Genome-Based Analysis and Gene Dosage Studies Provide New Insight into 3-Hydroxy-4-Methylvalerate Biosynthesis in Ralstonia eutropha
Genome-Based Analysis and Gene Dosage Studies Provide New Insight into 3-Hydroxy-4-Methylvalerate Biosynthesis in Ralstonia eutropha
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基于基因组的分析和基因剂量研究为富养罗尔斯顿菌的 3-羟基-4-甲基戊酸生物合成提供了新的见解
DOI：
10.1128/jb.02474-14
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发表时间：
2015
影响因子：
3.2
通讯作者：
Takeharu Tsuge
中科院分区：
文献类型：
--
作者：
Azusa Saika;Kazunori Ushimaru;Shoji Mizuno;Takeharu Tsuge
表达来自假单胞菌属的广泛底物特异性聚羟基脂肪酸酯 (PHA) 合酶 1 的重组真养产碱杆菌菌株 PHB−4。菌株 61-3 (PhaC1Ps) 合成含有支链侧链单元 3-羟基-4-甲基戊酸 (3H4MV) 的 PHA 共聚物，其碳主链与亮氨酸相同。突变菌株 1F2 通过化学诱变衍生自富养罗伯茨菌菌株 PHB-4，并且显示出比亲本菌株更高水平的 3H4MV 产量。在本研究中，为了了解3H4MV产量增加的机制，对菌株1F2进行了全基因组测序，并将草图基因组序列与亲本菌株PHB-4进行了比较。该分析发现了 1F2 基因组中的四个点突变。在ilvH基因的第36位氨基酸（A36T）处发现一个点突变。ilvH编码一种调节乙酰羟酸合酶III（AHAS III）的亚基蛋白。 AHAS 催化丙酮酸转化为 2-乙酰乳酸，这是亮氨酸和缬氨酸等支链氨基酸生物合成的第一个反应。因此，A36T IlvH突变可能表现出AHAS对支链氨基酸反馈抑制的耐受性，从而增加流向支链氨基酸和3H4MV生物合成的碳通量。此外，还进行了基因剂量研究和同位素示踪研究来研究 3H4MV 生物合成途径。基于这些研究的观察结果，我们提出了富养罗伯茨菌中的3H4MV生物合成途径，该途径涉及异丁酰辅酶A（异丁酰辅酶A）和乙酰辅酶A之间的缩合反应以形成3H4MV碳主链。
Recombinant Ralstonia eutropha strain PHB−4 expressing the broad-substrate-specificity polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas sp. strain 61-3 (PhaC1Ps) synthesizes a PHA copolymer containing the branched side-chain unit 3-hydroxy-4-methylvalerate (3H4MV), which has a carbon backbone identical to that of leucine. Mutant strain 1F2 was derived from R. eutropha strain PHB−4 by chemical mutagenesis and shows higher levels of 3H4MV production than does the parent strain. In this study, to understand the mechanisms underlying the enhanced production of 3H4MV, whole-genome sequencing of strain 1F2 was performed, and the draft genome sequence was compared to that of parent strain PHB−4. This analysis uncovered four point mutations in the 1F2 genome. One point mutation was found in theilvHgene at amino acid position 36 (A36T) of IlvH.ilvHencodes a subunit protein that regulates acetohydroxy acid synthase III (AHAS III). AHAS catalyzes the conversion of pyruvate to 2-acetolactate, which is the first reaction in the biosynthesis of branched amino acids such as leucine and valine. Thus, the A36T IlvH mutation may show AHAS tolerance to feedback inhibition by branched amino acids, thereby increasing carbon flux toward branched amino acid and 3H4MV biosynthesis. Furthermore, a gene dosage study and an isotope tracer study were conducted to investigate the 3H4MV biosynthesis pathway. Based on the observations in these studies, we propose a 3H4MV biosynthesis pathway in R. eutropha that involves a condensation reaction between isobutyryl coenzyme A (isobutyryl-CoA) and acetyl-CoA to form the 3H4MV carbon backbone.