Abstract:

A kind of universal enzyme that catalyzes a multi-step continuous reaction in an organism plays an important role in the biological metabolic process. As a typical representative, phytoene dehydrogenase (CrtI) can catalyze multi-step continuous dehydrogenation to produce products of great value such as lycopene. Herein, the catalytic function of CrtI was studied in Saccharomyces cerevisiae. Firstly, by combining design and screening of three heterologous enzymes CrtE, CrtB and CrtI in the lycopene synthesis pathway, CrtI was confirmed as the main limiting factor, and CrtI from Blakeslea trispora exerted excellent catalytic function. Through bioinformatics and protein structural analysis the key residue S311 of BtCrtI was explored, which linked and maintained the key secondary structure of active center domain. Subsequently, the results of saturation mutation showed that the type of amino acid residue mutated at S311 had a significant effect on the structure and function of the active center domain. This provided a novel structural point for the design and modification of enzymes. Meanwhile, another interesting finding is that the various activity of CrtI mutants did not disturbing the carotenoid metabolic flow in our biosynthesis pathway. Therefore, CrtI is crucial to improve the yield and purity of lycopene.

Key words: enzyme, synthetic biology, biocatalysis, phytoene dehydrogenase(CrtI), multi-step dehydrogenation, enzyme active center structure

摘要：

在生物体内存在可催化多步连续反应的通用酶，对生物代谢过程具有重要作用。八氢番茄红素脱氢酶（CrtI）作为典型代表,可以催化多步连续脱氢反应,生成番茄红素等具有重要价值的产物。本文以酿酒酵母为底盘研究CrtI的催化功能特征。首先通过组合设计与筛选番茄红素合成路径中的三种外源酶CrtE、CrtB和CrtI，发现CrtI是主要的限制因素，且三孢布拉氏霉菌来源的CrtI(BtCrtI)表现出优异的催化功能。通过生物信息学与蛋白结构分析发现BtCrtI的S311残基是连接和稳定活性中心结构的关键。随后通过分析该位点的饱和突变结果，揭示了该位点的氨基酸残基类型对活性中心结构和功能的显著作用，为酶的设计和改造提供了新的思路。同时发现CrtI的活性差异未对合成路径中的类胡萝卜素的代谢流造成扰动，表明CrtI是番茄红素的产量和纯度的决定因素。

关键词: 酶, 合成生物学, 生物催化, 八氢番茄红素脱氢酶, 多步脱氢反应, 酶的活性中心结构