CN101541967A - 源自假单胞菌6-19的pha合酶的突变体和使用该突变体制备乳酸酯均聚物或共聚物的方法 - Google Patents
源自假单胞菌6-19的pha合酶的突变体和使用该突变体制备乳酸酯均聚物或共聚物的方法 Download PDFInfo
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- CN101541967A CN101541967A CNA2007800433773A CN200780043377A CN101541967A CN 101541967 A CN101541967 A CN 101541967A CN A2007800433773 A CNA2007800433773 A CN A2007800433773A CN 200780043377 A CN200780043377 A CN 200780043377A CN 101541967 A CN101541967 A CN 101541967A
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- acid
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- lactate
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Abstract
本发明涉及源自假单胞菌6-19(KCTC11027BP)的聚羟基链烷酸酯合酶(PHA合酶)突变体,其可以通过使用乳酰辅酶A作为底物来制备乳酸酯聚合物和/或共聚物。本发明涉及一种使用所述合酶突变体制备乳酸酯聚合物和/或共聚物的方法。源自假单胞菌6-19的本发明聚羟基链烷酸酯合酶突变体能够通过使用用常规聚羟基链烷酸酯合酶难以用作底物的乳酰辅酶A作为底物有效地制备乳酸酯聚合物和/或共聚物。
Description
技术领域
本发明涉及源自假单胞菌6-19(Pseudomonas sp.6-19)的聚羟基链烷酸酯合酶突变体,其中,该突变体可以使用乳酰辅酶A(lactyl-CoA)作为底物来生产乳酸酯聚合物和/或乳酸酯共聚物。本发明还涉及一种制备乳酸酯聚合物和/或乳酸酯共聚物的方法,其中,该方法使用所述聚羟基链烷酸酯合酶突变体。
背景技术
聚乳酸酯(PLA)是一种源自乳酸酯的常见生物可降解聚合物,其作为一种普通或者医用聚合物具有多种应用。目前,PLA通过聚合由微生物发酵产生的乳酸酯而制备,但是通过乳酸酯的直接聚合只能产生低分子量PLA(1000-5000道尔顿)。为了合成高分子量(>100,000道尔顿)的PLA,可以使用通过链偶联剂聚合由乳酸酯的直接聚合得到的低分子量PLA的方法。然而,其具有如下缺点:如由于加入溶剂或者链偶联剂而使高分子量PLA的制备方法复杂,且还不容易除去该溶剂或者链偶联剂。目前,在制备高分子量的商业上可用的PLA的方法中,正使用一种其中将乳酸酯转化成交酯以通过交酯环的环化脱水作用合成PLA的方法。
PLA均聚物可以由使用乳酸酯的化学合成方法容易地获得,但是难于制备具有多种单体单元的乳酸酯共聚物,并且其商业可用性非常低。
同时,聚羟基链烷酸酯(PHA)是一种在例如磷、氮、镁、氧的其它营养元素缺乏而碳源过量时在微生物中积累作为碳和能量储存化合物的聚酯。由于PHA与源于石油的合成聚合物具有相似的性质,并且同时呈现出优异的生物降解性质,所以其被认为是合成塑料的替代性材料。
为了在微生物中产生PHA,需要将微生物代谢物转化成PHA单体的酶和使用PHA单体合成PHA聚合物的PHA合酶。当使用微生物生产PLA和乳酸酯共聚物时,需要相同的体系,并且除了提供羟酰辅酶A(PHA合酶的原始底物)以外,还需要能够提供乳酰辅酶A的酶。
因此,本发明发明人开发了一种使用源自丙酸梭菌(Clostridiumpropionicum)的丙酰辅酶A转移酶来提供乳酰辅酶A的体系,并且成功地制备了PLA和乳酸酯共聚物(韩国专利申请公开第10-2006-0121555号)。但是,其对于在2-位羟基化的羟基链烷酸酯具有较小的PHA合酶活性。在体外检测的对乳酰辅酶A的PHA合酶活性已经有报道,但是对乳酰辅酶A的PHA合酶活性据报道如上述一样非常低(Zhang等人,Appl.Microbiol.Biotechnol.,56:131,2001;Valentin和Steinbuchel,Appl.Microbiol.Biotechnol.,40:699,1994;Yuan等人,Arch Biochem Biophys.,394:87,2001)。因此,如果PHA合酶不能够有效地利用乳酰辅酶A且使用PHA合酶来生产PLA和乳酸酯共聚物,则合成的效率必定低。也即是说,因为乳酸酯(在2-碳位羟基化的羟基链烷酸酯)不是PHA合酶的适合底物,所以能够有效使用乳酰辅酶A的PHA合酶对于有效地合成PLA和乳酸共聚物是非常重要的。
发明内容
技术问题
因此,本发明的一个目的是提供能够有效使用乳酰辅酶A作为底物的PHA合酶。
本发明的另一个目的是提供一种制备PLA和乳酸酯共聚物的方法,其中,所述方法使用含有能够使用乳酰辅酶A作为底物的PHA合酶基因和丙酰辅酶A转移酶基因的细胞或者植物。
技术方案
为了实现所述目的,本发明提供一种聚羟基链烷酸酯合酶突变体,该突变体使用乳酰辅酶A作为底物生产乳酸酯聚合物或者乳酸酯共聚物并且具有SEQ.ID No:10的氨基酸序列,其中,在SEQ.ID No:10的氨基酸序列的第481位的谷氨酰胺发生突变。
优选地,本发明提供聚羟基链烷酸酯合酶突变体,其中,选自在第130位的谷氨酸、在第325位的丝氨酸和在第477位的丝氨酸中的至少一个氨基酸进一步发生突变。
更优选地,本发明提供一种聚羟基链烷酸酯合酶突变体,该突变体使用乳酰辅酶A作为底物生产乳酸酯聚合物或者乳酸酯共聚物并且具有SEQ.ID No:10的氨基酸序列,其中,所述氨基酸序列具有如下任何一种突变:
a)S325T和Q481M;
b)E130D和Q481K;
c)S325T和Q481K;
d)E130D和Q481M;
e)E130D和Q481R;
f)E130D、S325T和Q481M;
g)E130D、S325T和Q481K;
h)E130D、S477R和Q481K;
i)E130D、S477R和Q481M;
j)E130D、S477R和Q481R;
k)E130D、S477H和Q481K;
l)E130D、S477H和Q481M;
m)E130D、S477H和Q481R;
n)E130D、S477F和Q481K;
o)E130D、S477F和Q481M;
p)E130D、S477F和Q481R;
q)E130D、S477Y和Q481K;
r)E130D、S477Y和Q481M;
s)E130D、S477Y和Q481R;
t)E130D、S325T、S477R和Q481M;
u)E130D、S325T、S477R和Q481K;
v)E130D、S325T、S477F和Q481M;
w)E130D、S325T、S477G和Q481M;或
x)E130D、S325T、S477F和Q481K。
本发明发明人证实假单胞菌6-19的聚羟基链烷酸酯合酶的一些突变体可以使用乳酰辅酶A作为底物并且非常有效地生产乳酸酯聚合物和/或共聚物,由此完成了本发明。
本发明还提供编码所述聚羟基链烷酸酯合酶突变体的基因。
本发明又提供包含用于合成乳酸酯聚合物或共聚物的基因的重组载体。
更优选地,本发明提供重组载体,其进一步包含编码丙酰辅酶A转移酶(pct)的基因。
本发明还提供用上述重组载体转化的细胞或者植物。
本发明又提供由使用上述重组载体转化而获得的细胞或者植物,其中,原始细胞或者植物没有编码丙酰辅酶A转移酶的基因。
本发明还提供一种制备乳酸酯聚合物或共聚物的方法,其中,该方法包括培养所述细胞或者植物。
更优选地,本发明提供所述方法,其中,所述培养是在含有3-羟基丁酸酯(3-HB)的培养基中进行的并且所制备的共聚物为含有3-羟基丁酸酯单体单元和乳酸酯单体单元的共聚物。
在这里使用的术语“共聚物”意图包括由两种不同单体构成的二元共聚物、由三种不同单体构成的三元共聚物或者由四种不同单体构成的四元共聚物。
在本发明中,所述羟基链烷酸酯为选自下列化合物中的至少一种:3-羟基丁酸酯、3-羟基戊酸酯、4-羟基丁酸酯、中链长(C6~14)的(D)-3-羟基羧酸、3-羟基丙酸、3-羟基己酸、3-羟基庚酸、3-羟基辛酸、3-羟基壬酸、3-羟基癸酸、3-羟基十一烷酸、3-羟基十二烷酸、3-羟基十四烷酸、3-羟基十六烷酸、4-羟基戊酸、4-羟基己酸、4-羟基庚酸、4-羟基辛酸、4-羟基癸酸、5-羟基戊酸、5-羟基己酸、6-羟基十二烷酸、3-羟基-4-戊烯酸、3-羟基-4-反-己烯酸、3-羟基-4-顺-己烯酸、3-羟基-5-己烯酸、3-羟基-6-反-辛烯酸、3-羟基-6-顺-辛烯酸、3-羟基-7-辛烯酸、3-羟基-8-壬烯酸、3-羟基-9-癸烯酸、3-羟基-5-顺-十二碳烯酸、3-羟基-6-顺-十二碳烯酸、3-羟基-5-顺-十四碳烯酸、3-羟基-7-顺-十四碳烯酸、3-羟基-5,8-顺-顺-十四碳烯酸、3-羟基-4-甲基戊酸、3-羟基-4-甲基己酸、3-羟基-5-甲基己酸、3-羟基-6-甲基庚酸、3-羟基-4-甲基辛酸、3-羟基-5-甲基辛酸、3-羟基-6-甲基辛酸、3-羟基-7-甲基辛酸、3-羟基-6-甲基壬酸、3-羟基-7-甲基壬酸、3-羟基-8-甲基壬酸、3-羟基-7-甲基癸酸、3-羟基-9-甲基癸酸、3-羟基-7-甲基-6-辛烯酸、苹果酸、3-羟基琥珀酸甲酯、3-羟基己二酸甲酯、3-羟基辛二酸甲酯、3-羟基壬二酸甲酯、3-羟基癸二酸甲酯、3-羟基辛二酸乙酯、3-羟基癸二酸乙酯、3-羟基庚二酸丙酯、3-羟基癸二酸苄酯、3-羟基-8-乙酰氧基辛酸、3-羟基-9-乙酰氧基壬酸、苯氧基-3-羟基丁酸、苯氧基-3-羟基戊酸、苯氧基-3-羟基庚酸、苯氧基-3-羟基辛酸、对氰基苯氧基-3-羟基丁酸、对氰基苯氧基-3-羟基戊酸、对氰基苯氧基-3-羟基己酸、对硝基苯氧基-3-羟基己酸、3-羟基-5-苯基戊酸、3-羟基-5-环己基丁酸、3,12-二羟基十二烷酸、3,8-二羟基-5-顺-十四碳烯酸、3-羟基-4,5-环氧癸酸、3-羟基-6,7-环氧十二烷酸、3-羟基-8,9-环氧-5,6-顺-十四烷酸、7-氰基-3-羟基庚酸、9-氰基-3-羟基壬酸、3-羟基-7-氟庚酸、3-羟基-9-氟壬酸、3-羟基-6-氯己酸、3-羟基-8-氯辛酸、3-羟基-6-溴己酸、3-羟基-8-溴辛酸、3-羟基-11-溴十一烷酸、3-羟基-2-丁烯酸、6-羟基-3-十二碳烯酸、3-羟基-2-甲基丁酸、3-羟基-2-甲基戊酸和3-羟基-2,6-二甲基-5-庚烯酸。
本发明还提供一种制备乳酸酯聚合物或乳酸酯共聚物的方法,其中,该方法包括培养所述细胞或者植物。
在这里使用的术语“载体”指的是一种DNA构造,其包含DNA序列,该DNA序列可操作地连接至在适合的宿主内能够表达该DNA的适合控制序列。在本发明中,载体可以为质粒、噬菌体或者简单的基因组插入。当用其转化适合的宿主时,载体可以不顾宿主基因组自我复制并运行,或者在某些情况下,载体并入宿主基因组。因为目前最常使用质粒作为载体,所以在这里可替换地使用质粒代替载体。但是,根据本发明的载体包括具有相同功能的不同类型载体,其已经或者将为本领域技术人员所知。
术语“表达控制序列”指的是用于在特定宿主中表达可操作连接的编码序列所必需的DNA序列。这种控制序列包含:用于进行转录的启动子;用于控制转录的任意操纵基因序列;编码适合mRNA核糖体的结合位点的序列;和用于控制转录和转译的终止的序列。例如,适合于原核生物的控制序列包含启动子、任意操纵基因序列和核糖体结合位点。对于真核生物,包含启动子、多聚腺苷酸信号和增强子。在质粒中,启动子是对基因的表达量影响最严重的一个因素。优选,使用SRα启动子、源于巨细胞病毒的启动子等作为高度表达的启动子。
在各种表达控制序列中的任何一种都可以用在载体中表达本发明的DNA序列。例如,有用的表达控制序列的实例包括SV40或者腺病毒的早期或者后期启动子、lac体系、trp体系、TAC或TRC体系、T3和T7启动子、噬菌体λ的主要操纵基因和启动子区域、fd编码蛋白的控制区域、用于3-磷酸甘油酸酯激酶或者其它糖酵解酶的启动子、例如Pho5的磷酸酶的启动子、酵母α交配系统的启动子和已知控制真核生物、原核生物或其病毒的基因表达的结构序列,和它们的各种组合。
当将核酸置入与其它核酸序列的功能关系中时,其被“可操作地连接”。这可能意味着基因和控制序列连接的方式,因为当适合的分子(例如转录激活蛋白)与控制序列结合时,基因的表达是可能的。例如,当前肽序列或分泌前导的DNA表达为参与多肽的分泌的前蛋白时,其可操作地连接至该多肽的DNA;当启动子或者增强子影响编码序列的转录时,其可操作地连接至该编码序列;或者当设置核糖体结合位点以便有利于翻译时,其可操作地连接至编码序列。总体而言,“可操作地连接”指的是被连接的DNA序列为毗邻的,而且在分泌前导的情况下,是毗邻的且在阅读相(readingphase)内。但是,增强子不必为毗邻的。连接是通过在方便的限制位点的连接实现的。如果不存在这些位点,则根据常规实践使用合成的寡核苷酸接头或者连接体。
这里使用的术语“表达载体”一般指的是作为重组载体的双链DNA片段,其中插入了典型的异源DNA片段。所述异源DNA指的是不是在宿主细胞中天然产生的异型DNA。一旦将该表达载体引入到宿主细胞中,其可以不顾宿主染色体DNA进行复制产生几个拷贝和插入它们中的异源DNA。
如相关领域中的技术人员所已知的,为了增加转染入宿主细胞中的基因的表达水平,相应的基因应当可操作地连接至用于控制转录和解码表达的序列,其在选择的表达宿主中起作用。优选地,将表达控制序列和相应的基因包含在一个表达载体中,所述载体同时包含病毒选择标记和复制起点。如果表达宿主是真核生物,则表达载体应当进一步包含在真核生物表达宿主中有用的表达标记。
在本发明中,例如质粒载体、噬菌体载体、粘粒载体或者YAC(酵母人工染色体)载体的各种载体都可以用作上述载体。对于本发明来说,优选使用质粒载体。可用于这些目的的典型质粒载体具有:(a)复制起点,以便其导致有效的复制使得各个宿主细胞包含几百个拷贝的质粒载体;(b)抗生素抗性基因,以使得可以选择用质粒载体转化的宿主细胞;和(c)序列,含有将要插入外源DNA片段的限制酶位点。即使在没有适合的限制酶位点的情况下,根据常规方法通过使用合成的寡核苷酸接头或者连接体也可以容易地连接载体或者外源DNA。
本发明的重组载体可以根据本领域中的已知方法转化到适合的宿主细胞中。在本发明中优选的宿主细胞是原核细胞,更优选的是大肠杆菌。优选的大肠杆菌菌株包括:大肠杆菌DH5a、大肠杆菌JM101、大肠杆菌K12、大肠杆菌W3110、大肠杆菌X1776、大肠杆菌XL1-Blue(Stratagene)和大肠杆菌B。但是,也可以使用例如FMB101、NM522、NM538和NM539的其它大肠杆菌菌株和其它原核细胞种属。除了上述大肠杆菌以外,在这里还可以使用例如农杆菌A4的农杆菌属、例如枯草杆菌的杆菌属、例如鼠伤寒沙门氏菌或粘质沙雷菌的各种肠细菌和各种假单胞菌属作为宿主细胞,但是本发明的范围并不限于上述实例。
另外,原核细胞的转化可以根据描述在Sambrook等人(supra)的章节1.82中的氯化钙法容易地进行。或者,也可以使用电穿孔法(Neumann等人,EMBO J.,1:841(1982))来转化这些类型的细胞。
制备含有转移酶基因和合酶基因的植物的植物的转化可以通过使用农杆菌或者病毒载体的常规方法实现。例如,通过用含有本发明的基因的重组载体转化农杆菌并且用转化的农杆菌感染目标植物的组织等而获得转化的植物。更具体而言,可以通过如下步骤制备转化的植物:(a)预培养目的植物的外植体,然后通过共培养该外植体和转化的农杆菌来转化该外植体;(b)培养所述感染的外植体以诱导愈伤组织;以及(c)切下所得到的愈伤组织,并且将其在生芽培养基(shoot-inducing medium)中培养。
这里使用的术语“外植体”指的是从植物上切下的组织片段,并且包括子叶或者下胚轴。子叶或者下胚轴可以用作本发明的外植体。更优选使用通过消毒并清洗植物的种子而在MS培养基中使其发芽而得到的子叶。
可以用于本发明的转化的植物包括,但不限于,烟草、番茄、辣椒、豆、米和玉米。此外,即使转化的植物为有性繁殖的,对于本领域技术人员来说显而易见的是,也可以通过使用植物组织培养等无性繁殖这种植物。
附图说明
图1是显示包含源自假单胞菌6-19的聚羟基链烷酸酯合酶基因的重组表达载体的制备方法的图。
图2是在能够合成PHB的条件下,在培养用含有phaC1Ps6-19合酶和SCL突变体(phaC1Ps6-19200和phaC1Ps6-19300)的重组载体(pPs619C1-ReAB、pPs619C1200-ReAB和pPs619C1300-ReAB)转化的大肠杆菌之后的FACS(荧光激活细胞分选)结果。
图3是同时表达PHA合酶和CP-PCT的组成型表达载体的简图。
图4是显示包含源自假单胞菌6-19的PHA合酶基因、SCL突变体基因和CP-PCT基因的重组表达载体的制备方法的图。
具体实施方式
以下将更详细地描述本发明。下面提供的实施例以说明的方式来帮助本领域中的技术人员理解本发明,但是并非意图限制本发明的范围。
特别地,虽然在下面的实施例中公开了当用PHA合酶突变体制备乳酸酯共聚物时通过加入3-羟基丁酸酯(3-HB)获得的聚(3-羟基丁酸酯-共-乳酸酯)(P(3HB-共-LA))的合成,但是对于本发明技术领域中的技术人员来说,显而易见的是,通过加入非3-HB的其它羟基链烷酸酯可以制备含有不同羟基链烷酸酯和乳酸酯的多种共聚物。
<实施例1>源自假单胞菌6-19的PHA合酶基因的克隆和表达载体的构建
为了分离源自假单胞菌6-19(KCTC 11027BP)的PHA合酶(phaC1Ps6-19)基因,提取假单胞菌6-19的全部DNA,并根据phaC1Ps6-19基因的序列制备SEQ ID NO:1和2的引物(Ae-jin Song,Master′s Thesis,Department ofChemical and Biomolecular Engineering,KAIST,2004)以及进行PCR来得到phaC1Ps6-19的基因。
SEQ ID NO:1:5-GAG AGA CAA TCA AAT CAT GAG TAA CAA GAGTAA CG-3
SEQ ID NO:2:5-CAC TCA TGC AAG CGT CAC CGT TCG TGC ACGTAC-3
作为用琼脂凝胶电泳检测PCR反应产物的结果,观察到对应于phaC1Ps6-19基因的1.7Kbp的基因片段。为了表达phaC1Ps6-19合酶,构建了表达供应单体的酶和合酶的组成型表达系统的操纵子(图1)。
含有源自富养罗尔斯通氏菌(Ralstonia eutropha)H16的PHB-合成操纵子的DNA片段用BamHI/EcoRI从pSYL105载体中切除出来(Lee等人,Biotech.Bioeng.,1994,44:1337-1347),并被插入到pBluescript II(Stratagene)的BamHI/EcoRI-识别位点来构建pReCAB重组载体。
已知pReCAB载体通过PHB操纵子启动子组成型表达PHA合酶(phaCRE)和供应单体的酶(phaARE和phaBRE),并且也已知在大肠杆菌中很好地工作(Lee等人,Biotech.Bioeng.,1994,44:1337-1347)。pReCAB载体用BstBI/SbfI切开以删除富养罗尔斯通氏菌H16PHA合酶(phaCRE),然后将上述phaC1Ps6-19基因插入到BstBI/SbfI位点以制备pPs619C1-ReAB重组载体(图1)。
在没有氨基酸突变的情况下用SDM(定点诱变)方法去除包含在内部的BstBI位点来制备在两端具有两个BstBI/SbfI位点的phaC1Ps6-19合酶基因片段,并用SEQ ID NO:3和4、SEQ ID NO:5和6以及SEQ ID NO:7和8的引物进行重叠PCR来加入BstBI/SbfI-识别位点
SEQ ID NO:3:5-atg ccc gga gcc ggt tcg aa-3
SEQ ID NO:4:5-CGT TAC TCT TGT TAC TCA TGA TTT GAT TGT CTCTC-3
SEQ ID NO:5:5-GAG AGA CAA TCA AAT CAT GAG TAA CAA GAG TAACG-3
SEQ ID NO:6:5-CAC TCA TGC AAG CGT CAC CGT TCG TGC ACG TAC-3
SEQ ID NO:7:5-GTA CGT GCA CGA ACG GTG ACG CTT GCA TGA GTG-3
SEQ ID NO:8:5-aac ggg agg gaa cct gca gg-3
通过测序验证pPs619C1-ReAB重组载体的phaC1Ps6-19基因序列,并将结果显示在SEQ ID NO:9中,使用其编码的氨基酸序列显示在SEQ ID NO:10中。
这些序列的相似性测试表明该基因与源自假单胞菌属菌株61-3的phaC1(Matsusaki等人,J.Bacteriol.,180:6459,1998)具有84.3%的核苷酸序列同一性和88.9%的氨基酸序列同一性,由此证实这两种合酶是非常相似的。由这些结果证实,在本发明中得到的phaC1Ps6-19合酶是II型PHA合酶。
为了证实phaC1Ps6-19合酶是否合成了PHB,用pPs619C1-ReAB重组载体转化大肠杆菌XL-1Blue(Stratagene),并将其在PHB检测培养基(LB琼脂,葡萄糖20g/L,尼罗红0.5ug/ml)中进行培养。该检测的结果是,没有观察到PHB的合成。
<实施例2>源自假单胞菌6-19的PHA合酶的底物-特异性突变体的制备
在各种类型的PHA合酶中,已知II型PHA合酶为MCL-PHA(中链长PHA)合酶,能够聚合具有相对较长碳链的底物。预期这种MCL合酶可以用于制备乳酸酯聚合物。即使源自假单胞菌61-3的phaC1合酶(其与本发明的phaC1Ps6-19合酶具有高度同一性)是II型合酶,但是据报道phaC1合酶具有相对较宽范围的底物特异性(Matsusaki等人,J.Bacteriol.,180:6459,1998),并且已经报道了对其适于制备SCL-PHA(短链长PHA)的突变体的研究(Takase等人,Biomacromolecules,5:480,2004)。基于这些结果,通过序列对比分析发现了影响SCL活性的氨基酸的三个位置,并通过SDM方法使用SEQ ID NO:11~14的引物制备了在下面表1中所示的phaC1Ps6-19合酶突变体。
表1
SEQ ID NO:11:5-CTG ACC TTG CTG GTG ACC GTG CTT GAT ACCACC-3
SEQ ID NO:12:5-GGT GGT ATC AAG CAC GGT CAC CAG CAA GGTCAG-3
SEQ ID NO:13:5-CGA GCA GCG GGC ATA TC A TGA GCA TCC TGAACC CGC-3
SEQ ID NO:14:5-GCG GGT TCA GGA TGC TCA TGA TAT GCC CGCTGC TCG-3
SEQ ID NO:15:5-atc aac ctc atg acc gat gcg atg gcg ccg acc-3
SEQ ID NO:16:5-ggt cgg cgc cat cgc atc ggt cat gag gtt gat-3
用这些重组载体转化大肠杆菌XL-1Blue,并将其在PHB检测培养基(LB琼脂,葡萄糖20g/L,尼罗红0.5ug/ml)中进行培养。在用pPs619C1200-ReAB转化的大肠杆菌XL-1Blue和用pPs619C1300-ReAB转化的大肠杆菌XL-1Blue中都观察到了PHB的合成。也即是说,用phaARE和phaBRE的供应单体的酶从葡萄糖制备了3HB-辅酶A,并通过phaC1Ps6-19合酶SCL突变体(phaC1Ps6-19200和phaC1Ps6-19300)使用3HB-辅酶A作为底物来制备PHB。为了进行定量分析,在含有葡萄糖(20g/L)的LB培养基中在37℃培养转化后的重组大肠杆菌XL1-Blue4天。将培养后的重组大肠杆菌进行蔗糖休克(sucrose shock)并用尼罗红染色,将其用FACS(荧光激活细胞分选)法进行分析(图2)。
用包含野生型合酶的pPs619C1-ReAB载体转化的大肠杆菌XL1-Blue没有被尼罗红染色,而用pPs619C1200-ReAB或pPs619C1300-ReAB转化的大肠杆菌XL1-Blue因为在细胞中被尼罗红染色的PHB而显示出高荧光性。此外,通过收集培养的微生物经由离心,将其在80℃的干燥器中干燥48小时,然后进行气相色谱分析来评价在细胞中制备的PHB的含量。在用pPs619C1200-ReAB和pPs619C1300-ReAB转化的大肠杆菌XL1-Blue中的PHB的含量分别为29.7%(w/w)和43.1%(w/w),而在pPs619C1-ReAB的情况下没有检测到PHB。
<实施例3>能够表达源自假单胞菌6-19的PHA合酶和丙酰辅酶A转移酶的重组大肠杆菌的构建,以及使用其制备PLA或乳酸酯共聚物
在这个实施例中,使用源自丙酸梭菌(CP-PCT)的丙酰辅酶A转移酶用来供应乳酰辅酶A,一种合成PLA和乳酸酯共聚物所需的单体。如图3所示构建同时表达PHA合酶和CP-PCT的组成型表达体系的操纵子。CP-PCT对微生物具有毒性是众所周知的。也就是说,在由IPTG诱导的tac启动子或者T7启动子表达体系(该体系广泛用于重组蛋白的表达)中,在加入诱导物之后,所有微生物都很快死亡。因此,认为使用其中较弱表达但是根据微生物的生长连续表达的表达体系是适当的。将通过用丙酸梭菌的染色体DNA和SEQ ID NO:17和18的引物进行的PCR得到的片段用作cp-pct,并且为了容易克隆而通过SDM方法去除野生型CP-CPT的NdeI位点(图4)。
SEQ ID NO:17:5-ggaattcATGAGAAAGGTTCCCATTATTACCGCAGATGA
SEQ ID NO:18:5-gc tctaga tta gga ctt cat ttc ctt cag acc cat taa gcc ttc tg
另外,使用SEQ ID NO:19和20的引物进行重叠PCR来添加SbfI/NdeI识别位点。
SEQ ID NO:19:5-agg cct gca ggc gga taa caa ttt cac aca gg-3
SEQ ID NO:20:5-gcc cat atg tct aga tta gga ctt cat ttc c-3
用SbfI/NdeI切开含有phaC1Ps6-19合酶SCL突变体(phaC1Ps6-19300)的pPs619C1300-ReAB载体来去除源自富养罗尔斯通氏菌H16的供应单体的酶(phaARE和phaBRE),然后将该PCT-克隆的cp-pct基因插入到SbfI/NdeI位点来制备pPs619C1300-CPPCT重组载体(图4)。
另外,使用上述相同方法制备含有其它phaC1Ps6-19合酶SCL突变体(phaC1Ps6-19200)的pPs619C1200-CPPCT重组载体和含有野生型phaC1Ps6-19合酶的pPs619C1-CPPCT重组载体。为了证实是否cp-pct通过供应单体制备了聚合物,用pPs619C1200-CPPCT和pPs619C1300-CPPCT重组载体转化大肠杆菌XL-1Blue,并将其在PHB检测培养基(LB琼脂,葡萄糖20g/L,3HB 2g/L,尼罗红0.5μg/ml)中进行培养。该检测的结果是,在两者中都没有观察到PHB的合成。
使用pPs619C1300-CPPCT重组载体,在多种条件下进行玻瓶培养来制备PLA和乳酸酯共聚物。结果示于下面的表2中。
表2
菌株 | 介质 | 培养 | 底物 | PHB含量(%) | PLA含量(%) |
XL1-Blue | LB葡萄糖(10g/L) | 需氧 | 乳酸酯(5g/L) | - | 0.6 |
XL1-Blue | LB→MR葡萄糖(20g/L) | 2步骤(需氧→厌氧) | - | - | 1 |
Top10 | LB→MR葡萄糖(20g/L) | 2步骤(需氧→厌氧) | - | - | 0.3 |
XL1-Blue | LB→MR葡萄糖(20g/L) | 2步骤(需氧→厌氧) | 3HB(2g/L) | 3.19 | 2.75 |
Top10 | LB→MR葡萄糖(20g/L) | 2步骤(需氧→厌氧) | 3HB(2g/L) | 2.84 | 3.1 |
Top10 | LB葡萄糖(20g/L) | 需氧 | 3HB(0.05g/L)乳酸酯(5g/L) | 0.07 | 1.97 |
Top10 | LB葡萄糖(20g/L) | 需氧 | 3HB(0.2g/L)乳酸酯(5g/L) | 0.31 | 3.18 |
通过用MR培养基代替培养基然后进行厌氧培养进行所述2步骤培养来在细胞内产生乳酸酯。作为在多种条件下培养重组大肠杆菌的结果,基于干燥细胞的重量,所制备的PLA均聚物约为1%,且通过添加3HB作为底物制备的乳酸酯共聚物约为6%。在根据本发明的补料分批培养中使用的MR培养基的组成示于下面的表3中。
表3
组分 | 改进的R(MR)(/L) |
KH2PO4 | 6.67g |
(NH4)2HPO4 | 4g |
柠檬酸盐 | 0.8g |
MgSO4·H2O | 0.8g |
3HB | 0或0.2g |
葡萄糖 | 50g |
微量组分* | 5mL |
*微量组分(/L):FeSO4·H2O,10g;ZnSO4·H2O,2.25g;CuSO4·H2O,1g;MnSO4·H2O,0.5g;CaCl2·H2O,2g;Na2B4O7·H2O,0.23g;(NH4)6Mo7O24,0.1g;35%HCl,10mL。
<实施例4>通过重组大肠杆菌的补料分批培养制备P(3HB-共-LA)共聚物
将重组大肠杆菌(用pPs619C1300-CPPCT载体转化了的)在3mL含有20g/L的葡萄糖、100mg/L的氨苄西林等的LB培养基中,以200rpm的搅拌速度在37℃培养12小时。将该培养物接种至100mL的相同培养基中并在相同条件下培养6小时。使用最终培养基作为补料分批培养的种子培养物。使用MR培养基作为补料分批培养的起始培养基。通过将100mL的种子培养物接种到2.4L的MR培养基中开始进行补料分批培养。培养基的温度为37℃,且用14%的氨水溶液将pH调节至6.8-6.9,以及通过控制空气供应和搅拌速度将溶解氧保持为大于饱和空气的20%。此时,供应空气的速度是1vvm。当在起始培养基中含有的葡萄糖耗尽时,加入20g的葡萄糖和5g的3-羟基丁酸酯(3-HB),并同时将搅拌速度调节至200rpm并且将空气供应速度降低至0.1vvm以将需氧条件改变为厌氧条件。对于整个培养,供应葡萄糖5次。在这5次中,在第一次和第三次同时供应5g的3HB。在终止培养后,通过离心收集细胞并冷冻干燥。
为了精制所制备的聚合物,使用氯仿通过索氏提取器(Soxhlet extractor)从冷冻干燥的细胞中提取聚合物。然后,用旋转蒸发仪从溶解聚合物的氯仿溶液中去除大部分的氯仿,并向剩余溶液中加入甲醇来沉淀聚合物。过滤出沉淀的聚合物并在真空干燥器中干燥12小时。
另外,将一部分用离心得到的细胞在80℃的干燥器中干燥48小时,并用气相色谱分析在细胞中的P(3HB-共-LA)共聚物的含量。使用PLA均聚物和其中3HV的含量为12wt%的P(3HB-共-3HV)共聚物作为标准。
结果,基于干燥细胞的重量,在大肠杆菌中合成的P(3HB-共-LA)的含量为约10%,而在共聚物中PLA的含量为88mol%。
由这个对最终得到的聚合物进行气相色谱分析的实施例,证明所获得的聚合物为P(3HB-共-LA)共聚物且在共聚物中PLA的含量为88mol%。
<实施例5>通过重组大肠杆菌的补料分批培养制备PLA均聚物
按照实施例4的方法,进行用pPs619C1300-CPPCT载体转化的重组大肠杆菌的种子培养,并通过将100mL的种子培养物接种至2.4L的MR培养基中开始补料分批培养。培养基的温度为37℃,且用14%的氨水溶液将pH调节至6.8-6.9,以及通过控制空气供应和搅拌速度将溶解氧保持为大于饱和空气的20%。此时,供应空气的速度是1vvm。当在起始培养基中含有的葡萄糖耗尽时,加入20g的葡萄糖,同时将搅拌速度调节至200rpm并且将空气供应速度降低至0.1vvm以将需氧条件改变为厌氧条件。对于整个培养,供应葡萄糖5次。在终止培养后,通过离心收集细胞并冷冻干燥。根据实施例4的方法收集聚合物。
另外,将一部分用离心得到的细胞在80℃的干燥器中干燥48小时,并用气相色谱分析在细胞中的PLA的含量。使用4-羟基丁酸甲酯、PLA均聚物和其中3HV的含量为12wt%的P(3HB-共-3HV)共聚物作为标准。
结果,没有检测到3HB和4HB,并且只检测到PLA。基于干燥细胞的重量,在大肠杆菌中合成的PLA的含量为约10%。由最终得到的聚合物的气相色谱结果,检测到所获得的聚合物为聚乳酸酯聚合物,其中PLA的含量为99.1mol%。
<实施例6>多种突变体的制备
使用下面的引物根据在上述实施例2中公开的相同方法制备多种PHA合酶突变体。所得到的突变体示于表4、5、6和7中。
E130D
SEQ ID NO:15:5’-atc aac ctc atg acc gat gcg atg gcg ccg acc-3’
SEQ ID NO:16:5′-ggt cgg cgc cat cgc atc ggt cat gag gtt gat-3′
S325T
SEQ ID NO:11:5′-CTG ACC TTG CTG GTG ACC GTG CTT GAT ACCACC-3′
SEQ ID NO:12:5′-GGT GGT ATC AAG CAC GGT CAC CAG CAA GGTCAG-3′
S477R
SEQ ID NO:21:5′-gaattc gtg ctg tcg agc cgc ggg cat atc-3′
SEQ ID NO:22:5′-gat atg ccc gcg gct cga cag cac gaa ttc-3′
S477H
SEQ ID NO:23:5′-gaa ttc gtg ctg tcg agc cat ggg cat atc-3′
SEQ ID NO:24:5′-gat atg ccc atg gct cga cag cac gaa ttc-3′
S477F
SEQ ID NO:25:5′-gaa ttc gtg ctg tcg agc ttt ggg cat atc-3′
SEQ ID NO:26:5′-gat atg ccc aaa gct cga cag cac gaa ttc-3′
S477Y
SEQ ID NO:27:5′-gaa ttc gtg ctg tcg agc tat ggg cat atc-3′
SEQ ID NO:28:5′-gat atg ccc ata gct cga cag cac gaa ttc-3′
S477G
SEQ ID NO:29:5′-gaa ttc gtg ctg tcg agc ggc ggg cat atc-3′
SEQ ID NO:30:5′-gat atg ccc gcc gct cga cag cac gaa ttc-3′
Q481K
SEQ ID NO:31:5′-ggg cat atc aaa agc atc ctg aac ccg c-3′
SEQ ID NO:32:5′-gcg ggt tca gga tgc ttt tga tat gcc c-3′
Q481M
SEQ ID NO:33:5′-ggg cat atc atg agc atc ctg aac ccg c-3′
SEQ ID NO:34:5′-gcg ggt tca gga tgc tca tga tat gcc c-3′
Q481R
SEQ ID NO:35:5′-ggg cat atc cgc agc atc ctg aac ccg c-3′
SEQ ID NO:36:5′-gcg ggt tca gga tgc tgc gga tat gcc c-3′
表4
表5
表6
表7
<实施例7>使用多种突变体合成P(3HB-共-LA)
根据在上述实施例3中描述的相同方法,构建了能够表达源自假单胞菌6-19的PHA合酶突变体和丙酰辅酶A转移酶的重组大肠杆菌,并使用该重组大肠杆菌通过与在上述实施例4中描述的相同方法制备P(3HB-共-LA)。结果示于表8、9和10中。
表8
表9
表10
如表8、9和10中所示,本发明的PHA合酶突变体能够有效地使用乳酰辅酶A作为底物制备乳酸酯共聚物。
[工业适用性]
如上所示,源自假单胞菌6-19的本发明聚羟基链烷酸酯合酶突变体能够通过使用用常规聚羟基链烷酸酯合酶难以用作底物的乳酰辅酶A作为底物有效地制备乳酸酯聚合物和/或共聚物。
序列表
<110>LG化学株式会社
韩国科学技术院
<120>源自假单胞菌6-19的PHA合酶的突变体和使用该突变体制备乳酸酯均聚物或共聚物的方法
<130>F2007-0088PC(PCT07-069)
<150>KR10-2006-0116234
<151>2006-11-23
<160>36
<170>KopatentIn 1.71
<210>1
<211>35
<212>DNA
<213>人工序列
<220>
<223>引物
<400>1
gagagacaat caaatcatga gtaacaagag taacg 35
<210>2
<211>33
<212>DNA
<213>人工序列
<220>
<223>引物
<400>2
cactcatgca agcgtcaccg ttcgtgcacg tac 33
<210>3
<211>20
<212>DNA
<213>人工序列
<220>
<223>引物
<400>3
atgcccggag ccggttcgaa 20
<210>4
<211>35
<212>DNA
<213>人工序列
<220>
<223>引物
<400>4
cgttactctt gttactcatg atttgattgt ctctc 35
<210>5
<211>35
<212>DNA
<213>人工序列
<220>
<223>引物
<400>5
gagagacaat caaatcatga gtaacaagag taacg 35
<210>6
<211>33
<212>DNA
<213>人工序列
<220>
<223>引物
<400>6
cactcatgca agcgtcaccg ttcgtgcacg tac 33
<210>7
<211>33
<212>DNA
<213>人工序列
<220>
<223>引物
<400>7
gtacgtgcac gaacggtgac gcttgcatga gtg 33
<210>8
<211>20
<212>DNA
<213>人工序列
<220>
<223>引物
<400>8
aacgggaggg aacctgcagg 20
<210>9
<211>1677
<212>DNA
<213>假单胞菌6-19(KCTC 11027BP)
<400>9
atgagtaaca agagtaacga tgagttgaag tatcaagcct ctgaaaacac cttggggctt 60
aatcctgtcg ttgggctgcg tggaaaggat ctactggctt ctgctcgaat ggtgcttagg 120
caggccatca agcaaccggt gcacagcgtc aaacatgtcg cgcactttgg tcttgaactc 180
aagaacgtac tgctgggtaa atccgggctg caaccgacca gcgatgaccg tcgcttcgcc 240
gatccggcct ggagccagaa cccgctctat aaacgttatt tgcaaaccta cctggcgtgg 300
cgcaaggaac tccacgactg gatcgatgaa agtaacctcg cccccaagga tgtggcgcgt 360
gggcacttcg tgatcaacct catgaccgaa gcgatggcgc cgaccaacac cgcggccaac 420
ccggcggcag tcaaacgctt ttttgaaacc ggtggcaaaa gcctgctcga cggcctctcg 480
cacctggcca aggatctggt acacaacggc ggcatgccga gccaggtcaa catgggtgca 540
ttcgaggtcg gcaagagcct gggcgtgacc gaaggcgcgg tggtgtttcg caacgatgtg 600
ctggaactga tccagtacaa gccgaccacc gagcaggtat acgaacgccc gctgctggtg 660
gtgccgccgc agatcaacaa gttctacgtt ttcgacctga gcccggacaa gagcctggcg 720
cggttctgcc tgcgcaacaa cgtgcaaacg ttcatcgtca gctggcgaaa tcccaccaag 780
gaacagcgag agtggggcct gtcgacctac atcgaagccc tcaaggaagc ggttgacgtc 840
gttaccgcga tcaccggcag caaagacgtg aacatgctcg gggcctgctc cggcggcatc 900
acttgcactg cgctgctggg ccattacgcg gcgattggcg aaaacaaggt caacgccctg 960
accttgctgg tgagcgtgct tgataccacc ctcgacagcg acgtcgccct gttcgtcaat 1020
gaacagaccc ttgaagccgc caagcgccac tcgtaccagg ccggcgtact ggaaggccgc 1080
gacatggcga aggtcttcgc ctggatgcgc cccaacgatc tgatctggaa ctactgggtc 1140
aacaattacc tgctaggcaa cgaaccgccg gtgttcgaca tcctgttctg gaacaacgac 1200
accacacggt tgcccgcggc gttccacggc gacctgatcg aactgttcaa aaataaccca 1260
ctgattcgcc cgaatgcact ggaagtgtgc ggcaccccca tcgacctcaa gcaggtgacg 1320
gccgacatct tttccctggc cggcaccaac gaccacatca ccccgtggaa gtcctgctac 1380
aagtcggcgc aactgtttgg cggcaacgtt gaattcgtgc tgtcgagcag cgggcatatc 1440
cagagcatcc tgaacccgcc gggcaatccg aaatcgcgct acatgaccag caccgaagtg 1500
gcggaaaatg ccgatgaatg gcaagcgaat gccaccaagc atacagattc ctggtggctg 1560
cactggcagg cctggcaggc ccaacgctcg ggcgagctga aaaagtcccc gacaaaactg 1620
ggcagcaagg cgtatccggc aggtgaagcg gcgccaggca cgtacgtgca cgaacgg 1677
<210>10
<211>559
<212>PRT
<213>假单胞菌6-19(KCTC 11027BP)
<400>10
Met Ser Asn Lys Ser Asn Asp Glu Leu Lys Tyr Gln Ala Ser Glu Asn
1 5 10 15
Thr Leu Gly Leu Asn Pro Val Val Gly Leu Arg Gly Lys Asp Leu Leu
20 25 30
Ala Ser Ala Arg Met Val Leu Arg Gln Ala Ile Lys Gln Pro Val His
35 40 45
Ser Val Lys His Val Ala His Phe Gly Leu Glu Leu Lys Asn Val Leu
50 55 60
Leu Gly Lys Ser Gly Leu Gln Pro Thr Ser Asp Asp Arg Arg Phe Ala
65 70 75 80
Asp Pro Ala Trp Ser Gln Asn Pro Leu Tyr Lys Arg Tyr Leu Gln Thr
85 90 95
Tyr Leu Ala Trp Arg Lys Glu Leu His Asp Trp Ile Asp Glu Ser Asn
100 105 110
Leu Ala Pro Lys Asp Val Ala Arg Gly His Phe Val Ile Asn Leu Met
115 120 125
Thr Glu Ala Met Ala Pro Thr Asn Thr Ala Ala Asn Pro Ala Ala Val
130 135 140
Lys Arg Phe Phe Glu Thr Gly Gly Lys Ser Leu Leu Asp Gly Leu Ser
145 150 155 160
His Leu Ala Lys Asp Leu Val His Asn Gly Gly Met Pro Ser Gln Val
165 170 175
Asn Met Gly Ala Phe Glu Val Gly Lys Ser Leu Gly Val Thr Glu Gly
180 185 190
Ala Val Val Phe Arg Asn Asp Val Leu Glu Leu Ile Gln Tyr Lys Pro
195 200 205
Thr Thr Glu Gln Val Tyr Glu Arg Pro Leu Leu Val Val Pro Pro Gln
210 215 220
Ile Asn Lys Phe Tyr Val Phe Asp Leu Ser Pro Asp Lys Ser Leu Ala
225 230 235 240
Arg Phe Cys Leu Arg Asn Asn Val Gln Thr Phe Ile Val Ser Trp Arg
245 250 255
Asn Pro Thr Lys Glu Gln Arg Glu Trp Gly Leu Ser Thr Tyr Ile Glu
260 265 270
Ala Leu Lys Glu Ala Val Asp Val Val Thr Ala Ile Thr Gly Ser Lys
275 280 285
Asp Val Asn Met Leu Gly Ala Cys Ser Gly Gly Ile Thr Cys Thr Ala
290 295 300
Leu Leu Gly His Tyr Ala Ala Ile Gly Glu Asn Lys Val Asn Ala Leu
305 310 315 320
Thr Leu Leu Val Ser Val Leu Asp Thr Thr Leu Asp Ser Asp Val Ala
325 330 335
Leu Phe Val Asn Glu Gln Thr Leu Glu Ala Ala Lys Arg His Ser Tyr
340 345 350
Gln Ala Gly Val Leu Glu Gly Arg Asp Met Ala Lys Val Phe Ala Trp
355 360 365
Met Arg Pro Asn Asp Leu Ile Trp Asn Tyr Trp Val Asn Asn Tyr Leu
370 375 380
Leu Gly Asn Glu Pro Pro Val Phe Asp Ile Leu Phe Trp Asn Asn Asp
385 390 395 400
Thr Thr Arg Leu Pro Ala Ala Phe His Gly Asp Leu Ile Glu Leu Phe
405 410 415
Lys Asn Asn Pro Leu Ile Arg Pro Asn Ala Leu Glu Val Cys Gly Thr
420 425 430
Pro Ile Asp Leu Lys Gln Val Thr Ala Asp Ile Phe Ser Leu Ala Gly
435 440 445
Thr Asn Asp His Ile Thr Pro Trp Lys Ser Cys Tyr Lys Ser Ala Gln
450 455 460
Leu Phe Gly Gly Asn Val Glu Phe Val Leu Ser Ser Ser Gly His Ile
465 470 475 480
Gln Ser Ile Leu Asn Pro Pro Gly Asn Pro Lys Ser Arg Tyr Met Thr
485 490 495
Ser Thr Glu Val Ala Glu Asn Ala Asp Glu Trp Gln Ala Asn Ala Thr
500 505 510
Lys His Thr Asp Ser Trp Trp Leu His Trp Gln Ala Trp Gln Ala Gln
515 520 525
Arg Ser Gly Glu Leu Lys Lys Ser Pro Thr Lys Leu Gly Ser Lys Ala
530 535 540
Tyr Pro Ala Gly Glu Ala Ala Pro Gly Thr Tyr Val His Glu Arg
545 550 555
<210>11
<211>33
<212>DNA
<213>人工序列
<220>
<223>引物
<400>11
ctgaccttgc tggtgaccgt gcttgatacc acc 33
<210>12
<211>33
<212>DNA
<213>人工序列
<220>
<223>引物
<400>12
ggtggtatca agcacggtca ccagcaaggt cag 33
<210>13
<211>36
<212>DNA
<213>人工序列
<220>
<223>引物
<400>13
cgagcagcgg gcatatcatg agcatcctga acccgc 36
<210>14
<211>36
<212>DNA
<213>人工序列
<220>
<223>引物
<400>14
gcgggttcag gatgctcatg atatgcccgc tgctcg 36
<210>15
<211>33
<212>DNA
<213>人工序列
<220>
<223>引物
<400>15
atcaacctca tgaccgatgc gatggcgccg acc 33
<210>16
<211>33
<212>DNA
<213>人工序列
<220>
<223>引物
<400>16
ggtcggcgcc atcgcatcgg tcatgaggtt gat 33
<210>17
<211>39
<212>DNA
<213>人工序列
<220>
<223>引物
<400>17
ggaattcatg agaaaggttc ccattattac cgcagatga 39
<210>18
<211>46
<212>DNA
<213>人工序列
<220>
<223>引物
<400>18
gctctagatt aggacttcat ttccttcaga cccattaagc cttctg 46
<210>19
<211>32
<212>DNA
<213>人工序列
<220>
<223>引物
<400>19
aggcctgcag gcggataaca atttcacaca gg 32
<210>20
<211>31
<212>DNA
<213>人工序列
<220>
<223>引物
<400>20
gcccatatgt ctagattagg acttcatttc c 31
<210>21
<211>30
<212>DNA
<213>人工序列
<220>
<223>引物
<400>21
gaattcgtgc tgtcgagccg cgggcatatc 30
<210>22
<211>30
<212>DNA
<213>人工序列
<220>
<223>引物
<400>22
gatatgcccg cggctcgaca gcacgaattc 30
<210>23
<211>30
<212>DNA
<213>人工序列
<220>
<223>引物
<400>23
gaattcgtgc tgtcgagcca tgggcatatc 30
<210>24
<211>30
<212>DNA
<213>人工序列
<220>
<223>引物
<400>24
gatatgccca tggctcgaca gcacgaattc 30
<210>25
<211>30
<212>DNA
<213>人工序列
<220>
<223>引物
<400>25
gaattcgtgc tgtcgagctt tgggcatatc 30
<210>26
<211>30
<212>DNA
<213>人工序列
<220>
<223>引物
<400>26
gatatgccca aagctcgaca gcacgaattc 30
<210>27
<211>30
<212>DNA
<213>人工序列
<220>
<223>引物
<400>27
gaattcgtgc tgtcgagcta tgggcatatc 30
<210>28
<211>30
<212>DNA
<213>人工序列
<220>
<223>引物
<400>28
gatatgccca tagctcgaca gcacgaattc 30
<210>29
<211>30
<212>DNA
<213>人工序列
<220>
<223>引物
<400>29
gaattcgtgc tgtcgagcgg cgggcatatc 30
<210>30
<211>30
<212>DNA
<213>人工序列
<220>
<223>引物
<400>30
gatatgcccg ccgctcgaca gcacgaattc 30
<210>31
<211>28
<212>DNA
<213>人工序列
<220>
<223>引物
<400>31
gggcatatca aaagcatcct gaacccgc 28
<210>32
<211>28
<212>DNA
<213>人工序列
<220>
<223>引物
<400>32
gcgggttcag gatgcttttg atatgccc 28
<210>33
<211>28
<212>DNA
<213>人工序列
<220>
<223>引物
<400>33
gggcatatca tgagcatcct gaacccgc 28
<210>34
<211>28
<212>DNA
<213>人工序列
<220>
<223>引物
<400>34
gcgggttcag gatgctcatg atatgccc 28
<210>35
<211>28
<212>DNA
<213>人工序列
<220>
<223>引物
<400>35
gggcatatcc gcagcatcct gaacccgc 28
<210>36
<211>28
<212>DNA
<213>人工序列
<220>
<223>引物
<400>36
gcgggttcag gatgctgcgg atatgccc 28
Claims (11)
1、一种聚羟基链烷酸酯合酶突变体,其使用乳酰辅酶A作为底物生产乳酸酯聚合物或者乳酸酯共聚物并且具有SEQ.ID No:10的氨基酸序列,其中,在SEQ.ID No:10的氨基酸序列的第481位的谷氨酰胺发生突变。
2、如权利要求1所述的聚羟基链烷酸酯合酶突变体,其中,选自在第130位的谷氨酸、在第325位的丝氨酸和在第477位的丝氨酸中的至少一个氨基酸进一步发生突变。
3、如权利要求2所述的聚羟基链烷酸酯合酶突变体,其中,所述氨基酸序列具有如下任何一种突变:
a)S325T和Q481M;
b)E130D和Q481K;
c)S325T和Q481K;
d)E130D和Q481M;
e)E130D和Q481R;
f)E130D、S325T和Q481M;
g)E130D、S325T和Q481K;
h)E130D、S477R和Q481K;
i)E130D、S477R和Q481M;
j)E130D、S477R和Q481R;
k)E130D、S477H和Q481K;
l)E130D、S477H和Q481M;
m)E130D、S477H和Q481R;
n)E130D、S477F和Q481K;
o)E130D、S477F和Q481M;
p)E130D、S477F和Q481R;
q)E130D、S477Y和Q481K;
r)E130D、S477Y和Q481M;
s)E130D、S477Y和Q481R;
t)E130D、S325T、S477R和Q481M;
u)E130D、S325T、S477R和Q481K;
v)E130D、S325T、S477F和Q481M;
w)E130D、S325T、S477G和Q481M;或
x)E130D、S325T、S477F和Q481K。
4、一种编码权利要求1所述的聚羟基链烷酸酯合酶突变体的基因。
5、一种重组载体,其含有用于合成乳酸酯聚合物或共聚物的权利要求4所述的基因。
6、如权利要求5所述的重组载体,其进一步包含编码丙酰辅酶A转移酶(pct)的基因。
7、一种用权利要求5所述的重组载体转化的细胞或者植物。
8、一种使用权利要求6所述的重组载体转化而获得的细胞或者植物,其中,原始细胞或者植物没有编码丙酰辅酶A转移酶的基因。
9、一种制备乳酸酯聚合物或乳酸酯共聚物的方法,其中,该方法包括培养权利要求7或8所述的细胞或者植物。
10、如权利要求9所述的方法,其中,所述培养是在含有羟基链烷酸酯的培养基中进行的,并且所述共聚物为含有羟基链烷酸酯单体单元和乳酸酯单体单元的共聚物。
11、如权利要求10所述的方法,其中,所述羟基链烷酸酯是选自下列化合物中的至少一种:3-羟基丁酸酯、3-羟基戊酸酯、4-羟基丁酸酯、中链长(C6~14)的(D)-3-羟基羧酸、3-羟基丙酸、3-羟基己酸、3-羟基庚酸、3-羟基辛酸、3-羟基壬酸、3-羟基癸酸、3-羟基十一烷酸、3-羟基十二烷酸、3-羟基十四烷酸、3-羟基十六烷酸、4-羟基戊酸、4-羟基己酸、4-羟基庚酸、4-羟基辛酸、4-羟基癸酸、5-羟基戊酸、5-羟基己酸、6-羟基十二烷酸、3-羟基-4-戊烯酸、3-羟基-4-反-己烯酸、3-羟基-4-顺-己烯酸、3-羟基-5-己烯酸、3-羟基-6-反-辛烯酸、3-羟基-6-顺-辛烯酸、3-羟基-7-辛烯酸、3-羟基-8-壬烯酸、3-羟基-9-癸烯酸、3-羟基-5-顺-十二碳烯酸、3-羟基-6-顺-十二碳烯酸、3-羟基-5-顺-十四碳烯酸、3-羟基-7-顺-十四碳烯酸、3-羟基-5,8-顺-顺-十四碳烯酸、3-羟基-4-甲基戊酸、3-羟基-4-甲基己酸、3-羟基-5-甲基己酸、3-羟基-6-甲基庚酸、3-羟基-4-甲基辛酸、3-羟基-5-甲基辛酸、3-羟基-6-甲基辛酸、3-羟基-7-甲基辛酸、3-羟基-6-甲基壬酸、3-羟基-7-甲基壬酸、3-羟基-8-甲基壬酸、3-羟基-7-甲基癸酸、3-羟基-9-甲基癸酸、3-羟基-7-甲基-6-辛烯酸、苹果酸、3-羟基琥珀酸甲酯、3-羟基己二酸甲酯、3-羟基辛二酸甲酯、3-羟基壬二酸甲酯、3-羟基癸二酸甲酯、3-羟基辛二酸乙酯、3-羟基癸二酸乙酯、3-羟基庚二酸丙酯、3-羟基癸二酸苄酯、3-羟基-8-乙酰氧基辛酸、3-羟基-9-乙酰氧基壬酸、苯氧基-3-羟基丁酸、苯氧基-3-羟基戊酸、苯氧基-3-羟基庚酸、苯氧基-3-羟基辛酸、对氰基苯氧基-3-羟基丁酸、对氰基苯氧基-3-羟基戊酸、对氰基苯氧基-3-羟基己酸、对硝基苯氧基-3-羟基己酸、3-羟基-5-苯基戊酸、3-羟基-5-环己基丁酸、3,12-二羟基十二烷酸、3,8-二羟基-5-顺-十四碳烯酸、3-羟基-4,5-环氧癸酸、3-羟基-6,7-环氧十二烷酸、3-羟基-8,9-环氧-5,6-顺-十四烷酸、7-氰基-3-羟基庚酸、9-氰基-3-羟基壬酸、3-羟基-7-氟庚酸、3-羟基-9-氟壬酸、3-羟基-6-氯己酸、3-羟基-8-氯辛酸、3-羟基-6-溴己酸、3-羟基-8-溴辛酸、3-羟基-11-溴十一烷酸、3-羟基-2-丁烯酸、6-羟基-3-十二碳烯酸、3-羟基-2-甲基丁酸、3-羟基-2-甲基戊酸和3-羟基-2,6-二甲基-5-庚烯酸。
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CN201610252102.1A Pending CN105754966A (zh) | 2006-11-23 | 2007-11-21 | 源自假单胞菌6-19的pha合酶的突变体和使用该突变体制备乳酸酯均聚物或共聚物的方法 |
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EP (1) | EP2089522B1 (zh) |
JP (1) | JP5550066B2 (zh) |
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CN (2) | CN101541967A (zh) |
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JP3990880B2 (ja) * | 2001-07-10 | 2007-10-17 | キヤノン株式会社 | ポリヒドロキシアルカノエート被覆リポソームの製造方法 |
KR100979694B1 (ko) | 2005-05-24 | 2010-09-02 | 한국과학기술원 | 폴리락테이트 또는 그 공중합체 생성능을 가지는 세포 또는식물 및 이를 이용한 폴리락테이트 또는 그 공중합체의제조방법 |
AU2007322529B2 (en) * | 2006-11-21 | 2013-07-04 | Korea Advanced Institute Of Science And Technology | Copolymer containing 3-hydroxyalkanoate unit and lactate unit, and its manufacturing method |
EP2084209B1 (en) * | 2006-11-21 | 2021-06-02 | LG Chem, Ltd. | Copolymer comprising 4-hydroxybutyrate unit and lactate unit and its manufacturing method |
WO2009031762A2 (en) | 2007-09-07 | 2009-03-12 | Lg Chem, Ltd. | Recombinant microorganism capable of producing polylactate or polylactate copolymer from sucrose and method for producing polylactate or polylactate copolymer from sucrose using the same |
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2007
- 2007-11-21 BR BRPI0719342-4A patent/BRPI0719342A2/pt not_active Application Discontinuation
- 2007-11-21 EP EP07834164.1A patent/EP2089522B1/en active Active
- 2007-11-21 CN CNA2007800433773A patent/CN101541967A/zh active Pending
- 2007-11-21 KR KR1020070119435A patent/KR100957777B1/ko active IP Right Grant
- 2007-11-21 AU AU2007322532A patent/AU2007322532B2/en active Active
- 2007-11-21 CN CN201610252102.1A patent/CN105754966A/zh active Pending
- 2007-11-21 JP JP2009538328A patent/JP5550066B2/ja active Active
- 2007-11-21 WO PCT/KR2007/005858 patent/WO2008062999A1/en active Application Filing
- 2007-11-21 US US12/312,676 patent/US8541652B2/en active Active
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103173472A (zh) * | 2013-03-28 | 2013-06-26 | 中国科学院微生物研究所 | 经过密码子优化的点突变丙酰辅酶a转移酶基因及其应用 |
Also Published As
Publication number | Publication date |
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BRPI0719342A2 (pt) | 2014-02-04 |
US8541652B2 (en) | 2013-09-24 |
CN105754966A (zh) | 2016-07-13 |
EP2089522B1 (en) | 2017-11-01 |
EP2089522A1 (en) | 2009-08-19 |
KR20080047279A (ko) | 2008-05-28 |
JP5550066B2 (ja) | 2014-07-16 |
US20100050298A1 (en) | 2010-02-25 |
EP2089522A4 (en) | 2010-02-17 |
WO2008062999A1 (en) | 2008-05-29 |
KR100957777B1 (ko) | 2010-05-12 |
AU2007322532A1 (en) | 2008-05-29 |
JP2010510775A (ja) | 2010-04-08 |
AU2007322532B2 (en) | 2012-02-02 |
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