Activation of cryptic biosynthetic gene clusters by fungal artificial chromosomes to produce novel secondary metabolites

Chengcang C. Wu; Andrea A. Stierle; Donald B. Stierle; Hongyu Chen; Michael Swyers; Timothy Decker; Emili Borkowski; Peter Korajczyk; Rosa Ye; Niel Mondava; Chengcang C. Wu; Andrea A. Stierle; Donald B. Stierle; Hongyu Chen; Michael Swyers; Timothy Decker; Emili Borkowski; Peter Korajczyk; Rosa Ye; Niel Mondava

doi:10.3934/microbiol.2023039

AIMS Microbiology

2023, Volume 9, Issue 4: 757-779. doi: 10.3934/microbiol.2023039

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Research article

Activation of cryptic biosynthetic gene clusters by fungal artificial chromosomes to produce novel secondary metabolites

1.
Intact Genomics, Inc. 1100 Corporate Square Drive, Suite 257, St Louis, Missouri, 63132, USA
2.
Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, Montana 59812, USA
These authors contributed equally to this work.

Received: 25 August 2023 Revised: 07 December 2023 Accepted: 08 December 2023 Published: 18 December 2023

In 2017, we reported the discovery of Berkeleylactone A (BPLA), a novel, potent antibiotic produced exclusively in co-culture by two extremophilic fungi, Penicillium fuscum and P. camembertii/clavigerum, which were isolated from the Berkeley Pit, an acid mine waste lake, in Butte, Montana. Neither fungus synthesized BPLA when grown in axenic culture. Recent studies suggest that secondary metabolites (SMs) are often synthesized by enzymes encoded by co-localized genes that form “biosynthetic gene clusters” (BGCs), which might remain silent (inactive) under various fermentation conditions. Fungi may also harbor cryptic BGCs that are not associated with previously characterized molecules.

We turned to the tools of Fungal Artificial Chromosomes (FAC)-Next-Gen-Sequencing (NGS) to understand how co-culture activated cryptic biosynthesis of BPLA and several related berkeleylactones and to further investigate the true biosynthetic potential of these two fungi. FAC-NGS enables the capture of BGCs as individual FACs for heterologous expression in a modified strain of Aspergillus nidulans (heterologous host, FAC-AnHH). With this methodology, we created ten BGC-FACs that yielded fourteen different SMs, including strobilurin, which was previously isolated exclusively from basidiomycetes. Eleven of these compounds were not detected in the extracts of the FAC-AnHH. Of this discrete set, only the novel compound citreohybriddional had been isolated from either Penicillium sp. before and only at very low yield. We propose that through heterologous expression, FACs activated these silent BGCs, resulting in the synthesis of new natural products (NPs) with yields as high as 50%–60% of the crude organic extracts.
Citation: Chengcang C. Wu, Andrea A. Stierle, Donald B. Stierle, Hongyu Chen, Michael Swyers, Timothy Decker, Emili Borkowski, Peter Korajczyk, Rosa Ye, Niel Mondava. Activation of cryptic biosynthetic gene clusters by fungal artificial chromosomes to produce novel secondary metabolites[J]. AIMS Microbiology, 2023, 9(4): 757-779. doi: 10.3934/microbiol.2023039

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Abstract

In 2017, we reported the discovery of Berkeleylactone A (BPLA), a novel, potent antibiotic produced exclusively in co-culture by two extremophilic fungi, Penicillium fuscum and P. camembertii/clavigerum, which were isolated from the Berkeley Pit, an acid mine waste lake, in Butte, Montana. Neither fungus synthesized BPLA when grown in axenic culture. Recent studies suggest that secondary metabolites (SMs) are often synthesized by enzymes encoded by co-localized genes that form “biosynthetic gene clusters” (BGCs), which might remain silent (inactive) under various fermentation conditions. Fungi may also harbor cryptic BGCs that are not associated with previously characterized molecules.

We turned to the tools of Fungal Artificial Chromosomes (FAC)-Next-Gen-Sequencing (NGS) to understand how co-culture activated cryptic biosynthesis of BPLA and several related berkeleylactones and to further investigate the true biosynthetic potential of these two fungi. FAC-NGS enables the capture of BGCs as individual FACs for heterologous expression in a modified strain of Aspergillus nidulans (heterologous host, FAC-AnHH). With this methodology, we created ten BGC-FACs that yielded fourteen different SMs, including strobilurin, which was previously isolated exclusively from basidiomycetes. Eleven of these compounds were not detected in the extracts of the FAC-AnHH. Of this discrete set, only the novel compound citreohybriddional had been isolated from either Penicillium sp. before and only at very low yield. We propose that through heterologous expression, FACs activated these silent BGCs, resulting in the synthesis of new natural products (NPs) with yields as high as 50%–60% of the crude organic extracts.

Acknowledgments

We thank the Keller lab at University of Wisconsin-Madison and the Kelleher lab at Northwestern University for their collaboration in the development of the initial FAC technology. This work was supported in part by the US National Institutes of Health (National Institute of Allergy and Infectious Diseases SBIR award in the form of grant R44 AI138765 to C.C.W. and A.A.S

Author contributions

C.C.W. and A.A.S. conceived of, designed and supervised the study. C.C.W., A.A.S., D.B.S. and H.G.C. wrote the manuscript. R.Y., C.C.W., M.S., E.B. and P.K. worked on the FAC library creation, assembly, FAC end sequencing, FAC DNA preparation and FAC engineering. M.S., C.C.W. and H.G.C. carried out fungal secondary metabolite gene cluster prediction, FAC bioinformatic analyses and FAC next-generation sequencing assembly and annotation; performed the A. nidulans transformation with BGC-FACs. A.A.S., D.B.S. and N.M. prepared the FAC fermentation and sample extractions for metabolite identification. D.B.S. ran all of the LC/MS and NMR experiments; A.A.S. and D.B.S. purified individual compounds and determined structures using spectral methods; all authors contributed to the preparation of the manuscript.

Additional information

Supplementary Information accompanies this paper.

Competing interests

C.C.W., R.Y., H.Y.C., M.S. E.B. and P.K. are employees of Intact Genomics, which sells the unbiased Random Shear BAC and FAC libraries and services for genome discovery and DNA research, as well as BAC cloning, DNA end repairing, E. coli competent cells and other enzyme kits for DNA and protein research.

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