Genome sequence of Kitasatospora setae NBRC 14216T: an evolutionary snapshot of the family Streptomycetaceae.

Kitasatospora setae NBRC 14216(T) (=KM-6054(T)) is known to produce setamycin (bafilomycin B1) possessing antitrichomonal activity. The genus Kitasatospora is morphologically similar to the genus Streptomyces, although they are distinguishable from each other on the basis of cell wall composition and the 16S rDNA sequence. We have determined the complete genome sequence of K. setae NBRC 14216(T) as the first Streptomycetaceae genome other than Streptomyces. The genome is a single linear chromosome of 8,783,278 bp with terminal inverted repeats of 127,148 bp, predicted to encode 7569 protein-coding genes, 9 rRNA operons, 1 tmRNA and 74 tRNA genes. Although these features resemble those of Streptomyces, genome-wide comparison of orthologous genes between K. setae and Streptomyces revealed smaller extent of synteny. Multilocus phylogenetic analysis based on amino acid sequences unequivocally placed K. setae outside the Streptomyces genus. Although many of the genes related to morphological differentiation identified in Streptomyces were highly conserved in K. setae, there were some differences such as the apparent absence of the AmfS (SapB) class of surfactant protein and differences in the copy number and variation of paralogous components involved in cell wall synthesis.

Stereoselective synthesis of (R)-3-quinuclidinol through asymmetric reduction of 3-quinuclidinone with 3-quinuclidinone reductase of Rhodotorula rubra.

A novel nicotinamide adenine dinucleotide phosphate-dependent carbonyl reductase, 3-quinuclidinone reductase, was isolated from Rhodotorula rubra JCM3782. The enzyme catalyzes the asymmetric reduction of 3-quinuclidinone to (R)-3-quinuclidinol. The gene encoding the enzyme was also cloned and sequenced. A 819-bp nucleotide fragment was confirmed to be the gene encoding the 3-quinuclidinone reductase by agreement of the internal amino acid sequences of the purified enzyme. The gene encodes a total of 272 amino acid residues, and the deduced amino acid sequence shows similarity to those of several short-chain dehydrogenase/reductase family proteins. An expression vector, pWKLQ, which contains the full length 3-quinuclidinone reductase gene was constructed. Using Escherichia coli cells coexpressing the 3-quinuclidinone reductase and glucose dehydrogenase (cofactor regeneration enzyme) genes, 618 mM 3-quinuclidinone was almost stiochiometrically converted to (R)-3-quinuclidinol with an >99.9% enantiomeric excess within 21 h of reaction.

Enhancement of activity of lipase-displaying yeast cells and their application to optical resolution of (R,S)-1-benzyloxy-3-chloro-2-propyl monosuccinate.

Rhizopus oryzae lipase (ROL) was displayed on the cell surface of Saccharomyces cerevisiae via the Flo1 N-terminal region (1100 amino acids), which corresponds to a flocculation functional domain. The activity of lipase-displaying yeast whole-cell biocatalysts was enhanced 7.3-fold by incubation of the yeast cells at 20 degrees C in distilled water for 8 days after 8 day cultivation. The amount of lipase molecules present in cell wall and intracellular fractions was found to be increased 4.5- and 1.8-fold, respectively, by incubation, which proves that ROL molecules are expressed during incubation. The ROL-displaying yeast whole-cell biocatalyst with enhanced activity was successfully catalyzed by optical resolution of the pharmaceutical precursor (R,S)-1-benzyloxy-3-chloro-2-propyl monosuccinate. Moreover, it showed stable activity through at least eight reaction cycles. These results demonstrate that ROL-displaying yeast cells with enhanced activity by incubation in distilled water are very effective in industrial bioconversion processes.