SBIR-STTR Award

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 highly random 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 gene searching 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 that represent 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. The budding 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 highly expressed 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 a partial 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 Pichia genome sequence will pave the way for creating strains that synthesize more "mammalian-like" oligosaccharides. A genome database will also stimulate basic researchers to study Pichia cell biology and to develop improved techniques for manipulating this yeast. Therefore, sequencing the Pichia genome is vitally important for the future development of this organism as a protein expression system.

Keywords:
Pichia Pastoris, Sequencing, Strain Development, Yeast Expression System

Pichia pastoris is a budding yeast used by hundreds of academic and industrial researchers to synthesize a variety of eukaryotic recombinant proteins. P. pastoris is also a model system used to investigate certain areas of eukaryotic cell biology, such as peroxisome biogenesis and degradation, and Golgi organization. The goals of this project are: (1) to determine, with high fidelity, the DNA sequence of the genome of P. pastoris by a combination of "shot-gun" sequencing and "primer-walking" gap closing; (2) to annotate the expected 6,000 plus ORFs; (3) to develop a publicly accessible web site dedicated to the dissemination of information on the sequencing and annotation results; (4) to construct and make available DNA microarrays focused on ~500 P. pastoris genes whose expression is relevant to recombinant protein production; and (5) to construct certain modified P. pastoris strains for improved production of recombinant proteins. The sequencing, assembly, and preliminary gene annotation work will be performed by Integrated Genomics (IG), an experienced microbial sequencing and analysis company. The hosting of the web site, DNA microarray, and host strain constructions will be performed at the Keck Graduate Institute (KGI). KGI is a new educational and research institution focusing on bioinformatics and genomics technologies. The availability of the DNA sequence of the P. pastoris genome with associated materials and information as proposed in this application will be of enormous benefit to the many commercial and academic researchers utilizing this novel but important yeast. Because of the similarity in amino acid sequence between most P. pastoris proteins and their Saccharomyces cerevisiae counterparts, much of the great wealth of information generated over the years for S. cerevisiae genes and products will be transferred directly to those of P. pastoris. Users of P. pastoris will be able to rapidly identify genes of interest and, in conjunction with PCR methodology, have these genes in hand for further study literally overnight. Thus, the P. pastoris genomic database will be a commercially valuable product in itself. The focused DNA microarrays will be a useful tool to commercial laboratories that wish to monitor gene expression as part of their bioprocess development efforts for new recombinant protein production strains. Finally, the P. pastoris strains constructed for this project are expected to lead to improved levels of recombinant protein production from P. pastoris and therefore, to be valuable to the many companies and academic labs endeavoring to improve the efficiency of their recombinant processes.