Synthesis and characterization of novel enzymes from extreme environments

The broad application of enzymes in industry is hindered by the very limited repertoire of available enzymes, their low activity and stability, and high costs. The main goal of the BEEM project is to discover novel enzymes for new applications in a sustainable bio-based economy, specifically for applications in bioremediation, bioenergy and bioproducts.

To identify such enzymes, existing DNA libraries and those generated through BEEM research into fermentation and bioremediation will be analyzed. The existing DNA libraries to be analyzed include many from BEEM's European partner MArine Metagenomes for Biotechnology Applications (MAMBA) from extreme environments including the deep anoxic hypersaline basins of the Mediterranean Sea. These include metagenomic fosmid libraries (~120,000 clones) prepared from various microbial communities and several more fosmid libraries (~240,000 clones) that will be available to BEEM researchers in the next two years. Metagenome sequences will also be provided by the US Department of Energy Joint Genome Institute.

The key to enzyme discovery is to broadly screen these libraries to identify constructs that have interesting activities. These are then sequenced and the data used to identify all of the metabolic genes in the construct. Both sequence-based (i.e. based on DNA hybridization) and activity-based screens will be used to characterize the potential catalytic function of cloned DNA fragments. In addition, BEEM researchers will take advantage of new analytical capabilities developed by Surface Interface Ontario.

The BEEM team includes researchers from Structural Proteomics in Toronto who have unparalleled experience in developing the rapid, high throughput assays for enzyme activity required for this work. They will develop a series of novel screening assays specifically targeting anaerobic redox processes and solid substrates such as cellulosic materials.

Promising genes will then be cloned, expressed and purified in BEEM's high throughput pipeline. The enzyme activity of the purified proteins will then be tested using 19 general screens established in our labs. Proteins showing catalytic activity will be subjected to more specific studies, including secondary screens with more stringent reaction conditions, in order to determine their function. The characterized proteins will also be tested for activity against an array of various industrial and natural substrates, including lignin, lignocellulose, residual fibre from biofuel production, organohalogens, oil, heavy metals and phosphates. Since many enzymes are known to cooperate synergistically to degrade complex substrates, the degradation activity of various combinations of novel enzymes on polymeric substrates, and industrial and natural samples will be tested. This will lead to the identification of novel enzymes related to biotransformation and biodegradation activities.

Publications

Structure and activity of the Saccharomyces cerevisiae deoxyuridine triphosphate pyrophosphatase DUT1, an essential housekeeping enzyme Tchigvintsev, A., Singer, A.U., Flick, R., Petit, P., Brown, G., Evdokimova, E., Savchenko, A., and A.F. Yakunin

Sequence- and activity-based screening of microbial genomes for novel dehalogenases. Chan, W. Y., Wong, M., Guthrie, J., Savchenko, A. V., Yakunin, A. F., Pai, E. F., and E. A. Edwards

Evolution and classification of CRISPR-Cas systems Makarova, K.S., Haft, D.H., Barrangou, R., Brouns, S.J.J., Charpentier, E., Horvath, P., Moineau, S., Mojica, F.J.M., Wolf, Y.I., Yakunin, A.F., van der Oost, J., and Eugene V. Koonin

A dual function of the CRISPR–Cas system in bacterial antivirus immunity and DNA repair Babu, M., Beloglazova, N., Flick, R., Graham, C., Skarina, T., Nocek, B., Gagarinova, A., Pogoutse, O., Brown, G., Binkowski, A., Phanse, S., Joachimiak, A., Koonin, EV., Savchenko, A., Emili, A., Greenblatt, J., Edwards, A.M. and AF Yakunin

Riboneogenesis in yeast Clasquin, M.F., Melamud, E., Singer, A., Gooding, J.R., Xu, X., Aiping, D., Cui, H., Campagna, S.R., Savchenko, A., Yakunin, A.F., Rabinowitz, J.D., and A. A. Caudy

Characterization of reductive dehalogenases in a chlorinated ethene-degrading bioaugmentation culture Chan, W. W. M.

Biochemical and structural studies of the uncharacterized protein PA0743 from Pseudomonas aeruginosa revealed a NAD+-dependent L-serine dehydrogenase Tchigvintsev, A., Singer, A., Brown, G., Flick, R., Evdokimova, E., Tan, K., Gonzalez, C.F., Savchenko, A., and A.F. Yakunin

Mode of coniferous wood decay by the white rot fungus Phanerochaete carnosa as elucidated by FTIR and ToF-SIMS Mahajan, S., Jeremic, D., Goacher, R.E., and E.R. Master

Structure and activity of the Pseudomonas aeruginosa hotdog-fold thioesterases PA5202 and PA2801 Gonzalez, C.F., Tchigvintsev, A., Brown, G., Flick, R., Evdokimova, E., Xu, X., Osipiuk, J., Cuff, M., Lynch, S., Joachimiak, A., Savchenko, A., and A. Yakunin

Application of ToF-SIMS for the Detection of Enzyme Activity on Solid Wood Substrates Goacher, R.E., Edwards, E.A., Yakunin, A.F., Mims, C.A., and E.R. Master

Structure and activity of the cold-active and anion-activated carboxyl esterase OLEI01171 from the oil-degrading marine bacterium Oleispira antarctica Lemak, S., Tchigvintsev, A., Petit, P., Flick, R., Singer, A.U., Brown, G., Evdokimova, E., Egorova, O., Gonzalez, C.F., Chernikova, T.N., Yakimov, M.M., Kube, M., Reinhardt, R., Golyshin, P.N., Savchenko, A., and A. Yakunin

Single residues dictate the co-evolution of dual esterases: MCP hydrolases from the α/β hydrolase family Alcaide, M., Tornes, J., Stogios, P.J., Xu, X., Gertler, C., Di Leo, R., Bargiela, R., Lafraya, A., Guazzaroni, M.E., Lopez-Cortes, N., Chernikova, T.N., Golyshina, O.V., Nechitaylo, T.Y., Plumeier, I., Pieper, D.H., Yakimov, M.M., Savchenko, A., Golyshin, P.N., and M. Ferrer

Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica Kube, M., Chernikova, T.N., Al-Ramahi, Y., Beloqui, A., Lopez-Cortez, N., Guazzaroni, M.E., Heipieper HJ, Klages S, Kotsyurbenko OR, Langer, I., Nechitaylo, T.Y., Lünsdorf, H., Fernández, M., Juárez, S., Ciordia, S., Singer, A., Kagan, O., Egorova, O., Alain Petit, P., Stogios, P., Kim, Y., Tchigvintsev, A., Flick, R., Denaro, R., Genovese, M., Albar, J.P., Reva, O.N., Martínez-Gomariz, M., Tran, H., Ferrer, M., Savchenko, A., Yakunin, A.F., Yakimov, M.M., Golyshina, O.V., Reinhardt, R., and P.N. Golyshin

Characterization of hydrolytic dehalogenases: Substrate specificity and isotope fractionation Tran, C.

Structure and activity of the NAD(P)+-dependent succinate semialdehyde dehydrogenase YneI from Salmonella typhimurium Zheng, H., Beliavsky, A., Tchigvintsev, A., Brunzelle, J.S., Brown, G., Flick, R., Evdokimova, E., Wawrzak, Z., Mahadevan, R., Anderson, W.F., Savchenko, A., and A.F. Yakunin

Biochemical diversity of carboxyl esterases and lipases from Lake Arreo -- a metagenomic approach Martínez-Martínez, M., Alcaide, M., Tchigvintsev, A., Reva, O., Polaina, J., Bargiela, R., Guazzaroni, M.E., Chicote A, Canet A, Valero F, Eguizabal, E.R., Guerrero, M.D., Yakunin, A.F., and M. Ferrer