A Microfluidic Platform for the Discovery and Functional Annotation of Metagenomic Enzymes

Through the creation of carbon neutral biofuels and environmentally friendly methods for chemical and pharmaceutical synthesis, synthetic biology hopes to produce biology-based solutions to some of the worlds grand challengesdisease, energy supply, and pollution. Many of these challenging problems require the discovery of genes, proteins, and metabolic pathways with new and different chemistries. Nature is a vast repository of genetic and chemical diversity, and by extracting the genomes of microbial communities (i.e. the metagenome) from environmental samples, it is possible to identify genes for these biotechnology applications. However, gene identification currently requires both large sequencing efforts and massive screens of recombinant bacterial cells using slow and labor-intensive plate based assays. This platform technology uses an innovative microfluidic cell-sorting technology that will revolutionize the process of gene annotation for synthetic biology. This device physically implements simple yet powerful computer search algorithms as fluidic circuits in a microfluidic chip, and has the potential to screen a single strain out of billions based on the activity of secreted enzymes. This will enable us to rapidly search libraries of millions of E. coli expressing diverse metagenomic DNA for useful secreted enzymes and other gene products. During Phase I we demonstrated proof of concept of the device by using our prototype device to sort a single luciferase secreting yeast cell out of a heterogenous population that was not producing the enzyme. During the grant period we built and tested the prototype microfluidic device, engineered luciferase secreting yeast and e coli strains and used them to demonstrate the power of the microfluidic device. Lastly we created an E. coli library expressing diverse metagenomic DNA from sites likely to contain bacteria that produce enzymes with lignocellulose degrading capabilities. As part of our initial proof of concept for our device, we are using our device to discover metagenomic genes for enzymes that degrade cellulose, and more specifically lignocellulose. These enzymes are essential for cellulosic ethanol biofuel productionefficient lignocellulose catalysis is currently the bottleneck in development of this technology. We created metagenomic libraries created from soil samples and use our device to identify variants producing enzymes for these two applications. During the Phase II grant, we have three aims: (i) develop our core sorting technology, (ii) use it to identify microbial genes for enzymes catalyzing lignocellulose breakdown for use in cellulosic biofuel production, and (iii) develop the technology as a standalone system. Commercial Applications and Other Benefits: Our product and our service will both address the need for research and development of enzymes with novel functions for the $3 billion enzyme market. We will meet this growing need by disrupting the current paradigm of one colony at a time plate-based assays; accelerating the pace of scientific discovery.