HEx: a heterologous expression platform for the discovery of fungal natural products
Colin J. B. HarveyiD, Man‐Cheng TangiD, Ulrich SchlechtiD, Joe Horecka, Curt R. FischeriD, Hsiao‐Ching LiniD, Jian LiiD, Brian Naughton, J. Michael CherryiD, Molly MirandaiD, Yong Fuga LiiD, Angela M. ChuiD, James R Hennessy, Gergana A VandovaiD, Diane O. InglisiD, Raeka S. AiyariD, Lars Michael SteinmetziD, Ronald Wayne DavisiD, Marnix H. MedemaiD, Elizabeth S. SattelyiD, Chaitan KhoslaiD, Robert P. St.OngeiD, Yi TangiD, Maureen E. Hillenmeyer
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.\n\nSummaryHere 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.
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In-depth summary
Claude Sonnet 4.6 · max effort · 8 Nov 2026 · from the full text of version 1 (15 Jan 2018) · CC BY 4.0
A yeast-based refactoring platform (HEx) with new inducible promoters, an engineered host strain, and homology-based assembly expressed 41 cryptic fungal gene clusters; 22 yielded non-native compounds.
Most fungal biosynthetic gene clusters are silent or sit in hosts that cannot be cultured or manipulated, which limits native-host activation. Earlier complete-refactoring work in fungal hosts covered only a single cluster; this work scales the approach to dozens.
Key results
ShownOf 41 cryptic fungal clusters refactored and expressed in S. cerevisiae, a majority produced compounds not native to yeast, while the rest gave no detectable product.
22 of 41 clusters (54%) produced non-native compounds; 19 of 41 produced no detectable compounds. The set was 28 PKS-core clusters (3 to 11 genes) and 13 UbiA-type terpene cyclase (UTC) clusters. · Main Text; Expression of Cryptic BGCs
ShownA panel of sequence-divergent, delayed-induction PADH2-like promoters (HEx promoters) increased titers over strong constitutive promoters in test clusters.
48 candidates screened; PADH2, PMLS1, PPCK1 and PICL2 matched or exceeded PTDH3/PFBA1 at 24 h. 30 PADH2-like promoters in total, 7 strong. Compound 1 detected only with HEx promoters; compound 2 titer 4.5-fold higher; TC1 20-fold, TC3 100-fold (n=3). · Characterization of Genetic Parts; Figure legends
ShownThe engineered DHY host raised sporulation and mitochondrial stability relative to S288c-derived strains, improved respiratory growth over BY4741 and BJ5464, and increased PADH2- and PPCK1-driven GFP expression across tested backgrounds.
Sporulation rose from 2% to 62% after two days; petite frequency fell from 52% to 2.5%. Growth curves represent six biological replicates; a non-fluorescent cell population seen in BY4741 was undetectable in DHY. · Improved Host Strains; Figure legends
ShownA homologous-recombination assembly workflow with yeast-direct plasmid sequencing verification supported routine assembly of large multi-fragment constructs.
Assemblies of up to 14 unique fragments (78 unique assemblies); in the cir0 strain Y800, more than 75% of reads mapped to the target plasmid; 41 clusters assembled and over 1000 yeast clones sequenced. · High-throughput DNA Assembly; Figure legends
ShownPhylogeny-guided selection yielded compounds from under-studied clades, including polyketide-amino acid hybrids from basidiomycete clusters lacking NRPS genes. Correcting a predicted intron rescued a silent UTC cluster (TC5).
Basidiomycete PKS: 3 of 7 clusters produced compounds (PKS16, PKS17, PKS28); ascomycete PKS: 13 of 21 produced compounds; UTC: 5 of 13 clusters. Selection drew on 581 genomes, 3512 PKS BGCs and 326 UTC-containing BGCs. · Expression of Cryptic BGCs
Methods, data and reproducibility
PKS and UbiA-type terpene cyclase clusters were prioritized with antiSMASH2 and phylogenetics across 581 fungal genomes, synthesized, assembled by yeast homologous recombination with HEx promoters in the DHY host, and profiled by LC/HRMS, with NMR for structures.
- Publicly available sequenced fungal genomes mined for PKS and UTC biosynthetic gene clusters (GenBank, as of July 2015) · fungi (ascomycetes and basidiomycetes) · n = 581 genomes; 3512 PKS BGCs; 326 UTC BGCs
- Refactored cryptic fungal BGCs expressed in engineered S. cerevisiae (28 PKS-core, 13 UTC-core) · S. cerevisiae host; fungal gene donors · n = 41 clusters
- Candidate yeast promoters screened for delayed induction with single-copy GFP reporter integrations · Saccharomyces cerevisiae · n = 48 candidate genes; 30 PADH2-like promoters retained
- Assembly verification by sequencing of plasmids prepared from yeast clones · Saccharomyces cerevisiae · n = 78 unique assemblies; over 1000 clones sequenced
- Methods & tools
- HEx platform, antiSMASH2, MIBiG database, GenBank (NCBI), Yeast homologous recombination assembly, GFP reporter fluorescence assays, LC/HRMS untargeted metabolomics, NMR structure elucidation, MiSeq sequencing, DHY host strain background
- Reagents & environment
- DHY was built from S288c-derived BY4741 and BY4742, with BJ5464 as a comparison strain and Y800 as a cir0 strain. DHY adds SAL1, CAT5(91M), MIP1(661T), HAP1, MKT1(30G), RME1(INS-308A), TAO3(1493Q), deletes PEP4 and PRB1, and carries npgA and ATEG_05064.
- Experimental conditions
- Sequencing threshold set for 192 samples on a single MiSeq run; metabolite and fluorescence assays used three biological replicates; growth curves used six replicates. No computational hardware stated.
- Reproducibility gaps
- No code repository or data accession printed; strain lists, gene sequences and spectra are in supplementary tables and figures not included in this text.
Limitations (4)
- 19 of 41 clusters gave no detectable compounds. The authors flag incorrect intron prediction as a likely common failure mode and note that transcript and protein levels and ACP phosphopantetheinylation were not examined. · stated by the authors
- The authors suggest that producing several variants of every refactored cluster with different promoter strengths, to alter protein stoichiometry, might revive nonfunctional clusters or improve conversion of intermediates; this remains future work. · stated by the authors
- The HEx versus constitutive promoter comparison covers only four clusters (two controls, TC1 and TC3), so the titer gains may not generalize to the other clusters. · noted in this summary
- Only PKS and UTC core clusters were tested, and no native-host or alternative-host baseline success rate is given, so the 54% hit rate cannot be benchmarked against other expression approaches. · noted in this summary
Who should read:Read this if you want a scalable pipeline for refactoring and expressing silent fungal biosynthetic gene clusters in yeast, including promoter, host and assembly design and success and failure rates.
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