Application of MALDI‐TOF MS to lysine‐producing Corynebacterium glutamicum

Abstract

In the present work, a novel comprehensive approach of 13 C-tracer studies with labeling measurements by MALDI-TOF MS, and metabolite balancing was developed to elucidate key fluxes in the central metabolism of lysine producing Corynebacterium glutamicum during batch culture. MALDI-TOF MS methods established allow the direct quantification of labeling patterns of low molecular mass Corynebacterium products from 1 mL of diluted culture supernatant. A mathematical model of the central Corynebacterium metabolism was developed, that describes the carbon transfer through the network via matrix calculations in a generally applicable way and calculates steady state mass isotopomer distributions of the involved metabolites. The model was applied for both experimental planning of tracer experiments and parameter estimation. Metabolic fluxes were calculated from stoichiometric data and from selected mass intensity ratios of lysine, alanine, and trehalose measured by MALDI-TOF MS in tracer experiments either with 1-13 C glucose or with mixtures of 13 C 6 / 12 C 6 glucose. During the phase of maximum lysine production C. glutamicum ATCC 21253 exhibited high relative fluxes into the pentose phosphate pathway of 71%, a highly reversible glucose-6-phosphate isomerase, significant backfluxes from the tricarboxylic acid cycle to the pyruvate node consuming the lysine precursor oxaloacetate, 36% net flux of anaplerotic carboxylation and 63% contribution of the dehydrogenase branch in the lysine biosynthetic pathway. Due to the straightforward and simple measurements of selected labeling patterns by MALDI-TOF MS sensitively reflecting the flux parameters of interest, the presented approach has an excellent potential to extend metabolic flux analysis from single experiments with enormous experimental effort to a broadly applied technique.Keywords: MALDI-TOF mass spectrometry; metabolic flux analysis; Corynebacterium glutamicum; lysine; 13 C tracer.Production of amino acids by Corynebacterium glutamicum belongs to the major processes in industrial biotechnology. The world market for amino acids amounts to about 3 billion US dollars, whereby lysine with a worldwide production of about 250 000 tonnes per annum is one of the most important products [1]. Optimization of cultivation strategies and producer strains have led to increased yields and rates of amino-acid production by Corynebacteria. To a large extent, the strain improvement was based on random mutagenesis and subsequent selection [2,3]. In the last decade the powerful tools of genetic engineering allowed the targeted amplification and disruption of selected genes, which led to a number of mutants with increased capabilities of lysine secretion [4]. However, practically achieved yields are still far from theoretical optima, leaving a significant potential for further improvement. To achieve such improvements in a targeted and efficient way, detailed knowledge of intracellular carbon flux distributions and their regulation is needed. Data on genome, transcriptome and proteome ...

Genetic dissection of trehalose biosynthesis in Corynebacterium glutamicum: inactivation of trehalose production leads to impaired growth and an altered cell wall lipid composition

“…As described by Vallino & Stephanopoulos (1993) and Wittmann & Heinzle (2001), lysine-overproducing mutants of C. glutamicum accumulate up to 6 g trehalose l 21 in the culture broth under conditions close to those used for industrial lysine production. Attempts to connect this trehalose accumulation with changes in the osmolarity of the growth medium, using the type strain of C. glutamicum and NaCl addition to increase the osmolarity, were not successful (data not shown).…”

“…Significant amounts of free trehalose are observed in C. glutamicum cells as a response to hyperosmotic stress (Skjerdal et al, 1996). Also, it is notable that trehalose was found as one of the by-products excreted into the growth medium during lysine overproduction by C. glutamicum (Vallino & Stephanopoulos, 1993;Wittmann & Heinzle, 2001). …”

Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC‐MS

“…Such analytically deduced flux ratios were also used successfully as constraints for metabolic flux analysis [13][14][15]. Based on probabilistic equations, a more general methodology was developed to simultaneously identify network topology and multiple flux partitioning ratios [16,17].…”

Analysis and Engineering of Metabolic Pathway Fluxes in Corynebacterium glutamicum

“…In C. glutamicum, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate 13 C flux analysis: [112] (white circles), [125] (gray circles), [126] (black circles), [127] (white squares), [111] (gray squares), [111] (black squares), [128] (white triangles), [116] (gray triangles), [26] (black triangles), [129] (white diamonds), [115] (gray diamonds), [120] (black diamonds), [76] (white hexagons), and [118] (gray hexagons). The flux reversibility at the pyruvate node is defined as the ratio of the back flux to the anaplerotic net flux [130] 36 C. Wittmann dehydrogenase, and malic enzyme catalyze NADPH generating reactions, whereas NADPH consuming reactions comprise growth with a stoichiometric demand of 16.4 mmol NADPH (g biomass) À1 [34] and overproduction.…”

“…Lysine production flux (%) Fig. 6 Metabolic flux analysis linking lysine production with NADPH metabolism involving the pentose phosphate pathway (a) and isocitrate dehydrogenase (b) in different C. glutamicum strains investigated under various cultivation conditions by 13 C flux analysis: [125] (white circles), [129] (gray circles), [126] (black circles), [127] (white squares), [112] (gray squares), [126] (black squares), [128] (white triangles), [116] (gray triangles), [26,76] (black triangles), [115] (white diamonds), and [120] (gray diamonds)…”