Application of MALDI-TOF MS to lysine-producing Corynebacterium glutamicum- A novel approach for metabolic flux analysis

In the present work, a novel comprehensive approach of C-13-tracer studies with labeling measurements by MALDI-TOF MS, and metabolite balancing was de veloped to elucidate key fluxes in the central metabolism of lysine produci ng Corynebacterium glutamicum during batch culture. MALDI-TOF MS methods es tablished allow the direct quantification of labeling patterns of low molec ular mass Corynebacterium products from 1 muL of diluted culture supernatan t. A mathematical model of the central Corynebacterium metabolism was devel oped, that describes the carbon transfer through the network via matrix cal culations in a generally applicable way and calculates steady state mass is otopomer distributions of the involved metabolites. The model was applied f or both experimental planning of tracer experiments and parameter estimatio n, Metabolic fluxes were calculated from stoichiometric data and from selec ted mass intensity ratios of lysine, alanine, and trehalose measured by MAL DI-TOF MS in tracer experiments either with 1-C-13 glucose or with mixtures of C-13(6)/C-12(6) glucose. During the phase of maximum lysine production C. glutamicum ATCC 21253 exhibited high relative fluxes into the pentose ph osphate pathway of 71%, a highly reversible glucose-6-phosphate isomerase, significant backfluxes from the tricarboxylic acid cycle to the pyruvate no de consuming the lysine precursor oxaloacetate, 36% net flux of anaplerotic carboxylation and 63% contribution of the dehydrogenase branch in the lysi ne biosynthetic pathway. Due to the straightforward and simple measurements of selected labeling patterns by MALDI-TOF MS sensitively reflecting the f lux parameters of interest, the presented approach has an excellent potenti al to extend metabolic flux analysis from single experiments with enormous experimental effort to a broadly applied technique.