本文選題：聚酮合酶　切入點(diǎn)：阿維菌素　出處：《武漢大學(xué)》2017年碩士論文

【摘要】：阿維菌素(Avermectin)為阿維鏈霉菌(Streptomyces avermimitilis)產(chǎn)生的一類具有殺蟲、殺螨和殺線蟲活性的十六元大環(huán)內(nèi)酯化合物,其因C5、C22-C23、C25結(jié)構(gòu)不同共分為8個(gè)組份,分別為A1a,A1b,A2a,A2b,B1a,B1b,B2a,B2b,主要活性組分為阿維菌素B1a�，F(xiàn)市售伊維菌素是在阿維菌素的基礎(chǔ)上,利用化學(xué)催化的方式選擇性地對(duì)其C22-C23位加氫得到的二代衍生物。多拉菌素與阿維菌素的區(qū)別則在于其起始單元為環(huán)己烷甲酰-CoA。本課題組已經(jīng)通過構(gòu)建細(xì)菌人工染色體(Bacterial Artificial Chromosome,BAC)文庫成功獲取了阿維菌素生物合成基因簇,并在變鉛青鏈霉菌(Streptomyces lividans)中異源表達(dá)。通過利用梅嶺霉素聚酮合成酶Mei A1 M2上的DH-ER-KR結(jié)構(gòu)域替換阿維菌素聚酮合成酶Ave A1 M2上的DH-KR結(jié)構(gòu)域,成功構(gòu)建了雜合的聚酮合酶以生產(chǎn)伊維菌素。在此基礎(chǔ)上敲除aveD基因,得到了只產(chǎn)生伊維菌素B組份的重組菌株S.avermitilis XL316。阿維菌素的起始單元2-甲基丁酰-CoA和異丁酰-CoA分別由L-異亮氨酸和L-纈氨酸經(jīng)支鏈氨基酸分解代謝途徑產(chǎn)生。L-異亮氨酸和L-纈氨酸首先經(jīng)支鏈氨基酸轉(zhuǎn)氨酶催化,生成α-酮酸,再經(jīng)支鏈氨基酸脫氫酶(branched-chaina-ketoaciddehydrogenase,BCDH)作用發(fā)生氧化脫羧反應(yīng),生成2-甲基丁酰-CoA和異丁酰-CoA。由于2-甲基丁酰-CoA和異丁酰-CoA由同一支鏈氨基酸分解代謝途徑合成,為進(jìn)一步得到單一組份的阿維菌素衍生物,本課題組嘗試?yán)寐宸ニ∩锖铣苫虼刂械膌ovF,對(duì)阿維菌素生物合成基因簇中的起始模塊進(jìn)行替換,以期得到的單一的伊維菌素組份。阿維鏈霉菌的支鏈氨基酸脫氫酶僅僅含有E1α、E1β和E2三個(gè)亞基,而在某些真核生物體內(nèi)還存在兩個(gè)BCDH調(diào)控蛋白,即支鏈氨基酸脫氫酶激酶(BCDH Kinase)和支鏈氨基酸脫氫酶磷酸酶(BCDH phosphatase)。這兩個(gè)蛋白通過對(duì)支鏈氨基酸脫氫酶磷酸化失活,去磷酸化恢復(fù)活性,嚴(yán)格控制生物體內(nèi)的支鏈氨基酸分解代謝水平。我們通過從真菌中篩選,得到了一個(gè)可以在阿維鏈霉菌體內(nèi)發(fā)揮活性的支鏈氨基酸脫氫酶激酶(BCDH Kinase),在BCDHKinase1調(diào)控的基礎(chǔ)上,發(fā)酵重組菌株S.avermitilis DQ319,喂養(yǎng)環(huán)己基-(N-乙酰-)氨基-乙基-硫酯,成功檢測(cè)到了雙氫多拉菌素,其C22-C23為飽和鍵,起始單元為環(huán)己烷甲酰-CoA,具備伊維菌素和多拉菌素的雙重特征。本實(shí)驗(yàn)成功篩選了一個(gè)可以在原核生物體內(nèi)發(fā)揮活性的BCDHKinase,為真核生物基因在原核生物體內(nèi)表達(dá)提供了實(shí)踐基礎(chǔ)。在BCDH Kinase發(fā)揮活性的基礎(chǔ)上,首次發(fā)酵產(chǎn)生了新化合物雙氫多拉菌素。許多天然藥物的合成起始單元或者延伸單元都來源于支鏈氨基酸降解途徑�；诖�,將BCDH Kinase導(dǎo)入不同的宿主菌,通過特異性地喂養(yǎng)相關(guān)前體物或者轉(zhuǎn)入某些前體物的生物合成基因簇,就可以得到多種相關(guān)衍生物,為發(fā)現(xiàn)活性更好的新藥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. 偽 -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 偽 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.
【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
【分類號(hào)】：R915

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