Subcellular Engineering and Optogenetics to Control Metabolisms in Space and Time
Dr. Jose Avalos
Department of Chemical and Biological Engineering
and the Andlinger Center for Energy and the Environment
Subcellular localization and dynamic control of metabolic pathways have received much attention in metabolic engineering in recent years. Each subcellular compartment in yeast offers a unique physicochemical environment as well as distinct metabolite, enzyme, and cofactor compositions, which may benefit the activity of metabolic pathways. Furthermore, the spatial separation of organelles from cytosol offers unique opportunities to reduce the toxicity of intermediates, eliminate metabolic crosstalk, and enhance the efficiency of compartmentalized pathways. In the first part of my talk I will show how interesting and unexpected behaviors arise when organelles are involved in metabolic pathways, and present new data on how compartmentalizing the Ehrlich pathway in yeast mitochondria boosts isobutanol production.
In the second part of my talk I will show how optogenetics can be applied to metabolic engineering for dynamic control. Metabolic pathway optimization requires fine-tuning the timing and levels of expression of metabolic enzymes. Optogenetic controls are ideal for this, as light can be applied and removed instantly without complex media changes. I will present a new technological platform that utilizes a light-sensitive transcription factor to achieve unprecedented control over engineered metabolic pathways in yeast. Using this technology, we achieve robust and homogeneous transcriptional control at cell densities as high as 50 OD600 in 5L bioreactors. I will show how optogenetics enables a new mode of bioreactor operation, in which periodic light pulses are used to tune the levels and timing of enzyme expression during the fermentation to boost yields.
Combining mitochondrial engineering with dynamic regulation of metabolic pathways allows us to produce up to 8.49 ± 0.31 g/L of isobutanol and 2.38 ± 0.06 g/L of 2-methyl-1-butanol microaerobically from glucose in lab-scale bioreactors, which is more than a 10-fold improvement over strains lacking optogenetic controls. These results make a compelling case for the application of subcellular engineering and optogenetics to metabolic engineering to develop not only new strategies for metabolic pathway optimization, but also new capabilities for operating, optimizing, and automating bioreactors.
José Avalos is an assistant professor in the Department of Chemical and Biological Engineering and the Andlinger Center for Energy and the Environment at Princeton University. He is also an associated faculty member in the Princeton Environmental Institute and the Department of Molecular Biology. His research focuses on the use of biotechnology to address challenges in renewable energy, sustainable manufacturing, the environment, and human health. His lab works primarily in metabolic engineering, synthetic biology, protein engineering, systems biology, and structural biology.
Avalos earned a B.E. in chemical engineering from Universidad Iberoamericana in Mexico City. He then received an MSc in biochemical research from Imperial College in London, and a Ph.D. in biochemistry and biophysics from Johns Hopkins University. He did postdoctoral research at The Rockefeller University in membrane biophysics; and at Massachusetts Institute of Technology, in the Department of Chemical Engineering, and the Whitehead Institute for Biomedical Research in metabolic engineering and synthetic biology. He has received several awards, including the Damon Runyon Cancer Research Fellowship, the Ruth L. Kirschstein National Research Service Award, the Alfred P. Sloan Foundation Research Fellowship Award in Computational and Evolutionary Molecular Biology, the Pew scholarship, and the NSF CAREER Award.
Thursday, January 24 at 2:00pm to 3:00pm
Earle Hall, 100
206 S. Palmetto Blvd., Clemson, SC 29634