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

Abstract

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.

Keywords: Cliona sp.; LC-MS/MS; Penicillium chrysogenum; anticancer activity; camptothecin.

Figure 1

Screening for camptothecin producing fungal endophytes inhabiting marine sponges Cliona sp. and Hymedesmia sp. (A), General view of Cliona sp. and Hymedesmia sp. collected from the Red Sea, 20 km away from Sharm El sheikh, Egypt. (B), The collected fungal isolates from both sponges were grown on PDB for 10 days at 30 °C, then camptothecin was extracted by methylene chloride, checked by TLC, and visualized by UV at wavelength 254 nm comparing to authentic one. (C), TLC chromatogram of methylene chloride extracts from the fungal isolates (Upper panel), and the putative concentration of camptothecin calculated by the Image J software package, normalized to known concentration of authentic camptothecin (Lower panel). (D), HPLC chromatogram of extracted camptothecin and authentic one.

Figure 2

Identification of the potent camptothecin producing fungal isolate from Cliona sp. (A) Macromorphological features of the potent camptothecin producing isolate. (B) Microscopical features of the conidial heads of the fungal isolate at 1000× magnification. (C) Molecular phylogenetic analysis of the ITS sequence of the target isolate by Maximum Likelihood method.

Figure 3

Chemical analysis and anticancer activity of P. chrysogenum extracted camptothecin. After growing P. chrysogenum under standard conditions, camptothecin was extracted and checked by TLC, putative spots scraped off, and camptothecin extracted and chemically analyzed. (A) FTIR chromatogram of extracted camptothecin. (B) LC-MS/MS analyses of extracted camptothecin. The antiproliferative activity of extracted camptothecin against human larynx carcinoma (HEP-2) and colon carcinoma cell lines as revealed from the viability plot (C) and IC₅₀ values (D).

Figure 4

Chemical validation of the extracted camptothecin from P. chrysogenum. The putative spots of camptothecin were scraped-off from the TLC, and its purity was checked by HPLC. (A), LC-MS/MS fragmentation pattern of putative camptothecin from P. chrysogenum. (B), LC MS/MS spectra of the putative purified camptothecin with the onset chemical structure of camptothecin. (C), Scheme of molecular fragmentation pattern of camptothecin as revealed by the LC-MS/MS spectra.

Figure 5

The main effects of different variables on camptothecin production according to the Plackett-Burman experimental design. The normal probability plots of the variables for camptothecin production by P. chrysogenum as determined by the first order polynomial equation. (A) Pareto chart illustrates the order of significance of each variable. (B) Plot of correlation between predicted and actual camptothecin yield of P. chrysogenum. (C), Box-Cox power transform. (D) Plot of standardized effect with normal probability. Plot of standardized effect with normal residuals (E) and desirability (F).

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.

Figure 7

Restoring the biosynthetic potency of camptothecin of the 6-month stored P. chrysogenum upon addition of methylene chloride of Cliona sp. The 6-month stored P. chrysogenum was grown on the optimized medium, and methylene chloride extracts of Cliona sp. were amended to the culture after 5 days of incubation under standard conditions, then camptothecin was extracted and checked by LC-MS/MS. The LC-MS/MS pattern of extracted camptothecin from the control culture of P. chrysogenum (6-months) (A) and culture amended with methylene chloride extracts of Cliona sp. (B). (C) Morphological features showing conidial pigmentation of control culture (upper panel) and amended with methylene chloride extracts of Cliona sp. (lower panel). (D) Yield of camptothecin from the cultures of P. chrysogenum as determined by HPLC.