Pyrazine Biosynthesis in Corynebacterium glutamicum
Pyrazine Biosynthesis in Corynebacterium glutamicum
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DOI：
10.1002/ejoc.201000155
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发表时间：
2010-05-01
影响因子：
2.8
通讯作者：
Wittmann, Christoph
中科院分区：
文献类型：
--
作者：
Dickschat, Jeroen S.;Wickel, Susanne;Wittmann, Christoph
谷氨酸棒杆菌释放的挥发性化合物收集通过使用CLSA技术（闭环汽提装置），并通过GC-MS分析。顶空提取物含有几个偶姻和吡嗪，确定其合成或比较商业标准。用[H-2（7）]乙偶姻进行的饲喂实验导致标记掺入三甲基吡嗪和四甲基吡嗪中。构建了几个针对初级代谢基因的缺失突变体，以详细阐明吡嗪类化合物的生物合成途径。酮醇酸还原异构酶的缺失突变体不能将乙偶姻前体（S）-2-乙酰乳酸转化为途径中间体（R）-2，3-二羟基-3-甲基丁酸至支链氨基酸。该突变体需要缬氨酸、亮氨酸和异亮氨酸生长，并产生显著更高的量和更多不同的偶姻和吡嗪类化合物。乙酰乳酸合酶（AS）的基因缺失导致不能将丙酮酸转化为（S）-2-乙酰乳酸的突变体。该突变体还需要支链氨基酸，并且可能经由以相反顺序操作的缬氨酸生物合成途径从缬氨酸仅产生非常少量的吡嗪。构建完全不产生任何吡嗪的Δ AS Δ KR双突变体。这些结果为通过偶姻形成烷基化吡嗪提供了详细的生物合成模型。
The volatile compounds released by Corynebacterium glutamicum were collected by use of the CLSA technique (closed-loop stripping apparatus) and analysed by GC-MS. The headspace extracts contained several acyloins and pyrazines that were identified by their synthesis or comparison to commercial standards. Feeding experiments with [H-2(7)]acetoin resulted in the incorporation of labelling into trimethylpyrazine and tetramethylpyrazine. Several deletion mutants targeting genes of the primary metabolism were constructed to elucidate the biosynthetic pathway to pyrazines in detail. A deletion mutant of the ketol-acid reducto-isomerase was not able to convert the acetoin precursor (S)-2-acetolactate into the pathway intermediate (R)-2,3-dihydroxy-3-methylbutanoate to the branched amino acids. This mutant requires valine, leucine, and isoleucine for growth and produces significantly higher amounts and more different compounds of the acyloin and pyrazine classes. Gene deletion of the acetolactate synthase (AS) resulted in a mutant that is not able to convert pyruvate into (S)-2-acetolactate. This mutant also requires branched amino acids and produces only very small amounts of pyrazines likely from valine via the valine biosynthetic pathway operating in reverse order. A Delta AS Delta KR double mutant was constructed that does not produce any pyrazines at all. These results open up a detailed biosynthetic model for the formation of alkylated pyrazines via acyloins.