Preliminary analysis and annotation of the partial genome sequence of Francisella tularensis strain Schu 4.

Francisella tularensis, the aetiological agent of tularemia, is an important pathogen throughout much of the Northern hemisphere. We have carried out sample sequencing of its genome in order to gain a greater insight into this organism about which very little is known, especially at the genetic level. Nucleotide sequence data from a genomic DNA shotgun library of the virulent F. tularensis strain Schu 4 has been partially assembled to provide 1.83 Mb of the genome sequence. A preliminary analysis of the F. tularensis genome sequence has been performed and the data compared with 20 fully sequenced and annotated bacterial genomes. Plasmid-encoded genes, previously isolated from low virulence strains of F. tularensis, were not identified. A total of 1289 potential coding ORFs were identified in the data set., An analysis of this data revealed 413 ORFs which would encode proteins with no homology to known proteins. ORFs which could encode proteins involved in amino acid and purine biosynthesis were also identified. These biosynthetic pathways provide targets for the construction of a defined attenuated mutant of F. tularensis for use as a vaccine against tularemia

Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity*

Surface display is effectively utilized to construct a whole-cell biocatalyst. Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast. Here, the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae, and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor, recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed. Compared with the wild-type ROL-displaying yeast, the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate. To our knowledge, this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction. Consequently, the yeast whole-cell ROL biocatalyst was constructed with high activity. The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C. Furthermore, this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h