Materials and reagents
PCR amplification for plasmid construction was performed using Phusion High-Fidelity polymerase (New England Biolabs, NEB) and Prime STAR GXL DNA polymerase (TaKaRa Bio, Inc.), except for colony PCR, for which 2 × Taq Plus Master Mix (Vazyme) was used. The PCR primers were synthesized using GenScript. Fast digestion restriction enzymes were purchased from Thermo Fisher Scientific. Enzymes for Goldengate and USER cloning were purchased from NEB. Cloning was performed using the chemically competent E. coli strain DH10B (Invitrogen). All yeast strains were cultured in selective uracil-dropout media. AO transformants were selected under triple auxotrophic culture (arg-, ade- and met-) conditions. Yeast extracts and tryptone used for preparing Luria-Bertani (LB) medium were purchased from Oxoid. All salts and reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. The animal care and use were adhered to the Chinese National Guidelines for Ethical Review of Animal Welfare. Animals were handled according to the Guidelines of the China Animal Welfare Legislation, and the study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Renmin Hospital of Wuhan University Human Research Ethics Committee (IACUC issue no. 20201220A).
Preparation of fragments for automated plasmid construction
Functional genes were amplified from the genomes of the five filamentous fungi. For each gene, 40 base pairs overlapping sequences were added to the 5′ and 3′ terminals, corresponding to the specific promoter and terminator, respectively. Regulator cassettes containing promoters and terminators were amplified from the plasmids described in Supplementary Table 12. The primers used in this study are listed in Supplementary Table 11. The optimized vector backbones (pTAex3-, pUSA- and pAdeA-optimized) were constructed by inserting the amplification and screening cassettes of S. cerevisiae into the original vectors of pTAex3, pUSA and pAdeA34, respectively. Except for the amplification and screening cassettes of E. coli (pBR322_ori and ampicillin resistance gene) and S. cerevisiae (CEN_ori and uracil synthesis gene), the pTAex3-, pUSA- and pAdeA-optimized vector backbones contained the screening cassettes Arg, Sc and Ade of AO, respectively (Extended Data Fig. 2). The vector backbones containing three types of amplification and screening cassettes enabled the plasmids to be shuttled between E. coli, S. cerevisiae and AO. All amplified fragments were purified using VAHTS DNA Clean Beads (N411-01, Vazyme).
Plasmid construction using an automated yeast assembly method
For high-throughput assembly of plasmids, the LiAc/ss carrier DNA/polyethylene glycol (PEG) yeast transformation protocols were modified as previously reported35. S. cerevisiae CEN.PK2-1D (EUROSCARF) with the genotype MATaura3-52trp1-289leu2-3 112his3Δ1 MAL2-8C SUC2 was used for all yeast heterologous recombination experiments. For each assembled plasmid, 300 ng of each coding sequence fragment was combined with 300 ng of each required regulator cassette and 300 ng of the appropriate linearized expression vector. Using the Biomek FXP Laboratory Automation Workstation (Beckman Coulter) equipped with an MP200 96-Tip Tool for liquid-handling operations, this DNA mix was transformed into the CEN.PK2-1D strain by using the LiAc/PEG protocol. Detailed procedures are presented in Supplementary Fig. 2. Biomek Software runs the robotics platform based on the specified programs for various steps along the yeast assembly cycle, such as adding DNA mixture combined with LiAc/PEG solution into competent cells for transformation. Each program outlines a series of basic functions that do not change between assembly sets, such as ‘load P200 tips’, ‘aspirate’, ‘mix’ or ‘shake’. Detailed information about constructing plasmids is provided in Supplementary Table 12.
E. coli transformation for plasmid enrichment and verification
E. coli DH10B competent cells were dispensed into 96-well deep plates at 70 μl per well, mixed with 10 μl of yeast plasmids and incubated at 4 °C for 30 min. The plates were then transferred into an incubator preheated to 42 °C and incubated for 3 min; next, 0.8 ml of the LB medium was added to each well and incubated at 37 °C for 45 min. Cell cultures were harvested by centrifugation at 1,500g for 8 min and the supernatants were discarded. The remaining 50 μl of the cell culture was transferred onto LA medium containing the appropriate antibiotic. The colonies were picked with Molecular Device Qpix 460 and inoculated into LB medium for plasmid enrichment. The plasmids obtained were further verified using the appropriate restriction enzymes. Detailed procedures are shown in Supplementary Fig. 3.
Plasmid extraction
Yeast colonies were placed in 1.5 ml of uracil-dropout medium, seeded onto 96-well deep-well plates and incubated with shaking at 998 r.p.m. and 30 °C for 16–18 h. Cell cultures were harvested by centrifugation at 3,500 r.p.m. for 8 min, and the supernatants were discarded. Plasmid DNA was extracted using the magnetic bead method with a modified version of the MagPure Plasmid LQ Kit (Magen Bio). Lyticase (500 U ml−1, Sigma-Aldrich, 20210108) combined with 250 μl of buffer S1 was added to the cell culture and incubated at 25 °C for 2 h. For the detailed procedures, see Supplementary Fig. 4.
Construction of AO-strains for fungal BGC expression
We selected AO NSAR1 (niaD−, sC−, ΔargB, adeA−) as the heterologous expression host, and used the protoplast-polyethylene glycol method for protoplast transformation. The mycelium with spores was inoculated in 100 ml of dextrin-peptone-yeast liquid medium (2% dextrin, 1% polypeptone, 0.5% yeast extract, 0.5% KH2PO4, 0.05% MgSO4·7H2O), and cultivated for 2 d at 30 °C and shaking at 140 r.p.m. The protoplasts were obtained by digestion with yatalase (TaKaRa, 2.0 mg ml−1) and lysing enzymes (sigma, 3.0 mg ml−1).
For the high-throughput transformation of AO, the following liquid-handling operations were performed using the Biomek FXP Laboratory Automation Workstation (Beckman Coulter) equipped with MP200 96-Tip Tool and flexible 8 channels. Plasmid mixtures (10–15 μg each) were automatically aspirated using the P200 tips at the speed of 100 and dispensed into 96-well deep-well plates at 2 mm from the bottom of the wells, in which containing 100 μl of protoplast suspension that was picked in advance by the Thermo Scientific Multidrop Combi SMART Dispenser. After mixing five times at 100 μl s−1, the aliquots were incubated at 4 °C for 1 h, following which 1.25 ml of PEG solution (25% PEG 6000, 100 mM CaCl2, 0.6 M KCl, 10 mM Tris-HCI, pH 7.5) was automatically aspirated using P200 tips at a low speed of 50 and dispensed into the pellets at a speed of 100, as well as set stick wall mode of tips to reduce the loss of the ropy PEG solution. After incubation at 25 °C for 30 min, the mixture was centrifuged at 420g for 25 min. The supernatant was partially discarded, and the resulting 200 μl of solution was diluted with 1 ml of STC buffer (1.2 M Sorbitol, 10 mM CaCl2, 10 mM Tris-HCI, pH 7.5). After centrifugation at 420g for 25 min, the supernatant was partially discarded resulting about 100 μl of the mixture that was automatically aspirated at the speed of 100 using flexible 8 channels with P200 tips, dispensed at 10 mm above the wells and coated with shaking at 700 r.p.m. on a triple auxotrophic (arg-, ade- and met-) solid medium (2% glucose, 1.2 M sorbitol, 0.2% NH4Cl, 0.1% (NH4)2SO4, 0.05% KCl, 0.05% MgSO4·7H2O, 0.15% KH2PO4, 1.6% agar, pH 5.5) in 24-well deep plates (5 ml of medium per well); the plates were incubated at 30 °C for 2–5 d. Detailed procedures are shown in Supplementary Fig. 5.
For the verification of AO transformants, 0.1–1 μg mycelia of AO colonies were picked manually and suspended in 100 μl of NaOH solution (25 mM) in 96-well PCR plates and lysed by heating at 100 °C for 10 min in a PCR machine for genomic DNA extraction. The supernatant of the mixture was used as a template for PCR verification using 2 × Taq Plus Master Mix (Vazyme). All the constructed AO-strains are listed in Supplementary Table 13.
Fermentation and extraction of terpenoids
Authenticated AO transformants were initially cultured on dextrin-peptone-yeast agar plates at 30 °C for 5 d to obtain fresh mycelia and spores, which were then inoculated in 24-well deep plates containing solid medium (1 g of rice and 1.5 ml of distilled H2O per well) for product detection. Alternatively, they were inoculated in plastic lunch boxes containing rice medium (120 g of rice and 100 ml of distilled H2O per box) for product structure elucidation. Each culture was incubated at 30 °C for 2 weeks.
Fermented cultures in 24-well deep-well plates were extracted twice by successively soaking in acetone and ethyl acetate (5 ml per well) at room temperature for 2 h each. The organic solvent was combined and evaporated in a fume hood to obtain crude extracts (10–20 mg per well). Fermented cultures in a plastic lunch box were extracted three times using ethyl acetate. The organic phases were combined and concentrated to dryness using a rotary evaporator.
In vitro and in vivo anti-inflammatory assays of terpenoids
Inhibition of NO production by LPS-stimulated RAW 264.7 murine macrophages purchased from the American Type Culture Collection (ATCC, TIB-71) was used to evaluate the functions of the extracted terpenoids. The cells (2.0 × 105 cells per well) were seeded onto 24-well plates and treated with 1 μg ml−1 LPS in the absence or presence of the tested products for 16 h. Then, 50 μl of supernatant was transferred to a new 96-well plate, and 50 μl of Griess reagent I and 50 μl of Griess reagent II (Nitric Oxide Assay Kit, S0021, Beyotime) were added sequentially. Absorbance was measured at 540 nm on an EnSpire Multimode Plate Reader. The procedure details are shown in Supplementary Fig. 6. IMC and l-NMMA were used as positive controls.
The cytotoxicity of mangicol compounds was tested on RAW 264.7 cells using the Cell Counting Kit-8 (CCK-8) (Dojindo) method as follows. The exponentially growing cells were seeded onto 96-well plates at a density of 2.0 × 104 cells per well and incubated for 24 h at 37 °C in 5% CO2. The tested products at the indicated concentrations were added to each well and incubated for 24 h. Subsequently, the CCK-8 solution (10 μl) was added to each cell and incubated for another 1 h. The absorbance was measured at 450 nm using an EnSpire Multimode Plate Reader. Wells without drugs were used as blank controls.
Eight-week-old female ICR (CD-1) mice (Harlan Sprague Dawley Inc.) were divided into three groups with four mice per group, housed with 12 h:12 h dark/light cycle, temperature (22–25 °C), humidity (50–60%), provided with food and water ad libitum and acclimatized for 1 week. Each mouse received 2 μg per ear phorbol-12-myristate-13-acetate (PMA, 2 μg in 20 μl of acetone) in the right ear. Initially, the right ears were treated with DMSO (20 μl, vehicle control), IMC (100 μg in 20 μl of DMSO) or mangicol J (100 μg in 20 μl of DMSO), while the left ears received vehicle (20 μl of DMSO). One hour later, the phlogistic agent was applied using an automatic pipette in 10 μl volumes to both the inner and outer surfaces of the right ear. The left ear (control) received the vehicle (20 μl of acetone). Ear oedema was measured at 6 h post-PMA treatment using a digital calliper and calculated by subtracting the thickness of the left ear from the right ear. The data obtained are expressed as the mean ± s.d.; the unpaired Student’s t-test was used to determine statistical significance.
The anti-inflammatory activity of mangicol J based on an acute inflammation model was tested in mice using flow cytometry. Seven-week-old male C57BL/6J mice (Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd) were divided into three groups (three mice in the negative group, and five mice each in the model and experimental groups), housed with 12 h:12 h dark/light cycle, temperature (22–25 °C), humidity (50–60%), provided with food and water ad libitum and acclimatized for 1 week. The mice were stimulated with an intraperitoneal injection of 20 mg kg−1 LPS. After 1 h, the mice in which inflammation was induced were treated with compound 70 by intraperitoneal injection at a dose of 10 mg kg−1. Blood samples were collected after 6 h and analysed; the presence of interleukin (IL)-6, IL-10, IFN-γ, IL-17A and TNF-α was detected using a BD FACSCaliburM Flow Cytometer by FCAP Array Software using a Mouse Th1/Th2/Th17 CBA Kit (560485; BD Biosciences).
Inhibition of p-STAT3 protein level in JAK2-STAT3 signalling by IL-6 and interleukin-6 receptor subunit alpha (IL-6Rα)-stimulated human umbilical vein endothelial cells purchased from the ATCC (PCS-100-010) was used to evaluate the anti-inflammatory activity of mangicol J. The cells (2.0 × 105 cells per well) were seeded onto 12-well plates at a density of 2.0 × 104 cells per well and incubated for 24 h at 37 °C in 5% CO2. The negative group did not have treatment with IL-6 and IL-6Rα. The model group was treated with IL-6 (5 ng ml−1) and IL-6Rα (25 ng ml−1) simultaneously. The experimental group was treated with compound 70 (2 μg ml−1) for 3, 6 and 12 h after treatment with IL-6 (5 ng ml−1) and IL-6Rα (25 ng ml−1). Cells from each group were respectively gathered and lysed for cleavage protein for western blotting to analyse the concentration of β-actin, STAT3 and p-STAT3 proteins. The antibodies used are as follows, first antibody: β-Actin (mouse source, catalogue no. ab8226, dilution ratio. 1:10,000, abcam), p-STAT3 (rabbit source, catalogue no. 9145s, dilution ratio. 1:1,000, CST) and STAT3 (rabbit source, catalogue no. 12640s, dilution ratio. 1:1,000, CST); second antibody: hRP-goat anti-rabbit (goat source, catalogue no. E-AB-1003, dilution ratio. 1:3,000, Elabscience) and hRP-goat anti-mouse (goat source, catalogue no. E-AB-1001, dilution ratio. 1:3,000, Elabscience). The relative expression levels of the target proteins were calculated by dividing the intensity ratio of the target proteins by that of STAT3. The data obtained are presented as mean values ± s.d. and the unpaired Student’s t-test was used to determine statistical significance.
Plasmids for overproduction of mangicdiene and mangicol J
To overproduce mangicdiene, we constructed a series of plasmids to overexpress the MVA pathway genes. MVA pathway genes, promoters and terminators (Supplementary Table 10) were amplified using the corresponding primers from AO complementary DNA listed in Supplementary Table 14. A series of plasmids (Supplementary Table 15) was constructed using the yeast assembly method. To obtain additional copies of tHMG1 and FgMS, PhlyA-tHMG1-Tnos was amplified from pSC44 using the primer pair 59-1F/59-1R and inserted into SacI–digested pSC44 to generate pSC59. To overexpress mangicol J, pSC111 was constructed using a yeast assembly. To obtain an additional copy of mgcE, PhlyA-mgcE-Tnos was amplified from pSC111 using the primer pair 112-1 NotI F /112-2 HpaI PacI R and inserted into NotI/PacI–digested pSC111 to generate pSC112.
Plasmids for Cas9 genome editing
To achieve a high efficiency of CRISPR–Cas9-mediated homologous integration, we systematically tested and optimized the CRISPR–Cas9 method and constructed a series of plasmids. Specific genomic RNA sequences targeting pyrG, HS201, HS401, HS601 and HS801 were obtained using the open-source tool http://www.biootools.com/index.html (Extended Data Fig. 10)51. A series of plasmids were constructed to evaluate the influence of the number of single-guide RNAs, the introduction of AMA1 sequence52 and the concentration of sgRNAs towards genome-editing efficiency. The yeast replication element, CEN, was selected as an arbitrary sequence, amplified from pSC01 using the primer pair 78-1F/78-1R and inserted into XhoI/BamHI-digested pET28a to generate pSC78. Next, using the Goldengate method, sgRNA expression cassettes were digested with BsaI and inserted into pSC78 to generate the plasmids pSC93-pSC96, pSC98 and pSC252. PAf U3-transfer RNA-ligD sgRNA-tRNA-TAf U3 was synthesized and inserted into pET28 to generate pSC92. The PvuI/NotI-digested fragments of pSC92, pSC93 and pSC94 were inserted into pSC121(PacI/NotI-digested) to generate pSC131, pSC132 and pSC133, respectively. The PacI/NotI-digested fragments of pSC92, pSC93 and pSC94 were inserted into pSC60 (PacI/NotI-digested) to generate pSC65, pSC69 and pSC70, respectively. To assess the PU6-based CRISPR–Cas9 system, 96F1 was amplified from the NSAR1 genome by primer pair 96-1F/96-R, 116F2 was amplified from pUCm-gRNAscaffold-eGFP by primer pair 96-2F/116-2R and 116F3 was amplified from the NSAR1 genome using primer pair 116-3F/116-3R; three fragments were assembled using primer pair 96-1F/116-3R through overlap extension-PCR (OE-PCR) and inserted into pSC109 (NotI/PacI-digested) to generate pSC116; 117F2 was amplified from pSC116 by primer pair 96-2F/117-2R; 96F1 and 117F2 were assembled by OE-PCR; 117F3 and 117F4 were amplified from pSC116 by primer pairs 117-3F/117-3R and 117-4F/116-3R, respectively, and then assembled by OE-PCR. The two resulting PCR fragments NotI/BcuI and BcuI/PacI were digested and inserted into pSC109 (NotI/PacI-digested) to generate pSC117. The pSC98 NotI/PacI-digested fragment 3xPU6-pyrG sgRNA-TU6 was inserted into pSC109 (NotI/PacI-digested) to generate pSC118.
The pSC98 NotI/SmaI-digested fragment 3xPU6-pyrG sgRNA-TU6 was inserted into pSC87 (NotI/SmaI digested) to generate pSC184. To knock down the FgMS gene in strain AO-S84, pSC252 NotI/PacI-digested fragment 3xPU6-FgMS sgRNA-TU6 was inserted into pSC251 (NotI/PacI-digested) to generate pSC253. To overproduce mangicol J, 24-F1 and F2 were assembled using the primer pair 24-1F/254-2R. Then, PacI/NotI-digested 24-F1 + F2 was inserted into pSC134 (NotI/PacI-digested) to generate pSC24.
Strains for the overproduction of mangicol J in AO
To build a more efficient AO chassis for genome mining and overproduction of mangicol J, the native MVA pathway and the downstream mangicol J-forming pathway were engineered under the control of the desired promoters. Finally, a series of AO-strains were constructed, which are listed in Supplementary Table 16. AO-S84 was constructed by transforming the corresponding plasmid into AO NSAR1. AO-S184 was constructed by transforming pSC184 into AO NSAR1. AO-S95 was constructed using three genome-editing cycles. First, pSC249 and pSC246 were cotransformed into AO-S184, and the constructed strains were PCR-confirmed to contain inserted flanks at the HS401 site. After two or three subcultivations under non-selective conditions, the conidial suspension (104 conidia in 5 μl) of the strain was spotted onto a plate containing uracil and 5-FOA and a plate containing uracil without 5-FOA; strains that can grow under the two culture conditions were screened to serve as the starting strain for the next round of gene editing. The plasmids used in the second and third rounds of gene editing were pSC263, pSC260, pSC248 and pSC262. The construction of AO-S96 to AO-S98 is described in Supplementary Table 16. The engineered plasmids and strains were verified by PCR amplification to ensure that all the plasmids were correctly constructed and all the genes were correctly integrated into the genome of AO, as required.
Reporting Summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
