Modified carbon flux during oxygen limited growth of Corynebacterium glutamicum and the consequences for amino acid overproduction

Abstract

The amino acid producing bacterium Corynebucferirun glufumicum accumulated lactate, succinate and acetate under oxygen-limited growth conditions. Significant restructuring of carbon flux through the central metabolic pathways occurred with a notable decrease in pentose pathway flux and the operation of the TCA cycle in a reductive mode. Simultaneous consumption of residual sugar and organic acids took place when oxygen sufficient conditions were restored though amino acids yields were signiticantly perturbed. lntrodllction For several decades, Corynebacterium glutamicum or related species, have been used industrially for the production of various amino acids. Under certain conditions in which cell membrane permeability is modified via biotine limitation or surfactant addition these bacteria accumulate glutamic acid to high concentrations in the medium broth (Kikuchi and Nakao, 1986). Empirical optimisation of the fermentation strategy has enabled the final concentrations achieved to exceed 100 g/l while traditional genetic techniques have enabled the range of amino acids produced under appropriate fermentation conditions to be extended. Further progress to improve either product yields or specific production rates would now seem to depend on the capacity to modify carbon flux distribution within the central metabolic network to overcome specific limitations. A major limitation to the pragmatic application of this type of metabolic engineering approach is the relative absence of any real data concerning the regulatory mechanisms controlling carbon distribution through the diverse pathways of intermediary metabolism. The stoechiometric modelling techniques used by Vallino & Stephanopoulos (1991) postulated a high carbon flux through the pen&se pathway (5560% of carbon flux from glucose-6-P) during glucose catabolism: figure recently confirmed by NTvIR studies (A Guyonvarch, pers comm). The central metabolism of C.gluramicum is therefore somewhat different to the 'normal metabolism' model, based mostly on E. co/i drtti.in which carbon flux through the pentose pathway is generally believed to be only 1540% of the total G6P available. Such a difference has been attributed to the apparent absence of the NADmADP transhydrogenase enzyme in C. glutamicum and hence the necessity for a direct production of NADPH to satisfy the requirement for anabolic reactions. While such a metabolism is well adapted to the biotechnologists' requirements it must also be born in mind that such modified carbon flux implies a greatly modified control structure and hence the need to establish a number of regulatory details before the rational improvement of the micro-organism can proceed along defined directives. At the moment, much of 449

The Acetate Switch

“…glutamicum, a gram-positive bacterium used widely for the production of amino acids (267), can grow on either glucose or acetate as its sole carbon and energy source. Unlike E. coli or B. subtilis, wild-type cells do not accumulate substantial amounts of acetate during aerobic growth on glucose (470), although they may excrete acetate during anaerobic growth (120). Like E. coli, C. glutamicum uses the PTA-ACK pathway to assimilate high concentrations of acetate (200 mM).…”

Carbon Flux Analysis by 13 C Nuclear Magnetic Resonance To Determine the Effect of CO 2 on Anaerobic Succinate Production by Corynebacterium glutamicum

“…Under aerobic conditions, C. glutamicum is able to grow relatively fast to high cell densities in minimal medium. In contrast, in the absence of oxygen and nitrate, cell growth is negligible, while the ability to metabolize sugars is retained (5,6). Thus, cells of C. glutamicum can be grown aerobically and subsequently used for fermentation of desired products under anoxic conditions, as demonstrated for L-alanine, L-valine, or isobutanol (7)(8)(9)(10).…”

Identification of Two prpDBC Gene Clusters in Corynebacterium glutamicum and Their Involvement in Propionate Degradation via the 2-Methylcitrate Cycle

“…Up to now, a variety of genes primarily involved in amino acid production were characterized at the molecular level, and subsequently, the synthesis of selected amino acids was improved by genetic engineering of the biosynthetic pathways of this organism (29). Additionally, knowledge of central metabolic pathways in C. glutamicum is of growing interest for the deduction of metabolic fluxes in this organism (9,11,40). One of the most important pathways in the central metabolism is the citric acid cycle, which provides several metabolic precursors for amino acid production.…”