Improved NADPH supply for xylitol production by engineered Escherichia coli with glycolytic mutations

Abstract

Escherichia coli engineered to uptake xylose while metabolizing glucose was previously shown to produce high levels of xylitol from a mixture of glucose and xylose when expressing NADPH-dependent xylose reductase from Candida boidinii (CbXR) (Cirino et al., Biotechnol Bioeng. 2006;95:1167-1176). We then described the effects of deletions of key metabolic pathways (e.g., Embden-Meyerhof-Parnas and pentose phosphate pathway) and reactions (e.g., transhydrogenase and NADH dehydrogenase) on resting-cell xylitol yield (Y RPG: moles of xylitol produced per mole of glucose consumed) (Chin et al., Biotechnol Bioeng. 2009;102:209-220). These prior results demonstrated the importance of direct NADPH supply by NADP+-utilizing enzymes in central metabolism for driving heterologous NADPH-dependent reactions. This study describes strain modifications that improve coupling between glucose catabolism (oxidation) and xylose reduction using two fundamentally different strategies. We 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 Y RPG from 3.4 ± 0.6 to 5.4 ± 0.4. The second strategy focused on coupling aerobic growth on glucose to xylitol production by deleting pgi (encoding phosphoglucose isomerase) and sthA. Impaired growth due to imbalanced NADPH metabolism (Sauer et al., J Biol Chem. 2004;279:6613-6619) was alleviated upon expressing CbXR, resulting in xylitol production similar to that of the growth-uncoupled precursor strains but with much less acetate secretion and more efficient utilization of glucose. 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 vs. presence of xylose showed a strong correlation to the resulting Y RPG.

Cofactor engineering for advancing chemical biotechnology

“…The altered glycolysis in pfk mutants routed glucose through pentose phosphate pathway and led to higher NADPH dependent biosynthesis in systems sensitive to NADPH availability reported in [49][50][51] and found by our group [52]. Xylitol formation using NADPH-dependent xylose reductase from Candida boidinii was improved in an E. coli pfkA and sthA mutant [51]. Deletion of pgi and sthA also showed increased glucose utilization and lowered acetate formation.…”

“…In an engineered xylitol producing E. coli strain, double deletions of phosphofructokinase pfk and transhydrogenase sthA or phosphoglucose isomerase pgi and sthA genes were made and both combinations showed increased xylitol yield [47]. The NADPH supply in an engineered E. coli strain was improved by overexpression of fructose 1,6-bisphosphatase II glpX or co-expression of glpX and glucose-6-phosphate 1-dehydrogenase zwf [48].…”

“…We have shown that replacement of the E. coli native NAD + dependent GAPDH gapA by NADP + dependent GAPDH gapB from Bacillus subtilis increased product formation and the strain showed further increased NADPH dependent biosynthesis when NAD kinase was co-expressed (unpublished results). In another strategy, phosphofructokinase has been deleted from E. coli [49][50][51] and studied in various combinations with other alterations such as GAPDH overexpression, NAD kinase, glucose uptake systems and transhydrogenase alterations. The altered glycolysis in pfk mutants routed glucose through pentose phosphate pathway and led to higher NADPH dependent biosynthesis in systems sensitive to NADPH availability reported in [49][50][51] and found by our group [52].…”

“…In another strategy, phosphofructokinase has been deleted from E. coli [49][50][51] and studied in various combinations with other alterations such as GAPDH overexpression, NAD kinase, glucose uptake systems and transhydrogenase alterations. The altered glycolysis in pfk mutants routed glucose through pentose phosphate pathway and led to higher NADPH dependent biosynthesis in systems sensitive to NADPH availability reported in [49][50][51] and found by our group [52]. Xylitol formation using NADPH-dependent xylose reductase from Candida boidinii was improved in an E. coli pfkA and sthA mutant [51].…”

Improvement of NADPH bioavailability in Escherichia coli through the use of phosphofructokinase deficient strains

“…It is possible that these differences in enzyme properties contribute to variation in metabolism in individual mutant strains under certain conditions. Previous studies have shown that deleting pfkA alone improved NADPH-dependent xylitol production from xylose (Chin and Cirino 2011). We are studying the effects of blocking this step entirely by deleting both PFK genes, pfkA and pfkB, and consider that disrupting glycolysis at this point will then force fructose-6-phosphate to undergo the reversible isomerization reaction that proceeds in the direction of glucose 6-phosphate.…”