L-DOPA is a key pharmaceutical agent for treating Parkinson's, and market demand has exploded due to the aging population. There are several challenges associated with the chemical synthesis of L-DOPA, including complicated operation, harsh conditions, and serious pollution. A biocatalysis route for L-DOPA production is promising, especially via a route catalyzed by tyrosine phenol lyase (TPL). In this study, using TPL derived from Erwinia herbicola (Eh-TPL), a mutant Eh-TPL was obtained by integrating enzyme evolution and high-throughput screening methods. L-DOPA production using recombinant Escherichia coli BL21 (DE3) cells harbouring mutant Eh-TPL was enhanced by 36.5% in shake flasks, and the temperature range and alkali resistance of the Eh-TPL mutant were promoted. Sequence analysis revealed two mutated amino acids in the mutant (S20C and N161S), which reduced the length of a hydrogen bond and generated new hydrogen bonds. Using a fed-batch mode for whole-cell catalysis in a 5 L bioreactor, the titre of L-DOPA reached 69.1 g L-1 with high productivity of 11.52 g L-1 h-1, demonstrating the great potential of Eh-TPL variants for industrial production of L-DOPA.

中文翻译：

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整合酶进化和高通量筛选，可有效合成L-DOPA。

L-DOPA是治疗帕金森氏症的关键药物，由于人口老龄化，市场需求激增。L-DOPA的化学合成存在许多挑战，包括操作复杂，条件恶劣和严重污染。L-DOPA生产的生物催化途径很有希望，特别是通过酪氨酸酚裂解酶（TPL）催化的途径。在这项研究中，使用源自草生欧文氏菌的TPL（Eh-TPL），通过整合酶进化和高通量筛选方法获得了突变的Eh-TPL。使用带有突变Eh-TPL的重组大肠杆菌BL21（DE3）细胞在摇瓶中生产L-DOPA的速度提高了36.5％，并且促进了Eh-TPL突变体的温度范围和耐碱性。序列分析揭示了突变体中的两个突变氨基酸（S20C和N161S），它们减少了氢键的长度并产生了新的氢键。使用分批补料模式在5 L生物反应器中进行全细胞催化，L-DOPA的效价达到69.1 g L-1，高生产率为11.52 g L-1 h-1，证明了Eh-TPL的巨大潜力L-DOPA的工业生产变体。