CN114717208A - 一种酰基CoA合成酶及其应用 - Google Patents
一种酰基CoA合成酶及其应用 Download PDFInfo
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Abstract
本发明公开了一种酰基CoA合成酶及其应用，涉及基因工程、酶工程及组合生物合成领域，包括酰基CoA合成酶UkaQ及其突变体，酰基酰基CoA合成酶UkaQ氨基酸序列如SEQ ID NO：1所示；酰基CoA合成酶在高效生产天然产物聚酮化合物的应用。本发明为一种具有高效催化活性，底物谱宽泛且不受酰基化修饰调控的新型酰基‑CoA合成酶，该酰基‑CoA合成酶的鉴定和突变体的获取不仅扩展了腺苷合成酶超家族的多样性，而且为聚酮化合物及其他以酰基CoA为前体的天然产物的合成和碳骨架改造提供强有力的、通用的生物催化剂，对多种天然产物代谢过程的工程改造和工业化规模生产具有重要的应用和经济价值。
Description
技术领域
本发明涉及基因工程、酶工程及组合生物合成领域，尤其涉及一种酰基CoA合成酶及其应用。
背景技术
在生物体初级和次级代谢产物生物合成过程中，酰基CoA充当不同种类天然产物的前体，包括脂肪酸、聚酮化合物(polyketides)、聚醚(polyethers)、多烯(polyenes)、黄酮类(flavonoids)、芪类(stilbenes)生物碱和异戊二烯类等，上述化合物是发掘具有药用价值的天然产物的重要来源。在微生物生物体内酰基CoA的合成由酰基-CoA合成酶活化不同的羧酸底物生成，因此酰基-CoA合成酶是上述天然产物合成途径中的关键酶，被认为是调节相关产物合成的理想靶基因。
酰基-CoA合成酶属于腺苷合成酶家族(ANL superfamily)，其催化两步反应：腺苷形成反应(Adenylate Forming Reaction)和硫酯形成反应(Thioester FormingReaction)合成相应的硫酯。同时，酰基CoA合成酶拥有非常保守的10个基序，命名为MotifA1-A10。其中Motif 10中的Lys在腺苷形成酶家族中高度保守是催化腺苷化的关键活性位点。此外，酰基CoA合成酶也是最为典型的酰基化修饰底物，其中Motif 10中的Lys是发生酰基化修饰的关键赖氨酸位点。该位点的酰基化使得酰基-CoA合成酶失去催化活性，从而导致酰基CoA供应异常、胞内能量失衡、甚至还会引发细胞的生长缺陷。
由于该酶的重要性，许多酰基-CoA合成酶(acyl-CoA synthetase)的结构和功能已被解析，但其中大多数酰基-CoA合成酶的底物识别范围仅限于结构同源的衍生物，例如识别丙二酸以及甲基丙二酸的酰基-CoA合成酶(MatB)、识别肉桂酸及其衍生物的酰基-CoA合成酶(4CLs)、识别苯甲酸及其衍生物的酰基-CoA合成酶(BZLs)以及识别苯乙酸及其衍生物的酰基-CoA合成酶(PCLs)。但尚未鉴定对不同羧酸类的底物均具有高效催化的酰基CoA合成酶，而不依赖于Motif 10中的Lys的酰基-CoA合成酶更是未曾有过报道。因此开发具有高效催化活性，底物谱宽泛且不受酰基化修饰调控的新型酰基-CoA合成酶，具有重要的应用价值。
因此，本领域的技术人员致力于开发一种具有高效催化活性，底物谱宽泛且不受酰基化修饰调控的新型酰基-CoA合成酶。
发明内容
有鉴于现有技术的上述缺陷，本发明所要解决的技术问题是开发一种具有高效催化活性，底物谱宽泛且不受酰基化修饰调控的新型酰基-CoA合成酶及其突变体，并对其进行工程改造，将其应用到以酰基-CoA为前体的天然产物的组合生物合成中。
为实现上述目的，本发明提供了一种酰基CoA合成酶，包括酰基CoA合成酶UkaQ及其突变体，酰基CoA合成酶UkaQ氨基酸序列如SEQ ID NO：1所示。
进一步地，突变体包括突变体Q1、双突变株Q2和三突变株Q3；突变体Q1是酰基CoA合成酶UkaQ的第L526位置的亮氨酸(L)变成缬氨酸(V)突变体所得；双突变株Q2是突变体Q1第281位置的组氨酸(H)变成苯丙氨酸(F)所得；三突变株Q3是双突变株Q2第490位置的苯丙氨酸(F)变成丙氨酸(A)所得。
进一步地，突变体Q1的氨基酸序列如SEQ ID NO：2所示；双突变株Q2的氨基酸序列如SEQ ID NO：3所示；三突变株Q3的氨基酸序列如SEQ ID NO：4所示。
进一步地，酰基CoA合成酶与反应底物接触，进行催化反应，从而获得酰基-CoA产物；反应底物包括肉桂酸及其衍生物、3-苯基丙酸及其衍生物、苯甲酸及其衍生物、苯乙酸及其衍生物、饱和和不饱和脂肪酸及其衍生物、2-萘甲酸和喹哪啶酸双环芳香底物。
本发明还提供了一种酰基CoA合成酶在高效生产天然产物聚酮化合物的应用。
进一步地，包括以下步骤：
步骤1、制备聚酮化合物生产菌株；
步骤2、构建重组质粒，并将重组质粒回补，即通过接合转移的方法将重组质粒分别整合到步骤1得到的生产菌株的基因组上,得到相应的重组菌株；
步骤3、对步骤2得到的重组菌株通过喂养底物发酵；得到发酵产物；
步骤4、对步骤1得到的发酵产物进行分离和结构鉴定。
进一步地，步骤1中聚酮化合物生产菌株为生产脱酰基抗霉素的链霉菌。
进一步地，步骤2中重组质粒为包含酰基CoA合成酶UkaQ或/和其突变株Q3的整合质粒。
进一步地，步骤3中喂养底物为肉桂酸衍生物。
进一步地，步骤4中产物为新的抗霉素类似物DA18-DA23，抗霉素类似物DA18-DA23的结构式如结构式1-6所示：所述新的抗霉素类似物D18为7-(2-氯-苄基)-脱酰基抗霉素(7-(2-Cl-benzyl)-deacyl-ANT)、DA19为7-(3-氯-苄基)-脱酰基抗霉素(7-(3-Cl-benzyl)-deacyl-ANT)、DA20为7-(4-氯-苄基)-脱酰基抗霉素(7-(4-Cl-benzyl)-deacyl-ANT)、DA21为7-(2-溴-苄基)-脱酰基抗霉素(7-(2-Br-benzyl)-deacyl-ANT)、DA22为7-(3-溴-苄基)-脱酰基抗霉素(7-(3-Br-benzyl)-deacyl-ANT)、DA23为7-(4-溴-苄基)-脱酰基抗霉素(7-(4-Br-benzyl)-deacyl-ANT)。
本发明还提供了一种重组工程菌，包含酰基CoA合成酶及其突变体的核苷酸或在基因组中整合有酰基CoA合成酶UkaQ氨基酸序列如SEQ ID NO：1所示的核苷酸。
优选地，该工程菌为微生物(原核生物或真核生物)或植物；
更优选地，原核生物包括大肠杆菌，链霉菌，分枝杆菌等；
更优选地，真核生物包括酵母菌，丝状真菌等；
进一步地，植物工程菌包括烟草细胞或其他用于生产苯丙烷素化合物的植物工程菌。
在本发明的较佳实施方式1中，详细说明酰基-CoA合成酶UkaQ的序列分析；
在本发明的另一较佳实施方式2中，详细说明酰基-CoA合成酶UkaQ的表达载体构建和表达；
在本发明的较佳实施方式3中，详细说明酰基-CoA合成酶UkaQ的的生化功能鉴定；
在本发明的另一较佳实施方式4中，详细说明酰基-CoA合成酶UkaQ突变株的构建、蛋白表达和活性测试；
在本发明的较佳实施方式5中，详细说明酰基-CoA合成酶UkaQ及其突变体在天然产物生物合成中的应用。
本发明的效果和益处在于首次鉴定了一种具有新的催化机理的酰基CoA合成酶UkaQ，该酶不仅催化性能优良且不受酰基化修饰调控。同时本发明还构建了具有高稳定性和高催化性能的突变体Q1、Q2和Q3，该酰基-CoA合成酶的鉴定和突变体的获取不仅扩展了腺苷合成酶超家族的多样性，而且为聚酮化合物及其他以酰基CoA为前体的天然产物的合成和碳骨架改造提供强有力的、通用的生物催化剂，对多种天然产物代谢过程的工程改造和工业化规模生产具有重要的应用和经济价值。
1.本发明首次对酰基CoA合成酶UkaQ的氨基酸序列进行深入分析，揭示了其一级结构的独特性，是迄今鉴定的唯一一个不依赖MotifA10中的赖氨酸来完成腺苷化反应的酰基-CoA合成酶。
2.本发明鉴定的酰基CoA合成酶UkaQ打破了该家族酶经典的催化机制，为ANL家族增添了新的成员。
3.本发明构建的单点突变株Q4(L526V+E588K)失去催化活性，证明该酰基合成酶-CoA是依赖于Motif A10中谷氨酰胺(Glu)而不是赖氨酸来完成腺苷化反应，首次揭示了酰基CoA合成酶的这一新型的催化机制。
4.本发明首次对酰基CoA合成酶UkaQ进行体外表达和生化功能进行验证，公开了其底物识别范围，其催化底物谱展现出前所未有的宽泛性。进一步地，UkaQ可以高效的催化的底物包括肉桂酸及其衍生物、3-苯基丙酸及其衍生物、苯甲酸及其延伸物、苯乙酸及其衍生物、饱和和不饱和脂肪酸及其衍生物及其2-萘甲酸和喹哪啶酸等双环芳香底物，该酶底物谱是现有的酰基CoA合成酶所不能达到的。
5.本发明通过同源建模，对酰基CoA合成酶UkaQ的结构进行进一步的分析。合理设计其突变，其中突变株Q1(L526V)大大提升了蛋白的稳定性。
6.本发明构建的双突变株Q2(L526V+H281F)增强了该酶对肉桂酸及其衍生物的催化活性。
7.本发明构建的三突变株Q3(L526V+H281F+F490A)进一步拓宽了UkaQ的底物识别范围，是迄今鉴定的第一个可识别4-咪唑丙烯酸合成相应硫酯产物的酰基-CoA合成酶。
8.本发明还提供了上述酰基CoA合成酶UkaQ和其突变株Q3在高效生产天然产物聚酮化合物的应用,获得了新的脱酰基抗霉素类似物，极大地丰富了抗霉素的结构多样性，为开发具有更好生物活性的聚酮化合物提供了候选库。同时含酰基CoA合成酶UkaQ和其突变株Q3的重组菌株与不含UkaQ的菌株相比，产物的产量大幅度提高。
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明，以充分地了解本发明的目的、特征和效果。
附图说明
图1是本发明的一个较佳实施例1的一个新型酰基-CoA合成酶UkaQ和其他经典的酰基-CoA合成酶多序列比对结果；
图2是本发明的一个较佳实施例2的新型酰基-CoA合成酶UkaQ和其较佳突变体Q1、Q2和Q3和Q4的蛋白SDS-PAGE电泳结果图；
图3是本发明的一个较佳实施例3的UkaQ及其突变体催化的38个羧酸底物的结构式；
附图4是本发明的一个较佳实施例4的较佳突变株Q1、Q2和Q3的催化活力测试结果；
附图5是本发明的一个较佳实施例5的质粒构建图谱；
附图6是本发明的一个较佳实施例5的产DA-18,DA-19,DA-20,DA-21,DA-22,DA-23的结构式和质谱分析结果；
附图7是本发明的一个较佳实施例5的重组工程菌株mWHU2489的发酵结果；
附图8是本发明的一个较佳实施例5的重组工程菌株mWHU2490的发酵结果；
附图9是本发明的一个较佳实施例5的重组工程菌株mWHU2491的发酵结果；
附图10是本发明的一个较佳实施例5的化合物DA-18的核磁和结构解析；
附图11是本发明的一个较佳实施例5的化合物DA-19的核磁和结构解析；
附图12是本发明的一个较佳实施例5的化合物DA-20的核磁和结构解析；
附图13是本发明的一个较佳实施例5的化合物DA-22的核磁和结构解析；
附图14是本发明的一个较佳实施例5的化合物DA-23的核磁和结构解析；
附图15是本发明的一个较佳实施例5的化合物DA-18的氢谱图；
附图16是本发明的一个较佳实施例5的化合物DA-18的碳谱图；
附图17是本发明的一个较佳实施例5的化合物DA-18的1H的异核多碳相关谱(HMBC)；
附图18是本发明的一个较佳实施例5的化合物DA-18的1H的异核多量子关系图(HMQC)；
附图19是本发明的一个较佳实施例5的化合物DA-18的1H的同核化学位移谱图(COSY)；
附图20是本发明的一个较佳实施例5的化合物DA-19的氢谱图；
附图21是本发明的一个较佳实施例5的化合物DA-19的碳谱图；
附图22是本发明的一个较佳实施例5的化合物DA-19的1H的异核多碳相关谱(HMBC)；
附图23是本发明的一个较佳实施例5的化合物DA-19的1H的异核多量子关系图(HMQC)；
附图24是本发明的一个较佳实施例5的化合物DA-19的1H的同核化学位移谱图(COSY)；
附图25是本发明的一个较佳实施例5的化合物DA-20的氢谱图；
附图26是本发明的一个较佳实施例5的化合物DA-20的碳谱图；
附图27是本发明的一个较佳实施例5的化合物DA-20的1H的异核多碳相关谱(HMBC)；
附图28是本发明的一个较佳实施例5的化合物DA-20的1H的异核多量子关系图(HMQC)；
附图29是本发明的一个较佳实施例5的化合物DA-20的1H的同核化学位移谱图(COSY)；
附图30是本发明的一个较佳实施例5的化合物DA-22的氢谱图；
附图31是本发明的一个较佳实施例5的化合物DA-22的碳谱图；
附图32是本发明的一个较佳实施例5的化合物DA-22的1H的异核多碳相关谱(HMBC)；
附图33是本发明的一个较佳实施例5的化合物DA-22的1H的异核多量子关系图(HMQC)；
附图34是本发明的一个较佳实施例5的化合物DA-22的1H的同核化学位移谱图(COSY)；
附图35是本发明的一个较佳实施例5的化合物DA-23的氢谱图分析结果；
附图36是本发明的一个较佳实施例5的化合物DA-23的碳谱图分析结果；
附图37是本发明的一个较佳实施例5的化合物DA-23的1H的异核多碳相关谱(HMBC)分析结果；
附图38是本发明的一个较佳实施例5的化合物DA-23的1H的异核多量子关系图(HMQC)；
附图39是本发明的一个较佳实施例5的化合物DA-23的1H的同核化学位移谱图(COSY)；
附图40是本发明的一个较佳实施例5的化合物DA-21的二级质谱图(MS/MS)。
具体实施方式
以下参考说明书附图介绍本发明的多个优选实施例，使其技术内容更加清楚和便于理解。本发明可以通过许多不同形式的实施例来得以体现，本发明的保护范围并非仅限于文中提到的实施例。
下述实施例中涉及的大肠杆菌菌株E.coli BL21(DE3)和E.coli DH5α购买自北京全式金生物技术有限公司。
下述实施例中培养基配方：
LB液体培养基：10g/LNaCl、10g/L蛋白胨和5g/L酵母粉，定容至1L，0.1Mpa压力，121℃下灭菌20min。下述实施例中缓冲液(Hepes buffer)配方：50mM Hepes,50mM NaCl,PH＝7.5。
实施例1.UkaQ的序列分析
(1)UkaQ的序列分析
将UkaQ与已被解析的酰基CoA合成酶的氨基酸序列利用多序列比对软件ClustalW进行比对，结合该类酶保守基序MotifA1-MotifA10的特征，找到UkaQ的保守基序MotifA1-MotifA10。其中用来做对比的酰基CoA合成酶序列是来自毛白杨的4-香豆酸-CoA连接酶3A9U、来自烟草的4-香豆酸-CoA连接酶5U95、来自沼泽红假单胞菌的苯甲酸-CoA连接酶4EAT、来自天蓝色链霉菌的丙二酰CoAA3NYQ、来自新洋葱伯克霍尔德氏菌的苯乙酸-CoA连接酶2Y4O。多序列比对结果如图1所示，其中标注的“Lysing的位置”是酰基辅酶A合成酶保守基序(MotifA10)中负责催化腺苷化反应的关键位点。
序列必比对结果显示，UkaQ的保守基序MotifA1-MotifA9的特征和同功能酶的特征一致，但其MotifA10中不含负责催化腺苷化反应的关键活性位点赖氨酸(Lysing)，取而代之的是一个提供负电子的谷氨酰胺残基(Glu)。这是迄今鉴定的唯一一个不依赖MotifA10中的赖氨酸来完成腺苷化反应的酰基-CoA合成酶。
实施例2.UkaQ的表达载体构建和表达
(1)采用以下步骤获得编码UkaQ的基因
以UKaQ的氨基酸序列SEQ ID NO:1(GenBank:AQN08598.1)为模板，通过反向翻译转换成基因的核酸序列，全合成UkaQ基因DNA，合成在通用载体PUC57上。
(2)表达载体p-UkaQ和重组菌株的构建
将UkaQ基因DNA和pET28a质粒分别用NdeI/XhoI酶切并回收，酶连后转化至EcoliDH5α感受态，并筛选正确的重组菌，提取质粒验证成功后命名为p-UkaQ。
(3)采用以下步骤对p-UkaQ进行体外表达
将实施例1中重组质粒p-UkaQ转化至E.coli BL21(DE3)感受态，于37℃倒置过夜培养，从上述培养过夜的平板上挑取单克隆至装有5ml含终浓度为50μg/ml硫酸卡那霉素的LB液体培养基(配方为Tryptone 10g/L,Yeast Extract 5g/L,氯化钠10g/L)，于37℃、220rpm振荡培养过夜。次日，从上述过夜培养的菌液吸取3ml培养液，加入到新鲜的300ml含终浓度为50μg/ml硫酸卡那霉素的LB液体培养基，于37℃，220rpm振荡培养至OD值约为0.6时，冷却至18℃加入终浓度为0.1mM的IPTG诱导，于18℃，220rpm继续振荡培养20h。离心(4000rpm，10min，4℃)收集菌体。将细胞重新悬浮在30mL裂解缓冲液(25mM HEPES、300mMNaCl、10％甘油，pH7.5)中并通过超声裂解。高速离心(12000rpm，30min，4℃)后，将1mLNi-NTA琼脂糖树脂添加到上清液中，并将溶液在0℃下振荡1h。将蛋白质树脂混合物加载到重力流柱上，并用含不同浓度咪唑的缓冲液A(25mM HEPES、300mM NaCl、10％甘油、pH值7.5)进行洗脱。通过12％丙烯酰胺SDS-PAGE验证每管洗脱液中蛋白的表达情况，收集目的仅含目的蛋白的洗脱液到PD-10脱盐柱中，并使用缓冲液B(25mM HEPES、50mM NaCl、10％甘油，pH 7.5)进行脱盐，并使用Nanodrop 2000分光光度计(Thermo Scientific)基于280nm的吸光度确定浓度。蛋白表达结果显示：酰基CoA合成酶UkaQ的纯化后的电泳结果如图2所示，其在表达过程中发生部分降解，降解后的蛋白大小比原蛋白小10KDa左右。
实施例3：UkaQ的生化功能鉴定
UkaQ的体外活性测定体系组成如下：1.5mM羧酸底物、2mMATP、1.5mM COA、5mM Mg2 +、50mM Hepes Buffer(pH 7.5)和1μM UkaQ，总体积200μl，反应在1.5mL的EP管中进行，以不添加UkaQ或其突变蛋白的反应作为对照进行。反应在25℃下孵育1h，加入等体积的甲醇淬灭,12,000rmp下离心20min去除蛋白后，取1μl样品上清液进行UPLC分析。所有样品的UPLC分析均在Eclipse XDB-C18分析柱上进行，流速为0.2mL min-1，分析程序如下：T＝0min，5％B；T＝3min，5％B；T＝10min，65％B；T＝12min，5％B；T＝15min，5％B(溶剂A:含10mM CH3COONH4的H2O，溶剂B:CH3CN)。对于LC-HRMS分析，条件与UPLC分析相同。其中测试底物的结构如图3所示，测试底物包括：
A)部分所示的肉桂酸系列，底物1a-14a，分别为：肉桂酸、邻氯-肉桂酸、间氯-肉桂酸、对氯-肉桂酸、对氟-肉桂酸、邻溴-肉桂酸、间溴-肉桂酸、对溴-肉桂酸、间羟基-肉桂酸、对羟基-肉桂酸、2-甲基-肉桂酸、4-甲基-肉桂酸、4-氨基-肉桂酸、对甲氧基-肉桂酸；
B)部分所示的脂肪酸系列，底物15a-26a,分别为：正丁酸、正戊酸、正己酸、正庚酸、正辛酸、正癸酸、巴豆酸、反-戊烯酸、2-己烯酸、正壬酸、2-辛烯酸、2-癸烯酸；
C)部分所示的其他系列的羧酸,底物27a-38a,分别为：3-苯基丙酸、环己基丙烯、环己基丙酸、2-萘甲酸、喹哪啶酸、3-吲哚-丙烯酸、4-咪唑-丙烯酸、苯甲酸、环己甲酸、对-羟基苯乙酸、对甲基苯乙酸、2-甲基苯乙酸。
活性测试结果显示，除底物苯甲酸(34a)对羟基苯乙酸(36a)和4-咪唑丙烯酸(33a)外，其余底物均可被UkaQ催化生成相应的酰基-CoA。
实施例4：UkaQ突变株的构建、蛋白表达和活性测试
1)UkaQ突变株Q1(L526V)的构建
以p-UkaQ质粒作为模板，以L526V-F/R为引物进行滚环突变PCR，引物由上海擎科生物科技公司进行合成。
定点突变引物如下：
L526V-F：GCTGGCTTGGGTGGATCCCGCTG
L526V-R：CAGCGGGATCCACCCAAGCCAGC
得到定点突变目标质粒片段，取2μL PCR产物用经琼脂糖凝胶电泳验证条带大小。PCR产物经DpnI酶消化3h后，进行大肠化转，转化方法如实施例2所示，分别挑取转化子经小量LB培养基培养后提取质粒，送去上海擎科生物科技有限公司进行测序鉴定。将鉴定正确的突变体质粒命名为Q1(L526V)。
2)UkaQ突变株Q2(L526V+H281F)的构建
以质粒Q1作为模板，以H281F-F/RR为引物进行滚环突变PCR，定点突变引物如下：
H281F-F:GGGGGCAACTTCAACGTCAATTTAG
H281F-R：CCCCCGTTGAAGTTGCAGTTAAATC
将鉴定正确的突变体质粒命名为Q2(L526V+H281F)
3)UkaQ突变株Q3(L526V+H281F+F490A)的构建
以质粒Q2作为模板，以F490A-F/R为引物进行滚环突变PCR，定点突变引物如下：
F490A-F：GACCGGCACCGCGGTCCATGTCG
F490A-R：CTGGCCGTGGCGCCAGGTACAGC
将鉴定正确的突变体质粒命名为Q3(L526V+H281F+F490A)
4)UkaQ突变株Q4(L526V+E588K)的构建
以质粒Q1作为模板，以E588K-F/R为引物进行滚环突变PCR，定点突变引物如下：
E588K-F：GACCTCGACGCGGGCGAAATTACGGACAAG
E588K-R：CTTGTCCGTAATTTCGCCCGCGTCGAGGTC
将鉴定正确的突变体质粒命名为Q4(L526V+E588K)
5)突变株Q1、Q2和Q3和Q4的表达
采用实施例1中的表达方法对突变株Q1、Q2和Q3和Q4进行蛋白表达。
蛋白纯化结果显示，与野生株相比，突变株Q1展现出较佳的稳定性，在表达过程中基本未发生降解。此外，在Q1的基础上进行突变得到的双突变株Q2、Q4和三突变株Q3也表现出较好的稳定性。结果如图2所示。
6)UkaQ突变株Q1、Q2和Q3的Q4催化活性测试
活性测试比较通过无机焦磷酸盐试剂盒(EnzChek PyrophosphateAssay Kit：E-6645，Invitrogen)结合连续分光光度法测定。反应体系(300μL)包含1x缓冲液和焦磷酸盐测定试剂盒的两种酶，10mM ATP、10mM COA、5mM MgCl2和0.1μM Q1或Q2或Q3或Q4。将反应体系混合物(底物除外)在22℃下孵育10分钟。通过向混合物中加入相应羧酸底物开始反应，当反应开始时，立即对混合物进行紫外/可见分光光度计测定读取360nm处的吸光度。比活力按公式7计算：
公式7：
活性测试结果如图4所示，突变体Q2对肉桂酸及其衍生物的活性相比Q1大大提高。突变体Q3在Q2的基础上拓宽了该酰基-CoA合成酶的底物谱，其可以催化底物苯甲酸(34a)对羟基苯乙酸(36a)和4-咪唑丙烯酸(33a)生成相应的酰基-CoA产物，进一步地，突变体Q4则失去了催化活性，说明，该酰基-CoA合成酶为了抵御自身被酰基化修饰，进化出一种新的催化机制，使得催化腺苷化反应不再依赖于Motif中的赖氨酸，从而避免在生物体内因酰基化修饰而失去催化活性。
实施例5：UkaQ及其突变体在天然产物生物合成中的应用
本发明还提供了上述酰基-CoA合成酶及其突变体在生物合成聚酮化合物脱酰基抗霉素中的应用，包括以下步骤：
1)脱酰基抗霉素生产菌株的改造
我们以在前期研究中构建的一株脱酰基抗霉素的高产菌株(mWHU2987)为目的菌株，为了拓宽其合成途径相关酶的底物宽泛性从而将目的延伸单元引入到抗霉素的碳骨架中，制备菌株mWHU2488，作为重组菌株构建的底盘菌株。
2)含UkaQ或其突变体的重组工程菌的构建
分别用相应的引物扩增目的基因antEV350G、spnD和ukaQ以及Q3，并将其连在带有启动子kasOp整合质粒PIB139-kasOp*上，得到3个基因盒，如图5所示，分别是pWHU2051(kasOp*-antEV350G-spnD),pWHU2052(kasOp*-antEV350G-spnD-ukaQ)和pWHU2053(kasOp*-antEV350G-spnD-Q3)，并进行测序验证。将上述3个重组质粒转化至大肠杆菌ET12567中，通过接合转移的方法将这3个质粒分别整合到菌株mWHU2488的基因组上,得到相应的重组菌株mWHU2489(ΔantD-antE::kasOp*-antEV350G-spnD),mWHU2490(ΔantD-antE::kasOp*-antEV350G-spnD-ukaQ)和mWHU2491(ΔantD-antE::kasOp*-antEV350G-spnD-Q3)。其中不含酰基-CoA合成酶UkaQ或其突变株基因的重组菌株mWHU2489作为对照菌株。
3)重组菌株的发酵和喂养
具体分为以下3步：
1)液体培养：将上述重组菌株mWHU2489,mWHU2490和mWHU2491分别接种于A3发酵培养基(棉子饼粉10g，可溶性淀粉10g，(NH4)2SO43g，CaCO35g，NaCl3g，琼脂粉20g，酵母提取物0.35g，胰蛋白胨0.35g，MgCl20.95 g，调节pH至7.5，去离子水定容至1L)各7瓶，30℃，220rpm振荡培养24h。
2)底物喂养：将相应的羧酸底物(2a-4a,6a-8a)用DMSO溶解，分别投喂到上述发酵培养基中，终浓度1mM。30℃，220rpm继续振荡培养72h。以不加底物的发酵液作为各自的对照组。
3)样品处理，发酵液进行HPLC检测:将上述发酵液用等体积的乙酸乙酯萃取，萃取液旋干后用1mL的甲醇溶解，过0.22μm有机滤膜至液相瓶中进行HPLC检测。其中，DA-18,DA-19,DA-20,DA-21,DA-22,DA-23的结构式和质谱分析结果如图6所示。
HPLC检测发酵结果显示：产物DA-18和DA-21仅在菌株含有酰基-CoA合成酶Q3基因的菌株mWHU2491中产生且DA-18的产量达5.7mg/L。对于产物DA-19，mWHU2491的最终产量达到9.1mg/L，是mWHU2489(4.5mg/L)的2倍和mWHU2489(6.7mg/L)的1.4倍。通过喂养4-氯肉桂酸(4a)，产生产物DA-20，且在mWHU2491的产量约为14.3mg/L，mWHU2490为9.8mg/L，mWHU2489为8.1mg/L。通过喂养3-溴肉桂酸(7a)，从重组菌株mWHU2491和mWHU2490中分别以13.6mg/L和10.2mg/L的产量分离到产物DA-22，而在菌株mWHU2489中未检测到产物。当喂养4-溴肉桂酸(8a)时，重组菌株mWHU2491产生8.4mg/L DA-23，是菌株mWHU2489(2.4mg/L)产量的3.5倍和菌株mWHU2489(2.6mg/L)产量的3.2倍。重组工程菌株mWHU2489的发酵结果如图7所示；I)为未改造的菌株2487的发酵液相分析图，II)为菌株mWHU2489未喂养底物的发酵结果，III)-VII)是菌株2489分别喂养底物2a-8a的发酵结果。
重组工程菌株mWHU2490的发酵结果如图8所示；I)为未改造的菌株2487的发酵液相分析图，II)为菌株mWHU2490未喂养底物的发酵结果，III)-VII)是菌株2490分别喂养底物2a-8a的发酵结果。
重组工程菌株mWHU2491的发酵结果如图9所示。I)为未改造的菌株2487的发酵液相分析图，II)为菌株mWHU2491未喂养底物的发酵结果，III)-VII)是菌株2491分别喂养底物2a-8a的发酵结果。
将产物进行核磁和结构解析，化合物DA-18的核磁和结构解析分析结果如图10所示；化合物DA-19的核磁和结构解析分析结果如图11所示；化合物DA-20的核磁和结构解析分析结果如图12所示；化合物DA-22的核磁和结构解析分析结果如图13所示；化合物DA-23的核磁和结构解析分析结果如图14所示；化合物DA-18的氢谱图分析结果如图15所示；化合物DA-18的碳谱图分析结果如图16所示；化合物DA-18的1H的异核多碳相关谱(HMBC)分析结果如图17所示；化合物DA-18的1H的异核多量子关系图(HMQC)分析结果如图18所示；化合物DA-18的1H的同核化学位移谱图(COSY)分析结果如图19所示；化合物DA-19的氢谱图分析结果如图20所示；化合物DA-19的碳谱图分析结果如图21所示；化合物DA-19的1H的异核多碳相关谱(HMBC)分析结果如图22所示；化合物DA-19的1H的异核多量子关系图(HMQC)分析结果如图23所示；化合物DA-19的1H的同核化学位移谱图(COSY)分析结果如图24所示；化合物DA-20的氢谱图分析结果如图25所示；化合物DA-20的碳谱图分析结果如图26所示；化合物DA-20的1H的异核多碳相关谱(HMBC)分析结果如图27所示；化合物DA-20的1H的异核多量子关系图(HMQC)分析结果如图28所示；化合物DA-20的1H的同核化学位移谱图(COSY)分析结果如图29所示；化合物DA-22的氢谱图分析结果如图30所示；化合物DA-22的碳谱图分析结果如图31所示；化合物DA-22的1H的异核多碳相关谱(HMBC)分析结果如图32所示；化合物DA-22的1H的异核多量子关系图(HMQC)分析结果如图33所示；化合物DA-22的1H的同核化学位移谱图(COSY)分析结果如图34所示；化合物DA-23的氢谱图分析结果如图35所示；化合物DA-23的碳谱图分析结果如图36所示；化合物DA-23的1H的异核多碳相关谱(HMBC)分析结果如图37所示；化合物DA-23的1H的异核多量子关系图(HMQC)分析结果如图38所示；化合物DA-23的1H的同核化学位移谱图(COSY)分析结果如图39所示；化合物DA-21的二级质谱图(MS/MS)分析结果如图40所示。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。
序列表
<110> 上海交通大学
<120> 一种酰基CoA合成酶及其应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 628
<212> PRT
<213> 人工序列 (Artifical sequence)
<220>
<221> MUTAGEN
<222> (586)
<223> UkaQ
<400> 1
Met Pro His Pro Thr Thr Arg Thr Ala Pro His Ser Ala Ala Gly Gly
1 5 10 15
Thr Thr Ala Gly Glu Thr Ser Ser Pro Leu Phe Ala Pro Ala Arg Thr
20 25 30
Val Arg Arg Asp Arg Pro Asp Gly Thr Val Leu Leu Ser Ser Ala Gln
35 40 45
Pro Leu Gly Val Tyr Pro Ala Ser Val Thr Asp His Leu Arg Thr Trp
50 55 60
Ala Gln Ala Gly Pro Asp Arg Pro Leu Val Ala Glu Arg Gly Ala Asp
65 70 75 80
Gly Arg Trp Gly His Arg Thr Tyr Gly Glu Val Leu Ala Ala Ala Glu
85 90 95
Ala Val Gly Gln Ala Leu Leu Asp Arg Gly Leu Ser Ala Arg Arg Pro
100 105 110
Leu Met Val Leu Ser Gly Asn Ser Thr Gly His Leu Leu Met Thr Leu
115 120 125
Gly Ala Leu Ser Ala Gly Ile Pro Val Ala Pro Val Ser Val Ala Tyr
130 135 140
Ser Leu Leu Ser Arg Asp His Ala Arg Ile Arg Ala Ile Ala Glu Leu
145 150 155 160
Leu Arg Pro Gly Ala Val Tyr Ala Glu Asp Ala Gly Pro Phe Gly Pro
165 170 175
Ala Leu Ala Ala Ala Gly Gly Gly Ala Ile Val Val Ala Ala Arg Gly
180 185 190
Gly Pro Ala Glu His Ser Leu Asp Ala Leu Leu Arg Thr Val Pro Gly
195 200 205
Arg Ala Phe Glu Ala Ala Arg Ala Gly Val Thr Ser Ala Thr Val Ala
210 215 220
Lys Val Leu Phe Thr Ser Gly Ser Thr Gly Ala Pro Lys Gly Val Val
225 230 235 240
Thr Thr His Gly Met Leu Cys Ala Asn Gln Arg Met Met Arg Gln Val
245 250 255
Trp Pro Phe Leu Ala Gly Glu Arg Pro Val Leu Leu Asp Trp Leu Pro
260 265 270
Trp Ser His Thr Phe Gly Gly Asn His Asn Val Asn Leu Val Leu Ala
275 280 285
Asn Gly Gly Thr Leu Tyr Leu Asp Asp Gly Arg Pro Thr Pro Glu Leu
290 295 300
Phe Gly Arg Thr Leu Ala Asn Leu Arg Glu Val Ser Pro Thr Leu Ala
305 310 315 320
Phe Asn Val Pro Ala Gly Tyr Ala Arg Leu Val Pro Ala Leu Glu Arg
325 330 335
Asp Arg Glu Leu Ala Glu Arg Phe Phe Ala Arg Leu Arg Leu Val Phe
340 345 350
Asn Ala Ala Ala Ala Leu Ala Pro Ala Leu Arg Glu Arg Leu Arg Ala
355 360 365
Leu Gly Arg Glu Val Thr Gly Arg Asp Val Pro Val Thr Gly Ser Trp
370 375 380
Gly Ala Thr Glu Thr Ser Pro Ala Ser Thr Ser Ala His Phe Pro Phe
385 390 395 400
Thr Asp Pro Arg Cys Ile Gly Val Pro Leu Pro Gly Val Glu Leu Lys
405 410 415
Leu Val Pro Ala Glu Gly Asp Gly Tyr Glu Val Arg Val Arg Gly Pro
420 425 430
His Val Thr Pro Gly Tyr Leu Gly Arg Pro Asp Leu Asp Ala Arg Ala
435 440 445
Phe Asp Glu Glu Gly Tyr Tyr Arg Pro Gly Asp Ala Val Ala Phe Ala
450 455 460
Asp Pro Gly Asp Ala Gly Ala Gly Leu Val Phe Arg Gly Arg Leu Thr
465 470 475 480
Glu Asp Phe Lys Leu Ser Thr Gly Thr Phe Val His Val Glu Ala Val
485 490 495
Arg Gly Ala Leu Leu Ser Ala Ala Pro Val Leu Ser Asp Ala Val Ile
500 505 510
Thr Gly Glu His Arg Asp Ala Val Cys Ala Leu Ala Trp Leu Asp Pro
515 520 525
Ala Glu Ala Glu Arg Leu Leu Gly Arg Arg Pro Ala Ala Asp Gly Gly
530 535 540
Val Leu Tyr Ser Asp Ala Leu Ala Ala His Leu Gly Ala Ala Leu Glu
545 550 555 560
Arg Leu Asn Arg Gly Ala Gly Ser Ala Ser Arg Val Gln Arg Leu Leu
565 570 575
Val Leu Ala Asp Pro Pro Asp Leu Asp Ala Gly Glu Ile Thr Asp Lys
580 585 590
Gly Tyr Val Asn Gln Arg Arg Val Leu Ala Ala Arg Ala Pro Leu Val
595 600 605
Ala Arg Leu His Ala Asp Pro Ala Pro Arg His Val Ile Thr Pro Arg
610 615 620
Ser Gly Leu Thr
625
<210> 2
<211> 628
<212> PRT
<213> 人工序列 (Artifical sequence)
<220>
<221> MUTAGEN
<222> (526)
<223> Q1
<400> 2
Met Pro His Pro Thr Thr Arg Thr Ala Pro His Ser Ala Ala Gly Gly
1 5 10 15
Thr Thr Ala Gly Glu Thr Ser Ser Pro Leu Phe Ala Pro Ala Arg Thr
20 25 30
Val Arg Arg Asp Arg Pro Asp Gly Thr Val Leu Leu Ser Ser Ala Gln
35 40 45
Pro Leu Gly Val Tyr Pro Ala Ser Val Thr Asp His Leu Arg Thr Trp
50 55 60
Ala Gln Ala Gly Pro Asp Arg Pro Leu Val Ala Glu Arg Gly Ala Asp
65 70 75 80
Gly Arg Trp Gly His Arg Thr Tyr Gly Glu Val Leu Ala Ala Ala Glu
85 90 95
Ala Val Gly Gln Ala Leu Leu Asp Arg Gly Leu Ser Ala Arg Arg Pro
100 105 110
Leu Met Val Leu Ser Gly Asn Ser Thr Gly His Leu Leu Met Thr Leu
115 120 125
Gly Ala Leu Ser Ala Gly Ile Pro Val Ala Pro Val Ser Val Ala Tyr
130 135 140
Ser Leu Leu Ser Arg Asp His Ala Arg Ile Arg Ala Ile Ala Glu Leu
145 150 155 160
Leu Arg Pro Gly Ala Val Tyr Ala Glu Asp Ala Gly Pro Phe Gly Pro
165 170 175
Ala Leu Ala Ala Ala Gly Gly Gly Ala Ile Val Val Ala Ala Arg Gly
180 185 190
Gly Pro Ala Glu His Ser Leu Asp Ala Leu Leu Arg Thr Val Pro Gly
195 200 205
Arg Ala Phe Glu Ala Ala Arg Ala Gly Val Thr Ser Ala Thr Val Ala
210 215 220
Lys Val Leu Phe Thr Ser Gly Ser Thr Gly Ala Pro Lys Gly Val Val
225 230 235 240
Thr Thr His Gly Met Leu Cys Ala Asn Gln Arg Met Met Arg Gln Val
245 250 255
Trp Pro Phe Leu Ala Gly Glu Arg Pro Val Leu Leu Asp Trp Leu Pro
260 265 270
Trp Ser His Thr Phe Gly Gly Asn His Asn Val Asn Leu Val Leu Ala
275 280 285
Asn Gly Gly Thr Leu Tyr Leu Asp Asp Gly Arg Pro Thr Pro Glu Leu
290 295 300
Phe Gly Arg Thr Leu Ala Asn Leu Arg Glu Val Ser Pro Thr Leu Ala
305 310 315 320
Phe Asn Val Pro Ala Gly Tyr Ala Arg Leu Val Pro Ala Leu Glu Arg
325 330 335
Asp Arg Glu Leu Ala Glu Arg Phe Phe Ala Arg Leu Arg Leu Val Phe
340 345 350
Asn Ala Ala Ala Ala Leu Ala Pro Ala Leu Arg Glu Arg Leu Arg Ala
355 360 365
Leu Gly Arg Glu Val Thr Gly Arg Asp Val Pro Val Thr Gly Ser Trp
370 375 380
Gly Ala Thr Glu Thr Ser Pro Ala Ser Thr Ser Ala His Phe Pro Phe
385 390 395 400
Thr Asp Pro Arg Cys Ile Gly Val Pro Leu Pro Gly Val Glu Leu Lys
405 410 415
Leu Val Pro Ala Glu Gly Asp Gly Tyr Glu Val Arg Val Arg Gly Pro
420 425 430
His Val Thr Pro Gly Tyr Leu Gly Arg Pro Asp Leu Asp Ala Arg Ala
435 440 445
Phe Asp Glu Glu Gly Tyr Tyr Arg Pro Gly Asp Ala Val Ala Phe Ala
450 455 460
Asp Pro Gly Asp Ala Gly Ala Gly Leu Val Phe Arg Gly Arg Leu Thr
465 470 475 480
Glu Asp Phe Lys Leu Ser Thr Gly Thr Phe Val His Val Glu Ala Val
485 490 495
Arg Gly Ala Leu Leu Ser Ala Ala Pro Val Leu Ser Asp Ala Val Ile
500 505 510
Thr Gly Glu His Arg Asp Ala Val Cys Ala Leu Ala Trp Val Asp Pro
515 520 525
Ala Glu Ala Glu Arg Leu Leu Gly Arg Arg Pro Ala Ala Asp Gly Gly
530 535 540
Val Leu Tyr Ser Asp Ala Leu Ala Ala His Leu Gly Ala Ala Leu Glu
545 550 555 560
Arg Leu Asn Arg Gly Ala Gly Ser Ala Ser Arg Val Gln Arg Leu Leu
565 570 575
Val Leu Ala Asp Pro Pro Asp Leu Asp Ala Gly Glu Ile Thr Asp Lys
580 585 590
Gly Tyr Val Asn Gln Arg Arg Val Leu Ala Ala Arg Ala Pro Leu Val
595 600 605
Ala Arg Leu His Ala Asp Pro Ala Pro Arg His Val Ile Thr Pro Arg
610 615 620
Ser Gly Leu Thr
625
<210> 3
<211> 628
<212> PRT
<213> 人工序列 (Artifical sequence)
<220>
<221> MUTAGEN
<222> (281)
<223> Q2
<400> 3
Met Pro His Pro Thr Thr Arg Thr Ala Pro His Ser Ala Ala Gly Gly
1 5 10 15
Thr Thr Ala Gly Glu Thr Ser Ser Pro Leu Phe Ala Pro Ala Arg Thr
20 25 30
Val Arg Arg Asp Arg Pro Asp Gly Thr Val Leu Leu Ser Ser Ala Gln
35 40 45
Pro Leu Gly Val Tyr Pro Ala Ser Val Thr Asp His Leu Arg Thr Trp
50 55 60
Ala Gln Ala Gly Pro Asp Arg Pro Leu Val Ala Glu Arg Gly Ala Asp
65 70 75 80
Gly Arg Trp Gly His Arg Thr Tyr Gly Glu Val Leu Ala Ala Ala Glu
85 90 95
Ala Val Gly Gln Ala Leu Leu Asp Arg Gly Leu Ser Ala Arg Arg Pro
100 105 110
Leu Met Val Leu Ser Gly Asn Ser Thr Gly His Leu Leu Met Thr Leu
115 120 125
Gly Ala Leu Ser Ala Gly Ile Pro Val Ala Pro Val Ser Val Ala Tyr
130 135 140
Ser Leu Leu Ser Arg Asp His Ala Arg Ile Arg Ala Ile Ala Glu Leu
145 150 155 160
Leu Arg Pro Gly Ala Val Tyr Ala Glu Asp Ala Gly Pro Phe Gly Pro
165 170 175
Ala Leu Ala Ala Ala Gly Gly Gly Ala Ile Val Val Ala Ala Arg Gly
180 185 190
Gly Pro Ala Glu His Ser Leu Asp Ala Leu Leu Arg Thr Val Pro Gly
195 200 205
Arg Ala Phe Glu Ala Ala Arg Ala Gly Val Thr Ser Ala Thr Val Ala
210 215 220
Lys Val Leu Phe Thr Ser Gly Ser Thr Gly Ala Pro Lys Gly Val Val
225 230 235 240
Thr Thr His Gly Met Leu Cys Ala Asn Gln Arg Met Met Arg Gln Val
245 250 255
Trp Pro Phe Leu Ala Gly Glu Arg Pro Val Leu Leu Asp Trp Leu Pro
260 265 270
Trp Ser His Thr Phe Gly Gly Asn Phe Asn Val Asn Leu Val Leu Ala
275 280 285
Asn Gly Gly Thr Leu Tyr Leu Asp Asp Gly Arg Pro Thr Pro Glu Leu
290 295 300
Phe Gly Arg Thr Leu Ala Asn Leu Arg Glu Val Ser Pro Thr Leu Ala
305 310 315 320
Phe Asn Val Pro Ala Gly Tyr Ala Arg Leu Val Pro Ala Leu Glu Arg
325 330 335
Asp Arg Glu Leu Ala Glu Arg Phe Phe Ala Arg Leu Arg Leu Val Phe
340 345 350
Asn Ala Ala Ala Ala Leu Ala Pro Ala Leu Arg Glu Arg Leu Arg Ala
355 360 365
Leu Gly Arg Glu Val Thr Gly Arg Asp Val Pro Val Thr Gly Ser Trp
370 375 380
Gly Ala Thr Glu Thr Ser Pro Ala Ser Thr Ser Ala His Phe Pro Phe
385 390 395 400
Thr Asp Pro Arg Cys Ile Gly Val Pro Leu Pro Gly Val Glu Leu Lys
405 410 415
Leu Val Pro Ala Glu Gly Asp Gly Tyr Glu Val Arg Val Arg Gly Pro
420 425 430
His Val Thr Pro Gly Tyr Leu Gly Arg Pro Asp Leu Asp Ala Arg Ala
435 440 445
Phe Asp Glu Glu Gly Tyr Tyr Arg Pro Gly Asp Ala Val Ala Phe Ala
450 455 460
Asp Pro Gly Asp Ala Gly Ala Gly Leu Val Phe Arg Gly Arg Leu Thr
465 470 475 480
Glu Asp Phe Lys Leu Ser Thr Gly Thr Phe Val His Val Glu Ala Val
485 490 495
Arg Gly Ala Leu Leu Ser Ala Ala Pro Val Leu Ser Asp Ala Val Ile
500 505 510
Thr Gly Glu His Arg Asp Ala Val Cys Ala Leu Ala Trp Val Asp Pro
515 520 525
Ala Glu Ala Glu Arg Leu Leu Gly Arg Arg Pro Ala Ala Asp Gly Gly
530 535 540
Val Leu Tyr Ser Asp Ala Leu Ala Ala His Leu Gly Ala Ala Leu Glu
545 550 555 560
Arg Leu Asn Arg Gly Ala Gly Ser Ala Ser Arg Val Gln Arg Leu Leu
565 570 575
Val Leu Ala Asp Pro Pro Asp Leu Asp Ala Gly Glu Ile Thr Asp Lys
580 585 590
Gly Tyr Val Asn Gln Arg Arg Val Leu Ala Ala Arg Ala Pro Leu Val
595 600 605
Ala Arg Leu His Ala Asp Pro Ala Pro Arg His Val Ile Thr Pro Arg
610 615 620
Ser Gly Leu Thr
625
<210> 4
<211> 628
<212> PRT
<213> 人工序列 (Artifical sequence)
<220>
<221> MUTAGEN
<222> (490)
<223> Q3
<400> 4
Met Pro His Pro Thr Thr Arg Thr Ala Pro His Ser Ala Ala Gly Gly
1 5 10 15
Thr Thr Ala Gly Glu Thr Ser Ser Pro Leu Phe Ala Pro Ala Arg Thr
20 25 30
Val Arg Arg Asp Arg Pro Asp Gly Thr Val Leu Leu Ser Ser Ala Gln
35 40 45
Pro Leu Gly Val Tyr Pro Ala Ser Val Thr Asp His Leu Arg Thr Trp
50 55 60
Ala Gln Ala Gly Pro Asp Arg Pro Leu Val Ala Glu Arg Gly Ala Asp
65 70 75 80
Gly Arg Trp Gly His Arg Thr Tyr Gly Glu Val Leu Ala Ala Ala Glu
85 90 95
Ala Val Gly Gln Ala Leu Leu Asp Arg Gly Leu Ser Ala Arg Arg Pro
100 105 110
Leu Met Val Leu Ser Gly Asn Ser Thr Gly His Leu Leu Met Thr Leu
115 120 125
Gly Ala Leu Ser Ala Gly Ile Pro Val Ala Pro Val Ser Val Ala Tyr
130 135 140
Ser Leu Leu Ser Arg Asp His Ala Arg Ile Arg Ala Ile Ala Glu Leu
145 150 155 160
Leu Arg Pro Gly Ala Val Tyr Ala Glu Asp Ala Gly Pro Phe Gly Pro
165 170 175
Ala Leu Ala Ala Ala Gly Gly Gly Ala Ile Val Val Ala Ala Arg Gly
180 185 190
Gly Pro Ala Glu His Ser Leu Asp Ala Leu Leu Arg Thr Val Pro Gly
195 200 205
Arg Ala Phe Glu Ala Ala Arg Ala Gly Val Thr Ser Ala Thr Val Ala
210 215 220
Lys Val Leu Phe Thr Ser Gly Ser Thr Gly Ala Pro Lys Gly Val Val
225 230 235 240
Thr Thr His Gly Met Leu Cys Ala Asn Gln Arg Met Met Arg Gln Val
245 250 255
Trp Pro Phe Leu Ala Gly Glu Arg Pro Val Leu Leu Asp Trp Leu Pro
260 265 270
Trp Ser His Thr Phe Gly Gly Asn Phe Asn Val Asn Leu Val Leu Ala
275 280 285
Asn Gly Gly Thr Leu Tyr Leu Asp Asp Gly Arg Pro Thr Pro Glu Leu
290 295 300
Phe Gly Arg Thr Leu Ala Asn Leu Arg Glu Val Ser Pro Thr Leu Ala
305 310 315 320
Phe Asn Val Pro Ala Gly Tyr Ala Arg Leu Val Pro Ala Leu Glu Arg
325 330 335
Asp Arg Glu Leu Ala Glu Arg Phe Phe Ala Arg Leu Arg Leu Val Phe
340 345 350
Asn Ala Ala Ala Ala Leu Ala Pro Ala Leu Arg Glu Arg Leu Arg Ala
355 360 365
Leu Gly Arg Glu Val Thr Gly Arg Asp Val Pro Val Thr Gly Ser Trp
370 375 380
Gly Ala Thr Glu Thr Ser Pro Ala Ser Thr Ser Ala His Phe Pro Phe
385 390 395 400
Thr Asp Pro Arg Cys Ile Gly Val Pro Leu Pro Gly Val Glu Leu Lys
405 410 415
Leu Val Pro Ala Glu Gly Asp Gly Tyr Glu Val Arg Val Arg Gly Pro
420 425 430
His Val Thr Pro Gly Tyr Leu Gly Arg Pro Asp Leu Asp Ala Arg Ala
435 440 445
Phe Asp Glu Glu Gly Tyr Tyr Arg Pro Gly Asp Ala Val Ala Phe Ala
450 455 460
Asp Pro Gly Asp Ala Gly Ala Gly Leu Val Phe Arg Gly Arg Leu Thr
465 470 475 480
Glu Asp Phe Lys Leu Ser Thr Gly Thr Ala Val His Val Glu Ala Val
485 490 495
Arg Gly Ala Leu Leu Ser Ala Ala Pro Val Leu Ser Asp Ala Val Ile
500 505 510
Thr Gly Glu His Arg Asp Ala Val Cys Ala Leu Ala Trp Val Asp Pro
515 520 525
Ala Glu Ala Glu Arg Leu Leu Gly Arg Arg Pro Ala Ala Asp Gly Gly
530 535 540
Val Leu Tyr Ser Asp Ala Leu Ala Ala His Leu Gly Ala Ala Leu Glu
545 550 555 560
Arg Leu Asn Arg Gly Ala Gly Ser Ala Ser Arg Val Gln Arg Leu Leu
565 570 575
Val Leu Ala Asp Pro Pro Asp Leu Asp Ala Gly Glu Ile Thr Asp Lys
580 585 590
Gly Tyr Val Asn Gln Arg Arg Val Leu Ala Ala Arg Ala Pro Leu Val
595 600 605
Ala Arg Leu His Ala Asp Pro Ala Pro Arg His Val Ile Thr Pro Arg
610 615 620
Ser Gly Leu Thr
625
Claims (10)
1.一种酰基CoA合成酶，其特征在于，包括酰基CoA合成酶UkaQ及其突变体，所述酰基CoA合成酶UkaQ氨基酸序列如SEQ ID NO：1所示。
2.如权利要求1所述的酰基CoA合成酶，其特征在于，所述突变体包括突变体Q1、双突变株Q2和三突变株Q3；所述突变体Q1是所述酰基CoA合成酶UkaQ的第L526位置的亮氨酸(L)变成缬氨酸(V)突变体所得；所述双突变株Q2是所述突变体Q1第281位置的组氨酸(H)变成苯丙氨酸(F)所得；所述三突变株Q3是所述双突变株Q2第490位置的苯丙氨酸(F)变成丙氨酸(A)所得。
3.如权利要求2所述的酰基CoA合成酶，其特征在于，所述突变体Q1的氨基酸序列如SEQID NO：2所示；所述双突变株Q2的氨基酸序列如SEQ ID NO：3所示；所述三突变株Q3的氨基酸序列如SEQ ID NO：4所示。
4.如权利要求1所述的酰基CoA合成酶，其特征在于，所述酰基CoA合成酶与反应底物接触，进行催化反应，从而获得酰基-CoA产物；所述反应底物包括肉桂酸及其衍生物、3-苯基丙酸及其衍生物、苯甲酸及其衍生物、苯乙酸及其衍生物、饱和和不饱和脂肪酸及其衍生物、2-萘甲酸和喹哪啶酸双环芳香。
5.如权利要求1所述的酰基CoA合成酶在高效生产天然产物聚酮化合物的应用。
6.如权利要求5所述的应用，其特征在于，包括以下步骤：
步骤1、制备聚酮化合物生产菌株；
步骤2、构建重组质粒，并将所述重组质粒回补，即通过接合转移的方法将所述重组质粒分别整合到步骤1得到的生产菌株的基因组上,得到相应的重组菌株；
步骤3、对步骤2得到的重组菌株通过喂养底物发酵；得到发酵产物；
步骤4、对步骤1得到的发酵产物进行分离和结构鉴定。
7.如权利要求6所述的应用，其特征在于，所述步骤1中所述聚酮化合物生产菌株为生产脱酰基抗霉素的链霉菌。
8.如权利要求6所述的应用，其特征在于，所述步骤2中所述重组质粒为包含酰基CoA合成酶UkaQ或/和其突变株Q3的整合质粒。
9.如权利要求6所述的应用，其特征在于，所述步骤3中所述喂养底物为肉桂酸衍生物。
10.如权利要求6所述的应用，其特征在于，所述步骤4中所述产物为新的抗霉素类似物DA18-DA23，所述抗霉素类似物DA18-DA23的结构式如结构式1-6所示：
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1334181A2 (en) * | 2000-11-17 | 2003-08-13 | Metabolix, Inc. | Production of medium chain length polyhydroxyalkanoates from fatty acid biosynthetic pathways |
| US20090117629A1 (en) * | 2007-08-27 | 2009-05-07 | Claudia Schmidt-Dannert | Isoprenoid wax ester synthases, isoprenoid acyl coa-synthetases, and uses thereof |
| CN104427870A (zh) * | 2012-07-09 | 2015-03-18 | 明治制果药业株式会社 | Uk-2生物合成基因和使用其提高uk-2生产率的方法 |
| US20200131522A1 (en) * | 2017-07-13 | 2020-04-30 | Radici Chimica S.P.A. | Biological methods for modifying cellular carbon flux |
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2022
- 2022-05-18 CN CN202210547914.4A patent/CN114717208A/zh active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1334181A2 (en) * | 2000-11-17 | 2003-08-13 | Metabolix, Inc. | Production of medium chain length polyhydroxyalkanoates from fatty acid biosynthetic pathways |
| US20090117629A1 (en) * | 2007-08-27 | 2009-05-07 | Claudia Schmidt-Dannert | Isoprenoid wax ester synthases, isoprenoid acyl coa-synthetases, and uses thereof |
| CN104427870A (zh) * | 2012-07-09 | 2015-03-18 | 明治制果药业株式会社 | Uk-2生物合成基因和使用其提高uk-2生产率的方法 |
| US20200131522A1 (en) * | 2017-07-13 | 2020-04-30 | Radici Chimica S.P.A. | Biological methods for modifying cellular carbon flux |
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| Title |
|---|
| 汪信东: "樟树长链脂肪酰基CoA合成酶基因9克隆与表达分析", 《广西植物》, vol. 38, no. 10, pages 1335 - 1345 * |
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