MICROBIAL CATALYSTS TO PRODUCE FUEL ETHANOL AND VALUE ADDED PRODUCTS
Location: National Center for Agricultural Utilization Research
Title: A PLASMID-BASED FUNCTIONAL PROTEOMIC WORKCELL ASSAY DESIGNED TO SCREEN ORPINOMYCES CELLULASE F NNY MUTAGENIZED LIBRARIES IN HIGH THROUGHTPUT
Submitted to: Great Lakes Regional American Chemical Society Symposium
Publication Type: Abstract Only
Publication Acceptance Date: October 20, 2004
Publication Date: October 20, 2004
Citation: Hughes, S.R., Li, X., Cotta, M.A., Bischoff, K.M., Riedmuller, S., Farrelly, P. 2004. A plasmid-based functional proteomic workcell assay designed to screen Orpinomyces cellulase F NNY mutagenized libraries in high throughtput [abstract]. 36th Great Lakes Regional American Chemical Society Symposium, October 17-20, 2004, Peoria, Illinois. Poster 361.
We are developing a high throughput plasmid-based functional proteomic workcell that integrates in a robotic platform the operations of media filling, colony picking, plate sealing, advanced liquid handling, incubation and shaking, and assay data collection into a custom database, with 2D bar code tracking of plates, clones, strains, and datapoints for rapid optimization of clones and expression of full-length cDNA libraries to produce improved fuel ethanol yeast strains.
To test this platform, we have designed an assay to screen cellulase F mutations in high throughput from the anaerobic fungus Orpinomyces. To produce protein from the plasmid-based expression libraries of full-length cellulase F, we are using the expression bacterium E. coli BL21 DE3. The last four codons are randomized using an NNY motif, with changes for all possible nucleotides in the 1st and 2nd positions of the codons and any pyrimidine in the third position of each of the codon. This type of directed mutagenesis gives rise to combinations of all 20 amino acids at each of the last four codons.
Using this high throughput assay, it will be possible to obtain 160,000 different versions of cellulase F for screening on a plate containing AZO-cellulose. These 160,000 clones are within the screening parameters of this workcell when the samples are multiplexed before plating. Several novel cellulase F enzymes have been identified that are catalytic at a pH < 5. Further screening should produce the numbers of clones needed to find enzymes that work at even lower pH and higher temperature. Several versions that are not as active as the wild type highlight the importance of these last four amino acids to cellulase F processivity.