School of Chemical and Biomedical Engineering, College of Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, Singapore.
Metab Eng. 2013 Mar;16:95-102. doi: 10.1016/j.ymben.2013.01.003. Epub 2013 Jan 23.
Microbial production of biofuel has attracted significant attention in recent years. The fatty acids are important precursors for the production of fuels and chemicals, and its biosynthesis is initiated by the conversion of acetyl-CoA to malonyl-CoA which requires acetyl-CoA as key substrate. Herein, the yeast Saccharomyces cerevisiae was proposed to be metabolically engineered for cytosol acetyl-CoA enhancement for fatty acid synthesis. By gene disruption strategy, idh1 and idh2 genes involved in citrate turnover in tricarboxylic acid cycle (TCA cycle) were disrupted and the citrate production level was increased to 4- and 5-times in mutant yeast strains. In order to convert accumulated citrate to cytosol acetyl-CoA, a heterologous ATP-citrate lyase (ACL) was overexpressed in yeast wild type and idh1,2 disrupted strains. The wild type strain expressing acl mainly accumulated saturated fatty acids: C14:0, C16:0 and C18:0 at levels about 20%, 14% and 27%, respectively. Additionally, the idh1,2 disrupted strains expressing acl mainly accumulated unsaturated fatty acids. Specifically in Δidh1 strain expressing acl, 80% increase in C16:1 and 60% increase in C18:1 was detected. In Δidh2 strain expressing acl, 60% increase in C16:1 and 45% increase in C18:1 was detected. In Δidh1/2 strain expressing acl, there was 92% increase in C16:1 and 77% increase in C18:1, respectively. The increased fatty acids from our study may well be potential substrates for the production of hydrocarbon molecules as potential biofuels.
近年来，微生物生产生物燃料引起了人们的极大关注。脂肪酸是燃料和化学品生产的重要前体，其生物合成由乙酰辅酶 A 转化为丙二酰辅酶 A 起始，这需要乙酰辅酶 A 作为关键底物。在此，提出对酵母酿酒酵母进行代谢工程改造，以增强细胞质乙酰辅酶 A 用于脂肪酸合成。通过基因敲除策略，敲除了三羧酸循环（TCA 循环）中涉及柠檬酸转化的 idh1 和 idh2 基因，使突变酵母菌株中的柠檬酸产量增加了 4-5 倍。为了将积累的柠檬酸转化为细胞质乙酰辅酶 A，在酵母野生型和 idh1、2 敲除菌株中过表达了一种异源 ATP-柠檬酸裂解酶（ACL）。表达 acl 的野生型菌株主要积累饱和脂肪酸：C14:0、C16:0 和 C18:0，分别约为 20%、14%和 27%。此外，表达 acl 的 idh1、2 敲除菌株主要积累不饱和脂肪酸。具体来说，在表达 acl 的Δidh1 菌株中，C16:1 增加了 80%，C18:1 增加了 60%。在表达 acl 的Δidh2 菌株中，C16:1 增加了 60%，C18:1 增加了 45%。在表达 acl 的Δidh1/2 菌株中，C16:1 增加了 92%，C18:1 增加了 77%。我们研究中增加的脂肪酸很可能是作为潜在生物燃料的烃分子生产的潜在底物。
