The beta-lactam biosynthesis genes for isopenicillin N epimerase and deacetoxycephalosporin C synthetase are expressed from a single transcript in Streptomyces clavuligerus.
Isopenicillin N isomerase (epimerase) has been purified from Streptomyces clavuligerus, and the amino acid sequence of the N-terminus has been determined. By using single oligonucleotide probes based on high GC codon bias ("guessmers"), the translation start codons were determined for two successive genes in the beta-lactam-biosynthetic pathway and mapped within a 3.6-kilobase-pair KpnI restriction fragment. The epimerase gene (cefD) was located immediately upstream of the deacetoxycephalosporin C synthetase (expandase) gene (cefE) that was characterized previously. cefD was sequenced and expressed in Escherichia coli; the resulting cell extracts contained epimerase activity. Western immunoblots demonstrated that a protein comigrated with purified S. clavuligerus epimerase at 44 kilodaltons. cefD and cefE were separated by an 81-base-pair segment. The DNA sequence upstream of the epimerase gene had a high AT content, suggestive of a promoter region. Primer extension analysis of S. clavuligerus mRNA showed that the start of transcription occurred approximately 130 base pairs upstream of the epimerase translation start site; Northern (RNA blot) analysis revealed a hybridization signal large enough to code for both epimerase and expandase, and nuclease S1 protection assays showed that a single message may code for epimerase, expandase, and another unknown protein. When cefD and cefE were placed in an expression vector, concomitant synthesis of both epimerase and expandase occurred in E. coli.
Kovacevic
S
S
Department of Molecular Genetics Research, Lilly Corporate Center, Indianapolis, Indiana 16285.
Tobin
M B
MB
Miller
J R
JR
D000591:Amino Acid Isomerases / Q000235:genetics*; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001426:Bacterial Proteins; D001482:Base Composition; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D019751:Intramolecular Transferases; D007535:Isomerases / Q000235:genetics*; D008969:Molecular Sequence Data; D009710:Nucleotide Mapping; D015345:Oligonucleotide Probes; D046915:Penicillin-Binding Proteins; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic; D047090:beta-Lactams
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D015345:Oligonucleotide Probes; D046915:Penicillin-Binding Proteins; D012329:RNA, Bacterial; D047090:beta-Lactams; D015719:Single-Strand Specific DNA and RNA Endonucleases; D007535:Isomerases; D000591:Amino Acid Isomerases; C040886:isopenicillin N epimerase; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase