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Overexpression of Global Regulator LaeA Induced Secondary Metabolite Production in Aspergillus versicolor 0312

This experiment investigated the effect of the global regulator laeA on the secondary metabolites of Aspergillus versicolor. The laeA gene was cloned from Aspergillus versicolor 0312 to introduce pSilent I overexpression vector, and the recombinant plasmid was introduced into Aspergillus versicolor 0312 by PEG guided protoplast transformation. Then, the over-expressing strain 0312-laeA was screened and verified by metabolic level and gene level. A new compound versicolor A (1) was obtained by liquid fermentation of the over-expressing strain, together with four known compounds, acetylapoaranotin (2), acetylaranotin (3), diisobutyl phthalate (4) and ergosterol (5). The structure of the new compound was elucidated by using various spectroscopic techniques inclusive of HRESIMS, 1Dand 2D-NMR data. Compound 1 showed cytotoxicity against MOLT-4 cell lines with IC50 values of 29.6 μM, while 2 showed significant cytotoxicities against MOLT-4, MCF-7, and CaCo-2 cell lines with IC50 values of 7.8, 19.9, and 15.9 μM, respectively. The results further supported that laeA is a global regulator that could up-regulate and/or activate cryptic gene clusters to produce new secondary metabolites. This study provides an important basis for exploring the mechanism of laeA gene on the secondary metabolites of Aspergillus versicolor.


Introduction
Natural products, produced by bacteria, fungi and plants, provide important resources for the development of new drugs [1][2][3][4]. Filamentous fungi can secrete secondary metabolites with biological activity. Some important clinical drug molecules are derived from filamentous fungi, such as penicillin (antibiotic) and cyclosporine (immunosuppressant) [5]. However, in the past two decades, it has been more and more difficult to find novel active substances from filamentous fungi, especially the compounds with a new backbone, by traditional separation methods. Recently, genome sequencing technology and bioinformatics research, researchers [6][7] have drawn more and more attention in the discovery of bioactive natural molecules, but also in the development of synthetic drug. However, many biosynthetic gene clusters are not expressed in the host organism, or not activated in the laboratory cultural conditions. Therefore, activating the silent gene clusters provide a promising strategy to yield novel or therapeutic metabolites [8][9][10]. LaeA gene is a global regulator in filamentous fungi, and could regulate expression of a large number of secondary metabolite gene clusters. Previous studies reveals that overexpression of the laeA regulator in Aspergillus nidulans increases the production of terrequinone A [11]; deletion of laeA in Aspergillus flavus inhibits aflatoxin gene transcription [12].
Aspergillus versicolor 0312, a filamentous fungus, was isolated from the stems of Paris polyphylla var. yunnanensis. The whole genome sequencing analysis of the target strain showed that the strain contained a large number of natural product biosynthetic gene clusters. However, in our preliminary cultural experiment on potato glucose medium, chase medium, and DPY medium, limited number of secondary metabolites were produced in A. versicolor 0312, as shown by UPLC analysis (Figure 1), implying that many of a large number of natural product biosynthetic genes or gene clusters in this fungus were not, or were only minimally, expressed. A heteroditerpenoid biosynthetic gene cluster in A. versicolor 0312 gene clusters was successfully introduced into Aspergillus oryzae NSAR1 and a series of novel heteroditerpenoids were discovered [13]. Therefore, overexpression of the global regulator laeA in A. versicolor 0312 was performed in order to activate or up-regulate the silent gene clusters and produce novel compounds. In this study, overexpression of the global regulator laeA in A. versicolor 0312 led to isolation of a new acetaminobutyric acid substance versicolor A (1) and four known compounds (2)(3)(4)(5). The known compounds were identified as acetylapoaranotin (2) [14], acetylaranotin (3) [14], diisobutyl phthalate (4) [15], and ergosterol (5) [16] by comparison with the reported data ( Figure 2). All the isolated compounds were tested for their cytotoxic activity by CCK-8 assay in vitro. Herein, we report the isolation, structure elucidation, as well as cytotoxic activities of these compounds.

General Experimental Procedures
The 1D and 2D NMR spectra were recorded on a Bruker DRX-600 MHz spectrometer with TMS as an internal standard. The HRESIMS data were recorded on an API QSTAR time-of-flight spectrometer. Optical rotations were measured with a JASCO P-1020 polarimeter. The IR spectra were obtained by a Bruker FT-IR Tensor 27 with KBr pellets. UV spectra were obtained with a Shimadzu UV-2401A spectrophotometer. Column chromatography was performed with silica gel (200-300 mesh, Qingdao marine), Spherical C18 Monomeric, 120A (50 μm, SILICYCLE), and Sephadex LH-20 gel (40-70 μm, GE Healthcare). Semipreparative HPLC was performed on a liquid chromatograph with a 250 × 10 mm column (Reprosil-Pur Basic C18). Fractions were monitored by thin layer chromatography, spots were visualized by UV light (254 nm and 365 nm) and by heating silica gel plates sprayed with 10% H2SO4 in EtOH. All solvents used in column chromatography were distilled.

Strains and Media
Aspergillus versicolor 0312 was isolated from the stems of Paris polyphylla var. yunnanensis collected in Kunming, Yunnan Province, People's Republic of China in 2017 and identified by ITS analysis (GenBank Accession No. MK311351). The strain cells were cultivated at 30 ℃, 200 rpm in DPY medium (2% dextrin, 1% hipolypepton (Nihon Pharmaceutical Co.,Ltd.), 0.5% yeast extract (OXOID), 0.5% KH2PO4, and 0.05% MgSO4•7H2O) for three days, and used as a source for genomic DNA extracting [Quick Plant Genomic DNA Kit (TIANGEN)] and cloning of laeA gene. Standard DNA engineering experiments were carried out using Escherichia coli DH5α (Tsingke, Beijing, China). E. coli cells harboring each plasmid were cultivated in Luria-Bertani (LB) medium and were selected with ampicillin.

Cloning of the LaeA Gene and Construction of the LaeA Overexpression Vector
pSilent-I [17] is a fungal expression vector containing the trpC promoter and trpC terminator. LaeA gene was amplified from A. versicolor 0312 genomic DNA with the primers 0312-laeA-F (aggtacgtacaagctATGTTTGGAAAGGGTCCGG) and 0312-laeA-R (taccacaggccttagTTATCTCGCG-ACGGTTTTCGCG) using the high-fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech Co., Ltd). The full-length laeA genes was purified, and introduced into the HindIII and SphI double digested pSilent-I vector to afford pSilent-I-laeA overexpression vector using an In-Fusion® HD Cloning Kit (Clontech Laboratories, Inc.) according to the manufacturer's protocol.

Transformation of Aspergillus versicolor 0312
Transformation of A. versicolor 0312 protoplasts was done using polyethylene glycol. The pSilent-I-laeA plasmid was used for the transformation to construct the laeA overexpressed A. versicolor 0312 strain. To construct the other strains without laeA gene, the corresponding empty vector of pSilentI harboring a single gene were used. The preparation of A. versicolor 0312 protoplasts and the PEG-mediated transformations were performed as previously described [18][19]. The A. versicolor 0312 cells transformed with the pSilent-I empty vector or pSilent-I-laeA plasmid for the overexpression of laeA were selected on plates containing hygromycin (ca. 200 μg/mL), prepared as described previously [20]; 20 μg of DNA were thus used for the transformation, and 20-30 transformants were obtained. Of these transformants were chemically examined to afford the versicolor A (1) production strain.

Fermentation, Isolation, and Purification
The over-expressing strain was used to inoculate three Erlenmeyer flasks (500 mL), each containing 200 mL of media (potato glucose medium). Three flasks of the inoculated media were incubated at 28 ℃ on a rotary shaker at 200 rpm for 2 days to prepare the seed culture. Fermentation was carried out in a fermentation tank (10 L), incubated at 30 ℃, 200 rpm for 3 day. The fermented whole broth was filtered through multilayer gauze to remove the mycelia, and the broth was then extracted 4 times with EtOAc. The mycelia were extracted 3 times with acetone under ultrasonicassisted extraction method. The solvent was evaporated under reduced pressure to afford a crude residue. The crude residue was extracted 4 times with EtOAc. Finally, the filtrates were combined and evaporated in vacuum to yield a crude extract (6 g).
The human cancer cell lines was used in the cytotoxic activity assay and the cell were cultured in DMEM or RPMI-1640 medium (Hyclone, USA) supplemented with 10% fetal bovine serum (Hyclone, USA) and 1% antibiotic mixture of penicillin (100 U/mL) and streptomycin (100 mg/mL) in a humidified environment with 5% CO2 at 37 ℃. Cells viability was assessed using the CCK-8 method in 96-well microplates. Cells in logarithmic growth phase were selected, were seeded just before sample addition with an initial density of 1×10 6 cells/mL in 100 μL medium. Each tumour cell line was exposed to each test compound at various concentrations in triplicate for 48 h, with cisplatin as a positive control. In this study we set the blank group, negative control group and 5 samples (30, 20, 10, 5, 2.5 μg/mL) to be tested at different concentrations, each concentration has three parallel. The blank group consisted of 110 µL of medium. The negative control group consisted of 100 µL of cells suspension and 10 µL of medium. The sample group consisted of 100 µL of cells suspension and 10 µL of different compounds. After incubation for 44 h under growth conditions, then 10 µL of the CCK-8 solution was added to each well, and the incubation of cells was continued for 4 h. Absorbance was then determined on a Spectra Max Plus plate reader at 450 nm. The IC50 value of each sample was calculated following Reed and Muench's method [22].

Structure Elucidation
Compound 1 was isolated as a white amorphous powder. The molecular formula of 1 was established as C22H22N2O5S2 on the basis of sodium adduction at m/z 481.0864 [M + Na] + (calcd for 481.0868) in the positive HRESIMS spectrum, in association with 1 H and 13 C NMR spectra, indicating thirteen indices of hydrogen deficiency. The IR spectrum showed characteristic absorption peak signals at 1738 cm -1 and 1670 cm -1 for carbonyl and amide carbonyl functional groups. Its 13 C NMR and DEPT spectra exhibited 22 carbon signals assignable for three methyls, two methylenes, nine methines including two peroxymethine groups (δC 137.4 and 139.6), five olefinic (δC 105.2, 117.3, 124.7, 125.5 and 127.2), one dioxymethine (δC 72.0), and one diazomethine (δC 60.3), and eight quaternary carbons (two amide carbonyls at δC 163.1 and 164.9, one ester carbonyl at δC 170.3). The full assignment of the 1 H and 13 C NMR spectra was confirmed by the analysis of HSQC and HMBC spectroscopic data. The 1 H and 13 C NMR data (Table 1) [23], indicating that the configurations of C-8 and C-9 are consistent with acetylapoaranotin, of which the absolute configuration was confirmed by a crystal analysis [25]. Thus the configurations of the C-8 and C-9 positions are 8S and 9S, respectively ( Figure 3). Thus, the structure of compound 1 was established and named as versicolor A.