A gene with a high-nucleotide sequence homology to the edeine B1 amidinohydrolase gene of Bacillus brevis was identified in the database of the Bacillus subtilis genome. The gene was isolated, expressed in Escherichia coli, and the gene product was analyzed with regard to the characteristics of its enzyme activity. A 32-kDa protein encoded by the ywhG gene showed a 69.8% amino acid sequence-homology to the edeine B1 amidinohydrolase of B. brevis. Among various guanidino-compounds, edeine B1 and agmatine were both efficiently hydrolyzed by the protein encoded by the ywhG gene, although edeine B1 was a more potent substrate than agmatine in this assay system. These data indicate that the protein encoded by the ywhG gene is an agmatinase that is essential for polyamine biosynthesis in B. subtilis.

The first step of C-P compound biosynthesis is a C-P bond formation reaction catalyzed by phosphoenolpyruvate phosphomutase, but this reaction favors the cleavage of the C-P bond. This C-P bond forming reaction is driven by the following reaction catalyzed by phosphonopyruvate (PnPy) decarboxylase. We have cloned and sequenced the gene (bcpC) encoding PnPy decarboxylase, a key enzyme of C-P compound biosynthesis, from the bialaphos (BA) producing microorganism Streptomyces hygroscopicus by complementation methods using Streptomyces wedmorensis NP-7, which is a mutant of a fosfomycin producing strain deficient in this step. The location of this gene in the BA biosynthetic gene cluster was determined by using the expression system in Streptomyces lividans. DNA sequencing of this gene revealed a 1203-bp open reading frame encoding a polypeptide of 401 amino acids.

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The fosfomycin resistance gene, fosC, has been cloned from the fosfomycin-producing organism, Pseudomonas syringae PB-5123. Sequence analysis upstream of this gene found a new ORF showing significant homology to 2-hydroxypropylphosphonic acid epoxidase from fosfomycin-producing Streptomyces wedmorensis. The purified recombinant protein of this ORF converted 2-hydroxypropylphosphonic acid to fosfomycin. This result clearly showed the ORF to encode 2-hydroxypropylphosphonic acid epoxidase in PB-5123.

The biosynthetic step following the phosphoenolpyruvate (PEP) phosphomutase reaction which forms a C-P bond of bialaphos was proven by the identification of phosphonopyruvate (PnPy) and phosphonoacetaldehyde (PnAA) as intermediates in the culture broth of Streptomyces hygroscopicus, a producing organism of bialaphos, and by detection of enzymatic decarboxylation of PnPy to PnAA. Purified PnPy decarboxylase turned out to require thiamine diphosphate and Mg2+ as cofactors. PnPy decarboxylase drives the unfavorable forward reaction to form PnPy catalyzed by PEP phosphomutase and is suggested to be essential to C-P compound biosynthesis.

Fig. 1. Structures of FMand FMderivatives inactivated by gene products offomA andfomB. analysis revealed the presence of six open reading frames with unknownfunctions in the sequenced fragment, in addition to the four genes necessary to FMbiosynthesis. So far studied, most antibiotic-producing Streptomyces possessed resistance genes and regulatory genes in their antibiotic production gene cluster. Antibiotic resistance genes have been cloned from various organisms using antibiotic sensitive hosts, Streptomyces lividans or Escherichia coll. We investigated the FMgene cluster to identify a gene(s) which confers FMresistance to E. coli. Deletion analysis of various fragments in the FMbiosynthetic gene cluster revealed that bothfomA andfomB, formerly called as orfA and orfB9\ respectively, and characterized as FM-resistance genes, were required for complete

The retinoblastoma protein (pRB) is inactivated during the development of a wide variety of human cancers. In the course of our screening for antitumor antibiotics by using pRB-inactivated cells, an actinomycete strain was found to produce two active substances, which were elucidated to be new members of the leptomycin-anguinomycin family by NMR spectral analysis and were designated anguinomycins C and D. The anguinomycins induced growth arrest against normal cells and induced cell death against transformed cells, in which pRB was inactivated by viral oncoproteins such as human papillomavirus E7, adenovirus E1A and simian virus 40 large T antigen.

catalyzed by PnAAmethyltransferase. Based on the finding that fosfomycin non-producing mutants defective in the biosynthesis of vitamin B12 incorporated [methyl14C]methylcobalamin to fosfomycin, we proposed that PnAAwas directly methylated by a nucleophilic attack of the methyl anion derived from methylcobalamin catalyzed by PnAAmethyltransferase5). More recently we have determined the nucleotide sequences of the genes encoding P-methyltransferase6)

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.

Thiotipin was isolated from the mycelium of Streptomyces sp. DT31 as a tipA promoter inducing substance. Based on various NMR studies including H-1-H-1 COSY, HSQC, FG-HMBC, phase-sensitive C-13-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.

An enzymatic activity catalyzing P-methylation of N-acetyldemethylphosphinothricin, a biosynthetic intermediate of the herbicide bialaphos, was detected in a cell extract of Streptomyces hygroscopicus SF-1293, a bialaphos producing organism. The gene coding for this P-methylation enzyme in the bialaphos biosynthetic gene cluster was also expressed in Streptomyces lividans. The methyl donor of the reaction was determined to be methylcobalamin. The P-methylation enzyme utilized both N-acetyldemethylbialaphos and N-acetyldemethylphosphinothricin as substrates.

The 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.

Bialaphos (BA) is a tripeptide produced by Streptomyces hygroscopicus SF-1293 and is characterized by the presence of a unique C-P-C bond. Through the biosynthetic studies on BA we have revealed the involvement of three enzymes catalyzing different C-P bond formation mechanisms. Two of them, phosphoenolpyruvate phosphomutase (PEP phosphomutase) and carboxyphosphonoenolpyruvate phosphonomutase (CPEP phosphonomutase), have been purified and characterized to catalyze the reactions of a similar type, i.e., intramolecular rearrangements of phosphate esters to form a C-P bond. On the other hand, the remaining one catalyzes P-methylation of phosphinic acid derivatives. In order to reveal these enzymatic properties in more detail, the corresponding genes were identified in the biosynthetic cluster of BA and then expressed in S. lividans. Introduction of PEP phosphomutase or CPEP phosphonomutase gene into S. lividans using pIJ680 resulted in efficient expression of the corresponding enzymes. On the other hand, the P-methylation enzyme was only expressed in S. lividans when the gene was under control of a thiostrepton-inducible strong promoter of pAK114.

During the course of a screening program for new antitumor antibiotics from microorganisms using a unique assay system, we have found that Streptomyces avallaneus 02-3 produced new chromomycin antibiotics, 02-3D and 02-3G in addition to several known related antibiotics. In this paper, we wish to report the isolation and structural studies of 02-3D and 02-3G