Abstract
Terpenoids, critical components of human medicine, are the largest family of natural products. Fungi are an important source of terpenoids, but many of the corresponding biosynthetic gene clusters (BGCs) are silent in laboratory conditions. Strategies such as homologous activation and heterologous expression were usually used to active a single cluster, making them low efficiency. Here we developed an automated and high-throughput (auto-HTP) biofoundry workflow using Aspergillus oryzae as a chassis that enables efficient genome mining, characterization of BGCs and identification of bioactive fungal terpenoids. We simultaneously refactored 39 BGCs into 208 engineered strains, producing 185 distinct terpenoids. An anti-inflammatory screen returned the sesterterpenoid mangicol J; re-examination of our engineered strains revealed the likely biosynthetic pathway. Finally, we optimized the mevalonate pathway in A. oryzae to provide a more efficient chassis for overproduction of terpenoids. The auto-HTP biofoundry workflow together with the optimized A. oryzae chassis can accelerate the discovery and development of terpenoid natural products.
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Data availability
Data supporting the findings of this study are available within the article and its Supplementary Information files. The accession numbers (NMDCN0000QH2 to NMDCN0000QH9, NMDCN0000QHA to NMDCN0000QHV, and NMDCN0000QI0 to NMDCN0000QI9) and nucleotide sequences for the characterized enzymes were deposited in the National Microbiology Data Center (https://nmdc.cn/en) will be available after publication. Source data are provided with this paper. All other data that support the findings of this study are available from the corresponding author upon reasonable request.
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Acknowledgements
We thank H. Liu (Wuhan Institute of Biotechnology) for his technical support in setting up the automatic high-throughput biofoundry workflow. This work was financially supported by the National Key R&D Program of China (grant nos. 2018YFA0900400 and 2021YFC2102600), the National Natural Science Foundation of China (grant nos. 31670090, 31800032 and 32070063) and the Medical Science Advancement Program (Clinical Medicine) of Wuhan University (grant no. TFLC2018002).
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Contributions
T.L., G.B. and Z.D. designed the experiments. Y.Y., Z.M. and P.Y. refactored terpenoid BGCs. Y.Y., S.C. and W.D. performed in vitro and in vivo screening of anti-inflammatory activity. S.C., H.H. and H.C. constructed the AO chassis and increased the titre of mangicol J. R.C. and Y.C. characterize the structure of terpenoids. Y.Y., S.C., S.F., G.B. and T.L. analysed the data. G.B. and T.L. wrote the paper.
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T.L., Y.Y., S.C., G.B., P.Y., Z.M. and R.C. have filed four patent applications (Chinese Patent Application Nos. 202010936790.X, 202111306130.4, 202111306130.4 and 202111261216.X) on the basis of this contribution. The remaining authors declare no competing interests.
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Nature Catalysis thanks Nancy Keller, Jose A Carrasco Lopez and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Strength evaluation of selected promoters in AO-NSAR1.
The strength of each promoter (black bars) was assessed based on GUS activity. All data are mean values of three independent experiments, and error bars indicate the standard deviation.
Extended Data Fig. 2 Principles for plasmid and AO-strain construction.
a, Strength-evaluated promoters and BGC library. b, Three-host shuttle plasmid library. c, Principle of plasmid and AO-strain construction and the strain library. Abbreviations: DME, Downstream modification enzyme.
Extended Data Fig. 3 Overview of Biomek FXP Laboratory Automation Workstation.
The workstation is integrated with different functional devices as follows: 1) Biomek FXp Liquid Handling System in the core center (yellow, H), which is equipped with dual arms (a fixed AP96 module and a flexible 8 channel); 2) Biometra T-Robot PCR (wathet, L); 3) Thermo Multidrop Combi Dispenser (green, N); 4) Kbiosystems WASP Sealer (gray, O); 5) Thermo Cytomat MPH (purple, P); 6) Nexus XPEEL: automated plate seal removal system (dusty blue, Q); 7) Biomek 405 L SUV Washer (transparent square, M); 8) Ronata 46 RSC Centrifuge (red, I); 9) Thermo Cytomat CO2 Incubator (orange, J); 10) PE Envision, Multi-mode Microplate Reader (mazarine, K).
Extended Data Fig. 4 Venn diagram of terpenoids detection number.
The number terpenoids detected from Aspergillus oryzae strains using gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) coupled with high-resolution electrospray ionization mass spectrometry (HR-ESI-MS).
Extended Data Fig. 5 Summary of terpenoids produced by terpenoid BGCs.
26 BGCs were detected the terpenoids, among which 16 sesquiterpenoid BGCs (yellow), 8 diterpenoid BGCs (blue) and 2 sesterterpenoid BGCs (red) were included, respectively. The number in, for example, “sesterterpenoid-12 products”, was represented the total products detected in each BGC.
Extended Data Fig. 6 Cell viability evaluation of compounds produced by AO-strains.
a, Cell viability evaluation of RAW 264.7 microphage cells treated with crude products (final concentration, 500 μg/mL) produced by AO-strains. b, Influence of different concentrations (25 μM, 50 μM, 100 μM) of mangicols (68, 69, 70, 72 and 73) and L-NMMA and IMC towards the cell viability of RAW264.7 macrophage cells. L-NMMA, NG-Monomethyl-L-arginine, monoacetate salt (an inhibitor of nitric oxide synthase); IMC, indomethacin. Data (a and b) are presented as mean values ± SD (n = 6).
Extended Data Fig. 7 Functional characterization of MgcC by in vitro assay.
a, The expression verification of MgcC by SDS-PAGE. b, In vitro reaction system for functional verification of MgcC. The reaction included 0.5 μM MgcC enzyme, 1 mM Mg2+, 200 μM substrate (70) and 20 mM phosphate buffer (pH 7.4) was performed at 30 °C for 8 h. c, HR-ESI-MS detection of compound 71 produced by MgcC. Compound 71 were detected in the reaction contained MgcC and substrate 70 (i). The reactions, without MgcC (ii) or without substrate(iii), were unable to produce 71. Three independent experiments were repeated with similar results.
Extended Data Fig. 8 The strain for production of mangicdiene and mangicol J.
Engineered strains via the endogenous MVA or overexpressed mevalonate pathways. Strains shaded in green were attained by gene randomly insertion, strains shaded in pink were attained by gene hotspot site integration. Circles, triangles, squares, and diamonds represent different plasmids, and different colors represent different genes.
Extended Data Fig. 9 Flowchart of AO mutant construction in this study.
Blue represents the production of mangicdiene, red represents the production of mangicol J, purple represents overexpressed genes.
Extended Data Fig. 10 Protospacers used in this study.
a, Protospacer sequences used in this study. b, High expression loci (hot spots, HS) and genetic information. The blue shaded area represents the sgRNA site. The gray shaded area represents the genes flanking the sgRNA site.
Supplementary information
Supplementary Information
Supplementary Methods, Figs. 1–62, Tables 1–10 and References.
Supplementary Video 1
PCR fragments preparation and plasmid construction using the yeast assembly method with an automatic workstation.
Supplementary Video 2
E. coli transformation using the automatic workstation for plasmid propagation and plasmid extraction.
Supplementary Video 3
Construction of AO-strains for fungal terpenoid BGC expression and high-throughput screening of anti-inflammatory activity.
Supplementary Tables
Supplementary Tables 11–17.
Source data
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Source Data Extended Data Fig. 1
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Yuan, Y., Cheng, S., Bian, G. et al. Efficient exploration of terpenoid biosynthetic gene clusters in filamentous fungi. Nat Catal 5, 277–287 (2022). https://doi.org/10.1038/s41929-022-00762-x
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DOI: https://doi.org/10.1038/s41929-022-00762-x
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