發(fā)布時間：2018-03-21 07:18

  本文選題：乙酰乳酸合酶　切入點：極端嗜鹽古菌　出處：《河北大學》2017年碩士論文　論文類型：學位論文

【摘要】：乙酰乳酸合酶(Acetohydroxyacid Synthase,AHAs)是一種硫胺素二磷酸依賴性酶,是催化纈氨酸(Val)、亮氨酸(Leu)和異亮氨酸(Ile)等支鏈氨基酸生物合成的關鍵酶,該酶的底物為酮酸類物質。乙酰乳酸合酶可以縮合兩分子丙酮酸產生乙酰乳酸并最終合成纈氨酸和亮氨酸,也可以催化一分子丙酮酸和一分子2-酮丁酸合成乙酰羥基丁酸用以合成異亮氨酸。乙酰乳酸合酶在細菌真菌中研究較多,但在極端嗜鹽古菌中還未有報道,本論文以地中海富鹽菌Haloferax mediterranei作為研究菌株,對該酶進行了鑒定并研究了其對聚羥基脂肪酸酯(PHAs)積累的影響。首先,通過生物信息學分析在H.mediterranei中發(fā)現了編碼AHAs的3個候選基因,包括編碼小亞基的HFX_1571(ilvN)、編碼大亞基的HFX_1572(ilvB)和HFX_6032(ilvB3)。利用本實驗室已建立的pyrF高效基因敲除系統(tǒng)和多糖敲除菌株DF50?eps,構建了上述3個候選基因的相關突變株:DF50?eps?ilvN、DF50?eps?ilvB、DF50?eps?ilvB3以及DF50?eps?ilvN?ilvB。然后,對突變株進行發(fā)酵培養(yǎng)從而探索上述基因在嗜鹽古菌支鏈氨基酸合成中的作用和對PHAs積累的影響。在不添加氨基酸的發(fā)酵培養(yǎng)基中,與對照菌株DF50?eps相比,突變株DF50?eps?ilvN、DF50?eps?ilvB和DF50?eps?ilvN?ilvB的生長受到明顯抑制,而DF50?eps?ilvB3的生長速率與對照株相當。該結果暗示,地中海富鹽菌的乙酰乳酸合酶可能由兩個相鄰基因HFX_1571和HFX_1572共同編碼,而HFX_6032為冗余基因。當培養(yǎng)基中分別添加不同濃度的支鏈氨基酸(亮氨酸、纈氨酸、異亮氨酸)后,可以明顯緩解DF50?eps?ilvN、DF50?eps?ilvB和DF50?eps?ilvN?ilvB生長被抑制的現象,如同時添加濃度分別為0.1 g/L的亮氨酸、纈氨酸和異亮氨酸時,DF50?eps?ilvN?ilvB的菌體濃度與對照DF50?eps相似。該結果進一步說明HFX_1571和HFX_1572共同編碼了有功能的嗜鹽古菌乙酰乳酸合酶。同時發(fā)現,當HFX_1571或者HFX_1572敲除后,導致菌體基礎代謝受阻,嚴重降低了PHAs的積累。本論文的研究工作,鑒定了地中海富鹽菌中負責支鏈氨基酸合成的關鍵酶---乙酰乳酸合酶。該酶由小亞基IlvN和大亞基IlvB組成,分別由HFX_1571和HFX_1572編碼,而HFX_6032為冗余基因。
[Abstract]:Acetyllactate synthase (Acetohydroxyacid SynthaseAHAsA) is a thiamine diphosphate dependent enzyme that catalyzes the biosynthesis of branched amino acids such as valine valine, leucine, leucine and Ile. Acetyllactate synthase condenses two molecules of pyruvate to produce acetyllactic acid and ultimately to synthesize valine and leucine. It can also catalyze the synthesis of acetyl hydroxybutyric acid from pyruvate and 2-ketobutyric acid. In this paper, Haloferax mediterranei was used as the study strain to identify the enzyme and its effect on the accumulation of polyhydroxyfatty acid ester. Firstly, three candidate genes encoding AHAs were found in H. mediterranei by bioinformatics analysis. HFXs encoding small subunits and HFXs encoding large subunits (HFX1 572 / ilvB) and HFX6032 / ilvB3s are included. Using the pyrF efficient gene knockout system and polysaccharide knockout strain DF50? EPS, a mutant of the three candidate genes: DF50? Eps? IlvN,DF50? Eps? IlvB,DF50? Eps? IlvB3 and DF50? Eps? IlvN? Then the mutant was fermented to explore the role of the above genes in the biosynthesis of branched chain amino acids and the effect on the accumulation of PHAs. In the fermentation medium without amino acids, the mutant was compared with the control strain DF50? Compared with eps, the mutant DF50? Eps? IlvN,DF50? Eps? IlvB and DF50? Eps? IlvN? The growth of ilvB was significantly inhibited, while DF50? Eps? The growth rate of ilvB3 is similar to that of the control strain. The results suggest that the acetyllactate synthase of Salt-rich Mediterranean bacteria may be encoded by two adjacent genes, HFX_1571 and HFX_1572. HFX_6032 is a redundant gene. When different concentrations of branched amino acids (leucine, valine, isoleucine) were added to the medium, DF50? Eps? IlvN,DF50? Eps? IlvB and DF50? Eps? IlvN? The inhibition of ilvB growth, such as the addition of 0.1 g / L leucine, valine and isoleucine at the same time. Eps? IlvN? The bacterial concentration of ilvB and control DF50? Eps is similar. The results further indicate that HFX_1571 and HFX_1572 co-encode the functional acyllactic synthase of paleococcus halophilus. It was also found that when HFX_1571 or HFX_1572 was knocked out, the basal metabolism of the bacteria was blocked. In this paper, the key enzyme responsible for the synthesis of branched-chain amino acids in Salt-rich bacteria, -acetyllactate synthase, was identified. The enzyme consists of small subunit IlvN and large subunit IlvB, which are encoded by HFX_1571 and HFX_1572, respectively. HFX_6032 is a redundant gene.
【學位授予單位】：河北大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q55

【參考文獻】

相關期刊論文 前2條

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2 ;Development of pyrF-based gene knockout systems for genome-wide manipulation of the archaea Haloferax mediterranei and Haloarcula hispanica[J];遺傳學報;2011年06期

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本文編號：1642823