CN105505892B - 2,4-二羟基丁酸的生产方法 - Google Patents
2,4-二羟基丁酸的生产方法 Download PDFInfo
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- CN105505892B CN105505892B CN201610007101.0A CN201610007101A CN105505892B CN 105505892 B CN105505892 B CN 105505892B CN 201610007101 A CN201610007101 A CN 201610007101A CN 105505892 B CN105505892 B CN 105505892B
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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Abstract
本发明涉及通过合成途径生产2,4‑二羟基丁酸(2,4‑DHB)的方法,包括使用苹果酸激酶将苹果酸转化为4‑磷酸‑苹果酸,使用苹果酸半醛脱氢酶将所述4‑磷酸苹果酸转化为苹果酸‑4‑半醛,并使用DHB脱氢酶将所述苹果酸‑4‑半醛转化为2,4‑DHB。
Description
本发明涉及通过使用合成途径从苹果酸生产2,4-二羟基丁酸的新方法,合成途径包含分别具有苹果酸激酶,苹果酸半醛脱氢酶,和2,4-二羟基丁酸脱氢酶活性的酶。
本申请中引用的羧酸在其盐(如2,4-二羟基丁酸盐)或酸(如2,4-二羟基丁酸)形式下是等同命名的。
2,4-二羟基丁酸(等同2,4-DHB或DHB)是有相当经济效益的化合物。通过调整适合的pH,可以在水介质中容易地将DHB转化为α-羟基-γ-丁内酯。α-羟基-γ-丁内酯是生产在动物营养中有很大市场的甲硫氨酸物2-羟基-4-(甲硫基)-丁酸(HMTB)的重要前体(Decket al.,2008)。目前,α-羟基-γ-丁内酯通过包含γ-丁内酯在α位置的卤化,并随后在碱性介质中以羟基替代卤素原子的多阶段反应衍生自γ-丁内酯(Deck et al.,2008)。
由于油价的增长,从可再生资源生产DHB的需要升高。微生物能够将源自生物质的原料,如糖或有机酸,转化为大量各种不同的化学化合物(Werpy&Petersen,2004)。随着生物化学和基因组学信息量的增长,可以修饰微生物以使其能以高产量和产率过量生产天然发生的代谢中间体(Bailey,1991)。生产微生物的优化经常需要合理的代谢网络工程,这确保其中目标代谢物的生物合成所需的酶过量表达,并减轻产物的反馈抑制。另外的可能性是使用能催化目标代谢物生产的新酶***。
代谢工程方法和酶催化需要生化和产生目标代谢物的代谢途径调节方面的详细知识。在生产DHB的情况下,这些信息是无法获得的。仅有少量研究报道了琥珀酸半醛脱氢酶缺乏症患者中的DHB发生(Shinka et al.2002),然而没有确定DHB生产中涉及的酶反应。因此,DHB的发酵或酶生产需要(i)确定能将易得到的前体转化为DHB的热力学可行途径,(ii)确定或构建途径中能够催化各反应步骤的酶和(iii)在适合的生物生产中,途径酶的功能表达。
本发明目的是为满足这些需求。
因此,本发明的一个目的是生产2,4-DHB的方法,包括使用苹果酸激酶将苹果酸转化为4-磷酸-苹果酸的第一步骤,使用苹果酸半醛脱氢酶将4-磷酸-苹果酸转化为苹果酸-4-半醛的第二步骤,使用DHB脱氢酶将苹果酸-4-半醛转化为2,4-DHB的第三步骤。
在第一个反应中(见图1(i)),通过具有苹果酸激酶活性的酶的作用,苹果酸(1)被转化为4-磷酸-苹果酸(2)(A)。在第二个反应(B)中,通过具有苹果半醛脱氢酶活性的酶的作用,4-磷酸-苹果酸被转化为苹果酸-4-半醛(3)。更准确地,反应(B)是由在生物合成途径意义中具有脱磷酸4-磷酸-苹果酸还原酶活性的酶来催化。在第三反应(C)中,通过具有DHB脱氢酶活性的酶的作用,苹果酸-4-半醛被转化为DHB。更准确地,反应(C)是由在生物合成途径意义中具有苹果酸-4-半醛还原酶活性的酶来催化。
目前为止在活细胞中既没有描述过也没有确认过上面引用的酶和中间产物。这样苹果酸激酶,苹果酸半醛脱氢酶,DHB脱氢酶和4-磷酸-苹果酸是本发明进一步的目的。
在本发明另外一个方面中,生产2,4-DHB方法的第一步骤包括苹果酸激酶,其特征在于其能将苹果酸转化为4-磷酸-苹果酸。所述酶可以通过酶的至少一个突变获得,所述突变改进了突变酶对苹果酸的活性和/或底物亲和力。
在本发明中,表述“改进活性和/或底物亲和力”意思是突变前的酶,要么是
-不能使用底物(苹果酸,4-磷酸-苹果酸或苹果酸-4-半醛),和/或
-以低于最大特定速率的至少3倍的速率(at a maximum specific rate atleast three times lower)来合成反应产物(4-磷酸-苹果酸或苹果酸-4-半醛或DHB),和/或
-对苹果酸,4-磷酸-苹果酸,或苹果酸-4-半醛具有低于至少3倍(at least threetimes lower)的亲和力,和/或
-对天然底物(天冬氨酸,4-磷酸-天冬氨酸,天冬氨酸-4-半醛)具有高于至少3倍(at least 3times higher)的亲和力。
在其另外一个方面,本发明涉及苹果酸激酶将苹果酸转化为4-磷酸-苹果酸的用途。
苹果酸激酶的活性可以通过实施例1中描述的酶测试来测量(见“酶测定”)。
根据本发明另外一个方面,苹果酸激酶可以通过突变天冬氨酸激酶获得。
图2显示了不同生物来源天冬氨酸激酶的氨基酸序列比对。所有氨基酸位置的参照是基于大肠杆菌LysC基因编码的天冬氨酸激酶氨基酸序列(SEQ ID No.4所示)来制作。通过与下列酶如图2中所示的简单序列比对,本领域技术人员可以容易地发现来自不同生物的其他天冬氨酸激酶中对应保守区域的相对位置:
-AKIII-大肠杆菌天冬氨酸激酶III(SEQ ID No.4),
-AKI(SEQ ID No.87)大肠杆菌天冬氨酸激酶I,
-AKII(SEQ ID No.88)-大肠杆菌天冬氨酸激酶II,
-MJ-詹氏甲烷球菌(Methanococcus jannaschii)(SEQ ID No.89),
-TT-嗜热栖热菌(Thermus thermophilus)(SEQ ID No.90),
-CG-谷氨酸棒杆菌(Corynebacterium glutamicum)(SEQ ID No.91),
-AT-拟南芥(Arabidopsis thaliana)(SEQ ID No.92),
-SC-酿酒酵母(Saccharomyces cerevisiae)(SEQ ID No.93)。
所述比对可以以ClustalW2软件完成。例如,SEQ ID No.4所示天冬氨酸激酶的E119残基对应拟南芥天冬氨酸激酶的E207残基(SEQ ID No.50)或对应于酿酒酵母天冬氨酸激酶的E147残基(SEQ ID No.51)。
与野生型酶相比,根据本发明的突变天冬氨酸激酶包括至少一个在至少一个下列位置的突变:S39,T45,V115,E119,F184和/或S201,其中所述位置天然发生的氨基酸被其他19个天然存在的蛋白氨基酸的任一个取代,即被丙氨酸,精氨酸,天冬酰胺,天冬氨酸,半胱氨酸,谷氨酸,谷氨酰胺,甘氨酸,组氨酸,异亮氨酸,亮氨酸,赖氨酸,甲硫氨酸,苯丙氨酸,脯氨酸,丝氨酸,苏氨酸,色氨酸,酪氨酸,或缬氨酸任一所取代。
在非排他的例子中,使用大肠杆菌天冬氨酸激酶Lys C作为模板验证了通过定点突变构建苹果酸激酶。根据本发明的一个方面,通过以天冬酰胺,谷氨酰胺,半胱氨酸,脯氨酸,丝氨酸,苏氨酸,缬氨酸或甘氨酸任一来替代119位上的谷氨酸,LysC的底物特异性向苹果酸改变。
在本发明进一步的方面中,苹果酸激酶由SEQ ID No.9,更具体的由SEQ IDNo.12,SEQ ID No.14,SEQ ID No.16,SEQ ID No.18,SEQ ID No.20,SEQ ID No.22,SEQ IDNo.24或SEQ ID No.26表示。
天冬氨酸激酶通常受甲硫氨酸,苏氨酸或赖氨酸的抑制。因此,通过天冬氨酸激酶随机或定点突变来构建的苹果酸激酶也会受所述氨基酸的抑制。在本发明进一步的方面中,苹果酸激酶受甲硫氨酸,苏氨酸或赖氨酸的抑制被苹果酸激酶的突变减轻。
在本发明具体的方面中,至少一个下列氨基酸E250,M318,S321,V339,S338,F324,L325,V339,S345,E346,D340,T344和/或T352的突变使得上面描述的突变的LysC(苹果酸激酶)对赖氨酸抑制不敏感(见实施例3)。
本发明也包括这样修饰过的酶,和更具体的由SEQ ID No.39,SEQ ID No.41,SEQID No.43或SEQ ID No.45所示的那些酶。
在再进一步的方面中,根据本发明的生产2,4-DHB方法的第二步骤包括苹果酸半醛脱氢酶,其特征在于其能将4-磷酸-苹果酸转化为苹果酸-4-半醛,所述酶具有在生物合成途径意义中的脱磷酸4-磷酸-苹果酸还原酶活性。
苹果酸半醛脱氢酶活性可以通过实施例4中描述的酶测试来测量(见“酶测定”)。
该酶可以通过酶的至少一个突变得到,所述突变改进了突变酶对4-磷酸-苹果酸的活性和/或底物亲和力。
根据另外一个方面,可以通过突变具有报导的半醛脱氢酶活性,更具体的具有反应还原意义中的脱磷酸活性,更具体的作用于由3,4,或5个碳原子组成的有机分子的酶来获得本发明的苹果酸半醛脱氢酶。在本发明具体的方面中,所述苹果酸半醛脱氢酶是通过突变天冬氨酸半醛脱氢酶来获得。
天冬氨酸半醛脱氢酶,大肠杆菌的Asd和酿酒酵母的Hom2天然地显现对4-磷酸-苹果酸2的脱氢酶活性。
根据本发明另外一个方面,可以通过突变天冬氨酸半醛脱氢酶来改进苹果酸半醛脱氢酶。
图3显示了不同生物来源天冬氨酸半醛脱氢酶的氨基酸序列比对。所有氨基酸位置的参照是基于大肠杆菌Asd基因编码的天冬氨酸脱氢酶(如SEQ ID No.20所示)来制作。通过与下列酶如图4中所示的简单序列比对,本领域技术人员可以容易地发现来自不同生物的其他天冬氨酸半醛酶中对应保守区域的相对位置:
-EC-大肠杆菌(SEQ ID No.49),
-MJ-詹氏甲烷球菌(SEQ ID No.94),
-TT-嗜热栖热菌(SEQ ID No.95),
-BS-枯草芽孢杆菌(SEQ ID No.96)
-CG-谷氨酸棒杆菌(SEQ ID No.97),
-AT-拟南芥(SEQ ID No.98),
-SC-酿酒酵母(SEQ ID No.99)。
所述比对可以以ClustalW2软件容易地完成。
具有改进的苹果酸半醛脱氢酶活性的酶的构建可以如下完成。
根据本发明的苹果酸半醛脱氢酶在具体方面对应天冬氨酸半醛脱氢酶,与野生型酶相比,其包括至少一个在至少一个T136,Q162,I230,E241和/或H274位置的突变,其中所述位置天然发生的氨基酸被其他19个天然存在的蛋白氨基酸的任一个取代,即被丙氨酸,精氨酸,天冬酰胺,天冬氨酸,半胱氨酸,谷氨酸,谷氨酰胺,甘氨酸,组氨酸,异亮氨酸,亮氨酸,赖氨酸,甲硫氨酸,苯丙氨酸,脯氨酸,丝氨酸,苏氨酸,色氨酸,酪氨酸,或缬氨酸任一所取代。
如实施例5所验证,大肠杆菌asd的定向诱变可以改进突变的酶对4-磷酸-苹果酸的活性和底物亲和力,同时减少酶对其天然底物4-磷酸-天冬氨酸的偏好。
为了改进Asd对4-磷酸-苹果酸的活性,并根据本发明的一个方面,通过定向诱变,E241被谷氨酰胺,丙氨酸,半胱氨酸,甘氨酸,组氨酸,异亮氨酸或甲硫氨酸残基所替代(实施例5)。
在本发明进一步的方面中,苹果酸半醛脱氢酶由SEQ ID No.68,和更具体的由SEQID No.54,SEQ ID No.56,SEQ ID No.58,SEQ ID No.60,SEQ ID No.62,SEQ ID No.64或SEQ ID No.66表示。
在其另外一个方面中,本发明涉及苹果酸半醛脱氢酶将4-磷酸-苹果酸转化为苹果酸-4-半醛的用途。
在另外一个方面中,根据本发明的生产2,4-DHB方法的第三步骤包括DHB脱氢酶,其特征在于其将苹果酸-4-半醛转化为2,4-DHB,所述酶具有在生物合成途径意义中的苹果酸-4-半醛还原酶活性。
已经潜在具有DHB脱氢酶活性的候选DHB脱氢酶可以从作用于C3,C4,或C5化合物的β-羟酸脱氢酶类中选择。
根据本发明再进一步的方面,所述DHB脱氢酶可以是与β-羟酸脱氢酶,如羟基丙二酸半醛还原酶,琥珀酸半醛还原酶,丙二酸半醛还原酶,甲基丁醛还原酶,锌型醇脱氢酶,L-苏氨酸-3-脱氢酶,或高丝氨酸还原酶,在结构和机制上相关的酶。
本发明也涉及甲基丁醛还原酶或琥珀酸半醛还原酶将苹果酸-4-半醛转化为2,4-DHB的用途。在具体实施方式中,所述甲基丁醛还原酶由SEQ ID No.74所示,而所述琥珀酸半醛还原酶由SEQ ID No.76所示。DHB还原酶的活性可以通过实施例6中描述的酶测试来测量(见“酶测定”)。
可以通过酶的至少一个突变来增加DHB脱氢酶对苹果酸-4-半醛的亲和力,所述突变增加突变的酶对苹果酸-4-半醛的活性和/或底物亲和力,和/或通过至少因子2(factor2)降低对其天然底物的活性或亲和力。
依照本发明的DHB脱氢酶在具体方面对应嗜热金属球菌琥珀酸半醛还原酶(SEQID No.76),与野生型酶相比,其包括至少一个在至少一个S40,N43,H39,T49,F85,Q108,L281和N305位置的突变,其中所述位置天然发生的氨基酸被其他19个天然存在的蛋白氨基酸的任一个取代,即被丙氨酸,精氨酸,天冬酰胺,天冬氨酸,半胱氨酸,谷氨酸,谷氨酰胺,甘氨酸,组氨酸,异亮氨酸,亮氨酸,赖氨酸,甲硫氨酸,苯丙氨酸,脯氨酸,丝氨酸,苏氨酸,色氨酸,酪氨酸,或缬氨酸任一所取代。
如非排他性实施例中所验证,如SEQ ID No.81所示,通过以定向诱变导入的双突变H39R,N43H增加了嗜热金属球菌琥珀酸半醛还原酶对(L)-苹果酸-4-半醛的亲和力。简单的突变体H39R(SEQ ID No.225)和N43H(SEQ ID No.227)可包括在本发明中(实施例7)。
DHB脱氢酶可以被用于将苹果酸-4-半醛转化为2,4-DHB,其组成了本发明进一步的方面。
可以调整基因的核酸序列以适应宿主生物所使用的密码子,从而增加异种表达的蛋白的生产,其组成了本发明进一步的方面。
编码嗜热金属球菌琥珀酸半醛还原酶H39R N43H的合成基因的合成是本发明进一步的方面,如SEQ ID No.228所示的在大肠杆菌中表达所述酶优化了合成基因的核苷酸序列。
在再进一步的方面中,本发明也涉及核酸,更具体的涉及编码如上所述的苹果酸激酶的分离的核酸序列。
在另一个方面中,所述核酸由SEQ ID No.13,SEQ ID No.15,SEQ ID No.17,SEQID No.19,SEQ ID No.21,SEQ ID No.23,SEQ ID No.25,SEQ ID No.27,SEQ ID No.38,SEQID No.40,SEQ ID No.42或SEQ ID No.44所示。
在再进一步的方面中,本发明也涉及编码如上所述的苹果酸半醛脱氢酶的分离的核酸序列。
更具体的,所述核酸优选的由SEQ ID No.55,SEQ ID No.57,SEQ ID No.59,SEQID No.61,SEQ ID No.63,SEQ ID No.65或SEQ ID No.67所示。
在再进一步的方面中,本发明也涉及编码如上所述的DHB脱氢酶的分离的核酸序列。
在另外一个方面中,所述核酸由SEQ ID No.73或SEQ ID No.75,SEQ ID No.224,SEQ ID No.226或SEQ ID No.82所示。
依照本发明,“核酸序列”是指单链或双链形式的DNA或RNA分子,优选为DNA分子。“分离的DNA”,如本发明中所用,是指非自然发生的或者在其最初存在的自然环境中不再存在的DNA,如与嵌合基因其他调节元件相关的DNA编码序列,被转入另外的宿主细胞的DNA,或者与任何自然发生的DNA序列相比,具有不同核苷酸序列的,人工,合成制造的DNA序列。
本发明也涉及嵌合基因,包括相互功能连接的,至少一个在宿主生物中有功能的启动子,编码根据本发明的任一苹果酸激酶,苹果酸半醛脱氢酶或DHB脱氢酶的多核苷酸,以及在相同宿主生物中有功能的终止子元件。嵌合基因可能包含的各种元件为,首先,调节蛋白的转录,翻译和突变的元件,如启动子,编码信号肽或转运肽的序列,或者组成多聚腺苷酸化信号的终止元件,和第二,编码蛋白的多核苷酸。表述“相互功能连接”意思是嵌合基因的所述元件以这些元件中一个的功能受另一个功能影响的方式相互连接。举例说,当启动子能够影响编码序列表达时,启动子被功能连接到该编码序列。可以使用本领域技术人员公知的技术来进行依照本发明的嵌合基因的构建及其各种元件的组装。组成嵌合基因的调节元件的选择基本上取决于其必须在其中起作用的宿主生物,并且本领域技术人员能够选择在给定宿主生物中起作用的调节元件。术语“有功能的”是表示能够在给定宿主生物中起作用。
依照本发明的嵌合基因可能包含的启动子是组成型或诱导型的。举例说,用于在细菌中表达的启动子可以从下面提到的启动子中选择。对于在大肠杆菌中的表达,提及的启动子可以由lac,trp,lpp,phoA,recA,araBAD,prou,cst-l,tetA,cadA,nar,tac,trc,lpp-lac,Psyn,cspA,PL,PL-9G-50,PR-PL,T7,[λ]PL-PT7,T3-lac,T5-lac,T4基因32,nprM-lac,VHb和蛋白A启动子或其他Ptrp启动子组成(WO 99/64607)。对于在革兰氏阳性菌如棒状杆菌或链霉菌中的表达,提及的启动子可以由PtipA或PS1和PS2(FR91/09870)启动子或者申请EP0629699A2中描述的那些启动子组成。对于在酵母和真菌中的表达,提及的启动子可以由乳酸克鲁维酵母PLAC4启动子或者乳酸克鲁维酵母Ppgk启动子(专利申请FR 91/05294),木霉tef1或cbh1启动子(WO94/04673),青霉his,csl或apf启动子(W0 00/68401)和曲霉gla启动子组成。
依照本发明,嵌合基因可以包含位于启动子和编码序列之间的其他调节序列,如转录激活子(增强子)。
这样,在特定实施方式中本发明的嵌合基因包括至少,在转录方向功能连接的,在宿主生物中有功能的启动子调控序列,编码本发明苹果酸激酶,苹果酸半醛脱氢酶的核酸序列,以及在所述宿主生物中有功能的终止子调节序列。
本发明也涉及克隆和/或表达载体,其包括依照本发明的嵌合基因或本发明的核酸序列。依照本发明的载体可用于转化宿主生物和在该生物中表达任一种苹果酸激酶,苹果酸半醛脱氢酶和/或DHB脱氢酶。该载体可以是质粒,粘粒,噬菌体或病毒。优选的,依照本发明的转化载体是质粒。通常,该载体的主要能力应该是在宿主生物细胞中保持自身并自我复制,特别是凭借复制起点的存在下,和表达本发明中任一苹果酸激酶,苹果酸半醛脱氢酶和/或DHB脱氢酶的能力。为了稳定转化宿主生物的目的,载体也可以被整合入基因组中。这样载体的选择,以及将依照本发明的嵌合基因***该载体的技术为本领域技术人员常规知识的一部分。有利的是,除了依照本发明的嵌合基因外,用于本发明的载体也包含编码选择标记的嵌合基因。该选择标记使得能够选择出被有效转化的宿主生物,即包含了载体的那些宿主生物。依照本发明特定的实施方式,被转化的宿主生物为细菌,酵母,真菌。在可以使用的选择标记中,提及的选择标记可以由含有抗生素抗性基因,如,例如潮霉素磷酸转移酶基因的标记组成。其他标记可以是补充营养缺陷型的基因,如pyrA,pyrB,pyrG,pyr4,arg4,argB和trpC基因,钼蝶呤合成酶基因或乙酰胺酶基因。提及的基因可以由编码容易确认的酶如GUS酶的基因,或者编码色素或在转化细胞中调节色素生产的酶的基因组成。这样的选择标记基因在专利申请WO 91/02071,WO95/06128,WO 96/38567和WO 97/04103中特别描述。
本发明也涉及包含至少一种依照本发明的嵌合基因的转化的宿主生物,嵌合基因整合入其基因组或者在染色体外遗传元件,例如质粒上被携带。在本发明更具体的方面中,转化的宿主生物包含,编码苹果酸激酶的本发明核酸,或者含有编码苹果酸激酶核酸的嵌合基因,或者含有编码苹果酸激酶核酸的表达载体,和/或编码苹果酸半醛酶的核酸,或者含有编码苹果酸半醛酶的核酸的嵌合基因,或者含有编码苹果酸半醛酶的核酸的表达载体,和/或编码DHB脱氢酶的核酸,或者含有编码DHB脱氢酶的核酸的嵌合基因,或者含有编码DHB脱氢酶的核酸的表达载体。
在本发明具体的方面中,编码苹果酸激酶的核酸由SEQ ID No.13,SEQ ID No.15,SEQ ID No.17,SEQ ID No.19,SEQ ID No.21,SEQ ID No.23,SEQ ID No.25,SEQ IDNo.27,SEQ ID No.38,SEQ ID No.40,SEQ ID No.42或SEQ ID No.44所示,编码苹果酸半醛酶的核酸由SEQ ID No.55,SEQ ID No.57,SEQ ID No.59,SEQ ID No.61,SEQ ID No.63,SEQ ID No.65,或SEQ ID No.67所示,而编码DHB脱氢酶的核酸由SEQ ID No.73,SEQ IDNo.75,SEQ ID No.224,SEQ ID No.226或SEQ ID No.82所示。
术语“宿主生物”是表示任何较低等的单细胞生物,依照本发明的嵌合基因,核酸,或载体可以被导入其中以生产2,4-DHB。优选的,宿主生物是微生物,具体是真菌,例如青霉,曲霉和更具体的黄曲霉,金孢子菌属或木霉属,酵母,具体是酵母属,克鲁维酵母属或毕赤酵母属和更具体的鲁氏接合酵母,细菌,例如埃希氏菌属,具体是大肠杆菌,或棒状杆菌属,更具体的谷氨酸棒杆菌,或链霉菌属,或者杆状病毒。
宿主生物可以是从糖,如葡萄糖天然过量生产苹果酸或琥珀酸的宿主生物,或者被工程化以从糖,如葡萄糖过量生产苹果酸或琥珀酸的宿主生物,其中促进有机酸,如苹果酸,丙酮酸,琥珀酸,富马酸运出的所有潜在的膜转运蛋白已被缺失。宿主生物可以是被工程化以过量生产DHB的生物,其中促进有机酸,如苹果酸,丙酮酸,琥珀酸,富马酸运出的所有膜转运蛋白已被缺失。促进苹果酸和其他有机酸运出的透性酶的例子为来自粟酒裂殖酵母的Mae1(Camarasa et al.,2001;Grobler et al.,1995),来自枯草芽孢杆菌的DctA(Groeneveld et al.,2010),来自大肠杆菌的Dct1-4,来自酿酒酵母的Jen1(Akita et al,2000)。专家可以基于序列同源性确定其他微生物中的候选透性酶。这些构造会作用以将苹果酸或其他有机酸保持在细胞中以使其可用于DHB生产。
表述“转化的宿主生物”是表示宿主生物,其已经将至少一种依照本发明的嵌合基因整合入其基因组中,或者染色体外遗传元件,例如质粒里,并从而在其组织中,或在培养基中生产苹果酸激酶,苹果酸半醛脱氢酶和/或DHB脱氢酶中任一种。为获得依照本发明的宿主生物,本领域技术人员可以使用许多已知的转化技术中的一种。
这些技术中的一种包括将要转化的宿主细胞与聚乙二醇(PEG)和依照本发明的载体相接触。电穿孔是另外一种方法,其包括使要转化的细胞和本发明的载体经受电场。另外的方法包括通过显微注射直接将载体注入细胞或组织。也可以使用“基因枪”方法。其包含以其上吸附有本发明载体的微粒轰击细胞或组织(美国专利号4,945,050)。
文献中描述了用于大肠杆菌和其他革兰氏阴性菌的几种转化细菌的方法。可以使用接合。对于革兰氏阳性菌,可以使用电穿孔,也可以使用原生质体转化,特别是对于链霉菌属的细菌。
文献中描述了几种转化真菌的方法。EP0260762中描述了用于曲霉的以PEG做的原生质体转化,而WO 00/36120中描述了该方法对绳状青霉种的调整方案。通过限制性酶介导整合,或REMI的转化也已知,使用农杆菌属细菌的原生质体转化同样已知。文献中也描述了转化酵母的技术。
在进一步的方面,本发明涉及2,4-DHB的生产过程,包括培养本发明的转化微生物的步骤。
为生产DHB,各种碳水化合物可以被单独使用或者作为混合物使用,例如葡萄糖,果糖,蔗糖,糖蜜,麦芽糖,赤糖糊,淀粉水解物(葡萄糖,寡糖),乳糖,麦芽糖,淀粉和淀粉水解物,纤维素,纤维素水解物,甘油和确定的烃,油和脂肪如豆油,葵花籽油,花生油和椰子油,以及脂肪酸,如例如棕榈酸,硬脂酸和亚油酸。这些物质可以被单独使用或作为混合物使用。
各种氮源可以被单独或作为混合物使用来进行商业或中试规模生产,包括无机化合物如气态氨和氨水,无机酸或有机酸的铵盐,如硫酸铵,硝酸铵,磷酸铵,氯化铵,乙酸铵和碳酸铵。此外,天然含氮有机材料,如大豆水解物,大豆蛋白HCl水解物(总氮约7%),豆粕,豆饼水解物,玉米浆,酪蛋白水解物,酵母提取物,肉提取物,麦芽提取物,尿素,蛋白胨和氨基酸也可以被使用。
生产过程可以在有氧,厌氧和限制氧条件下进行。其可以作为分批补料过程或按批次过程进行。
可以通过在培养基中培养宿主生物来进行所述2,4-DHB的生产,培养基中单独的或者与能够确保生长的其他碳源一起加入了苹果酸(或者另外的有机酸如丙酮酸,琥珀酸,或富马酸)。苹果酸(和其他有机酸)可以被直接加入,或者通过设计两阶段发酵过程加入,两阶段发酵过程中,苹果酸(或其他有机酸)在第一过程阶段中由过量生产苹果酸的微生物产生,而2,4-DHB生产在随后的阶段中由依照本发明的宿主生物实现。
产物的分离和纯化是非常重要的因素,其极大地影响整个过程的效率和产物成本。产物回收的方法一般包括分别的细胞分离,以及产物纯化,浓缩和干燥步骤。
细胞分离
可以使用超滤和离心来从发酵培养基中分离细胞。高培养基粘度使得从发酵培养基中分离细胞变得复杂。因此,我们可以加入添加剂如无机酸或碱的盐,或者加热培养液以优化细胞分离。
产物回收
多种离子交换色谱方法可以用于在去除生物质前或后分离DHB。这些包括依照其等电点使用初级阳离子交换树脂促进产物分离。通常,以溶液填充树脂,而随着洗脱液pH值的增加(如通过加入氢氧化铵),保留的产物被单独洗脱。另外的可能性是以固定的或仿移动的树脂床来使用离子交换树脂。可能需要联合不同的色谱步骤以达到足够的产品纯度。与昂贵的结晶步骤相比那些纯化方法更经济,在终产物形式方面也提供额外的优势和灵活性。
产物浓缩和干燥
纯化过程也可能包括干燥步骤,其可以包含适合的干燥工具如喷雾造粒机,喷雾干燥器,筒式烘干机,回转烘干机,隧道式烘干机。可以通过使用多功能浓缩器或薄膜蒸发器,以130℃蒸汽在减压下加热发酵液来获得浓缩DHB溶液。
通过优化宿主生物代谢网络中的碳通量再分配和通过确保DHB途径三种酶NADPH和ATP的充足供应,可以确保DHB的高效生产。通常通过缺失或者减少竞争的自然发酵途径来促进碳通量引导进入所需的代谢途径以及NAD(P)H辅助因子的供应。非排他性的例子为:
-通过阻碍乙醇的形成(通过缺失丙酮酸脱羧酶)来优化酿酒酵母中苹果酸的生产(Zelle et al.,2008;Zelle et al.,2010)。
-通过阻碍乳酸的形成(如缺失IdhA),乙酸的形成(如缺失pta,ackA),乙醇的形成(如缺失adhE),甲酸的形成(如缺失pflB,focA),丙酮酸的氧化(如缺失poxB),苹果酸的降解(如缺失maeB和scfA),琥珀酸的形成(如缺失frdBC),甲基乙二醛的形成(缺失mgsA)来优化大肠杆菌中的琥珀酸或苹果酸生产(Jantama et al.,2008a;Jantama et al.,2008b;Lin et al.,2005;Sanchez et al.,2005a;Zhang et al.,2011)。
增加有机酸生产碳通量和ATP供应的另一种可能方式是工程化磷酸烯醇式丙酮酸(PEP)/丙酮酸/草酰乙酸的分支节点(综述于(Sauer&Eikmanns 2005))。确保从磷酸烯醇式丙酮酸到草酰乙酸碳通量增加的代谢工程化策略的非排他性例子为:
-通过阻碍丙酮酸激酶的作用和增加PEP羧化激酶的活性来优化酿酒酵母中苹果酸的生产(Zelle et al.,2010)。
-通过增加天然或异种表达的PEP羧化酶,PEP羧化激酶,或丙酮酸羧化酶的活性,来优化大肠杆菌中琥珀酸的生产(Millard et al.,1996;Sanchez et al.,2005b;Zhanget al.,2009)。
在使用PEP消耗磷酸转移酶***(PTS)进行葡萄糖初始磷酸化步骤的大肠杆菌和其他细菌中,增加有机酸生产碳通量和ATP供应的另一种可能方式为缺失PTS***的基本元件(例如ptsl或ptsG)(Lin et al.,2005;Zhang et al.,2009)。在携带有PTS***缺失突变的突变体中,为确保进一步的葡萄糖摄入,必须保证另外的葡萄糖摄入***(如GalP)的活性。
增加有机酸生产中进入所需途径的碳通量的另一种可能方式是工程化柠檬酸和乙醛酸循环。非排他性例子为:
-通过增加异柠檬酸裂解酶活性(缺失转录阻遏子iclR)来优化大肠杆菌中琥珀酸的生产(Lin et al.,2005;Sanchez et al.,2005a)。
-通过缺失异柠檬酸脱氢酶,和/或琥珀酸脱氢酶来优化琥珀酸的生产(Lin etal.,2005)。
增加DHB生产中进入所需途径的碳通量的另一种可能方式是在生产生物中表达适合的丙酮酸脱氢酶和柠檬酸合成酶。大肠杆菌的天然丙酮酸脱氢酶和柠檬酸合成酶被细胞内高NADH浓度抑制,这使得这些酶在厌氧条件下活力较低。在大肠杆菌中,对NADH不敏感的丙酮酸脱氢酶突变体的表达导致了厌氧条件下乙酰-CoA的过量生产和发酵终产物之间改变的碳通量再分配(乙酸,乳酸,乙醇,甲酸和丙酮酸)(Wang et al.2010)。对NADH不敏感的枯草芽孢杆菌柠檬酸合成酶的异种表达增加了工程化的大肠杆菌菌株中琥珀酸的生产(Sanchez et al.,2005a)。结合上述突变,使用适合的丙酮酸脱氢酶和柠檬酸合成酶(NADH敏感或不敏感的),能调整厌氧和有氧条件下乙醛酸与柠檬酸循环反应和发酵途径之间的碳通量再分配。
增加通过DHB途径的碳通量的另一种可能方式是缺失可能降解途径中间体4-磷酸苹果酸,4-苹果酸半醛的酶促反应。可能降解苹果酸半醛的候选酶是琥珀酸半醛脱氢酶(sad,gabD),以及其他能以末端醛基氧化C4分子的脱氢酶。
增加宿主生物中DHB产率的另一种可能方式是缺失降解DHB的代谢反应。DHB是苹果酸酶的竞争性抑制物,因此,具有对该酶的活性位点的竞争性高亲和力(Rognstad&Katz,1979)。因此,DHB可以被其他酶识别并可能被降解。这些酶可以被识别并从宿主生物中缺失。
当2,4-DHB生产是基于苹果酸或其他有机酸的加入时,生产2,4-DHB的微生物应该功能性表达能促进苹果酸(或其他有机酸如丙酮酸,琥珀酸等)摄入的膜输送蛋白。
下面的实施例举例说明了本发明。这些实施例仅是为了举例说明的目的,而不应被解释为以任何方式限制本发明的范围。
附图简述
图1:(i)描述将(L)-苹果酸转化为(L)-2,4-二羟基丁酸(DHB)的反应方案,和(ii)将(L)-天冬氨酸转化为(L)-高丝氨酸的类似转化。
图2:来自不同生物的天冬氨酸激酶氨基酸序列比对(Ec_AKIII-天冬氨酸激酶III(SEQ ID No.4),LysC,Ec_AKI(SEQ ID No.87)-大肠杆菌天冬氨酸激酶I,ThrA,Ec_AKII(SEQ ID No.88)-大肠杆菌天冬氨酸激酶II,MetL,Mj-詹氏甲烷球菌(SEQ ID No.89),Tt-嗜热栖热菌(SEQ ID No.90),Cg-谷氨酸棒杆菌(SEQ ID No.91),At-拟南芥(SEQ IDNo.92),Sc-酿酒酵母(SEQ ID No.93)),图使用ClustalW2生成(Larkin et al.,2007)。
图3:来自不同生物的天冬氨酸半醛脱氢酶氨基酸序列比对(Ec-大肠杆菌(SEQ IDNo.49),Mj-詹氏甲烷球菌(SEQ ID No.94),Tt-嗜热栖热菌(SEQ ID No.95),Bs-枯草芽孢杆菌(SEQ ID No.96),Cg-谷氨酸棒杆菌(SEQ ID No.97),At-拟南芥(SEQ ID No.98),Sc-酿酒酵母(SEQ ID No.99)),图使用ClustalW2生成(Larkin et al.,2007)。
图4:对应DHB保留时间的区域上气相色谱放大图,显示:(A)DHB标准品(浓度=1mM);(B)包含苹果酸激酶(MK),苹果酸半醛脱氢酶(MSA-Dh),和苹果酸半醛还原酶(MSA-red)的反应A组合物;(C)对照反应B组合物(与A相同但缺少MSA-red);(D)对照反应C组合物(与A相同但缺少MSA-Dh)。
图5:纯化的LysC E119G,LysC E119G E250K,LysC E119G T344M,LysC E119GS345L,LysC E119G T344M,和LysC E119G T3521突变体对反应缓冲液中赖氨酸浓度的相对活性。
实施例
实施例1:分别来自大肠杆菌和酿酒酵母的天冬氨酸激酶LysC和Hom3的天冬氨酸和苹果酸激酶活性测试。
含有野生型天冬氨酸激酶基因的质粒的构建:构建质粒pLYSCwt是通过使用高保真聚合酶PhusionTM(Finnzymes)和能分别在起始密码子上游和终止密码子下游导入NdeI和BamHI限制性位点的正向及反向引物5'CACGAGGTACATATGTCTGAAATTGTTGTCTCC3'(SEQ IDNo.1)和5'CTTCCAGGGGATCCAGT-ATTTACTCAAAC3'(SEQ ID No.2),以PCR扩增lysC基因。使用大肠杆菌DH5α的基因组DNA作为模板。PCR产物以NdeI和BamHI消化,使用T4DNA连接酶(Biolabs)连接到pET8a表达载体(Novagen)的对应位点,并转化入大肠杆菌DH5α细胞。得到的pAKIIIwt质粒被分离并通过DNA测序显示其包含具有正确序列(SEQ ID No.3)的全长lysC基因。对应的蛋白由SEQ ID No.4所示。
构建质粒pHOM3wt是通过使用高保真聚合酶PhusionTM(Finnzymes)和能分别在起始密码子上游和终止密码子下游导入NheI和EcoRI限制性位点的正向及反向引物5'TATAATGCTAGCATGCCAATGGATTTCCAACC 3'(SEQ ID No.5)和5'TATAATGAATTCTTAAATTCCAAGTCTTTTCAATTGTTC 3'(SEQ ID No.6),以PCR扩增HOM3基因。使用酿酒酵母BY4741的基因组DNA作为模板。PCR产物以NheI和EcoRI消化,使用T4DNA连接酶(Biolabs)连接到pET8a表达载体(Novagen)的对应位点,并转化入大肠杆菌DH5α细胞。得到的pHOM3wt质粒被分离并通过DNA测序显示其包含具有全长HOM3基因的正确序列(SEQ ID No.7)。对应的蛋白由SEQ ID No.8所示。
酶的表达:以适合的质粒转化大肠杆菌BL21D3星细胞。在250mL LB培养基中表达有N-末端六组氨酸标记的酶,在通过向培养基中加入1mM异丙基-β-D-硫代吡喃半乳糖苷(IPTG)来诱导蛋白表达之前,LB培养基在OD600为0.1的条件下过夜培养并生长至OD600为0.6。表达蛋白3小时之后,以13000g离心10分钟收集细胞并将其储存在-20℃下直至进一步的分析。生长和蛋白表达在37℃下进行。培养基含有50μg/L的卡那霉素。
酶的纯化:将冷冻的表达培养物的细胞团块重悬浮在0.5mL破碎缓冲液中(50mMHepe,300mM NaCl,pH 7.5),通过设定至30%能量输出的四个连续声波处理循环(BioblockScientific,VibraCellTM72437)破碎开。通过在4℃下13000g离心粗提物15分钟来去除细胞碎片并保留澄清的上清。通过加入15mg/mL的链霉素(Sigma),在4℃下13000g离心样品10分钟并保留上清来从提取物中去除RNA和DNA。澄清的蛋白提取物与0.75mL床体积的TalonTM钴亲和树脂(Clontech)在4℃下温育1h。在台式离心机上700g离心悬液并去除上清。在以0.5mL洗脱缓冲液(50mM Hepe,300mM NaCl,500mM咪唑,pH 7.5)洗脱天冬氨酸激酶之前,以10个床体积的清洗缓冲液(50mM Hepe,300mM NaCl,15mM咪唑,pH 7.5)清洗树脂。用SDS-PAGE分析来判断洗脱的酶的纯度。
酶测试:通过在磷酸烯醇式丙酮酸,丙酮酸激酶,和乳酸脱氢酶的存在下,将激酶反应中ADP产物与NADH氧化耦合来测试天冬氨酸或苹果酸激酶的活性。
反应方案:
天冬氨酸(或苹果酸)激酶
天冬氨酸(或苹果酸)+ATP→4-磷酸-(L)-天冬氨酸(或者4-磷酸-(L)-苹果酸)+ADP
丙酮酸激酶
ADP+磷酸烯醇式丙酮酸→ATP+丙酮酸
乳酸脱氢酶
丙酮酸+NADH→NAD++乳酸
测试混合物含有50mM Hepes(pH 7.5),50mM KCl,5mM MgCl2,0.24mM NADH,0.96mM ATP,0.96mM PEP,9μg/mL乳酸脱氢酶(Sigma,L2500),12.4μg/mL丙酮酸脱氢酶(Sigma,P1506),以及适量纯化的天冬氨酸(苹果酸)激酶。通过加入50mM(L)-天冬氨酸或(L)-苹果酸来起始反应。酶测试在30℃下96孔平底微量滴定板中以250μL终体积进行。随后在酶标仪(BioRad 680XR)中检测反应物340nm处NADH的特征吸收。
羟肟酸测试:为判断通过野生型或突变天冬氨酸激酶处理的底物的磷酸化,即酰基磷酸酐的形成,将激酶反应产物与羟胺温育以形成对应的天冬氨酸或苹果酸异羟肟酸衍生物。测试混合物含有120mM Hepes(pH 8),200mM KCl,10mM ATP,200mM羟胺,10mM天冬氨酸或苹果酸,以及适量的纯化蛋白。30分钟后通过加入等体积的1.7%(w/v)FeCl31M盐酸溶液来终止反应。异羟肟酸-铁复合物的形成是通过在酶标仪中测量其540nm处特征吸收来判断。使用含有除ATP外所有组分的测试混合物作为空白。
结果:如异羟肟酸测试所判断,纯化的LysC(无组氨酸标签,SEQ ID No.4)和Hom3(无组氨酸标签,SEQ ID No.7)酶显示出天冬氨酸激酶活性但不能够磷酸化苹果酸(Keng&Voila,1996)。LysC和Hom3对天冬氨酸的最大活性分别为4.5μmol/(min*mgprot)和1.6μmol/(min*mgprot)。通过测量不同底物浓度(c)下的初始反应速率(v)并通过提取v对v/c曲线的斜率,以Eadie和Hofstee的方法估算对天冬氨酸的Km值。纯化的组氨酸标签化的LysC的Km被估算为约0.6mM,显示组氨酸标签化蛋白具有与Km被报导为0.6mM的非组氨酸标签化的纯化酶相同的底物亲和力(Marco-Marin et al.,2003)。
实施例2:大肠杆菌天冬氨酸激酶LysC的定向诱变和突变体酶苹果酸激酶活性的测试。
使用表1中所列寡核苷酸对并使用pLYSCwt质粒(SEQ ID No.3)作为模板来进行定向诱变。使用表1中所列寡核苷酸对通过PCR(Phusion 1U,HF缓冲液20%(v/v),dNTPs2.5mM,正向和反向引物每种1μM,模板质粒200ng,水)导入改变氨基酸序列的点突变。除了促进突变克隆识别的功能突变外,PCR创建的质粒包含新的Nco1限制性位点(使用沉默突变导入)。PCR产物被以Dpnl在37℃消化1h以去除模板DNA,并转化入NEB 5α感受态大肠杆菌细胞(NEB)。通过限制性位点分析来识别突变的质粒并通过DNA测序判断其携带的目标突变。
表1:用于在E119位置突变大肠杆菌天冬氨酸激酶lysC的寡核苷酸。
显示119位置突变的序列可以由SEQ ID No.9所示,其中119位置的残基为X,X是19个天然产生的氨基酸中的任一个(除谷氨酰胺)。
如实施例1所述,突变体酶被表达,纯化并测试天冬氨酸和苹果酸激酶活性。结果总结于表2中。
表2:突变体酶苹果酸激酶活性的表征。值对应来自至少两个独立试验的平均值。
表2中所列突变体对天冬氨酸均没有活性。
结果显示通过以半胱氨酸,甘氨酸,天冬酰胺,脯氨酸,谷氨酰胺,丝氨酸,苏氨酸或缬氨酸替换119位置处的保守谷氨酸,天冬氨酸激酶可以被转化为苹果酸激酶。
表2中所列酶对应的核酸序列为SEQ ID No.13,SEQ ID No.15,SEQ ID No.17,SEQID No.19,SEQ ID No.21,SEQ ID No.23,SEQ ID No.25和SEQ ID No.27。
实施例3:构建对赖氨酸抑制的敏感性大幅降低的苹果酸激酶
使用表3中所列寡核苷酸对并使用pLYSC_E119G质粒作为模板来进行定向诱变(如实施例2所描述的,通过将下列改变导入lysC基因的DNA序列:(SEQ ID No.15)来获得pLYSC_E119G质粒)。使用表1中所列寡核苷酸对通过PCR(Phusion 1U,HF缓冲液20%(v/v),dNTPs 2.5mM,正向和反向引物每种1μM,模板质粒200ng,水)导入改变氨基酸序列的点突变。当可能时,除了促进突变克隆识别的功能突变外,PCR创建的质粒包含新的限制性位点(使用沉默突变导入)。PCR产物被以Dpnl在37℃消化1h以去除模板DNA,并转化入NEB 5α感受态大肠杆菌细胞(NEB)。通过限制性位点分析来识别突变的质粒并通过DNA测序判断其携带目标突变。
表3:用于突变大肠杆菌苹果酸激酶lysC E119G的寡核苷酸。
蛋白LysC E119G的核酸序列包含另外的突变,其对应(i)以赖氨酸替换250位置上的谷氨酸,如SEQ ID No.38所示;其对应氨基酸序列由SEQ ID No.39所示;(ii)以甲硫氨酸替换344位置上的苏氨酸,如SEQ ID No.40所示;其对应氨基酸序列由SEQ ID No.41表示;(iii)以异亮氨酸替换352位置上的苏氨酸,如SEQ ID No.42表示;其对应氨基酸序列由SEQID No.43表示;(iv)以亮氨酸替换345位置上的丝氨酸,如SEQ ID No.44表示;其对应氨基酸序列由SEQ ID No.45表示。
酶的表达和纯化:如实施例1所述进行组氨酸标签化酶LysC E119G,LysC E119GE250K,LysC E119G T344M,LysC E119G S345L,LysC E119G T3521的蛋白表达。
酶测试:如实施例1所述测试苹果酸激酶活性。改变反应缓冲液中的赖氨酸浓度。
结果:将突变E250K,T344M或S345L导入LysC E119G,很大程度上使得苹果酸激酶活性对升高的赖氨酸浓度不敏感(见图4)。
实施例4:来自大肠杆菌的天冬氨酸半醛脱氢酶Asd的天冬氨酸和苹果酸半醛脱氢酶活性测试。
含有野生型天冬氨酸半醛脱氢酶基因的质粒的构建:构建质粒pASDwt是通过使用高保真聚合酶PhusionTM(Finnzymes)和能分别在起始密码子上游和终止密码子下游导入NheI和BamHI限制性位点的正向及反向引物5'TATAATGCTAGCATGAAAAATGTTGGTTTTATCGG 3'(SEQ IDNo.46)和5'TATAATGGATCCTTACGCCAGTTGACGAAGC 3'(SEQ ID No.47),以PCR扩增大肠杆菌asd基因。使用大肠杆菌DH5α的基因组DNA作为模板。PCR产物以NheI和BamHI消化,使用T4DNA连接酶(Biolabs)连接到pET8a表达载体(Novagen)的对应位点,并转化入大肠杆菌DH5α细胞。得到的pASDwt质粒被分离并通过DNA测序显示其包含具有正确序列(SEQ IDNo.48)的全长asd基因。所述酶的对应氨基酸序列由SEQ ID No.49所示。
酶的表达和纯化:如实施例1所述进行组氨酸标签化酶Asd的蛋白表达。
酶测试:在反向生物合成方向上,通过下面在将天冬氨酸或苹果酸半醛分别氧化为4-磷酸-(L)-天冬氨酸或4-磷酸-(L)-苹果酸期间的NADP还原来测试天冬氨酸或苹果酸半醛脱氢酶活性(Robert et al,.2003)。
(L)-天冬氨酸半醛(或(L)-苹果酸半醛)+NADP+Pi→4-磷酸-(L)-天冬氨酸(或4-磷酸-(L)-苹果酸)+NADPH。
测试混合物含有200mM Hepes(pH 9),50mM K2HPO4,0.25mM NADP。通过加入(L)-天冬氨酸半醛或(L)-苹果酸半醛来起始反应。(L)-天冬氨酸半醛以L-天冬氨酸β-半醛水合三氟乙酸(保持在pH3以防降解)的形式加入,该形式是高丝氨酸脱氢酶和天冬氨酸半醛脱氢酶酶测试的适合底物(Robert et al,.2003)。酶测试之前通过将稳定的苹果酸半醛衍生物2-[(4S)-2,2-二甲基-5-氧-1,3-二氧戊环-4-基]乙醛(DMODA)脱保护来新鲜生产不稳定的苹果酸半醛。通过将DMODA在2M盐酸中25℃温育15分钟来获得苹果酸半醛,并蒸发释放的丙酮(35℃,50mbar)。使用碳酸氢钠将苹果酸半醛溶液的pH固定在3。
酶测试在30℃下96孔平底微量滴定板中以250μL终体积进行。随后在酶标仪(BioRad 680XR)中检测反应物340nm处NADPH的特征吸收。
结果:组氨酸标签化的野生型天冬氨酸半醛脱氢酶,Asd,以160μmol/(min*mgprot)的最大特定活性将(L)-天冬氨酸半醛氧化为4-磷酸-(L)-天冬氨酸。(L)-苹果酸半醛酶具有0.01μmol/(min*mgprot)的活性。
实施例5:大肠杆菌天冬氨酸脱氢酶Asd的定向诱变和突变体酶苹果酸半醛脱氢酶活性的测试。
使用pASDwt质粒作为模板并按照实施例2所述方案导入Asd氨基酸序列中的点突变。使用表4中所列寡核苷酸对来突变241位置的谷氨酸残基或者136位置的苏氨酸残基。通过限制性位点分析来识别析突变的质粒并通过DNA测序判断其携带目标突变。
241位置突变的Asd蛋白可以由SEQ ID No.68所示,其中241位置的残基为X,X是其他19个生物产生的氨基酸中的任一个(除谷氨酰胺)。
表4:用于在E241和T136位置突变大肠杆菌天冬氨酸半醛脱氢酶Asd的寡核苷酸。
结果:在E241位置突变Asd的活性和Km总结于表5中。谷氨酸241由丙氨酸,半胱氨酸,甘氨酸,组氨酸,异亮氨酸,甲硫氨酸,或谷氨酰胺取代的Asd突变体以明显高于野生型酶的最大特定活性将(L)-天冬氨酸-4-半醛氧化成4-磷酸-(L)-天冬氨酸。双突变体AsdE241Q T136N(SEQ ID No.231)具有0.25μmol/(min*mgprot)的最大特定活性和0.25mM的Km。
表5:突变体酶苹果酸半醛脱氢酶活性的表征。值对应来自至少两个独立试验的平均值。
*仅估算所选突变体的Km值。
对应核酸由SEQ ID No.55,SEQ ID No.57,SEQ ID No.48,SEQ ID No.59,SEQ IDNo.61,SEQ ID No.63,SEQ ID No.65和SEQ ID No.67所示。
双突变体Asd E241Q T136N具有由SEQ ID No.230所示的核酸序列。
实施例6:2,4DHB脱氢酶的识别
为识别适合的2,4DHB脱氢酶,测试了不同生物来源的β-羟酸脱氢酶还原苹果酸半醛的能力。测试的酶中有来自酿酒酵母的甲基丁醛还原酶,Ypr1(Ford&Elis,2002)(SEQ IDNo.73和SEQ ID No.74);和来自嗜热金属球菌的琥珀酸半醛还原酶Ms-Ssr(Kockelkom&Fuchs,2009)(SEQ ID No.75和SEQ ID No.76)。使用表6中所列引物扩增YPR1和Ms-SSR基因并将其克隆入载体pET28(限制性酶见表3),分别得到质粒pYPR1和pMs-SSR。如实施例1中所述表达和纯化蛋白。
表6:用于克隆候选β-羟酸脱氢酶的引物和限制性酶。
测试苹果酸半醛还原酶活性:
反应:
(L)-苹果酸半醛+NAD(P)H→(L)-2,4-二羟基丁酸+NAD(P)
测试混合物含有200mM Hepes(pH 7.5),50mM KCl,5mM MgCl2,0.24mM NADH或NADPH,以及适量纯化的酶。通过加入10mM(L)-苹果酸半醛来起始反应(每个测试的苹果酸半醛即时制备,见实施例4)。酶测试在30℃下96孔平底微量滴定板中以250μL终体积进行。随后在酶标仪(BioRad 680XR)中检测反应物340nm处NAD(P)H的特征吸收。结果列于表7中。
表7:所选β-羟酸脱氢酶对苹果酸半醛的还原活性(结果表示至少两个独立试验的平均值)。
嗜热金属球菌琥珀酸半醛脱氢酶和酿酒酵母甲基丁醛还原酶具有苹果酸半醛还原酶活性。Ms-SSR对苹果酸半醛的Km为1.1mM。
实施例7:嗜热金属球菌琥珀酸半醛还原酶的定向诱变
使用表8中所列寡核苷酸对并使用pMs-SSR质粒作为模板来进行定向诱变。通过PCR(Phusion 1U,HF缓冲液20%(v/v),dNTPs 2.5mM,正向和反向引物每种1μM,模板质粒200ng,水)导入改变氨基酸序列的点突变。当可能时,除了促进突变克隆识别的功能突变外,PCR创建的质粒包含新的限制性位点(使用沉默突变导入)。PCR产物被以Dpnl在37℃消化1h以去除模板DNA,并转化入NEB 5α感受态大肠杆菌细胞(NEB)。通过限制性位点分析来识别突变的质粒并通过DNA测序判断其携带目标突变。表9总结了突变体的动力学参数。结果表明当与野生型酶相比时,双突变体Ms-SSR H39R N43H(SEQ ID No.81,SEQ ID No.82)具有改进的苹果酸半醛亲和力。
表8:用于突变嗜热金属球菌琥珀酸半醛还原酶(Ms-SSR)的引物对
表9:嗜热金属球菌琥珀酸半醛还原酶(Ms-SSR)突变体的动力学参数总结(结果表示至少两个独立试验的平均值)。
对应核酸序列由SEQ ID No.224,SEQ ID No.226和SEQ ID No.82所示。
实施例8:DHB的体外生产
如实施例1中所述表达并纯化酶苹果酸激酶(LysC E119G,SEQ ID No.15),苹果酸半醛脱氢酶(Asd E241Q;SEQ ID No.67)和苹果酸半醛还原酶(Ms SSrR,SEQ ID No.76)。通过向含有50mM Hepes(pH 7.5),50mM KCl,5mM MgCl2,1mM NADPH,180μg/mL苹果酸激酶(Lys E119G),325μg/mL苹果酸半醛脱氢酶(Asd E241Q),和130μg/mL苹果酸半醛还原酶(Ms_Ssr)的反应混合物中加入50mM苹果酸来证实体外DHB的生产(反应A)。对照反应含有所有组分但缺少苹果酸半醛还原酶(反应B)或苹果酸半醛脱氢酶(反应C)。在30℃温育30分钟之后,通过气相色谱分析反应混合物[CPG Varian Series 430;装有FID检测器;自动进样器CP8400;无分流注射器1177(230℃);柱:CPWAX58/FFAP,30m×0.53mm,df0.50μm;和衬层:入口套,鹅颈6.5×78.5×4mm GWOL(Varian)]。载气为流速25mL/min的氮气。使用空气-氢气混合物进行火焰离子化(流速分别为300mL/min和30mL/min)。检测器温度为240℃。注入的样品体积为1μL。温度程序在表10中提供。
表10:反应混合物分析的温度程序
在反应A中(存在所有酶)检测到DHB产出,但在对照反应B和C中没有DHB产出(图5)。
实施例9:优化嗜热金属球菌琥珀酸半醛还原酶编码序列以在大肠杆菌中表达。
使用软件,优化包括突变体H39R和N43H的嗜热金属球菌琥珀酸半醛还原酶的编码序列以在大肠杆菌中最大表达。合成基因由基因合成仪(lnvitrogen Life Technologie)生产。Nhel和EcoRI限制性位点被分别引入到起始密码子上游和终止密码子下游,这允许直接克隆入pET28a+(Novagen)。
得到的pSSR-H39RN43H-opt质粒被分离并以DNA测序显示其包含具有正确序列(SEQ ID No.228)的全长嗜热金属球菌SSR H39R N43H基因。
实施例10:使用大肠杆菌作为宿主生物构建促进苹果酸激酶(大肠杆菌lysC基因突变体),苹果酸半醛脱氢酶(大肠杆菌asd基因突变体),和DHB脱氢酶(嗜热金属球菌琥珀酸半醛还原酶基因突变体)同时表达的质粒。
质粒pLYSC-E119G E250K(SEQ ID No.38)被用作操纵子构建的骨架。使用pASD-E241Q(SEQ ID No.55)作为模板,并使用能分别在rbs上游和终止密码子下游导入BamHI和EcoRI限制性位点的正向及反向引物5'TATAAGGATCCGTTTAACTTTAAGAAGGAGATATACCATGGG 3'(SEQ ID No.83)和5'TATAAGAATTCTTACGCCAGTTGACGAAG 3'(SEQ ID No.84),通过PCR(高保真聚合酶PhusionTM(Finnzymes))获得含有pET28(Novagen)核糖体结合位点(rbs)和ASD-E241Q编码区的DNA片段。PCR产物以BamHI和EcoRI消化,使用T4DNA连接酶(Biolabs)连接到pLYSC-E119G E250K的对应位点,并转化入大肠杆菌DH5α细胞。得到的pLYSC-E119G-E250K_ASD-E241Q质粒被分离并以DNA测序显示其具有正确序列。
使用pSSR-H39RN43H-opt作为模板,并使用能分别在rbs上游和终止密码子下游导入notl和PspXI限制性位点的正向及反向引物5'TATAAGCGGCCGCGTTTAACTTTAAGAAGGAGATAT3'(SEQ ID No.85)和5'TATAAACTCGAGCTTACGGAATAATCAGG 3'(SEQ ID No.86),通过PCR获得含有pET28核糖体结合位点(rbs)和优化编码的Ms-SSR-H39RN43H-opt编码区的DNA片段。PCR产物以notl和PspXI消化,使用T4DNA连接酶(Biolabs)连接到pLYSC-E119G-E250K_ASD-E241Q的对应位点,并转化入大肠杆菌DH5α细胞。得到的pET28-DHB质粒(SEQ ID No.229)被分离并以DNA测序显示其具有正确序列。
通过以Sphl和Xbal消化pET28-DHB并克隆有适合限制性位点的另外启动子区域,可以用任何其他诱导型或组成型启动子替换同时调控三个基因表达的5’上游启动子区域(即pET28-DHB中的T7启动子)。作为使用诱导型启动子的例子,pET28-DHB骨架的T7启动子被tac启动子替换,其特性允许葡萄糖存在下的蛋白表达(de Boer et al.,1983)。通过以Sphl和Xbal消化质粒,从质粒pEXT20(Dykxhoom et al.,1996)获得tac启动子。包含启动子的DNA片段被纯化并克隆入Sphl/Xbal消化的pET28-DHB质粒。得到的pTAC-DHB质粒被分离并以DNA测序显示其具有正确序列。
表11:本研究中构建的质粒列表
实施例11:构建优化DHB发酵生产的碳通量再分配和NADPH辅助因子供应的大肠杆菌菌株
阻断了大肠杆菌菌株MG1655中的数个基因以优化DHB生产的碳通量再分配和辅助因子供应。依照Datsenko et al.(Datsenko&Wanner,2000)使用λ红重组酶方法进行基因缺失。
使用高保真聚合酶PhusionTM(Finnzymes),并使用pKD4质粒的FRT侧翼卡那霉素抗性基因(kan)作为模板,通过PCR制备缺失盒(Datsenko&Wanner,2000)。有义引物含有对应每个目标基因5’末端的序列(下划线),随后有对应pKD4的FRT-kan-FRT盒的20bp。反义引物含有对应每个目标基因3’末端的序列(下划线),随后有对应盒的20bp。引物在表12中描述。在转化之前,PCR产物被以Dpnl消化并纯化。
通过在LB培养基中37℃将细胞培养至OD600为0.6,浓缩细胞100倍并以冰冷的10%甘油清洗两次,大肠杆菌MG1655株系被制成电转感受态。通过电穿孔(2.5kV,200Ω,25μF,在2mm间隙的转化杯中)以质粒pKD46转化细胞(Datsenko&Wanner,2000)。30℃下在氨苄青霉素(100μg/mL)LB固体培养基上选择转化体。
阻断盒被转化入有λ红重组酶表达的质粒pKD46的电转感受态大肠杆菌菌株中。细胞30℃下在含有氨苄青霉素(100μg/mL)的SOB液体培养基上培养。当培养物的OD600达到0.1时,通过加入10mM***糖诱导λ红重组酶***。在细胞被离心收获,以冰冷的10%甘油清洗两次,并通过电穿孔以阻断盒转化之前,细胞被进一步培养至OD600为0.6。在30℃下LB液体培养基中过夜表型表达之后,细胞被接种在含25μg/mL卡那霉素的固体LB培养基上。培养后在30℃选择转化体。
使用Crimson Taq聚合酶(NEB),以菌落PCR判断基因替换。以侧翼座特异性引物(见表13)进行第一反应以判断同时的母片段的缺失和新突变特异片段的获得。通过使用FRT-卡那霉素抗性盒中与各自的通用测试引物k1rev,或k2for(见表13)接近的座特异性引物(有义座引物/k1rev和k2for/反向座引物)完成另外的两个反应。
随后使用有FLP重组酶的质粒pCP20将抗性基因(FRT-kan-FRT)从染色体上切下(Cherepanov&Wackernagel,1995),留下含有一个FRT位点的创痕区域。pCP20是显示温度敏感复制和FLP重组酶合成热诱导的氨苄青霉素和CmR质粒。以pCP20转化卡那霉素抗性突变体,并在30℃下选择氨苄青霉素抗性转化体。随后在固体LB培养基上37℃培养突变体并测试所有抗生素抗性的缺失。使用Crimson Taq聚合酶和侧翼座特异性引物(表13),以菌落PCR分析FRT-卡那霉素盒的切除。通过重复上述步骤获得多个缺失。
携带单个或多个缺失的菌株被如上所述制成电转感受态,以允许转化DHB途径酶IPTG诱导表达的pTAC-DHB质粒(见实施例10),并在含50μg/mL卡那霉素的固体LB培养基上选择。
使用大肠杆菌MG1655基因组DNA作为模板并使用分别为5'TATAATCCCGGGATGCGCGTTAACAATGGTTTGACC3'(SEQ ID No.100)和5'TATAATTCTAGATTACAGTTTCGGACCAGCCG 3'(SEQ IDNo.101)的正向和反向引物,通过扩增pck编码序列来构建拥有大肠杆菌PEP羧化激酶编码pck基因的质粒pACT3-pck。DNA片段以Xmal和Xbal消化,使用T4DNA连接酶(Biolabs)连接带pACT3表达载体的对应位点,并转化入大肠杆菌DH5α细胞。在含氯霉素(25μg/mL)的固体LB培养基上选择转化体。得到的质粒被分离并通过测序判断pck基因的正确***。使用表14中所列引物类似地构建分别拥有aceA,ppc,galP,或pykA(所有大肠杆菌)或乳酸乳球菌pycA的质粒pACT3-aceA,pACT3-ppc,pACT3-gaIP,pACT3-pykA和pACT3-pyc。
上面提及的pACT3衍生质粒和pTAC-DHB质粒被转入携带表12中所列缺失组合的大肠杆菌MG1655突变体。在含氯霉素(25μg/mL)和卡那霉素(50μg/mL)的固体LB培养基上选择包含两种质粒的转化体。构建菌株的实例列于表15中。
表12:用于基因阻断的引物。与靶基因同源的序列加下划线。
表13:用于判断基因阻断的引物对。
表14:用于基因过量表达的引物。用于克隆入pACT3的限制性位点加下划线。
表15:构建用以生产DHB的菌株的实例
实施例12:通过发酵葡萄糖来生产2,4-二羟基丁酸
菌株和培养条件:使用从质粒pTAC-DHB共表达的苹果酸激酶,苹果酸半醛脱氢酶和DHB脱氢酶的大肠杆菌菌株ECE1(见实施例11),以及仅含有空质粒(即无上面提及酶的编码序列的pTAC骨架)的同基因对照菌株来进行试验。1升培养基含有20g葡萄糖,18gNa2HPO4*12H2O,3g KH2PO4,0.5g NaCl,2g NH4Cl,0.5g MgSO4*7H2O,0.015CaC12*2H2O,1毫升0.06moI/L的在100倍稀释的浓缩HCl中制备的FeC13储备液,2mL 10mM的HCl硫胺素储备液,20g MOP3,50μg硫酸卡那霉素,以及1mL微量元素溶液(每升含0.04g Na2EDTA*2H2O,0.18gCoC12*6H2O,ZnSO4*7H2O,0.04g Na2MoO4*2H2O,0.01g H3BO3,0.12g MnSO4*H2O,0.12g CuCl2*H2O)。调节pH至7并对培养基过滤灭菌。所有培养在37℃下,在170rpm的Infors旋转振荡器上进行。过夜培养物(测试管中3mL培养基)从甘油储备物中接种并将500mL摇瓶中培养的100mL生长培养物的初始OD600调整为0.05。当生长培养物中的OD600达到0.2时,加入1mmol/L浓度的IPTG。
通过LC-MS/MS分析来估算DHB浓度:
通过离心从细胞中分离培养基(Beckmann-Coulter Allegra 21R,RotorBeckmann S4180,10min,4800rpm)。澄清的上清被储存在-20℃下直至进一步分析。使用装有ACQUITY UPLC BEH柱(C18,1.7μm,100×2.1mm,Waters)的,与质量敏感检测器(TQ,Waters,ESI模式,毛细电压:2.5kV,锥孔电压25V,提取器电压:3V,源温度:150℃,脱溶剂温度:450℃,锥孔气流:50L/h,脱溶剂气流:750L/h)的HPLC(Waters)来定量DHB含量。柱温度被保持在30℃。流动相为88%的0.08%四正丁基氢氧化铵溶液,和12%乙腈的混合物。流动速率固定在0.4mL/min。样品注入体积为5μL。
结果:
在加入IPTG诱导苹果酸激酶,天冬氨酸半醛脱氢酶和DHB脱氢酶表达8h和24h之后,估算菌株大肠杆菌ECE1和对照菌株培养基中DHB的含量。如表16中所见,表达DHB通路酶的菌株ECE1产生比对照菌株明显更高量的DHB,证明了通过图1(i)中所示代谢途径发酵生产DHB的可行性。
表16:大肠杆菌ECE1和对照菌株培养基中DHB的浓度。
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Claims (18)
1.苹果酸半醛脱氢酶,其由SEQ ID No.54,SEQ ID No.56,SEQ ID No.58,SEQ IDNo.60,SEQ ID No.62,SEQ ID No.64,SEQ ID No.66或SEQ ID No.231所示。
2.分离的核酸序列,其编码根据权利要求1的苹果酸半醛脱氢酶。
3.权利要求2的分离的核酸序列,其由SEQ ID No.55,SEQ ID No.57,SEQ ID No.59,SEQ ID No.61,SEQ ID No.63,SEQ ID No.65,SEQ ID No.67,或SEQ ID No.230所示。
4.嵌合基因,包括至少在转录方向功能连接的,在宿主生物中有功能的启动子调控序列,根据权利要求2或3核酸序列,以及在所述宿主生物中有功能的终止子调节序列。
5.权利要求4的嵌合基因,包括在转录方向功能连接的:
-在宿主生物中有功能的启动子调节序列,
-根据权利要求2或3的核酸序列,
-由SEQ ID No.73或SEQ ID No.75或SEQ ID No.82所示的核酸序列,和
-在所述宿主生物中有功能的终止子调节序列。
6.权利要求5的嵌合基因,其由SEQ ID No.229所示。
7.表达载体,包含如权利要求2或3所要求的核酸,或者根据权利要求4到6中任一项所述的嵌合基因。
8.宿主微生物,其表达根据权利要求1的苹果酸半醛脱氢酶。
9.根据权利要求8的宿主微生物,其被用至少一种权利要求2到3的核酸和/或用至少一种根据权利要求4-6中任一项的嵌合基因来转化。
10.根据权利要求8或9的宿主微生物,其为细菌,酵母或真菌。
11.权利要求10的宿主微生物,更具体的其从大肠杆菌,酿酒酵母,谷氨酸棒杆菌,鲁氏酵母或黄曲霉中选择。
12.权利要求11的宿主微生物,其为大肠杆菌,进一步包含在表12所列至少一个基因中的断裂,和/或过量表达表14所列的至少一个基因。
13.生产2,4-DHB的方法,包括培养根据权利要求8到12任一项的,能表达苹果酸半醛脱氢酶的宿主微生物的步骤。
14.根据权利要求13的生产2,4-DHB的方法,其中在加入苹果酸或另外的有机酸培养基中培养宿主生物。
15.根据权利要求14的生产2,4-DHB的方法,其中有机酸为丙酮酸、琥珀酸或富马酸。
16.权利要求14的方法,其中培养基进一步包含另外的碳源。
17.根据权利要求13的生产2,4-DHB的方法,包括以过量生产苹果酸的微生物生产苹果酸或其他有机酸的第一步骤。
18.根据权利要求1苹果酸半醛脱氢酶将4-磷酸-苹果酸转化为苹果酸-4-半醛的用途。
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