K. Clevenger, J.W. Bok, R. Ye, G. Miley, T. Velk, C. Chen, K.H. Yang, P. Gao, M. Lamprecht, P. Thomas, M.D. Islam, N. Keller, N. Kelleher, C. Wu
Intact Genomics, Inc.,
Keywords: FAC, SM gene cluster, gene/gene cluster editing, natural product/drug discovery
Summary:We have developed the fungal artificial chromosome [FAC, or autonomously replicating E. coli- fungal shuttle bacterial artificial chromosome (BAC)] vector and system. The FAC technology enables unbiased capturing of 263 full-length large secondary metabolite (SM) clusters (100kb or larger each) as individual FAC clones, from 6 sequenced fungal genomes for one-step transformation and heterologous expression. The FAC technology also enables precise SM gene and gene cluster editing and activating silent and cryptic SM gene clusters. We have demonstrated the FAC technology is extremely effective for natural product and drug discovery by using a liquid chromatography-fourier transform mass spectrometer (LC-FTMS) and novel data analysis pipeline. This novel FAC platform provides a major advance in the study of fungal biosynthesis, enabling rapid detection of novel SMs, identification of their biosynthetic machinery, and dissection of their biosyntheses. As an example, three SM compounds, benzomalvin A, ophiobolin G, and a novel lipopeptide has been discovered with a FAC clone: AtFAC9J20 containing a super-SM gene cluster. This disruptive technology could revive natural product and drug discovery from sequenced fungi, fungal metagenomes and environmental microbial metagenomes.