Genetics and biochemistry of coenzyme A biosynthesis in the yeast Saccharomyces cerevisiae

由于辅酶A （CoA）及其酰化产物（主要是乙酰辅酶A）在所有生物体的代谢中起着至关重要的作用，因此辅酶A的生物合成是必不可少的细胞功能。通过结构基因CAB1- cab5（辅酶A生物合成）的鉴定和关键酶泛酸激酶高活性变体（CAB1基因产物）的构建，我们获得了与野生型相比乙酰辅酶A浓度增加约6倍的酵母细胞。由于乙酰辅酶a在代谢中具有基础作用，并作为启动新细胞分裂周期的信号分子，我们希望研究乙酰辅酶a含量增加对细胞蛋白质组及其部分代谢组（重点是脂肪酸和麦角甾醇）的影响。通过改变ACS2（乙酰辅酶a合成酶）和LEU5（线粒体辅酶a输入源）基因的表达，我们计划改变辅酶a和乙酰辅酶a的比例以及辅酶a在细胞内的分布。这些研究的结果应该使我们能够确定生物技术中辅酶a依赖过程的有利条件。我们能够证明基因产物Cab2-Cab5彼此相互作用，并且它们形成coa合成蛋白复合物（CoA-SPC），其中Cab3作为介导相互作用的关键亚基。在未来，我们计划更精确地研究CoA-SPC，可能定义尚未确定的亚基及其化学计量学。为了做到这一点，Cab3将被表位标记并用于复合物的亲和纯化，随后将通过质谱分析。最后将酵母中辅酶a - spc的测定结果与高等真核生物进行比较。

Since coenzyme A (CoA) and its acylation products (mainly acetyl-CoA) are of central importance for metabolism in all organisms, biosynthesis of CoA is an essential cellular function. By identification of structural genes CAB1-CAB5 (coenzyme A biosynthesis) and construction of a hyperactive variant of key enzyme pantothenate kinase (CAB1 gene product) we obtained yeast cells with a concentration of acetyl-CoA which was about 6-fold increased, compared to a wild-type. Since acetyl-CoA has a fundamental role in metabolism and functions as a signalling molecule for initiation of a new cell division cycle we wish to investigate the implications of the increased amount of acetyl-CoA for the cellular proteome and part of its metabolome (focusing on fatty acids and ergosterol). By varying gene expression of genes ACS2 (acetyl-CoA synthetase) and LEU5 (mitochondrial CoA importer) we plan to modify the ratio of CoA and acetyl-CoA as well as the intracellular distribution of CoA. Results of these studies should allow us to define favourable conditions for CoA-dependent processes in biotechnology. We were able to demonstrate that gene products Cab2-Cab5 interact with each other and that they form a CoA-synthesizing protein complex (CoA-SPC) with Cab3 as a key subunit for mediating interactions. In the future we plan to investigate CoA-SPC more precisely, possibly defining subunits not yet identified and its stoichiometry. To do this, Cab3 will be epitope-tagged and used for affinity-purification of the complex which will be subsequently analyzed by mass spectrometry. Results obtained for CoA-SPC of yeast will be finally compared with the situation in higher eukaryotes.