Parallel gene editing of the cepacin biosynthetic gene cluster in yeast identifies key branch point in cepacin A and collimonins C/D biosynthesis Jinlian Zhao 1 , Chuan Huang 1 , Matthew Jenner 1,2 , Lona M. Alkhalaf 1 and Gregory L. Challis 1,2,3 1 Department of Chemistry, University of Warwick, UK; 2 Warwick Integrative Synthetic Biology Centre, University of Warwick, UK, 3 Monash University, Australia Cepacin A is the first example of bacterial polyyne metabolites. 1 It has promising antibacterial activity and is a major component of Burkholderia ambifaria biological control. 1,2 The biosynthetic gene cluster (BGC) encoding cepacin A was defined recently from B. ambifaria BCC0191 by comparative genomic mining. 2 However, the biosynthetic pathway of this highly unstable bacterial polyyne has remained unclear. In a previous study, we cloned cepacin BGC into a yeast-adapted Burkholderia - Escherichia shuttle vector (pMLBAD_yeast) and successfully expressed it in Paraburkholderia phytofirmans PsJN. 3 To investigate the biosynthesis of cepacin A, in this ongoing study we carried out in-frame gene deletion to explore intermediates accumulated in mutants for gene function characterization, where 12 genes were knocked out by editing cepacin BGC in Saccharomyces cerevisiae based on the strategy of transformation associated homologous recombination (TAR) cloning. 4 Inactivation of genes ccnJ – ccnO all abolished the production of cepacin A without enrichment of intermediates or related metabolites; Deletions of ccnE , ccnF , and ccnH led to the accumulation of shunt metabolites collimonins C/D; 5 Knock-out of ccnG accumulated an intermediate with same structure as massilin C; 6 while in mutants of Δ ccnD and Δ ccnP , cepacin A was found produced with a significantly decreased yield. These mutagenesis results, together with gene complementation data, demonstrated that six conserved genes ( ccnJ – ccnO ) are essential to form cepacin backbone with electron-rich consecutive alkyne moiety at early step and four tailoring genes ( ccnE – ccnH ) are responsible for structure modification at later step, and ccnD / ccnP genes are crucial but not essential to cepacin biosynthesis. A plausible biosynthetic pathway of cepacin A was therefore deduced, and specifically a key branch point in pathways of cepacin A and collimonins C/D was uncovered for the first time, and CcnG was identified as the key enzyme involved in the branch point. References 1. L. Parker, M. L. Rathnum, V. Seiner, W. H. Trejo, P. A. Principe, R. B. Sykes, J . Antibiot ., 1984, 5, 431–440. 2. J. Mullins, J. A. H. Murray, M. J. Bull, M. Jenner, C. Jones, G. Webster, A. E. Green, D. R. Neill, T. R. Connor, J. Parkhill, G. L. Challis, E. Mahenthiralingam, Nat . Microbiol ., 2019, 4, 996–1005. 3. D. Petrova, J. Zhao, G. Webster, A. J. Mullins, K. Williams, A. S. Alswat, G. L. Challis, A. Bailey, E. Mahenthiralingam, Microb . Biotechnol ., 2022, under review. 4. J. Zhang, K. Yamanaka, X. Tang, B. S. Moore, Meth . Enzymol ., 2019, 621, 87–110. 5. Kai, M. Sogame, F. Sakurai, N. Nasu, M. Fujita, Org . Lett ., 2018, 20, 3536–3540. 6. Lin, S. Y. Hoo, L. Ma, C. Lin, K. Huang, Y. Ho, C. Sun, H. Lee, P. Chen, L. Shu, B. Wang, W. Hsu, T. Ko, Y. Yang, 2022, 10.21203/rs.3.rs-1110265/v1.
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