Metabolic Function of Corynebacterium glutamicum Aminotransferases AlaT and AvtA and Impact on l -Valine Production

Abstract

Aminotransferases (ATs) interacting with L-alanine are the least studied bacterial ATs. Whereas AlaT converts pyruvate to L-alanine in a glutamate-dependent reaction, AvtA is able to convert pyruvate to L-alanine in an L-valine-dependent manner. We show here that the wild type of Corynebacterium glutamicum with a deletion of either of the corresponding genes does not exhibit an explicit growth deficiency. However, a double mutant was auxotrophic for L-alanine, showing that both ATs can provide L-alanine and that they are the only ATs involved. Kinetic studies with isolated enzymes demonstrate that the catalytic efficiency, k cat /K m , of AlaT is higher than 1 order of magnitude in the direction of L-alanine formation (3.5 ؋ 10 4 M ؊1 s ؊1 ), but no preference was apparent for AvtA, suggesting that AlaT is the principal L-alanine-supplying enzyme. This is in line with the cytosolic L-alanine concentration, which is reduced in the exponential growth phase from 95 mM to 18 mM by a deletion of alaT, whereas avtA deletion decreases the L-alanine concentration only to 76 mM. The combined data show that the presence of both ATs has subtle but obvious consequences on balancing intracellular amino acid pools in the wild type. The consequences are more obvious in an L-valine production strain where a high intracellular drain-off of the L-alanine precursor pyruvate prevails. We therefore used deletion of alaT to successfully reduce the contaminating L-alanine in extracellular accumulated L-valine by 80%.Among the numerous transformations of amino acids performed by pyridoxal-5Ј-phosphate (PLP)-dependent enzymes, the reversible transfer of amino groups from the amino acids to 2-oxo-acids is predominant. Such reactions are catalyzed by aminotransferases (ATs), which play a vital role in the synthesis of amino acids as well as in amino acid interconversions (5).A functional study of ATs in a single organism may encounter problems due to the large number of ATs usually present, their closely related structure, and overlapping substrate specificities (10,19). This is evident, for instance, for Escherichia coli and its three ATs encoded by tyrB, aspC, and ilvE, which are involved in the synthesis of aromatic amino acids. Here, the in vivo function could be studied only when the other two genes were inactivated (8). Nonetheless, we succeeded in identifying the function of 11 ATs of 20 putative PLP-dependent proteins encoded in the Corynebacterium glutamicum genome, thus gaining a global view of the transamination activities in this bacterium (16,17). This bacterium is of particular interest due to its excellent amino acid production properties. Currently, most of the 2 ϫ 10 6 tons of amino acids produced per year are synthesized by fermentation with C. glutamicum (13), and a recent monograph discusses a number of features of this useful bacterium (6).Among the amino acids made with C. glutamicum are L-isoleucine and L-valine, which are mostly used for pharmaceutical purposes and therefore are required to have the highest purity....

Metabolic Engineering of Corynebacterium glutamicum for 2-Ketoisovalerate Production

“…4B) with a volumetric productivity of 5.0 mM h Ϫ1 and showed a maximal Y P/S of 0.56 mol of 2-ketoisovalerate per mol of glucose between 16 and 31 h. Furthermore, the cells excreted about 20 mM L-valine and about 5 mM both L-alanine and pyruvate after 56 h of fermentation. The remaining L-alanine and L-valine formation by both C. glutamicum ⌬aceE ⌬ilvE (pJC4ilvBNCD) and C. glutamicum ⌬aceE ⌬pqo ⌬ilvE (pJC4ilvBNCD) might be due to amination of pyruvate and of 2-ketoisovalerate by aminotransferases AlaT and AvtA, respectively (31,32). Figure 5 summarizes the averages of 2-ketoisovalerate accumulation, the molar Y P/S values, and the volumetric productivities in the overall production phases of both strains calculated for three independent fermentations.…”

Improvement of the Redox Balance Increasesl-Valine Production by Corynebacterium glutamicum under Oxygen Deprivation Conditions

“…Indeed, LeuDH increased L-valine production (Table 5) by improvement of the intracellular redox balance (Table 7) as well as through a much more efficient amination than TA (Tables 5 and 6). This is presumably due to differences in amino-group donors: LeuDH uses NH 3 , whereas the donor for TA is primary L-glutamate (26,27), and NADPH is required for regeneration of L-glutamate (5). In other words, by using LeuDH instead of TA, an amino-group donor and a reduced cofactor needed for L-valine synthesis were changed from L-glutamate and NADPH to NH 3 and NADH, respectively (Fig.…”

“…However, drastic improvement such as the 20-fold (from 54 mM to 1,170 mM) increase in L-valine production realized in this study has not previously been attributed to improved cofactor imbalance. Several by-products were coproduced, but these could be reduced by genetic disruption based on previous analysis of each biosynthetic pathway (e.g., for alanine, alaT and avtA [26,27]; for succinate, ppc [18]; for acetate, pta-ack and ctfA [50]), resulting in further increased L-valine yield. In conclusion, our bioprocess of C. glutamicum under oxygen deprivation was able to produce considerable amounts of L-valine and can be applied to the efficient production of many kinds of commodity chemicals, including other amino acids.…”

“…1, there exists an imbalance in cofactor production versus consumption in the L-valine synthesis pathway: the synthesis of one mole of L-valine from one mole of glucose generates two moles of NADH via glycolysis but consumes a total of two moles of NADPH, one at the AHAIR reaction and the other at the regeneration of L-glutamate (5) as an amino-group donor for the TA reaction (26,27) (Fig. 1).…”

Metabolic Engineering of Escherichia coli for the Synthesis of the Plant Polyphenol Pinosylvin

“…E. coli cells were separated from the medium and inactivated by silicone oil centrifugation for the determination of cytoplasmic concentrations of transcinnamic acid, trans-cinnamoyl CoA, malonyl-CoA, and pinosylvin as described previously (22). The resulting upper phase was used for further analysis by LC-MS/MS.…”