CN114990086B - 酶组合物及其应用 - Google Patents
酶组合物及其应用 Download PDFInfo
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- CN114990086B CN114990086B CN202210679823.6A CN202210679823A CN114990086B CN 114990086 B CN114990086 B CN 114990086B CN 202210679823 A CN202210679823 A CN 202210679823A CN 114990086 B CN114990086 B CN 114990086B
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Abstract
本发明涉及生物技术领域,尤其涉及酶组合物及其应用。本发明提供了酶组合物包括:查尔酮合成酶CHS和查尔酮异构酶样蛋白CHIL;查尔酮合成酶包括灯盏花EbCHS;查尔酮异构酶样蛋白包括:高粱SbCHIL和/或大豆GmCHIL。本发明提供了在酿酒酵母中将CHIL引入对香豆酸到柚皮素的合成过程,有效的提高了柚皮素的合成能力,这为柚皮素的工业化生产奠定了基础。
Description
技术领域
本发明涉及生物技术领域,尤其涉及酶组合物及其应用。
背景技术
柚皮素(Naringenin),化学名为4’,5,7-三羟基黄酮,属于二氢黄酮类化合物。柚皮素具有抗菌、抗炎、抗癌和抗氧化等生理药理活性,广泛应用于食品、保健品及医药领域。柚皮素作为黄酮类化合物的骨架结构,可经多步反应生成黄酮、异黄酮、黄烷酮、黄酮醇、花色素等化合物。
以对香豆酸为前体,首先经4-香豆酰辅酶A连接酶(4-coumarate-CoA ligase,4CL)催化生成对香豆酰-CoA,然后1分子对香豆酰-CoA与3分子丙二酰-CoA经查尔酮合成酶(Chalcone synthase,CHS)催化发生克莱森缩合反应生成柚皮素查尔酮,进而经查耳酮异构酶(Chalcone isomerase,CHI)作用发生立体异构闭环生成柚皮素,如图4所示。
查耳酮异构酶样蛋白(CHI-Like protein)CHIL,是一种IV型CHI,但缺乏CHI的活性。在植物中,CHIL与同物种的CHS共表达,可以纠正CHS催化的混杂性,进而促进类黄酮化合物的合成。如已鉴定的拟南芥来源的AtCHIL与同物种的CHS共表达,可以增强植物中花色素和黄酮醇的合成,但尚未有CHIL在酿酒酵母中合成柚皮素的报道。
发明内容
有鉴于此,本发明提供了酶组合物及其应用。本发明提供了在酿酒酵母中将CHIL引入对香豆酸到柚皮素的合成过程,有效的提高了柚皮素的合成能力。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了酶组合物包括:查尔酮合成酶和查尔酮异构酶样蛋白;
所述查尔酮合成酶包括:灯盏花EbCHS;
所述查尔酮异构酶样蛋白包括:高粱SbCHIL和/或大豆GmCHIL。
具体的,在本发明的一些实施方案中,上述酶组合物中:
由所述灯盏花EbCHS和所述高粱SbCHIL组成;或
由所述灯盏花EbCHS和所述大豆GmCHIL组成。
在本发明的一些实施方案中,上述酶组合物中所述灯盏花EbCHS具有:
(1)、如SEQ ID NO:1所示的氨基酸序列;或
(2)、在如(1)所示的氨基酸序列的基础上经取代、缺失、添加和/或替换1个或多个氨基酸的序列;或
(3)、与(1)或(2)所示的氨基酸序列同源性90%以上的序列;和
所述高粱SbCHIL具有:
(4)、如SEQ ID NO:2所示的氨基酸序列;或
(5)、在如(4)所示的氨基酸序列的基础上经取代、缺失、添加和/或替换1个或多个氨基酸的序列;或
(6)、与(4)或(5)所示的氨基酸序列同源性90%以上的序列;或
所述大豆GmCHIL具有:
(7)、如SEQ ID NO:3所示的氨基酸序列;或
(8)、在如(7)所示的氨基酸序列的基础上经取代、缺失、添加和/或替换1个或多个氨基酸的序列;或
(9)、与(7)或(8)所示的氨基酸序列同源性90%以上的序列。
具体的,在本发明的一些实施方案中,上述SEQ ID NO:1的序列为:MASSIDIAAIREAQRAQGPATILAIGTATPSNCVYQADYPDYYFRITKSEHMVDLKEKFKRMCDKSMIRKRYMHLTEEYLKENPSLCEYMAPSLDARQDVVVVEVPKLGKEAATKAIKEWGQPKSKITHLIFCTTSGVDMPGADYQLTKLLGLRPSVKRFMMYQQGCFAGGTVLRLAKDLAENNKGARVLVVCSEITAVTFRGPNDTHLDSLVGQALFGDGAAAVIVGSDPDLTTERPLFEMISAAQTILPDSEGAIDGHLREVGLTFHLLKDVPGLISKNIEKALTQAFSPLGISDWNSLFWIAHPGGPAILDQVELKLGLKEEKMRATRHVLSEYGNMSSACVLFIIDEMRKKSAEDGAATTGEGLDWGVLFGFGPGLTVETVVLHSLPTTTAIAT。
具体的,在本发明的一些实施方案中,上述SEQ ID NO:2的序列为:MLISAVGSETKTITFEGIPFPAEITAAGNPLSLLATGITDIEIHFLQIKYNAIGVYLHSNDDSDLLTTHLGAWKGKTAEDLLADAAFWSALVSSPVEKLLRVVVIKEIKGSQYGVQLESSVRDRLAAVDLYEDDEEEALEKVAEFFQAKYFKPGSVITFHFPATPGPAEITFVTEGKADAKITVENEHVAGMIQKWYLGGDNAVSPTTVRSLADRFAALLAA。
具体的,在本发明的一些实施方案中,上述SEQ ID NO:3的序列为:MATEEVLVDEITYPTKITTTKPLSLLGHGITDMEIHFIHVKFYSIGVYLEPEVVGHLDQFKGKSAKELEDNEEFFNALISAPVEKFIRLVVIKEIKGAQYGVQIETAVRDRLAAEDKYEEEEEEALEKVIEFFQSKYFKKLSVITYHFPANSATAEIVVSLEGKEDSKYVIENANVVEAIKKWYLGGSSAVSSSTIQSLASTFSQELSK。
在本发明的一些实施方案中,上述酶组合物还包括:向日葵Ha4CL和/或细枝真杆菌ErCHI;
所述向日葵Ha4CL具有:
(10)、如SEQ ID NO:4所示的氨基酸序列;或
(11)、在如(10)所示的氨基酸序列的基础上经取代、缺失、添加和/或替换1个或多个氨基酸的序列;或
(12)、与(10)或(11)所示的氨基酸序列同源性90%以上的序列;或
所述细枝真杆菌ErCHI具有:
(13)、如SEQ ID NO:5所示的氨基酸序列;或
(14)、在如(13)所示的氨基酸序列的基础上经取代、缺失、添加和/或替换1个或多个氨基酸的序列;或
(15)、与(13)或(14)所示的氨基酸序列同源性90%以上的序列。
具体的,在本发明的一些实施方案中,上述SEQ ID NO:4的序列为:MAPEKEIIFRSKLPDIYIPKHLPLHSYCFENISTFNNRPCLIDGATGVVHTYADVELTSRKVASAFHQHGINKGDVIMILLPNSPEFVYSFFGASYLGAVSTMANPFFTSAEIIKQAKASNAKIIVTQASHVPKIKEYAAENSIKIVCIDSAPEGCLHFTELVSGDETKLPEVEISSDDVVALPYSSGTTGLPKGVMLTHKGLVTSVAQQVDGENPNLWIHSEDVLMCVLPLFHIYSLNSILLCGLRAGAAILIMQKFDIVPFLELIEKYKVTIGPFVPPIVLAIANNEEVVDKYDLSSMRTVMSGAAPLGKELEDMVRRKFTNAKLGQGYGMTEAGPVLAMCLAFAKEPYEIKSGACGTVVRNAEMKIVDPETGLSLPRNQRGEICIRGDQIMKGYLNDPESTKTTIDSDGWLHTGDIGLIDDDDELFIVDRLKELIKYKGFQVAPAELEALLLTHPQISDVAVVPMVNEAAGEVPVAFVVKTKDSSVTEDDIKQFVHKQVVFYKRINRVFFIDTIPKSPAGKILRKELRAKLAAGVPN。
具体的,在本发明的一些实施方案中,上述SEQ ID NO:5的序列为:MADFKFEPMRSLIYVDCVSEDYRPKLQRWIYKVHIPDSISQFEPYVTKYAFYPSFPIPPQGDRFGYARMQLTEHHWLVSDLDPRLEIKAIAETFPMDVLVWQGQIPAAAHTDAQIDSDGDAGNAARKSNNAEGNPFIFAFLPMWWEKDLKGKGRTIEDGANYRFNMTIGFPEGVDKAEGEKWLFEKVVPILQAAPECTRVLASAVKKDINGCVMDWVLEIWFENQSGWYKVMVDDMKALEKPSWAQQDAFPFLKPYHNVCSAAVADYTPSNNLANYRGYITMR。
本发明还提供了编码上述酶组合物的核酸分子,包括:
编码所述灯盏花EbCHS的核酸分子;或
编码所述向日葵Ha4CL的核酸分子;或
编码所述细枝真杆菌ErCHI的核酸分子;和
编码所述高粱SbCHIL的核酸分子;或
编码所述大豆GmCHIL的核酸分子。
在本发明的一些实施方案中,上述核酸分子中
编码所述灯盏花EbCHS的核酸分子具有:
(16)、如SEQ ID NO:6所示的核苷酸序列;或
(17)、如(16)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列,且与(16)所示的核苷酸序列功能相同或相似的核苷酸序列;或
(18)、与(16)或(17)所示的核苷酸序列至少有80%同一性的核苷酸序列;或
编码所述向日葵Ha4CL的核酸分子具有:
(19)、如SEQ ID NO:7所示的核苷酸序列;或
(20)、如(19)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列,且与(19)所示的核苷酸序列功能相同或相似的核苷酸序列;或
(21)、与(19)或(20)所示的核苷酸序列至少有80%同一性的核苷酸序列;或
编码所述细枝真杆菌ErCHI的核酸分子具有:
(22)、如SEQ ID NO:8所示的核苷酸序列;或
(23)、如(22)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列,且与(22)所示的核苷酸序列功能相同或相似的核苷酸序列;或
(24)、与(22)或(23)所示的核苷酸序列至少有80%同一性的核苷酸序列;和
编码所述高粱SbCHIL的核酸分子具有:
(25)、如SEQ ID NO:9所示的核苷酸序列;或
(26)、如(25)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列,且与(25)所示的核苷酸序列功能相同或相似的核苷酸序列;或
(27)、与(25)或(26)所示的核苷酸序列至少有80%同一性的核苷酸序列;或
编码所述大豆GmCHIL的核酸分子具有:
(28)、如SEQ ID NO:10所示的核苷酸序列;或
(29)、如(28)所示的核苷酸序列经取代、缺失或添加一个或多个碱基获得的核苷酸序列,且与(28)所示的核苷酸序列功能相同或相似的核苷酸序列;或
(30)、与(28)或(29)所示的核苷酸序列至少有80%同一性的核苷酸序列。
具体的,在本发明的一些实施方案中,上述SEQ ID NO:6的序列为:ATGGCTTCTTCTATCGACATCGCTGCTATCAGAGAAGCTCAAAGAGCTCAAGGTCCAGCTACTATCTTGGCTATCGGTACTGCTACTCCATCTAACTGTGTTTACCAAGCTGACTACCCAGACTACTACTTCAGAATCACTAAGTCTGAACACATGGTTGACTTGAAGGAAAAGTTCAAGAGAATGTGTGACAAGTCTATGATCAGAAAGAGATACATGCACTTGACTGAAGAATACTTGAAGGAAAACCCATCTTTGTGTGAATACATGGCTCCATCTTTGGACGCTAGACAAGACGTTGTTGTTGTTGAAGTTCCAAAGTTGGGTAAGGAAGCTGCTACTAAGGCTATCAAGGAATGGGGTCAACCAAAGTCTAAGATCACTCACTTGATCTTCTGTACTACTTCTGGTGTTGACATGCCAGGTGCTGACTACCAATTGACTAAGTTGTTGGGTTTGAGACCATCTGTTAAGAGATTCATGATGTACCAACAAGGTTGTTTCGCTGGTGGTACTGTTTTGAGATTGGCTAAGGACTTGGCTGAAAACAACAAGGGTGCTAGAGTTTTGGTTGTTTGTTCTGAAATCACTGCTGTTACTTTCAGAGGTCCAAACGACACTCACTTGGACTCTTTGGTTGGTCAAGCTTTGTTCGGTGACGGTGCTGCTGCTGTTATCGTTGGTTCTGACCCAGACTTGACTACTGAAAGACCATTGTTCGAAATGATCTCTGCTGCTCAAACTATCTTGCCAGACTCTGAAGGTGCTATCGACGGTCACTTGAGAGAAGTTGGTTTGACTTTCCACTTGTTGAAGGACGTTCCAGGTTTGATCTCTAAGAACATCGAAAAGGCTTTGACTCAAGCTTTCTCTCCATTGGGTATCTCTGACTGGAACTCTTTGTTCTGGATCGCTCACCCAGGTGGTCCAGCTATCTTGGACCAAGTTGAATTGAAGTTGGGTTTGAAGGAAGAAAAGATGAGAGCTACTAGACACGTTTTGTCTGAATACGGTAACATGTCTTCTGCTTGTGTTTTGTTCATCATCGACGAAATGAGAAAGAAGTCTGCTGAAGACGGTGCTGCTACTACTGGTGAAGGTTTGGACTGGGGTGTTTTGTTCGGTTTCGGTCCAGGTTTGACTGTTGAAACTGTTGTTTTGCACTCTTTGCCAACTACTACTGCTATCGCTACTTAA。
具体的,在本发明的一些实施方案中,上述SEQ ID NO:7的序列为:ATGGCTCCAGAAAAGGAAATCATCTTCAGATCCAAGTTGCCAGACATTTACATCCCAAAGCACCTCCCCCTACATTCTTACTGTTTCGAAAACATCTCTACTTTCAACAACAGACCATGCTTGATCGATGGTGCTACCGGTGTCGTCCACACTTACGCTGACGTTGAACTAACATCTCGTAAGGTTGCCTCTGCTTTCCACCAACACGGTATCAACAAGGGTGATGTTATCATGATTTTATTGCCAAACTCTCCAGAATTTGTTTACTCTTTCTTCGGTGCCTCCTATTTGGGTGCCGTATCAACTATGGCTAACCCTTTTTTCACTTCCGCTGAAATCATTAAACAAGCTAAGGCTTCCAATGCTAAGATCATTGTCACTCAAGCCTCTCATGTTCCAAAGATCAAGGAATACGCTGCCGAAAATTCCATTAAGATCGTCTGTATCGATTCTGCTCCAGAAGGTTGTTTGCACTTCACTGAATTGGTCTCCGGTGACGAAACCAAGTTGCCGGAAGTCGAAATTTCCTCCGACGATGTTGTTGCTTTGCCATACTCCTCTGGTACCACTGGGTTGCCAAAGGGTGTTATGTTGACTCACAAAGGCTTGGTCACTTCTGTCGCTCAACAAGTTGACGGTGAAAACCCAAACTTGTGGATTCACTCTGAAGACGTTCTGATGTGTGTTCTGCCATTGTTCCATATCTACTCTTTGAACAGCATCTTGTTGTGTGGTTTGCGTGCTGGTGCTGCTATTTTGATTATGCAAAAGTTCGATATTGTGCCTTTCTTGGAATTGATTGAAAAGTATAAGGTTACCATCGGTCCATTCGTTCCACCAATTGTTTTGGCCATTGCTAACAATGAAGAAGTTGTTGACAAGTACGATTTGTCTTCCATGAGAACCGTCATGTCTGGTGCTGCTCCATTAGGTAAAGAATTGGAAGACATGGTTAGAAGAAAGTTTACCAACGCTAAGCTAGGTCAAGGTTACGGTATGACTGAAGCCGGTCCAGTTTTGGCTATGTGTTTGGCTTTCGCCAAGGAACCATACGAAATCAAGTCTGGTGCTTGTGGTACTGTCGTCAGAAACGCCGAAATGAAAATCGTTGATCCAGAAACTGGTTTGAGTTTACCAAGAAACCAAAGAGGTGAAATCTGTATCAGAGGTGACCAAATCATGAAGGGTTACTTGAACGACCCAGAATCTACCAAGACCACAATTGATTCCGATGGTTGGTTGCACACCGGTGACATTGGTTTGATTGATGACGACGATGAATTATTTATTGTCGACAGATTGAAGGAGTTAATCAAGTACAAGGGTTTCCAAGTTGCTCCAGCTGAATTGGAAGCCTTGTTGTTGACCCACCCACAAATTTCTGACGTTGCTGTCGTTCCAATGGTTAACGAAGCTGCTGGAGAAGTCCCAGTCGCCTTTGTTGTCAAGACTAAGGACTCTAGTGTTACTGAAGATGACATCAAGCAATTCGTTCACAAGCAAGTCGTCTTCTACAAGAGAATCAACAGAGTTTTCTTCATCGACACTATTCCAAAATCCCCTGCTGGTAAGATTTTGAGAAAGGAATTACGTGCTAAATTAGCCGCCGGTGTCCCAAACTGA。
具体的,在本发明的一些实施方案中,上述SEQ ID NO:8的序列为:ATGGCTGATTTCAAGTTCGAACCAATGAGATCCTTGATTTACGTTGATTGTGTTTCCGAAGATTACAGACCAAAGTTGCAAAGATGGATTTACAAGGTTCATATTCCAGATTCCATTTCCCAATTCGAACCATACGTTACCAAGTACGCTTTCTACCCATCCTTCCCAATTCCACCACAAGGTGATAGATTCGGTTACGCTAGAATGCAATTGACCGAACATCATTGGTTGGTTTCCGATTTGGACCCTAGATTGGAAATTAAGGCTATTGCTGAAACCTTCCCAATGGATGTTTTGGTTTGGCAAGGTCAAATTCCAGCTGCTGCTCATACCGATGCTCAAATTGATTCCGATGGTGATGCTGGTAACGCTGCTAGAAAGTCCAACAACGCTGAAGGTAACCCATTCATTTTCGCTTTCTTGCCAATGTGGTGGGAAAAGGATTTGAAGGGTAAGGGTAGAACCATTGAAGATGGTGCTAACTACAGATTCAACATGACCATTGGTTTCCCAGAAGGTGTTGATAAGGCTGAAGGTGAAAAGTGGTTGTTCGAAAAGGTTGTTCCAATTTTGCAAGCTGCTCCAGAATGTACCAGAGTTTTGGCTTCCGCTGTTAAGAAGGATATTAACGGTTGTGTTATGGATTGGGTTTTGGAAATTTGGTTCGAAAACCAATCCGGTTGGTACAAGGTTATGGTTGATGATATGAAGGCTTTGGAAAAGCCATCCTGGGCTCAACAAGATGCTTTCCCATTCTTGAAGCCATACCATAACGTTTGTTCCGCTGCTGTTGCTGATTACACCCCATCCAACAACTTGGCTAACTACAGAGGTTACATTACCATGAGATAA。
具体的,在本发明的一些实施方案中,上述SEQ ID NO:9的序列为:ATGTTGATCTCCGCTGTCGGCTCTGAAACCAAGACCATTACTTTCGAAGGTATCCCATTCCCAGCTGAAATCACAGCTGCTGGTAACCCATTGTCCTTGTTGGCTACTGGTATTACCGACATTGAAATTCACTTCTTGCAAATCAAATACAACGCTATTGGTGTTTATTTGCACTCTAACGATGACTCCGATTTATTAACTACTCACTTAGGTGCTTGGAAGGGTAAGACTGCTGAAGATTTGTTGGCAGATGCTGCTTTCTGGTCTGCTTTGGTTTCATCCCCTGTCGAAAAGTTGCTAAGAGTTGTCGTCATCAAGGAGATCAAGGGTTCTCAATACGGTGTTCAATTGGAATCTTCTGTTAGAGACAGATTGGCCGCTGTTGACTTGTACGAAGACGATGAAGAAGAAGCTTTAGAAAAAGTTGCTGAATTTTTCCAAGCCAAGTACTTCAAGCCAGGTTCCGTTATCACCTTTCATTTCCCAGCCACTCCAGGTCCAGCTGAAATTACCTTTGTCACTGAAGGTAAGGCTGATGCCAAGATCACTGTTGAAAACGAACACGTTGCCGGTATGATTCAAAAGTGGTACTTGGGTGGTGACAATGCTGTCTCTCCAACCACCGTCCGTTCTTTGGCTGACAGATTCGCTGCTTTGCTCGCCGCCTGA。
具体的,在本发明的一些实施方案中,上述SEQ ID NO:10的序列为:ATGGCGACCGAGGAAGTGCTGGTTGACGAAATCACCTACCCGACCAAAATCACCACCACCAAACCGCTGAGCCTGCTGGGCCACGGCATCACCGACATGGAGATCCACTTCATTCACGTGAAGTTTTACAGCATTGGTGTTTATCTGGAGCCGGAAGTGGTTGGTCACCTGGACCAGTTCAAGGGCAAAAGCGCGAAGGAGCTGGAAGATAACGAGGAGTTCTTCAACGCGCTGATCAGCGCGCCGGTGGAGAAATTTATTCGTCTGGTGGTTATCAAGGAAATTAAAGGTGCGCAGTACGGCGTGCAAATCGAAACCGCGGTGCGTGACCGTCTGGCGGCGGAAGATAAATATGAGGAAGAGGAAGAGGAAGCGCTGGAGAAAGTGATTGAGTTCTTCCAAAGCAAATACTTCAAGAAACTGAGCGTTATCACCTATCACTTTCCGGCGAACAGCGCGACCGCGGAAATTGTGGTTAGCCTGGAGGGCAAGGAAGATAGCAAATACGTTATCGAGAACGCGAACGTGGTTGAAGCGATTAAGAAATGGTATCTGGGTGGCAGCAGCGCGGTTAGCAGCAGCACCATTCAGAGCCTGGCGAGCACCTTTAGCCAAGAACTGAGCAAATAA。
本发明还提供了表达载体包括上述核酸分子。
本发明还提供了宿主,转化和/或转染上述表达载体。
具体的,在本发明的一些实施方案中上述宿主包括:酵母菌。
最重要的,在本发明的一些实施方案中上述宿主包括:酿酒酵母。
本发明还提供了上述酶组合物、上述核酸分子、上述表达载体和/或上述宿主在合成柚皮素中的应用。
具体的,本发明还提供了合成柚皮素的方法,包括取上述宿主与对香豆酸底物混合,发酵,获得所述柚皮素。
本发明还提供了上述酶组合物、上述核酸分子、上述表达载体和/或上述的宿主在制备柚皮素或所述柚皮素衍生物中应用。
本发明提供了酶组合物包括:查尔酮合成酶和查尔酮异构酶样蛋白;
所述查尔酮合成酶包括:灯盏花EbCHS;所述查尔酮异构酶样蛋白包括:高粱SbCHIL和/或大豆GmCHIL。
本发明的有益效果包括:
(1)本发明提供了在酿酒酵母中将CHIL引入对香豆酸到柚皮素的合成过程,有效的提高了柚皮素的合成能力。
(2)以文献报道的AtCHIL作为对照,本发明提供的SbCHIL使柚皮素的产量提高了23.32%,GmCHIL使柚皮素产量提高了15.25%。
(3)与现有技术相比,其中最优的菌株ZNG2发酵72h结束后,对香豆酸到柚皮素的转化率达到34.87%。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示菌株ZNG1;
图2示表达载体pZNG1的图谱;
图3示不同植物源CHIL对柚皮素产量的影响;
图4示柚皮素生物合成途径。
具体实施方式
本发明公开了酶组合物及其应用。
应该理解,表述“……中的一种或多种”单独地包括每个在所述表述后叙述的物体以及所述叙述的物体中的两者或更多者的各种不同组合,除非从上下文和用法中另有理解。与三个或更多个叙述的物体相结合的表述“和/或”应该被理解为具有相同的含义,除非从上下文另有理解。
术语“包括”、“具有”或“含有”,包括其语法同义语的使用,通常应该被理解为开放性和非限制性的,例如不排除其他未叙述的要素或步骤,除非另有具体陈述或从上下文另有理解。
应该理解,只要本发明仍可操作,步骤的顺序或执行某些行动的顺序并不重要。此外,两个或更多个步骤或行动可以同时进行。
本文中的任何和所有实例或示例性语言如“例如”或“包括”的使用,仅仅打算更好地说明本发明,并且除非提出权利要求,否则不对本发明的范围构成限制。本说明书中的任何语言都不应解释为指示任何未要求保护的要素对于本发明的实践是必不可少的。
此外,用以界定本发明的数值范围与参数皆是约略的数值,此处已尽可能精确地呈现具体实施例中的相关数值。然而,任何数值本质上不可避免地含有因个别测试方法所致的标准偏差。因此,除非另有明确的说明,应当理解本公开所用的所有范围、数量、数值与百分比均经过“约”的修饰。在此处,“约”通常是指实际数值在一特定数值或范围的正负10%、5%、1%或0.5%之内。
本发明酿酒酵母细胞培养及产物检测以及菌株的构建中,所用原料及试剂均可由市场购得。
下面结合实施例,进一步阐述本发明:
实施例1酿酒酵母细胞培养及产物检测
(1)酿酒酵母SC选择性培养基
SC选择性培养基主要用于酿酒酵母细胞的培养、筛选及发酵。
SC液体选择性培养基配方为:葡萄糖20g/L,酵母基本氮源(YNB)6.7g/L,氨基酸缺省混合粉末2g/L(配方见表1)。用10M的NaOH溶液调节pH值至6.5。SC固体培养基再额外添加1.5%(w/v)的琼脂粉。配制完成后于0.1Mpa,115℃下灭菌15min。在使用前根据具体情况补充所需的氨基酸母液,母液终浓度分别为:尿嘧啶(Ura)20mg/L、色氨酸(Trp)20mg/L、组氨酸(His)20mg/L以及亮氨酸(Leu)100mg/L。
表1氨基酸缺省混合粉末成分
Table 1Composition of amino acid drop-out mixture
(2)酿酒酵母发酵
酿酒酵母发酵均为摇瓶水平,使用(1)中的SC培养基进行发酵,具体操作如下:
1)一级种子培养:挑取单菌落接种至3mL相应缺陷型的SC液体培养基中,30℃,250rpm下培养20h左右,OD600达到6左右;
2)二级种子培养:将步骤1)中获得的一级种子转接到4~5mL新鲜的相应缺陷型的SC培养基中,初始OD600为0.1左右,30℃,250rpm下培养20h左右,细胞生长周期到达指数生长中期;
3)摇瓶发酵:将步骤2)获得的二级种子转接至50mL相应缺陷型的SC发酵培养基中,控制初始OD600为0.1,同时加入200mg/L的对香豆酸底物,30℃,250rpm下培养72h。
(3)产物提取
4)发酵结束后,取3mL(2)获得的发酵液于15mL离心管中,加入5mL乙酸乙酯萃取;
5)在MIX-2500迷你混合仪上充分振荡2h,7500rpm离心8min;
6)取3mL上清至50mL旋蒸小烧瓶中,在旋转蒸发仪上蒸干;
7)加入500μL无水甲醇溶解,过0.22μm孔径的有机系滤膜后进行高效液相色谱(HPLC)检测。
(4)产物检测
HPLC测定的检测器为紫外检测器,色谱柱为Thermo公司的Hypersil GOLDTM AqC18柱,柱型号250mm×4.6mm,5μm。检测波长290nm;流速1mL/min;进样量10μL;柱温30℃。流动相成分:30%乙腈-70%水-0.5%乙酸。
实施例2 ZNG1菌株的构建
以酿酒酵母菌株CEN.PK2-1C为底盘细胞,首先重构柚皮素合成途径。将灯盏花(Erigeron breviscapus)来源的EbCHS(如SEQ ID NO:1所示)、向日葵(Helianthusannuus)来源的Ha4CL(如SEQ ID NO:4所示)和细枝真杆菌(Eubacterium ramulus)来源ErCHI(如SEQ ID NO:5所示)与LEU2筛选标记基因整合到染色体的YPRCΔ15位点,构建菌株ZNG1,整合示意图见图1。
具体构建步骤如下:所用基因按照酿酒酵母密码子偏好性进行优化,交由金斯瑞化学合成。以BY4741基因组为模板,扩增启动子PHXT7、PTEF1和PT EF2,终止子TADH1、TGPM1和TENO2,整合位点上下游同源臂片段YPRCΔ15UP和YPRCΔ15DOWN。通过多轮重叠延伸PCR(OE-PCR)组装得到三个整合片段YPRCΔ15UP-loxp-LEU2-loxp、PHXT7-EbCHS-TADH1和PTEF1-Ha4CL-TGPM1-PTE F2-ErCHI-TCYC1-YPRCΔ15DOWN。将这三个片段通过醋酸锂转化法转入CEN.PK 2-1C感受态细胞,涂布SC-Leu固体平板。筛选阳性克隆,得到菌株ZNG1。
产物检测
在SC-Leu培养基中添加终浓度200mg/L的对香豆酸底物,30℃,250rpm摇瓶发酵培养72h,经HPLC检测分析,柚皮素产量达到80.45mg/L。
实施例3 ZNG2-ZNG6菌株的构建
由实施例2结果表明,我们以菌株ZNG1为底盘细胞,引入CHIL来提高柚皮素的合成能力。筛选了3种植物源的CHILs,分别为高粱(Sorghum bicolor)来源的SbCHIL(如SEQ IDNO:2所示)、大豆(Glycine max)来源的GmCHIL(如SEQ ID NO:3所示)、拟南芥(Arabidopsisthaliana)来源的AtCHIL(GeneBank序列号为:NP_568154.1)。
(1)表达载体的构建
将上述CHILs分别构建在pRS426质粒上,得到表达载体pZNG1-pZNG3。以表达载体pZNG1的构建为例(图2),首先以BY4741基因组为模板,扩增启动子PPGK1,终止子TGPD。通过OE-PCR扩增得到表达盒PPGK1-SbCHIL-TGPD。将其与pRS426线性化载体利用ClonExpress II同源重组试剂盒进行快速一步组装,并转化E.coli DH5α感受态细胞,涂布LB+Amp的固体平板。筛选阳性克隆,提质粒得到表达载体pZNG1。同样的,按照上述同样的实验步骤分别得到表达载体pZNG2和pZNG3。
(2)菌株的构建
将(1)获得的质粒pZNG1、pZNG2和pZNG3通过醋酸锂转化法分别转入ZNG1感受态细胞,涂布SC-Leu-Ura固体平板。筛选阳性克隆,构建酵母工程菌ZNG2、ZNG3和ZNG4。
(3)产物检测
将(2)获得的菌株ZNG2、ZNG3和ZNG4在SC-Leu-Ura培养基添加200mg/L的对香豆酸底物,30℃,250rpm摇瓶发酵培养72h,发酵结果见图3,根据柚皮素的产量衡量CHIL的催化效果。
结果如图3和表2所示,携带SbCHIL的菌株ZNG2合成柚皮素的量最高,达到99.21mg/L,相比于无CHIL的对照菌株ZNG1提高了23.32%(p<0.1)。携带GmCHIL的菌株ZNG3次之,产生了92.72mg/L的柚皮素,相比于对照菌株ZNG1提高了15.25%(p<0.1)。携带AtCHIL的菌株ZNG4合成了84.61mg/L的柚皮素,相比于对照菌株ZNG1也有轻微的提高,但效果不显著。
表2
菌株 | 滴度(mg/L) |
ZNG1 | 80.45 |
ZNG2 | 99.21 |
ZNG3 | 92.72 |
ZNG4 | 84.61 |
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 天津大学佐治亚理工深圳学院
<120> 酶组合物及其应用
<130> MP22014167
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 398
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Ala Ser Ser Ile Asp Ile Ala Ala Ile Arg Glu Ala Gln Arg Ala
1 5 10 15
Gln Gly Pro Ala Thr Ile Leu Ala Ile Gly Thr Ala Thr Pro Ser Asn
20 25 30
Cys Val Tyr Gln Ala Asp Tyr Pro Asp Tyr Tyr Phe Arg Ile Thr Lys
35 40 45
Ser Glu His Met Val Asp Leu Lys Glu Lys Phe Lys Arg Met Cys Asp
50 55 60
Lys Ser Met Ile Arg Lys Arg Tyr Met His Leu Thr Glu Glu Tyr Leu
65 70 75 80
Lys Glu Asn Pro Ser Leu Cys Glu Tyr Met Ala Pro Ser Leu Asp Ala
85 90 95
Arg Gln Asp Val Val Val Val Glu Val Pro Lys Leu Gly Lys Glu Ala
100 105 110
Ala Thr Lys Ala Ile Lys Glu Trp Gly Gln Pro Lys Ser Lys Ile Thr
115 120 125
His Leu Ile Phe Cys Thr Thr Ser Gly Val Asp Met Pro Gly Ala Asp
130 135 140
Tyr Gln Leu Thr Lys Leu Leu Gly Leu Arg Pro Ser Val Lys Arg Phe
145 150 155 160
Met Met Tyr Gln Gln Gly Cys Phe Ala Gly Gly Thr Val Leu Arg Leu
165 170 175
Ala Lys Asp Leu Ala Glu Asn Asn Lys Gly Ala Arg Val Leu Val Val
180 185 190
Cys Ser Glu Ile Thr Ala Val Thr Phe Arg Gly Pro Asn Asp Thr His
195 200 205
Leu Asp Ser Leu Val Gly Gln Ala Leu Phe Gly Asp Gly Ala Ala Ala
210 215 220
Val Ile Val Gly Ser Asp Pro Asp Leu Thr Thr Glu Arg Pro Leu Phe
225 230 235 240
Glu Met Ile Ser Ala Ala Gln Thr Ile Leu Pro Asp Ser Glu Gly Ala
245 250 255
Ile Asp Gly His Leu Arg Glu Val Gly Leu Thr Phe His Leu Leu Lys
260 265 270
Asp Val Pro Gly Leu Ile Ser Lys Asn Ile Glu Lys Ala Leu Thr Gln
275 280 285
Ala Phe Ser Pro Leu Gly Ile Ser Asp Trp Asn Ser Leu Phe Trp Ile
290 295 300
Ala His Pro Gly Gly Pro Ala Ile Leu Asp Gln Val Glu Leu Lys Leu
305 310 315 320
Gly Leu Lys Glu Glu Lys Met Arg Ala Thr Arg His Val Leu Ser Glu
325 330 335
Tyr Gly Asn Met Ser Ser Ala Cys Val Leu Phe Ile Ile Asp Glu Met
340 345 350
Arg Lys Lys Ser Ala Glu Asp Gly Ala Ala Thr Thr Gly Glu Gly Leu
355 360 365
Asp Trp Gly Val Leu Phe Gly Phe Gly Pro Gly Leu Thr Val Glu Thr
370 375 380
Val Val Leu His Ser Leu Pro Thr Thr Thr Ala Ile Ala Thr
385 390 395
<210> 2
<211> 222
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Leu Ile Ser Ala Val Gly Ser Glu Thr Lys Thr Ile Thr Phe Glu
1 5 10 15
Gly Ile Pro Phe Pro Ala Glu Ile Thr Ala Ala Gly Asn Pro Leu Ser
20 25 30
Leu Leu Ala Thr Gly Ile Thr Asp Ile Glu Ile His Phe Leu Gln Ile
35 40 45
Lys Tyr Asn Ala Ile Gly Val Tyr Leu His Ser Asn Asp Asp Ser Asp
50 55 60
Leu Leu Thr Thr His Leu Gly Ala Trp Lys Gly Lys Thr Ala Glu Asp
65 70 75 80
Leu Leu Ala Asp Ala Ala Phe Trp Ser Ala Leu Val Ser Ser Pro Val
85 90 95
Glu Lys Leu Leu Arg Val Val Val Ile Lys Glu Ile Lys Gly Ser Gln
100 105 110
Tyr Gly Val Gln Leu Glu Ser Ser Val Arg Asp Arg Leu Ala Ala Val
115 120 125
Asp Leu Tyr Glu Asp Asp Glu Glu Glu Ala Leu Glu Lys Val Ala Glu
130 135 140
Phe Phe Gln Ala Lys Tyr Phe Lys Pro Gly Ser Val Ile Thr Phe His
145 150 155 160
Phe Pro Ala Thr Pro Gly Pro Ala Glu Ile Thr Phe Val Thr Glu Gly
165 170 175
Lys Ala Asp Ala Lys Ile Thr Val Glu Asn Glu His Val Ala Gly Met
180 185 190
Ile Gln Lys Trp Tyr Leu Gly Gly Asp Asn Ala Val Ser Pro Thr Thr
195 200 205
Val Arg Ser Leu Ala Asp Arg Phe Ala Ala Leu Leu Ala Ala
210 215 220
<210> 3
<211> 209
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Ala Thr Glu Glu Val Leu Val Asp Glu Ile Thr Tyr Pro Thr Lys
1 5 10 15
Ile Thr Thr Thr Lys Pro Leu Ser Leu Leu Gly His Gly Ile Thr Asp
20 25 30
Met Glu Ile His Phe Ile His Val Lys Phe Tyr Ser Ile Gly Val Tyr
35 40 45
Leu Glu Pro Glu Val Val Gly His Leu Asp Gln Phe Lys Gly Lys Ser
50 55 60
Ala Lys Glu Leu Glu Asp Asn Glu Glu Phe Phe Asn Ala Leu Ile Ser
65 70 75 80
Ala Pro Val Glu Lys Phe Ile Arg Leu Val Val Ile Lys Glu Ile Lys
85 90 95
Gly Ala Gln Tyr Gly Val Gln Ile Glu Thr Ala Val Arg Asp Arg Leu
100 105 110
Ala Ala Glu Asp Lys Tyr Glu Glu Glu Glu Glu Glu Ala Leu Glu Lys
115 120 125
Val Ile Glu Phe Phe Gln Ser Lys Tyr Phe Lys Lys Leu Ser Val Ile
130 135 140
Thr Tyr His Phe Pro Ala Asn Ser Ala Thr Ala Glu Ile Val Val Ser
145 150 155 160
Leu Glu Gly Lys Glu Asp Ser Lys Tyr Val Ile Glu Asn Ala Asn Val
165 170 175
Val Glu Ala Ile Lys Lys Trp Tyr Leu Gly Gly Ser Ser Ala Val Ser
180 185 190
Ser Ser Thr Ile Gln Ser Leu Ala Ser Thr Phe Ser Gln Glu Leu Ser
195 200 205
Lys
<210> 4
<211> 540
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Ala Pro Glu Lys Glu Ile Ile Phe Arg Ser Lys Leu Pro Asp Ile
1 5 10 15
Tyr Ile Pro Lys His Leu Pro Leu His Ser Tyr Cys Phe Glu Asn Ile
20 25 30
Ser Thr Phe Asn Asn Arg Pro Cys Leu Ile Asp Gly Ala Thr Gly Val
35 40 45
Val His Thr Tyr Ala Asp Val Glu Leu Thr Ser Arg Lys Val Ala Ser
50 55 60
Ala Phe His Gln His Gly Ile Asn Lys Gly Asp Val Ile Met Ile Leu
65 70 75 80
Leu Pro Asn Ser Pro Glu Phe Val Tyr Ser Phe Phe Gly Ala Ser Tyr
85 90 95
Leu Gly Ala Val Ser Thr Met Ala Asn Pro Phe Phe Thr Ser Ala Glu
100 105 110
Ile Ile Lys Gln Ala Lys Ala Ser Asn Ala Lys Ile Ile Val Thr Gln
115 120 125
Ala Ser His Val Pro Lys Ile Lys Glu Tyr Ala Ala Glu Asn Ser Ile
130 135 140
Lys Ile Val Cys Ile Asp Ser Ala Pro Glu Gly Cys Leu His Phe Thr
145 150 155 160
Glu Leu Val Ser Gly Asp Glu Thr Lys Leu Pro Glu Val Glu Ile Ser
165 170 175
Ser Asp Asp Val Val Ala Leu Pro Tyr Ser Ser Gly Thr Thr Gly Leu
180 185 190
Pro Lys Gly Val Met Leu Thr His Lys Gly Leu Val Thr Ser Val Ala
195 200 205
Gln Gln Val Asp Gly Glu Asn Pro Asn Leu Trp Ile His Ser Glu Asp
210 215 220
Val Leu Met Cys Val Leu Pro Leu Phe His Ile Tyr Ser Leu Asn Ser
225 230 235 240
Ile Leu Leu Cys Gly Leu Arg Ala Gly Ala Ala Ile Leu Ile Met Gln
245 250 255
Lys Phe Asp Ile Val Pro Phe Leu Glu Leu Ile Glu Lys Tyr Lys Val
260 265 270
Thr Ile Gly Pro Phe Val Pro Pro Ile Val Leu Ala Ile Ala Asn Asn
275 280 285
Glu Glu Val Val Asp Lys Tyr Asp Leu Ser Ser Met Arg Thr Val Met
290 295 300
Ser Gly Ala Ala Pro Leu Gly Lys Glu Leu Glu Asp Met Val Arg Arg
305 310 315 320
Lys Phe Thr Asn Ala Lys Leu Gly Gln Gly Tyr Gly Met Thr Glu Ala
325 330 335
Gly Pro Val Leu Ala Met Cys Leu Ala Phe Ala Lys Glu Pro Tyr Glu
340 345 350
Ile Lys Ser Gly Ala Cys Gly Thr Val Val Arg Asn Ala Glu Met Lys
355 360 365
Ile Val Asp Pro Glu Thr Gly Leu Ser Leu Pro Arg Asn Gln Arg Gly
370 375 380
Glu Ile Cys Ile Arg Gly Asp Gln Ile Met Lys Gly Tyr Leu Asn Asp
385 390 395 400
Pro Glu Ser Thr Lys Thr Thr Ile Asp Ser Asp Gly Trp Leu His Thr
405 410 415
Gly Asp Ile Gly Leu Ile Asp Asp Asp Asp Glu Leu Phe Ile Val Asp
420 425 430
Arg Leu Lys Glu Leu Ile Lys Tyr Lys Gly Phe Gln Val Ala Pro Ala
435 440 445
Glu Leu Glu Ala Leu Leu Leu Thr His Pro Gln Ile Ser Asp Val Ala
450 455 460
Val Val Pro Met Val Asn Glu Ala Ala Gly Glu Val Pro Val Ala Phe
465 470 475 480
Val Val Lys Thr Lys Asp Ser Ser Val Thr Glu Asp Asp Ile Lys Gln
485 490 495
Phe Val His Lys Gln Val Val Phe Tyr Lys Arg Ile Asn Arg Val Phe
500 505 510
Phe Ile Asp Thr Ile Pro Lys Ser Pro Ala Gly Lys Ile Leu Arg Lys
515 520 525
Glu Leu Arg Ala Lys Leu Ala Ala Gly Val Pro Asn
530 535 540
<210> 5
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Ala Asp Phe Lys Phe Glu Pro Met Arg Ser Leu Ile Tyr Val Asp
1 5 10 15
Cys Val Ser Glu Asp Tyr Arg Pro Lys Leu Gln Arg Trp Ile Tyr Lys
20 25 30
Val His Ile Pro Asp Ser Ile Ser Gln Phe Glu Pro Tyr Val Thr Lys
35 40 45
Tyr Ala Phe Tyr Pro Ser Phe Pro Ile Pro Pro Gln Gly Asp Arg Phe
50 55 60
Gly Tyr Ala Arg Met Gln Leu Thr Glu His His Trp Leu Val Ser Asp
65 70 75 80
Leu Asp Pro Arg Leu Glu Ile Lys Ala Ile Ala Glu Thr Phe Pro Met
85 90 95
Asp Val Leu Val Trp Gln Gly Gln Ile Pro Ala Ala Ala His Thr Asp
100 105 110
Ala Gln Ile Asp Ser Asp Gly Asp Ala Gly Asn Ala Ala Arg Lys Ser
115 120 125
Asn Asn Ala Glu Gly Asn Pro Phe Ile Phe Ala Phe Leu Pro Met Trp
130 135 140
Trp Glu Lys Asp Leu Lys Gly Lys Gly Arg Thr Ile Glu Asp Gly Ala
145 150 155 160
Asn Tyr Arg Phe Asn Met Thr Ile Gly Phe Pro Glu Gly Val Asp Lys
165 170 175
Ala Glu Gly Glu Lys Trp Leu Phe Glu Lys Val Val Pro Ile Leu Gln
180 185 190
Ala Ala Pro Glu Cys Thr Arg Val Leu Ala Ser Ala Val Lys Lys Asp
195 200 205
Ile Asn Gly Cys Val Met Asp Trp Val Leu Glu Ile Trp Phe Glu Asn
210 215 220
Gln Ser Gly Trp Tyr Lys Val Met Val Asp Asp Met Lys Ala Leu Glu
225 230 235 240
Lys Pro Ser Trp Ala Gln Gln Asp Ala Phe Pro Phe Leu Lys Pro Tyr
245 250 255
His Asn Val Cys Ser Ala Ala Val Ala Asp Tyr Thr Pro Ser Asn Asn
260 265 270
Leu Ala Asn Tyr Arg Gly Tyr Ile Thr Met Arg
275 280
<210> 6
<211> 1197
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atggcttctt ctatcgacat cgctgctatc agagaagctc aaagagctca aggtccagct 60
actatcttgg ctatcggtac tgctactcca tctaactgtg tttaccaagc tgactaccca 120
gactactact tcagaatcac taagtctgaa cacatggttg acttgaagga aaagttcaag 180
agaatgtgtg acaagtctat gatcagaaag agatacatgc acttgactga agaatacttg 240
aaggaaaacc catctttgtg tgaatacatg gctccatctt tggacgctag acaagacgtt 300
gttgttgttg aagttccaaa gttgggtaag gaagctgcta ctaaggctat caaggaatgg 360
ggtcaaccaa agtctaagat cactcacttg atcttctgta ctacttctgg tgttgacatg 420
ccaggtgctg actaccaatt gactaagttg ttgggtttga gaccatctgt taagagattc 480
atgatgtacc aacaaggttg tttcgctggt ggtactgttt tgagattggc taaggacttg 540
gctgaaaaca acaagggtgc tagagttttg gttgtttgtt ctgaaatcac tgctgttact 600
ttcagaggtc caaacgacac tcacttggac tctttggttg gtcaagcttt gttcggtgac 660
ggtgctgctg ctgttatcgt tggttctgac ccagacttga ctactgaaag accattgttc 720
gaaatgatct ctgctgctca aactatcttg ccagactctg aaggtgctat cgacggtcac 780
ttgagagaag ttggtttgac tttccacttg ttgaaggacg ttccaggttt gatctctaag 840
aacatcgaaa aggctttgac tcaagctttc tctccattgg gtatctctga ctggaactct 900
ttgttctgga tcgctcaccc aggtggtcca gctatcttgg accaagttga attgaagttg 960
ggtttgaagg aagaaaagat gagagctact agacacgttt tgtctgaata cggtaacatg 1020
tcttctgctt gtgttttgtt catcatcgac gaaatgagaa agaagtctgc tgaagacggt 1080
gctgctacta ctggtgaagg tttggactgg ggtgttttgt tcggtttcgg tccaggtttg 1140
actgttgaaa ctgttgtttt gcactctttg ccaactacta ctgctatcgc tacttaa 1197
<210> 7
<211> 1623
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atggctccag aaaaggaaat catcttcaga tccaagttgc cagacattta catcccaaag 60
cacctccccc tacattctta ctgtttcgaa aacatctcta ctttcaacaa cagaccatgc 120
ttgatcgatg gtgctaccgg tgtcgtccac acttacgctg acgttgaact aacatctcgt 180
aaggttgcct ctgctttcca ccaacacggt atcaacaagg gtgatgttat catgatttta 240
ttgccaaact ctccagaatt tgtttactct ttcttcggtg cctcctattt gggtgccgta 300
tcaactatgg ctaacccttt tttcacttcc gctgaaatca ttaaacaagc taaggcttcc 360
aatgctaaga tcattgtcac tcaagcctct catgttccaa agatcaagga atacgctgcc 420
gaaaattcca ttaagatcgt ctgtatcgat tctgctccag aaggttgttt gcacttcact 480
gaattggtct ccggtgacga aaccaagttg ccggaagtcg aaatttcctc cgacgatgtt 540
gttgctttgc catactcctc tggtaccact gggttgccaa agggtgttat gttgactcac 600
aaaggcttgg tcacttctgt cgctcaacaa gttgacggtg aaaacccaaa cttgtggatt 660
cactctgaag acgttctgat gtgtgttctg ccattgttcc atatctactc tttgaacagc 720
atcttgttgt gtggtttgcg tgctggtgct gctattttga ttatgcaaaa gttcgatatt 780
gtgcctttct tggaattgat tgaaaagtat aaggttacca tcggtccatt cgttccacca 840
attgttttgg ccattgctaa caatgaagaa gttgttgaca agtacgattt gtcttccatg 900
agaaccgtca tgtctggtgc tgctccatta ggtaaagaat tggaagacat ggttagaaga 960
aagtttacca acgctaagct aggtcaaggt tacggtatga ctgaagccgg tccagttttg 1020
gctatgtgtt tggctttcgc caaggaacca tacgaaatca agtctggtgc ttgtggtact 1080
gtcgtcagaa acgccgaaat gaaaatcgtt gatccagaaa ctggtttgag tttaccaaga 1140
aaccaaagag gtgaaatctg tatcagaggt gaccaaatca tgaagggtta cttgaacgac 1200
ccagaatcta ccaagaccac aattgattcc gatggttggt tgcacaccgg tgacattggt 1260
ttgattgatg acgacgatga attatttatt gtcgacagat tgaaggagtt aatcaagtac 1320
aagggtttcc aagttgctcc agctgaattg gaagccttgt tgttgaccca cccacaaatt 1380
tctgacgttg ctgtcgttcc aatggttaac gaagctgctg gagaagtccc agtcgccttt 1440
gttgtcaaga ctaaggactc tagtgttact gaagatgaca tcaagcaatt cgttcacaag 1500
caagtcgtct tctacaagag aatcaacaga gttttcttca tcgacactat tccaaaatcc 1560
cctgctggta agattttgag aaaggaatta cgtgctaaat tagccgccgg tgtcccaaac 1620
tga 1623
<210> 8
<211> 852
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atggctgatt tcaagttcga accaatgaga tccttgattt acgttgattg tgtttccgaa 60
gattacagac caaagttgca aagatggatt tacaaggttc atattccaga ttccatttcc 120
caattcgaac catacgttac caagtacgct ttctacccat ccttcccaat tccaccacaa 180
ggtgatagat tcggttacgc tagaatgcaa ttgaccgaac atcattggtt ggtttccgat 240
ttggacccta gattggaaat taaggctatt gctgaaacct tcccaatgga tgttttggtt 300
tggcaaggtc aaattccagc tgctgctcat accgatgctc aaattgattc cgatggtgat 360
gctggtaacg ctgctagaaa gtccaacaac gctgaaggta acccattcat tttcgctttc 420
ttgccaatgt ggtgggaaaa ggatttgaag ggtaagggta gaaccattga agatggtgct 480
aactacagat tcaacatgac cattggtttc ccagaaggtg ttgataaggc tgaaggtgaa 540
aagtggttgt tcgaaaaggt tgttccaatt ttgcaagctg ctccagaatg taccagagtt 600
ttggcttccg ctgttaagaa ggatattaac ggttgtgtta tggattgggt tttggaaatt 660
tggttcgaaa accaatccgg ttggtacaag gttatggttg atgatatgaa ggctttggaa 720
aagccatcct gggctcaaca agatgctttc ccattcttga agccatacca taacgtttgt 780
tccgctgctg ttgctgatta caccccatcc aacaacttgg ctaactacag aggttacatt 840
accatgagat aa 852
<210> 9
<211> 669
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atgttgatct ccgctgtcgg ctctgaaacc aagaccatta ctttcgaagg tatcccattc 60
ccagctgaaa tcacagctgc tggtaaccca ttgtccttgt tggctactgg tattaccgac 120
attgaaattc acttcttgca aatcaaatac aacgctattg gtgtttattt gcactctaac 180
gatgactccg atttattaac tactcactta ggtgcttgga agggtaagac tgctgaagat 240
ttgttggcag atgctgcttt ctggtctgct ttggtttcat cccctgtcga aaagttgcta 300
agagttgtcg tcatcaagga gatcaagggt tctcaatacg gtgttcaatt ggaatcttct 360
gttagagaca gattggccgc tgttgacttg tacgaagacg atgaagaaga agctttagaa 420
aaagttgctg aatttttcca agccaagtac ttcaagccag gttccgttat cacctttcat 480
ttcccagcca ctccaggtcc agctgaaatt acctttgtca ctgaaggtaa ggctgatgcc 540
aagatcactg ttgaaaacga acacgttgcc ggtatgattc aaaagtggta cttgggtggt 600
gacaatgctg tctctccaac caccgtccgt tctttggctg acagattcgc tgctttgctc 660
gccgcctga 669
<210> 10
<211> 630
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atggcgaccg aggaagtgct ggttgacgaa atcacctacc cgaccaaaat caccaccacc 60
aaaccgctga gcctgctggg ccacggcatc accgacatgg agatccactt cattcacgtg 120
aagttttaca gcattggtgt ttatctggag ccggaagtgg ttggtcacct ggaccagttc 180
aagggcaaaa gcgcgaagga gctggaagat aacgaggagt tcttcaacgc gctgatcagc 240
gcgccggtgg agaaatttat tcgtctggtg gttatcaagg aaattaaagg tgcgcagtac 300
ggcgtgcaaa tcgaaaccgc ggtgcgtgac cgtctggcgg cggaagataa atatgaggaa 360
gaggaagagg aagcgctgga gaaagtgatt gagttcttcc aaagcaaata cttcaagaaa 420
ctgagcgtta tcacctatca ctttccggcg aacagcgcga ccgcggaaat tgtggttagc 480
ctggagggca aggaagatag caaatacgtt atcgagaacg cgaacgtggt tgaagcgatt 540
aagaaatggt atctgggtgg cagcagcgcg gttagcagca gcaccattca gagcctggcg 600
agcaccttta gccaagaact gagcaaataa 630
Claims (6)
1.酶组合物,其特征在于,包括:查尔酮合成酶和查尔酮异构酶样蛋白;
所述查尔酮合成酶包括:灯盏花EbCHS、向日葵Ha4CL和细枝真杆菌ErCHI;
所述查尔酮异构酶样蛋白包括:高粱SbCHIL和/或大豆GmCHIL;
所述灯盏花EbCHS的氨基酸序列如SEQ ID NO:1所示;
所述向日葵Ha4CL的氨基酸序列如SEQ ID NO:4所示列;
所述细枝真杆菌ErCHI的氨基酸序列如SEQ ID NO:5所示;
所述高粱SbCHIL的氨基酸序列如SEQ ID NO:2所示;
所述大豆GmCHIL的氨基酸序列如SEQ ID NO:3所示。
2.编码如权利要求1所述酶组合物的核酸分子,其特征在于,
编码所述灯盏花EbCHS的核酸分子的核苷酸序列如SEQ ID NO:6所示;
编码所述向日葵Ha4CL的核酸分子的核苷酸序列如SEQ ID NO:7所示;
编码所述细枝真杆菌ErCHI的核酸分子的核苷酸序列如SEQ ID NO:8所示;
编码所述高粱SbCHIL的核酸分子的核苷酸序列如SEQ ID NO:9所示的核苷酸序列;
编码所述大豆GmCHIL的核酸分子的核苷酸序列如SEQ ID NO:10所示。
3.表达载体,其特征在于,包括如权利要求2所述的核酸分子。
4.宿主,其特征在于,转化和/或转染如权利要求3所述的表达载体。
5.如权利要求4所述的宿主,其特征在于,包括:酵母菌。
6.如权利要求1所述的酶组合物、如权利要求2所述的核酸分子、如权利要求3所述的表达载体和/或如权利要求4或5所述的宿主在合成柚皮素中的应用。
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