METABOLIC AND PHYSIOLOGICAL-STUDIES OF CORYNEBACTERIUM-GLUTAMICUM MUTANTS

The physiology and central carbon metabolism of Corynebacterium glutam icum was investigated through the study of specific disruption mutants . Mutants deficient in phosphoenolpyruvate carboxylase (PPC) and/or py ruvate kinase (PK) activity were constructed by disrupting the corresp onding gene(s) via transconjugation. Standard batch fermentations were carried out: with these mutants and results were evaluated in the con text of intracellular flux analysis. The following were determined. (a ) There is a significant reduction in the glycolytic pathway flux in t he pyruvate kinase deficient mutants during growth on glucose, also ev idenced by secretion of dihydroxyacetone and glyceraldehyde. The resul ting metabolic overflow is accommodated by the pentose phosphate pathw ay (PPP) acting as mechanism for dissimilating, in the form of CO2, la rge amounts of accumulated intermediates. (b) The high activity throug h the PPP causes an overproduction of reducing power in the form of NA DPH. The overproduction of biosynthetic reducing power, as well as the shortage of NADPH produced via the tricarboxylic acid cycle (as evide nced by a reduced citrate synthase flux), are compensated by an increa sed activity of the transhydrogenase (THD) enzyme catalyzing the react ion NADPH + NAD(+) < - > NADP(+) + NADH. The presence of active THD wa s also confirmed directly by enzymatic assays. (c) Specific glucose up take rates declined during the course of fermentation and this decline was more pronounced in the case of a double mutant strain deficient i n both PPC and PK. Specific ATP consumption rates similarly declined d uring the course of the batch. However, they were approximately the sa me for all strains, indicating that energetic requirements for biosynt hesis and maintenance are independent of the specific genetic backgrou nd of a strain. The above results underline the importance of intracel lular flux analysis, not only for producing a static set of intracellu lar flux estimates, but also for uncovering changes occurring in the c ourse of a batch fermentation or as result of specific genetic modific ations. (C) 1997 John Wiley & Sons, Inc.