HEx: A heterologous expression platform for the discovery of fungal natural products

Abstract

For decades, fungi have been a source of U.S. Food and Drug Administration-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.

Fig. 1. Standard workflow for heterologous expression.

Aspects in green italics are addressed in this study.

Fig. 2. Tools developed for the HEx platform.

(A) eGFP expression from a series of P_ADH2-like promoters in cultures grown under both fermentative (YPD) and respiratory (YPE) conditions. All fluorescence intensities are reported as the mean of three biological replicates. Error bars represent 1 SD (n = 3). (B) Four fungal BGCs, two controls and two previously uncharacterized systems, each produce improved titers when heterologously expressed using P_ADH2-like promoters as compared to strong constitutive promoters. ND, not detected. Error bars represent 1 SD (n = 3). Quantitation for TC1 and TC3 was based on the sum of the integrations of extracted ion counts corresponding to the oxidized sesquiterpenoids outlined in table S4.

Fig. 3. Description and characterization of DHY strains.

(A) Annotated genotype of the DHY yeast background. (B) DHY-derived yeast strain JHY702 shows improved growth, particularly after diauxic shift. Growth curves are representative of six biological replicates. Density plots for fluorescence intensity in multiple backgrounds show significantly improved eGFP expression when driven by both (C) P_ADH2 and (D) P_PCK1. Density plots represent the fluorescence intensity of 10⁴ individual cells.

Fig. 4. DNA assembly by yeast homologous recombination.

(A) DNA assembly from commercially synthesized fragments and genetic parts using yeast homologous recombination. (B) Modified yeast plasmid preparation (exo+) leads to increased number of sequencing reads mapping to plasmid DNA. Dotted line marks the efficiency threshold to allow sequencing of 192 samples on a single MiSeq run. (C) Efficient assembly of up to 14 unique DNA parts can be achieved using the protocol outlined here. Data based on 78 unique assemblies.

Fig. 5. PKS BGCs examined for this study.

All putative gene function abbreviations are listed in table S9. Cladogram was constructed as described in Materials and Methods. All plots are the chromatograms of the specified extracted ion in three biological replicates each of both the strain expressing the BGC and an empty vector control strain. Chromatograms are data collected with electrospray ionization in either positive (ESI+), negative (ESI−), or rapid polarity switching (RPS) mode or with multimode ionization in positive mode (MMI). Expression strains are outlined in table S10, and EICs of novel products are shown in the figs. S7 to S23.

Fig. 6. UbiA-type cyclases represent a general class of biosynthetic enzymes.

Putative enzyme activity abbreviations are listed in table S9. Cladogram generated using UTC cyclase sequence. The cyclases associated with all clusters examined in this study are denoted by orange tips in the cladogram.