Catabolism of lysine in Penicillium chrysogenum leads to formation of 2-aminoadipic acid, a precursor of penicillin biosynthesis

Abstract

Penicillium chrysogenum L2, a lysine auxotroph blocked in the early steps of the lysine pathway before 2-aminoadipic acid, was able to synthesize penicillin when supplemented with lysine. The amount of penicillin produced increased as the level of lysine in the media was increased. The same results were observed in resting-cell systems. Catabolism of [U-_4CJlysine by resting cells and batch cultures of P. chrysogenum L2 resulted in the formation of labeled saccharopine and 2-aminoadipic acid. Formation of [14C] saccharopine was also observed in vitro when cell extracts of P. chrysogenum L2 and Wis 54-1255 were used. Saccharopine dehydrogenase and saccharopine reductase activities were found in cell extracts of P. chrysogenum, which indicates that lysine catabolism may proceed by reversal of the two last steps of the lysine biosynthetic pathway. In addition, a high lysine:2-ketoglutarate-6-aminotransferase activity, which converts lysine into piperideine-6-carboxylic acid, was found in cell extracts of P. chrysogenum. These results suggest that lysine is catabolized to 2-aminoadipic acid in P. chrysogenum by two different pathways. The relative contribution of lysine catabolism in providing 2-aminoadipic acid for penicillin production is discussed. 2-Aminoadipic acid is a key intermediate in the biosynthesis of lysine and penicillin in P-lactam-producing fungi (Fig. 1) (2, 16, 21), as well as in the catabolism of lysine in mammals (9, 13) and in 3-lactam-producing actinomycetes (17). The catabolism of lysine in a wide range of microorganisms, including Pseudomonas (10), Flavobacterium (35), Streptomyces (23), Candida (8, 33), Neurospora (34) and Rhodotorula (18) spp.,

Molecular Regulation of β-Lactam Biosynthesis in Filamentous Fungi

“…For P. chrysogenum, a similar observation was made (99). A lysine-auxotrophic mutant blocked in a step before L-␣-AAA was also able to synthesize penicillin when L-lysine was added to the medium.…”

Gene Targeting inPenicillium chrysogenum: Disruption of thelys2Gene Leads to Penicillin Overproduction

“…Wis 54-1255, a low-level penicillin-producing strain, and a P. chrysogenum pyrG1 mutant, a uridine auxotroph obtained from Wis 54-1255 by mutation with nitrosoguanidine and selection for 5Ј-fluoroorotic acid resistance (8), were used as recipient strains in transformation experiments. P. chrysogenum L2, a lysine auxotroph blocked in the first part of the ␣-aminoadipate pathway (9), was used as the control strain in nutritional experiments. Escherichia coli DH5␣ was used for high-frequency plasmid transformation (10 7 to 10 8 transformants/g of DNA).…”

“…For penicillin production studies, spores from one plate were inoculated in defined inoculation (DI) medium (solution A: 10 g of citric acid, 2.5 g of acetic acid, 3 g of ethylamine, 5 g of (NH 4 ) 2 SO 4 , 1 g of KH 2 PO 4 , 0.5 g of MgSO 4 ⅐ 7H 2 O, 0.05 g of FeSO 4 ⅐ 7H 2 O, 0.01 g of ZnSO 4 ⅐ 7H 2 O, 0.01 g of CuSO 4 ⅐ 5H 2 O, 0.01 g of MnSO 4 ⅐ 4H 2 O, 0.005 g of CoSO 4 , 0.001 g of NaCl in 800 ml of distilled water, pH 5.5; solution B: 20% glucose in 200 ml of distilled water; both solutions were sterilized separately and mixed before use [80 ml of solution A with 20 ml of solution B in a 500-ml flask]). After 48 h of incubation at 25°C and at 250 rpm, 10 ml of the culture in DI medium was added to a 500-ml flask containing 100 ml of defined production (DP) medium (9). The cultures were incubated for 168 h at 25°C and at 250 rpm; 1-ml samples were taken every 24 h to measure penicillin production.…”

Post-translational enzyme modification by the phosphopantetheinyl transferase is required for lysine and penicillin biosynthesis but not for roquefortine or fatty acid formation in Penicillium chrysogenum

“…The P. chrysogenum Wis54-1255 pyrGstrain (an uridine auxotroph derived from the Wis54-1255 strain) and the high penicillin producing strains AS-P-78 and AS-P-99 were used to study the gene copy number. The lysine auxotroph L2/lys1strain, a derivative of the L2 mutant strain [34] disrupted in the lys1 gene (F. Teves et al, unpublished data) was used as control strain of lysine auxotrophy, respectively. Uridine auxotrophs were grown in presence of 140 g/ml of uridine.…”