Improved oxygen transfer in cultures of Nocardia lactamdurans maintains the cephamycin biosynthetic proteins for prolonged time and enhances the conversion of deacetylcephalosporin into cephamycin C

Δ-1-Piperideine-6-carboxylate (P6C) dehydrogenase activity, which catalyses the conversion of P6C into α-aminoadipic acid, has been studied in the cephamycin C producer Streptomyces clavuligerus by both spectrophotometric and radiometric assays. The enzyme has been purified 124-fold to electrophoretic homogeneity with a 26% yield. The native protein is a monomer of 56.2 kDa that efficiently uses P6C (apparent Km 14 μM) and NAD+ (apparent Km 115 μM), but not NADP+ or other electron acceptors, as substrates. The enzyme activity was inhibited (by 66%) by its end product NADH at 0.1 mM concentration. It did not show activity towards pyrroline-5-carboxylate and was separated by Blue-Sepharose chromatography from pyrroline-5-carboxylate dehydrogenase, an enzyme involved in the catabolism of proline. P6C dehydrogenase reached maximal activity later than other early enzymes of the cephamycin pathway. The P6C dehydrogenase activity was decreased in ammonium (40 mM)-supplemented cultures, as was that of lysine 6-aminotransferase. P6C dehydrogenase activity was also found in other cephamycin C producers (Streptomyces cattleya and Nocardia lactamdurans) but not in actinomycetes that do not produce β-lactams, suggesting that it is an enzyme specific for cephamycin biosynthesis, involved in the second stage of the two-step conversion of lysine to α-aminoadipic acid.

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Inducing Effect of Diamines on Transcription of the Cephamycin C Genes from the lat and pcbAB Promoters inNocardia lactamdurans

Journal of Bacteriology ◽

10.1128/jb.181.8.2379-2384.1999 ◽

1999 ◽

Vol 181 (8) ◽

pp. 2379-2384 ◽

Cited By ~ 7

Author(s):

Ana Lucia Leitão ◽

Francisco J. Enguita ◽

Juan Luis De La Fuente ◽

Paloma Liras ◽

Juan F. Martin

Keyword(s):

Cell Growth ◽

Antibiotic Production ◽

Protein A ◽

Transcriptional Level ◽

Intracellular Accumulation ◽

Low Resolution ◽

Cephamycin C ◽

Nocardia Lactamdurans ◽

Shake Flasks ◽

Acid Precursor

ABSTRACT The diamines putrescine, cadaverine, and diaminopropane stimulate cephamycin biosynthesis in Nocardia lactamdurans, in shake flasks and fermentors, without altering cell growth. Intracellular levels of the P7 protein (a component of the methoxylation system involved in cephamycin biosynthesis) were increased by diaminopropane, as shown by immunoblotting studies. Lysine-6-aminotransferase and piperideine-6-carboxylate dehydrogenase activities involved in biosynthesis of the α-aminoadipic acid precursor were also greatly stimulated. The diamine stimulatory effect is exerted at the transcriptional level, as shown by low-resolution S1 protection studies. The transcript corresponding to the pcbAB gene and to a lesser extent also the lat transcript were significantly increased in diaminopropane-supplemented cultures, whereas transcription from the cefD promoter was not affected. Coupling of the lat and pcbABpromoters to the reporter xylE gene showed that expression from the lat and pcbAB promoters was increased by addition of diaminopropane in Streptomyces lividans. Intracellular accumulation of diamines in Nocardia may be a signal to trigger antibiotic production.

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