Shi Shuobo, Chen Yun, Siewers Verena, Nielsen Jens
Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
mBio. 2014 May 6;5(3):e01130-14. doi: 10.1128/mBio.01130-14.
ABSTRACT Acetyl coenzyme A (acetyl-CoA) carboxylase (ACCase) plays a central role in carbon metabolism and has been the site of action for the development of therapeutics or herbicides, as its product, malonyl-CoA, is a precursor for production of fatty acids and other compounds. Control of Acc1 activity in the yeast Saccharomyces cerevisiae occurs mainly at two levels, i.e., regulation of transcription and repression by Snf1 protein kinase at the protein level. Here, we demonstrate a strategy for improving the activity of ACCase in S. cerevisiae by abolishing posttranslational regulation of Acc1 via site-directed mutagenesis. It was found that introduction of two site mutations in Acc1, Ser659 and Ser1157, resulted in an enhanced activity of Acc1 and increased total fatty acid content. As Snf1 regulation of Acc1 is particularly active under glucose-limited conditions, we evaluated the effect of the two site mutations in chemostat cultures. Finally, we showed that our modifications of Acc1 could enhance the supply of malonyl-CoA and therefore successfully increase the production of two industrially important products derived from malonyl-CoA, fatty acid ethyl esters and 3-hydroxypropionic acid. IMPORTANCE ACCase is responsible for carboxylation of acetyl-CoA to produce malonyl-CoA, which is a crucial step in the control of fatty acid metabolism. ACCase opened the door for pharmaceutical treatments of obesity and diabetes as well as the development of new herbicides. ACCase is also recognized as a promising target for developing cell factories, as its malonyl-CoA product serves as a universal precursor for a variety of high-value compounds in white biotechnology. Yeast ACCase is a good model in understanding the enzyme's catalysis, regulation, and inhibition. The present study describes the importance of protein phosphorylation in regulation of yeast ACCase and identifies potential regulation sites. This study led to the generation of a more efficient ACCase, which was applied in the production of two high-value compounds derived from malonyl-CoA, i.e., fatty acid ethyl esters that can be used as biodiesel and 3-hydroxypropionic acid that is considered an important platform chemical.
摘要 乙酰辅酶A（acetyl-CoA）羧化酶（ACCase）在碳代谢中起核心作用，并且一直是治疗药物或除草剂开发的作用靶点，因为其产物丙二酰辅酶A是脂肪酸和其他化合物生产的前体。酿酒酵母中Acc1活性的控制主要发生在两个水平，即转录调控以及Snf1蛋白激酶在蛋白质水平的抑制作用。在此，我们展示了一种通过定点诱变消除Acc1的翻译后调控来提高酿酒酵母中ACCase活性的策略。研究发现，在Acc1中引入两个位点突变，即Ser659和Ser1157，会导致Acc1活性增强以及总脂肪酸含量增加。由于Acc1的Snf1调控在葡萄糖受限条件下特别活跃，我们在恒化器培养中评估了这两个位点突变的效果。最后，我们表明对Acc1的修饰可以增强丙二酰辅酶A的供应，因此成功提高了两种源自丙二酰辅酶A的重要工业产品，脂肪酸乙酯和3-羟基丙酸的产量。重要性 ACCase负责将乙酰辅酶A羧化生成丙二酰辅酶A，这是脂肪酸代谢控制中的关键步骤。ACCase为肥胖症和糖尿病的药物治疗以及新型除草剂的开发打开了大门。ACCase也被认为是开发细胞工厂的一个有前景的靶点，因为其丙二酰辅酶A产物是白色生物技术中多种高价值化合物的通用前体。酵母ACCase是理解该酶的催化、调控和抑制作用的良好模型。本研究描述了蛋白质磷酸化在酵母ACCase调控中的重要性，并确定了潜在的调控位点。这项研究产生了一种更高效的ACCase，并将其应用于两种源自丙二酰辅酶A的高价值化合物的生产，即可用作生物柴油的脂肪酸乙酯和被认为是重要平台化学品的3-羟基丙酸。
