Does the detoxification of penicillin side-chain precursors depend on microsomal monooxygenase and glutathione S-transferase in Penicillium chrysogenum?

The glutathione (GSH) S-conjugation of 1,2-epoxy-3-(4'-nitrophenoxy)propane was catalysed predominantly by microsomal glutathione S-transferase (mGST) in Penicillium chrysogenum. The specific mGST activity unlike the cytosolic GST (cGST) activity increased substantially when the penicillin side-chain precursor phenoxyacetic acid (POA) was included in the culture medium. Therefore, a microsomal monooxygenase (causing possible release of epoxide intermediates) and mGST-dependent detoxification pathway may exist for the side-chain precursors as an alternative to microsomal activation to acyl-CoA and subsequent transfer to beta-lactam molecules. The P. chrysogenum pahA and Aspergillus nidulans phacA gene products, which are cytochrome p450 monooxygenases and are able to hydroxylate phenylacetic acid (PA) at position 2 on the aromatic ring, are unlikely to release toxic epoxide intermediates but epoxidation of PA and POA due to the action of other microsomal monooxygenases cannot be excluded. The GSH-dependent detoxification of POA was provoked by a well-controlled transient lowering of pH (down to 5.0) at the beginning of the production phase in a fed-batch fermentation system. Both the specific GST and gammaGT activities were increased but the intracellular GSH concentrations remained unaltered unless the pH of the feed was transiently lowered below 5.0. At pH 4.6, the GSH pool was depleted rapidly but no antibiotic production was observed. Although sucrose was taken up effectively by the cells, cell death and autolysis were progressing. Therefore, the industrial exploitation of the GSH-dependent detoxification of penicillin side-chain precursors to reduce intracellular GSH-levels in order to avoid the GSH inhibition of the beta-lactam biosynthetic enzymes seems to be rather unlikely. P. chrysogenum mGST and cGST were separated using GSH-Sepharose 6B affinity chromatography. The purified cGST possessed a homodimer (alpha(2)) tertiary structure with M(r) (, alpha) = 29500.

Autolysis and aging of Penicillium chrysogenum cultures under carbon starvation: Chitinase production and antifungal effect of allosamidin.

In carbon-depleted cultures of Penicillium chrysogenum, age-related chitinases were shown to play a crucial role in both autolysis and fragmentation as indicated by in vivo enzyme inhibition experiments using allosamidin. This pseudotrisaccharide even hindered significantly the outgrowth of new hyphal tips from the surviving yeastlike fragments after glucose supplementation. The antifungal effect of allosamidin on autolyzing P. chrysogenum mycelia was fungistatic rather than fungicidal. In growing hyphae, membrane-bound microsomal chitinase zymogen(s) were detected, which may be indicative of some compartmentalization of these hydrolases. Later, during autolysis, no zymogenic chitinase was detected in any enzyme fraction studied, including microsomes. These observations may explain the different sensitivity of growing and autolyzing mycelia to allosamidin. Chitinases taking part in the age-related fragmentation of hyphae and the outgrowth of surviving hyphal fragments seem to be potent targets for future antifungal drug research.