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

Schmidt-Dannert 0332478基因组测序项目产生的大量新基因序列为在后基因组领域设计新的途径提供了巨大的机会。然而，已知生物合成酶的未表征序列同源物可以用于与表征同源物的体外重组，以获得更大的序列空间(而不是仅使用表征序列)，以在这样创建的文库中发现新的代谢途径。本申请旨在扩展独特的一组新的类胡萝卜素途径，可用于在大肠杆菌中首次创建用于合成类胡萝卜素衍生香料和风味化合物的新的生物合成路线。通过用基因组序列中发现的新的、未鉴定的细菌Ccd同源物来扩展先前设计的类胡萝卜素途径，将产生新的生物合成途径，用于生产各种不同的天然和非天然香气和有色化合物。比较底物的特异性和同源物的切割产物将有助于我们更好地理解类胡萝卜素双加氧酶的切割机制。为了获得基因组序列中大量未鉴定的基因同源物所呈现的序列多样性，以用于新催化功能的体外进化，将开发一种新的序列制导的体外重组方法，允许大量相当不同的序列重组。

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.