Engineering more thermostable metabolic enzymes for improving CBP organisms
Monday, April 28, 2014
Exhibit/Poster Hall, lower level (Hilton Clearwater Beach)
Noah Kastelowitz1, Deanne W. Sammond2, Markus Alahuhta2, Hui Wei2, Paul Lin3, Adam Guss4, James C. Liao3, Michael E. Himmel2, Hang Yin1 and Yannick Bomble2, (1)Department of Chemistry and Biochemistry, University of Boulder, Boulder, CO, (2)Biosciences Center, National Renewable Energy Laboratory, Golden, CO, (3)University of California, Los Angeles, (4)BioEnergy Science Center, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN
Ethanol and butanol yields from Clostridium thermocellum could be improved by inclusion of new heterologous metabolic pathways.  Several of these pathways are well characterized and the enzymatic steps well defined.  It was reported that the expression of Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase in the mesophile, C. cellulolyticum, increased ethanol yields by about 60%.  Incorporation of the same combination of metabolic enzymes; as well as other metabolic enzymes (such as a ketoacid decarboxylase) could increase the production of ethanol and enable the production butanol in the thermophile, Clostridium thermocellum.  However, there exists no thermophilic equivalent of these decarboxylases, thus these metabolic enzymes can only be used in C. thermocellum if their optimal operating temperature (Topt) can be increased.  We have used existing and generated new protein structures of the pyruvate decarboxylase and ketoacid decarboxylase and now intend to produce more thermostable mutants using rosetta design and molecular dynamics.  We will accomplish this goal by improving the stability of the protein monomer; as well as engineering the interfaces between them.  Promisingly, several in silico mutations show an increase in stability and are being tested in vitro.