Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan; PRESTO, Japan Science and Technology Agency (JST), Saitama 332-0012, Japan.
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan.
Bioresour Technol. 2024 Aug;406:130927. doi: 10.1016/j.biortech.2024.130927. Epub 2024 Jun 1.
2-Phenylethanol, known for its rose-like odor and antibacterial activity, is synthesized via exogenous phenylpyruvate by the sequential reaction of phenylpyruvate decarboxylase (PDC) and aldehyde reductase. We first targeted ARO10, a phenylpyruvate decarboxylase gene from Saccharomyces cerevisiae, and identified a suitable aldehyde reductase gene. Co-expression of ARO10 and yahK in E. coli transformants yielded 1.1 g/L of 2-phenylethanol in batch culture. We hypothesized that there might be a bottleneck in PDC activity. The computer-based enzyme evolution was utilized to enhance production. The introduction of an amino acid substitution in ARO10 (ARO10 I544W) stabilized the aromatic ring of the phenylpyruvate substrate, increasing 2-phenylethanol yield 4.1-fold compared to wild-type ARO10. Cultivation of ARO10 I544W-expressing E. coli produced 2.5 g/L of 2-phenylethanol with a yield from glucose of 0.16 g/g after 72 h. This approach represents a significant advancement, achieving the highest yield of 2-phenylethanol from glucose using microbes to date.
2-苯乙醇具有玫瑰般的香气和抗菌活性，可通过外源苯丙酮酸经苯丙酮酸脱羧酶（PDC）和醛还原酶的连续反应合成。我们首先针对来自酿酒酵母的苯丙酮酸脱羧酶基因 ARO10，并鉴定出合适的醛还原酶基因。ARO10 和 yahK 在大肠杆菌转化体中的共表达在分批培养中产生了 1.1 g/L 的 2-苯乙醇。我们假设可能存在 PDC 活性的瓶颈。基于计算机的酶进化被用于提高生产。在 ARO10 中引入一个氨基酸取代（ARO10 I544W）稳定了苯丙酮酸底物的芳环，与野生型 ARO10 相比，2-苯乙醇的产量增加了 4.1 倍。ARO10 I544W 表达的大肠杆菌培养物在 72 小时后产生了 2.5 g/L 的 2-苯乙醇，葡萄糖得率为 0.16 g/g。这种方法是一个重大的进展，是迄今为止利用微生物从葡萄糖中获得 2-苯乙醇最高产量的方法。
