Genes for cysteine biosynthesis and metabolism in Streptomyces venezuelae ISP5230: Cloning, sequencing, functional analysis and relevance to chloramphenicol biosynthesis.

摘要

The chloramphenicol (Cm) biosynthesis gene cluster (cml) in Streptomyces venezuelae is flanked by auxotrophic markers (pdx and cys) about 45 kb apart. By cloning pdx and walking the chromosome from this marker, a 4.0-kb SacI fragment hybridizing with bca (a putative Cm chlorination gene) was discovered about 30-kb from pdx. Use of the 4.0-kb SacI sequence as a marker to orient chromosome walking vis-a-vis pdx was anticipated, but was abandoned when analysis of the sequence failed to confirm the presence of an ORF associated with chlorination. As an alternative marker to orient chromosome walking, the cys-28 mutation flanking the cml cluster was chosen, and its phenotype was characterized. The gene could not be cloned by complementation because of the high reversion frequency of available cys mutants, and attempts to isolate S. venezuelae Cm production mutants by transposon mutagenesis with Tn4560 gave disappointing results, attributed to non-random transposition of Tn4560 in the S. venezuelae genome.; Indications that the cys-28 phenotype was due to loss of cysteine synthase (CS) activity directed attention towards cloning the CS gene (cysK) of S. venezuelae. A pair of PCR primers designed from the conserved amino acid sequences of CSs in the GenBanK database was used to amplify a cysK fragment from the S. venezuelae genome. Amino acid sequence deduced from the PCR product showed high similarity to CSs from both prokaryotes and eukaryotes, and to cystathionine beta-synthases (CBSs) of eukaryotes. A genomic library of S. venezuelae constructed in the phagemid pBluescript II SK (+) was screened by colony hybridization using the PCR product as a probe. From a transformant that hybridized strongly to the probe, pJV207 containing a 9.2-kb insert of S. venezuelae DNA was isolated, and a 7.0-kb fragment subcloned from pJV207 was sequenced. Analysis of the 7.0 kb sequence detected two ORFs (ORF1 and ORF2). The deduced amino acid sequence of ORF1 resembled the sequences of both CS and CBS from various organisms. It showed evolutionary links to the CSs of prokaryotes and eukaryotes, and to the CBS of eukaryotes. In length it resembled CBS. Disruption of ORF1 in S. venezuelae blocked the pathway from homocysteine to cysteine, leading to the conclusion that ORF1 encodes cystathionine beta-synthase. The product of ORF2 showed significant sequence similarity to the acetyl-CoA transferase (thiolase) of various organisms. Since disruption of ORF2 had no effect on Cm production, the possible role of this gene as a dichloroacetyl transferase in Cm biosynthesis was discounted.; Cell extracts of S. venezuelae strains were assayed for enzymes (O-acetyl-L-serine sulfhydrylase, cystathionine beta- and gamma-synthases, cystathionine beta- and gamma-lyases, and homocysteine synthase) potentially involved directly or by transsulfuration in cysteine synthesis. The results indicated that the structural gene for O-acetyl-L-serine sulfhydrylase was intact in the strain VS263 carrying the cys-28 mutation, but was not expressed in minimal medium; both cystathionine beta- and gamma-lyase activities were detected. S. venezuelae contained high O-acetylhomoserine sulfhydrylase and cystathionine beta-synthase (with O-succinylhomoserine as a substrate) activities, but CBS activity was undetected by HPLC and was dispensable for cysteine biosynthesis in the wild-type strain.