L-Methionine Production with Recombinant E. Coli

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. Obviously, this is the consequence of the demanding precursor, energy and reduction equivalent needs for the biosynthesis of L-methionine using e.g. glucose as carbon source.

This contribution considers metabolic engineering studies for L-methionine formation by glucose-based fermentation. Analyzing network topologies, first, the coupling of cellular energy management and metabolic performance for L-methionine biosynthesis is outlined considering recombinant C. glutamicum and E. coli strains. Constraints for metabolic and biochemical engineering are derived.

Promising L-methionine producers were investigated by sophisticated stimulus response experiments triggering product synthesis in vivo. Rapid sampling technologies 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—QQQ-MS studies 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 and studied. Tools of metabolic control analysis were applied to unravel details of metabolic regulation. Thereof strategies for further strain improvement are derived.

L-methionine producers were tested in lab-scale fermentations applying e.g. different process scenarios and using various sulphur sources. Process phases were analyzed by metabolic flux analysis. Fermentation performance data revealing e.g molar product/substrate yields >20% will be presented that underline the feasibility and attractiveness of glucose-based L-methionine production.