Alkaloid diversity expansion of a talent fungus Penicillium raistrichii through OSMAC-based cultivation

Introduction Alkaloidal natural products are attractive for their broad spectrum of pharmaceutical bioactivities. In the present work, the highly productive saline soil derived fungus, Penicillium raistrichii, was subjected to the strategy of OSMAC (one strain many compounds) with changes of cultivation status. Then, the work-flow led to the expansion of the alkaloid chemical diversity and subsequently induced the accumulation of four undescribed alkaloids, named raistrimides A–D (1–4), including three β-carbolines (1–3), one 2-quinolinone (4), and one new natural product, 2-quinolinone (5), along with five known alkaloid chemicals (6–10). Methods A set of NMR techniques including 1H, 13C, HSQC and HMBC, along with other spectroscopic data of UV-Vis, IR and HRESIMS, were introduced to assign the plain structures of compounds 1–10. The absolute configuration of 1–3 were elucidated by means of X-ray crystallography or spectroscopic analyses on optical rotation values and experimental electronic circular dichroism (ECD) data. In addition, it was the first report on the confirmation of structures of 6, 7 and 9 by X-ray crystallography data. The micro-broth dilution method was applied to evaluate antimicrobial effect of all compounds towards Staphylococcus aureus, Escherichia coli, and Candida albicans. Results and discussion The results indicated compounds 1, 3 and 4 to be bioactive, which may be potential for further development of anti-antimicrobial agents. The finding in this work implied that OSMAC strategy was a powerful and effective tool for promotion of new chemical entities from P. raistrichii.


Introduction
It is clearly understood that the secondary metabolite (SM) production capacity of microbes lies on biosynthetic gene clusters (BGCs) (Bills and Gloer, 2016;Genilloud, 2019;Zhang J. J. et al., 2019;Ke and Yoshikuni, 2020;Alexander, 2023).Yet, the traditional workflow involving laboratory cultivation of microbes, followed by extraction of fermentation, SMs purification, and structure elucidation, often results in the silent or weak expression of BGCs and thus confines the occurrences and number of the new chemical entities, due to failure of resembling the nature environment with intense microbial competition.There is a growing consensus that unlocking the silent BGCs is a key channel to counteract the dramatic decline in the quantity of novel naturally-produced chemical structures (Reen et al., 2015;Zhang X. et al., 2019).Zhong et al. 10.3389/fmicb.2023.1279140Frontiers in Microbiology 02 frontiersin.org During the recent several decades, in order to tap the biosynthetic potential and strengthen silent gene expression, molecular methodologies (Reen et al., 2015;Palys et al., 2020;Wang et al., 2020;Han et al., 2020a,b) like gene manipulation, including knocking down, introduction or heterologous expression, regulation of promoters, and induction of mutations, as well as approaches based on cultivation conditions variation, have been employed and have achieved great successes in mining cryptic SMs.Of these, the one strain many compounds (OSMAC) strategy, covering medium variation, cultivation condition change, co-cultivation, or addition of epigenetic modifiers, has been efficiently applied to promote new therapeutic agents discovery, highlighting it as an easy and productive approach to trigger the reaction of the crypt BGCs and enhance the natural SMs production according to the extensive literature reports (Bode et al., 2002;Romano et al., 2018;Pan et al., 2019).

General experimental procedures
An uncorrected XRC-1 micro melting point apparatus was used to measure the melting points.Uv-Vis and IR data were performed on a TU-1091 spectrophotometer and a Thermo Nicolet 6,700 infrared spectrophotometer, respectively.A Rudolph Autopol V Plus digital polarimeter, a Bruker Smart 1,000 CCD X-ray diffractometer, and an Applied Photophysics Chirascan spectropolarimeter were used for the determination of optical rotations, X-ray crystal data, and electron circular dichroism (ECD) spectra, respectively.Varian Inova 600 and Bruker AV-400/AVIII 500 spectrometers were used to measure 1D and 2D NMR data in different deuterium solvents using tetramethylsilane as an internal standard (IS).HRESIMS spectra were recorded using an Agilent 1200RRLC-6520 Accurate-Mass Q-TOF LC/MS mass spectrometer or a Waters Q-TOF Ultima GLOBAL GAA076 LC.A Shimadzu LC-6 AD and LC-20A Liquid Chromatography were applied for the preparative isolation and analysis on HyperClone C 18 columns (5 μm, 10 × 250 mm, 5 μm, 4.6 × 250 mm, respectively).Open column chromatography (CC) isolations were performed on silica gel (200-300 mesh, Qingdao Marine Chemical, China), reverse phase (RP) C 18 material (Pharmacia Fine Chemical Co., Sweden), and Sephadex LH-20 (Ge Healthcare Bio-Sciences AB, Sweden).

Fungal material
Saline soil samples were collected from the coast in the Circum-Bohai-Sea region in Zhanhua county, from which a talent strain of Penicillium raistrickii with GenBank Accession NO.HQ717799 was isolated in August 2008.The voucher specimen was preserved at the laboratory of Natural Drug Discovery and Research, Binzhou Medical University.

Cultivation and extraction
The spores of P. raistrickii growing well on potato dextrose agar (PDA) were incubated in 500-mL Erlenmeyer flasks which contained 180 mL of culture medium comprising 2% glucose, 1% maltose, 0.03% yeast extract, 0.003% MgSO 4 •7H 2 O, and 0.005% KH 2 PO 4 with the natural pH value in seawater gathered from offshore areas near Yantai, and fermented under static condition at about 28°C for 60 days.A whole volume of 40 L culture broth was isolated into supernatant and mycelia through cheesecloth.The supernatant was extracted with ethyl acetate (3 × 30 L), and the mycelia was extracted with methanol (3 × 10 L) to afford crude aqueous extract followed by partitioning with ethyl acetate, which yielded an extract of 45 g.

Biological assay
The antimicrobial activity of the isolated compounds was examined by the broth microdilution method according to the method as previously described (Ma et al., 2019).Microorganisms evaluated in this work included Staphylococcus aureus, Escherichia coli, and Candida albicans, and chloramphenicol or ketoconazole was applied as positive control.

Results and discussion
Compound 1 was obtained as a yellowish needle.The molecular formula of C 17 H 15 N 3 O 4 was established from the deprotonated-ion HRESIMS at m/z 324.0993 (calcd for C 17 H 14 N 3 O 4 , 324.0990).It indicated a β-carboline chromophore by the feature of the UV absorptions at 377, 286 and 220 nm (Chen et al., 2010).The 1 H NMR spectrum in Table 1 showed four aromatic hydrogen signals at δ H 8.46 (d, J = 7.5 Hz), 7.85 (d, J = 8.2 Hz), 7.64 (t, J = 7.5 Hz), and 7.35 (t, J = 7.5 Hz).The coupling patterns of these four aromatic hydrogens indicated the characteristics of an ο-disubstituted benzene ring in the indole moiety (Tangella et al., 2018).The doublet methyl at δ H 1.52, multiplet methine at δ H 4.59, and the doublet imino proton at δ H 8.85 were suggestive of an alanine residue functionality.The 13 C NMR data showed eleven aromatic carbon signals from δ C 142.4 to 113.3 (Table 1), which featured the β-carboline skeleton.The signals of keto carbonyl at δ C 200.9 and the methyl at δ C/H 25.9/2.93,along with the HMBC crosspeaks from the methyl to the carbonyl, implied the existence of an acetyl group (Figure 2).It was located at C-1 on the basis of correlation from H-15 to C-1.Further interactions from H-17 to C-16 and C-19, and from H-4 to C-16 suggested the linkage of C-3 and the alanine residue through C-16.Thus, the plain structure of 1 was accomplished.Considering that alanine moiety plays a crucial role in the specific optical rotation in compound 1 (the same condition also applied to compounds 2, 3, and 10), the consistent specific rotation behaviors of 1 {[α ] D 20 + 10.8 (c 0.062, MeOH)}and oldhamiaine A {[α ] D

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+ 14.0 (c 0.062, MeOH)}suggested the absolute configuration of C-19 to be S (Zhang et al., 2015).Additionally, the experimental and calculated ECD curves (Figure 3) further supported the absolute configuration assignment.The absolute stereochemistry of 1 was established to be 19S.Compound 2 was purified as light yellow needles with a molecular formula of C 21 H 23 N 3 O 4 given by the positive HRESIMS at m/z 382.1762 [M + H] + (calcd for C 21 H 24 N 3 O 4 , 382.1761).The UV and IR spectra of 2 also showed the absorption features of a β-carboline skeleton.The 1 H and 13 C NMR spectroscopic data of 2 (Table 1) in the downfield regions were almost identical with those of 1, indicative of the same β-carboline moiety.Furthermore, the signals in the upfield of two methines at δ H 4.60 and 2.10, one diastereotopic methylene at δ H 1.54 and 1.31, two methyls at δ H 0.98 and 0.95, and the carbon resonances at δ C 56.8, 37.2, 25.4, 16.6, 11.7 (Table 1), suggested the fragment of an isoleucine residue, which was supported by the HMBC correlations (Figure 2).Finally, the X-ray analyses unambiguously determined the absolute configuration of 2 (Figure 4).
Compound 3, light yellow needles, was determined to have a molecular formula of C 19 H 19 N 3 O 4 by the negative HRESIMS data (m/z 352.1302, calcd for C 19 H 18 N 3 O 4 , 352.1303).Its UV and IR absorption bands were almost the same as those of compounds 1 and 2. The spectroscopic data covering 1 H and 13 C NMR (Table 1) closely resembled those of 1, except for additional appearances of one methine and one methyl signals.Careful analyses of the chemical shifts and the splitting behaviors of a double doublets methine at δ H 4.52, a multiplet methine at δ H 2.34, two doublet methyl groups at δ H 1.03, and a doublet NH at δ H 8.59 were suggestive of a valine motif, which was supported by the corresponding 1 H-1 H COSY data Key HMBC correlations of 1-5 and 9, and key COSY for 3. (Figure 2).The position of the valine motif was accomplished according to the HMBC interactions from H-4 and H-17 to C-16 (Figure 2).The acetyl group linked at C-1 was elucidated by the correlations from H-15 to C-1 and C-14.The similar ECD absorptions (ESI Supplementary Figure S24) together with evidence of the same optical rotation direction with that of 10 confirmed the absolute configuration of 3.
Compound 4, colorless needles, exhibited a deprotonated ion signal at m/z 280.0973 (calcd for C 17 H 14 NO 3 , 280.0968) in negative HRESIMS, which corresponded with its molecular formula of C 17 H 15 NO 3 .In 1 H NMR spectroscopic data, two aromatic protons at δ H 7.44 and 7.18 (Table 2) which were presented in an orthocoupled doublet fashion, indicated the presence of a p-disubstituted ring fragment.Four aromatic protons at δ H 7.60, 7.48, 7.39, and 7.15 (Table 2) represented the existence of an o-disubstituted phenyl ring moiety.Evidence of the HMBC data from H-17 to C-14 and from H-12 to C-4 and C-14 revealed the methoxyl and C-4 anchored at the p-disubstituted phenyl ring (Figure 2).The NH group and C-4 were located at the o-disubstituted phenyl ring, which was supported by HMBC cross peaks from H-6 to C-4 and C-10, along with the chemical shift of C-10.The remaining three carbon signals, including a carbonyl at δ C 160.3, a quaternary carbon at δ C 147.0, and a methoxyl at δ C 60.3 (Table 2) were assembled to complete the whole structure.Finally, the proposed structure of 3 was further undoubtedly defined by the X-ray diffraction analysis (Figure 4).
The structure of 5 was finally determined by 1D and 2D NMR data (Figure 2) along with the data from a previously reported synthetic intermediate (He et al., 2015;Zhang et al., 2015), and it was purified from natural for the first time.
Compound 9 was named dehydrocyclopeptin (Hayashi et al., 1997;Ma et al., 2012).It was first isolated from Penicillium cyclopium (Framm et al., 1973), but the chemical shift assignments of some signals and the configuration of its double band needed to be discussed.In the current report, HSQC and HMBC spectra (Figure 2) helped to provide the 1 H and 13 C NMR data (Table 3), and X-ray diffraction analysis unambiguously built up an E configuration of double band (Figure 4).
The antimicrobial activity of compounds 1-10 was examined against S. aureus, E. coli, and C. albicans with chloramphenicol or ketoconazole as the positive control.As the results show in Table 4, compound 1 demonstrated an obvious antimicrobial effect, while 3 and 4 exhibited activity toward S. aureus and E. coli.

Conclusion
In summary, an OSMAC-based strategy applied to P. raistrickii of changing cultivation status from shaking fermentation to a static condition in a liquid medium expanded the alkaloidal SM diversity and thus induced the accumulation of four unreported alkaloids, including three β-carbolines (1-3) and one 2-quinolinone (4), along with one new natural compound 2-quinolinone (5) and five known β-Carboline alkaloids possess the fundamental framework of tricyclic pyrido [3,4-b] indole and are mainly obtained from plants and marine organisms, with only a few from microbes to our knowledge.2-quinolinones, or named 2-quinolones, are quinoline derivatives with a carbonyl group inserted in the 2 position, most of which are produced by fungal genera (Neff et al., 2012).Benzodiazepine alkaloids are seven-membered cyclodipeptides composed of an anthranilic acid and another α-amino acid or its deoxygenated analogs.They are metabolites of filamentous fungi or actinomycetes, with one exception from sea hare up to now, and have not been found in higher plants (Roos, 1990;Ojika et al., 1993).These three kinds of alkaloids have a strong attraction to scientists due to their structural diversities and extensive scope of pharmacological effects, such as antitumor, antidepressant, antimicrobial, antimalarial, anti-inflammatory, antioxidant, insecticidal and analgesic effects (Roos, 1990;Cao et al., 2007;Cui et al., 2009;Wang et al., 2014;Simonetti et al., 2016;Reddy et al., 2018).
The structures of 1-10 were constructed by comprehensive analyses of the spectroscopic data.Measures including X-ray diffraction data, ECD, and optical rotation values helped to solve the absolute configurations of the new compounds.In addition, the configuration of the double band in compound 9 was unambiguously revised by X-ray diffraction data, and its 1 H and 13 C NMR data were reassigned in this work.Furthermore, it was the first report of the X-ray diffraction analyses on the absolute stereochemistry of compounds 7.
All of the compounds (1-10) in the work were screened for their antimicrobial activity against microorganisms like S. aureus, E. coli, and C. albicans.Compounds 1, 3, and 4 showed antimicrobial effects.As for the β-carbolines obtained in the work, compound 1 exhibited an obvious pharmaceutical effect against tested microorganisms with MIC values of 8.0, 5.0, and 2.0 μg/mL, and compound 3 also showed some activity.The results implied that the amino acid residues and a free carboxyl group in this type of β-carboline alkaloids might play a pivotal part in the antimicrobial effect.

FIGURE 2
FIGURE 2 FIGURE 3Experimental and calculated ECD spectra of compound 1.
The author(s) declare financial support was received for the research, authorship, and/or publication of this article.The National Natural Science Foundation of China (No. 31270082), Science and Technology Development Plan of Yantai (No. 2020XDRH111) gave financial support to this work.