Biomolecular engineering

 

Protein and cellular engineering are powerful approaches to enhance the efficiency of biological processes. Our current focus is on chromate bioremediation. Hexavalent chromate is a carcinogen which is a wide-spread environmental pollutant. Bacteria can remediate chromate, but several improvements are needed to make them efficient agents in this respect. We have cloned several genes that encode chromate reductase activity, and using pure enzyme preparations, have identified suitable candidates (the "safe enzymes" as opposed to many others that the cell contains) for improvement through enzyme evolution. The improvements we seek concern greater affinity for chromate, decreased reactive oxygen species (ROS) generation during chromate reduction (which is the main reason for chromate toxicity to the remediating bacteria), and a broader range, so that the same enzyme can detoxify also other contaminants. We have recently shown that several "safe" chromate reductases can also reduce uranyl [U(VI)] to its insoluble U(IV) valence state. We have improved both the chromate and uranyl reducing activity of one of the "safe" enzymes for both chromate and uranyl up to several hundred fold. Furthermore, collaborations are in place to: combine rational approaches with DNA shuffling to improve the enzymes; screen the shuffled libraries for improvement in remediating capability for various actinides; and for structural studies on the wild type and improved enzymes.

At the organismal level, our work is directed to ensure maximal expression of chromate reductase activity with minimal bacterial growth. Extensive growth produces large amounts of biomass that hampers bioremediation. In addition, attempts are directed towards engineering bacteria better equipped to function under the harsh conditions of polluted sites.

Biological role of 'chromate reductases': While many enzymes that we and others have characterized have been referred to as chromate reductases, we have shown in the case of one of them that it is its quinone reductases activity that has the greatest biological significance. This enzyme (ChrR) reduces quinones by simultaneous two-electron transfer, avoiding formation of semiquinone, and producing quinols that promote tolerance to oxidative stress. This was demonstrated by a variety of appproaches including assesment of intracellular oxidative stress by protein carbonylation assays and FACS analysis.

Barak, Y., D. F. Ackerley, C. J. Dodge, B. Lal, A. Cheng, A. J. Francis, and A. Matin. 2006. Analysis of novel soluble Cr(VI) and U(VI) reductases and generation of improved enzymes using directed evolution. Applied and Environmental Microbiology  72 (11): 7074-708 [PDF]

Ackerley, D.F., Y. Barak, S.V. Lynch, J. Curtin, and A. Matin. 2006. Effect of chromate stress on Escherichia coli K12. Journal of Bacteriology 188 (9): 3371-338. [PDF]

Gonzalez C.F., D.F. Ackerley, S.V. Lynch, and A. Matin. 2005. ChrR, A soluble quinone reductase of Pseudomonas Putida that defends against H2O2The Journal of Biological Chemistry. 280(24): 22590-22595. [PDF]

Gonzalez C., D. Ackerley, M. Keyhan, and A. Matin. Evaluation of Class II Chromate Reductases and their Bioremediation Potential. In Press 2005. [PDF]

Ackerley, D.F., C.F. Gonzalez, C.H. Park, R. Blake II, M. Keyhan, and A. Matin. 2004. Mechanism of chromate reduction by the Escherichia coli protein, NfsA, and the role of different chromate reductases in minimizing oxidative stress during chromate reduction. Environmental Microbiology 6 (8): 851-860 [PDF]

Ackerley, D.F., C.F. Gonzalez, C.H. Park, R. Blake II, M. Keyhan, and A. Matin. 2004. Chromate reducing properties of soluble flavoproteins from Pseudomonas putida and Escherichia coli. Applied & Environmental Microbiology 70(2):873-882 [PDF]

Keyhan M., D. F. Ackerley, and A. Matin. Targets of Improvement in Bacterial Chromate Bioremediation. Paper E-06, in: M. Pellei and A. Porta (Eds.), Remediation of Contaminated Sediments—2003. Proceedings of the Second International Conference on Remediation of Contaminated Sediments (Venice, Italy; 30 Sep–3 Oct 2003). ISBN 1-57477- 143-4, published by Battelle Press, Columbus, OH [PDF]

Park, C.H., C. Gonzales, D. Ackerley, M. Keyhan, and A. Matin. 2002. Molecular Engineering of Soluble Bacterial Proteins with Chromate Reductase Activity. In: R.E. Hinchee, A. Porta, and M. Pellei (eds.), Remediation and Reuse of Contaminated Sediments, pp. 103-111. Proceedings of the First International Conference on Remediation of Chlorinated Sediments (Venice, Italy, 10-12 October 2001), Volume S1-3. Battelle Press, Columbus, Ohio, USA.

Park, C-H., M. Keyhan, B. Wielinga, S. Fendorf, and A. Matin. 2000. Purification to homogeneity and characterization of a novel Pseudomonas putida chromate reductase. Applied & Environmental Microbiology 66 (5): 1788-1795 [PDF]

Patent

Matin, A., Barak, Y., and Ackerley, D. 2006. Improved nitroreductase enzymes for bioremediation. S06-391 (STAN-449US2)