Engineering Carotenoid Derived Aroma Compound Pathways into E. coli by Exploring the Sequence Diversity of Putative Carotenoid Dioxygenases Present in Microbial Genome Sequences

基因组测序项目产生的大量新基因序列为后基因组领域新途径的设计提供了巨大的机会。然而，已知生物合成酶的非特征序列同源物可以用于与特征同源物的体外重组，以获得更大的序列空间（而不是仅使用特征序列），从而在这些已创建的文库中发现新的代谢途径。该应用程序旨在扩展独特的新型类胡萝卜素途径，首次在大肠杆菌中创建新的生物合成途径，用于合成类胡萝卜素衍生的香气和风味化合物。通过在基因组序列中鉴定出新的、未表征的细菌CCD同源物，扩展先前工程的类胡萝卜素途径，将产生新的生物合成途径，用于生产各种天然和非天然的香气和颜色化合物。比较CCD同源物的底物特异性和裂解产物将提高我们对类胡萝卜素双加氧酶裂解机理的理解。为了利用基因组序列中发现的大量未表征的基因同源物所呈现的序列多样性来进行新的催化功能的体外进化，将开发一种新的序列引导体外重组方法，允许重组大量相当不同的序列。

Schmidt-Dannert 0332478The vast number of new gene sequences generated by genome sequencing projects provides an enormous opportunity for the design of new pathways in the post-genomic area. Yet uncharacterized sequence homologs to known biosynthetic enzymes could be used for in vitro recombination with the characterized homologs to access a much larger sequence space (as opposed to using only characterized sequences) for the discovery of new metabolic pathways in such created libraries. This application seeks to expand on the unique set of novel carotenoid pathways available to create for the first time new biosynthetic routes in E. coli for the synthesis of carotenoid derived aroma and flavor compounds. By extending previously engineered carotenoid pathways with novel, uncharacterized bacterial CCD homologs identified in genome sequences, novel biosynthetic pathways for the production of a diverse array of natural and non-natural aroma and color compounds will be generated. Comparison of substrate specificities and cleavage products of CCD homologs will improve our understanding of the mechanistic aspects of carotenoid dioxygenase cleavage. To access the sequences diversity presented by numerous uncharacterized gene homologs identified in genome sequences for in vitro evolution of novel catalytic functions, a novel sequence guided in vitro recombination approach allowing recombination of a large number of fairly diverse sequences will be developed.