Transformation associated recombination (TAR) cloning of promising antibiotic producing biosynthetic gene clusters (BGCs)
Fayrouz El Maddah 1 , Lona Alkhalaf 1 , Greg Challis 1,2 1 University of Warwick, UK, 2 Monash University, Australia
Transformation-associated recombination (TAR) cloning is one of the most reliable methods for selective isolation of large regions from complex genomic DNA, employing the highly recombinant nature of Saccharomyces cerevisiae . 1 TAR cloning has accelerated the heterologous expression of natural product biosynthetic gene clusters (BGCs), enabling biosynthetic pathways to be rapidly elucidated and engineered, and has been applied to the capture of large BGCs from microbial genomes. 2 We aimed to employ this technique to clone the BGCs for the antibiotics enacyloxin II and vibroxin, which have promising activity against multidrug-resistant Acinetobacter baumannii . 3,4 These antibiotics are biosynthesised by the bacteria Burkholderia ambifaria and Vibrio rhizospherae , respectively. Their biosynthesis involves a modular polyketide synthase (PKS), a multienzyme that assembles and modifies acetate-derived units to create a structurally complex hydrocarbon chain. References 1. Kouprina, N.; Larionov, V. Chromosoma 2016 , 125 (4), 621–632. 2. Zhanga, J. J.; Yamanakab, K.; Tanga, X.; Moore, B. S. Methods Enzym. 2019 , 621 (3), 87–110. 3. Mahenthiralingam, E.; Song, L.; Sass, A.; White, J.; Wilmot, C.; Marchbank, A.; Boaisha, O.; Paine, J.; Knight, D.; Challis, G. L. Chem. Biol. 2011 , 18 (5), 665–677. 4. Challis, G.; Griffiths, D.; Masschelein, J. Antimicrobial Agents, WO 2017 /182828 A1
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