Patrick C. Cirino
Assistant Professor, Chemical Engineering
The Pennsylvania State University
Whole-cell biocatalysis is often preferred over in vitro or non-biological chemical transformations. Of primary interest are transformations requiring regeneration of cofactors and/or incorporating several enzymes in a metabolic pathway. An important classification of whole-cell transformations receiving increasing attention is those which utilize pathways for product formation that are not growth coupled, but instead in competition with growth-related pathways. Our representative transformation of this type is the NADPH-dependent reduction of xylose to xylitol in engineered E. coli, where reducing equivalents are derived from glucose oxidation via central carbon metabolism. An important parameter that describes the efficiency of this process is the yield, defined as moles of xylose reduced per mole of glucose oxidized. In batch cultures, xylitol yield is lowest during the growth phase (where NADPH is required for cell growth) and is increased during stationary phase. Yield on xylitol is significantly improved in cultures of “resting” cells, which are not growing but still metabolically active. Important parameters that affect the activity and viability of resting cells include the initial growth conditions (e.g., rich versus minimal medium), the growth stage at which they are harvested, and the protocol for implementing the cells (e.g., glucose limited or excess). In combination with stoichiometric network models of E. coli metabolism, yield measurements for a variety of gene deletion strains (e.g., pgi, zwf, sthA, pntA), are being used to elucidate the contributions of the pentose phosphate pathway, citric acid cycle, and transhydrogenase reaction to NADPH-dependent xylose reduction. The important role of xylose transport energetics is also taken into consideration. The implications of our results in improving the efficiency of biocatalytic strains employing NAD(P)H-dependent transformations will be discussed.