NADPH Supply (nadph + supply)

Distribution by Scientific Domains


Selected Abstracts


Analysis of NADPH supply during xylitol production by engineered Escherichia coli

BIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009
Jonathan W. Chin
Abstract Escherichia coli strain PC09 (,xylB, cAMP-independent CRP (crp*) mutant) expressing an NADPH-dependent xylose reductase from Candida boidinii (CbXR) was previously reported to produce xylitol from xylose while metabolizing glucose [Cirino et al. (2006) Biotechnol Bioeng 95(6): 1167,1176]. This study aims to understand the role of NADPH supply in xylitol yield and the contribution of key central carbon metabolism enzymes toward xylitol production. Studies in which the expression of CbXR or a xylose transporter was increased suggest that enzyme activity and xylose transport are not limiting xylitol production in PC09. A constraints-based stoichiometric metabolic network model was used to understand the roles of central carbon metabolism reactions and xylose transport energetics on the theoretical maximum molar xylitol yield (xylitol produced per glucose consumed), and xylitol yields (YRPG) were measured from resting cell biotransformations with various PC09 derivative strains. For the case of xylose-proton symport, omitting the Zwf (glucose-6-phosphate dehydrogenase) or PntAB (membrane-bound transhydrogenase) reactions or TCA cycle activity from the model reduces the theoretical maximum yield from 9.2 to 8.8, 3.6, and 8.0 mol xylitol (mol glucose),1, respectively. Experimentally, deleting pgi (encoding phosphoglucose isomerase) from strain PC09 improves the yield from 3.4 to 4.0 mol xylitol (mol glucose),1, while deleting either or both E. coli transhydrogenases (sthA and pntA) has no significant effect on the measured yield. Deleting either zwf or sucC (TCA cycle) significantly reduces the yield from 3.4 to 2.0 and 2.3 mol xylitol (mol glucose),1, respectively. Expression of a xylose reductase with relaxed cofactor specificity increases the yield to 4.0. The large discrepancy between theoretical maximum and experimentally determined yield values suggests that biocatalysis is compromised by pathways competing for reducing equivalents and dissipating energy. The metabolic role of transhydrogenases during E. coli biocatalysis has remained largely unspecified. Our results demonstrate the importance of direct NADPH supply by NADP+ -utilizing enzymes in central metabolism for driving heterologous NADPH-dependent reactions, and suggest that the pool of reduced cofactors available for biotransformation is not readily interchangeable via transhydrogenase. Biotechnol. Bioeng. 2009;102: 209,220. 2008 Wiley Periodicals, Inc. [source]


Importance of NADPH supply for improved L -valine formation in Corynebacterium glutamicum

BIOTECHNOLOGY PROGRESS, Issue 2 2010
Tobias Bartek
Abstract Cofactor recycling is known to be crucial for amino acid synthesis. Hence, cofactor supply was now analyzed for L -valine to identify new targets for an improvement of production. The central carbon metabolism was analyzed by stoichiometric modeling to estimate the influence of cofactors and to quantify the theoretical yield of L -valine on glucose. Three different optimal routes for L -valine biosynthesis were identified by elementary mode (EM) analysis. The modes differed mainly in the manner of NADPH regeneration, substantiating that the cofactor supply may be crucial for efficient L -valine production. Although the isocitrate dehydrogenase as an NADPH source within the tricarboxylic acid cycle only enables an L -valine yield of YVal/Glc = 0.5 mol L -valine/mol glucose (mol Val/mol Glc), the pentose phosphate pathway seems to be the most promising NADPH source. Based on the theoretical calculation of EMs, the gene encoding phosphoglucoisomerase (PGI) was deleted to achieve this EM with a theoretical yield YVal/Glc = 0.86 mol Val/mol Glc during the production phase. The intracellular NADPH concentration was significantly increased in the PGI-deficient mutant. L -Valine yield increased from 0.49 0.13 to 0.67 0.03 mol Val/mol Glc, and, concomitantly, the formation of by-products such as pyruvate was reduced. 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source]