A Pseudomonas sp. strain IOCa11 exhibiting broad substrate profile for polycyclic aromatic hydrocarbons (PAHs) degradation was isolated by enrichment techniques from oil-contaminated soil. We applied genome shuffling of Pseudomonas sp. strain IOCa11 to achieve improved degradation of PAHs. The initial mutant population was generated by nitrosoguanidine treatment and population exhibiting improved phenotype was subjected to multiple round of protoplast fusion in order to allow recombination between genomes. Mutant, designated as SF-IOC11-16A, obtained after recursive protoplast fusion showed substantial improvement in ability to degrade PAHs in liquid media. It could degrade 98% DBT in 72 hours in comparison to 74% by the wild parent strain. Similar improvement in degradation of the naphthalene (NAP), phenanthrene (PHE), and benzo (α) pyrene (BAP) was also observed in shuffled strain. The shuffled strain was also able to grow at higher concentration of PAHs and degrade them efficiently. The results indicate that genome shuffling can successfully be used to improve the PAHs degradation capability of bacteria.
References
[1]
K. C. Jones, J. A. Stratford, K. S. Waterhouse, E. T. Furlong, W. Giger, R. A. Hites, C. Schaffner and A. E. Johnston, “Increases in the Polynuclear Aromatic Hydrocarbon Content of an Agricultural Soil over the Last Century,” Environment Science Technnology, Vol. 23, No. 1, 1989, pp. 95-101. doi:10.1021/es00178a012
[2]
C. E. Cerniglia, “Biodegradation of Polycyclic Aromatic Hydrocarbons,” Current Opinion in Biotechnology, Vol. 4, No. 3, 1993, pp. 331-338.
doi:10.1016/0958-1669(93)90104-5
[3]
G. Fuchs, M. Boll and J. Heider, “Microbial Degradation of Aromatic Compounds—From One Strategy to Four,” Nature Review Microbiology, Vol. 9, No. 11, 2011, pp. 803-816. doi:10.1038/nrmicro2652
[4]
R. A. Kanaly and S. Harayama, “Biodegradation of High- Molecular-Weight Polycyclic Aromatic Hydrocarbons by Bacteria,” Journal of Bacteriology, Vol. 182, No. 8, 2000, pp. 2059-2067. doi:10.1128/JB.182.8.2059-2067.2000
[5]
W. P. Stemmer, “DNA Shuffling by Random Fragmentation and Reassembly: In Vitro Recombination for Molecular Evolution,” Proceedings of Notational. Academy of Sciences USA, Vol. 91, No. 2, 1994, pp. 10747-10751.
doi:10.1073/pnas.91.22.10747
[6]
Y. Z. Zhan, K. Perry, V. A. Vinci, K. Powell, W. P. C. Stemmer and S. del Cardayre, “Genome Shuffling Leads to Rapid Phenotypic Improvement in Bacteria,” Nature, Vol. 415, 2002, pp. 644-646. doi:10.1038/415644a
[7]
M. H. Dai, S. Ziesman, T. Ratcliffe, R. T. Gill and S. D. Copley, “Visualization of Protoplast Fusion and Quantitation of Recombination in Fused Protoplasts of Auxotrophic Strains of Escherichia coli,” Metabolic Engineering, Vol. 7, No. 1, 2005, pp. 45-52.
doi:10.1016/j.ymben.2004.09.002
[8]
D. R. Singleton, L. G. Ramirez and M. D. Aitken, “Characterization of a Polycyclic Aromatic Hydrocarbon Deg- radation Gene Cluster in a Phenanthrene-Degrading Acidovorax Strain,” Applied Environmental Microbiology, Vol. 75, No. 9, 2009, pp. 2613-2620.
doi:10.1128/AEM.01955-08
[9]
M. H. Dai and S. Copley, “Genome Shuffling Improves Degradation of the Anthropogenic Pesticide Pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723,” Applied Environmental Microbiolology, Vol. 70, No. 4, 2004, pp. 2391-2397.
doi:10.1128/AEM.70.4.2391-2397.2004
[10]
J. Gong, H. Zheng, Z. Wu, T. Chen and X. Zhao, “Genome Shuffling: Progress and Applications for Phenotype Improvement,” Biotechnology Advances, Vol. 27, No. 6, 2009, pp. 996-1005. doi:10.1016/j.biotechadv.2009.05.016
[11]
D. Pinel, F. D’Aoust, S. B. del Cardayre, P. K. Bajwa, H. Lee and V. J. Martin, “Saccharomyces cerevisiae Genome Shuffling through Recursive Population Mating Leads to Improved Tolerance to Spent Sulfite Liquor,” Applied Environmental Microbiology, Vol. 77, No. 14, 2011, pp. 4736-4743. doi:10.1128/AEM.02769-10
[12]
M. Kumar, V. Leon, A. De Sisto, O. A. Ilzins, I. Galindo-Castro and S. L. Fuenmayor, “Polycyclic Aromatic Hydrocarbon Degradation by Biosurfactant-Producing Pseudomonas sp. IR1,” Zeitschrift für Naturforschung, Vol. 61, No. 3-4, 2006, pp. 203-212.
[13]
M. Kumar, V. Leon, A. De Sisto, O. A. Ilzins and L. Luis, “Biosurfactant Production and Hydrocarbon-Degradation by Halotolerant and Thermotolerant Pseudomonas sp.,” World Journal of Microbiology and Biotechnology, Vol. 24, No. 7, 2008, pp. 1047-1057.
doi:10.1007/s11274-007-9574-5
[14]
A. R. Clemente, A. T. Anazawa and L. R. Durrant, “Biodegradation of Polycyclic Aromatic Hydrocarbons by Soil Fungi,” Brazilian Journal of Microbiology, Vol. 32, No. 4, 2001, pp. 255-261.
doi:10.1590/S1517-83822001000400001
[15]
M. Arbabi, N. Simin and C. Anyakora, “Biodegradation of Polycyclic Aromatic Hydrocarbons (PAHs) in Petroleum Contaminated Soils,” Iran Journal Chemistry Chemical Engineering, Vol. 28, No. 3, 2009, pp. 53-59.
[16]
S. D. Haemmerli, M. S. A. Leisola, D. Sanglard and A. Fiechter, “Oxidation of Benzo (α) Pyrene by Extracellular Ligninases of Phanerochaete chrysosporium,” Journal Biological Chemistry, Vol. 261, No. 15, 1986, pp. 6900- 6903.
[17]
J. Sambrook, E. F. Fritsch and T. Maniatis, “Molecular Cloning: A Laboratory Manual,” 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989.
[18]
M. Kumar, S. C. Agrawal and R. K. Upreti, “An improved Method for Preparation of Spheroplast from Ecscherichia coli,” In: P. C. Jain, Ed., Microbiology and Biotechnology for Sustainable Development, CBS Publishers and Distributors, New Delhi, 2004, pp. 314-325.
[19]
R. Patnaik, S. Louie, V. Gavrilovic, K. Perry, W. P. C. Stemmer, C. M. Ryan and S. del Cardayre, “Genome Shuffling of Lactobacillus for Improved Acid Tolerance,” Na- ture Biotechnology, Vol. 20, 2002, pp. 707-712.
doi:10.1038/nbt0702-707
[20]
M. H. Dai and S. Copley, “Genome Shuffling Improves Degradation of Anthropogenic Pesticide Pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723,” Applied Environmental Microbiolology, Vol. 70, No. 4, 2004, pp. 2391-2397. doi:10.1128/AEM.70.4.2391-2397.2004
[21]
H. Hida, T. Yamada and Y. Yamada, “Genome Shuffling of Streptomyces sp. U121 for Improved Production of Hydroxycitric Acid,” Applied Microbiology and Biotechnology, Vol. 73, No. 6, 2007, pp. 1387-1399.
doi:10.1007/s00253-006-0613-1
[22]
M. Kumar, “Improving PAH Degradation by Genome Shuffling,” Asian Journal of Microbiology Biotechnology and Environmental Sciences, Vol. 9, No. 1, 2007, pp. 145- 149.
