Generierung einer NADP+-abhängligen Glykolat-Dehydrogenase mit Hilfe von "directed evolution" und in vivo Analyse ihrer Einsetzbarkeit zur Reduzierung der pflanzlichen Photorespiration
Zusammenfassung der Projektergebnisse
The overall goal in my proposed project was to engineer an NADP+ -dependent GDH with high in vivo activity in plants and to test its efficiency for improving certain metabolic pathways aimed at decreasing photorespiration. One enzyme, glyoxylate reductase 1 from Arabidopsis thaliana (GR1) slowly catalyzes the desired reaction. However, experiments that I performed using purified GR1 enzyme, growth experiments using bacteria expressing GR1, as well as theoretical predictions show that the NADP+ -dependent conversion of glycolate to glyoxylate is limited by the thermodynamic equilibrium rather than by the enzyme catalyst. The energetics of this reaction overwhelmingly favors glycolate formation such that there is about 109 times more glycolate than glyoxylate at equilibrium. Since an enzyme can only change the rate at which this equilibrium is reached, not the equilibrium itself, the NADP+ -dependent oxidation of glycolate to glyoxylate is fundamentally limited and too inefficient for use in plants. The in vivo experimental work I performed while exploring the possibilities of generating a glycolate dehydrogenase peaked my interest in how membrane potential (membrane voltage) relates to growth and survival of the cell. However, there are very few tools available to experimentally measure and perturb the membrane potential in living organisms in high throughput. I therefore started studying rhodopsins which are interesting for light- harvesting applications in bioenergy production and as fluorescent sensors of membrane potential. First, I performed a comprehensive study of amino acid substitutions in the retinal binding pocket of Gloeobacter violacaeus rhodopsin (GR) that tune its absorption spectrum (λmax). Screening 7,216 clones for shifted λmax resulted in 70 unique shifted variants. The two most extreme variants - GRb3 and GRr3 - differ by only seven mutations, yet their λmax are 161 nm apart. A subset of red-shifted GRs exhibit high levels of fluorescence relative to wild-type GR. Second, I transferred some of the GR mutations to Archaerhopsin-3 from Halorubrum sodomense (Arch). I then collaborated to test the applicability of these variants as sensors in vivo. One of these variants exhibited improved characteristics which enabled simultaneous monitoring of multiple neurons and - for the first time - the in vivo application in Caenorhabditis elegans worms. Finally, I performed directed evolution on Arch to increase its fluorescence intensity >20-fold over that of the wild type protein. These bright Arch variants enable labeling of biological membranes in the far-red/infrared and exhibit the furthest red-shifted fluorescence emission thus far reported for a fluorescent protein (maximal excitation/emission at ~620 nm/730 nm). The fluorescent proteins developed in this project should prove immensely useful for answering questions in cell biology, neurology and optogenetics.
Projektbezogene Publikationen (Auswahl)
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Archaerhodopsin variants with enhanced voltage-sensitive fluorescence in mammalian and Caenorhabditis elegans neurons. Nature Communications, 5, Article number: 4894 (2014), 9 S.
Flytzanis NC, Bedbrook CN, Chiu H, Engqvist MKM, Xiao C, Chan KY, Sternberg PW, Arnold FH, Gradinaru V
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“Directed Evolution of a Far-Red Fluorescent Rhodopsin” Proceedings of the National Academy of Sciences PNAS, September 9, 2014, 111 (36) 13034-13039
McIsaac RS, Engqvist MKM, Wannier T, Rosenthal AZ, Herwig L, Flytzanis NC, Imasheva ES, Lanyi JK, Balashov SP, Gradinaru V and Arnold FH
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“Directed Evolution of Rhodopsin Spectral Properties.” Journal of Molecular Biology, 2014 Jun 27. (Epub ahead of print)
Engqvist MKM, McIsaac RS, Dollinger P, Flytzanis NC, Abrams M, Schor S and Arnold FH
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“Mitochondrial 2-hydroxyglutarate metabolism” Mitochondrion. 2014 Feb 21. (Epub ahead of print)
Engqvist MKM, Eßer C, Maier A, Lercher MJ and Maurino VG
