EXPLORING THE EFFECTS OF OVEREXPRESSING AN NADP+-DEPENDENT GAPDH AND THE NAD+ SALVAGE PATHWAY ON COFACTOR AVAILABILITY FOR NADPH-DEPENDENT BIOTRANSFORMATION IN ESCHERICHIA COLI

Open Access
Author:
Patch, Renae Christine
Area of Honors:
Chemical Engineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Patrick Cirino, Thesis Supervisor
  • Patrick C Cirino, Thesis Supervisor
  • Themis Matsoukas, Honors Advisor
Keywords:
  • biocatalysis
  • cofactor regeneration
Abstract:
Improving the efficiency of biocatalytic production of xylitol has been studied by attempting to improve the availability of NADPH, which is a necessary co-substrate in the CbXR-catalyzed reduction of xylose to xylitol in Escherichia coli [1]. This research analyzes the impact of coexpressing (along with CbXR) three different enzymes proposed to improve NADPH availability on overall xylitol production, xylitol yield (moles of xylitol produced per mole of glucose consumed, YRPG). Three novel bicistronic plasmids were constructed in which CbXR is immediately upstream of either GDP1, an NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase, pncB, encoding nicotinate phosphoribosyltransferase, or nadK, encoding nicotinamide adenine dinucleotide kinase. Xylitol production in the early stages of batch culturing generally showed an increase with the addition of nadK or GDP1 to the system, compared to the expression of only CbXR, while pncB caused a decrease in the total concentration of xylitol. Because the standard deviation was so large, the YRPG values for each enzyme were considered statistically the same as those seen previously with CbXR expression. However, the average YRPG increased with both GDP1 and nadK while decreasing with pncB, and with more data these values may be proven to be statistically significant. Although the cofactor concentration levels generally increased and decreased as expected according to their associated reactions, the cofactor analysis saw no trends in NADPH/NADP+ ratios when compared to xylitol yields for each enzyme. GDP1 ratios were much lower, even though the YRPG increased, and the ratios seen with pncB expression were the same as CbXR. The only positive correlation between NADPH/NADP+ ratios and xylitol yield was seen with nadK expression, but the standard deviation on the reported ratios was too large to make any reliable conclusions regarding the specific impact of this enzyme.