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

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