Increased NADPH concentration obtained by metabolic engineering of the pentose phosphate pathway in Aspergillus niger

Abstract

The pentose phosphate pathway (PPP) and glycolysis comprise the most central pathways in primary metabolism (Fig. 1). The PPP is believed to be the major source of NADPH required for many biosynthetic and detoxification reactions. The flux through this pathway has been reported to increase at high NADPH requirements, for Many biosynthetic reactions and bioconversions are limited by low availability of NADPH. With the purpose of increasing the NADPH concentration and ⁄ or the flux through the pentose phosphate pathway in Aspergillus niger, the genes encoding glucose 6-phosphate dehydrogenase (gsdA), 6-phosphogluconate dehydrogenase (gndA) and transketolase (tktA) were cloned and overexpressed in separate strains. Intracellular NADPH concentration was increased two-to ninefold as a result of 13-fold overproduction of 6-phosphogluconate dehydrogenase. Although overproduction of glucose 6-phosphate dehydrogenase and transketolase changed the concentration of several metabolites it did not result in increased NADPH concentration. To establish the effects of overexpression of the three genes, wild-type and overexpressing strains were characterized in detail in exponential and stationary phase of bioreactor cultures containing minimal media, with glucose as the carbon source and ammonium or nitrate as the nitrogen source and final cell density limiting substrate. Enzymes, intermediary metabolites, polyol pools (intra-and extracellular), organic acids, growth rates and rate constant of induction of acid production in postexponential phase were measured. None of the modified strains had a changed growth rate. Partial least square regressions showed the correlations between NADPH and up to 40 other variables (concentration of enzymes and metabolites) and it was possible to predict the intracellular NADPH concentration from relatively easily obtainable data (the concentration of enzymes, polyols and oxalate). This prediction might be used in screening for high NADPH levels in engineered strains or mutants of other organisms.

Polyol synthesis inAspergillus niger: Influence of oxygen availability, carbon and nitrogen sources on the metabolism

“…Based on these findings, it was concluded that the accumulation of erythritol was due to excess activity of the PP pathway in excess compared with the cellular demand for the pathway intermediates (Röhr et al, 1987). Recently, Poulsen et al (2005) have also observed high PP pathway enzyme activities during stationary phase in A. niger. In the present work, the production yields of erythritol and arabitol increased during oxygen limitation when the biomass production was low and the production of CO 2 was high.…”

“…For each glucose-6-phosphate entering the PP pathway two NADP molecules are reduced by the enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. When nitrate is used as nitrogen source, an increase of these two enzyme activities has been noticed in A. nidulans and A. niger (Habison et al, 1983;Hankinson and Cove, 1972;Kinghorn and Unkles, 1994;Poulsen et al, 2005;Singh et al, 1988). More specifically, Poulsen et al (2005) have shown that, in A. niger, the activities of three enzymes belonging to the PP pathway increase when nitrate is added to the medium: glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and transketolase.…”

“…When nitrate is used as nitrogen source, an increase of these two enzyme activities has been noticed in A. nidulans and A. niger (Habison et al, 1983;Hankinson and Cove, 1972;Kinghorn and Unkles, 1994;Poulsen et al, 2005;Singh et al, 1988). More specifically, Poulsen et al (2005) have shown that, in A. niger, the activities of three enzymes belonging to the PP pathway increase when nitrate is added to the medium: glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and transketolase. This phenomenon would most likely result in an increased flux through the PP pathway (Pedersen et al, 1999) and could explain the increase of the erythritol and xylitol production when nitrate is used as nitrogen source, as these two compounds are synthesized from intermediates of the PP pathway.…”

Improved product‐per‐glucose yields in P450‐dependent propane biotransformations using engineered Escherichia coli

“…Increasing the carbon flux through the PP pathway is often pursued (Chin et al, 2009;Lim et al, 2002;Poulsen et al, 2005;Skorupa Parachin et al, 2009) or proposed (Walton and Stewart, 2004) as a strategy to increase the efficiency of NADPH-dependent reactions in whole-cell systems. Our results indicate that while the Dpgi mutation improves the Y PPG (2.6-fold), this benefit is counterbalanced by a decrease in the specific in vivo activity of the biocatalyst (Fig.…”

Thermococcus kodakarensis : the key to affordable biohydrogen production

“…Although both strategies have been applied successfully in various prokaryotes, they are not effective in every organism [215,216]. Moreover, as mentioned above, G6PDH is a key enzyme not only for control of the PPP flux but also for control of the ED pathway.…”