Bridging-type changes facilitate successive oxidation steps at about 1 V in two binuclear manganese complexes - implications for photosynthetic water-oxidation

The redox behavior of two synthetic manganese complexes illustrates a mechanistic aspect of importance for light-driven water oxidation in Photosystem 11 (PSII) and design of biomimetic systems (artificial photosynthesis). The coupling between changes in oxidation state and structural changes was investigated for two binuclear manganese complexes (1 and 2), which differ in the set of first sphere ligands to Mn (N3O3 in 1, N2O4 in 2). Both complexes were studied by electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy (XAS) in three oxidation states which had been previously prepared either electro- or photochemically. The following bridging-type changes are suggested. In 1: Mn-II-(mu-OR)(mu-OCO)(2)-Mn-II double left right arrow Mn-II-(mu-OR)(mu-OCO)(2)-Mn-III double right arrow Mn-III-(mu-OR)(mu-OCO)-(mu-O)-Mn-IV. In 2: Mn-II-(mu-OR)(mu-OCO)(2)-Mn-III double left right arrow Mn-III-(mu-OR)(mu-OCO)(2)-Mn-III double right arrow Mn-III-(mu-OR)([mu-OCO)(mu-O)-Mn-IV. In both complexes, the first one-electron oxidation proceeds without bridging-type change, but involves a redox-potential increase by 0.5-1 V. The second one-electron oxidation likely is coupled to mu-oxo-bridge (or mu-OH) formation which seems to counteract a further potential increase. In both complexes, mu-O(H) bridge formation is associated with a redox transition proceeding at similar to 1 V, but the mu-O(H) bridge is observed at the Mn-2(III,III) level in I and at the Mn-III,Mn-IV level in 2, demonstrating modulation of the redox behavior by the terminal ligands. It is proposed that also in PSII bridging-type changes facilitate successive oxidation steps at approximately the same potential. (c) 2006 Elsevier Inc. All rights reserved

Expression of the Escherichia coli pntAB genes encoding a membrane-bound transhydrogenase in Corynebacterium glutamicum improves L-lysine formation

Kabus A, Georgi T, Wendisch VF, Bott M. Expression of the Escherichia coli pntAB genes encoding a membrane-bound transhydrogenase in Corynebacterium glutamicum improves L-lysine formation. Applied Microbiology and Biotechnology. 2007;75(1):47-53.A critical factor in the biotechnological production of (L)-lysine with Corynebacterium glutamicum is the sufficient supply of NADPH. The membrane-integral nicotinamide nucleotide transhydrogenase PntAB of Escherichia coli can use the electrochemical proton gradient across the cytoplasmic membrane to drive the reduction of NADP(+) stop via the oxidation of NADH. As C. glutamicum does not possess such an enzyme, we expressed the E. coli pntAB genes in the genetically defined C. glutamicum lysine-producing strain DM1730, resulting in membrane-associated transhydrogenase activity of 0.7 U/mg protein. When cultivated in minimal medium with 10% (w/v) carbon source, the presence of transhydrogenase slightly reduced glucose consumption, whereas the consumption of fructose, glucose plus fructose, and, in particular, sucrose was stimulated. Biomass was increased by pntAB expression between 10 and 30% on all carbon sources tested. Most importantly, the lysine concentration was increased in the presence of transhydrogenase by similar to 10% on glucose, similar to 70% on fructose, similar to 50% on glucose plus fructose, and even by similar to 300% on sucrose. Thus, the presence of a proton-coupled transhydrogenase was shown to be an efficient way to improve lysine production by C. glutamicum. In contrast, pntAB expression had a negative effect on growth and glutamate production of C. glutamicum wild type

Impact of overexpressing NADH kinase on glucose and xylose metabolism in recombinant xylose-utilizing Saccharomyces cerevisiae

During growth of Saccharomyces cerevisiae on glucose, the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis, respectively. A deviation from the optimal level of these cofactors often results in major changes in the substrate uptake and biomass formation. However, the metabolism of xylose by recombinant S. cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH and creates cofactor imbalance during growth on xylose. As one possible solution to overcoming this imbalance, the effect of overexpressing the native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains was also evaluated during growth on glucose. Overexpressing NADH kinase in the cytosol redirected carbon flow from CO2 to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose. However, cytosolic NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption by channeling carbon flow from ethanol to xylitol. In contrast, overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent. Overall, although NADH kinase did not increase the rate of xylose consumption, we believe that it can provide an important source of NADPH in yeast, which can be useful for metabolic engineering strategies where the redox fluxes are manipulated

Overexpression of NADH-dependent fumarate reductase improves d-xylose fermentation in recombinant Saccharomyces cerevisiae

Deviation from optimal levels and ratios of redox cofactors NAD(H) and NADP(H) is common when microbes are metabolically engineered. The resulting redox imbalance often reduces the rate of substrate utilization as well as biomass and product formation. An example is the metabolism of D-xylose by recombinant Saccharomyces cerevisiae strains expressing xylose reductase and xylitol dehydrogenase encoding genes from Scheffersomyces stipitis. This pathway requires both NADPH and NAD+. The effect of overexpressing the glycosomal NADH-dependent fumarate reductase (FRD) of Trypanosoma brucei in D-xylose-utilizing S. cerevisiae alone and together with an endogenous, cytosol directed NADH-kinase (POS5∆17) was studied as one possible solution to overcome this imbalance. Expression of FRD and FRD + POS5∆17 resulted in 60 and 23 % increase in ethanol yield, respectively, on D-xylose under anaerobic conditions. At the same time, xylitol yield decreased in the FRD strain suggesting an improvement in redox balance. We show that fumarate reductase of T. brucei can provide an important source of NAD+ in yeast under anaerobic conditions, and can be useful for metabolic engineering strategies where the redox cofactors need to be balanced. The effects of FRD and NADH-kinase on aerobic and anaerobic D-xylose and D-glucose metabolism are discussed