Abstract:

The currently reported β-tyrosine production method requires complex substrates and mostly rely on precious metal catalyst. To solve this, an artificially designed cascade reaction was set up by cascading TPL tyrosine phenol lyase (TPL) and tyrosine amino mutase (TAM) to synthesize (R)-β-tyrosine, with cheap compounds (phenol, pyruvate acid and ammonium salt) as substrates. These two enzymes were screened by gene mining and the catalytic efficiency of rate limiting enzyme was improved by protein engineering. The screened enzymes were co-expressed in E. coli host, and the best (R)-β-tyrosine synthesis strain E. coli S10 was obtained after optimization. In 1 L scale reaction, (R)-β-tyrosine was synthesized with 78% conversion and >99% ee. The molecular weight and structure of the purified product were identified by high resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR), which further verified that the cascade system can be used for the synthesis of (R)-β-tyrosine. This study provides a theoretical guidance for the green enzymatic production of (R)-tyrosine.

Key words: enzyme, biocatalysis, biotechnology, cascade catalysis, (R)-β-tyrosine

摘要：

目前报道的β-酪氨酸生产方法需要较为复杂的底物而且大多依赖于贵金属催化剂。为了实现生物法绿色合成β-酪氨酸，通过人工设计的级联反应，将酪氨酸酚裂解酶和酪氨酸氨基变位酶进行级联，以苯酚、丙酮酸和铵盐等廉价化合物为底物合成(R)-β-酪氨酸。通过基因挖掘筛选了所需的酶元件，并采用蛋白质工程改造，提升了限速酶的催化效率。在大肠杆菌宿主中对所筛选的酶进行共同表达，经优化获得了(R)-β-酪氨酸合成菌株E. coli S10。在1 L规模反应中，菌株E. coli S10合成(R)-β-酪氨酸的转化率达到78%，ee值>99%。利用高分辨质谱(HRMS)和核磁共振图谱(NMR)分别鉴定了纯化产物的分子量和结构，结果表明通过该级联路径可成功合成目标产物(R)-β-酪氨酸。研究工作可为(R)-β-酪氨酸的绿色酶法生产提供理论指导。

关键词: 酶, 生物催化, 生物技术, 多酶级联催化, (R)-β-酪氨酸