Localization of the lysine epsilon-aminotransferase (lat) and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (pcbAB) genes from Streptomyces clavuligerus and production of lysine epsilon-aminotransferase activity in Escherichia coli.
Lysine epsilon-aminotransferase (LAT) in the beta-lactam-producing actinomycetes is considered to be the first step in the antibiotic biosynthetic pathway. Cloning of restriction fragments from Streptomyces clavuligerus, a beta-lactam producer, into Streptomyces lividans, a nonproducer that lacks LAT activity, led to the production of LAT in the host. DNA sequencing of restriction fragments containing the putative lat gene revealed a single open reading frame encoding a polypeptide with an approximately Mr 49,000. Expression of this coding sequence in Escherichia coli led to the production of LAT activity. Hence, LAT activity in S. clavuligerus is derived from a single polypeptide. A second open reading frame began immediately downstream from lat. Comparison of this partial sequence with the sequences of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D valine (ACV) synthetases from Penicillium chrysogenum and Cephalosporium acremonium and with nonribosomal peptide synthetases (gramicidin S and tyrocidine synthetases) found similarities among the open reading frames. Since mapping of the putative N and C termini of S. clavuligerus pcbAB suggests that the coding region occupies approximately 12 kbp and codes for a polypeptide related in size to the fungal ACV synthetases, the molecular characterization of the beta-lactam biosynthetic cluster between pcbC and cefE (approximately 25 kbp) is nearly complete.
Department of Molecular Genetics Research, Lilly Research Laboratories, Indianapolis, Indiana 46285.
M B
MB
Tobin
S
S
Kovacevic
K
K
Madduri
J A
JA
Hoskins
P L
PL
Skatrud
L C
LC
Vining
C
C
Stuttard
J R
JR
Miller
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D008040:Genetic Linkage; D005822:Genetic Vectors; D051306:L-Lysine 6-Transaminase; D008969:Molecular Sequence Data; D010088:Oxidoreductases; D010453:Peptide Synthases / Q000235:genetics*; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*; D000637:Transaminases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*
D010088:Oxidoreductases; C038370:isopenicillin N synthetase; D000637:Transaminases; D051306:L-Lysine 6-Transaminase; D010453:Peptide Synthases; C095368:alpha-aminoadipyl-cysteinyl-valine synthetase