Phaseolotoxin合成中AmtA的功能研究与Bottromycin合成中间体的挖掘

发布时间：2018-03-31 04:19

本文选题：肽类天然产物　切入点：菜豆菌毒素　出处：《武汉大学》2017年硕士论文

【摘要】：肽类天然产物由氨基酸组成,分子量介于氨基酸与蛋白质之间,结构多种多样。自然界中存在许多肽类天然产物,具有抗病毒、抗菌、杀虫和免疫抑制等丰富的生物活性。本文主要以ATP-grasp酶催化的肽类天然产物菜豆菌毒素(Phaseolotoxin,PHT)和核糖体合成的肽类(RiPPs)天然产物波卓霉素(Bottromycin,BTM)为重点,围绕这两种肽类天然产物的生物合成展开研究。Phaseolotoxin是一种磷酸酰胺类天然产物,由PSOrn、Ala以及hArg组成。其中PSOrn和hArg为非天然氨基酸,需要从头合成。我们首先发酵丁香假单胞菌,检测到了Phaseolotoxin,然后超量表达了PHT基因簇中的amtA基因。amtA编码脒基转移酶,该酶可催化L-Lys生成L-hArg。与其它已知的脒基转移酶相比,体外实验证明AmtA具有特殊的底物特异性:精氨酸和刀豆氨酸可作为脒基供体,赖氨酸和鸟氨酸可作为脒基受体。定点突变实验显示突变株AmtAM243P H244N仍具催化转移脒基反应的活性,但Met246对于该反应中脒基的转移是十分重要的。同时,我们研究了波卓霉素的生物合成,期望找到其生物合成中间体。波卓霉素是由核糖体翻译后经多步修饰(RiPP)形成的,具有多重抗菌活性。该天然产物含有的大脒环和β-甲基化氨基酸残基结构在已知的天然产物中极其少见。BTM基因簇中具有后修饰功能的酶可将其前体肽(BtmD)转化为波卓霉素,但由于该过程的中间体不易获得,导致其合成途径一直没有被诠释清楚。为了得到BTM生物合成中间体,一方面我们将btmD基因单独构建到在链霉菌中可诱导的表达型载体上形成重组质粒pWDZ54来提高前体肽的产量。另一方面,我们去掉BTM基因簇里的btmA(transporter),btmD(precursorpeptide),btmH(peptidase)基因,将剩下的基因簇构建到链霉菌整合型载体pSET152上形成重组质粒pWDZ55,这样避免前体肽被切断和转运至细胞外。然后再将这两个构建好的载体同时转到合适的宿主中去,以期获得产量较高且易纯化的波卓霉素生物合成中间体。
[Abstract]:Peptide natural products are composed of amino acids with molecular weight between amino acids and proteins. Insecticidal and immunosuppressive activities are abundant in this paper. In this paper, the main focus of this paper is ATP-grasp enzyme catalyzed peptide toxin Phaseolotoxin (PHT) and ribosomal peptide rips (Bottromycininine), a natural product of ribosome synthesis. Studies on the biosynthesis of these two natural peptide products .Phaseolotoxin, a natural product of phosphate amides, consists of PSOrnum Ala and hArg, in which PSOrn and hArg are non-natural amino acids and need to be synthesized from scratch. Phase olotoxin was detected, and then the amtA gene. AmtA encoded by amidotoxin in PHT gene cluster was overexpressed. This enzyme can catalyze L-Lys to produce L-hArg. compared with other known aminotransferases. In vitro experiments showed that AmtA had special substrate specificity: arginine and concanavalin could be used as amidine donors, and lysine and ornithine could act as amidine receptors. Site-directed mutagenesis showed that AmtAM243P H244N still had the activity of catalyzing the transfer of amidine. However, Met246 is very important for the transfer of amidine in this reaction. At the same time, we have studied the biosynthesis of berthromycin and hope to find its biosynthesis intermediate. This natural product contains large amidine ring and 尾 -methylated amino acid residues, which are rare in known natural products. The enzyme with post-modification function in .BTM gene cluster can transform its precursor peptide, BtmD), into bentorubicin. However, because the intermediate in this process is not easily available, the synthetic pathway has not been clearly interpreted. In order to obtain the intermediate of BTM biosynthesis, On the one hand, we constructed the btmD gene into an inducible expression vector in Streptomyces to form a recombinant plasmid pWDZ54 to increase the yield of precursor peptides. On the other hand, we removed the BtmAX transporter from the BTM gene cluster. The remaining gene clusters were constructed into the streptomyces integrated vector pSET152 to form the recombinant plasmid pWDZ55. in order to prevent the precursor peptide from being cut off and transported out of the cell, the two constructed vectors were then transferred to the appropriate host at the same time. In order to obtain a high yield and easy to purify the intermediate for the biosynthesis of poplomycin.
【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】：R914

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