Bacchus Bio innovation Co. Ltd, 6-3-7-505 Minatojima Minamimachi, Chuo-ku, Kobe, 650-0047, Japan.
Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.
Microb Cell Fact. 2024 Jun 15;23(1):178. doi: 10.1186/s12934-024-02453-7.
Computational mining of useful enzymes and biosynthesis pathways is a powerful strategy for metabolic engineering. Through systematic exploration of all conceivable combinations of enzyme reactions, including both known compounds and those inferred from the chemical structures of established reactions, we can uncover previously undiscovered enzymatic processes. The application of the novel alternative pathways enables us to improve microbial bioproduction by bypassing or reinforcing metabolic bottlenecks. Benzylisoquinoline alkaloids (BIAs) are a diverse group of plant-derived compounds with important pharmaceutical properties. BIA biosynthesis has developed into a prime example of metabolic engineering and microbial bioproduction. The early bottleneck of BIA production in Escherichia coli consists of 3,4-dihydroxyphenylacetaldehyde (DHPAA) production and conversion to tetrahydropapaveroline (THP). Previous studies have selected monoamine oxidase (MAO) and DHPAA synthase (DHPAAS) to produce DHPAA from dopamine and oxygen; however, both of these enzymes produce toxic hydrogen peroxide as a byproduct.
In the current study, in silico pathway design is applied to relieve the bottleneck of DHPAA production in the synthetic BIA pathway. Specifically, the cytochrome P450 enzyme, tyrosine N-monooxygenase (CYP79), is identified to bypass the established MAO- and DHPAAS-mediated pathways in an alternative arylacetaldoxime route to DHPAA with a peroxide-independent mechanism. The application of this pathway is proposed to result in less formation of toxic byproducts, leading to improved production of reticuline (up to 60 mg/L at the flask scale) when compared with that from the conventional MAO pathway.
This study showed improved reticuline production using the bypass pathway predicted by the M-path computational platform. Reticuline production in E. coli exceeded that of the conventional MAO-mediated pathway. The study provides a clear example of the integration of pathway mining and enzyme design in creating artificial metabolic pathways and suggests further potential applications of this strategy in metabolic engineering.
计算挖掘有用的酶和生物合成途径是代谢工程的一种强大策略。通过系统地探索包括已知化合物和从已建立反应的化学结构推断出的化合物在内的所有可能的酶反应组合，我们可以发现以前未发现的酶促过程。新的替代途径的应用使我们能够通过绕过或强化代谢瓶颈来提高微生物生物生产。苯并异喹啉生物碱（BIAs）是一组具有重要药物性质的植物衍生化合物。BIAs 生物合成已成为代谢工程和微生物生物生产的一个主要范例。BIA 生产在大肠杆菌中的早期瓶颈是 3,4-二羟基苯乙醛（DHPAA）的生产和转化为四氢罂粟碱（THP）。以前的研究已经选择单胺氧化酶（MAO）和 DHPAA 合酶（DHPAAS）从多巴胺和氧气中产生 DHPAA；然而，这两种酶都会产生有毒的过氧化氢作为副产物。
在本研究中，通过计算机模拟途径设计来缓解合成 BIA 途径中 DHPAA 生产的瓶颈。具体来说，细胞色素 P450 酶，酪氨酸 N-单加氧酶（CYP79）被鉴定为通过替代的芳基乙醛肟途径绕过已建立的 MAO 和 DHPAAS 介导的途径，以一种与过氧化物无关的机制生成 DHPAA。该途径的应用有望减少有毒副产物的形成，与传统的 MAO 途径相比，导致延胡索酸（高达 60mg/L 在瓶规模）的产量提高。
本研究使用 M-path 计算平台预测的旁路途径显示出延胡索酸产量的提高。大肠杆菌中延胡索酸的产量超过了传统的 MAO 介导途径。该研究为在创建人工代谢途径中整合途径挖掘和酶设计提供了一个清晰的范例，并表明该策略在代谢工程中的进一步潜在应用。
