Tomomi Hidaka

CLONING AND NUCLEOTIDE SEQUENCE OF FOSFOMYCIN BIOSYNTHETIC GENES OF STREPTOMYCES WEDMORENSIS

The biosynthetic pathway for production of the antibiotic fosfomycin by Streptomyces wedmorensis consists of four steps including the formation of a C-P bond and an epoxide. Fosfomycin production genes were cloned from genomic DNA using S. wedmorensis mutants blocked at different steps of the biosynthetic pathway. Four genes corresponding to each of the biosynthetic steps were found to be clustered in a DNA fragment of about 5 kb. Nucleotide sequencing of a large fragment revealed the presence of ten open reading frames, including the four biosynthetic genes and six genes with unknown functions.

Sequence of a P-.methyltransferase-encoding gene isolated from a bialaphos-producing Streptomyces hygroscopicus

The nucleotide sequence of the Streptomyces hygroscopicus gene encoding P-methyltransferase, catalyzing the formation of a carbon-phosphorus bond, involved in bialaphos biosynthesis, has been determined. The amino-acid sequence deduced from the nt sequence, shows homology with those of magnesium-protoporphyrin IX monomethyl ester oxidative cyclase (Mg-ProtoMe cyclase) of Rhodobacter capsulatus and the enzyme catalyzing the methylation of the aldehyde carbon of phosphonoacetaldehyde in fosfomycin biosynthesis.

Nucleotide sequence of a carboxyphosphonoenolpyruvate phosphonomutase gene isolated from a bialaphos-producing organism, Streptomyces hygroscopicus, and its expression in Streptomyces lividans

SummaryThe carboxyphosphonoenolpyruvate (CPEP) phosphonomutase gene of bialaphos-producing Streptomyces hygroscopicus, which encodes a C-P bond forming enzyme was cloned into Streptomyces lividans and sequenced. The amino acid composition of the protein coded in an open reading frame of 295 codons and its calculated molecular mass, 32,800 Da, coincided well with those of the purified enzyme. Introduction of the CPEP phosphonomutase gene, the expression of which is controlled by the promoter of the aph gene, into S. lividans resulted in the production of this enzyme at a level almost equivalent to that in the parent strain.

Leptin changes Ca2+/calmodulin-dependent response and upregulates the gene expression of calcineurin in rat hypothalamus

To understand the leptins action on hypothalamic arcuate nucleus (ARC), Ca2+/calmodulin (CaM)-dependent response in leptin-treated rat ARC was investigated. The in vitro phosphorylation of ARC extracts from leptin-treated rats did not respond to CaM. Although CaM-dependent protein kinase II activity was not affected by leptin, the gene expression of calcineurin, CaM-dependent phosphatase, increased in leptin-treated rats.

Thiotipin, a novel thiopeptide with a tipA promoter inducing activity produced by Streptomyces sp. DT31

Abstract Thiotipin was isolated from the mycelium of Streptomyces sp. DT31 as a tipA promoter inducing substance. Based on various NMR studies including 1 H- 1 H COSY, HSQC, FG-HMBC, phase-sensitive 13 C-decoupled HMBC (D-HMBC) and NOE, its structure was established as a thiopeptide with oxazoles, thiazoles and several modified amino acids. Minimum induction concentration of thiotipin for tipA promoter was 80 ng/ml.

Biochemical Mechanism of C–P Bond Formation of Bialaphos: Use of Gene Manipulation for the Analysis of the C–P Bond Formation Step

One of the three C-P bond formation steps, defined as step 5 in the bialaphos (BA) biosynthetic pathway, was analyzed using a new BA non-producing mutant NP71. The mutant was derived from a BA producer by gene replacement of an unidentified region next to the gene responsible for the step 5 deficiency of the mutant NP213, obtained by conventional mutation procedures. Biochemical analysis of these two mutants indicated that NP71 was defective in the formation of carboxyphosphonoenolpyruvate (CPEP), while NP213 lacked the enzyme CPEP phosphonomutase, which catalyzed the intramolecular rearrangement of CPEP.