Monday, November 5, 2007 - 4:45 PM

Optimization Of Resting Cell Parameters For Growth-Uncoupled Product Formation

Jonathan W. Chin and Patrick C. Cirino. Chemical Engineering, Penn State University, 222 Fenske Lab, University Park, PA 16802

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 in which product formation is 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. A key 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 increases during stationary phase. Yield on xylitol is significantly improved in cultures of resting cells, which are not growing but still metabolically active. Parameters that affect the activity and viability of resting cells include the initial growth conditions (e.g., temperature, rich versus minimal medium), the growth stage at which cells are harvested, use of protein synthesis inhibitor and/or mode of nutrient deprivation, and the protocols for cell handling and implementation (e.g., degree of aeration, glucose limited or excess). In this study we are systematically considering these and other experimental parameters in order to identify and understand optimal resting cell conditions with respect to glucose uptake rate, culture lifetime and xylitol yield. For example, we find that changes in protein expression during cell harvest/preparation can significantly affect resting cell activity, and yield is highest using cells from late stationary phase. The role of maintenance energy requirements in resting cells will also be addressed.