Biofuels Production projects
Engineering Fluorescent Biofuel Sensors
The high-throughput construction of metabolically engineered organisms is hindered by our inability to rapidly assay the presence of most small molecules. Recently, biosensors fashioned around the green fluorescent protein (GFP) have been developed to sense a number of important chemicals such as calcium and ATP, but little attempt has been made to develop this work in the biofuel space. A fluorescent biosensor of biofuels or biofuel intermediates will have an enormous impact on our ability to construct and evolve production strains with superior yields and rates. This project is engineering GFP-based biosensors of various biofuels (medium and long-chain esters, alcohols, and alkanes) and their intermediates in fatty acid metabolism. A small number of the best biosensors will be optimized and used in a proof of principle experiment to screen for novel mutations to improve the flux and yield of biofuel-producing strains.
The goal of this project is to develop fluorescent sensors of biofuels and their metabolic intermediates for use in both in vivo and diagnostic applications. It is conceptually based on biosensors developed for neural imaging, which demonstrated that circularly permuted variants of the green fluorescent protein fused to ligand-binding domains can act as quantitative fluorescent sensors of ligand or ion concentration. The majority of our effort in 2014 was focused on the development of a transposon-based system for creating diverse libraries of biosensors and a fluorescence-activated cell-sorting (FACS) assay for identifying functional biosensors from this large library, which is >106 in size. This platform has been used to construct biosensors for the model sugar of glucose and maltose, and we continue to refine its use general use in screening. One significant hurdle to emerge during this effort is the potential for libraries with significant biases, wherein non-functional members populate the library faster than functional members can be isolated. Recent results suggest some of these biases can be eliminated through careful optimization of expression and FACS protocols. One new molecule of interest has emerged as a target -- the disaccharide trehalose, which is often produced by yeast during times of stress. Trehalose and maltose are isomers, and many maltose-binding proteins bind trehalose with similar affinity. Given the role of trehalose in fermentation stress and our previous construction of a maltose biosensor, we have initiated a new effort to construct high affinity sensors of trehalose for use as a diagnostic.
The goal of our work is to develop high-throughput fluorescent sensors of biofuel molecules and their biosynthetic intermediates and to use these sensors in high-throughput strain construction. Developing such sensors has historically been a slow, laborious process. To this end, we have developed a novel transposon and flow cytometry-based approach to construct and evaluate extremely large (~10^7) biosensor libraries. Using this and related approaches we have developed fluorescent sensors for maltose, glucose, and fatty acyl-CoAs. We are currently screening a panel of ligand-binding domains to develop fluorescent sensors for a number of additional biofuel-related molecules.
The goal of our project is to construct genetically encoded fluorescent sensors of biofuels and biofuel-like molecules as a tool to facilitate the rapid construction of biofuel-producing microbes. Historically, the construction of fluorescent sensors has been hampered by the ability to engineer them in a rational and predicable manner. To overcome this historical problem, we have instead developed a screening pipeline for rapid construction and assay. In 2012, we developed a cell-free expression platform that enables us to biochemically evaluate 102-103 engineered sensors per day, in order to eliminate traditional bottlenecks in in vivo protein expression, and a high-throughput directed evolution screening strategy fashioned around fluorescence activated cell-sorting. We are currently using both strategies in parallel to develop sensors of fatty acids and their biosynthetic intermediates, higher alcohols, and disaccharides associated with lignocellulosic biofuels.
Published in 2013
The Bacterial Carbon-Fixing Organelle Is Formed by Shell Envelopment of Preassembled Cargo, A. H. Chen, A. Robinson-Mosher, D. F. Savage, P. A. Silver, J. K. Polka, PLoS One, doi: 10.1371/journal.pone.0076127, 2013.