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

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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.