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. 2022 Jul 5;12(8):158. doi: 10.1007/s13205-022-03219-x

Chemical mutagenesis and high throughput media optimization in Tolypocladium inflatum MTCC-3538 leads to enhanced production of cyclosporine A

Vidushi Abrol 1,2, Manoj Kushwaha 1, Sharada Mallubhotla 2, Sundeep Jaglan 1,3,✉
PMCID: PMC9256877  PMID: 35814036

Abstract

Diethyl sulphate-based mutagenesis was performed on fungal strain Tolypocladium inflatum MTCC-3538. Two mutant morphotypes MT1-3538 and MT2-3538 were selected for further chemo-profiling studies. LCMS/MS profiling of fungal crude extract confirmed that the wild-type and mutant strains (MT1-3538, MT2-3538) were competent to produce cyclosporine A. MT2-3538 produced 2.1 fold higher cyclosporine A in comparison to the wild type. Further, LCMS-based high throughput media optimization was performed for MT2-3538 in 20 different media combinations to increase cyclosporine A yield. On the basis of ion-intensity profiling, media combination consisting of Glucose 0.1 g/L; Peptone 0.005 g/L and Valine 0.005 g/L was selected and used for up-scaling purpose. Mutant MT2-3538 with optimized media combination increased cyclosporine yield 16 fold and could potentially be exploited for commercial outcomes.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-022-03219-x.

Keywords: Cyclosporine A, Tolypocladium inflatum, Chemical mutagenesis, Diethyl sulphate (DES), Direct Infusion-Tandem Mass Spectrometry (DI-MS/MS)

Introduction

Cyclosporine A possesses broad-spectrum pharmacological properties and is used as an anti-inflammatory (Gilger et al. 2010; Stiller and Opelz 1991), an antiparasitic (Zeligs 2016; Weiser and Matha 1988), an immunosuppressive (El-Kashef et al. 2017; Řeháček and De-xiu 1991), and an antifungal (Shinde et al. 2012) agent. Cyclosporine A is generally used to treat or cure graft and host complexity in bone marrow and organ transplantation (Miach PJ. 1986). Tolypocladium inflatum (Aarnio and Agathos 1989; Yang et al. 2018), Penicillium fellutanum (Anjum et al. 2012), Fusarium solani (Huang-jian et al. 2017), and Neocosmospora arinfecta (Nakajima et al. 1988, 1989) are well known for large-scale submerged fermentation of cyclosporine A. To increase cyclosporine A yields, different parameters which includes fermentation conditions (Anjum and Iram 2017) and nutrient composition of the culture medium have been explored (Agathos and Parekh 1990; Irum and Anjum 2012; Manonmani et al. 2016; Patocka et al. 2020), fermentation conditions (Ko et al. 2001; Demir and Tari 2016), ( Chun and Agathos 1989; Ooijkaas et al. 2000; Blount 2018). It is evident from the above cited literature that the media optimization strategy plays a significant role in the enhancement of secondary metabolites in fungi. Different statistical techniques to optimize fermentation conditions for T. inflatum have concluded that varying media components, including glucose, peptone, etc., enhanced the cyclosporine A yield in comparison to the primary medium (Survase et al. 2009a, 2009b). In recent studies, glucose has proven to be an effective carbon source for cyclosporine A production (Anjum and Iram 2017). Literature also reveals the suitability of peptone as a nitrogen source and amino acids that impart vitamins and minerals to drug molecules like cyclosporine A in bioprocesses. Moreover, L-valine amino acid acts as a gene inducer in transcription during cyclosporine A synthesis (Tanseer and Anjum 2011). From previous studies, for the enhanced production of cyclosporine A, six Penicillium fungal strains were investigated using submerged fermentation strategy with media composition of peptone, glucose, KH2PO4 and KCl, it was reported that Penicillium fellutanum emerged as a new fungal source of cyclosporine A (Anjum et al. 2012).

To obtain a competitive commercial advantage other non-culture-based methods such as the use of chemical mutagenesis have also been applied. Domratcheva et al. 2018 reported that N-nitroso-N-methylurea (NMU), a chemical mutagen targeting the metabolites of T. inflatum increased the yield of cyclosporine A by 1.6 times.

In the present study, to enhance the yield of cyclosporine A, various combinations of glucose (carbon), peptone (nitrogen), and amino acid (L-valine) were used during cultivation of T. inflatum by the selected fungal organism. The 24-microwell plate-based microbial culturing strategy was used as a high-throughput tool for bioprocess development (Duetz et al. 2000; Ferreira-Torres et al. 2005; Vervoort et al. 2017), whereas the DI-MS and high-performance liquid chromatography (HPLC) complimented the entire process of rapidly identifying and quantifying cyclosporine A yields from T. inflatum. In addition, diethyl sulphate (DES), a chemical mutagen was used to mutate T. inflatum MTCC-3538 and which increased the yield of cyclosporine A. DES behaves like an alkylating agent and reacts with DNA, and alters the guanine residue leading to inaccuracy in DNA replication and repair (Li et al. 2016; Wu et al. 2019; Abrol et al. 2021).

Experimental section

Microorganism and chemicals

Fungal strain T. inflatum MTCC-3538 (= ATCC34921 = NRRL8044) was procured from Microbial Type Culture Collection (MTCC), Chandigarh, India; after revival, the culture was maintained on potato dextrose agar (PDA) slants. The standards of cyclosporine A and DES were procured from Sigma-Aldrich, India. The media components that are glucose, casein peptone, L-valine, L-leucine, casein acid hydrolysate, malt extract, peptone, α-amino butyric acid, glycerol, ammonium sulphate, potassium dihydrogen phosphate and potassium chloride were procured from the Hi-Media Laboratories, India.

Chemical mutagenesis of MTCC-3538 and mutant selection

Mutants of T. inflatum were produced using the method reported by Fang et al. 2014 with minor alterations of DES concentration and incubation period. The fungal strain was grown on PDA media for 7 days, consequently the fungal was suspended in a normal saline solution containing 0.01% Tween 20. The experimental vials having different concentrations of DES (5.0 to 640.0 mM) were prepared in 20% dimethyl sulfoxide (DMSO). The 5 × 106 CFU mL−1 fungal culture was inoculated in each vial containing different DES concentrations, and the final volume was kept at 5.0 mL (Table S1). The vials with varying densities of DES and T. inflatum were incubated for 32 days at 4 °C. Further, 100 μL of cell suspension from each vial was top spread on petri dishes containing PDA media and incubated at 28 °C for 7 days. The petri dishes were subjected to visual inspection for the presence of morphologically distinct colonies compared to that of the wild-type. The visually distinct colonies (mutants) were picked and obtained as pure culture.

Large scale culture and preparation of extracts

For seed culture of T. inflatum and their mutant’s, agar plugs were inoculated in Erlenmeyer flasks (2 × 500 mL), each containing 150 mL potato dextrose broth (PDB). The flasks were kept in a shaking incubator for 3 days in dark (28 °C at 150 rpm). A set of 30 Erlenmeyer flasks (1 L), each containing 400 mL of sterile PDB were inoculated with 10% of (v/v) seed culture. The flasks were incubated for 10 days (28 °C at 150 rpm). The broth culture was terminated by addition of 10% of methanol (v/v) and filtered using a cheese cloth. The filtrate was extracted twice with an equal volume of ethyl acetate and dried using a rotary evaporator to obtain crude extract (8 g). The remaining cell mass was homogenized with methanol and centrifuged at 5000 rpm for 10 min, and the supernatant was subjected to a rotary evaporator to obtain the extract (5 g).

HPLC profiling of crude extracts

The HPLC analysis of the crude extract of wild-type (MTCC-3538) and mutant strains (MT1-3538 and MT2-3538) was performed using the Shimadzu UFLC system. Chromatographic separation was achieved using Merck RP18 HPLC column (4.6 mm × 250 mm, 5 µm). Mobile phase A was water with 0.1% formic acid, and mobile phase (B) contained acetonitrile (ACN). The elution gradient program was followed as 0 min (30% B), 25 min (90% B), 35 min (90% B), 37 min (30% B) and 40 min (30% B). The total run time was 40 min, whereas the flow rate was 0.7 mL/min. The crude extract (10 mg) was dissolved in 1 mL of methanol and filtered through a 0.2 µm syringe filter. Subsequently, 10 μL of the sample was taken and subjected for HPLC analysis.

Semi-preparative isolation of crude extracts

Semi-preparative HPLC isolation was performed using the Shimadzu UFLC system. The chromatographic separation was achieved using the Agilent RP-18 column (RP-18, 10 × 250 mm, 5 μm). The mobile phase A composed of water with 0.1% formic acid and a mobile phase (B) composed of ACN, the gradient system was 20 to 80% B in 0 to 20 min, hold at 80% of B for 20 to 30 min, 80 to 20% B in 35 min and hold at 20% B for next 5 min with a total run time of 40 min. The flow rate was 1.5 mL/min. Crude extract (100 mg) was dissolved in 1 mL of methanol and filtered through 0.2 µm syringe filter and 100 μL of sample was injected for each HPLC run.

LC–MS profiling of crude extracts

LC–MS analysis was done using an Agilent mass spectrometer (Agilent technologies, LCMS-QQQ, 6410B, Santa Clara, CA, USA). The samples were analyzed with positive and negative polarity switching mode from 100 to 1700 m/z, with 0.8 cycles/s of scan rate. The chromatographic separation was achieved using an RP18 HPLC column (4.6 mm × 100 mm, 3.5 µm, Agilent ZORBAX Eclipse). The mobile phase (A) contained 0.1% formic acid in water and mobile phase (B) of ACN. An elution gradient program, i.e., at 0 min (20% B), 5 min (80% B), 10 min (80% B), 12 min (20% B) and 18 min (20% B) was followed in each HPLC run. Sample volume (10 μL) was injected for every HPLC run and following were the mass spectrometric conditions: capillary voltage 3500 V, vaporizer 200 V, gas temperature 300 °C, drying gas 12.0 L·min−1, voltage charge 2000 V, nebulizer pressure 30 psi; negative-ion mode capillary voltage 2500 V, fragmentor 175 V, skimmer1 65.0 V. All the data processing was executed and evaluated using Agilent mass hunter qualitative software (version B.05.00, Agilent Technologies, Santa Clara, CA, USA).

Rapid optimization of medium in 24-well culture plate

In preliminary screening, the confirmation of cyclosporine A production by wild-type and mutant cultures (MT1-3538 and MT2-3538) was assessed by growing them on PDB at a small scale (5 mL). The cultures producing cyclosporine A on PDB were grown on already reported various media for cyclosporine A production (Table S2). Type-V media (Table S2) was found promising for cyclosporine A production, which was subjected to further optimization by varying the components, i.e., glucose, peptone, and valine (Table S3). Different concentrations of glucose, peptone, and valine were tried in a 24-well plate to produce cyclosporine A. Each well of the culture plate contained 1 mL of media and 100 μL of seed inoculum, incubated at 28 °C for 10 days, in dark and static condition. After 10 days the broth was pipetted out from each well and transferred into a glass tube containing 1 mL of methanol. The glass tube containing broth was subjected to 5-min sonication, followed by centrifugation at 10,000 rpm for 10 min. The supernatant was retrieved and subjected to LCMS analysis. The peak obtained at m/z 1202.8 [M+H]+ belonged to cyclosporine A, whereas different peak intensities were obtained with different media. The media combination possessing the most abundant peak at m/z 1202.8 [M+H]+, was selected for scale-up.

Quantification of cyclosporine A

The quantification of cyclosporine A was carried out on Shimadzu UFLC. A HPLC method was developed and optimized to quantify cyclosporine A in the crude extract. HPLC column LiChroCART®, RP18e, 4.6 × 250 mm, 5 μm was used for the chromatographic separation. Eluent A composed of 0.1% formic acid in water whereas B comprised of ACN gradient programme followed at 20–90% of B in 0–15 min, 90% B hold for next 10 min, 90–20% of B in 25–30 min and hold for 5 min at the initial composition of the mobile phase. The flow rate was 0.8 mL/min and the chromatographic data were acquired at 190 nm. Different concentrations of standard cyclosporine A prepared in methanol and used for quantitative analysis. The calibration equation of cyclosporine A was derived by analyzing the peak area (y-axis) versus the concentration (x-axis, mg/mL) with trend line as y = 4 × 106 x-20243 (R2 = 0.99) (Figure S2).

Results

Fungal strain MTCC-3538

The fungal strain MTCC-3538 was observed to have hyaline and septate mycelia microscopically. The mycelial morphology and its phenotypic characteristics were similar, as reported earlier (Dreyfuss et al. 1976; Lin et al. 2011). In the first instance, the microscopic images look similar, but if we carefully observe the mycelia of the MT2-3538 (Figure S9, c), it seems sparse and has almost double the thickness of mycelia as compared to that of the wild-type and MT1-3538 strain (Figure S9).

Effect of DES mutagen on T. inflatum

Previously reported literature suggests that DMSO does not cause any mutation effect on fungal spores/mycelia at lower temperatures because DMSO may enhance DES penetration in the cell. The DES-based mutagenesis protocol was standardized for treating the fungal cultures with different concentrations of DES, prepared in 20% (v/v) DMSO in water. The visual observations recorded for the treatment are given in Table S1. After the treatment, different morphological variations were observed between mutant and wild-type strains; based on these observations; two mutants were obtained from T. inflatum (Fig. 1). The DMSO-treated fungal culture has similar morphological characteristics with the wild-type strain (without DES). The combination of DMSO and DES causes mutagenesis, which appears as a different morphological character.

Fig. 1.

Fig. 1

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Visual observation (Morphological) of DES-treated Tolypocladium inflatum

Chemo-profiling-based selection of the mutant strain

HPLC chemo-profiling was performed for the mutants and wild-type strain. Based on chromatographic data, it was observed that the two mutants (MT1-3538, MT2-3538) (DES derived) of T. inflatum show a significant difference of peak in the HPLC chromatogram, which might produce different metabolites in comparison to the wild-type strain. Chemical mutagenesis encourages the future discovery to obtain new bioactive metabolites or known compounds with higher yields (Figure S3 and S3).

Identification, isolation and quantification of cyclosporine A

The LCMS/MS-based de-replication strategy was applied to identify metabolites from fungal crude extract. Further, the wild-type strain was searched at the species level in the dictionary of natural products (DNP). Their results confirmed the presence of cyclosporine A with m/z 1202.84 upon comparision with standard cyclosporine A LC-MS chromatogram. The DNP search results with m/z 1202.84 are shown in Figure S4.

After the comparative analysis, the mass of cyclosporine A was detected in the crude extracts of control and mutant strains of T. inflatum as shown in Fig. 2.

Fig. 2.

Fig. 2

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LCMS analysis of crude extract obtained from MT2-3538 and wild type, a  +ESI mass spectrum, peak area of m/z 1202.8 [M+H]+ from control crude extract b +ESI mass spectrum, peak area of m/z 1202.8 [M+H]+ from MT2-3538 crude extract. The peak area of peak at m/z 1202.8 was 482,449 in control but in mutant strain the peak area of peak at m/z 1202.8 was 1,027,819, which is 2.12 time higher, hence MT2-3538 was selected for further media engineering studies

Further, an investigation of wild-type and mutant (MT2-3538) strains of T. inflatum was done and it was found that the peak area of LCMS chromatogram with m/z 1202.84 in MT2-3538 was 2.1 times higher than that of the control (Figure S5). LCMS data are represented in Figure S6. Subsequently, media optimization was performed for the up-scaling of cyclosporine A using 20 different media compositions and their crude extracts were analyzed by mass spectrometry, the (11th media containing Glucose 0.1 g/L; Peptone 0.005 g/L and Valine 0.005 g/L) having the maximum ion intensity of peak at m/z 1225.0, [M+H]+ cyclosporine A was 3.3 × 104 (Fig. 3 and Figure S7) was selected for further studies. Based on ion intensity of peak at m/z 1225.0, [M+H]+ cyclosporine A, the 11th media were further used for the up-scaling purpose. Later, MT2-3538 fermentation was performed up to 10 L using the optimized media. The extract was prepared and subjected to semi-preparative isolation. The peak at tR 37.9 min was collected and cyclosporine A content was determined using mass spectrometric analysis (Figure S6 and S8).

Fig. 3.

Fig. 3

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Ion intensity of cyclosporine A (m/z 1224.9, [M+Na]+) in crude extracts of fungal cultures grown on 20 media types

A comparative analysis of control and mutant strain for the production of cyclosporine A revealed that applying a micro-titer plate-based approach for microbial system can act as a high-throughput tool for bioprocess development and integration for the production of cyclosporine A at a very small scale (1 mL). In this experimental setup, 24-well culture plate was used for media optimization, signifies the most economical and manageable technique for media optimization. Despite the uniform culture conditions during fermentation the quantitative analysis reveals different ion intensity of cyclosporine A in mutant MT2 (319.1 mg/L) and wild strain (20.9 mg/L). This shows that the yield of cyclosporine A in mutant strain (MT2-3538) was 16 times higher than that of control strain T. inflatum (Figure S8). The results reveal that this is a new viable option for increasing the yield of cyclosporine A for future industrial and commercial use.

Discussion

The filamentous fungi viz. T. inflatum has been found as a biological source of important bioactive metabolites like cyclosporine A in our study. Cyclosporine A is a non-polar cyclic peptide earlier identified as a major product of Trichoderma polysporum strain and further isolated from T. inflatum (MTCC-3538) (Casareto et al. 1998). Cyclosporine A and its derivatives are well-known metabolites for their antifungal and antibacterial activity (Marudhu et al. 2007). Recently, cyclosporine derivatives have also been reported in clinical studies as anti-hepatitis C (HCV) drugs (Membreno et al. 2013). Since T. inflatum has been reported for the presence of so many important metabolites, it is more interesting to diversify its metabolite spectrum with a practical approach.

Interestingly, the DES-based chemical mutagenesis has been found suitable for activating silent metabolites in the fungal strains absent in wild types, which ultimately leads to an extensive variety of chemical diversity. It is also observed that the mutagenicity of DES acts as an alkylating agent that reacts chemically with DNA to modify the base (primarily guanine) (Hoffmann 1980; Kahn et al. 1994). Therefore, the most prominent and feasible strategy which is prone to increase the production of different important secondary metabolites by activating silent pathways and modifying the biochemical nature of the compounds is DES-based chemical mutagenesis ( Barrios-Gonzalez et al. 2003; Survase et al. 2011). This strategy is highly applicable to overcome various biological threats and develop different pharmacological treatments (Chai et al. 2012; Dong et al. 2014; Yabutani et al. 2017).

In the present study, we have isolated two mutants using DES-based chemical mutagenesis of MTCC-3538 (MT1-3538, MT2-3538). It can be presumed that the chemical mutagenesis in MTCC-3538 would affect the peptide synthetase pathways which would be responsible for the improved yield of cyclosporine A in mutants of T. inflatum. In our initial studies, comparative analysis through HPLC data of mutants and wild-type strains shows that the peak area of cyclosporine A is enhanced 2.1 times for the mutant strains. This preliminary observation raised our expectations for a high yield of cyclosporine A. Considering the above, studies depict that MTCC-3538 can utilize glucose as a carbon source, while, hexose monosaccharide was the most efficient sugar, which acts as a major energy source for maximum living beings, including fungi. Peptones are formed from animal milk or meat, broken down by proteolytic digestion, and have small peptides linked with fats, salts, vitamins, and other biological entities. This nitrogen source also provides metals, vitamins, and amino acids like valine and proves to be the best supplement for cyclosporine A enhancement in relation to other combinations. Valine acts as a promoter to enhance the transcription of genes for cyclosporine A synthetase or other associated structural genes connected to cyclosporine A synthesis in MTCC-3538 and gives the positive impact as an additional supplement when included in the fermentation medium. It is also possible that this amino acid may cause cell modification to favor the induction of secondary metabolites by targeting transcription genes during vegetative cell development (Keller et al. 2005).

Furthermore, the 24-well plate-based culture system proved that it works as a high-throughput strategy for bioprocess development and media engineering. In the optimized media containing Glucose 0.1 g/L; Peptone 0.005 g/L and Valine 0.005 g/L the maximum ion intensity peak was observed at m/z 1225.0, [M+H]+ cyclosporine A, i.e., 3.3 × 104 (Fig. 3 and S7). In conclusion, on engineered media, the production of cyclosporine A in the mutant strain was 16 times higher than that of the control strain of T. inflatum. Hence, our outcomes revealed the best alternative strategy for submerged fermentation on a micro-titer plate for enhanced cyclosporine A production for future commercial exploitation.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOC 1577 KB) (1.5MB, doc)

Acknowledgements

Sundeep Jaglan gratefully acknowledges the Department of Science Technology (DST), Govt. of India for the financial assistance with Grant No. ECR/2017/001381

Author contributions

VA executed the experimental work and prepared the original draft of the manuscript. MK did the analytical studies. SM did the formal analysis, supervision, review and editing of the manuscript. SJ contributed for the conceptualization, validation, formal analysis, supervision, editing of the manuscript and funding acquisition. All the authors read and approved the final manuscript.

Funding

This article is funded by SERB DST INDIA, ECR/2017/001381, Sundeep Jaglan.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest in the publication.

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