Genetic Manipulation Of Amycolatopsis Mediterrarei S699 For Production Of Rifamycin Analogs

This project aims in genetic manipulation of A. mediterrarei S699 to produce rifamycin analogs which will be effective against multi drug resistant tuberculosisstrain. This project aims in genetic manipulations of polyketide synthase (PKS) gene cluster of Rifamycin B producing A. mediterranei S699. The genetic manipulation involves domain deletion, addition or swapping that will result in various rifamycin analogs which will be effective against infecting strains. For this purpose, firstly we developed cloning vectors and standardized the transformation system. Our efforts of several years of research led to the development of plasmid pRL1 by manipulating an indigenous plasmid pA387 (isolated from Amycolatopsis benzoylytica) by cloning its origin of replication in E.coli. Subsequently, series of cloning vectors, developed was patented (Lal, R. US Patent 005985560A; Tuteja et al., 2000) and used for standardization of transformation protocol in A. mediterranei. Secondly, we also cloned and characterize the rifamycin polyketide synthase (rifPKS) gene cluster from A.mediterranei (Kaur et al., 2001) and picked up entire rifPKS cluster on cosmid clones. This was followed by manipulation of A. mediterranei by combinatorial approach to develop proof of concept for the production of rifamycin analogues effective against MDR strains. We were successful in swapping acyltransferase domain of module 6 of rifPKS (rifAT6), that adds propionate unit with that of acyltransferase domain of module 2 of rapamycin PKS (rapAT2) from Streptomyces hygroscopicus. This successful genetic manipulation of AT6 domain swapping led to the generation of novel analog 24-desmethylrifamycin B. The resulting analog 24-desmethylrifamycin B produced was further converted to 24-desmethylrifamycin S & 24-desmethylrifamycin SV that were found to have a better antibacterial activity than rifamycin B. Before this technology is commercialized, an Indian Patent followed by PCT is being filed. For details, please visit the Tab "Patent".

Prof. Rup Lal is also interested in the evolution of lin genes and genetic manipulation of rifamycin producer Amycolatopsis mediterranei. His group has already sequenced genomes of Amycolatopsis mediterranei S699, Thermus sp. RL, Acinetobacter sp. HA and Sphingobium indicum B90A. He has also explored metagenomics diversity of the HCH dumpsite and currently working on the metagenomic revelations of the hot water springs (with temp. of ~96 degrees Celcius) situated atop the Himalayan ranges at Manikaran, Himachal Pradesh.

Important Papers

• Nigam, A., Almabruk, K. H., Saxena, A., Yang, J., Mukherjee, U., Kaur, H., Kohli, P., Kumari, R., Singh, P., Zakharov, L. N., Singh, Y., Mahmud, T., and Lal, R. 2014. Modification of Rifamycin.
• Polyketide Backbone Leads to Improved Drug Activity Against Rifampicin-Resistant Mycobacterium tuberculosis . J Biochem . doi: 10.1074/jbc.M114.572636. http://www.jbc.org/content/early/2014/06/12/jbc.M114.572636.abstract
• Lal, R., Lal, S., Gründ E and Eichenlaub, R. Construction of a hybrid plasmid capable of replication in Amycolatopsis mediterranei . Appl. Environ. Microbiol. 1991, 57, 665-671.
• Lal, R., Khanna, R., Dhingra, N., Khanna, M and Lal, S. 1998. Development of an improved cloning vector and transformation system in Amycolatopsis mediterranei (Nocardia mediterranei). J. Antibiot. 51, 161- 169.
• Tuteja, D., Dua, M., Khanna, R., Dhingra, N., Khanna, M., Kaur, H., Saxena, D. and Lal, R. 2000. The importance of homologous recombination in the generation of large deletions in hybrid plasmids in Amycolatopsis mediterranei. Plasmid . 43, 1- 11.
• Verma, M., Kaur, J., Kumar, M., Kumari, K., Saxena, A., Anand, S., Nigam, A., Ravi, V., Raghuvanshi, S., Khurana, P., Tyagi, A.K., Khurana, J.P., and Lal, R. 2011. Whole Genome Sequence of Rifamycin B Producing Amycolatopsis mediterranei S699. J. Bacteriol. 193, 5562-5563