Fungal Genetics and Biology xxx (2015) xxx–xxx

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Structure and biosynthesis of fumosorinone, a new protein tyrosine phosphatase 1B inhibitor firstly isolated from the entomogenous fungus Isaria fumosorosea Linxia Liu a,1, Jun Zhang a,1, Chuan Chen a, Jitao Teng a, Chengshu Wang b,⇑, Duqiang Luo a,⇑ a

Hebei University, College of Life Science, Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, Baoding 071002, China Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences, Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai 200032, China b

a r t i c l e

i n f o

Article history: Received 23 September 2014 Revised 26 March 2015 Accepted 30 March 2015 Available online xxxx Keywords: Natural product Isaria fumosorosea Fumosorinone Protein tyrosine phosphatase 1B inhibitor PKS–NPRS Biosynthesis

a b s t r a c t Fumosorinone, isolated from the entomogenous fungus Isaria fumosorosea, is a new 2-pyridone alkaloid which is elucidated by HRESIMS 1D and 2DNMR. Fumosorinone is structurally similar to tenellin and desmethylbassianin but it differs in chain length and degree of methylation. It is characterized by a classic noncompetitive inhibitor of protein tyrosine phosphatase 1B (IC50 14.04 lM) which was implicated as a negative regulator of insulin receptor signaling and a potential drug target for the treatment of type II diabetes and other associated metabolic syndromes. For further study, we identified the biosynthetic gene cluster of fumosorinone from ongoing genome sequencing project, and it was verified by a direct knock-out strategy, reported for the first time in I. fumosorosea, using the Agrobacterium-mediated transformation in conjunction with linear deletion cassettes. The biosynthetic gene cluster includes a hybrid polyketide synthase–nonribosomal peptide synthetase gene, two cytochrome P450 enzyme genes, a trans-enoyl reductase gene, and other two transcription regulatory genes. Comparison of fumosorinone biosynthetic cluster with known gene clusters gives further insight into biosynthesis of pyridone alkaloids and provides the foundation for combinatorial biosynthesis for new fumosorinone derivatives. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction Entomogenous fungi are an ecologically highly specialized group of microorganism, which produce a plenty of insecticidal and pharmaceutical active compounds (Isaka et al., 2005; Molnar et al., 2010; Namatame et al., 1999; Takahashi et al., 1998). Especially, the insect fungal secondary metabolite Myriocin (ISPI) derived fingolimod (Gilenya; FTY720) was approved (September 2010) by the U.S. FDA as a new treatment for multiple sclerosis (Strader et al., 2011). Therefore, interest in searching for bioactive compounds from insect pathogenic fungi is highly increased in the world. From insect pathogenic fungi, a lot of bioactive compounds have been found including polyketides and nonribosomal peptides, such as tenellin and bassianin (Jeffs and Khachatourians, 1997) from Beauveria bassiana which inhibited mammalian erythrocytes membrane ATPase activity, millitarinones (Schmidt et al., 2003)

⇑ Corresponding authors. 1

E-mail addresses: [email protected] (C. Wang), [email protected] (D. Luo). co-first author.

from Paecilomyces militaris which showed neuritogenic activity, aspyridone A (Bergmann et al., 2007) from Aspergillus nidulans which is a cytotoxin. So how the secondary metabolites are produced by insect pathogenic fungi is studied by more and more people. At present, several biosynthetic gene clusters of secondary metabolites such as Beauvericin (Xu et al., 2008), tenellin (Eley et al., 2007), desmethylbassianin (Heneghan et al., 2011) and NG391 (Donzelli et al., 2010) were functionally verified. The core genes, involved in the biosynthesis of secondary metabolites in insect pathogens, are dimethylallyl tryptophan synthase (DMAT), terpene cyclase (TC), terpene synthase (TS), fatty-acid synthase (FAS), geranylgeranyl diphosphate synthase (GGPS), non-ribosomal peptide synthetase (NRPS), polyketide synthase (PKS) and hybrid PKS–NRPS enzyme (Brakhage and Schroeckh, 2011; Xiao et al., 2012). Of all the core genes involved in the biosynthesis of secondary metabolites in insect pathogenic fungi, PKS, NRPS, and hybrid PKS–NRPS are paid more attention for their catalytic action of outstanding diversity of secondary metabolites. The highly reducing PKSs are megasynthases that function iteratively by using a set of individual catalytic domains repeatedly and programmedly to produce structurally diverse fungal

http://dx.doi.org/10.1016/j.fgb.2015.03.009 1087-1845/Ó 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: Liu, L., et al. Structure and biosynthesis of fumosorinone, a new protein tyrosine phosphatase 1B inhibitor firstly isolated from the entomogenous fungus Isaria fumosorosea. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.03.009

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L. Liu et al. / Fungal Genetics and Biology xxx (2015) xxx–xxx

metabolites (Ma et al., 2009; Xiao et al., 2012). Despite the complexity of PKSs, even more impressive biosynthetic machinerythe PKS–NRPS hybrid is found in nearly all filamentous fungi (Donzelli et al., 2010). A typical PKS–NRPS hybrid consists of an iterative PKS module followed by a single NRPS module. The hybrid prudently combines the versatile nature of acetate-derived polyketide chains with the more than 300 proteinogenic and nonproteinogenic amino acids that could be incorporated by NRPS modules (Boettger et al., 2012; Fisch, 2013). Among this class of natural products, an array of pyridones is well known to be produced by various fungi and more importantly even by entomogenous fungi (Haga et al., 2013; Hosoya et al., 2013; Ma et al., 2011; Peng et al., 2011). Several 2-pyridones with similar chemical structure are displayed in Scheme 1. The entomopathogenic hyphomycete, Isaria fumosorosea which is geographically widespread species (Tigano-milani et al., 1995), is commercially available for the biocontrol of important agricultural pests, such as whitefly (Fargues and Bon, 2004). In our search of new bioactive compounds from insect pathogenic fungus I. fumosorosea, we have isolated a new pyridone alkaloid compound, named fumosorinone (Scheme 1). Its structure is elucidated by HRESIMS (High Resolution Electrospray Ionization Mass Spectrometry), IR (Infrared Radiation), UV (Ultraviolet), 1D, and 2DNMR (1 and 2 dimension(s) Nuclear Magnetic Resonance). Further, we found it is an effective inhibitor of protein tyrosine phosphatase 1B (PTP1B) with IC50 (Concentration Causing 50% Inhibition) (Jarrott, 1970) value of 14.04 lM. Protein tyrosine phosphatases (PTPs) are a diverse family of enzymes encoded by 107 genes in the human genome. Specific PTPs including PTP1B, SHP2 and MEG2 have potential as drug targets for several diseases. It is evidenced that PTP1B is a potential drug target for the treatment of type II diabetes and other associated metabolic syndromes (Barr, 2010). Hence, fumosorinone is a new potential therapeutic agent for the treatment of type II diabetes. For better understanding how the fumosorinone is produced, herein, we studied the biosynthetic mechanism. Given its

structural similarity to tenellin and desmethylbassianin, fumosorinone is expected to be produced by a hybrid PKS–NRPS using a similar biosynthetic mechanism. To facilitate comprehensive understanding of gene clusters involved in the biosynthesis of fumosorinone, we proposed that a similar PKS–NRPS gene would be involved in fumosorinone biosynthesis and that we could test if the gene is responsible for the fumosorinone biosynthesis by Agrobacterium tumefaciens mediated gene knock out. 2. Materials and methods 2.1. General experimental procedures Column chromatography (CC): silica gel (SiO2; 200–300 mesh; Yantai Zhi Fu chemical Co., PR China), Thin layer chromatography (TLC): silica gel GF254 plates (Yantai Zhi Fu chemical Co., Ltd., PR China), and Sephadex LH-20 gel (25–100 lm, GE Healthcare Co., Ltd., Sweden). Optical rotations: Perkin–Elmer 341 spectropolarimeter. UV Spectra: Shimadzu UV-210 spectrometer, kmax (log e) in nm. IR Spectra: Perkin–Elmer 577 spectrometer; KBr pellets; in cm1. NMR Spectra: Bruker AM-600 spectrometer; d in ppm, J in Hz; Me4Si as internal standard. FT-MS Spectra: Bruker apex-ultra 7.0 T spectrometer in m/z. LC–MS:Acquity UPLC Xevo TQ-s (Waters, USA). 2.2. Fungal material and cultivation conditions I. fumosorosea was cultured on slants of potato dextrose agar (PDA, 20.0 g of glucose, 200.0 g of potato (peeled), 3.0 g of KH2PO4, 1.5 g of MgSO4, 0.1 g of citric acid, and 10.0 mg of thiamin hydrochloride, in 1 L of deionized H2O) at 28 °C for 7 days, and then inoculated into 500 mL Erlenmeyer flask containing 100 mL of PDA medium. The final pH of the media was adjusted to 6.5 before sterilization. After 7 days of incubation at 28 °C on rotary shakers at 150 rpm, about 25 mL of culture liquid were transferred into

Scheme 1. 2-Pyridones of natural products, similar in chemical structure.

Please cite this article in press as: Liu, L., et al. Structure and biosynthesis of fumosorinone, a new protein tyrosine phosphatase 1B inhibitor firstly isolated from the entomogenous fungus Isaria fumosorosea. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.03.009

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100 Erlenmeyer flasks, each containing 200.0 g Rice medium (88 g of rice, 110 mL distilled water), and the fermentation was carried out at 28 °C under light for 30 days. Escherichia coli DH5a and BL21 were grown on solid and in liquid LB (1% tryptone, 0.5% yeast extract, 0.5% NaCl, pH 7.0) media. I. fumosorosea cultures were grown on the Sabouraud’s dextrose agar medium supplemented with yeast extract (4% dextrose, 1% peptone, 1% yeast extract, 2% agar, pH6.5, SDAY) (Sun-Hee et al., 2013) at 28 °C for 5 days for DNA isolation and 3–4 days for transformation. A. tumefaciens LBA4404 was first grown on YM medium (0.04% (w/v) yeast extract, 1% mannitol, 0.01% NaCl, 0.02% MgSO47H20, and 0.05% K2HPO43H2O, pH 7.0, 1.5% agar for YM solid medium) and used for transformation of I. fumosorosea. During transformation, A. tumefaciens LBA4404 was cultivated in induction medium (IM) (10 mM K2HPO4, 10 mM KH2PO4, 2.5 mM NaCl, 2 mM MgSO4, 0.7 mM CaCl2, 9 lM FeSO4, 4 mM NH4SO4, 10 mM glucose, 40 mM 2-[N-morpholino]ethanesulfonic acid, pH 5.5, 0.5% glycerol (w/v), with or without 200 lM acetosyringone). The co-cultivation medium was same as IM, except containing 5 mM instead of 10 mM glucose and 1.5% agar.

2.3. Basic DNA manipulations Total DNA isolation from I. fumosorosea was performed as described previously (Nicholson et al., 2001). PCR primers used in this work are listed in the Supplementary Information. Oligonucleotide-specified sections of DNA were routinely amplified by PCR using Cobuddy Super Fidelity DNA Polymerase (CWBIO). Plasmid DNA preparation, restriction endonuclease digestion, and other DNA manipulations were performed according to standard procedures (Sambrook et al., 1989).

2.4. Extraction and isolation The culture broth (20 L) was extracted three times with 15L ethyl acetate (AcOEt) each time. Evaporation of the solvent in vacuo gave a yellow oily residue (200 g), which was subjected to CC (SiO2; petroleum ether (PE)/AcOEt 100:0, 95:5, 90:10, 80:20, 60:40, 50:50 (v/v)) to afford six fractions, Frs. 1–6. Fr.4 (35 g) eluted with PE/AcOEt 80:20 (v/v) was further purified by repeated CC (PE/acetone 5:1 (v/v)) to afford fumosorinone (40 mg). Fumosorinone: Isolated as yellow powder. [a]19 D = 175°, (c = 0.003, MeOH); UV (MeOH) kmax (log e) nm: 253 (4.25), 374 (4.66); IR (KBr) vmax cm1: 3267, 2960, 1643, 1516, 1446, 1268, 1218, 837, 758; Positive ion HR–ESI–MS [M + Na]+ m/z 500.2410 (calcd. for C29H35NNaO5 500.2407); 1H-NMR (600 MHz, CD3OD) dH 7.87 (1H, s, and H-6), 6.94 (1H, d, J = 9.8, and H-9), 6.68 (1H, dd, J = 14.6, 10.3, and H-10), 6.65 (1H, dd, J = 14.6, 9.5, and H-11), 6.41 (1H, dd, J = 15.3, 9.4, and H-12), 6.46 (1H, d, J = 15.3, and H13), 5.39 (1H, d, J = 9.8, and H-15), 2.68 (1H, m, and H-16), 1.16 (1H, overlapped, and Ha-17), 1.34 (1H, overlapped, and Hb-17), 1.34 (1H, m, and H-18), 1.16 (1H, overlapped, and Ha-19), 1.34 (1H, overlapped, and Hb-19), 0.91 (3H, t, J = 7.3, and H3-20), 0.88 (3H, d, J = 6.8, and H3-21), 0.98 (3H, d, J = 6.5, and H3-22), 1.84 (3H, s, and H3-23), 2.06 (3H, s, and H3-24), 7.32 (1H, d, J = 8.6, and H-20 ), 6.86 (1H, d, J = 8.6, and H-30 ), 6.86 (1H, d, J = 8.6, and H-50 ), 7.32 (1H, d, J = 8.6, and H-60 ); 13C-NMR (150 MHz, CD3OD) dc 157.6 (s, C-2), 109.6 (s, C-3), 165.1 (s, C-4), 115.0 (s, C-5), 136.5 (d, C-6), 198.4 (s, C-7), 135.6 (s, C-8), 140.4 (d, C-9), 126.8 (d, C-10), 141.6 (d, C-11), 126.2 (d, C-12), 142.0 (d, C-13), 132.6 (s, C-14), 142.7 (d, C-15), 30.4 (d, C-16), 44.6 (t, C-17), 32.3 (d, C18), 29.8 (t, C-19), 10.3 (q, C-20), 18.1 (q, C-21), 20.4 (q, C-22), 11.3 (q, C-23), 11.0 (q, C-24), 123.5 (s, C-10 ), 130.2 (d, C-20 ), 115.0 (d, C-30 ), 157.1 (s, C-40 ), 113.5 (d, C-50 ), 130.1 (d, C-60 ).

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2.5. Recombinant protein phosphatase 1B (PTP1B) Plasmids for expression of his-tag fusion proteins of human PTP were constructed in PET-28a by PCR subcloning techniques. Only the PTP domain of PTP1B (aa 1–280) was included. HIS-PTP fusion proteins were expressed in E. coli BL21 and affinity purified with Ni–NTA Magnetic Agarose Beads (Qiagen). The his-tag recombinant purification protocol was carried out according to the purification under native conditions in The QIAexpressÒ System. The purified proteins were dialyzed with dialysis buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 1 mM dithiothreitol plus 5% glycerol). 2.6. PTP1B assay Para-nitrophenyl phosphate (pNPP) was used in enzymatic reactions to determine the intrinsic catalytic activities of PTP1B (Pan et al., 2013). All assays using pNPP as the substrate were performed in a reaction buffer containing 10 mM of NaAc (sodium acetate), 1 mM of ethylene diamine tetraacetic acid, 1 mM of dithiothreitoll at 37 °C, pH 5.5. Every experiment was performed in triplicate. Enzyme activity was assayed by incubating 0.61 lM PTP1B recombinant protein at 37 °C, for 15 min. Absorbance at 405 nm was detected immediately after addition of varied concentrations of pNPP (0, 0.78, 1.56, 3.12, 6.25, 12.5, 25 and 50 mM). By the double-reciprocal plot of Michaelis–Menten equation, we concluded the vmax and km values (Karthik et al., 2014; Gacche et al., 2011). The inhibitory activity measurement of PTP1B (Doman et al., 2002) was performed with different concentrations (0, 2.5, 5, 10, 20, 40 lg mL1) of fumosorinone and 0.61 lM PTP1B. The reaction mixture was incubated at 37 °C for 15 min and the catalytic activity was detected by monitoring the absorbance at 405 nm immediately after adding pNPP (12.5 mM, based on Km value). IC50 (the concentration of an inhibitor that is required for 50% inhibition of an enzyme) data were derived from the experiment. The mode of inhibition of fumosorinone against PTP1B was measured with increasing concentrations of pNPP (0, 0.78, 1.56, 3.12, 6.25, 12.5, 25 and 50 mM) as a substrate in the absence and presence of fumosorinone at 10 lg mL1 and 20 lg mL1. Optimal amounts of fumosorinone were determined based on the enzyme inhibitory activity assay. Mode of inhibition of fumosorinone was determined by Lineweaver–Burk plot analysis of the data calculated following Michaelis–Menten kinetics. 2.7. Construction of the PKS–NRPS knock-out plasmid The benR gene from the plasmid pBT6 (McCluskey, 2003) obtained from the FGSC and the A. nidulans trpC promoter from the vector pATMT1 saved in our lab were amplified by PCR, respectively. The two fragments were ligated to produce a 2.6 kb trpCbenR fragment by recombinant PCR technology. The gene replacement cassette was assembled in ppzp111 by placing one of two nearly contiguous fragments from the PKS–NRPS coding region on each side of the trpC-benR expression cassette by Clontech’s In-Fusion HD Cloning Kit. We therefore amplified the 50 flanking region named ‘‘up’’ with primers F1-FW and F1-RV and 30 flanking region called ‘‘down’’ with primers F3-FW and F3-RV separately from I. fumosorosea genomic DNA to remove middle fragment approximately corresponding to the phosphopantetheine attachment site (see the Supplementary Information). To introduce compatible ends for cloning, 15 bp long overlap at their ends are included in the primers used for amplification of the fragments to be cloned followed the kit’s instructions. The vector ppzp111 was digested with BamHI and EcoRI to a linearized vector. The up, down and the trpC-benR fragments were ligated into the

Please cite this article in press as: Liu, L., et al. Structure and biosynthesis of fumosorinone, a new protein tyrosine phosphatase 1B inhibitor firstly isolated from the entomogenous fungus Isaria fumosorosea. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.03.009

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linearized vector to be a novel binary vector ppzp111-uttd (see the Supplementary Information) using infusion clone technology (Frandsen et al., 2012). This resulting vector was saved in E. coli HST08 that provided in the kit. 2.8. Electrotransformation of A. tumefaciens A. tumefaciens LBA4404 was grown at 28 °C for 2 days in YM medium plate supplemented with streptomycin sulfate (50 lg mL1) (Ooms et al., 1982). Then we used a well-separated colony to inoculate 50 mL of YM broth in a 500 mL flask, with vigorous shaking at 28 °C until OD550 = 0.8–1.2. The cells were harvested by centrifugation (4000 rpm, 10 min, 4 °C), washed twice in ice-cold 10% glycerol and resuspended the cell pellet in the same 10% glycerol to a final volume of 1.5 mL. Then, we kept the cells on ice before use. We added 30 lL of the electrocompetent cells and 2 lL (100 ng) of DNA in TE (10 mM Tris–HCl and 1 mM EDTA, pH 8.0) buffer to an ice-cold 1.5 mL tube. The suspension was gently mixed by tapping the tube several times and incubated on ice for 10 min. The samples were transferred to a microelectroporation chamber that had been cooled to 4 °C. Then we followed the instructions of the electroporation apparatus (Gene Pulser Xcell, BioRad, USA) and finally transfered the cells to the YM broth in a 15 mL Falcon tube. The remaining cells were resuspended in the microchamber with 200 lL of YM broth using a sterilized Pasteur pipet to remove as many cells as possible and incubated the cells at 28 °C for 3 h with shaking at 250 rpm. The cells were plated onto a 25 lg mL1 chloramphenicol selective YM plate. Antibiotic-resistant colonies were observed on the plate after incubation for 2– 3 days at 28 °C. 2.9. Agrobacterium-mediated transformation of I. fumosorosea The transformation procedure was based on a previously described protocol (Mullins et al., 2001; Michielse et al., 2008) with some modifications. A. tumefaciens strain LBA4404 harboring ppzp111-uttd vector was grown overnight in 5 mL of YM liquid medium supplemented with 25 lg mL1 chloramphenicol at 28 °C with shaking (240 rpm). The culture was then centrifuged at 4000 rpm for 10 min and the pelleted cells were resuspended to an optimal density of OD600 of 0.2–0.3 with induction medium (IM) with 200 lM acetosyringone (AS) (IM + AS) or without AS (IMAS). Then A. tumefaciens was incubated for 4–5 h at 28 °C with shaking at 200 rpm to reach an OD600 of 0.6–0.8 in (IM + AS) or (IM  AS) medium. I. fumosorosea was grown on SDAY medium (Sun-Hee et al., 2013) for 3 days and incubated at 28 °C. The conidia were collected in a 5 mL solution of sterile 0.9% (w/v) NaCl and passed through Miracloth. The spores were centrifuged for 10 min at 3000 rpm at 4 °C and discarded the supernatant. The conidia were determined using a hemocytometer and diluted to a final concentration of 1  106 conidia mL1 in IM. I. fumosorosea and these A. tumefaciens transformant cells were mixed together in 1:1, 1:2, 1:3, and 2:1 and 3:1 (v/v). Each mixture was placed at 200 lL onto membranes of cellulose (90 mm diameter) and plated on IM agar medium with or without 200 lM AS. Following cocultivation at 23 °C for 48– 72 h, the membranes were transferred to SDAY selection media supplemented with 100 lg mL1 ampicillin, 50 lg mL1 kanamycin, and 5 lg mL1 benomyl. The plates were incubated for 5 days at 28 °C until colonies appear. Control I. fumosorosea cells were treated the same way, except that they were cocultivated with strains of A. tumefaciens that had not been transformed with the constructed vector. The selectable marker insertion site was identified by PCR method (see the Supplementary Information).

2.10. Analysis of metabolite production For analysis, 4 mutants together with I. fumosorosea (wild type) were grown in SDY liquid medium (200 mL) in a 500 mL flask at 28 °C for 10 days. The mycelia were extracted with ethyl acetate (100 mL) three times. The extracts were analyzed by LC–MS by using a Waters Acquity UPLC coupled to a Xevo TQ-S mass spectrometer and equipped with an Agilent ZORBAX-XDB-C18 5 l 4.6  150 mm column. Solvent A: 0.1% methanoic acid in HPLCgrade water; solvent B: 90% HPLC-grade MeOH. 2.11. Insect pathogenesis assay Fungal spores were collected in 0.05% Tween 80 from 5 to 7 days well-sporulating SDAY plates. The spore concentrations were adjusted to 107 mL. Galleria mellonella larvae (Eley et al., 2007; Fargues and Bon, 2004) were obtained from Agriculture University of Hebei, China. Cohorts of fifth-instar (weight 0.21– 0.23 g) were dipped either into the spore suspension, or into 0.05% Tween 80 solution as a control, for 1 min. Excess spore solution was removed by placement on a dry paper towel, and the insects were put into petri dishes containing artificial diet (50 g wheat bran, 50 g wheat flour, 50 g maize flour, 25 g milk powder, 10 g yeast, 30 g glycerol, 25 g honey and 25 g wax). The cohorts were held in incubator to keep the humidity high enough for fungal spore germination. The incubation was performed at 28 °C and larval mortality was recorded daily for 9 days. All fungus-inoculated insects died over a 7-day period.

3. Results 3.1. Purification and identification of fumosorinone from I. fumosorosea Fumosorinone is a new found pyridine alkaloid which is extracted from I. fumosorosea. Fumosorinone was obtained as a yellow, amorphous solid with a molecular formula of C29H35NO5 (13 degrees of unsaturation), determined by HRESIMS (478.2588 [M + H]+ and 500.2410 [M + Na]+). Its 1H and 13C NMR spectra in CD3OD showed resonances for 32 nonexchangeable protons (H five methyl groups (CH3), two methylenes (CH2), thirteen methines (CH), and nine quaternary carbons (C) including one carboxylic carbon (dC 157.6), and one a, b unsaturated ketone carbon (dC 198.4). Three exchangeable protons were not seen in the 1H NMR spectrum. These data accounted for all the NMR resonances, suggesting a bicyclic compound. Analysis of the 1H and 13C NMR spectroscopic data revealed structural features similar to those of militarinone D (Schmidt et al., 2003). The difference between militarinone D and fumosorinone were: (a) the side chain of fumosorinone has one more propene group than militarinone D; (b) amide proton in militarinone D is replaced by a hydroxy group in fumosorinone. A proton spin system of an olefin chain, consisting of one doublet of H-9 (dH 6.94) and four doublets of doublets (H-10 to H-13), was readily detected by 1H–1H COSY signals. The methyl (dC 11.0, dH 2.06 (s)) was unambiguously assigned to H-24 by HMBC correlations with H3-24 to the C-7, C-8 and C-9. HMBC correlations from H3-24, H9 to C-7 indicated conjugation of the olefin with the bridge-carbony. Extension of the olefin chain with another double bond was deduced from HMBC correlations of H-13 (dH 6.46, d, J = 15.3), H-15 (dH 5.39, d, J = 9.8) and methyl signal of H3-23 (dH 1.84, s) with the quaterernary olefinic C-14 (dC 132.6) (see the Supplementary Information). Therefore, the planar structure of 1 was determined as shown in Scheme 1.

Please cite this article in press as: Liu, L., et al. Structure and biosynthesis of fumosorinone, a new protein tyrosine phosphatase 1B inhibitor firstly isolated from the entomogenous fungus Isaria fumosorosea. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.03.009

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The relative configuration of the olefin moiety in fumosorinone was determined by analysis of the 1H–1H coupling constants and NOESY data. The C-10/C-11 and C-12/C-13 double bonds were all assigned E-geometry based on a large coupling constant of 15.0 Hz observed for corresponding olefinic protons, and the same assignment was made for the C-14/C-15 olefin by NOESY correlations of H-12 with H3-23 and H-13 with H-15 (see the Supplementary Information). The relative configurations of C-16 and C-18 were deduced by comparison of the 13C NMR shift values of the C-21 and C-22 methyl groups with those of the model compounds, and a difference of 2.3 ppm revealed their syn-configuration. Thus, compound 1 was elucidated as shown in Scheme 1, named fumosorinone. 3.2. Identification of fumosorinone as a noncompetitive PTP1B Inhibitor We have isolated and identified 56 compounds including the new pyridine alkaloid fumosorinone from the extraction of entomogenous fungus I. fumosorosea. After high throughout activity screening, we found fumosorinone with the most potent inhibition activity of PTP1B in the tested compounds. To elucidate the inhibition style of fumosorinone, steady-state kinetic characterization was conducted. In the study, the initial velocities of PTP1B were measured at two selected concentrations of the inhibitor over a range of varied concentrations of the only substrate, pNPP. The data were plotted with velocities versus pNPP concentration and fitted to Michaelis– Menten equation (Fig. 1 Panel A), double-reciprocal plot was also shown with 1/velocity versus 1/[pNPP]. From the fitting we obtained a Km of 1.41 ± 0.04 mM and a series of varying Vmax of 24.03 ± X S1, 13.04 ± X S1 and 8.94 ± X S1 corresponding to the varied fumosorinone concentrations of 0, 10 and 20 lg mL1 while the double-reciprocal plot shows all the straight lines focus in the second quadrant and show a pattern of intersection close to the X-axis, which suggests that fumosorinone is a classic noncompetitive inhibitor for PTP1B (Panel B of Fig. 1). Fumosorinone potently inhibited PTP1B with an IC50 of 14.04 lM. 3.3. Isolation of fumosorinone biosynthetic gene cluster Fumosorinone (1) shared the high level of structural similarity with other 2-pyridones such as aspyridone A (2) (Xu et al., 2010),

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tenellin (3) (Eley et al., 2007) and desmethylbassianin (5) (Heneghan et al., 2011) which are synthesized by hybrid PKS– NPRS, suggesting that the biosynthetic core gene of fumosorinone should be controlled by hybrid PKS–NPRS as its analogs. Compared tenellin synthetase sequences to I. fumosorosea genome sequences (I. fumosorosea ARSEF 2679 Genome sequencing project, http:// www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA72737) by Basic Local Alignment Search Tool (BLAST), we identified the biosynthetic gene cluster of fumosorinone. Then a total 26,398 bp DNA segment from the genome of I. fumosorosea was isolated to be fumosorinone biosynthetic gene cluster.

3.4. The comparison of putative sequence with other PKS–NPRS gene clusters Sequence analysis of the entire contig revealed that the gene cluster structure is identical to the tenellin synthetase and desmethylbassianin synthetase. It contains a PKS/NRPS gene which is supposed to encode the fumosorinone synthetase (FUMOS). Further blast analysis showed that FUMOS is homologous to other fungal PKS–NRPS domains, especially tenellin synthase (TENS), desmethlbassianin synthase (DMBS) and putative Cordyceps militaris polyketide synthase. The entire putative FUMOS gene cluster has a 65.5% identity value to DMBS, and 63.8% identity value to TENS at the protein level (with Vector NTI software). Detailed sequence analysis based on homology to known TENS and DMBS sequences allowed us to identify two introns and six open reading frames (ORFs) in the 26 kb genomic DNA sequence (Table 1). In all introns, the 50 end of the introns contained the conserved splice sequence GT, while at the 30 end was AG. There are five introns in tenellin synthase (TENS). Interestingly, unlike TENS and DMBS we discovered that the FUMOS did not have introns (see the Supplementary Information). ORF1 and ORF2 were homologous to kinesin motor domain and fungal transcription factor regulatory region which possesses C6 zinc finger domain, respectively (Fernandes et al., 1998). The last four complete ORFs are arranged in the same orientation as tenellin and desmethlbassianin counterparts. ORF3 (fumoA) and ORF4 (fumoB) showed homology to cytochrome P450 enzymes, and ORF5 (fumoC) was homologous to trans enoyl-reductase, especially to tenC and dmbC (Eley et al., 2007; Heneghan et al., 2011).

Fig. 1. Kinetic analysis of PTP1B inhibition by fumosorinone. (A) is Michaelis–Menten plot, and (B) is double-reciprocal plot of initial velocities versus varied concentrations of pNPP. Concentration of fumosorinone was selected at 0 (j), 10 lg mL1 (d) and 20 lg mL1 (N).

Please cite this article in press as: Liu, L., et al. Structure and biosynthesis of fumosorinone, a new protein tyrosine phosphatase 1B inhibitor firstly isolated from the entomogenous fungus Isaria fumosorosea. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.03.009

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L. Liu et al. / Fungal Genetics and Biology xxx (2015) xxx–xxx

Table 1 The 26.4 kb I. fumosorosea genomic DNA showed the organization of six open reading frames and the positions of two introns (dark vertical bands). The boundary of each domain is not exactly.

Feature

Start bp

End bp

BLASTX homology

% Identity/positives

ORF1 ORF2 ORF3/fumoA Intron1 Intron2 ORF4/fumoB ORF5/fumoC ORF6/fumoS

1350 3671 8023 8608 9205 9990 12,207 13,982

2246 6460 9716 8678 9297 11,630 13,391 26,398

Beauveria bassiana kinesin motor domain Beauveria bassiana C6 zinc finger domain Cordyceps militaris cytochrome P450 – – Beauveria bassiana cytochrome P450 Beauveria bassiana DMBC Beauveria bassiana TENS

81/88 53/65 77/84 – – 59/73 68/81 64/76

3.5. Verification of PKS–NRPS by gene knockout using the Agrobacterium-mediated transformation To confirm the involvement of FUMOS in the biosynthesis of fumosorinone, inactivation of the PKS–NRPS gene was performed. We used a directed knockout experiment to prove that the PKS– NRPS gene is responsible for the biosynthesis of fumosorinone. To find a suitable selectable marker, we tested several antibiotic markers including herbicide glufosinate, hygromycin B, G418 and benomyl. The results have shown that I. fumosorosea is resistant to all antibiotics except benomyl with a concentration less than 5 lg mL1 (Song et al., 2011). A selective cassette (trpC-benR) which consists of the promoter trpC and benomyl resistance gene (benR) was constructed by ligation. The knock-out plasmid was

constructed based on the backbone of ppzp111 by placing one of two nearly continuous fragments from PKS–NRPS coding region on each side of the trpC-benR selective cassette. After A. tumefaciens-mediated transformation, four clones (ko1, ko2, ko3, ko4) were selected and verified by PCR analysis. All the mutants showed insertion of the benR cassettes (Fig. 2). We found that gene knockout strains showed different colonies morphology compared to wild type, which has more developed aerial mycelium. Gene knockout strains and wild type strain were cultured in SDY liquid media with or without 5 lg mL1 benomyl at 28 °C for 10 days, respectively. The mycelia were extracted with ethyl acetate (100 mL) for three times. The crude ethyl acetate extracts were used for UPLC–MS analysis. At the same condition, the peak of standard fumosorinone is at 4.14 min, and the wild type crude

Fig. 2. Genetic and chemical analysis of I. fumosorosea transformants: (A) PCR analysis of I. fumosorosea transformants, amplified with the 2.6 kb fragments of trpC-benR. Molecular weight marker was k-HindIII digest marker. Lanes 1–4 were mutants (ko1, ko2, ko3 and ko4); lane 5 is wild type as control. (B) Different colonies morphology between wild type (right) and transformed I. fumosorosea ko3 (left) after 5 days of incubation. (C) Standard fumosorinone LCMS analysis which is monitored at k365 nm, PDA trace; (D) crude extracts of wild type (wt) LC–MS analysis, k365 nm, PDA trace; (E) wild type (wt) LC–MS analysis, m/z trace at 476 Da (fumosorinone, [M–H]); (F) transformant ko3 LC–MS analysis, k365 nm, PDA trace.

Please cite this article in press as: Liu, L., et al. Structure and biosynthesis of fumosorinone, a new protein tyrosine phosphatase 1B inhibitor firstly isolated from the entomogenous fungus Isaria fumosorosea. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.03.009

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7

Scheme 2. Putative biosynthesis of fumosorinone.

extract has a peak corresponding at 4.11 min that proved it is the compound together with the evidence of m/z trace at 476 Da (fumosorinone, [M-H]). The mutants (ko1, ko2) produced fumosorinone, and no fumosorinone was detected in ko3 and ko4 extracts of gene knockout strains (Fig. 2 and see the Supplementary Information). The peak at 4.42 min in wild type and 4.46 min in mutant ko3 are another compound with m/z trace 309 ([M–H]) and a different UV absorption curve (see the Supplementary Information). These results indicated this PKS–NRPS is in charge of the biosynthesis of fumosorinone. Ectopic integration had occurred in ko1 and ko2 mutants. 3.6. Putative synthesis pathway of fumosorinone The genetic basis for the biosynthesis of tenellin is well established. The structure of fumosorinone is highly similar to tenellin. Based on the tenellin research (Eley et al., 2007; Halo et al., 2008b; Heneghan et al., 2010; Moore et al., 1998), we proposed the reactions involved in the biosynthesis of fumosorinone (Scheme 2). PKS modules are programmed and produce polyketide chains with different reduction and methylation patterns (Fig. 3) and NRPS modules are responsible for fusing the polyketide chain to an amino acid (tyrosine) (Boettger et al., 2012) and an offloading domain for release (Du and Lou, 2010). In this case, acetate is extended seven times with seven unites of malonate by PKS modules, programmed C-methylations occur after the first three and the sixth extensions, and cycles of full reduction occur after the first two extensions (Fisch et al., 2011).

3.7. Insect pathogenesis We examined the pathogenicity of I. fumosorosea toward larvae of Galleria mellonella. I. fumosorosea wild type and the mutant ko3 were grown on SDAY media in order to prepare and collect spores. Incubation of the larvae with spores resulted in death after about 7 days compared to the control (0.05% Tween 80 treatment only). No difference in pathogenicity was observed between the wild type and ko3 strain. 4. Discussion We discovered a novel compound fumosorinone from I. fumosorosea with IC50 value of 14.04 lM against PTP1B. PTP1B (encoded by the PTPN1 gene) was the first mammalian PTP to be purified and characterized (Feldhammer et al., 2013; Tonks et al., 1988). PTP1B is a negative insulin regulator and a target of insulin resistance which is a characteristic feature of type 2 diabetes mellitus and its increased activity and expression is implicated in the pathogenesis of insulin resistance (Jiang and Zhang, 2008). PTP1B inhibitors enhance the sensibility of insulin receptor (IR) and have favorable curing effect for insulin resistance-related diseases (Barr, 2010). Therefore, the inhibition of PTP1B is anticipated to become a potential therapeutic strategy to treat type 2 diabetes mellitus. A large number of PTP1B inhibitors, either synthetic or natural products played as protagonists for discovering new drugs, have developed and investigated for their ability to stimulate insulin signaling with favorable curing

Please cite this article in press as: Liu, L., et al. Structure and biosynthesis of fumosorinone, a new protein tyrosine phosphatase 1B inhibitor firstly isolated from the entomogenous fungus Isaria fumosorosea. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.03.009

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L. Liu et al. / Fungal Genetics and Biology xxx (2015) xxx–xxx

Fig. 3. Programming during the biosynthesis of the fumosorinone pentaketide.

Table 2 Domain organization and analysis of ORF6 (FUMOS) with 4138 amino acids. The domain boundaries are approximate.

Beta-ketoacyl synthase (KS) Acyl transferase (AT) Dehydratase (DH) Methyltransferase (MT) Beta-ketoacyl reductase (KR) Acyl carrier protein (ACP) Condensation (C) Adenylation (A) Peptidyl carrier protein (PCP) Dieckmanna Cyclase (DKC)

Superfamily

Boundaries

Active site

Condening enzymes Acyl-transf-1 Hot-dog AdoMet-Mtases NADB-Rossmann PP-binding Condensation AFD-class-I PP-binding NADB-Rossmann

E16-I453 V590-D911 H990-V1310 K1486-L1697 R2204-D2388 I2490-Q2590 E2699-V2987 R3166-A3622 E3687-V3755 I3810-R4092

Cysteine – – S-adenosylmethionine binding site NAD(P)-binding motif Phosphopantetheine attachment site – AMP binding site Phosphopantetheine attachment site NAD(P)-binding motif

effects (Tamrakar et al., 2014). The representative compound is MSI-1436 (Trodusquemine) which can suppress appetite and causes fat-specific weight loss in diet-induced obese mice and it is in phase I trial (Scott et al., 2010). Fumosorinone, a new natural

product, was found to inhibit PTP1B activity in this study. In our further research, we have found fumosorinone increased the insulin-provoked glucose uptake, decreased the expression of PTP1B in insulin-resistant HepG2 cells and augmented the insulin

Please cite this article in press as: Liu, L., et al. Structure and biosynthesis of fumosorinone, a new protein tyrosine phosphatase 1B inhibitor firstly isolated from the entomogenous fungus Isaria fumosorosea. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.03.009

L. Liu et al. / Fungal Genetics and Biology xxx (2015) xxx–xxx

signaling pathway. What’s more, fumosorinone apparently reduced blood glucose and lipid levels in serum of diabetic KKAy mice and it did not show overt symptoms of toxicity (unpublished data). As a consequence, fumosorinone has shown a treatment effect in mouse model for type 2 diabetes and it is a new potential therapeutic agent for the treatment of type 2 diabetes. We have applied for a patent: CN102675199 B. To facilitate further comprehensive understanding of gene clusters involved in the biosynthesis of fumosorinone, we searched the genome of I. fumosorosea. A 26,398 bp fragment of I. fumosorosea genomic DNA which contained a PKS–NPRS gene cluster was cloned and demonstrated to be involved in fumosorinone biosynthesis using the Agrobacterium-mediated gene knock out transformation. The annotated sequence has been deposited with GenBank (Accession No.: KP737857). FUMOS is a 12.4 kb PKS–NPRS gene that contains typical fungal iterative type I PKS, is followed by an NRPS module (Table 2). PKS has a general domain organization of b-ketoacyl synthase (KS), acyl transferase (AT), dehydratase (DH), methyltransferase (MT), b-ketoacyl-reductase (KR) and acyl carrier protein (ACP). The NRPS modules have the domain organization of condensation (C), adenylation (A), peptidyl carrier protein (PCP) and Dieckmann cyclase (DKC). ORF3 (fumoA) encodes a cytochrome P450 monooxygenase which maybe catalyzes an unprecedented oxidative ring expansion of prefumosorinone A to form the 2-pyridone core of fumosorinone (Halo et al., 2008a). ORF4 (fumoB) encodes an unusual cytochrome P450 monooxygenase which maybe hydroxylated the nitrogen of prefumosorinone B but not the acyltetramic acid prefumosorinone A to form fumosorinone. These need be further confirmed by experiments. ORF5 (fumoC) was homologous to trans-acting enoylreductase (ER) for enoyl reduction while in the fumoS the ER component is inactive. Rational domain swaps between TENS and DMBS showed the influence of single module on PKS programming (Fisch et al., 2011). C-methylation (CMeT) contributes significantly to its own programming and the KR domain appears to represent the chainlength-determining factor. But the mechanisms how the CMeT domain controls the numbers and positions of methyl groups and the KR domain programs the chain length are elusive. Compared with tenellin and desmethybassianin, fumosorinone has a longer side chain and more methyl groups. Rational domain swaps between FUMOS and TENS or DMBS might help to bring out the problem more clearly. We will explore the programming rules of different polyketide chains and methyl degrees by further sequence analyses and domain swaps. In conclusion, we isolated a novel compound fumosorinone as a novel kind of selective protein tyrosine phosphatase 1B inhibitor and identified its biosynthetic gene cluster by knock-out experiment. The detailed research on FUMOS could provide the basis of secondary metabolites biosynthesis in entomogenous fungi.

Acknowledgments The authors thank Prof. Jigang Li and Jingze Zhang for providing plasmid ppzp111 and A. tumefaciens LBA4404. The plasmid pBT6 was obtained from the Fungal Genetics Stock Center (Kansas City, Missouri USA). This work was supported by National Natural Science Foundation of China (31171885 and 31371957), Changjiang Scholars and Innovative Research Team in University (IRT1124), The Ph.D. Programs Foundation Ministry of Education of China (20121301110006), Key Projects in the Hebei Province Science & Technology (13226508D).

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Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fgb.2015.03.009.

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Please cite this article in press as: Liu, L., et al. Structure and biosynthesis of fumosorinone, a new protein tyrosine phosphatase 1B inhibitor firstly isolated from the entomogenous fungus Isaria fumosorosea. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.03.009

Structure and biosynthesis of fumosorinone, a new protein tyrosine phosphatase 1B inhibitor firstly isolated from the entomogenous fungus Isaria fumosorosea.

Fumosorinone, isolated from the entomogenous fungus Isaria fumosorosea, is a new 2-pyridone alkaloid which is elucidated by HRESIMS 1D and 2DNMR. Fumo...
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