Biofuels Toxicity & Tolerance Group
Engineering Solvent Tolerant Hosts
Cellular engineering in microbial hosts to generate host platforms that are tolerant to various stresses, toxic products, and byproducts of fuel production is an essential part of an effective biofuels program. Inhibitory compounds from lignocellulose and pretreatment procedures, metabolic burden, and stress from engineered pathways and accumulation of toxic byproducts and final target fuels are all potential sources of negative impact on the microbial system. Systems biology studies that involve measurements at the transcript, protein, and metabolite levels, provide an effective method to examine sources of such toxicity and provide candidates that provide mechanisms to alleviate this stress. Our projects also include large-scale bioprospecting for targeted mechanisms to alleviate solvent stresses, such as the use of efflux pumps in Escherichia coli. We explore both native and heterologous stress mechanisms in 'chassis' engineering of model host organisms E. coli and Saccharomyces cerevisiae. We also explore regulatory mechanisms that will provide better control of stress response in engineering robust microbial hosts for consumption of sugars from renewable sources and production of biofuel candidates.
Selected Projects
Study of biofuel stress in E. coli
Exposure to alcohols has been reported to impact bacterial growth via a variety of mechanisms, including increased membrane fluidity, ion leakage, changes in fatty acid composition, difficulties in translation, and elongated cells. In this study, we have characterized the cell-wide response of E.coli to exogenous n-butanol stress using transcript, protein, and metabolite analyses.
Analysis of the data indicates that n-butanol stress has components common to other stress responses, including perturbation of respiratory functions (nuo, cyo operons), oxidative stress (sodA, sodC, yqhD), heat shock and cell envelope stress (rpoE, depP, cpxP), and metabolite transport and biosynthesis. This data is available via microbesonline.org. We are now exploring chassis engineering based on these cues so as to generate a high production titer, butanol-producing host with the Synthetic Biology Group at JBEI.
Fuels Transport for Efficient Fuel Production and Tolerance
For microbial fuel production, the efficiency with which fuel can be exported from the cell is likely to have significant influence on production titer. Build-up of fuel molecules may directly reduce titer, and when toxic, also cause significant intracellular stress, leading to feedback inhibition of fuel production. Transport systems, such as efflux pumps and ABC-transport systems in bacteria and yeast, are documented to export a broad range of substrates including solvents and provide a direct engineering route to relieve fuel accumulation-related stress and improve production titer. To address our goal of improving solvent resistance using efflux pumps, we have used a high-throughput approach to create a library efflux pumps in E. coli that can confer tolerance to many candidate fuels.
Study of Transcriptional Factors in S. cerevisiae
Expression profiles of several hundred transcriptional factors have been measured in S. cerevisiae under numerous biofuels production relevant conditions so as to map their functional profiles. This data is being mined to better understand cell wide stresses in a variety of conditions to guide cellular engineering and to develop parts for more efficient control of engineered pathways and stress response mechanisms.
People
- Aindrila Mukhopadhyay
- Mario Ouellet
- Zain Dossani
- Heather Szmidt
- Marijke Frederix
- Mary Dunlop, Alumni
Selected References
- Dunlop, M. J., J. D. Keasling, et al. (2010). "A Model For Improving Microbial Biofuel Production Using A Synthetic Feedback Loop." Synthetic and Systems Biology DOI 10.1007/s11693-010-9052-5.
- Rutherford, B. J., R. H. Dahl, et al. (2010). "Functional genomic study of exogenous n-butanol stress in Escherichia coli." Applied and Environmental Microbiology 76(6): 1935-1945.
- Ito, J., C. J. Petzold, et al. (2010). "The Role of Proteomics in the Development of Cellulosic Biofuels." Current Proteomics 7(2): 121-134.
- Ouellet, M., P. D. Adams, et al. (2009). "A Rapid and Inexpensive Labeling Method for Microarray Gene Expression Analysis." BMC Biotechnology 9: 97.
- Fortman, J. L., S. Chhabra, et al. (2008). "Biofuel Alternatives to Ethanol: Pumping the Microbial Well." Trends in Biotechnology 26(7): 375-381.
- Mukhopadhyay, A., A. M. Redding, et al. (2008). "Importance of Systems Biology in Engineering Microbes for Biofuel Production." Current Opinion in Biotechnology 19(3): 228-234.
