Figure 5.3 Labeling experiments-based MFA of two isogenic glutamate dehydrogenase mutants (homologous, NADPH-dependent, and heterologous, NADH-dependent) of the lysine producer strain C. glutamicum MH20-22B. Flux values were converted to flux ratios and expressed as (NADH-dependent mutant)/(NADPH-dependent mutant). Numbers near the thick lines give the estimated net fluxes while those near the thin arrows give the measured fluxes required for biomass synthesis. Adapted from Marx et al. (20). Abbreviations, AKG, a-ketoglutarate; DL-DAP, DL-diaminopimelate; E4P, erythrose-4-P; FUM, fumarate; F16P, fructose-1-6-bisphosphate; F6P, fructose-6-phosphate; GAP, glyceraldehyde-3-phosphate; G6P, glucose-6-phosphate; ICIT, isocitrate; LL-DAP, LL-diaminopimelate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PYR, pyruvate; P5P, pentose-5-phosphate; S7P, sedoheptulose-7-phosphate.

resulted in increased isoleucine production (13). A second strategy could be the redirection of metabolic flux in a branch point. Ikeda and Katsumata (14) engineered a tryptophan-producing mutant of C. glutamicum to produce L-tyrosine or L-phenylalanine in abundance

(26 and 28 g/L, respectively) by overexpressing the branch-point enzymes (chorismate mutase and prephenate dehydratase), catalyzing the conversion of the common intermediate chorismate into tyrosine and phenylalanine. Using a similar approach, Katsumata et al. (15) produced threonine using a lysine-producing C. glutamicum strain, by amplifying a threo-nine biosynthetic operon. Other strategies could include introducing heterologous enzymes that use different cofactors than those used by the native enzyme as well as amplifying the enzyme that catalyzes the steps linking central metabolism and the biosynthetic pathway (6). Ikeda et al. (16) achieved a 61% increase in tryptophan yield (50 g/L of tryptophan) in a tryptophan-producing C. glutamicum KY10894 by coexpressing two enzymes catalyzing the initial steps in the biosynthesis of aromatic amino acids (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase) and serine (3-phosphoglycerate dehydrogenase) together with tryptophan biosynthetic enzymes.

5.2.3 Modification of Transport Systems

By altering amino acid transport systems, one could expect to decrease their intracellular concentration and avoid feedback inhibition. The following two examples illustrate that show the significance of this strategy. During the production of L-tryptophan by C. glutamicum, the accumulation of this product in the extracellular medium resulted in a backflow into the cells, which produced severe feedback inhibition in the biosynthesis of tryptophan (6). Ikeda and Katsumata (17) solved this problem by creating mutants with lower levels of tryptophan uptake, which resulted in an accumulation of 10 to 20% more tryptophan than in their parent. Another example of transport engineering is the increase in the fermentation yield of cysteine by overexpressing multidrug efflux genes (mar genes) in a cysteine-producing strain of E. coli (6).

5.2.4 Use of Analytical Tools in the ME Toolbox

In this section, I will give some examples of using MFA [See reference (2) for a detailed description of MFA] to improve the production of specific amino acids, including the two amino acids with the largest production volume worldwide (L-glutamic acid with 800,000 tons/year and L-lysine with 600,000 tons/year).

Glutamate. As with other amino acids, analysis and modification of central metabolic and biosynthetic pathways in glutamate-producing C. glutamicum strains have contributed to their improvement. MFA, including estimation of fluxes using labeling experiments, has elucidated the relative contribution of different pathways (e.g., Embden-Meyerhof and hexose monophosphate) under various physiological conditions and genetic backgrounds [(18) and references therein]. For example, MFA has established the relationship between the decline of oxoglutarate dehydrogenase complex and the flux distribution at the metabolic branch point of 2-oxoglutarate glutamate. This suggests that metabolic flux through anaplerotic pathways could be limiting the production of glutamate. Many of these findings have either been verified by genetic approaches or have led to a rational modification of metabolic pathways to improve the production of glutamate [(18) and references therein].

L-lysine. L-lysine production is another example of the application of labeling experiments-based MFA. Very comprehensive approaches have been used to assess all major fluxes in the central metabolism of C. glutamicum, which reveal patterns that can be used for designing ME strategies. In a comparison of six metabolic patterns, several metabolic fluxes that depend significantly on the physiological state of the cells were identified [(19) and references therein]. These included the coordinated flux through PPP and the tricarboxylic acid (TCA) cycle, the high capacity for the reoxidation of NADPH, and futile cycles between C3-compounds of glycolysis and C4-compounds of the TCA cycle. As an example, Figure 5.3 shows a comparison of the metabolic flux distribution between two isogenic glutamate dehydrogenase mutants (homologous, NADPH-dependent, and heterologous, NADH-dependent) of the L-lysine producer strain C. glutamicum MH20-22B (20). Metabolic flux patterns revealed that the PPP flux was high only for a high demand of NADPH and a low TCA cycle flux. The heterologous, NADH-dependent, glutamate dehydrogenase mutant required more NADH, resulting in an increased TCA cycle flux and then more NADPH supplied by the isocitrate dehydrogenase step in the TCA cycle. This led to a decreased flux of the PPP due to lower NADPH requirements (TCA cycle already produced part of it). The inverse is true for the homologous, NADPH-depen-dent, glutamate dehydrogenase mutant. There is a higher NADPH requirement, the PPP flux is higher, and the TCA cycle flux is lower.

5.2.5 Use of LAB in the Production of Amino Acids

LAB also has been used to produce different amino acids. Lactococcus lactis (21) has been engineered to produce L-alanine as the only end product of fermentation (more than 99%). Rerouting the carbon flux toward alanine was achieved by expressing Bacillus sphaericus alanine dehydrogenase (AlaDH) in lactate dehydrogenase (LDH) deficient strains. Finally, stereospecific production (> 99%) of L-alanine was achieved by disrupting the gene encoding alanine racemase.

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