Associate Scientist, Great Lakes Bioenergy Research Center
1552 University Avenue, Madison WI 53726
Post Doctoral Studies, Howard Hughes Medical Institute, Stanford University
Ph.D. University of Illinois (Microbiology)
M.S. University of Illinois (Microbiology)
B.S. University of Utah (Biology)
My group is part of the Microbial Synthetic Biology Lab (MiSynBio lab) within the Great Lakes Bioenergy Research Center (GLBRC). We are interested in understanding and improving the conversion of lignocellulose to biofuel. Much of our current studies are currently focused on the Gram-negative bacterium Escherichia coli, due to its sophisticated genetics, well-understood physiology, and widespread use as an industrial microbe. However, we are also interested in the physiology of the Gram-negative bacterium Cellvibrio japonicus, due to its capacity to degrade a variety of lignocellulosic substrates.
Our short-term research is focused on the construction of consolidated bioprocessing strains (CBP) capable of the degradation of lignocellulose, and subsequent fermentation of the liberated sugars to biofuel. We will then use these CBP strains to better understand the current enzymatic and metabolic bottlenecks in cellulosic biofuel production. The conversion of E. coli to a consolidated processor requires the introduction of genes encoding lignocellulases, as well as a mechanism for their secretion from the cell. To find bacteria that can serve as a source of lignocellulases and secretion systems, we developed liquid and solid media that facilitate the rapid identification of bacteria capable of biomass degradation. In collaboration with Cameron Currie’s group, we have identified novel Gram-negative bacterial strains isolated from cellulose-rich environments. Using the Gram-negative bacterium C. japonicus as a source of cellulose-degrading genes, we have constructed first generation E. coli strains capable of cellulose degradation. We are also engineering E. coli to efficiently produce and tolerate ethanol. We have taken our first-generation ethanologenic strains and subjected them to directed evolution to improve both anaerobic growth and ethanol tolerance. In both cases the directed evolution has led us in surprising directions, which have led to new approaches to strain improvement.
We are also interested in studying the physiology of cellulose-degrading organisms, using C. japonicus as a model. The genome of C. japonicus was recently sequenced, and we developed a genetic system that allows for the construction of directed gene disruptions. Using this genetic system, we have obtained evidence that the majority of cellulolytic activity produced by C. japonicus is secreted via the Type II secretion system. In addition to improving our understanding of C. japonicus, this information provides us with the foundation necessary to engineer CBP strains with improved lignocellulolytic capabilities.
- Sonti, R. V., Keating, D. H., and Roth, J. R. Lethal Transposition of Mud Phages in Rec- Strains of Salmonella typhimurium. Genetics 1992:133:17-28.
Keating, D. H., Rawlings-Carey, M. A., and Cronan, J. E., Jr. The Unmodified (Apo) Form of ACP Is an Inhibitor of Cell Growth. J. Biol. Chem. 1995;270:22229-22235.
Keating, D. H.,Zhang, Y., and Cronan, J. E., Jr. The Apparent Coupling Between Synthesis and Post-Translational Modification of Escherichia coli Acyl Carrier Protein is Due to Inhibition of Amino Acid Biosynthesis. J. Bacteriol. 1996;178:2662-2667.
Keating, D. H., and Cronan, J. E., Jr. An Isoleucine to Valine Substitution in Escherichia coli Acyl Carrier Protein Results in a Functional Protein of Decreased Molecular Radius. J. Biol. Chem. 1996;271:15905-15910.
Galibert F, Finan TM, Long SR, Puhler A, Abola P, Ampe F, Barloy-Hubler F, Barnett MJ, Becker A, Boistard P, Bothe G, Boutry M, Bowser L, Buhrmester J, Cadieu E, Capela D, Chain P, Cowie A, Davis RW, Dreano S, Federspiel NA, Fisher RF, Gloux S, Godrie T, Goffeau A, Golding B, Gouzy J, Gurjal M, Hernandez-Lucas I, Hong A, Huizar L, Hyman RW, Jones T, Kahn D, Kahn ML, Kalman S, Keating DH, Kiss E, Komp C, Lelaure V, Masuy D, Palm C, Peck MC, Pohl TM, Portetelle D, Purnelle B, Ramsperger U, Surzycki R, Thebault P, Vandenbol M, Vorholter FJ, Weidner S, Wells DH, Wong K, Yeh KC, Batut J. The composite genome of the legume symbiont Sinorhizobium meliloti. Science. 2001;293:668-672.
Barnett MJ, Fisher RF, Jones T, Komp C, Abola AP, Barloy-Hubler F, Bowser L, Capela D, Galibert F, Gouzy J, Gurjal M, Hong A, Huizar L, Hyman RW, Kahn D, Kahn ML, Kalman S, Keating DH, Palm C, Peck MC, Surzycki R, Wells DH, Yeh KC, Davis RW, Federspiel NA, Long SR. Nucleotide sequence and predicted functions of the entire Sinorhizobium meliloti pSymA megaplasmid. Proc Natl Acad Sci. 2001;98:9883-9888.
Keating, David H., Willits, Michael G., and Long, Sharon R. A Sinorhizobium meliloti Lipopolysaccharide Mutant Altered in Cell Surface Sulfation and Symbiosis. J Bacteriol. 2002;184:6681-9.
Wais, Rebecca, Keating, David H. and Long, Sharon R. Structure-Function Analysis of Nod Factor-Induced Root Hair Calcium Spiking in Rhizobium-Legume Symbiosis. Plant Physiol. 2002;129: 211-224.
Cronan, G. E. and D. H. Keating. Sinorhizobium meliloti Lipopolysaccharide Sulfotransferase. J. Bacteriol. 2004;186:168–4176.
G. E. Townsend II, and D. H. Keating. Mesorhizobium loti Produces nodPQ-Dependent Sulfated Cell-Surface Polysaccharides. J. Bacteriol. 2006. 188:8560-72.
D. H. Keating. Sinorhizobium meliloti ExoR Mediates a Global Transcriptional Response to Divalent Cations. 2007. FEMS Microbiol. Lett. 267:23–29.
D. H. Keating. Sinorhizobium meliloti SyrA is a Transcriptional Regulator of Genes Involved in LPS Sulfation and Exopolysaccharide Biosynthesis. 2007. J. Bacteriol. 189:2510-20.
Klein A., Shulla A., Reimann S. A., Keating D. H., Wolfe A. J. The Intracellular Concentration of Acetyl Phosphate in Escherichia coli Is Sufficient for Direct Phosphorylation of Two-Component Response Regulators. J Bacteriol. 2007. 189:5574-81.
D. H. Keating, A. H. Klein, and A. J. Wolfe. 2008. Optimized two-dimensional thin layer chromatography to monitor the intracellular concentration of acetyl phosphate and other small phosphorylated molecules. Biol. Prot. Online. 10:36-46.
Townsend, G. E. and D. H. Keating. 2008. Identification and Characterization of KpsS, a novel class of polysaccharide sulfotransferase in Mesorhizobium loti. Mol. Microbiology, Mol Microbiol. 68:1149-64.
Müller M.G, L. S. Forsberg, and D.H. Keating. 2009. The rkp-1 cluster is required for secretion of Kdo homopolymeric capsular polysaccharide in Sinorhizobium meliloti strain Rm1021. J Bacteriol. 2009.
Gardner, J. G., and D.H. Keating. 2010. Cellvibrio japonicus requires the Type II secretion system for utilization of cellulosic substrates. Applied Environmental Microbiology. In Press.