Pyruvate carboxylase as an anaplerotic enzyme in Corynebacterium glutamicum

Abstract

The recent discovery that phosphoenolpyruvate carboxylase (PEPCx) is dispensable for growth and lysine production in Corynebacterium glutamicum implies that this organism possesses (an) alternative anaplerotic enzyme(s). In permeabilized cells of C. glutamicum, w e detected pyruvate carboxylase (PCx) activity. This activity was effectively inhibited by low concentrations of ADP, AMP and acetyl-CoA. PCx activity was highest [45 2 5 nmol min'l (mg dry wt) ''] in cells grown on lactate or pyruvate, and was about two-to threefold lower when the cells were grown on glucose or acetate, suggesting that formation of PCx is regulated by the carbon source in the growth medium. In cells grown a t low concentrations of biotin (< 5 pg I-'), PCx activity was drastically reduced, indicating that the enzyme is a biotin protein. Growth experiments with the wild-type and a defined PEPCx-negative mutant of C. glutamicum on glucose showed that the mutant has a significantly higher demand for biotin than the wild-type, whereas both strains have the same high biotin requirement for growth on lactate and the same low biotin requirement for growth on acetate. These results indicate that (i) PCx is an essential anaplerotic enzyme for growth on glucose in the absence of PEPCx, (ii) PCx is an essential anaplerotic enzyme for growth on lactate even in the presence of PEPCx, and (iii) PCx has no anaplerotic significance for growth on acetate as the carbon source. In support of these conclusions, screening for clones unable to grow on a minimal medium containing lactate, but able to grow on a medium containing glucose or acetate, led to the isolation of PCx-defective mutants of C. glutamicum .~~~ ~~

Cometabolism of a Nongrowth Substrate:l-Serine Utilization byCorynebacterium glutamicum

“…The dd obtained for C-2 of aspartate was 43% Ϯ 1% in the mutant compared to 47% Ϯ 1% in the wild type. This indicates that the pyruvate resulting from L-serine is used to synthesize oxaloacetate by the anaplerotic pyruvate carboxylase (39,40), which is further converted to L-aspartate. These data confirm that besides L-serine dehydratase, further L-serine-converting reactions yielding pyruvate are operating.…”

Analysis and Engineering of Metabolic Pathway Fluxes in Corynebacterium glutamicum

“…Among the most striking findings was the identification of pyruvate carboxylase as the major anaplerotic enzyme in C. glutamicum [27]. Subsequently, overexpression of its gene has been shown to improve lysine production [150]. Knowing the importance of pyruvate carboxylase for lysine production, the point mutation P458S, identified in a classically derived producer, was introduced into the pyc gene and also resulted in strong increase of lysine production [136].…”

“…Concerning synthesis of glutamate in C. glutamicum which demands efficient replenishment of the TCA cycle, pyruvate carboxylase was identified as a major anaplerotic enzyme [118,150]. The flux through pyruvate carboxylase strongly increased under glutamate production induced by Tween 40 addition, while PEP carboxylase and PEP carboxykinase fluxes remained constant.…”

Enhanced Glutamic Acid Production by a H+-ATPase-Defective Mutant ofCorynebacterium glutamicum

“…H þ -ATPase-Defective Mutant of Corynebacterium glutamicumof anaplerotic carbon flux through Pcx, a biotin-binding enzyme, determines the biotin demand for growth, 19) and no expression of Pcx has been detected under conditions of biotin limitation. 19) Hence, the amounts of Pcx and AccBC were analyzed using the cells cultured under various biotin concentrations in Medium F3 0 by shake-flask culture (see the preceding section for conditions) to elucidate the mechanism(s) of enhanced glutamic acid production as well as the changes in the biotin requirement observed in strain F172-8.…”

“…19) It has been reported that enzymes in anaplerosis such as Pcx, 6) phosphoenolpyruvate carboxylase, 20) and phosphoenolpyruvate carboxykinase are deeply involved in glutamic acid production. 6) Also it has been found that the necessity The data shown in Fig.…”