本文選題：肽類(lèi)天然產(chǎn)物　切入點(diǎn)：菜豆菌毒素　出處：《武漢大學(xué)》2017年碩士論文

【摘要】：肽類(lèi)天然產(chǎn)物由氨基酸組成,分子量介于氨基酸與蛋白質(zhì)之間,結(jié)構(gòu)多種多樣。自然界中存在許多肽類(lèi)天然產(chǎn)物,具有抗病毒、抗菌、殺蟲(chóng)和免疫抑制等豐富的生物活性。本文主要以ATP-grasp酶催化的肽類(lèi)天然產(chǎn)物菜豆菌毒素(Phaseolotoxin,PHT)和核糖體合成的肽類(lèi)(RiPPs)天然產(chǎn)物波卓霉素(Bottromycin,BTM)為重點(diǎn),圍繞這兩種肽類(lèi)天然產(chǎn)物的生物合成展開(kāi)研究。Phaseolotoxin是一種磷酸酰胺類(lèi)天然產(chǎn)物,由PSOrn、Ala以及hArg組成。其中PSOrn和hArg為非天然氨基酸,需要從頭合成。我們首先發(fā)酵丁香假單胞菌,檢測(cè)到了Phaseolotoxin,然后超量表達(dá)了PHT基因簇中的amtA基因。amtA編碼脒基轉(zhuǎn)移酶,該酶可催化L-Lys生成L-hArg。與其它已知的脒基轉(zhuǎn)移酶相比,體外實(shí)驗(yàn)證明AmtA具有特殊的底物特異性:精氨酸和刀豆氨酸可作為脒基供體,賴(lài)氨酸和鳥(niǎo)氨酸可作為脒基受體。定點(diǎn)突變實(shí)驗(yàn)顯示突變株AmtAM243P H244N仍具催化轉(zhuǎn)移脒基反應(yīng)的活性,但Met246對(duì)于該反應(yīng)中脒基的轉(zhuǎn)移是十分重要的。同時(shí),我們研究了波卓霉素的生物合成,期望找到其生物合成中間體。波卓霉素是由核糖體翻譯后經(jīng)多步修飾(RiPP)形成的,具有多重抗菌活性。該天然產(chǎn)物含有的大脒環(huán)和β-甲基化氨基酸殘基結(jié)構(gòu)在已知的天然產(chǎn)物中極其少見(jiàn)。BTM基因簇中具有后修飾功能的酶可將其前體肽(BtmD)轉(zhuǎn)化為波卓霉素,但由于該過(guò)程的中間體不易獲得,導(dǎo)致其合成途徑一直沒(méi)有被詮釋清楚。為了得到BTM生物合成中間體,一方面我們將btmD基因單獨(dú)構(gòu)建到在鏈霉菌中可誘導(dǎo)的表達(dá)型載體上形成重組質(zhì)粒pWDZ54來(lái)提高前體肽的產(chǎn)量。另一方面,我們?nèi)サ鬊TM基因簇里的btmA(transporter),btmD(precursorpeptide),btmH(peptidase)基因,將剩下的基因簇構(gòu)建到鏈霉菌整合型載體pSET152上形成重組質(zhì)粒pWDZ55,這樣避免前體肽被切斷和轉(zhuǎn)運(yùn)至細(xì)胞外。然后再將這兩個(gè)構(gòu)建好的載體同時(shí)轉(zhuǎn)到合適的宿主中去,以期獲得產(chǎn)量較高且易純化的波卓霉素生物合成中間體。
[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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R914

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