Synthesis of (R)-1,3-butanediol by enantioselective oxidation using whole recombinant Escherichia coli cells expressing (S)-specific secondary alcohol dehydrogenase

The synthesis of (R)-1,3-butanediol (BDO) from its racemate was studied using whole cells of recombinant Escherichia coli expressing an (S)-specific secondary alcohol dehydrogenase (CpSADH) from Candida parapsilosis by enantioselective oxidation. Under the optimized conditions, the yield of (R)-1,3-BDO reached 72.6 g/l, with a molar recovery yield of 48.4% from a racemate of 15% and an optical purity of 95% ee.     

Cloning of the maltose phosphorylase gene from Bacillus sp. strain RK-1 and efficient production of the cloned gene and the trehalose phosphorylase gene from Bacillus stearothermophilus SK-1 in Bacillus subtilis

The maltose phosphorylase (MPase) gene of Bacillus sp. strain RK-1 was cloned by PCR with oligonucleotide primers designed on the basis of a partial N-terminal amino acid sequence of the purified enzyme. The MPase gene consisted of 2,655 bp encoding a theoretical protein with a Mr of 88,460, and had no secretion signal sequence, although most of the MPase activity was detected in the culture supernatant of RK-1. This cloned MPase gene and the trehalose phosphorylase (TPase) gene from Bacillus stearothermophilus SK-1 were efficiently expressed intracellularly under the control of the Bacillus amyloliquefaciens alpha-amylase promoter in Bacillus subtilis. The production yields were estimated to be more than 2 g of enzyme per liter of medium, about 250 times the production of the original strains, in a simple shake flask. About 60% of maltose was converted into trehalose by the simultaneous action of both enzymes produced in B. subtilis.     

Radical scavenging activities of niacin-related compounds

We investigated whether niacin-related compounds had radical-scavenging activity by electron spin resonance methods. Many compounds, but not trigonelline, had radical-scavenging activity against hydroxyl radicals. However, for the nitric oxide radical and 1,1-diphenyl-2-picrylhydrazyl radical, only nicotinic acid hydrazide and isonicotinic acid hydrazide had scavenging activities. These results suggest that the moiety of hydrazide might have an important role in scavenging abilities of various radicals.     

A genome-wide strategy for the identification of essential genes in Staphylococcus aureus

To address the need for new approaches to antibiotic drug development, we have identified a large number of essential genes for the bacterial pathogen, Staphylococcus aureus, using a rapid shotgun antisense RNA method. Staphylococcus aureus chromosomal DNA fragments were cloned into a xylose-inducible expression plasmid and transformed into S. aureus. Homology comparisons between 658 S. aureus genes identified in this particular antisense screen and the Mycoplasma genitalium genome, which contains 517 genes in total, yielded 168 conserved genes, many of which appear to be essential in M. genitalium and other bacteria. Examples are presented in which expression of an antisense RNA specifically reduces its cognate mRNA. A cell-based, drug-screening assay is also described, wherein expression of an antisense RNA confers specific sensitivity to compounds targeting that gene product. This approach enables facile assay development for high throughput screening for any essential gene, independent of its biochemical function, thereby greatly facilitating the search for new antibiotics.     

Regulation of Lck and Fyn tyrosine kinase activities by transmembrane protein tyrosine phosphatase leukocyte common antigen-related molecule

Leukocyte common antigen-related molecule (LAR) is a receptor-like protein tyrosine phosphatase (PTPase) with two PTPase domains. In the present study, we detected the expression of LAR in the brain, kidney, and thymus of mice using anti-LAR PTPase domain subunit monoclonal antibody (mAb) YU1. In the thymus, LAR was expressed on CD4(-)CD8(-) and CD4(-)CD8(low) thymocytes. The development of thymocytes in CD45 knockout mice is blocked partially in the maturation of CD4(-)CD8(-) to CD4(+)CD8(+). We postulated that LAR regulates Lck and Fyn in the immature thymocytes. Transfection of wild-type LAR activated extracellular signal-regulated kinase signal transduction pathway in CD45-deficient Jurkat cells stimulated with anti-CD3 mAb. LAR mutants, with Cys to Ser mutation in the catalytic center of PTPase D1, bound to tyrosine-phosphorylated Lck and Fyn, and LAR PTPase domain 2 was tyrosine phosphorylated by Fyn tyrosine kinase. The phosphorylated LAR was associated with Fyn Src homology 2 domain. Moreover, LAR dephosphorylated phosphorylated tyrosine residues in both the COOH terminus and kinase domain of Fyn in vitro. Our results indicate that Lck and Fyn would be substrates of LAR in immature thymocytes and that each LAR PTPase domain plays distinct functional roles in phosphorylation and dephosphorylation.     

Construction of BAC libraries derived from the ascidian Ciona intestinalis

Large insert genomic bacterial artificial chromosome (BAC) libraries were constructed from a basal chordate, the ascidian Ciona intestinalis. Insert analyses of randomly selected clones indicated that in the first library the mean insert size was 135 kb and predicted a 15-fold coverage of the Ciona genome, and in the second library the mean insert size was 165 kb and predicted a 5-fold coverage of the genome. These first large insert genomic libraries of the ascidian should increase the speed of genomic analyses of basal chordates.