地中海富盐菌乙酰乳酸合酶的鉴定

发布时间：2018-03-21 07:18

本文选题：乙酰乳酸合酶　切入点：极端嗜盐古菌　出处：《河北大学》2017年硕士论文　论文类型：学位论文

【摘要】：乙酰乳酸合酶(Acetohydroxyacid Synthase,AHAs)是一种硫胺素二磷酸依赖性酶,是催化缬氨酸(Val)、亮氨酸(Leu)和异亮氨酸(Ile)等支链氨基酸生物合成的关键酶,该酶的底物为酮酸类物质。乙酰乳酸合酶可以缩合两分子丙酮酸产生乙酰乳酸并最终合成缬氨酸和亮氨酸,也可以催化一分子丙酮酸和一分子2-酮丁酸合成乙酰羟基丁酸用以合成异亮氨酸。乙酰乳酸合酶在细菌真菌中研究较多,但在极端嗜盐古菌中还未有报道,本论文以地中海富盐菌Haloferax mediterranei作为研究菌株,对该酶进行了鉴定并研究了其对聚羟基脂肪酸酯(PHAs)积累的影响。首先,通过生物信息学分析在H.mediterranei中发现了编码AHAs的3个候选基因,包括编码小亚基的HFX_1571(ilvN)、编码大亚基的HFX_1572(ilvB)和HFX_6032(ilvB3)。利用本实验室已建立的pyrF高效基因敲除系统和多糖敲除菌株DF50?eps,构建了上述3个候选基因的相关突变株:DF50?eps?ilvN、DF50?eps?ilvB、DF50?eps?ilvB3以及DF50?eps?ilvN?ilvB。然后,对突变株进行发酵培养从而探索上述基因在嗜盐古菌支链氨基酸合成中的作用和对PHAs积累的影响。在不添加氨基酸的发酵培养基中,与对照菌株DF50?eps相比,突变株DF50?eps?ilvN、DF50?eps?ilvB和DF50?eps?ilvN?ilvB的生长受到明显抑制,而DF50?eps?ilvB3的生长速率与对照株相当。该结果暗示,地中海富盐菌的乙酰乳酸合酶可能由两个相邻基因HFX_1571和HFX_1572共同编码,而HFX_6032为冗余基因。当培养基中分别添加不同浓度的支链氨基酸(亮氨酸、缬氨酸、异亮氨酸)后,可以明显缓解DF50?eps?ilvN、DF50?eps?ilvB和DF50?eps?ilvN?ilvB生长被抑制的现象,如同时添加浓度分别为0.1 g/L的亮氨酸、缬氨酸和异亮氨酸时,DF50?eps?ilvN?ilvB的菌体浓度与对照DF50?eps相似。该结果进一步说明HFX_1571和HFX_1572共同编码了有功能的嗜盐古菌乙酰乳酸合酶。同时发现,当HFX_1571或者HFX_1572敲除后,导致菌体基础代谢受阻,严重降低了PHAs的积累。本论文的研究工作,鉴定了地中海富盐菌中负责支链氨基酸合成的关键酶---乙酰乳酸合酶。该酶由小亚基IlvN和大亚基IlvB组成,分别由HFX_1571和HFX_1572编码,而HFX_6032为冗余基因。
[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.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q55

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