Engineering and analysis of cofactor partitioning for NADPH-dependent xylitol production in Escherichia coli.

摘要

This research is focused on whole-cell biocatalysis as a way to regenerate reduced cofactors that are used to drive heterologous redox reactions of interest. Specifically, the focus is on engineering and understanding cofactor partitioning for heterologous production of xylitol in Escherichia coli. By replacing E. coli's native cyclic AMP receptor protein (CRP) with a cyclic AMP-independent mutant (CRP*), xylose uptake and xylitol production from mixtures of glucose and xylose was facilitated, with glucose serving as the growth substrate and source of reducing equivalents. Numerous xylose reductases (XRs) and xylulose dehydrogenases (XDHs) with varying nicotinamide cofactor specificities were screened in a crp*, Delta xylB double mutant strain (PC09). It was found that a NADPH-dependent xylose reductase from Candida boidinii (CbXR) consistently produced the highest concentration of xylitol in shake flask cultures (~275 mM in LB cultures, ~180 mM in minimal cultures). Use of non-growing, metabolically-active resting cells was next examined as a means of improving xylitol yield (Y RPG, mols of xylitol produced per mol of glucose consumed). An increase of YRPG in resting cells compared to batch cultures (~3.4 and ~1.8 respectively) was observed. By altering various conditions and parameters of resting cells, xylitol YRPG was further increased to ~4.0, while limiting fermentation product secretion (e.g. lactate, acetate, and ethanol).;It was then sought 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 suggested 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). These results were then compared to experimentally determined xylitol yields (YRPG), which 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), 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 of xylitol per mol of glucose, respectively. Experimentally, deleting pgi (encoding phosphoglucose isomerase) from strain PC09 improves the yield from 3.4 to 4.0, 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 of xylitol per mol of glucose, respectively. Although the metabolic role of transhydrogenases during E. coli biocatalysis has remained largely unspecified, these 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.;Finally, two fundamentally different strategies were studied to improve the coupling between glucose oxidation and xylose reduction based on the result that the pool of reduced cofactors is not readily interchangeable. It was first examined the effects of deleting the phosphofructokinase (pfk ) gene(s) on growth-uncoupled xylitol production and found that deleting both pfkA and sthA (encoding the E. coli soluble transhydrogenase) improved the xylitol YRPG from 3.4 to 5.4. The second strategy focuses on coupling growth with xylose reduction. Deleting the pgi and sthA genes resulted in a strain that was severely growth inhibited; however the growth was able to be partially restored upon expressing the NADPH-dependent CbXR to the double mutant strain (mumax = 0.12 from 0.06 hr -1) with concomitant xylitol production, which is a potential suitable strain for adaptive evolution. Intracellular nicotinamide cofactor levels were also quantified, and the magnitude of the change in the NADPH/NADP + ratio measured from cells consuming glucose in the absence versus presence of xylose showed a strong correlation to the resulting YRPG.