(107c) Cell Free Metabolic Engineering for Generation of Biohydrogen From Cellulosic Biomass

Cell Free Metabolic Engineering for Generation of Biohydrogen from Cellulosic Biomass

Franklin Lu, James Swartz

Unpurified cell extracts can be easily created from engineered cell lines through cell lysis while maintaining the majority of cellular proteins in their active forms. Consequently, these extracts have the capability of rapidly producing a large number of enzymes in a cost effective manner for in vitro catalysis. One application for these unpurified extracts is the conversion of cellulosic biomass to hydrogen, which has already been demonstrated in vitro using purified enzymes. While unpurified extracts are much cheaper to produce, they share similar problems with in vivo metabolic engineering in that side reactions can negatively impact product yield. However, they also differ in that the cell growth and maintenance requirements are removed. Therefore, cell free metabolic engineering can provide unique opportunities as well as challenges.

We have applied a cell free metabolic approach to inactivate competing reactions and improve the yield of a biological method to convert glucose to hydrogen. By generating a library of linear DNA templates, expressing mutants using cell free protein synthesis, and screening with spectroscopic activity assays, we selected mutant enzymes from key metabolic junctions with stable protease insertion sites. These enzymes were inserted into the genome of E.coli for in vivo expression in cell lines transformed with a plasmid for expression of the corresponding protease into the periplasm. After a growth phase, the cells were lysed, mixing the active protease and mutant enzymes, and thereby selectively inactivating undesirable metabolic pathways. These unpurified extracts were used in conjunction with a three enzyme synthetic pathway to produce hydrogen and improve the conversion yield of glucose to hydrogen.