We develop a novel synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and encoded metabolites.

Natural product biosynthetic pathways generate molecules of enormous structural complexity and exquisitely tuned biological activities. Studies of natural products have led to the discovery of many pharmaceutical agents, particularly antibiotics. Attempts to harness the catalytic prowess of biosynthetic enzyme systems, for both compound discovery and engineering, have been limited by a poor understanding of the evolution of the underlying gene clusters. We developed an approach to study the evolution of biosynthetic genes on a cluster-wide scale, integrating pairwise gene coevolution information with large-scale phylogenetic analysis. We used this method to infer the evolution of type II polyketide gene clusters, tracing the path of evolution from the single ancestor to those gene clusters surviving today. We identified 10 key gene types in these clusters, most of which were swapped in from existing cellular processes and subsequently specialized. The ancestral type II polyketide gene cluster likely comprised a core set of five genes, a roster that expanded and contracted throughout evolution. A key C24 ancestor diversified into major classes of longer and shorter chain length systems, from which a C20 ancestor gave rise to the majority of characterized type II polyketide antibiotics. Our findings reveal that (i) type II polyketide structure is predictable from its gene roster, (ii) only certain gene combinations are compatible, and (iii) gene swaps were likely a key to evolution of chemical diversity. The lessons learned about how natural selection drives polyketide chemical innovation can be applied to the rational design and guided discovery of chemicals with desired structures and properties.evolution | polyketide | natural products | gene cluster

Summary:Here we present the largest scale effort reported to date toward the complete refactoring and heterologous expression of fungal biosynthetic gene clusters utilizing HEx, a novel synthetic biology platform. Authors:Colin JB Harvey Abstract:For decades, fungi have been a source of FDA-approved natural products such as penicillin, cyclosporine, and the statins. Recent breakthroughs in DNA sequencing suggest that millions of fungal species exist on Earth with each genome encoding pathways capable of generating as many as dozens of natural products. However, the majority of encoded molecules are difficult or impossible to access because the organisms are uncultivable or the genes are transcriptionally silent. To overcome this bottleneck in natural product discovery, we developed the HEx (Heterologous EXpression) synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and their encoded metabolites in Saccharomyces cerevisiae . We applied this platform to 41 fungal biosynthetic gene clusters from diverse fungal species from around the world, 22 of which produced detectable compounds. These included novel compounds with unexpected biosynthetic origins, particularly from poorly studied species. This result establishes the HEx platform for rapid discovery of natural products from any fungal species, even those that are uncultivable, and opens the door to discovery of the next generation of natural products. Main Text:Natural products are indispensable to modern medicine, with 73% of antibiotics, 49% of anticancer compounds, and 32% of all new drugs approved by the FDA between 1980 and 2012 being natural products or derivatives thereof ( 1 ) . Fungi are prolific producers of therapeutically relevant natural products ( 2 , 3 ) , having yielded penicillin, the first widely used antibiotic; cyclosporine, the immunosuppressant that enabled widespread organ transplantation; and lovastatin, the progenitor of the statin class of cholesterol-lowering drugs. In all of these examples, compounds were isolated from laboratory cultures of single fungal isolates. Recent 2 . CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/247940 doi: bioRxiv preprint first posted online Jan. 15, 2018; advances in genome sequencing have revealed that more than 5 million fungal species likely exist on the earth ( 4 ) with each species encoding as many as 80 natural product biosynthetic pathways ( 5 , 6 ) . However, despite the increased ease of DNA sequencing, fungal cultivation remains a bottleneck: only a fraction of the fungi in any given environmental sample have been cultured under laboratory conditions ( 7 ) . Even within cultured species, the majority of biosynthetic gene clusters (BGCs) present in the genome are either transcriptionally silent or expressed at very low levels ( 8 ) . The identification and expression of these BGCs thus presents a major opportunity for the disco...

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