Regulation of the utilization of 4-hydroxybenzoate and 4-hydroxycinnamate in batch and continuous cultures of Pseudomonas testosteroni

Pseudomonas testosteroni metabolized 4-hydroxycinnamate by an initial cleavage of the side chain to yield acetate and the aromatic moiety, 4-hydroxybenzaldehyde. The latter was further oxidized via 4-hydroxybenzoate to protocatechuate, which underwent meta cleavage. During growth of the organism on 4-hydroxycinnamate, the for acetate showed an undulating pattern, which was attributed to alternating induction and repression of enzymes involved in the oxidation of acetate. Repression was caused either by 4-hydroxybenzoate or by its later metabolites, formate and pyruvate.In batch culture, P. testosteroni oxidized mixtures of 4-hydroxybenzoate and 4-hydroxycinnamate in a diauxic pattern. The capacity to oxidize 4-hydroxycinnamate appeared in the cells before 4-hydroxybenzoate was exhausted, indicating that the enzymes catalysing the conversion of 4-hydroxycinnamate into 4-hydroxybenzoate. were induced despite the presence of 4-hydroxybenzoate. The induction of these early enzymes of 4-hydroxycinnamate catabolism started when the molar concentration ratio of 4-hydroxybenzoate to 4-hydroxycinnamate fell below a value of 0.3.In continuous culture of P. testosteroni on a mixture of 4-hydroxybenzoate and 4-hydroxycinnamate, both substrates were almost completely utilized up to a dilution rate of about 0.5/h. At higher dilution rates, 4-hydroxycinnamate was decreasingly utilized so that eventually at a dilution rate of 0.74/h, its effluent concentration equalled its influent concentration. At D_M, a utilization ratio of 1.23 in favour of 4-hydroxybenzoate was found to become established in the culture. The of the cells for acetate was maximal at a dilution rate of 0.38/h and decreased before 4-hydroxycinnamate utilization was at its peak at 0.59/h. This suggested that it was mainly the aromatic moiety of 4-hydroxycinnamate which was metabolized at high dilution rates. The failure to utilize acetate at high dilution rates was apparently due to the repression of its catabolic enzymes by later metabolites of 4-hydroxybenzoate and to the relatively low concentration of acetate in the fermenter. This low concentration, due to the continuous washout of acetate, prevented it from relieving the repression.Abbreviations 4HB 4-hydroxybenzoate - 4HC 4-hydroxycinnamate - D_M dilution rate allowing maximal cell output rate - OD optical density