Abstract As a very long chain monounsaturated fatty acid, nervonic acid (NA) holds great application potential for brain diseases. 3-ketoacyl-CoA synthase (KCS) with substrate specificity plays a key role in the carbon chain elongation cycle for NA biosynthesis. A tunable and sensitive single-gene expression regulation is necessary to overproduce NA in yeast cell. Herein, β-estradiol inducible expression system (EIES) was employed to augment NA biosynthesis in Saccharomyces cerevisiae. EIES consisted of two recombinant vectors: pRS416-PGal1-GFP-TCYC1 and YEplac112-PTEF1-ZEV-TCYC1. The following was the optimized induction conditions of EIES in SD medium: 0.1 μM β-estradiol, 15 min of heat induction at 33 ℃ every 8 h within first 32 h. Using EIES to enhance KCS and ELOVL1 expression, NA contents in the recombinants increased by 332 % and 101 % respectively, compared with the control strain using strong PGK promoter. NA levels were further improved by knockout of elo2 in the recombinants expressing KCS or ELOVL1. This study suggested that EIES could be a promising tool for enhancing the biosynthesis of desired metabolites.

中文翻译：

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利用β-雌二醇诱导表达系统在酿酒酵母中过量产生神经酸

摘要 作为一种非常长链的单不饱和脂肪酸，神经酸（NA）在脑部疾病中具有巨大的应用潜力。具有底物特异性的 3-酮酰基辅酶 A 合酶 (KCS) 在 NA 生物合成的碳链延长循环中起关键作用。在酵母细胞中过度产生 NA 需要一个可调且灵敏的单基因表达调控。在此，β-雌二醇诱导表达系统 (EIES) 被用于增加酿酒酵母中 NA 的生物合成。EIES 由两个重组载体组成：pRS416-PGal1-GFP-TCYC1 和 YEplac112-PTEF1-ZEV-TCYC1。EIES在SD培养基中的优化诱导条件如下：0.1 μM β-雌二醇，前32 h内每8 h 33℃热诱导15 min。使用 EIES 增强 KCS 和 ELOVL1 表达，与使用强 PGK 启动子的对照菌株相比，重组体中的 NA 含量分别增加了 332% 和 101%。通过敲除表达 KCS 或 ELOVL1 的重组体中的 elo2，NA 水平进一步提高。该研究表明，EIES 可能是增强所需代谢物生物合成的有前途的工具。