Excretion of pyruvate by mutants of Alcaligenes eutrophus, which are impaired in the accumulation of poly(?-hydroxybutyric acid) (PHB), under conditions permitting synthesis of PHB

Abstract

Mutants of Alcaligenes eutrophus, which are defective in the intracellular accumulation of poly(β-hydroxybutyric acid), PHB, were cultivated in the presence of excess carbon source after growth had ceased due to depletion of ammonium, sulphate, phosphate, potassium, magnesium, or iron. Under these conditions all mutants excreted large amounts of pyruvate into the medium. Excretion of pyruvate occurred with lactate, gluconate or fructose as carbon sources; the highest rate of pyruvate excretion (8 mmol/1 per hour) was obtained with lactate. The rate of pyruvate excretion by strain N9A-PHB-02-HB-1 on gluconate amounted to 2.5–6.3 mmol/g protein per hour depending on the depleted nutrient. The ratios of the molar rates for the utilization of the substrates versus those for the excretion of pyruvate were 1.9, 1.0 or 0.7, respectively. Wild-type strains did not excrete even traces of pyruvate, but accumulated PHB. Depending on the limiting nutrient, strain N9A accumulated PHB at a rate of 0.21–0.49 g/g protein per hour. On a molar basis (β-hydroxybutyrate monomers versus pyruvate) the ratios for the rates for accumulation of PHB in the wild-type and for excretion of pyruvate in PHB-negative mutants were 0.9–1.4. The fermentation enzymes alcohol dehydrogenase and lactate dehydrogenase were not synthesized in cells starved of a nutrient; they were only detectable in cells cultivated under conditions of restricted oxygen supply. The latter conditions caused accumulation of PHB in the wild-type and excretion of pyruvate by the PHB-negative mutant.