General materials and methods
Biochemicals and media were purchased from Sinopharm Chemical Reagent Co., Ltd. (China), Shanghai Sangon Biotech Co. Ltd. (China), Oxoid Ltd. (U.K.) or Sigma‒Aldrich Corporation (USA) unless otherwise stated. Restriction endonucleases were purchased from New England Biolabs, Inc. Ltd. (USA). Chemical reagents were purchased from standard commercial sources. The primers, plasmids and strains used in this study are summarized in Additional file 1: Tables S1–S3.
Primer synthesis and DNA sequencing were performed at Shanghai Sangon Biotech Co. Ltd. (China). Plasmid purification kits and agarose gel DNA extraction kits were purchased from Omega Biotek (USA). PCR amplifications were carried out on an Applied Biosystems Veriti™ Thermal Cycler using either Taq DNA polymerase (Vazyme Biotech Co. Ltd, China) for routine genotype verification or Phanta® Max Super-Fidelity DNA Polymerase (Vazyme Biotech Co. Ltd, China) for high-fidelity amplification. ClonExpress® One Step Cloning Kits were purchased from Vazyme Biotech Co., Ltd. China. Transcriptome sequencing of Talaromyces sp. F08Z-0631 was performed at Novogene Co., Ltd. (China).
High-performance liquid chromatography (HPLC) analysis was conducted with Agilent 1260 Infinity II HPLC systems (Agilent Technologies Inc., USA). Electrospray ionization mass spectrometry (ESI–MS) was performed on an AB Sciex QTRAP® 6500+ mass spectrometer (AB Sciex Pte. Ltd., USA), and the data were analyzed using SCIEX OS Software. High resolution–ESI–MS (HR–ESI–MS) analysis was realized with an Agilent 6545 Accurate-Mass QTOF LC/MS System (Agilent Technologies Inc., USA), and the data were analyzed using Agilent MassHunter Qualitative Analysis software. Nuclear magnetic resonance (NMR) data were recorded on Bruker AVANCE NEO 600 M spectrometers (Bruker Co. Ltd, Germany).
Biosynthetic gene clusters were predicted with the fungal version of antiSMASH (https://fungismash.secondarymetabolites.org/). The deduced proteins were compared to other known proteins in databases using the available BLAST methods (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignments and the phylogenetic tree were created using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) with default settings. The visualization of sequence alignments was created using ESPript 3.0 (https://espript.ibcp.fr/ESPript/ESPript/), and the phylogenetic tree is a neighbour-joining tree without distance corrections.
Transcriptome sequencing of Talaromyces sp. F08Z-0631 under different culture conditions
Transcriptome sequencing was performed as previously described (Zhao et al. 2022). Talaromyces sp. F08Z-0631 was cultured under three different conditions: high-yield, low-yield and non-yield phlegmacin B1. The high-yield culture conditions are as described above. The medium for low yield was rice and soybean medium without trace elements (the yield of phlegmacin B1 was approximately 50 μg/g medium). The liquid fermentation with 40 mL starch–soybean powder medium (1% starch, 2% glucose, 1% hot-rolled soybean powder, 0.1% KH2PO4, 0.05% MgSO4, 0.2% CaCO3, pH not adjusted.) in a 250 mL flask, at 26 ℃, 200 rpm for 7 days (phlegmacin B1 was not detected). The experiments were conducted with three replicates and the mycelia were harvested and ground with liquid nitrogen. Total RNA was extracted with TRIzol (Invitrogen, USA) according to the manufacturer’s protocol. The quality and integrity of RNA samples were determined using a Nanodrop and a Qubit RNA assay kit (Invitrogen, USA). Sequencing libraries were generated and sequenced by Novogene Co., Ltd. (China) on an Illumina HiSeq platform.
Construction of fungal expression plasmids
The single gene fragments toaA, toaB and toaC were amplified from Talaromyces sp. F08Z-0631 genomic DNA. The corresponding primers used for gene cloning are listed in Additional file 1: Table S1. The PCR products were purified and cloned into the EcoRI and KpnI linearized pTAex3 vectors using the ClonExpress®II One Step Cloning Kit according to the manufacturer’s protocol to yield corresponding single gene expression plasmids.
For constructing plasmids coexpressing toaA and toaB, DNA fragments, including the amylase promoter and terminator, were amplified from corresponding pTAex3-based single gene expression plasmids with the primers listed in Additional file 1: Table S1 and then cloned into SpeI- and PstI-digested pAdeA using the ClonExpress® MultiS One Step Cloning Kit.
The constructed plasmids are listed in Additional file 1: Table S2, and representative plasmid maps are provided in Additional file 1: Fig. S5.
Construction and fermentation of A. oryzae NSAR1 heterologous expression strains
Similar to that previously described (Zhao et al. 2022), transformation of A. oryzae NSAR1 was performed by a protoplast–polyethylene glycol method. The spore suspension of A. oryzae NSAR1 was seeded in 10 mL DPY medium (2% dextrin, 1% polypeptone, 0.5% yeast extract, 0.05% MgSO4·7H2O, 0.5% KH2PO4, without pH adjustment), cultured for 2 days at 30 °C and 150 rpm, and then transferred into 100 mL DPY medium for another day. Mycelia were collected by filtration and digested with 1% Yatalase (Takara, Japan) in 0.6 M (NH4)2SO4, 50 mM maleic acid, pH 5.5, at 30 °C for 3 h. The residues were removed by filtration, and the protoplasts were collected by centrifugation at 1500 rpm for 10 min. Then, the protoplasts were washed with Solution 2 (1.2 M sorbitol, 50 mM CaCl2·2H2O, 35 mM NaCl, 10 mM Tris–HCl, pH 7.5) and resuspended to approximately 1 × 107 cells mL−1 in Solution 2. A total of 10 μL plasmids (5–10 μg) were added into a 200 μL protoplast suspension and incubated on ice for 30 min. Subsequently, 250 μL, 250 μL and 850 μL Solution 3 (60% PEG4000, 50 mM CaCl2·2H2O, 10 mM Tris–HCl, pH 7.5) was added and mixed successively. After incubation at room temperature for 20 min, 10 mL of Solution 2 was added, and the mixture was centrifuged at 1500 rpm for 10 min. The precipitates were suspended in 200 μL of Solution 2 and spread on M medium (0.2% NH4Cl, 0.1% (NH4)2SO4, 0.05% KCl, 0.05% NaCl, 0.1% KH2PO4, 0.05% MgSO4·7H2O, 0.002% FeSO4·7H2O, 2% glucose, 1.5% agar) with 1.2 M sorbitol and 0.15% methionine, 0.1%, arginine, which was selected for transformants harbouring the pAdeA-derived plasmid, and only 0.15% methionine at pH 5.5 for harbouring the pAdeA-derived and pTAex3-derived plasmids. Then, the plates were covered with the same upper medium containing 0.8% agar. The plates were incubated at 30 °C for 2–4 days, and the transformants were transferred to the corresponding selection medium without sorbitol for rejuvenation. All of the A. oryzae NSAR1 strains constructed in this work are listed in Additional file 1: Table S3.
The A. oryzae transformants were cultured in 10 mL DPY medium for 2 days, and then the seed broth was transferred into 100 mL Czapek–Dox (CD) medium (0.3% NaNO3, 0.2% KCl, 0.05% MgSO4·7H2O, 0.1% KH2PO4, 0.002% FeSO4·7H2O, 1% polypeptone, 2% starch, pH 5.5) in a 500 mL flask at 30 °C for 5 days and 150 rpm to induce the expression of heterologous genes under the PamyB promoter. To optimize fermentation conditions, 2% soybean meal and/or 2% peptone were added to CD medium, and then the pH was adjusted to 5.5. The fermentation broth was extracted with ethyl acetate. The extract was concentrated under reduced pressure and resuspended in acetonitrile for the analysis and isolation of metabolites.
Time-course analyses of compound 4 produced by AotoaA/toaB-toaC. Spores of AotoaA/toaB-toaC (~ 1 × 107) growing on PDA plates were inoculated into 10 mL DPY medium and cultured at 30 °C in shake flasks with rotary shaking at 150 rpm for 2 days. Then, the seed broth was transferred into 100 mL CD medium in a 500 mL flask at 30 °C for 5 days and 150 rpm. An 800 μL sample of each strain was individually collected each day. The broth was extracted with 800 μL ethyl acetate. The ethyl acetate extraction was dried under a nitrogen flow and redissolved in acetonitrile for the analysis and isolation of metabolites.
Analysis of metabolites and compound structural characterization
The ethyl acetate extract of fermentation broth and in vitro assays were used for HPLC analysis on an Agilent column (Zobrax SB-C18, 3.5 μm, 2.1 × 50 mm, Agilent, USA). The metabolites were eluted at a flow rate of 0.2 mL/min over a 35 min gradient as follows: T = 0 min, 40% B; T = 5 min, 40% B; T = 20 min, 100% B; T = 24 min, 100% B; T = 25 min, 40% B; T = 35 min, 40% B (solvent A, H2O + 0.1% HCOOH; solvent B, CH3CN + 0.1% HCOOH) with monitoring at 390 nm. For HPLC–ESI–MS analysis, the conditions were the same.
Ethyl acetate extraction was used to isolate compounds from the fermentation of A. oryzae transformant strains. Compounds 3 and 4 were isolated from the fermentation of AotoaA/toaB and AotoaA/toaB-toaC, respectively. The crude extracts were dried in vacuo and then subjected to C18 column (12 nm S-50 μm) chromatography using a methanol and H2O gradient system (from 20% to 100%). Fractions containing the target compound were combined for further purification using a semiprep HPLC with a C18 column (Agilent ZORBAX SB-C18, 5 μm, 9.4 × 250 mm). For structural characterization, HRESIMS, NMR, and optical rotation of compounds 3 and 4 were recorded. The structures of isolated compounds were determined by comparison with published data (Additional file 1: Text, Figs. S6, S7, and Tables S4, S5). The titres of compounds 3 and 4 were evaluated based on the HPLC peak area relative to pure compounds separated.
Exploration of the conversion of tetrahydroanthracene to anthraquinone compounds by alkali treatment
Compounds 3 and 4 (1 mg/mL) of dissolved in acetonitrile were divided into five 1.5 mL centrifuge tubes, each 100 µL, and dried with a termovap sample concentrator. Then, Tube 1: add 200 µL ddH2O; Tube 2: add 200 µL 0.1% NaOH; Tube 3, add 200 µL 1% NaOH; Tube 4, add 200 µL 5% NaOH; Tube 5, add 200 µL 5% NaOH. The centrifuge tubes were placed at room temperature for reaction for 3 h, and the pH value of tubes 2–4 was adjusted to 4–5 with 1% HCl. Subsequently, all 5 tubes of samples were fixed to 500 µL with ddH2O, extracted with 600 µL ethyl acetate and centrifuged at 12,000 rpm for 5 min. The supernatant was dried with a termovap sample concentrator and then dissolved in 500 µL acetonitrile for HPLC or HPLC–HRMS analysis.
Producing anthraquinone compounds by alkali treatment of the fermentation broth engineered A. oryzae NSAR1 strains
One hundred millilitres of the fermentation broth of AotoaA/toaB-toaC incubated in CD medium with 2% soybean meal at 30 °C, and 150 rpm for 2 days, then 5 g solid NaOH (final concentration: 50 g/L) was directly added to the broth, shaken and mixed, and left at room temperature for 24 h. Most of the mycelia were lysed, and the fermentation broth became dark reddish brown. The remaining mycelia were removed by filtration with filter paper, diluted with 1.5 times the volume of ddH2O, and then extracted twice with an equal volume of ethyl acetate. The extract was vacuumed, and the crude physcion was obtained. The titres of physcion were evaluated based on the HPLC peak area relative to their authentic standards. The HPLC purity of physion was calculated from the peak area of 390 nm wavelength. Three independent replicates were performed.
The experimental data were analyzed by GraphPad Prism 5, and the significant differences were analyzed using two-way ANOVA.