Strategy for efficient cloning of biosynthetic gene clusters from fungi
- 32 Downloads
Filamentous fungi are excellent sources for the production of a group of bioactive small molecules which are often called secondary metabolites (SMs). The advanced genome sequencing technology combined with bioinformatics analysis reveals a large number of unexplored biosynthetic gene clusters (BGCs) in the fungal genomes. To unlock this fungal SM treasure, many approaches including heterologous expression are being developed and efficient cloning of the BGCs is a crucial step to do this. Here, we present an efficient strategy for the direct cloning of fungal BGCs. This strategy consisted of Splicing by Overlapping Extension (SOE)-PCR and yeast assembly in vivo. By testing 14 BGCs DNA fragments ranging from 7 kb to 52 kb, the average positive rate was over 80%. The maximal insertion size for fungal BGC assembly was 52 kb. Those constructs could be used conveniently for the heterologous expression leading to the discovery of novel natural products. Thus, our results provide an efficient and quick method for the low cost direct cloning of fungal BGCs.
Keywordsbiosynthetic gene clusters Saccharomyces cerevisiae homologous recombination DNA assembly
Unable to display preview. Download preview PDF.
- Inglis, D.O., Binkley, J., Skrzypek, M.S., Arnaud, M.B., Cerqueira, G.C., Shah, P., Wymore, F., Wortman, J.R., and Sherlock, G. (2013). Comprehensive annotation of secondary metabolite biosynthetic genes and gene clusters of Aspergillus nidulans, A. fumigatus, A. niger and A. oryzae. BMC Microbiol 13, 91.CrossRefGoogle Scholar
- Mayorga, M.E., and Timberlake, W.E. (1990). Isolation and molecular characterization of the Aspergillus nidulans wA gene. Genetics 126, 73–79.Google Scholar
- Muller, H., Annaluru, N., Schwerzmann, J.W., Richardson, S.M., Dymond, J.S., Cooper, E.M., Bader, J.S., Boeke, J.D., and Chandra-segaran, S. (2012). Assembling large DNA segments in yeast. In Gene Synthesis: Methods and Protocols, J. Peccoud, ed. (New Jersey: Humana Press), pp. 133–150.CrossRefGoogle Scholar
- Paques, F., and Haber, J.E. (1999). Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 63, 349–404.Google Scholar
- Shimizu, K., and Keller, N.P. (2001). Genetic involvement of a cAMP-dependent protein kinase in a G protein signaling pathway regulating morphological and chemical transitions in Aspergillus nidulans. Genetics 157, 591–600.Google Scholar
- Wu, G., Zhou, H., Zhang, P., Wang, X., Li, W., Zhang, W., Liu, X., Liu, H.W., Keller, N.P., An, Z., et al. (2016). Polyketide production of pestaloficiols and macrodiolide ficiolides revealed by manipulations of epigenetic regulators in an endophytic fungus. Org Lett 18, 1832–1835.CrossRefGoogle Scholar
- Zhang, P., Wang, X., Fan, A., Zheng, Y., Liu, X., Wang, S., Zou, H., Oakley, B.R., Keller, N.P., and Yin, W.B. (2017). A cryptic pigment biosynthetic pathway uncovered by heterologous expression is essential for conidial development in Pestalotiopsis fici. Mol Microbiol 105, 469–483.CrossRefGoogle Scholar
- Zhang, A. P. Lu, A. M. Dahl-Roshak, P. S. Paress, S. Kennedy, J. S. Tkacz, and An, Z.Q. (2003). Efficient disruption of a polyketide synthase gene (pks1) required for melanin synthesis through Ag-robacterium-mediated transformation of Glarea lozoyeasis. Mol Genet Genomics 268, 645–655.Google Scholar
- Zhou, S., Zhang, P., Zhou, H., Liu, X., Li, S.M., Guo, L., Li, K., and Yin, W.B. (2019). A new regulator RsdA mediating fungal secondary metabolism has a detrimental impact on asexual development in Pestalotiopsis fici. Environ Microbiol 21, 4163–426.Google Scholar