggagta atgttgtttt tacaggctag tggaactctt gcgcgcgagg 660
ctgatcgagt ccggctggcg ggacgatctg aaggcattta caaaaggtac accactcggc 720
acttggcctg ttttgttgcc actagctacc aggccgtgac tgaccacgaa cccttcctct 780
ggagaataga aaggatccag tcccgggaga ctacaatgtc tgttgacgag gtcatcagag 840
aagtcggccc acatgctaga ggtaagcccc gcgagaacct tatatgctga ttagtgccat 900
ccgttgaaca tcgtctgatc tgcgacaagt tctggtaata cttgatagcc accgtcccag 960
acaaggtcaa gacagaactt cttgcgcaaa tcagtacttt tatcgaagac aacatgtaac 1020
gaaaaataaa aatgctgata tggaagggaa catcacgtac gccaggtacg tgagatgttt 1080
ttcctttttt ttttattttt tttgttgggg cttgagatgt tttgggtctt tcatgaagac 1140
gttcatgaca aacaagaaat acggacagcg agtttaccgc aatggaaaag tttacctcaa 1200
ccgaactttg aacgtttcgt gaaaacctgc gtgagaattc tttgagcatg gccataccta 1260
attgtcgccg gactgatctg tgatttgatc catggtccat cgctcctgac tcgctgcacg 1320
tcctgacaga gataacctcg catagtgtcg tacagaaacc aaaagcacgc gctcggtcca 1380
gtacagtaat ggggaactgg gtactgagga cagcttgaaa gtatatcctg taggggacga 1440
taacatgggc aatcttgatg gtgtggttga gtacagggta tatgcgttcc aggtaagcaa 1500
atatcacctt tgtgagacga tagggggcag ctatagtaaa tggccaggcg cttttatacg 1560
ggaagcaatt gcagcgatgg aggtgtgtac gctgcagccg cttgtatagc agaacgtaac 1620
cccaatcctc caactccggc aaggacaatg gggaaagaag gccggatata taaaatgctt 1680
gtcagcagaa ttaaagactg tgcggactga tctgccttaa tcggaaactt cagcagggct 1740
tctcgcagtg catgaaccct cggaccatct cctgtcccaa caacacgcca atgggcttat 1800
tattaatttt ttttggctgc cgttcaaaaa aaaaaagaaa aaaactgcca cttcttattt 1860
gagggctgca ggactgcttg ttaaggcgga taacctcaat ccgcccgatg ccttttaacg 1920
ctggcctgtt cttcctcttt tctttcttct ctcccttcac ccgtcttcac cctccctctt 1980
ccccccgttc ctacgtctac agccgttggc tcatcttgca gttgcttgtc tactatttgg 2040
tgccgaccta tattcctctg tcacccaacc taccgcactc acactcgcat aatggagtct 2100
ggacctatgc ctgccgggat ccccttccct gaatactatg actttttcat ggactggaag 2160
acacccctgg caattgctgc cacctacacc gccgctgttg ggctcttcaa ccccaaggtt 2220
ggc 2223
<210>18
<211>3557
<212>DNA
<213>高山被孢霉
<220>
<221>misc_feature
<222>(2092)..(2094)
<223>ATG翻译起始密码子
<220>
<221>misc_feature
<222>(2917)..(2919)
<223>’TAA’终止密码子
<400>18
actatagggc acgcgtggtc gacggcccgg gctggtccta cctctcaatc ttgttgccct 60
gtagtcctga aatgagctcg tcatgggtct ctgtcctctc ctcgttatgg tcaaacgcat 120
tggacccggt tttctttaga ggagcattct tcttgtttaa gccgactcct cttcccgtcc 180
cgaacgacaa cttgataggt ccaccatttc catttggtgg gtgaagcggg ctgtcctcag 240
cggacatgac tctgagctag aaggaagggt gtagaggacg aaggagtaag tgctaccgtt 300
cactggcagg tgaggagcgt cgggtctaag ccacatgcct gccaattgtc ctcgccaagt 360
gcttggaagg tgtattgatg ctcatgcagc tccactctgg acctggaaac gccaccgcat 420
cgcattctta atgagctctg ctacaaacga caatctgcga accagtttgc acagggagct 480
agtcgagtct ggaaagcggg accagtgagt gactcggcgg ggcttgcatc gctgaacatc 540
gccattggga tttcttagaa agcagctctt gctcttatac aaacaccttc ctgcgtgcct 600
caccttactt acgaggagta atgttgtttt tacaggctag tggaactctt gcgcgcgagg 660
ctgatcgagt ccggctggcg ggacgatctg aaggcattta caaaaggtac accactcggc 720
acttggcctg ttttgttgcc actagctacc aggccgtgac tgaccacgaa cccttcctct 780
ggagaataga aaggatccag tcccgggaga ctacaatgtc tgttgacgag gtcatcagag 840
aagtcggccc acatgctaga ggtaagcccc gcgagaacct tatatgctga ttagtgccat 900
ccgttgaaca tcgtctgatc tgcgacaagt tctggtaata cttgatagcc accgtcccag 960
acaaggtcaa gacagaactt cttgcgcaaa tcagtacttt tatcgaagac aacatgtaac 1020
gaaaaataaa aatgctgata tggaagggaa catcacgtac gccaggtacg tgagatgttt 1080
ttcctttttt ttttattttt tttgttgggg cttgagatgt tttgggtctt tcatgaagac 1140
gttcatgaca aacaagaaat acggacagcg agtttaccgc aatggaaaag tttacctcaa 1200
ccgaactttg aacgtttcgt gaaaacctgc gtgagaattc tttgagcatg gccataccta 1260
attgtcgccg gactgatctg tgatttgatc catggtccat cgctcctgac tcgctgcacg 1320
tcctgacaga gataacctcg catagtgtcg tacagaaacc aaaagcacgc gctcggtcca 1380
gtacagtaat ggggaactgg gtactgagga cagcttgaaa gtatatcctg taggggacga 1440
taacatgggc aatcttgatg gtgtggttga gtacagggta tatgcgttcc aggtaagcaa 1500
atatcacctt tgtgagacga tagggggcag ctatagtaaa tggccaggcg cttttatacg 1560
ggaagcaatt gcagcgatgg aggtgtgtac gctgcagccg cttgtatagc agaacgtaac 1620
cccaatcctc caactccggc aaggacaatg gggaaagaag gccggatata taaaatgctt 1680
gtcagcagaa ttaaagactg tgcggactga tctgccttaa tcggaaactt cagcagggct 1740
tctcgcagtg catgaaccct cggaccatct cctgtcccaa caacacgcca atgggcttat 1800
tattaatttt ttttggctgc cgttcaaaaa aaaaaagaaa aaaactgcca cttcttattt 1860
gagggctgca ggactgcttg ttaaggcgga taacctcaat ccgcccgatg ccttttaacg 1920
ctggcctgtt cttcctcttt tctttcttct ctcccttcac ccgtcttcac cctccctctt 1980
ccccccgttc ctacgtctac agccgttggc tcatcttgca gttgcttgtc tactatttgg 2040
tgccgaccta tattcctctg tcacccaacc taccgcactc acactcgcat aatggagtct 2100
ggacctatgc ctgccgggat ccccttccct gaatactatg actttttcat ggactggaag 2160
acacccctgg caattgctgc cacctacacc gccgctgttg ggctcttcaa ccccaaggtt 2220
ggcaaagtct cgcgcgtggt agccaagtcg gctaacgcca agccggcaga gcgcacgcag 2280
tccggcgccg ccatgaccgc ctttgtcttt gtccacaacc ttatcctctg cgtgtactct 2340
ggaatcacct tctactacat gttcccagcc atggtcaaga actttagaac acataccctc 2400
catgaggcct actgcgatac ggatcagagc ctgtggaaca acgcccttgg ctactggggc 2460
tacctcttct acctttcaaa gttttacgag gtcattgaca ccatcatcat catcttgaag 2520
gggcgccgct cgtccctgct ccagacctac caccacgccg gcgctatgat caccatgtgg 2580
tccggcatca actaccaggc aacgcccatt tggatttttg tcgtcttcaa ctcgttcatc 2640
cacaccatca tgtactgtta ctatgccttc acctcaatcg gcttccaccc cccaggcaag 2700
aagtacctca cctccatgca gatcacccag tttttggtcg gcatcactat cgccgtctct 2760
tatctcttcg tccctggatg tatccgcaca cccggtgctc agatggctgt ctggatcaac 2820
gtcggatacc tctttcccct cacttatctc tttgtggatt ttgccaagcg tacttactcc 2880
aagcgtagtg ccatcgccgc tcagaagaag gcccagtaaa gaggccactt cttgtcgttt 2940
actctactgt ttccaacatt tacatctctc aatggcagcc tcctccctct atccttcgca 3000
ctttcatttt agttccttgt attcataaaa rgtcaaaatt cattgtaact ccaaaagtgc 3060
caatggcttt atcatggatg agtgccatgt ttatggccca ttgcatcaat acaggaagat 3120
atgagcggtc agcgatagct attcctctga gttgtcctgg ccttgtatgc actctcgaag 3180
gaggaaatgg atcggagcat cagcactatt tcaggactag gagagataca tccaaaattt 3240
taaaggcatc tctggtcagt acaagagcga cttggcaaca agtctttagt caaacttcct 3300
cacctttgaa cttcctcaga ggtcgccgtg gcactgtgga cggagacccg tgaaaagaac 3360
tcatgcagca ggttaagcaa ccttggaaat ccatcgatga atgcctgatg taaaaattga 3420
gaagctgcaa gagccaaaag ttcagttcag cacatataag agactttggc caagagcgat 3480
gaagtcacat gggaaccacg taaacaaacc tcgtgcggag saccagcccg ggccgtcgac 3540
cacgcgtgcc ctatagt 3557
<210>19
<211>542
<212>DNA
<213>高山被孢霉
<220>
<221>内含子
<222>(1)..(542)
<400>19
cgtaagttga caacggttca aatttcgaaa cttgcactgt gggtttgtcc tggttttatt 60
cggcagcttt tttctccgaa gtcttggccc agggaggagg caagaagagc aattcagggt 120
aggggcgtcc ggagattgga ccctgccttg gactagcgaa tggaatacga gagcgaagct 180
gtctgcatgg gctgatgata atatttttta ctggagatga ttgtaaggtg cggcgcgagg 240
gtgtagtaca tcagtcgaca tggattcaac caaatgttct ctagcagcga taaccctggc 300
tgtcttttgt taatcacggc aatgctatcc ccaaagaaaa aaaaaagggt gcggttggca 360
cacggttgca caatggctta gccagacaat taacccaaaa aagtcaagag attccttttt 420
tttttttttt ttttttgggg ggggaacggg aaagtggggg ctgtgtgaaa ttggggcgaa 480
gtgaaccatg caactttgag ctacaaacac ctaattgttt ttgtcttgcc tgacattatt 540
ag 542
<210>20
<211>6457
<212>DNA
<213>人工序列
<220>
<223>质粒pKUNT2
<400>20
tttgaatcga atcgatgagc ctaaaatgaa cccgagtata tctcataaaa ttctcggtga 60
gaggtctgtg actgtcagta caaggtgcct tcattatgcc ctcaacctta ccatacctca 120
ctgaatgtag tgtacctcta aaaatgaaat acagtgccaa aagccaaggc actgagctcg 180
tctaacggac ttgatataca accaattaaa acaaatgaaa agaaatacag ttctttgtat 240
catttgtaac aattaccctg tacaaactaa ggtattgaaa tcccacaata ttcccaaagt 300
ccaccccttt ccaaattgtc atgcctacaa ctcatatacc aagcactaac ctaccgttta 360
aacaccacta aaaccccaca aaatatatct taccgaatat acagatctgc gacgacggaa 420
ttcctgcagc ccatctgcag aattcaggag agaccgggtt ggcggcgtat ttgtgtccca 480
aaaaacagcc ccaattgccc caattgaccc caaattgacc cagtagcggg cccaaccccg 540
gcgagagccc ccttcacccc acatatcaaa cctcccccgg ttcccacact tgccgttaag 600
ggcgtagggt actgcagtct ggaatctacg cttgttcaga ctttgtacta gtttctttgt 660
ctggccatcc gggtaaccca tgccggacgc aaaatagact actgaaaatt tttttgcttt 720
gtggttggga ctttagccaa gggtataaaa gaccaccgtc cccgaattac ctttcctctt 780
cttttctctc tctccttgtc aactcacacc cgaaatcgtt aagcatttcc ttctgagtat 840
aagaatcatt caccatggat tcgaccacgc agaccaacac cggcaccggc aaggtggccg 900
tgcagccccc cacggccttc attaagccca ttgagaaggt gtccgagccc gtctacgaca 960
cctttggcaa cgagttcact cctccagact actctatcaa ggatattctg gatgccattc 1020
cccaggagtg ctacaagcgg tcctacgtta agtcctactc gtacgtggcc cgagactgct 1080
tctttatcgc cgtttttgcc tacatggcct acgcgtacct gcctcttatt ccctcggctt 1140
ccggccgagc tgtggcctgg gccatgtact ccattgtcca gggtctgttt ggcaccggtc 1200
tgtgggttct tgcccacgag tgtggccact ctgctttctc cgactctaac accgtcaaca 1260
acgtcaccgg atgggttctg cactcctcca tgctggtccc ttactacgcc tggaagctga 1320
cccactccat gcaccacaag tccactggtc acctcacccg tgatatggtg tttgtgccca 1380
aggaccgaaa ggagtttatg gagaaccgag gcgcccatga ctggtctgag cttgctgagg 1440
acgctcccct catgaccctc tacggcctca tcacccagca ggtgtttgga tggcctctgt 1500
atctgctgtc ttacgttacc ggacagaagt accccaagct caacaaatgg gctgtcaacc 1560
acttcaaccc caacgccccg ctgtttgaga agaaggactg gttcaacatc tggatctcta 1620
acgtcggtat tggtatcacc atgtccgtca tcgcatactc catcaaccga tggggcctgg 1680
cttccgtcac cctctactac ctgatcccct acctgtgggt caaccactgg ctcgtggcca 1740
tcacctacct gcagcacacc gaccccactc tgccccacta ccacgccgac cagtggaact 1800
tcacccgagg agccgccgcc accatcgacc gagagtttgg cttcatcggc tccttctgct 1860
tccatgacat catcgagacc cacgttctgc accactacgt gtctcgaatt cccttctaca 1920
acgcccgaat cgccactgag aagatcaaga aggtcatggg caagcactac cgacacgacg 1980
acaccaactt catcaagtct ctttacactg tcgcccgaac ctgccagttt gttgaaggta 2040
aggaaggcat tcagatgttt agaaacgtca atggagtcgg agttgctcct gacggcctgc 2100
cttctaaaaa gtaggcggcc gcaagtgtgg atggggaagt gagtgcccgg ttctgtgtgc 2160
acaattggca atccaagatg gatggattca acacagggat atagcgagct acgtggtggt 2220
gcgaggatat agcaacggat atttatgttt gacacttgag aatgtacgat acaagcactg 2280
tccaagtaca atactaaaca tactgtacat actcatactc gtacccgggc aacggtttca 2340
cttgagtgca gtggctagtg ctcttactcg tacagtgtgc aatactgcgt atcatagtct 2400
ttgatgtata tcgtattcat tcatgttagt tgcgtacgaa gtcgtcaatg atgtcgatat 2460
gggttttgat catgcacaca taaggtccga ccttatcggc aagctcaatg agctccttgg 2520
tggtggtaac atccagagaa gcacacaggt tggttttctt ggctgccacg agcttgagca 2580
ctcgagcggc aaaggcggac ttgtggacgt tagctcgagc ttcgtaggag ggcattttgg 2640
tggtgaagag gagactgaaa taaatttagt ctgcagaact ttttatcgga accttatctg 2700
gggcagtgaa gtatatgtta tggtaatagt tacgagttag ttgaacttat agatagactg 2760
gactatacgg ctatcggtcc aaattagaaa gaacgtcaat ggctctctgg gcgtcgcctt 2820
tgccgacaaa aatgtgatca tgatgaaagc cagcaatgac gttgcagctg atattgttgt 2880
cggccaaccg cgccgaaaac gcagctgtca gacccacagc ctccaacgaa gaatgtatcg 2940
tcaaagtgat ccaagcacac tcatagttgg agtcgtactc caaaggcggc aatgacgagt 3000
cagacagata ctcgtcgacc ttttccttgg gaaccaccac cgtcagccct tctgactcac 3060
gtattgtagc caccgacaca ggcaacagtc cgtggatagc agaatatgtc ttgtcggtcc 3120
atttctcacc aactttaggc gtcaagtgaa tgttgcagaa gaagtatgtg ccttcattga 3180
gaatcggtgt tgctgatttc aataaagtct tgagatcagt ttggcgcgcc agctgcatta 3240
atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc 3300
gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 3360
ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 3420
aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 3480
ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 3540
aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 3600
gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 3660
tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 3720
tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 3780
gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 3840
cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 3900
cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 3960
agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 4020
caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 4080
ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc 4140
aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 4200
tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc 4260
agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac 4320
gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc 4380
accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg 4440
tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag 4500
tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc 4560
acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac 4620
atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag 4680
aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac 4740
tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg 4800
agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc 4860
gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact 4920
ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg 4980
atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa 5040
tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt 5100
tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg 5160
tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga 5220
tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag 5280
cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca 5340
ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag 5400
tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg 5460
gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt 5520
tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag 5580
agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc 5640
gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc 5700
gcttaatgcg ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa 5760
gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca 5820
aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc 5880
agtgaattgt aatacgactc actatagggc gaattgggcc cgacgtcgca tgcagtggtg 5940
gtattgtgac tggggatgta gttgagaata agtcatacac aagtcagctt tcttcgagcc 6000
tcatataagt ataagtagtt caacgtatta gcactgtacc cagcatctcc gtatcgagaa 6060
acacaacaac atgccccatt ggacagatca tgcggataca caggttgtgc agtatcatac 6120
atactcgatc agacaggtcg tctgaccatc atacaagctg aacaagcgct ccatacttgc 6180
acgctctcta tatacacagt taaattacat atccatagtc taacctctaa cagttaatct 6240
tctggtaagc ctcccagcca gccttctggt atcgcttggc ctcctcaata ggatctcggt 6300
tctggccgta cagacctcgg ccgacaatta tgatatccgt tccggtagac atgacatcct 6360
caacagttcg gtactgctgt ccgagagcgt ctcccttgtc gtcaagaccc accccggggg 6420
tcagaataag ccagtcctca gagtcgccct taattaa 6457
<210>21
<211>1936
<212>DNA
<213>解脂耶罗维亚酵母
<220>
<221>CDS
<222>(283)..(1539)
<223>δ-12脱饱和酶
<400>21
cgtagttata tacaagaggt agatgcgtgc tggtgttaga ggggctctca ggattaggag 60
gaaaatttga cattggccct caacatataa cctcgggtgt gcctctgttt accctcagct 120
tttgcttgtc cccaagtcag tcacgccagg ccaaaaaggt tggtggattg acagggagaa 180
aaaaaaaagc ctagtgggtt taaactcgag gtaagacatt gaaatatata ccggtcggca 240
tcctgagtcc ctttctcgta ttccaacaga ccgaccatag aa atg gat tcg acc 294
Met Asp Ser Thr
1
acg cag acc aac acc ggc acc ggc aag gtg gcc gtg cag ccc ccc acg 342
Thr Gln Thr Asn Thr Gly Thr Gly Lys Val Ala Val Gln Pro Pro Thr
5 10 15 20
gcc ttc att aag ccc att gag aag gtg tcc gag ccc gtc tac gac acc 390
Ala Phe Ile Lys Pro Ile Glu Lys Val Ser Glu Pro Val Tyr Asp Thr
25 30 35
ttt ggc aac gag ttc act cct cca gac tac tct atc aag gat att ctg 438
Phe Gly Asn Glu Phe Thr Pro Pro Asp Tyr Ser Ile Lys Asp Ile Leu
40 45 50
gat gcc att ccc cag gag tgc tac aag cgg tcc tac gtt aag tcc tac 486
Asp Ala Ile Pro Gln Glu Cys Tyr Lys Arg Ser Tyr Val Lys Ser Tyr
55 60 65
tcg tac gtg gcc cga gac tgc ttc ttt atc gcc gtt ttt gcc tac atg 534
Ser Tyr Val Ala Arg Asp Cys Phe Phe Ile Ala Val Phe Ala Tyr Met
70 75 80
gcc tac gcg tac ctg cct ctt att ccc tcg gct tcc ggc cga gct gtg 582
Ala Tyr Ala Tyr Leu Pro Leu Ile Pro Ser Ala Ser Gly Arg Ala Val
85 90 95 100
gcc tgg gcc atg tac tcc att gtc cag ggt ctg ttt ggc acc ggt ctg 630
Ala Trp Ala Met Tyr Ser Ile Val Gln Gly Leu Phe Gly Thr Gly Leu
105 110 115
tgg gtt ctt gcc cac gag tgt ggc cac tct gct ttc tcc gac tct aac 678
Trp Val Leu Ala His Glu Cys Gly His Ser Ala Phe Ser Asp Ser Asn
120 125 130
acc gtc aac aac gtc acc gga tgg gtt ctg cac tcc tcc atg ctg gtc 726
Thr Val Asn Asn Val Thr Gly Trp Val Leu His Ser Ser Met Leu Val
135 140 145
cct tac tac gcc tgg aag ctg acc cac tcc atg cac cac aag tcc act 774
Pro Tyr Tyr Ala Trp Lys Leu Thr His Ser Met His His Lys Ser Thr
150 155 160
ggt cac ctc acc cgt gat atg gtg ttt gtg ccc aag gac cga aag gag 822
Gly His Leu Thr Arg Asp Met Val Phe Val Pro Lys Asp Arg Lys Glu
165 170 175 180
ttt atg gag aac cga ggc gcc cat gac tgg tct gag ctt gct gag gac 870
Phe Met Glu Asn Arg Gly Ala His Asp Trp Ser Glu Leu Ala Glu Asp
185 190 195
gct ccc ctc atg acc ctc tac ggc ctc atc acc cag cag gtg ttt gga 918
Ala Pro Leu Met Thr Leu Tyr Gly Leu Ile Thr Gln Gln Val Phe Gly
200 205 210
tgg cct ctg tat ctg ctg tct tac gtt acc gga cag aag tac ccc aag 966
Trp Pro Leu Tyr Leu Leu Ser Tyr Val Thr Gly Gln Lys Tyr Pro Lys
215 220 225
ctc aac aaa tgg gct gtc aac cac ttc aac ccc aac gcc ccg ctg ttt 1014
Leu Asn Lys Trp Ala Val Asn His Phe Asn Pro Asn Ala Pro Leu Phe
230 235 240
gag aag aag gac tgg ttc aac atc tgg atc tct aac gtc ggt att ggt 1062
Glu Lys Lys Asp Trp Phe Asn Ile Trp Ile Ser Asn Val Gly Ile Gly
245 250 255 260
atc acc atg tcc gtc atc gca tac tcc atc aac cga tgg ggc ctg gct 1110
Ile Thr Met Ser Val Ile Ala Tyr Ser Ile Asn Arg Trp Gly Leu Ala
265 270 275
tcc gtc acc ctc tac tac ctg atc ccc tac ctg tgg gtc aac cac tgg 1158
Ser Val Thr Leu Tyr Tyr Leu Ile Pro Tyr Leu Trp Val Asn His Trp
280 285 290
ctc gtg gcc atc acc tac ctg cag cac acc gac ccc act ctg ccc cac 1206
Leu Val Ala Ile Thr Tyr Leu Gln His Thr Asp Pro Thr Leu Pro His
295 300 305
tac cac gcc gac cag tgg aac ttc acc cga gga gcc gcc gcc acc atc 1254
Tyr His Ala Asp Gln Trp Asn Phe Thr Arg Gly Ala Ala Ala Thr Ile
310 315 320
gac cga gag ttt ggc ttc atc ggc tcc ttc tgc ttc cat gac atc atc 1302
Asp Arg Glu Phe Gly Phe Ile Gly Ser Phe Cys Phe His Asp Ile Ile
325 330 335 340
gag acc cac gtt ctg cac cac tac gtg tct cga att ccc ttc tac aac 1350
Glu Thr His Val Leu His His Tyr Val Ser Arg Ile Pro Phe Tyr Asn
345 350 355
gcc cga atc gcc act gag aag atc aag aag gtc atg ggc aag cac tac 1398
Ala Arg Ile Ala Thr Glu Lys Ile Lys Lys Val Met Gly Lys His Tyr
360 365 370
cga cac gac gac acc aac ttc atc aag tct ctt tac act gtc gcc cga 1446
Arg His Asp Asp Thr Asn Phe Ile Lys Ser Leu Tyr Thr Val Ala Arg
375 380 385
acc tgc cag ttt gtt gaa ggt aag gaa ggc att cag atg ttt aga aac 1494
Thr Cys Gln Phe Val Glu Gly Lys Glu Gly Ile Gln Met Phe Arg Asn
390 395 400
gtc aat gga gtc gga gtt gct cct gac ggc ctg cct tct aaa aag 1539
Val Asn Gly Val Gly Val Ala Pro Asp Gly Leu Pro Ser Lys Lys
405 410 415
tagagctaga aatgttattt gattgtgttt taactgaaca gcaccgagcc cgaggctaag 1599
ccaagcgaag ccgaggggtt gtgtagtcca tggacgtaac gagtaggcga tatcaccgca 1659
ctcggcactg cgtgtctgcg ttcatgggcg aagtcacatt acgctgacaa ccgttgtagt 1719
ttccctttag tatcaatact gttacaagta ccggtctcgt actcgtactg atacgaatct 1779
gtgggaagaa gtcaccctta tcagaccttc atactgatgt ttcggatatc aatagaactg 1839
gcatagagcc gttaaagaag tttcacttaa tcactccaac cctcctactt gtagattcaa 1899
gcagatcgat aagatggatt tgatggtcag tgctagc 1936
<210>22
<211>419
<212>PRT
<213>解脂耶罗维亚酵母
<400>22
Met Asp Ser Thr Thr Gln Thr Asn Thr Gly Thr Gly Lys Val Ala Val
1 5 10 15
Gln Pro Pro Thr Ala Phe Ile Lys Pro Ile Glu Lys Val Ser Glu Pro
20 25 30
Val Tyr Asp Thr Phe Gly Asn Glu Phe Thr Pro Pro Asp Tyr Ser Ile
35 40 45
Lys Asp Ile Leu Asp Ala Ile Pro Gln Glu Cys Tyr Lys Arg Ser Tyr
50 55 60
Val Lys Ser Tyr Ser Tyr Val Ala Arg Asp Cys Phe Phe Ile Ala Val
65 70 75 80
Phe Ala Tyr Met Ala Tyr Ala Tyr Leu Pro Leu Ile Pro Ser Ala Ser
85 90 95
Gly Arg Ala Val Ala Trp Ala Met Tyr Ser Ile Val Gln Gly Leu Phe
100 105 110
Gly Thr Gly Leu Trp Val Leu Ala His Glu Cys Gly His Ser Ala Phe
115 120 125
Ser Asp Ser Asn Thr Val Asn Asn Val Thr Gly Trp Val Leu His Ser
130 135 140
Ser Met Leu Val Pro Tyr Tyr Ala Trp Lys Leu Thr His Ser Met His
145 150 155 160
His Lys Ser Thr Gly His Leu Thr Arg Asp Met Val Phe Val Pro Lys
165 170 175
Asp Arg Lys Glu Phe Met Glu Asn Arg Gly Ala His Asp Trp Ser Glu
180 185 190
Leu Ala Glu Asp Ala Pro Leu Met Thr Leu Tyr Gly Leu Ile Thr Gln
195 200 205
Gln Val Phe Gly Trp Pro Leu Tyr Leu Leu Ser Tyr Val Thr Gly Gln
210 215 220
Lys Tyr Pro Lys Leu Asn Lys Trp Ala Val Asn His Phe Asn Pro Asn
225 230 235 240
Ala Pro Leu Phe Glu Lys Lys Asp Trp Phe Asn Ile Trp Ile Ser Asn
245 250 255
Val Gly Ile Gly Ile Thr Met Ser Val Ile Ala Tyr Ser Ile Asn Arg
260 265 270
Trp Gly Leu Ala Ser Val Thr Leu Tyr Tyr Leu Ile Pro Tyr Leu Trp
275 280 285
Val Asn His Trp Leu Val Ala Ile Thr Tyr Leu Gln His Thr Asp Pro
290 295 300
Thr Leu Pro His Tyr His Ala Asp Gln Trp Asn Phe Thr Arg Gly Ala
305 310 315 320
Ala Ala Thr Ile Asp Arg Glu Phe Gly Phe Ile Gly Ser Phe Cys Phe
325 330 335
His Asp Ile Ile Glu Thr His Val Leu His His Tyr Val Ser Arg Ile
340 345 350
Pro Phe Tyr Asn Ala Arg Ile Ala Thr Glu Lys Ile Lys Lys Val Met
355 360 365
Gly Lys His Tyr Arg His Asp Asp Thr Asn Phe Ile Lys Ser Leu Tyr
370 375 380
Thr Val Ala Arg Thr Cys Gln Phe Val Glu Gly Lys Glu Gly Ile Gln
385 390 395 400
Met Phe Arg Asn Val Asn Gly Val Gly Val Ala Pro Asp Gly Leu Pro
405 410 415
Ser Lys Lys
<210>23
<211>24
<212>DNA
<213>人工序列
<220>
<223>引物MA ELONG 5’NcoI 3
<400>23
gatcccatgg agtctggacc tatg 24
<210>24
<211>30
<212>DNA
<213>人工序列
<220>
<223>引物MA ELONG 3’NotI 1
<400>24
gatcgcggcc gcttactggg ccttcttctg 30
<210>25
<211>5553
<212>DNA
<213>人工序列
<220>
<223>质粒pZF5T-PPC
<400>25
ggccgcattg atgattggaa acacacacat gggttatatc taggtgagag ttagttggac 60
agttatatat taaatcagct atgccaacgg taacttcatt catgtcaacg aggaaccagt 120
gactgcaagt aatatagaat ttgaccacct tgccattctc ttgcactcct ttactatatc 180
tcatttattt cttatataca aatcacttct tcttcccagc atcgagctcg gaaacctcat 240
gagcaataac atcgtggatc tcgtcaatag agggcttttt ggactccttg ctgttggcca 300
ccttgtcctt gctgtctggc tcattctgtt tcaacgcctt ttaattaatc gagcttggcg 360
taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 420
atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 480
ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 540
taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 600
tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 660
aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 720
aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 780
ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 840
acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 900
ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 960
tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 1020
tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 1080
gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 1140
agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 1200
tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 1260
agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 1320
tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 1380
acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1440
tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1500
agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1560
tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 1620
acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 1680
tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 1740
ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 1800
agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 1860
tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 1920
acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 1980
agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 2040
actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 2100
tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 2160
gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 2220
ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 2280
tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 2340
aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 2400
tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 2460
tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 2520
gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 2580
gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 2640
acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 2700
agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 2760
ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 2820
ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 2880
taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 2940
aacgcgaatt ttaacaaaat attaacgctt acaatttcca ttcgccattc aggctgcgca 3000
actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 3060
gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta 3120
aaacgacggc cagtgaattg taatacgact cactataggg cgaattgggt accgggcccc 3180
ccctcgaggt cgacgtttaa acagtgtacg cagtactata gaggaacatc gattgccccg 3240
gagaagacgg ccaggccgcc tagatgacaa attcaacaac tcacagctga ctttctgcca 3300
ttgccactag gggggggcct ttttatatgg ccaagccaag ctctccacgt cggttgggct 3360
gcacccaaca ataaatgggt agggttgcac caacaaaggg atgggatggg gggtagaaga 3420
tacgaggata acggggctca atggcacaaa taagaacgaa tactgccatt aagactcgtg 3480
atccagcgac tgacaccatt gcatcatcta agggcctcaa aactacctcg gaactgctgc 3540
gctgatctgg acaccacaga ggttccgagc actttaggtt gcaccaaatg tcccaccagg 3600
tgcaggcaga aaacgctgga acagcgtgta cagtttgtct taacaaaaag tgagggcgct 3660
gaggtcgagc agggtggtgt gacttgttat agcctttaga gctgcgaaag cgcgtatgga 3720
tttggctcat caggccagat tgagggtctg tggacacatg tcatgttagt gtacttcaat 3780
cgccccctgg atatagcccc gacaataggc cgtggcctca tttttttgcc ttccgcacat 3840
ttccattgct cggtacccac accttgcttc tcctgcactt gccaacctta atactggttt 3900
acattgacca acatcttaca agcggggggc ttgtctaggg tatatataaa cagtggctct 3960
cccaatcggt tgccagtctc ttttttcctt tctttcccca cagattcgaa atctaaacta 4020
cacatcacac aatgcctgtt actgacgtcc ttaagcgaaa gtccggtgtc atcgtcggcg 4080
acgatgtccg agccgtgagt atccacgaca agatcagtgt cgagacgacg cgttttgtgt 4140
aatgacacaa tccgaaagtc gctagcaaca cacactctct acacaaacta acccagctct 4200
ccatgggaac ggaccaagga aaaaccttca cctgggaaga gctggcggcc cataacacca 4260
aggacgacct actcttggcc atccgcggca gggtgtacga tgtcacaaag ttcttgagcc 4320
gccatcctgg tggagtggac actctcctgc tcggagctgg ccgagatgtt actccggtct 4380
ttgagatgta tcacgcgttt ggggctgcag atgccattat gaagaagtac tatgtcggta 4440
cactggtctc gaatgagctg cccatcttcc cggagccaac ggtgttccac aaaaccatca 4500
agacgagagt cgagggctac tttacggatc ggaacattga tcccaagaat agaccagaga 4560
tctggggacg atacgctctt atctttggat ccttgatcgc ttcctactac gcgcagctct 4620
ttgtgccttt cgttgtcgaa cgcacatggc ttcaggtggt gtttgcaatc atcatgggat 4680
ttgcgtgcgc acaagtcgga ctcaaccctc ttcatgatgc gtctcacttt tcagtgaccc 4740
acaaccccac tgtctggaag attctgggag ccacgcacga ctttttcaac ggagcatcgt 4800
acctggtgtg gatgtaccaa catatgctcg gccatcaccc ctacaccaac attgctggag 4860
cagatcccga cgtgtcgacg tctgagcccg atgttcgtcg tatcaagccc aaccaaaagt 4920
ggtttgtcaa ccacatcaac cagcacatgt ttgttccttt cctgtacgga ctgctggcgt 4980
tcaaggtgcg cattcaggac atcaacattt tgtactttgt caagaccaat gacgctattc 5040
gtgtcaatcc catctcgaca tggcacactg tgatgttctg gggcggcaag gctttctttg 5100
tctggtatcg cctgattgtt cccctgcagt atctgcccct gggcaaggtg ctgctcttgt 5160
tcacggtcgc ggacatggtg tcgtcttact ggctggcgct gaccttccag gcgaaccacg 5220
ttgttgagga agttcagtgg ccgttgcctg acgagaacgg gatcatccaa aaggactggg 5280
cagctatgca ggtcgagact acgcaggatt acgcacacga ttcgcacctc tggaccagca 5340
tcactggcag cttgaactac caggctgtgc accatctgtt ccccaacgtg tcgcagcacc 5400
attatcccga tattctggcc atcatcaaga acacctgcag cgagtacaag gttccatacc 5460
ttgtcaagga tacgttttgg caagcatttg cttcacattt ggagcacttg cgtgttcttg 5520
gactccgtcc caaggaagag taggcagcta agc 5553
<210>26
<211>5031
<212>DNA
<213>人工序列
<220>
<223>质粒pZF5T-PPC-E3
<400>26
atggagtctg gacctatgcc tgccgggatc cccttccctg aatactatga ctttttcatg 60
gactggaaga cacccctggc aattgctgcc acctacaccg ccgctgttgg gctcttcaac 120
cccaaggttg gcaaagtctc gcgcgtggta gccaagtcgg ctaacgccaa gccggcagag 180
cgcacgcagt ccggcgccgc catgaccgcc tttgtctttg tccacaacct tatcctctgc 240
gtgtactctg gaatcacctt ctactacatg ttcccagcca tggtcaagaa ctttagaaca 300
cataccctcc atgaggccta ctgcgatacg gatcagagcc tgtggaacaa cgcccttggc 360
tactggggct acctcttcta cctttcaaag ttttacgagg tcattgacac catcatcatc 420
atcttgaagg ggcgccgctc gtccctgctc cagacctacc accacgccgg cgctatgatc 480
accatgtggt ccggcatcaa ctaccaggca acgcccattt ggatttttgt cgtcttcaac 540
tcgttcatcc acaccatcat gtactgttac tatgccttca cctcaatcgg cttccacccc 600
ccargcaaga agtacctcac ctccatgcag atcacccagt ttttggtcgg catcactatc 660
gccgtctctt atctcttcgt ccctggatgt atccgcacac ccggtgctca gatggctgtc 720
tggatcaacg tcggatacct ctttcccctc acttatctct ttgtggattt tgccaagcgt 780
acttactcca agcgtagtgc catcgccgct cagaagaagg cccagtaagg ccgcattgat 840
gattggaaac acacacatgg gttatatcta ggtgagagtt agttggacag ttatatatta 900
aatcagctat gccaacggta acttcattca tgtcaacgag gaaccagtga ctgcaagtaa 960
tatagaattt gaccaccttg ccattctctt gcactccttt actatatctc atttatttct 1020
tatatacaaa tcacttcttc ttcccagcat cgagctcgga aacctcatga gcaataacat 1080
cgtggatctc gtcaatagag ggctttttgg actccttgct gttggccacc ttgtccttgc 1140
tgtctggctc attctgtttc aacgcctttt aattaatcga gcttggcgta atcatggtca 1200
tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 1260
agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 1320
cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 1380
caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 1440
tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 1500
cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 1560
aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 1620
gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 1680
agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 1740
cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 1800
cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 1860
ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 1920
gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 1980
tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg 2040
acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 2100
tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 2160
attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 2220
gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 2280
ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 2340
taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 2400
ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 2460
ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 2520
gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 2580
ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 2640
gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 2700
tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 2760
atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 2820
gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 2880
tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 2940
atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 3000
agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 3060
ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 3120
tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 3180
aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 3240
tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 3300
aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgcgccctgt 3360
agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 3420
agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc 3480
tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg 3540
cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga 3600
tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc 3660
caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg 3720
ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt 3780
aacaaaatat taacgcttac aatttccatt cgccattcag gctgcgcaac tgttgggaag 3840
ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa 3900
ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca 3960
gtgaattgta atacgactca ctatagggcg aattgggtac cgggcccccc ctcgaggtcg 4020
acgtttaaac agtgtacgca gtactataga ggaacatcga ttgccccgga gaagacggcc 4080
aggccgccta gatgacaaat tcaacaactc acagctgact ttctgccatt gccactaggg 4140
gggggccttt ttatatggcc aagccaagct ctccacgtcg gttgggctgc acccaacaat 4200
aaatgggtag ggttgcacca acaaagggat gggatggggg gtagaagata cgaggataac 4260
ggggctcaat ggcacaaata agaacgaata ctgccattaa gactcgtgat ccagcgactg 4320
acaccattgc atcatctaag ggcctcaaaa ctacctcgga actgctgcgc tgatctggac 4380
accacagagg ttccgagcac tttaggttgc accaaatgtc ccaccaggtg caggcagaaa 4440
acgctggaac agcgtgtaca gtttgtctta acaaaaagtg agggcgctga ggtcgagcag 4500
ggtggtgtga cttgttatag cctttagagc tgcgaaagcg cgtatggatt tggctcatca 4560
ggccagattg agggtctgtg gacacatgtc atgttagtgt acttcaatcg ccccctggat 4620
atagccccga caataggccg tggcctcatt tttttgcctt ccgcacattt ccattgctcg 4680
gtacccacac cttgcttctc ctgcacttgc caaccttaat actggtttac attgaccaac 4740
atcttacaag cggggggctt gtctagggta tatataaaca gtggctctcc caatcggttg 4800
ccagtctctt ttttcctttc tttccccaca gattcgaaat ctaaactaca catcacacaa 4860
tgcctgttac tgacgtcctt aagcgaaagt ccggtgtcat cgtcggcgac gatgtccgag 4920
ccgtgagtat ccacgacaag atcagtgtcg agacgacgcg ttttgtgtaa tgacacaatc 4980
cgaaagtcgc tagcaacaca cactctctac acaaactaac ccagctctcc a 5031
<210>27
<211>8462
<212>DNA
<213>人工序列
<220>
<223>质粒pZUF6S
<400>27
ggccgcaagt gtggatgggg aagtgagtgc ccggttctgt gtgcacaatt ggcaatccaa 60
gatggatgga ttcaacacag ggatatagcg agctacgtgg tggtgcgagg atatagcaac 120
ggatatttat gtttgacact tgagaatgta cgatacaagc actgtccaag tacaatacta 180
aacatactgt acatactcat actcgtaccc gggcaacggt ttcacttgag tgcagtggct 240
agtgctctta ctcgtacagt gtgcaatact gcgtatcata gtctttgatg tatatcgtat 300
tcattcatgt tagttgcgta cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 360
gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 420
tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 480
ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 540
cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 600
gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 660
tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 720
agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 780
tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 840
cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 900
ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 960
ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 1020
ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 1080
ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 1140
cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 1200
gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 1260
atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 1320
ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 1380
gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 1440
tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 1500
ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 1560
taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 1620
gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 1680
gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 1740
ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 1800
aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 1860
gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 1920
cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 1980
actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 2040
caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 2100
gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 2160
ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 2220
caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 2280
tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 2340
gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 2400
cccgaaaagt gccacctgac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 2460
ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct 2520
tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 2580
ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg 2640
atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 2700
ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 2760
tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc 2820
tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttccattc 2880
gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg 2940
ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc 3000
ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tatagggcga 3060
attgggtacc gggccccccc tcgaggtcga tggtgtcgat aagcttgata tcgaattcat 3120
gtcacacaaa ccgatcttcg cctcaaggaa acctaattct acatccgaga gactgccgag 3180
atccagtcta cactgattaa ttttcgggcc aataatttaa aaaaatcgtg ttatataata 3240
ttatatgtat tatatatata catcatgatg atactgacag tcatgtccca ttgctaaata 3300
gacagactcc atctgccgcc tccaactgat gttctcaata tttaaggggt catctcgcat 3360
tgtttaataa taaacagact ccatctaccg cctccaaatg atgttctcaa aatatattgt 3420
atgaacttat ttttattact tagtattatt agacaactta cttgctttat gaaaaacact 3480
tcctatttag gaaacaattt ataatggcag ttcgttcatt taacaattta tgtagaataa 3540
atgttataaa tgcgtatggg aaatcttaaa tatggatagc ataaatgata tctgcattgc 3600
ctaattcgaa atcaacagca acgaaaaaaa tcccttgtac aacataaata gtcatcgaga 3660
aatatcaact atcaaagaac agctattcac acgttactat tgagattatt attggacgag 3720
aatcacacac tcaactgtct ttctctcttc tagaaataca ggtacaagta tgtactattc 3780
tcattgttca tacttctagt catttcatcc cacatattcc ttggatttct ctccaatgaa 3840
tgacattcta tcttgcaaat tcaacaatta taataagata taccaaagta gcggtatagt 3900
ggcaatcaaa aagcttctct ggtgtgcttc tcgtatttat ttttattcta atgatccatt 3960
aaaggtatat atttatttct tgttatataa tccttttgtt tattacatgg gctggataca 4020
taaaggtatt ttgatttaat tttttgctta aattcaatcc cccctcgttc agtgtcaact 4080
gtaatggtag gaaattacca tacttttgaa gaagcaaaaa aaatgaaaga aaaaaaaaat 4140
cgtatttcca ggttagacgt tccgcagaat ctagaatgcg gtatgcggta cattgttctt 4200
cgaacgtaaa agttgcgctc cctgagatat tgtacatttt tgcttttaca agtacaagta 4260
catcgtacaa ctatgtacta ctgttgatgc atccacaaca gtttgttttg tttttttttg 4320
tttttttttt ttctaatgat tcattaccgc tatgtatacc tacttgtact tgtagtaagc 4380
cgggttattg gcgttcaatt aatcatagac ttatgaatct gcacggtgtg cgctgcgagt 4440
tacttttagc ttatgcatgc tacttgggtg taatattggg atctgttcgg aaatcaacgg 4500
atgctcaatc gatttcgaca gtaattaatt aagtcataca caagtcagct ttcttcgagc 4560
ctcatataag tataagtagt tcaacgtatt agcactgtac ccagcatctc cgtatcgaga 4620
aacacaacaa catgccccat tggacagatc atgcggatac acaggttgtg cagtatcata 4680
catactcgat cagacaggtc gtctgaccat catacaagct gaacaagcgc tccatacttg 4740
cacgctctct atatacacag ttaaattaca tatccatagt ctaacctcta acagttaatc 4800
ttctggtaag cctcccagcc agccttctgg tatcgcttgg cctcctcaat aggatctcgg 4860
ttctggccgt acagacctcg gccgacaatt atgatatccg ttccggtaga catgacatcc 4920
tcaacagttc ggtactgctg tccgagagcg tctcccttgt cgtcaagacc caccccgggg 4980
gtcagaataa gccagtcctc agagtcgccc ttaggtcggt tctgggcaat gaagccaacc 5040
acaaactcgg ggtcggatcg ggcaagctca atggtctgct tggagtactc gccagtggcc 5100
agagagccct tgcaagacag ctcggccagc atgagcagac ctctggccag cttctcgttg 5160
ggagagggga ctaggaactc cttgtactgg gagttctcgt agtcagagac gtcctccttc 5220
ttctgttcag agacagtttc ctcggcacca gctcgcaggc cagcaatgat tccggttccg 5280
ggtacaccgt gggcgttggt gatatcggac cactcggcga ttcggtgaca ccggtactgg 5340
tgcttgacag tgttgccaat atctgcgaac tttctgtcct cgaacaggaa gaaaccgtgc 5400
ttaagagcaa gttccttgag ggggagcaca gtgccggcgt aggtgaagtc gtcaatgatg 5460
tcgatatggg ttttgatcat gcacacataa ggtccgacct tatcggcaag ctcaatgagc 5520
tccttggtgg tggtaacatc cagagaagca cacaggttgg ttttcttggc tgccacgagc 5580
ttgagcactc gagcggcaaa ggcggacttg tggacgttag ctcgagcttc gtaggagggc 5640
attttggtgg tgaagaggag actgaaataa atttagtctg cagaactttt tatcggaacc 5700
ttatctgggg cagtgaagta tatgttatgg taatagttac gagttagttg aacttataga 5760
tagactggac tatacggcta tcggtccaaa ttagaaagaa cgtcaatggc tctctgggcg 5820
tcgcctttgc cgacaaaaat gtgatcatga tgaaagccag caatgacgtt gcagctgata 5880
ttgttgtcgg ccaaccgcgc cgaaaacgca gctgtcagac ccacagcctc caacgaagaa 5940
tgtatcgtca aagtgatcca agcacactca tagttggagt cgtactccaa aggcggcaat 6000
gacgagtcag acagatactc gtcgactcag gcgacgacgg aattcctgca gcccatctgc 6060
agaattcagg agagaccggg ttggcggcgt atttgtgtcc caaaaaacag ccccaattgc 6120
cccggagaag acggccaggc cgcctagatg acaaattcaa caactcacag ctgactttct 6180
gccattgcca ctaggggggg gcctttttat atggccaagc caagctctcc acgtcggttg 6240
ggctgcaccc aacaataaat gggtagggtt gcaccaacaa agggatggga tggggggtag 6300
aagatacgag gataacgggg ctcaatggca caaataagaa cgaatactgc cattaagact 6360
cgtgatccag cgactgacac cattgcatca tctaagggcc tcaaaactac ctcggaactg 6420
ctgcgctgat ctggacacca cagaggttcc gagcacttta ggttgcacca aatgtcccac 6480
caggtgcagg cagaaaacgc tggaacagcg tgtacagttt gtcttaacaa aaagtgaggg 6540
cgctgaggtc gagcagggtg gtgtgacttg ttatagcctt tagagctgcg aaagcgcgta 6600
tggatttggc tcatcaggcc agattgaggg tctgtggaca catgtcatgt tagtgtactt 6660
caatcgcccc ctggatatag ccccgacaat aggccgtggc ctcatttttt tgccttccgc 6720
acatttccat tgctcggtac ccacaccttg cttctcctgc acttgccaac cttaatactg 6780
gtttacattg accaacatct tacaagcggg gggcttgtct agggtatata taaacagtgg 6840
ctctcccaat cggttgccag tctctttttt cctttctttc cccacagatt cgaaatctaa 6900
actacacatc acacaatgcc tgttactgac gtccttaagc gaaagtccgg tgtcatcgtc 6960
ggcgacgatg tccgagccgt gagtatccac gacaagatca gtgtcgagac gacgcgtttt 7020
gtgtaatgac acaatccgaa agtcgctagc aacacacact ctctacacaa actaacccag 7080
ctctccatgg ctgccgctcc ctctgtgcga acctttaccc gagccgaggt tctgaacgct 7140
gaggctctga acgagggcaa gaaggacgct gaggctccct tcctgatgat catcgacaac 7200
aaggtgtacg acgtccgaga gttcgtccct gaccatcctg gaggctccgt gattctcacc 7260
cacgttggca aggacggcac cgacgtcttt gacacctttc atcccgaggc tgcttgggag 7320
actctcgcca acttctacgt tggagacatt gacgagtccg accgagacat caagaacgat 7380
gactttgccg ctgaggtccg aaagctgcga accctgttcc agtctctcgg ctactacgac 7440
tcctctaagg cctactacgc cttcaaggtc tccttcaacc tctgcatctg gggactgtcc 7500
accgtcattg tggccaagtg gggtcagacc tccaccctcg ccaacgtgct ctctgctgcc 7560
ctgctcggcc tgttctggca gcagtgcgga tggctggctc acgactttct gcaccaccag 7620
gtcttccagg accgattctg gggtgatctc ttcggagcct tcctgggagg tgtctgccag 7680
ggcttctcct cttcctggtg gaaggacaag cacaacactc accatgccgc tcccaacgtg 7740
catggcgagg atcctgacat tgacacccac cctctcctga cctggtccga gcacgctctg 7800
gagatgttct ccgacgtccc cgatgaggag ctgacccgaa tgtggtctcg attcatggtc 7860
ctgaaccaga cctggttcta cttccccatt ctctccttcg ctcgactgtc ttggtgcctc 7920
cagtccattc tctttgtgct gcccaacggt caggctcaca agccctccgg agctcgagtg 7980
cccatctccc tggtcgagca gctgtccctc gccatgcact ggacctggta cctcgctacc 8040
atgttcctgt tcatcaagga tcctgtcaac atgctcgtgt acttcctggt gtctcaggct 8100
gtgtgcggaa acctgctcgc catcgtgttc tccctcaacc acaacggtat gcctgtgatc 8160
tccaaggagg aggctgtcga catggatttc tttaccaagc agatcatcac tggtcgagat 8220
gtccatcctg gactgttcgc caactggttc accggtggcc tgaactacca gatcgagcat 8280
cacctgttcc cttccatgcc tcgacacaac ttctccaaga tccagcctgc cgtcgagacc 8340
ctgtgcaaga agtacaacgt ccgataccac accactggta tgatcgaggg aactgccgag 8400
gtcttctccc gactgaacga ggtctccaag gccacctcca agatgggcaa ggctcagtaa 8460
gc 8462
<210>28
<211>11046
<212>DNA
<213>人工序列
<220>
<223>质粒pZUF6S-E3WT
<400>28
ggccgcaagt gtggatgggg aagtgagtgc ccggttctgt gtgcacaatt ggcaatccaa 60
gatggatgga ttcaacacag ggatatagcg agctacgtgg tggtgcgagg atatagcaac 120
ggatatttat gtttgacact tgagaatgta cgatacaagc actgtccaag tacaatacta 180
aacatactgt acatactcat actcgtaccc gggcaacggt ttcacttgag tgcagtggct 240
agtgctctta ctcgtacagt gtgcaatact gcgtatcata gtctttgatg tatatcgtat 300
tcattcatgt tagttgcgta cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 360
gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 420
tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 480
ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 540
cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 600
gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 660
tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 720
agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 780
tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 840
cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 900
ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 960
ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 1020
ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 1080
ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 1140
cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 1200
gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 1260
atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 1320
ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 1380
gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 1440
tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 1500
ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 1560
taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 1620
gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 1680
gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 1740
ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 1800
aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 1860
gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 1920
cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 1980
actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 2040
caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 2100
gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 2160
ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 2220
caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 2280
tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 2340
gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 2400
cccgaaaagt gccacctgac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 2460
ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct 2520
tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 2580
ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg 2640
atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 2700
ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 2760
tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc 2820
tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttccattc 2880
gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg 2940
ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc 3000
ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tatagggcga 3060
attgggtacc gggccccccc tcgaggtcga tggtgtcgat aagcttgata tcgaattcat 3120
gtcacacaaa ccgatcttcg cctcaaggaa acctaattct acatccgaga gactgccgag 3180
atccagtcta cactgattaa ttttcgggcc aataatttaa aaaaatcgtg ttatataata 3240
ttatatgtat tatatatata catcatgatg atactgacag tcatgtccca ttgctaaata 3300
gacagactcc atctgccgcc tccaactgat gttctcaata tttaaggggt catctcgcat 3360
tgtttaataa taaacagact ccatctaccg cctccaaatg atgttctcaa aatatattgt 3420
atgaacttat ttttattact tagtattatt agacaactta cttgctttat gaaaaacact 3480
tcctatttag gaaacaattt ataatggcag ttcgttcatt taacaattta tgtagaataa 3540
atgttataaa tgcgtatggg aaatcttaaa tatggatagc ataaatgata tctgcattgc 3600
ctaattcgaa atcaacagca acgaaaaaaa tcccttgtac aacataaata gtcatcgaga 3660
aatatcaact atcaaagaac agctattcac acgttactat tgagattatt attggacgag 3720
aatcacacac tcaactgtct ttctctcttc tagaaataca ggtacaagta tgtactattc 3780
tcattgttca tacttctagt catttcatcc cacatattcc ttggatttct ctccaatgaa 3840
tgacattcta tcttgcaaat tcaacaatta taataagata taccaaagta gcggtatagt 3900
ggcaatcaaa aagcttctct ggtgtgcttc tcgtatttat ttttattcta atgatccatt 3960
aaaggtatat atttatttct tgttatataa tccttttgtt tattacatgg gctggataca 4020
taaaggtatt ttgatttaat tttttgctta aattcaatcc cccctcgttc agtgtcaact 4080
gtaatggtag gaaattacca tacttttgaa gaagcaaaaa aaatgaaaga aaaaaaaaat 4140
cgtatttcca ggttagacgt tccgcagaat ctagaatgcg gtatgcggta cattgttctt 4200
cgaacgtaaa agttgcgctc cctgagatat tgtacatttt tgcttttaca agtacaagta 4260
catcgtacaa ctatgtacta ctgttgatgc atccacaaca gtttgttttg tttttttttg 4320
tttttttttt ttctaatgat tcattaccgc tatgtatacc tacttgtact tgtagtaagc 4380
cgggttattg gcgttcaatt aatcatagac ttatgaatct gcacggtgtg cgctgcgagt 4440
tacttttagc ttatgcatgc tacttgggtg taatattggg atctgttcgg aaatcaacgg 4500
atgctcaatc gatgcagaat tcaggagaga ccgggttggc ggcgtatttg tgtcccaaaa 4560
aacagcccca attgccccaa ttgaccccaa attgacccag tagcgggccc aaccccggcg 4620
agagccccct tcaccccaca tatcaaacct cccccggttc ccacacttgc cgttaagggc 4680
gtagggtact gcagtctgga atctacgctt gttcagactt tgtactagtt tctttgtctg 4740
gccatccggg taacccatgc cggacgcaaa atagactact gaaaattttt ttgctttgtg 4800
gttgggactt tagccaaggg tataaaagac caccgtcccc gaattacctt tcctcttctt 4860
ttctctctct ccttgtcaac tcacacccga aatcgttaag catttccttc tgagtataag 4920
aatcattcac caggagagac cgggttggcg gcgtatttgt gtcccaaaaa acagccccaa 4980
ttgccccgga gaagacggcc aggccgccta gatgacaaat tcaacaactc acagctgact 5040
ttctgccatt gccactaggg gggggccttt ttatatggcc aagccaagct ctccacgtcg 5100
gttgggctgc acccaacaat aaatgggtag ggttgcacca acaaagggat gggatggggg 5160
gtagaagata cgaggataac ggggctcaat ggcacaaata agaacgaata ctgccattaa 5220
gactcgtgat ccagcgactg acaccattgc atcatctaag ggcctcaaaa ctacctcgga 5280
actgctgcgc tgatctggac accacagagg ttccgagcac tttaggttgc accaaatgtc 5340
ccaccaggtg caggcagaaa acgctggaac agcgtgtaca gtttgtctta acaaaaagtg 5400
agggcgctga ggtcgagcag ggtggtgtga cttgttatag cctttagagc tgcgaaagcg 5460
cgtatggatt tggctcatca ggccagattg agggtctgtg gacacatgtc atgttagtgt 5520
acttcaatcg ccccctggat atagccccga caataggccg tggcctcatt tttttgcctt 5580
ccgcacattt ccattgctcg gtacccacac cttgcttctc ctgcacttgc caaccttaat 5640
actggtttac attgaccaac atcttacaag cggggggctt gtctagggta tatataaaca 5700
gtggctctcc caatcggttg ccagtctctt ttttcctttc tttccccaca gattcgaaat 5760
ctaaactaca catcacacaa tgcctgttac tgacgtcctt aagcgaaagt ccggtgtcat 5820
cgtcggcgac gatgtccgag ccgtgagtat ccacgacaag atcagtgtcg agacgacgcg 5880
ttttgtgtaa tgacacaatc cgaaagtcgc tagcaacaca cactctctac acaaactaac 5940
ccagctatgg agtctggacc tatgcctgcc gggatcccct tccctgaata ctatgacttt 6000
ttcatggact ggaagacacc cctggcaatt gctgccacct acaccgccgc tgttgggctc 6060
ttcaacccca aggttggcaa agtctcgcgc gtggtagcca agtcggctaa cgccaagccg 6120
gcagagcgca cgcagtccgg cgccgccatg accgcctttg tctttgtcca caaccttatc 6180
ctctgcgtgt actctggaat caccttctac tacatgttcc cagccatggt caagaacttt 6240
agaacacata ccctccatga ggcctactgc gatacggatc agagcctgtg gaacaacgcc 6300
cttggctact ggggctacct cttctacctt tcaaagtttt acgaggtcat tgacaccatc 6360
atcatcatct tgaaggggcg ccgctcgtcc ctgctccaga cctaccacca cgccggcgct 6420
atgatcacca tgtggtccgg catcaactac caggcaacgc ccatttggat ttttgtcgtc 6480
ttcaactcgt tcatccacac catcatgtac tgttactatg ccttcacctc aatcggcttc 6540
caccccccar gcaagaagta cctcacctcc atgcagatca cccagttttt ggtcggcatc 6600
actatcgccg tctcttatct cttcgtccct ggatgtatcc gcacacccgg tgctcagatg 6660
gctgtctgga tcaacgtcgg atacctcttt cccctcactt atctctttgt ggattttgcc 6720
aagcgtactt actccaagcg tagtgccatc gccgctcaga agaaggccca gtaaattgat 6780
gattggaaac acacacatgg gttatatcta ggtgagagtt agttggacag ttatatatta 6840
aatcagctat gccaacggta acttcattca tgtcaacgag gaaccagtga ctgcaagtaa 6900
tatagaattt gaccaccttg ccattctctt gcactccttt actatatctc atttatttct 6960
tatatacaaa tcacttcttc ttcccagcat cgagctcgga aacctcatga gcaataacat 7020
cgtggatctc gtcaatagag ggctttttgg actccttgct gttggccacc ttgtccttgc 7080
tgtctggctc attctgtttc aacgcctttt aattaagtca tacacaagtc agctttcttc 7140
gagcctcata taagtataag tagttcaacg tattagcact gtacccagca tctccgtatc 7200
gagaaacaca acaacatgcc ccattggaca gatcatgcgg atacacaggt tgtgcagtat 7260
catacatact cgatcagaca ggtcgtctga ccatcataca agctgaacaa gcgctccata 7320
cttgcacgct ctctatatac acagttaaat tacatatcca tagtctaacc tctaacagtt 7380
aatcttctgg taagcctccc agccagcctt ctggtatcgc ttggcctcct caataggatc 7440
tcggttctgg ccgtacagac ctcggccgac aattatgata tccgttccgg tagacatgac 7500
atcctcaaca gttcggtact gctgtccgag agcgtctccc ttgtcgtcaa gacccacccc 7560
gggggtcaga ataagccagt cctcagagtc gcccttaggt cggttctggg caatgaagcc 7620
aaccacaaac tcggggtcgg atcgggcaag ctcaatggtc tgcttggagt actcgccagt 7680
ggccagagag cccttgcaag acagctcggc cagcatgagc agacctctgg ccagcttctc 7740
gttgggagag gggactagga actccttgta ctgggagttc tcgtagtcag agacgtcctc 7800
cttcttctgt tcagagacag tttcctcggc accagctcgc aggccagcaa tgattccggt 7860
tccgggtaca ccgtgggcgt tggtgatatc ggaccactcg gcgattcggt gacaccggta 7920
ctggtgcttg acagtgttgc caatatctgc gaactttctg tcctcgaaca ggaagaaacc 7980
gtgcttaaga gcaagttcct tgagggggag cacagtgccg gcgtaggtga agtcgtcaat 8040
gatgtcgata tgggttttga tcatgcacac ataaggtccg accttatcgg caagctcaat 8100
gagctccttg gtggtggtaa catccagaga agcacacagg ttggttttct tggctgccac 8160
gagcttgagc actcgagcgg caaaggcgga cttgtggacg ttagctcgag cttcgtagga 8220
gggcattttg gtggtgaaga ggagactgaa ataaatttag tctgcagaac tttttatcgg 8280
aaccttatct ggggcagtga agtatatgtt atggtaatag ttacgagtta gttgaactta 8340
tagatagact ggactatacg gctatcggtc caaattagaa agaacgtcaa tggctctctg 8400
ggcgtcgcct ttgccgacaa aaatgtgatc atgatgaaag ccagcaatga cgttgcagct 8460
gatattgttg tcggccaacc gcgccgaaaa cgcagctgtc agacccacag cctccaacga 8520
agaatgtatc gtcaaagtga tccaagcaca ctcatagttg gagtcgtact ccaaaggcgg 8580
caatgacgag tcagacagat actcgtcgac tcaggcgacg acggaattcc tgcagcccat 8640
ctgcagaatt caggagagac cgggttggcg gcgtatttgt gtcccaaaaa acagccccaa 8700
ttgccccgga gaagacggcc aggccgccta gatgacaaat tcaacaactc acagctgact 8760
ttctgccatt gccactaggg gggggccttt ttatatggcc aagccaagct ctccacgtcg 8820
gttgggctgc acccaacaat aaatgggtag ggttgcacca acaaagggat gggatggggg 8880
gtagaagata cgaggataac ggggctcaat ggcacaaata agaacgaata ctgccattaa 8940
gactcgtgat ccagcgactg acaccattgc atcatctaag ggcctcaaaa ctacctcgga 9000
actgctgcgc tgatctggac accacagagg ttccgagcac tttaggttgc accaaatgtc 9060
ccaccaggtg caggcagaaa acgctggaac agcgtgtaca gtttgtctta acaaaaagtg 9120
agggcgctga ggtcgagcag ggtggtgtga cttgttatag cctttagagc tgcgaaagcg 9180
cgtatggatt tggctcatca ggccagattg agggtctgtg gacacatgtc atgttagtgt 9240
acttcaatcg ccccctggat atagccccga caataggccg tggcctcatt tttttgcctt 9300
ccgcacattt ccattgctcg gtacccacac cttgcttctc ctgcacttgc caaccttaat 9360
actggtttac attgaccaac atcttacaag cggggggctt gtctagggta tatataaaca 9420
gtggctctcc caatcggttg ccagtctctt ttttcctttc tttccccaca gattcgaaat 9480
ctaaactaca catcacacaa tgcctgttac tgacgtcctt aagcgaaagt ccggtgtcat 9540
cgtcggcgac gatgtccgag ccgtgagtat ccacgacaag atcagtgtcg agacgacgcg 9600
ttttgtgtaa tgacacaatc cgaaagtcgc tagcaacaca cactctctac acaaactaac 9660
ccagctctcc atggctgccg ctccctctgt gcgaaccttt acccgagccg aggttctgaa 9720
cgctgaggct ctgaacgagg gcaagaagga cgctgaggct cccttcctga tgatcatcga 9780
caacaaggtg tacgacgtcc gagagttcgt ccctgaccat cctggaggct ccgtgattct 9840
cacccacgtt ggcaaggacg gcaccgacgt ctttgacacc tttcatcccg aggctgcttg 9900
ggagactctc gccaacttct acgttggaga cattgacgag tccgaccgag acatcaagaa 9960
cgatgacttt gccgctgagg tccgaaagct gcgaaccctg ttccagtctc tcggctacta 10020
cgactcctct aaggcctact acgccttcaa ggtctccttc aacctctgca tctggggact 10080
gtccaccgtc attgtggcca agtggggtca gacctccacc ctcgccaacg tgctctctgc 10140
tgccctgctc ggcctgttct ggcagcagtg cggatggctg gctcacgact ttctgcacca 10200
ccaggtcttc caggaccgat tctggggtga tctcttcgga gccttcctgg gaggtgtctg 10260
ccagggcttc tcctcttcct ggtggaagga caagcacaac actcaccatg ccgctcccaa 10320
cgtgcatggc gaggatcctg acattgacac ccaccctctc ctgacctggt ccgagcacgc 10380
tctggagatg ttctccgacg tccccgatga ggagctgacc cgaatgtggt ctcgattcat 10440
ggtcctgaac cagacctggt tctacttccc cattctctcc ttcgctcgac tgtcttggtg 10500
cctccagtcc attctctttg tgctgcccaa cggtcaggct cacaagccct ccggagctcg 10560
agtgcccatc tccctggtcg agcagctgtc cctcgccatg cactggacct ggtacctcgc 10620
taccatgttc ctgttcatca aggatcctgt caacatgctc gtgtacttcc tggtgtctca 10680
ggctgtgtgc ggaaacctgc tcgccatcgt gttctccctc aaccacaacg gtatgcctgt 10740
gatctccaag gaggaggctg tcgacatgga tttctttacc aagcagatca tcactggtcg 10800
agatgtccat cctggactgt tcgccaactg gttcaccggt ggcctgaact accagatcga 10860
gcatcacctg ttcccttcca tgcctcgaca caacttctcc aagatccagc ctgccgtcga 10920
gaccctgtgc aagaagtaca acgtccgata ccacaccact ggtatgatcg agggaactgc 10980
cgaggtcttc tcccgactga acgaggtctc caaggccacc tccaagatgg gcaaggctca 11040
gtaagc 11046
<210>29
<211>4099
<212>DNA
<213>高山被孢霉
<220>
<221>misc_feature
<222>(2092)..(2094)
<223>ATG翻译起始密码子
<220>
<221>内含子
<222>(2410)..(2951)
<220>
<221>misc_feature
<222>(3459)..(3461)
<223>’TAA’终止密码子
<400>29
actatagggc acgcgtggtc gacggcccgg gctggtccta cctctcaatc ttgttgccct 60
gtagtcctga aatgagctcg tcatgggtct ctgtcctctc ctcgttatgg tcaaacgcat 120
tggacccggt tttctttaga ggagcattct tcttgtttaa gccgactcct cttcccgtcc 180
cgaacgacaa cttgataggt ccaccatttc catttggtgg gtgaagcggg ctgtcctcag 240
cggacatgac tctgagctag aaggaagggt gtagaggacg aaggagtaag tgctaccgtt 300
cactggcagg tgaggagcgt cgggtctaag ccacatgcct gccaattgtc ctcgccaagt 360
gcttggaagg tgtattgatg ctcatgcagc tccactctgg acctggaaac gccaccgcat 420
cgcattctta atgagctctg ctacaaacga caatctgcga accagtttgc acagggagct 480
agtcgagtct ggaaagcggg accagtgagt gactcggcgg ggcttgcatc gctgaacatc 540
gccattggga tttcttagaa agcagctctt gctcttatac aaacaccttc ctgcgtgcct 600
caccttactt acgaggagta atgttgtttt tacaggctag tggaactctt gcgcgcgagg 660
ctgatcgagt ccggctggcg ggacgatctg aaggcattta caaaaggtac accactcggc 720
acttggcctg ttttgttgcc actagctacc aggccgtgac tgaccacgaa cccttcctct 780
ggagaataga aaggatccag tcccgggaga ctacaatgtc tgttgacgag gtcatcagag 840
aagtcggccc acatgctaga ggtaagcccc gcgagaacct tatatgctga ttagtgccat 900
ccgttgaaca tcgtctgatc tgcgacaagt tctggtaata cttgatagcc accgtcccag 960
acaaggtcaa gacagaactt cttgcgcaaa tcagtacttt tatcgaagac aacatgtaac 1020
gaaaaataaa aatgctgata tggaagggaa catcacgtac gccaggtacg tgagatgttt 1080
ttcctttttt ttttattttt tttgttgggg cttgagatgt tttgggtctt tcatgaagac 1140
gttcatgaca aacaagaaat acggacagcg agtttaccgc aatggaaaag tttacctcaa 1200
ccgaactttg aacgtttcgt gaaaacctgc gtgagaattc tttgagcatg gccataccta 1260
attgtcgccg gactgatctg tgatttgatc catggtccat cgctcctgac tcgctgcacg 1320
tcctgacaga gataacctcg catagtgtcg tacagaaacc aaaagcacgc gctcggtcca 1380
gtacagtaat ggggaactgg gtactgagga cagcttgaaa gtatatcctg taggggacga 1440
taacatgggc aatcttgatg gtgtggttga gtacagggta tatgcgttcc aggtaagcaa 1500
atatcacctt tgtgagacga tagggggcag ctatagtaaa tggccaggcg cttttatacg 1560
ggaagcaatt gcagcgatgg aggtgtgtac gctgcagccg cttgtatagc agaacgtaac 1620
cccaatcctc caactccggc aaggacaatg gggaaagaag gccggatata taaaatgctt 1680
gtcagcagaa ttaaagactg tgcggactga tctgccttaa tcggaaactt cagcagggct 1740
tctcgcagtg catgaaccct cggaccatct cctgtcccaa caacacgcca atgggcttat 1800
tattaatttt ttttggctgc cgttcaaaaa aaaaaagaaa aaaactgcca cttcttattt 1860
gagggctgca ggactgcttg ttaaggcgga taacctcaat ccgcccgatg ccttttaacg 1920
ctggcctgtt cttcctcttt tctttcttct ctcccttcac ccgtcttcac cctccctctt 1980
ccccccgttc ctacgtctac agccgttggc tcatcttgca gttgcttgtc tactatttgg 2040
tgccgaccta tattcctctg tcacccaacc taccgcactc acactcgcat aatggagtct 2100
ggacctatgc ctgccgggat ccccttccct gaatactatg actttttcat ggactggaag 2160
acacccctgg caattgctgc cacctacacc gccgctgttg ggctcttcaa ccccaaggtt 2220
ggcaaagtct cgcgcgtggt agccaagtcg gctaacgcca agccggcaga gcgcacgcag 2280
tccggcgccg ccatgaccgc ctttgtcttt gtccacaacc ttatcctctg cgtgtactct 2340
ggaatcacct tctactacat gttcccagcc atggtcaaga actttagaac acataccctc 2400
catgaggccc gtaagttgac aacggttcaa atttcgaaac ttgcactgtg ggtttgtcct 2460
ggttttattc ggcagctttt ttctccgaag tcttggccca gggaggaggc aagaagagca 2520
attcagggta ggggcgtccg gagattggac cctgccttgg actagcgaat ggaatacgag 2580
agcgaagctg tctgcatggg ctgatgataa tattttttac tggagatgat tgtaaggtgc 2640
ggcgcgaggg tgtagtacat cagtcgacat ggattcaacc aaatgttctc tagcagcgat 2700
aaccctggct gtcttttgtt aatcacggca atgctatccc caaagaaaaa aaaaagggtg 2760
cggttggcac acggttgcac aatggcttag ccagacaatt aacccaaaaa agtcaagaga 2820
ttcctttttt tttttttttt tttttggggg gggaacggga aagtgggggc tgtgtgaaat 2880
tggggcgaag tgaaccatgc aactttgagc tacaaacacc taattgtttt tgtcttgcct 2940
gacattatta gtactgcgat acggatcaga gcctgtggaa caacgccctt ggctactggg 3000
gctacctctt ctacctttca aagttttacg aggtcattga caccatcatc atcatcttga 3060
aggggcgccg ctcgtccctg ctccagacct accaccacgc cggcgctatg atcaccatgt 3120
ggtccggcat caactaccag gcaacgccca tttggatttt tgtcgtcttc aactcgttca 3180
tccacaccat catgtactgt tactatgcct tcacctcaat cggcttccac cccccaggca 3240
agaagtacct cacctccatg cagatcaccc agtttttggt cggcatcact atcgccgtct 3300
cttatctctt cgtccctgga tgtatccgca cacccggtgc tcagatggct gtctggatca 3360
acgtcggata cctctttccc ctcacttatc tctttgtgga ttttgccaag cgtacttact 3420
ccaagcgtag tgccatcgcc gctcagaaga aggcccagta aagaggccac ttcttgtcgt 3480
ttactctact gtttccaaca tttacatctc tcaatggcag cctcctccct ctatccttcg 3540
cactttcatt ttagttcctt gtattcataa aargtcaaaa ttcattgtaa ctccaaaagt 3600
gccaatggct ttatcatgga tgagtgccat gtttatggcc cattgcatca atacaggaag 3660
atatgagcgg tcagcgatag ctattcctct gagttgtcct ggccttgtat gcactctcga 3720
aggaggaaat ggatcggagc atcagcacta tttcaggact aggagagata catccaaaat 3780
tttaaaggca tctctggtca gtacaagagc gacttggcaa caagtcttta gtcaaacttc 3840
ctcacctttg aacttcctca gaggtcgccg tggcactgtg gacggagacc cgtgaaaaga 3900
actcatgcag caggttaagc aaccttggaa atccatcgat gaatgcctga tgtaaaaatt 3960
gagaagctgc aagagccaaa agttcagttc agcacatata agagactttg gccaagagcg 4020
atgaagtcac atgggaacca cgtaaacaaa cctcgtgcgg agsaccagcc cgggccgtcg 4080
accacgcgtg ccctatagt 4099
Claims (18)
1.一种编码C16/18脂肪酸延伸酶的分离的核酸分子,所述分离的核酸分子选自:
(a)编码SEQ ID NO:2所示氨基酸序列的分离的核酸分子,
(b)在以下杂交条件下与(a)杂交的分离的核酸分子:0.1×SSC,0.1% SDS,65℃,以及用2×SSC、0.1% SDS随后用0.1×SSC、0.1%SDS洗涤,或
与(a)或(b)完全互补的分离的核酸分子。
2.权利要求1的分离的核酸分子,其中所述分离的核酸分子选自SEQ ID NO:1、SEQ ID NO:18和SEQ ID NO:29。
3.由权利要求1的分离的核酸分子编码的多肽。
4.一种分离的核酸分子,其包含编码至少275个氨基酸的C16/18脂肪酸延伸酶的第一核苷酸序列,当根据BLAST比对方法与具有SEQID NO:2中所示序列的多肽相比较时,所述C16/18脂肪酸延伸酶具有至少90%的同一性,
或包含所述第一核苷酸序列的互补物的第二核苷酸序列。
5.一种嵌合基因,其包含与适合的调节序列可操纵地连接的权利要求1的分离的核酸分子。
6.包含权利要求5的嵌合基因的转化的宿主细胞。
7.权利要求6的转化的宿主细胞,其中所述宿主细胞选自藻类、细菌、真菌和酵母。
8.权利要求7的转化的宿主细胞,其中所述酵母是产油酵母。
9.权利要求8的转化的宿主细胞,其中所述产油酵母选自耶罗维亚酵母属(Yarrowia)、假丝酵母属(Candida)、红酵母属(Rhodotorula)、红冬孢酵母属(Rhodosporidium)、隐球菌属(Cryptococcus)、丝孢酵母属(Trichosporon)和油脂酵母属(Lipomyces)。
10.权利要求9的转化的宿主细胞,其中所述宿主细胞是解脂耶罗维亚酵母(Yarrowia lipolytica)。
11.权利要求10的转化的宿主细胞,其中所述解脂耶罗维亚酵母(Yarrowia lipolytica)是选自解脂耶罗维亚酵母(Yarrowia lipolytica)ATCC#20362、解脂耶罗维亚酵母(Yarrowia lipolytica)ATCC#8862、解脂耶罗维亚酵母(Yarrowia lipolytica)ATCC #18944、解脂耶罗维亚酵母(Yarrowia lipolytica)ATCC #76982、解脂耶罗维亚酵母(Yarrowialipolytica)ATCC #90812和解脂耶罗维亚酵母(Yarrowia lipolytica)LGAM S(7)1的菌株。
12.一种产生硬脂酸的方法,所述方法包括
a)提供产油酵母,所述产油酵母包含处在适合的调节序列的控制下的、编码如SEQ ID NO:2中所示的具有C16/18脂肪酸延伸酶活性的多肽的分离的核酸片段,
b)提供包含棕榈酸的延伸酶底物源,
c)在一定条件下与(b)的延伸酶底物一起生长步骤(a)的产油酵母,在所述条件中步骤(a)(i)或步骤(a)(ii)的核酸分子被表达并且产生硬脂酸,和
d)任选地回收步骤(c)的硬脂酸。
13.一种生产多不饱和脂肪酸的方法,所述方法包括
a)提供产油酵母,其包含
(i)编码如SEQ ID NO:2中所示具有C16/18脂肪酸延伸酶活性的多肽的分离的核酸片段,
(ii)编码功能性多不饱和脂肪酸生物合成途径的基因,
b)提供包含棕榈酸的延伸酶底物源,和
c)在一定条件下与(b)的延伸酶底物一起生长步骤(a)的产油酵母,在所述条件下产生多不饱和脂肪酸,和
d)任选地回收步骤(c)的多不饱和脂肪酸。
14.根据权利要求13的方法,其中所述多不饱和脂肪酸选自亚油酸、γ-亚麻酸、二十碳二烯酸、二高-γ-亚麻酸、花生四烯酸、α-亚麻酸、stearidonic acid、二十碳三烯酸、二十碳四烯酸、二十碳五烯酸、二十二碳五烯酸和二十二碳六烯酸。
15.根据权利要求13的方法,其中所述编码功能性多不饱和脂肪酸生物合成途径的基因编码选自Δ9脱饱和酶、Δ12脱饱和酶、Δ6脱饱和酶、C18/20脂肪酸延伸酶、Δ5脱饱和酶、Δ17脱饱和酶、C20/22脂肪酸延伸酶、Δ4脱饱和酶、Δ15脱饱和酶、Δ9脂肪酸延伸酶和Δ8脱饱和酶的酶。
16.根据权利要求12或13的任一项的方法,其中所述产油酵母选自耶罗维亚酵母属(Yarrowia sp.)、假丝酵母属(Candida sp.)、红酵母属(Rhodotorula sp.)、红冬孢酵母属(Rhodosporidium sp.)、隐球菌属(Cryptococcus sp.)、丝孢酵母属(Trichosporon sp.)和油脂酵母属(Lipomyces sp.)。
17.根据权利要求16的方法,其中所述耶罗维亚酵母属(Yarrowiasp.)选自解脂耶罗维亚酵母(Yarrowia lipolytica)ATCC #20362、解脂耶罗维亚酵母(Yarrowia lipolytica)ATCC #8862、解脂耶罗维亚酵母(Yarrowia lipolytica)ATCC #18944、解脂耶罗维亚酵母(Yarrowialipolytica)ATCC #76982和解脂耶罗维亚酵母(Yarrowia lipolytica)LGAM S(7)1。
18.通过权利要求12或13的任一项的方法产生的微生物油。
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US11/253,882 US7470532B2 (en) | 2005-10-19 | 2005-10-19 | Mortierella alpina C16/18 fatty acid elongase |
US11/253,882 | 2005-10-19 | ||
PCT/US2005/040141 WO2007046817A1 (en) | 2005-10-19 | 2005-11-04 | A mortierella alpina c16/18 fatty acid elongase |
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AU (1) | AU2005337476B2 (zh) |
CA (1) | CA2622398C (zh) |
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CN112592926A (zh) * | 2020-12-28 | 2021-04-02 | 江南大学 | 一种crispr系统及其在高山被孢霉中的应用 |
CN112661821A (zh) * | 2021-01-21 | 2021-04-16 | 江南大学 | 一种柠檬酸转运蛋白及其在脂质合成中的应用 |
CN112661821B (zh) * | 2021-01-21 | 2022-04-12 | 江南大学 | 一种柠檬酸转运蛋白及其在脂质合成中的应用 |
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