组合生物合成生产双氢多拉菌素

发布时间：2018-03-28 02:06

本文选题：聚酮合酶　切入点：阿维菌素　出处：《武汉大学》2017年硕士论文

【摘要】：阿维菌素(Avermectin)为阿维链霉菌(Streptomyces avermimitilis)产生的一类具有杀虫、杀螨和杀线虫活性的十六元大环内酯化合物,其因C5、C22-C23、C25结构不同共分为8个组份,分别为A1a,A1b,A2a,A2b,B1a,B1b,B2a,B2b,主要活性组分为阿维菌素B1a。现市售伊维菌素是在阿维菌素的基础上,利用化学催化的方式选择性地对其C22-C23位加氢得到的二代衍生物。多拉菌素与阿维菌素的区别则在于其起始单元为环己烷甲酰-CoA。本课题组已经通过构建细菌人工染色体(Bacterial Artificial Chromosome,BAC)文库成功获取了阿维菌素生物合成基因簇,并在变铅青链霉菌(Streptomyces lividans)中异源表达。通过利用梅岭霉素聚酮合成酶Mei A1 M2上的DH-ER-KR结构域替换阿维菌素聚酮合成酶Ave A1 M2上的DH-KR结构域,成功构建了杂合的聚酮合酶以生产伊维菌素。在此基础上敲除aveD基因,得到了只产生伊维菌素B组份的重组菌株S.avermitilis XL316。阿维菌素的起始单元2-甲基丁酰-CoA和异丁酰-CoA分别由L-异亮氨酸和L-缬氨酸经支链氨基酸分解代谢途径产生。L-异亮氨酸和L-缬氨酸首先经支链氨基酸转氨酶催化,生成α-酮酸,再经支链氨基酸脱氢酶(branched-chaina-ketoaciddehydrogenase,BCDH)作用发生氧化脱羧反应,生成2-甲基丁酰-CoA和异丁酰-CoA。由于2-甲基丁酰-CoA和异丁酰-CoA由同一支链氨基酸分解代谢途径合成,为进一步得到单一组份的阿维菌素衍生物,本课题组尝试利用洛伐他汀生物合成基因簇中的lovF,对阿维菌素生物合成基因簇中的起始模块进行替换,以期得到的单一的伊维菌素组份。阿维链霉菌的支链氨基酸脱氢酶仅仅含有E1α、E1β和E2三个亚基,而在某些真核生物体内还存在两个BCDH调控蛋白,即支链氨基酸脱氢酶激酶(BCDH Kinase)和支链氨基酸脱氢酶磷酸酶(BCDH phosphatase)。这两个蛋白通过对支链氨基酸脱氢酶磷酸化失活,去磷酸化恢复活性,严格控制生物体内的支链氨基酸分解代谢水平。我们通过从真菌中筛选,得到了一个可以在阿维链霉菌体内发挥活性的支链氨基酸脱氢酶激酶(BCDH Kinase),在BCDHKinase1调控的基础上,发酵重组菌株S.avermitilis DQ319,喂养环己基-(N-乙酰-)氨基-乙基-硫酯,成功检测到了双氢多拉菌素,其C22-C23为饱和键,起始单元为环己烷甲酰-CoA,具备伊维菌素和多拉菌素的双重特征。本实验成功筛选了一个可以在原核生物体内发挥活性的BCDHKinase,为真核生物基因在原核生物体内表达提供了实践基础。在BCDH Kinase发挥活性的基础上,首次发酵产生了新化合物双氢多拉菌素。许多天然药物的合成起始单元或者延伸单元都来源于支链氨基酸降解途径。基于此,将BCDH Kinase导入不同的宿主菌,通过特异性地喂养相关前体物或者转入某些前体物的生物合成基因簇,就可以得到多种相关衍生物,为发现活性更好的新药提供新的方法和思路。
[Abstract]:Avermectin (Avermectin) is a class of hexadecyclic lactones with insecticidal, acaricidal and nematicidal activities produced by Streptomyces avermitilisis. The main active components are Avermectin B1a.The main active component is abamectin B1a.Ivermectin is on sale on the basis of avermectin. The second generation derivatives obtained by selective hydrogenation of C22-C23 site by chemical catalysis. The difference between Dora and avermectin is that the initial unit is cyclohexane-formyl-CoA. our team has constructed bacterial artificial staining. The abamectin biosynthesis gene cluster was successfully obtained from the Bacterial Artificial Chromosome-BAC) library. The DH-KR domain of avermectin polyketone synthase (Ave A1 M2) was replaced by the DH-ER-KR domain of Melinomycin polyketone synthase (Mei A1 M2), which was expressed in Streptomyces lividanss (Streptomyces lividans). A heterozygous polyketone synthase was successfully constructed to produce ivermectin. On this basis, the aveD gene was knockout. A recombinant strain S.avermitilis XL316was obtained, which only produced ivermectin B component. The starting units of avermectin, 2-methylbutyl-CoA and isobutyl-CoA, were produced by branched-chain amino acid catabolism pathway from L- isoleucine and L-valine, respectively. Ammonia acid and L-valine were first catalyzed by branched amino acid aminotransferase. 伪 -ketoacid was synthesized by branched-chaina-ketoaciddehydrogenase (BCDH) to produce 2-methylbutyl-CoA and isobutyl-CoA. Because 2-methylbutyl-CoA and isobutyl-CoA were synthesized by the same branched-chains-ketoaciddehydrogenase (BCDHs) pathway, 2-methylbutyryl -CoA and isobutyl-CoA were synthesized by the same branched-chain amino acid catabolism pathway. In order to obtain a single component of avermectin derivatives, our team tried to replace the initiation module of abamectin biosynthesis gene cluster by using lovFin in lovastatin biosynthesis gene cluster. In order to obtain a single Ivermectin component, the branched amino acid dehydrogenase of Streptomyces avelicus contained only three subunits of E 1 伪 E 1 尾 and E 2, while there were two BCDH regulatory proteins in some eukaryotes. That is, BCDH kinase) and BCDH phosphatase (BCDH phosphatase). These two proteins restore their activity by dephosphorylation of branched-chain amino acid dehydrogenase (BCDH Kinase) and branched amino acid dehydrogenase (BCDH). Through screening from fungi, we have obtained a branched chain amino acid dehydrogenase kinase (BCDH Kinase), which can play an active role in Streptomyces avelicus. It is based on the regulation of BCDHKinase1. The recombinant strain S.avermitilis DQ319 was fed with cyclohexyl N-acetyl) amino-ethyl-thioester and dihydro#internal_person0# was successfully detected, and its C22-C23 was saturated. The initial unit is cyclohexanolformyl-CoA, which has the dual characteristics of Ivermectin and Dora. In this experiment, we successfully selected a BCD HKinase that can play an active role in prokaryotes, which can express eukaryote genes in prokaryotes. On the basis of the activity of BCDH Kinase, For the first time, a new compound, dihydro#internal_person0#, was produced. Many natural drugs are derived from branched-chain amino acid degradation pathways in the initial or extended units of synthesis. Based on this, BCDH Kinase is introduced into different host bacteria. A variety of related derivatives can be obtained by feeding related precursors specifically or by transferring them into a cluster of biosynthetic genes which can provide new methods and ideas for the discovery of new drugs with better activity.
【学位授予单位】：武汉大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R915

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