Physiological response of Corynebacterium glutamicum to oxidative stress induced by deletion of the transcriptional repressor McbR

Abstract

In the present work the metabolic response of Corynebacterium glutamicum to deletion of the global transcriptional regulator McbR, which controls, e.g. the expression of enzymes of Lmethionine and L-cysteine biosynthesis and sulfur assimilation, was studied. Several oxidative stress proteins were significantly upregulated among about 40 proteins in response to deletion of McbR. Linked to this oxidative stress, the mutant exhibited a 50 % reduced growth rate, a 30 % reduced glucose uptake rate and a 30 % reduced biomass yield. It also showed metabolic flux rerouting in response to the deletion. NADPH metabolism was strongly altered. In contrast to the wild-type, the deletion strain supplied significantly more NADPH than required for anabolism, indicating the activity of additional NADPH-consuming reactions. These involved enzymes of oxidative stress protection. Through redirection of metabolic carbon flux in the central catabolism, including a 40 % increased tricarboxylic acid (TCA) cycle flux, the mutant revealed an enhanced NADPH supply to provide redox power for the antioxidant systems. This, however, was not sufficient to compensate for the oxidative stress, as indicated by the drastically disturbed redox equilibrium. The NADPH/NADP + ratio in C. glutamicum DmcbR was only 0.29, and thus much lower than that of the wild-type (2.35). Similarly, the NADH/NAD + ratio was substantially reduced from 0.18 in the wild-type to 0.08 in the mutant. Deletion of McbR is regarded as a key step towards biotechnological L-methionine overproduction in C. glutamicum. C. glutamicum DmcbR, however, did not overproduce L-methionine; this was very likely linked to the low availability of NADPH. Since oxidative stress is often observed in industrial production processes, engineering of NADPH metabolism could be a general strategy for improvement of production strains. Unlike the wild-type, C. glutamicum DmcbR contained large granules with high phosphorus content. The storage of these energy-rich polyphosphates is probably the result of a large excess of formation of ATP, as revealed by estimation of the underlying fluxes linked to energy metabolism.

The TetR Family of Regulators

“…DNA binding by McbR is modulated by the small molecule S-adenosylhomocysteine, a byproduct of methylation reactions (185). Deletion of mcbR causes numerous pleiotropic effects on additional aspects of growth and metabolism, indicating that McbR plays a central role in regulating numerous physiological processes in C. glutamicum (186).…”

Large-Scale 13 C Flux Profiling Reveals Conservation of the Entner-Doudoroff Pathway as a Glycolytic Strategy among Marine Bacteria That Use Glucose

“…Marine bacteria possess various antioxidant mechanisms to eliminate reactive oxygen species (ROS), the inducers of oxidative stress (58). Particularly, NADPH is an oxidative stress protectant that is required by many important antioxidant defense mechanisms (75) and is important for counteracting oxidative stress (61,76,77). A recent study revealed the importance of the ED pathway for oxidative stress protection in P. putida (59).…”

“…Although the lack of high-level NADPH specificity for the glucose 6-phosphate dehydrogenase (Table 1) does not exclude at least a partial formation of NADH with the ED pathway in the corresponding strains, the nonspecificity of the enzyme surely enables the organism to cope with dynamic fluctuations in NADP ϩ and NADPH availability (73,75). Especially under conditions of oxidative stress, cells exhibit a drastically disturbed redox equilibrium and the NADPH/NADP ϩ ratio is reduced almost 10-fold (75). One can expect that this strongly promotes NADPH formation by glucose 6-phosphate dehydrogenase.…”

Analysis and Engineering of Metabolic Pathway Fluxes in Corynebacterium glutamicum

“…Recent developments provide the next level of systems biology studies, the parallel investigation of C. glutamicum on levels of gene expression, proteins, metabolites, and fluxes providing important links between the different functional components of cellular physiology. First examples of such systems-oriented studies already reveal a great potential [76,119,176]. Such approaches are especially promising for the targeted multidimensional alteration of complex regulatory networks towards better tolerance of production strains to high temperature or salt levels, or extreme pH values [137], but also reveal a great potential for efficient design of novel bioprocesses.…”