FLUX LIMITATIONS IN THE ORTHO PATHWAY OF BENZOATE DEGRADATION OF ALCALIGENES-EUTROPHUS - METABOLITE OVERFLOW AND INDUCTION OF THE META PATHWAY AT HIGH SUBSTRATE CONCENTRATIONS

The growth behaviour of Alcaligenes eutrophus using various concentrat ions of benzoate was investigated. In batch culture, growth was expone ntial and growth rate (mu) and yields (Y) were high [mu = 0.51 h(-1) a nd Y-X/benzoate = 0.56 mol carbon (mel carbon)(-1)] when low concentra tions of benzoate (< 5 mM) were used, These kinetic parameters were cl ose to the maxima determined in a benzoate-limited chemostat [mu(max) = 0.55 h(-1) and Y-X/benzoate(max) = 0.57 mol carbon (mel carbon)(-1)] and the part of the energy for maintenance was limited (m(ATP) = 4.3 +/- 2.2 mmol ATP g(-1) h(-1)). When higher concentrations of benzoate were used (up to 40 mM), several metabolic limitations appeared. the s pecific rate of benzoate consumption was not altered, whereas growth w as inhibited [K-i(benzoate) approximate to 27 mM], Furthermore, high c oncentrations of catechol together with some 1,2-dihydro-1,2-dihydroxy benzoate (DHB) transiently accumulated in the medium. The accumulation of catechol was attributed to limiting flux through catechol 1,2-diox ygenase estimated to be 5.2 mmol g(-1) h(-1), whereas that of DHB was provoked by an imbalance in the NADH/NAD(+) intracellular content, The direct consequence of DHB accumulation was the induction of the meta pathway for the degradation of catechol, and this pathway contributed up to 20% of the total flux of catechol to the central metabolism Fina lly, when very high concentrations of benzoate were used (55 mM), both growth and the specific rate of benzoate degradation were diminished due to a strong decrease in benzoate 1,2-dioxygenase specific activity .