Bioengineered Proteins for Chemical/Biological Defense, Protection, and Decontamination

The overall goal of Phase I is to generate preliminary sequence, functional, and pathway data for Pichia pastoris and to use that information to develop a strategy for strain improvement in Phase I This process will involve the construction of a highlyrandom plasmid library and generation of 30,000 sequencing reactions. Assembly of the contigs will be done using the Phred-Phrap-Consted suite of assembly tools. We will then identify open reading frames (ORFs) using both public and proprietary genesearching programs. We expect to identify 80-85% of the ORFs in the genome at this level of coverage. The identified ORFs will then be subjected to a FASTA search against IG's proprietary IG's non-redundant database containing over 650,000 ORFs thatrepresent 299 genomes currently. Functional assignments will then be made. Upon the identification of significant regions, strategies will be developed for strain improvements using gene disruption, gene replacement and heterologous gene expression. Thebudding yeast Pichia pastoris is widely used to produce foreign proteins for industrial, academic and military purposes. This yeast grows to high densities in fermenter cultures, and simple molecular biology procedures allow foreign genes to be highlyexpressed in Pichia from regulated or constitutive promoters. Unlike bacteria, Pichia is efficient at synthesizing, modifying and secreting eukaryotic proteins. Sequencing the Pichia genome will dramatically enhance the utility of this yeast. Even apartial genome sequence will allow Pichia to be reengineered to reduce endogenous proteolytic activity, increase the transcription and translation efficiency of foreign genes, and improve the folding and export of secreted proteins. Moreover, the Pichiagenome sequence will pave the way for creating strains that synthesize more