System Biological Studies of Microbial L-Methionine Production

M. Rahnert¹, A. Teleki¹, B. Bathe², H. Priefert², R. Takors¹

1 Institute of Biochemical Engineering, University of Stuttgart, Allmandring 31, 70569 Germany

2 Evonik Industries AG, Kantstr. 2, 33790 Halle

Goal 1: Process Optimisation of microbial L-Met-Producers focused on energetical aspects

The sulphur containing amino acid L-methionine (C₅H₁₁NO₂S) belongs to the small group of amino acids that are not (yet) produced by fermentation. While current market volumes of ~ 800.000 tons per year are highly attractive, the feed additive is still sold as chemically synthesized D/L-methionine or as the methionine hydroxy analog (C₅H₁₀O₃S) - and not as the pure L- enantiomer.

Microbial L-methionine synthesis is well known to be strongly linked to the synthesis of serine and assimilation of sulphur besides the central carbon metabolism. This is highly energy intensive and dependent on redox potential. Therefore seven ATP and eight NAD(P)H are required for the synthesis of one molecule of L-methionine with glucose as carbon source and sulphate as sulphur source. Consequently, an optimized process scenario that is mindful of this energy dependency is extremely important for maximizing methionine production.

The main goal of this research is to maximize production by identifying and optimizing process parameters relevant to energy utilisation in cultivations. This is achieved by developing diverse cultivation scenarios of production processes using different sulphur sources and limitation strategies. The effect of various input parameters are investigated on the metabolic level and analysed using metabolic flux analyses and flux balance analyses.

Goal 2: Stimulus-response based quantitative studies for a systems level understanding of model strains

Promising L-methionine producers were investigated by sophisticated stimulus response experiments triggering product synthesis. Rapid sampling methods with fast metabolism inactivation were applied to monitor metabolic dynamics as a function of external stimuli. As stimulators different compounds were tested that fulfilled the criteria of (i) fast cellular uptake and (ii) significantly strong perturbation of the complex L-methionine biosynthesis. Based on newly developed analytical protocols, LC-MS-QQQ studies (MRM) enabled the detailed monitoring of intracellular metabolism dynamics providing the basis for the model-based identification of metabolic engineering targets. In essence, dynamics of all intermediates of L-methionine biosynthesis were documented. Tools of metabolic control analysis were applied to unravel details of metabolic regulation. Thereof strategies for further strain improvement are derived.