Production, Bioprocessing and Anti-Proliferative Activity of Camptothecin from Penicillium chrysogenum, “An Endozoic of Marine Sponge, Cliona sp.”, as a Metabolically Stable Camptothecin Producing Isolate

Exploring the metabolic potency of fungi as camptothecin producers raises the hope of their usage as an industrial source of camptothecin, due to their short-life span and the feasibility of metabolic engineering. However, the tiny yield and loss of camptothecin productivity of fungi during storage and sub-culturing are challenges that counteract this approach. Marine fungi could be a novel source for camptothecin production, with higher yield and reliable metabolic sustainability. The marine fungal isolate Penicillium chrysogenum EFBL # OL597937.1 derived from the sponge “Cliona sp.” has been morphologically identified and molecularly confirmed, based on the Internal Transcribed Spacer sequence, exhibiting the highest yield of camptothecin (110 μg/L). The molecular structure and chemical identity of P. chrysogenum derived camptothecin has been resolved by HPLC, FTIR and LC-MS/MS analyses, giving the same spectroscopic profiles and mass fragmentation patterns as authentic camptothecin. The extracted camptothecin displayed a strong anti-proliferative activity towards HEP-2 and HCT-116 (IC50 values 0.33–0.35 µM). The yield of camptothecin was maximized by nutritional optimization of P. chrysogenum with a Plackett-Burman design, and the productivity of camptothecin increased by 1.8 fold (200 µg/L), compared to control fungal cultures. Upon storage at 4 °C as slope culture for 8 months, the productivity of camptothecin for P. chrysogenum was reduced by 40% compared to the initial culture. Visual fading of the mycelial pigmentation of P. chrysogenum was observed during fungal storage, matched with loss of camptothecin productivity. Methylene chloride extracts of Cliona sp. had the potency to completely restore the camptothecin productivity of P. chrysogenum, ensuring the partial dependence of the expression of the camptothecin biosynthetic machinery of P. chrysogenum on the chemical signals derived from the sponge, or the associated microbial flora. This is the first report describing the feasibility of P. chrysogenum, endozoic of Cliona sp., for camptothecin production, along with reliable metabolic biosynthetic stability, which could be a new platform for scaling-up camptothecin production.


Introduction
Cancer is the major cause of death worldwide, with an annual increase in the number of cases. With these elevated mortality rates, exploring novel approaches to cancer therapy

Collection of Marine Sponges, Isolation and Morphological Identification of the Endozoic Fungi
Two marine sponges, Cliona sp. and Hymedesmia sp. belonging to class Demospongiae, were collected from the Red Sea, 20 km away from Sharm El sheikh, Egypt [27 • 45 57.8 N 34 • 22 10.8 E] using scuba diving at a depth of 8:10 m off, during Nov-Dec /2018. The collected sponges were immediately frozen and kept at −20 • C to maintain the normal endogenous microbial flora. The sponges were identified by a staff member of the Marine Science Department, Faculty of Science, Suez Canal University. The sponges were brought to the laboratory in a sterile ice box, washed thoroughly with sterile sea water to eliminate adherent surface debris prior to isolation of their associated fungi, and sectioned into small segments of approximately 1 cm × 1 cm. The parts were surface sterilized with EtOH 70% (vol/vol) for 1 min, followed by 2.5% sodium hypochlorite for 2 min, then rinsed with sterile sea water to avoid epiphytic microbes [38][39][40][41]. The surface sterilized sponge parts were placed on the surface of potato dextrose agar (200 g potato extract, 20 g glucose, 20 g agar per liter), malt yeast agar (MYA), malt extract agar (MA) and Czapek's-Dox media (3.0 g NaNO 3 , 1.0 g KH 2 PO 4 , 0.5 g MgSO 4 ·7H 2 O, 0.5 g KCl, 0.01 g FeSO 4 .7H 2 O, 30 g glucose, 20 g agar) dissolved in 1 L of distilled water. Chloramphenicol (0.2 g) was added to the media and the plates with different media and sponge parts were incubated for 15 days at 30 • C [42][43][44]. The developed fungal colonies were purified by subculturing on the same media, and the purified fungal isolates were kept as slope cultures at 4 • C.

Screening and Chromatographic Analyses of Camptothecin from the Potent Fungi
The recovered endophytic fungi from the marine sponges were screened for camptothecin production by growing in potato dextrose broth (PDB) (BD Difco, Cat# DF0549-17-9) [48]. Each fungal isolate (2 agar plugs of 5 mm) of 6 days old PDA culture was inoculated into 50 mL PDB medium/250 mL Erlenmeyer flask. Three biological replicates of each fungal isolate were conducted. After incubation of the cultures at 30 • C for 10 days, the cultures were filtered by sterile cheesecloth, and the filtrates were centrifuged at 5000 rpm to remove any particulates. Camptothecin was extracted from the supernatant by CHCl 3 :MeOH (4:1), the organic phase was concentrated to a crude oily extract. The extract was fractionated on TLC (Merck 1 mm (20 cm × 20 cm), Silica gel 60 F254, Merck KGaA, Darmstadt, Germany) with the developing solvent system chloroform: methanol (9:1, v/v) [22,33,41]. The plates were visualized by UV illumination at 254 nm, the putative camptothecin spots with the same blue color and relative mobility as the authentic specimen (Cat. 7689-0 3-4) were considered, and their intensities were determined by Image J software package relative to the authentic example. The putative spots of silica containing camptothecin were scraped off, dissolved in methanol, and camptothecin was extracted as described by [22]. The purity and concentration of camptothecin were determined by HPLC (YOUNG In, Chromass, 9110+ Quaternary Pump, Korea) using a C18 reverse phase column (Eclipse Plus C18 4.6 mm × 150 mm, 3.5 µm, Cat. #959963-902) with isocratic mobile phase methanol/water (60:40 v/v) at a flow rate 1.0 mL/min for 20 min, scanned by photodiode array detector (DAD). The chemical identity and concentration of the putative camptothecin were confirmed from retention time and peak area of authentic example at λ 360 nm [22].

UV-Vis, FT-IR, and LC-MS Analyses
The putative spots of camptothecin, were scraped from the silica plate, dissolved in methanol, and scanned by UV-vis spectrophotometer (RIGOL, Ultra-3000 Series) at λ 300-400 nm. The concentration of the putative camptothecin was determined and compared to the authentic concentration of camptothecin, using methanol as blank baseline.
The FT-IR spectra of camptothecin were analyzed with a Bruker FT-IR Spectrometer in a range of 400-4000 cm −1 with KBr pellets.
The chemical structure of the extracted camptothecin was resolved from the 1 H and 13 C NMR analysis by JEOL (ECA-500II) 500 MHz NMR. The sample was dissolved in CDCl 3 , and the chemical shifts and coupling constants were expressed as parts per million (δ-scale) and hertz (Hz), respectively.
The chemical identity of putative camptothecin was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS), with a Thermo Scientific LCQ Deca mass spectrometer and Hypersil Gold aQ (C18 column), with an electrospray source in positiveion mode. The mobile phases A (0.1% formic acid), and B (acetonitrile in 0.1% formic acid) were used [22,33,48]. The gradient elution system was 2-98% mobile phase B over 30 min at a flow rate of 0.2 mL/min for 40 min. The chemical identity of the resolved signals was determined regarding their mass spectral fragmentation pattern and retention times by NIST mass spectral library.

Molecular Identification of the Recovered Endozoic Fungi
The recovered fungal endophytes from the sponges were morphologically identified regarding their micro-morphological and macromorphological features as adopted by the universal fungal identification keys [45][46][47]49]. The identity of the potent camptothecin producing fungi was molecularly confirmed from the sequence of their internal transcribed spacers (ITS) [40,[50][51][52][53]. Genomic DNA (gDNA) from the potent fungal isolates was extracted by cetyl-trimethyl-ammonium bromide (CTAB) reagent [40,52]. The purity and concentration of the extracted gDNA was checked and determined by 1.5% agarose gel. The fungal gDNA was used as a template for PCR with the primer set; ITS5 5 -TCCTCCGCTTATTGATATGC-3 , ITS4 5 -GAAGTAAAAGTCGTAA-CAAGG-3 . The PCR reaction contains 10 µL of 2 × PCR master mixture (i-Taq™, Cat. No. 25027), 1 µL of gDNA, 1 µL of primers (10 pmol) and completed to 20 µL total volume. The PCR was programed at initial denaturation 94 • C for 2 min, followed by 35 cycles at denaturation 94 • C for 30 s, annealing 55 • C for 20 s, extension 72 • C for 40 s, and final extension 72 • C for 2 min. The PCR products were analyzed by 2% agarose gel, sequenced by an Applied Biosystems Sequencer, HiSQV Bases. The sequences were non-redundantly BLAST searched on NCBI, imported into MEGA 7.0 software and aligned by Clustal W muscle algorithm [54] and the phylogenetic relatedness was created by the neighbor-joining method [54,55].

Bioprocess Optimization of Camptothecin Production by the Potent Fungal Isolate with Plackett-Burman and Faced Central Composite Designs (FCCD)
Various physicochemical parameters, acid whey, malt extract, potato starch, methyl jasmonate, tryptamine, peptone, dextrin, tryptone, glucose, salicylic acid, tryptophan, serine, cysteine, pyruvate, phenylalanine, and glutamate, were optimized by Plackett-Burman design to maximize the yield of camptothecin from the tested fungal isolate [22,33,56]. The sixteen parameters were screened by two variables of Plackett-Burman design, each represented by high (+) and low (−) levels, according to the first order reaction: Y is the predicted yield of camptothecin, Xi is an independent variable, βi is the linear coefficient, and β0 is the model intercept. Triplicates for each run were conducted, and the average camptothecin yield was used as the main response. The highest significant independent variables controlling camptothecin productivity by the selected fungal isolate were optimized by Faced Central Composite design (FCCD) to determine the individual interactions of the tested variables [57,58]. With the FCCD, each variable was represented by three levels, low (−1), medium (0), high (+1), and the center point. The regression model showing the linear quadratic and interaction coefficients follows the second-ordered polynomial equation: where Y is the predicted response, β0 is the regression coefficient, βi is the linear coefficient, βii is the quadratic coefficient, and Xi is the coded level of independent variable.

Effect of Fungal Growth Inhibitors/Elicitors on Camptothecin Production by the Potent Fungal Isolate
The influence of different fungal growth inhibitors on triggering the camptothecin productivity of the selected fungal isolate was evaluated [22,33,42,59,60]. The fungal cultures were grown in the optimal media for 5 days at 30 • C, amended with different concentrations of fluconazole, griseofulvin, lamifen, methyl-jasmonate and NaCl at 1.0, 5.0, and 10.0 mM final concentration, then the cultures were further incubated for 15 days at standard conditions. Camptothecin was extracted and quantified by TLC and HPLC and described above [22].

Stability of Camptothecin Productivity during Fungal Storage, and Effect of Sponge Extracts on Restoring Biosynthetic Machinery
The biosynthetic stability of camptothecin productivity by Penicillium chrysogenum in response to storage was evaluated. The axenic fungal isolate "first isolated" has been stored as slope culture at 4 • C, and the camptothecin productivity was evaluated monthly for 10 months by growing the fungus on the optimized medium, followed by extraction and quantification of camptothecin by TLC and HPLC [22,33].
The influence of different organic extracts of the marine sponge. i.e., chloroform, methanol, ethyl-acetate and methylene chloride, on the productivity of camptothecin from the tested fungus was assessed [22,33]. Five grams of fresh sponge materials were grinded in 50 mL of each solvent, and kept at 4 • C for 12 h. The extracts were filtered, centrifuged at 5000 rpm, and concentrated by evaporation till 10 mL. Different volumes (0.5, 2 and 4 mL) of each extract were amended for the 5 days old culture, and incubation continued for 14 days under standard conditions. The same solvent extracts of the sponge in fungus-free media were used as negative control, along with normal control of fungal cultures without sponge extracts. After incubation of the culture under standard conditions, camptothecin was extracted and quantified as described above.

Antiproliferative Activity of the Purified Camptothecin from Penicillium Chrysogenum
The antiproliferative activity of the extracted camptothecin CPT against human Larynx carcinoma (HEP-2) and colon carcinoma (HCT-116) cell lines was determined by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay [61]. The 96 microtiter plate was seeded with 10 3 cells/well, and incubated for 12 h at 37 • C, then amended with different concentrations of camptothecin, and the plates were re-incubated for 48 h. The MTT reagent (25 µL) was added, and the developed purple color of formazan complex was measured at λ 570 nm after 2 h. The IC 50 value was expressed by the amount of camptothecin suppressing the growth of 50% of the initial number of cells, normalizing to positive controls.

Statistical Analysis
The experiments were conducted in biological triplicates and the results were expressed by means ± STD. The significance and F-test were calculated using one-way ANOVA with Fisher's Least Significant Difference post hoc test.

Isolation, and Screening for Camptothecin from Marine Sponges-Derived Fungi
Two marine sponges, Cliona sp. and Hymedesmia sp., were isolated from the Red Sea at a depth of 10 m, and their fungal flora were isolated on PDA, Czapek's-Dox and malt extract media. Thirteen fungal isolates were recovered from the marine sponges; five isolates were from Cliona sp. and eight were isolates from Hymedesmia sp. (Table 1). These fungal isolates were morphologically identified based on their macroscopical and microscopical features according to the universal keys of fungal identification. These fungal flora belonged to two genera, Aspergillus and Penicillium, at 70% and 25%, respectively. The camptothecin productivity from the fungi isolates derived from the sponges was assessed by growing on PDB medium, incubated for 10 days under standard conditions, then camptothecin was extracted and quantified by TLC and HPLC ( Figure 1). From the screening paradigm, and the fungal isolates derived from Cliona sp, Penicillium chrysogenum was the highest camptothecin producer (110 µg/L), followed P. citrinum (38 µg/L), while the remaining fungal isolates exhibited a mild yield of camptothecin (0-10 µg/L). Practically, the productivity of the same species of the genus of Aspergillus derived from the different types of marine sponge was complete, ensuring the role of the fungal-animal interaction, and biological and physiological difference in manipulating the expression of camptothecin biosynthetic machinery of the potent fungus. Thus, the biological identity of marine sponge could make a significant contribution not only to the identity of their endogenous associated fungal flora, but also to the pattern of metabolic identity of the derived fungal isolates.

Molecular Identification of Potent Camptothecin Producing Fungi
The most potent camptothecin producing fungal isolate, Penicillium chrysogenum derived from Cliona sp. was further identified based on the sequence of their ITS regions. For the morphological description, the fungal isolate was grown on PDA and Malt Extract media for 10 days at 30 °C, and the macroscopical and microscopical features were observed daily. The fungal matter grew rapidly on PDA, with blue-brown, deep blue conidial pigmentation and mycelia color, with yellow edges of the colonies and absence of soluble exudates, and with a brown reverse on the colonies ( Figure 2). The fungus has a 2-4 divergent Penicilli, with bi-verticillate branching pattern, green conidia, globose, sub-gobose conidial heads and smoothed condidial cell walls. These morphological features are closely matched with the descriptions of P. chrysogenum [46,49,56,62,63]. The morphological identity of the isolate P. chrysogenum has been confirmed from the molecular sequence of the ITS region. The gDNA of the fungus was used as the template for PCR, and the PCR amplicon was around 650 bp ( Figure 2). The PCR amplicon was purified and sequenced, and BLAST searched non-redundantly on the GenBank. The target sequence displayed a 99.9% similarity with the deposited sequence of P. chrysogenum, with zero E value. The ITS sequence of the fungus was deposited in the GenBank with accession # OL597937.1. From the BLAST and phylogenic analysis, two cluster of Penicillium sp. arose, Cluster I and Cluster II ( Figure 2). The ITS sequence of P. chrysogenum EFBL displayed a 99.9% similarity with the P. chrysogenum isolates of accession numbers MK761052.

Molecular Identification of Potent Camptothecin Producing Fungi
The most potent camptothecin producing fungal isolate, Penicillium chrysogenum derived from Cliona sp. was further identified based on the sequence of their ITS regions. For the morphological description, the fungal isolate was grown on PDA and Malt Extract media for 10 days at 30 • C, and the macroscopical and microscopical features were observed daily. The fungal matter grew rapidly on PDA, with blue-brown, deep blue conidial pigmentation and mycelia color, with yellow edges of the colonies and absence of soluble exudates, and with a brown reverse on the colonies ( Figure 2). The fungus has a 2-4 divergent Penicilli, with bi-verticillate branching pattern, green conidia, globose, sub-gobose conidial heads and smoothed condidial cell walls. These morphological features are closely matched with the descriptions of P. chrysogenum [46,49,56,62,63]. The morphological identity of the isolate P. chrysogenum has been confirmed from the molecular sequence of the ITS region. The gDNA of the fungus was used as the template for PCR, and the PCR amplicon was around 650 bp ( Figure 2). The PCR amplicon was purified and sequenced, and BLAST searched nonredundantly on the GenBank. The target sequence displayed a 99.9% similarity with the deposited sequence of P. chrysogenum, with zero E value. The ITS sequence of the fungus was deposited in the GenBank with accession # OL597937.1. From the BLAST and phylogenic analysis, two cluster of Penicillium sp. arose, Cluster I and Cluster II ( Figure 2). The ITS sequence of P. chrysogenum EFBL displayed a 99.9% similarity with the P. chrysogenum isolates of accession numbers MK761052.

Chromatographic and Spectroscopic Analyses, and Antiproliferative Activity of Extracted Camptothecin
The chemical identity of the putative camptothecin from P. chrysogenum was confirmed from the TLC, HPLC, and LC-MS analyses, comparing to the authentic sample. Camptothecin was extracted, fractionated by TLC, and the silica gel spots containing camptothecin were scraped off and dissolved in methanol for chemical analysis. From the HPLC chromatogram, the putative sample of camptothecin gave the same retention time (7.42 min) as the authentic one ( Figure 3), ensuring its chemical proximity as camptothecin. From the UV-absorption spectra (Figure 3), the putative camptothecin sample from P. chrysogenum displayed the maximum absorption peak at absorbance 290 nm, identical to the standard absorption spectrum of camptothecin. The chemical identity of P. chrysogenum camptothecin was verified from the 1 HNMR, displaying the same resolved signals as the authentic one, distributed between 1.0 and 8.0 ppm, with three proton signals resolved at 1.0-2.5 ppm corresponding to methyl, acetate and acetylene groups. From the FTIR spectra, the extracted P. chrysogenum camptothecin had peaks at 3406.6 and 3393.3 cm −1 , assigned for the hydroxyl (OH) and amide group stretches, respectively. Peaks of 2923.56, 1729.83 and 1604.5 cm −1 were assigned to the aliphatic CH, ester groups and aromatic rings stretch, respectively. The COO stretching frequency peaked at 1268.9 cm −1 , while peak at 1029.8 cm −1 was assigned for the aromatic C and H blends ( Figure 3).

Chromatographic and Spectroscopic Analyses, and Antiproliferative Activity of Extracted Camptothecin
The chemical identity of the putative camptothecin from P. chrysogenum was confirmed from the TLC, HPLC, and LC-MS analyses, comparing to the authentic sample. Camptothecin was extracted, fractionated by TLC, and the silica gel spots containing camptothecin were scraped off and dissolved in methanol for chemical analysis. From the HPLC chromatogram, the putative sample of camptothecin gave the same retention time (7.42 min) as the authentic one ( Figure 3), ensuring its chemical proximity as camptothecin. From the UV-absorption spectra (Figure 3), the putative camptothecin sample from P. chrysogenum displayed the maximum absorption peak at absorbance 290 nm, identical to the standard absorption spectrum of camptothecin. The chemical identity of P. chrysogenum camptothecin was verified from the 1 HNMR, displaying the same resolved signals as the authentic one, distributed between 1.0 and 8.0 ppm, with three proton signals resolved at 1.0-2.5 ppm corresponding to methyl, acetate and acetylene groups. From the FTIR spectra, the extracted P. chrysogenum camptothecin had peaks at 3406.6 and 3393.3 cm −1 , assigned for the hydroxyl (OH) and amide group stretches, respectively. Peaks of 2923.56, 1729.83 and 1604.5 cm −1 were assigned to the aliphatic CH, ester groups and aromatic rings stretch, respectively. The COO stretching frequency peaked at 1268.9 cm −1 , while peak at 1029.8 cm −1 was assigned for the aromatic C and H blends ( Figure 3).

Bioprocess Optimization of Camptothecin Production by P. chrysogenum with Plackett-Burman Design
Among the tested isolates, P. chrysogenum was selected for further nutritional optimization to maximize its yield of camptothecin. The chemical identity of medium components and their interactions have a pivotal role in the regulation of the biosynthetic machineries of secondary metabolites by fungi. Consequently, the medium components were nutritionally optimized by response surface methodology with Plackett-Burman's design as "first order model equation" to maximize the yield of camptothecin. Sixteen runs were conducted to assess the effect of the different variables on camptothecin productivity for P. chrysogenum (Table 2). These variables include different carbon and nitrogen precursors of camptothecin in addition to growth modulators and elicitors. The significance of the tested independent variables influencing camptothecin productivity by P. chrysogenum, with the predicted and corresponding actual responses, was summarized in the Plackett-Burman design matrix ( Table 3). The actual and predicted yield of camptothecin by P. chrysogenum noticeably fluctuated from 50.3-203.5 μg/L, confirming the significance of the tested variables on camptothecin biosynthesis, and the efficiency of the Plackett-Burman design, the values of the coefficient of determination (R2 = 0.98) indicating the goodness-of-fit measure for the linear regression models ( Table 4). The variability in the response of camptothecin production was attributed to the selected independent variables

Bioprocess Optimization of Camptothecin Production by P. chrysogenum with Plackett-Burman Design
Among the tested isolates, P. chrysogenum was selected for further nutritional optimization to maximize its yield of camptothecin. The chemical identity of medium components and their interactions have a pivotal role in the regulation of the biosynthetic machineries of secondary metabolites by fungi. Consequently, the medium components were nutritionally optimized by response surface methodology with Plackett-Burman's design as "first order model equation" to maximize the yield of camptothecin. Sixteen runs were conducted to assess the effect of the different variables on camptothecin productivity for P. chrysogenum (Table 2). These variables include different carbon and nitrogen precursors of camptothecin in addition to growth modulators and elicitors. The significance of the tested independent variables influencing camptothecin productivity by P. chrysogenum, with the predicted and corresponding actual responses, was summarized in the Plackett-Burman design matrix ( Table 3). The actual and predicted yield of camptothecin by P. chrysogenum noticeably fluctuated from 50.3-203.5 µg/L, confirming the significance of the tested variables on camptothecin biosynthesis, and the efficiency of the Plackett-Burman design, the values of the coefficient of determination (R 2 = 0.98) indicating the goodness-of-fit measure for the linear regression models ( Table 4). The variability in the response of camptothecin production was attributed to the selected independent variables but the remaining variations were (0.01%). The values of the adjusted determination coefficient (Adj. R 2 = 0.92), F-value (16.524) and F-value (0.02 < 0.05) reveal the significance of this model. The analysis of variance (ANOVA) of the experimental design results was calculated and the coefficients, t Stat, p-value, and confidence levels were also recorded ( Table 4). The main effects and the normal probability of the tested variables were measured and plotted ( Figure 5), indicating that there are five different independent factors, Phenylalanine (X15), Pyruvate (X14), Methyl Jasmonate (X4), Salicylic acid (X10) and cysteine (X13), that positively influence camptothecin yield, while the other independent factors have a negative effect. The highest camptothecin yield (199 µg/L) was recorded at run 14, while the lowest value (52 µg/L) was observed in run 7. The significance of the variables affecting camptothecin productivity by P. chrysogenum is displayed in the Pareto Chart, as well as the probability plot of independent variables, actual and predicted yield of camptothecin ( Figure 5). The significance of each variable was assessed from the p-value and student's t-test as reported in Table 4. The arrangement of the points of residuals around the diagonal line shows the independent normal distribution of the variables, suggesting the perfect fitting of predicted and actual camptothecin yield. From the ANOVA analysis, the constructed model was highly significant, as shown from the values of Fisher's F-test 3.3 and probability p-value 0.0335. The first order polynomial equation of camptothecin production by P. chrysogenum regarding the significant independent variables was derived from the following equation:    The highest actual (199 µg/L) and predicted yield (203 µg/L) of camptothecin by P. chrysogenum was detected at run # 14, with the residual value-4.8. At run # 14, the maximum yield of camptothecin was detected at higher concentration variables of phenylalanine (5 g/L), pyruvate (20 g/L), methyl jasmonate (20 g/L), salicylic acid (4 g/L), cysteine (10 g/L), phenylalanine (8 g/L) on the 15th incubation day at 30 • C, with lower doses of other remaining variables (Table 4). Thus, upon using Plackett-Burman design, the yield of camptothecin by P. chrysogenum was increased by about 1.8 fold (200 µg/L) compared to control "non-optimized" fungal cultures (110.6 µg/L). A similar optimization protocol has been developed to optimize the camptothecin productivity by A. terreus and A. flavus (24), ensuring the successfulness of the optimization process by the response surface methodology of the cultural variables compared to classical one-one factor optimization.

Effect of Fungal Growth Inhibitors on Camptothecin Yield by P. chrysogenum
The effect of incorporation of fungal growth inhibitors griseofulvin, terbinafine, and fluconazole (1, 5, and 10 mM final conc.) on camptothecin yield by P. chrysogenum was assessed. The fungal isolate was grown on the optimized media, the different concentrations of each compound were added, and the cultures were continue incubated for 14 days; camptothecin was extracted and quantified by HPLC. From the obtained results ( Figure 6), there is no obvious inducing effect by inhibitors on the biosynthetic machinery of camptothecin for P. chrysogenum. The lack of inducing effect of these inhibitors on the biosynthetic machinery of camptothecin ensures the independence of camptothecin biosynthetic systems for this fungus on the external stimuli. Since the fungus has been isolated from the marine sponge, the effect of salinity might play a role on inducing the expression system for the biosynthesis of camptothecin. To validate this hypothesis, different salt concentrations were amended to the cultures, and the yield of camptothecin was evaluated as described above. From the results (Figure 6), an obvious decrease in the camptothecin yield by P. chrysogenum was recorded upon addition of NaCl. The highest yield of camptothecin was determined in the absence of NaCl, while the yield of camptothecin decreased by about 50% upon addition of 100 mM NaCl to the culture media of P. chrysogenum. The highest actual (199 μg/L) and predicted yield (203 μg/L) of camptothecin by P. chrysogenum was detected at run # 14, with the residual value-4.8. At run # 14, the maximum yield of camptothecin was detected at higher concentration variables of phenylalanine (5 g/L), pyruvate (20 g/L), methyl jasmonate (20 g/L), salicylic acid (4 g/L), cysteine (10 g/L), phenylalanine (8 g/L) on the 15th incubation day at 30 °C, with lower doses of other remaining variables (Table 4). Thus, upon using Plackett-Burman design, the yield of camptothecin by P. chrysogenum was increased by about 1.8 fold (200 μg/L) compared to control "non-optimized" fungal cultures (110.6 μg/L). A similar optimization protocol has been developed to optimize the camptothecin productivity by A. terreus and A. flavus (24), ensuring the successfulness of the optimization process by the response surface methodology of the cultural variables compared to classical one-one factor optimization.

Effect of Fungal Growth Inhibitors on Camptothecin Yield by P. chrysogenum
The effect of incorporation of fungal growth inhibitors griseofulvin, terbinafine, and fluconazole (1, 5, and 10 mM final conc.) on camptothecin yield by P. chrysogenum was assessed. The fungal isolate was grown on the optimized media, the different concentrations of each compound were added, and the cultures were continue incubated for 14 days; camptothecin was extracted and quantified by HPLC. From the obtained results ( Figure 6), there is no obvious inducing effect by inhibitors on the biosynthetic machinery of camptothecin for P. chrysogenum. The lack of inducing effect of these inhibitors on the biosynthetic machinery of camptothecin ensures the independence of camptothecin bio- synthetic systems for this fungus on the external stimuli. Since the fungus has been isolated from the marine sponge, the effect of salinity might play a role on inducing the expression system for the biosynthesis of camptothecin. To validate this hypothesis, different salt concentrations were amended to the cultures, and the yield of camptothecin was evaluated as described above. From the results (Figure 6), an obvious decrease in the camptothecin yield by P. chrysogenum was recorded upon addition of NaCl. The highest yield of camptothecin was determined in the absence of NaCl, while the yield of camptothecin decreased by about 50% upon addition of 100 mM NaCl to the culture media of P. chrysogenum.

Biosynthetic Stability of Camptothecin by P. chrysogenum in Response to Storage, and Effect of Sponge Extracts on Restoring Its Biosynthetic Machinery
The biosynthetic stability of camptothecin productivity by Penicillium chrysogenum in response to storage was evaluated. The axenic first fungal isolate, maintained as slope culture on PDA at 4 °C, was grown on the optimized media, and their camptothecin productivity over a 10 month interval was determined by standard conditions. From the results (Figure 6), it can be seen that the camptothecin productivity slightly decreased with storage of the fungus as slope culture at 4 °C. After 8 months of storage at 4 °C, the residual camptothecin productivity for P. chrysogenum was reduced to 119 μg/L, i.e., about a 40% loss, compared to zero culture (188 μg/L). A noticeable decrease in camptothecin productivity for P. chrysogenum was observed with fungal storage, consistent with the overall physiological features of secondary metabolites' attenuation by fungi [33,[40][41][42]56,62]. Remarkably, a visual fading to the mycelial pigmentation of P. chrysogenum was observed Figure 6. Effect of sea water concentration and growth inhibitors on camptothecin productivity for P. chrysogenum. The yield of camptothecin for P. chrysogenum in response to different sea water concentration (A), and growth inhibitors Grisofulvin, terbinafine (B). (C) The productivity of camptothecin for P. chrysogenum in response to fungal storage periods as slope culture. (D) The yield of camptothecin for P. chrysogenum in response to amendment with different extracts of Cliona sp.

Biosynthetic Stability of Camptothecin by P. chrysogenum in Response to Storage, and Effect of Sponge Extracts on Restoring Its Biosynthetic Machinery
The biosynthetic stability of camptothecin productivity by Penicillium chrysogenum in response to storage was evaluated. The axenic first fungal isolate, maintained as slope culture on PDA at 4 • C, was grown on the optimized media, and their camptothecin productivity over a 10 month interval was determined by standard conditions. From the results (Figure 6), it can be seen that the camptothecin productivity slightly decreased with storage of the fungus as slope culture at 4 • C. After 8 months of storage at 4 • C, the residual camptothecin productivity for P. chrysogenum was reduced to 119 µg/L, i.e., about a 40% loss, compared to zero culture (188 µg/L). A noticeable decrease in camptothecin productivity for P. chrysogenum was observed with fungal storage, consistent with the overall physiological features of secondary metabolites' attenuation by fungi [33,[40][41][42]56,62]. Remarkably, a visual fading to the mycelial pigmentation of P. chrysogenum was observed with fungal storage, strongly matched with the sequential loss of camptothecin productivity, ensuring the metabolic and genomic correlation of both biosynthetic pathways of camptothecin and melanin pigments [64].
The influence of different organic extracts, chloroform, methanol, ethyl-acetate and methylene chloride, for the marine sponge "Cliona sp." on restoring the productivity of camptothecin by P. chrysogenum was assessed. The extracts of Cliona sp. were amended to 6 month stored P. chrysogenum, and the fungal productivity of camptothecin was assessed by TLC, HPLC and LC-MS analyses. From the LC-MS profile (Figure 7), methylene chloride extracts of Cliona sp. have the maximum potency in restoring the camptothecin biosynthetic machinery of P. chrysogenum (Figure 6), with a lack of inducing effect in the other tested extracts. With methylene chloride extract, the yield of camptothecin was increased to 244 µg/L, i.e., by about 2.1 fold, compared to the 6 month stored P. chrysogenum culture (120 µg/L). From the profile of LC-MS, several metabolic intermediates were induced upon addition of sponge extracts, ensuring the dependence of the expression of a plethora of metabolites on the signals derived from the sponge, and these signals are mainly extracted from methylene chloride. Thus, it could be deduced that the expression of the camptothecin biosynthetic machinery of P. chrysogenum could be partially dependent on the chemical signals derived from the sponge, or from the associated microbial flora of the sponge. with fungal storage, strongly matched with the sequential loss of camptothecin productivity, ensuring the metabolic and genomic correlation of both biosynthetic pathways of camptothecin and melanin pigments [64]. The influence of different organic extracts, chloroform, methanol, ethyl-acetate and methylene chloride, for the marine sponge "Cliona sp." on restoring the productivity of camptothecin by P. chrysogenum was assessed. The extracts of Cliona sp. were amended to 6 month stored P. chrysogenum, and the fungal productivity of camptothecin was assessed by TLC, HPLC and LC-MS analyses. From the LC-MS profile (Figure 7), methylene chloride extracts of Cliona sp. have the maximum potency in restoring the camptothecin biosynthetic machinery of P. chrysogenum (Figure 6), with a lack of inducing effect in the other tested extracts. With methylene chloride extract, the yield of camptothecin was increased to 244 μg/L, i.e., by about 2.1 fold, compared to the 6 month stored P. chrysogenum culture (120 μg/L). From the profile of LC-MS, several metabolic intermediates were induced upon addition of sponge extracts, ensuring the dependence of the expression of a plethora of metabolites on the signals derived from the sponge, and these signals are mainly extracted from methylene chloride. Thus, it could be deduced that the expression of the camptothecin biosynthetic machinery of P. chrysogenum could be partially dependent on the chemical signals derived from the sponge, or from the associated microbial flora of the sponge.

Discussion
Camptothecin is one of the most powerful alkaloids for cancer therapy due to its unique affinity for binding with DNA topoisomerase I [9,65], blocking its various biological processes: DNA replication, RNA transcription and chromatin assembly. Camptothecin derivatives are one of the most commonly prescribed anticancer drugs, comparable to Taxol and vincristine, mainly extracted from the plant Camptotheca acuminata, inhabiting China and India [8]. However, the tiny yield of camptothecin from natural plant sources, difficulties in extraction, the vulnerability of the yield of this plant to environmental and ecological conditions, and the massive harvesting of the plant causing destruction to the ecological balance are all major challenges [1,66]. Nevertheless, with the emergence of endophytic fungi as an eminent producer of camptothecin, a new promising platform has been raised for the fungal fast growth, feasibility of mass production, independence from environmental conditions and feasibility of metabolic engineering [32,33,64,67]. However, the tiny yield and loss of the camptothecin biosynthetic potency of fungi during storage and subculture are the main challenges that halt the further industrial application of fungi [22,33,41,59,64,68]. Thus, searching for novel camptothecin producing fungal isolates, with metabolic/biosynthetic stability and sustainability for camptothecin biosynthesis, is the main objective of this work. Recently, marine fungi have been reported as an untapped repertoire for novel bioactive secondary metabolites, with diverse biological and pharmaceutical activities [34]. However, the biological and metabolic identities of marine fungi have received less attention and less exploration, compared to their terrestrial counterparts [35]. Thus, marine fungi could be a potential candidate for discoveries of novel compounds with unique chemical skeletons and scaffolds that can be modified to produce novel bioactivity and pharmaceutical activity. The rationality of producing a wide range of metabolites from marine fungi could be attributed to the diverse ecological, physical and biological factors that could be epigenetic regulators of the biosynthetic gene clusters of secondary metabolites. Sponges are one of the most recognized reservoirs of marine fungi, and about 40-60% of the sponge biomass mainly consists of associated microorganisms, along with sponge feeding [69]. Several marine fungi associated with the marine sponge were isolated and partially characterized [70,71]. Several types of Mediterranean marine sponges Ircinia variabilis, Suberites zeteki and Mycale armata were studied and their fungal associations were described [34,37,72]. Thus, isolating novel fungal isolates producing camptothecin, while evaluating their potential metabolic stability for camptothecin biosynthesis, is the objective of this study.
The marine sponges Cliona sp. and Hymedesmia sp. were collected from the Red Sea at a depth 10 m, and their fungal flora were isolated. Five isolates associated with Cliona sp. and eight isolates derived from Hymedesmia sp. were isolated. From the screening profile, Penicillium chrysogenum associated with Cliona sp. was the highest camptothecin producer, followed by P. citrinum. Practically, the yield of camptothecin of P. chrysogenum associated with Cliona sp. is much higher than that of the closely related isolate P. lilacinum associated Hymedesmia sp., ensuring the role of fungal-animal interaction and biological and physiological differences in manipulating the expression of the camptothecin encoding gene cluster. This is the first report describing the fungal flora of the marine sponges Cliona sp. and Hymedesmia sp., ensuring the metabolic uniqueness of their associated fungal flora. The morphological identity of P. chrysogenum EFBL, derived from Cliona sp., has been molecularly confirmed based on the ITS sequence, and deposited on GenBank with accession # OL597937.1. From the literature, this is the first report describing the metabolic potency of the marine fungal derived fungus P. chrysogenum as camptothecin producer that could produce novel biosynthetic machinery for sustainable camptothecin production. The yield of camptothecin for P. chrysogenum (110 µg/L), derived from Cliona sp., is higher than that of Aspergillus terreus (90 µg/L) and A. flavus (75 µg/L) endophytes of Ficus elastica, and A. fumigatus (35 µg/L) endophytes of Delonix regia [22,73]. The yield of camptothecin for P. chrysogenum is higher than that reported for A. flavus (51.7 µg/L) and A. flavus (37.2 µg/L), an endophyte of Astragalus fruticosus. The higher yield of endozoic associated P. chrysogenum from the sponge Cliona sp. could be ascribed to the diverse chemical signals due to fungal-animal interactions, or the identity of microbiome interactions and other related chemical signals that trigger the expression of camptothecin encoding genes. Similar results confirm the presence of the camptothecin biosynthetic machinery of A. flavus, an endophyte of Ficus elastica, using the same screening paradigm [22,59,62,73].
The yield of camptothecin by P. chrysogenum has been maximized by nutritional optimization of the medium components to assess their interactions in regulating the biosynthetic machinery of secondary metabolites by fungi [40,52,59,64,78,79]. The variables, including various proportions of carbon and nitrogen, as well as growth modulators and elicitors, were optimized by Plackett-Burman design to maximize the yield of camptothecin.
From the probability of the tested variables, independent factors, especially phenylalanine, pyruvate, methyl Jasmonate, salicylic acid and cysteine, have a positive influence on camptothecin yield. The maximum actual and predicted yield (203 µg/L) of camptothecin for P. chrysogenum was assessed at run # 14, with the residual value 4.8. The yield of camptothecin for P. chrysogenum increased by about 1.8 fold comparing to non-optimized fungal cultures. A similar optimization protocol has been developed to optimize the camptothecin productivity for A. terreus and A. flavus [22,33,64,80], ensuring the success of the optimization process by the response surface methodology of the cultural variables compared to classical one-one factor optimization. Similar optimization protocols have been developed to maximize camptothecin productivity by Fusarium solani, Nothapodytes nimmoniana and Trichoderma atroviride [18,69,81]. Upon addition of methyl-jasmonate, the yield of camptothecin was significantly increased by about 1.6 fold compared to control, this being consistent with the results of camptothecin production by Trichoderma atroviride [82] and A. terreus and A. flavus (El-Sayed et al. 2021). Methyl-jasmonate is the most recognized elicitor, causing crosstalk with plasma membrane receptors, inducing arrays of defense responses including reactive oxygen and nitrogen oxygen species that subsequently induce the expression of secondary metabolites, encoding genes [20,33,83]. The effect of various growth inhibitors on camptothecin productivity for P. chrysogenum was assessed. There is no obvious positive effect of these inhibitors on the biosynthetic machinery of camptothecin by P. chrysogenum, ensuring their lack in the biosynthetic machinery, independently of this fungus, of the external stimuli. The biosynthetic stability of camptothecin productivity by Penicillium chrysogenum in response to storage has been evaluated. The camptothecin productivity slightly decreased during the storage of the fungus; after 8 months of storage at 4 • C, the camptothecin productivity for P. chrysogenum decreased by about 40% compared to the first culture. Unlike the rapid attenuation of camptothecin productivity by A. terreus and A. flavus [33,64,82,83], the camptothecin biosynthetic machinery for P. chrysogenum was slightly stable. The overall biosynthetic attenuation of the secondary metabolites by fungi have been frequently reported [21,24,30,33,64,75,82,83]. The influence of different organic extracts, chloroform, methanol, ethyl-acetate and methylene chloride, from the marine sponge "Cliona sp." on restoring the productivity of camptothecin for P. chrysogenum was assessed. From the LC-MS analysis, it can be seen that methylene chloride extracts of Cliona sp. have the potency to restore the camptothecin biosynthetic machinery of P. chrysogenum, and the yield of camptothecin was increased to 244 µg/L. Several metabolic intermediates were induced upon addition of methylene chloride sponge extracts, ensuring the dependence of the expression of a plethora of metabolites on the signals derived from sponge, or from the sponge associated microbiome.
In conclusion, P. chrysogenum, endozoic of marine sponge Cliona sp., was a potent camptothecin producer. Remarkably, the biosynthetic machinery of camptothecin was relatively stable during fungal storage and subculturing. The slight attenuation of the fungal biosynthetic machinery has been completely restored with the addition of methylene chloride extract of sponge Cliona sp., ensuring the presence of specific signals derived from the sponge tissues, or signals from the associated microbiome of Cliona sp. Further studies are ongoing to uncover the molecular and metabolic identity of camptothecin and the biosynthetic machinery of P. chrysogenum using differential transcriptomic and proteomic approaches in order to stabilize their yield, paving the way for a novel platform for industrial camptothecin production.