2-40: Heterologous expression of multiple cellulolytic enzymes in Zymomonas mobilis

Consolidated bioprocessing (CBP) has the potential to reduce ethanol production costs from lignocellulosic biomass by reducing the production cost of saccharolytic enzymes.  CBP requires a microorganism that can carry out both the enzymatic depolymerization of plant cell wall polysaccharides and the fermentation of the resulting sugars to ethanol.  One microorganism that shows great promise in developing CBP is the facultative anaerobic gram-negative bacterium Zymomonas mobilis.   Z. mobilis can achieve a higher ethanol yield than most yeasts, has been metabolically engineered to use xylose and arabinose, has a naturally high tolerance to inhibitory compounds found in lignocellulosic hydrolysates, and has been successfully used to express heterologous proteins.  We describe here the expression of a hemi-cellulolytic enzyme (xynA from Thermomyces lanuginosus) and a cellulolytic enzyme (E1 from Acidothermus cellulolyticus) in both Z. mobilis and E. coli.  We use multiple promoters, terminators and plasmid backbones.  We additionally explore the use of codon optimization in enhancing heterologous expression in Z. mobilis.  We report here that Z. mobilis is capable of expressing both XynA and E1 protein.  Additionally, we show that both of these enzymes are catalytically active, providing a preliminary validation of using Z. mobilis as a CBP host.