Poster Title: Improving Xylitol Production and Cofactor Usage in E. coli Growing on Glucose or Xylose
Authors: Patrick C. Cirino, Jonathan W. Chin, Reza Khankal, Olubolaji Akinterinwa, Costas D. Maranas
Poster Abstract: Whole-cell biocatalysis is often preferred over in vitro enzyme systems requiring cofactor regeneration. One classification of whole-cell transformations is those which are not growth-coupled, but instead in competition with growth-related pathways. Our representative transformation of this type is the NAD(P)H-dependent reduction of xylose to xylitol via xylose reductase (XR) in engineered E. coli, where reducing equivalents are derived from glucose oxidation via central metabolism. Simultaneous transport of glucose and xylose is achieved either by the constitutive expression of xylose transporters or a CRP mutant. An important parameter to assess the efficiency of this process is the yield, defined as moles of xylose reduced per mole of glucose consumed. In batch cultures, xylitol yield is lowest during growth (where NADPH is required for cell growth) and is increased during stationary phase. Yield on xylitol is significantly improved in resting cells (~4.2) compared to growing cells (~1.5). The impact of any given metabolic engineering strategy depended on the E. coli host strain chosen (W3110, MG1655, E. coli B).
Stoichiometric models of E. coli metabolism help to understand the potential roles of enzymes and pathways in cofactor supply, and the effects of various genetic modifications on the theoretical maximum yield. Experimentally, we have quantified the effects of many single and double deletions in genes involved in central metabolism and respiration on the biocatalytic properties of xylitol-producing strains. We find that transhydrogenase plays little role in the cofactor yield, and that NADH availability is not readily translated to NADPH availability. NADPH-dependent transformations give higher yields than NADH-dependent ones, and genetic modifications that increase glucose oxidation through NADP+-dependent reactions (e.g., pgi deletion) directly improve yield.
In the presence of glucose, expression of CRP* is generally more favorable than direct expression of a xylose transporter (resulting in higher yields & xylitol titers, reduced acetate production). Microarray studies were performed to better understand how CRP* affects expression of other genes which may influence xylitol production and yield. As expected, gene expression in the context of CRP* is drastically different than that with wild-type CRP, including elevated expression of TCA cycle genes and anabolic pathways. Finally, xylitol phosphorylation by E. coli xylulokinase (XylB) was identified as a bottleneck to the use of xylose as an energy source during xylitol production, and this was overcome by replacement of xylB with XYL3 from Pichia stipitis.