Elsevier

Bioresource Technology

Volume 188, July 2015, Pages 251-257
Bioresource Technology

Enhanced camptothecin production by ethanol addition in the suspension culture of the endophyte, Fusarium solani

https://doi.org/10.1016/j.biortech.2014.12.106Get rights and content

Highlights

  • Ethanol as carbon and energy source for the endophyte, Fusarium solani.

  • Ethanol as an elicitor to enhance camptothecin production by the endophyte F. solani.

  • Using extract of a non-camptothecin producing plant for camptothecin production.

Abstract

Ethanolic extract of a non-camptothecin producing plant, Catharanthus roseus when added in the suspension culture of the endophyte Fusarium solani known to produce camptothecin, resulted in enhanced production of camptothecin by 10.6-fold in comparison to that in control (2.8 μg/L). Interestingly, addition of pure ethanol (up to 5% v/v) in the suspension culture of F. solani resulted in maximum enhancement in camptothecin production (up to 15.5-fold) from that obtained in control. In the presence of ethanol, a reduced glucose uptake (by ∼40%) and simultaneous ethanol consumption (up to 9.43 g/L) was observed during the cultivation period (14 days). Also, the total NAD level and the protein content in the biomass increased by 3.7- and 1.9-fold, respectively, in comparison to that in control. The study indicates a dual role of ethanol, presumably as an elicitor and also as a carbon/energy source, leading to enhanced biomass and camptothecin production.

Introduction

Camptothecin (CPT) is a pentacyclic quinoline alkaloid ((S)-4-ethyl-4-hydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14-(4H,12H)-dione) which serves as the lead molecule for Topotecan and Irinotecan, two of the FDA-approved, most in demand anti-cancer drugs. It is a highly effective anti-cancer molecule as it acts by binding to the topoisomerase I–DNA complex in cancer cells, thus preventing DNA replication and triggering cell death (Shweta et al., 2010). Several reports also suggest that CPT derivatives are effective in the treatment of AIDS (Sadaie et al., 2004), as anti-viral agents (Liu et al., 2010a), antifungal agents (Zhang et al., 2008), radiation sensitizers (Kvols, 2005), anti-parasitic agents against falciparum malaria (Chauhan and Srivastava, 2001) and trypanosomiasis (Bodley and Shapiro, 1995), and as insecticides (Liu et al., 2010b). Despite the ever-increasing demand, the only commercially available source of CPT is the natural plant extract mainly from Camptotheca acuminata and Nothapodytes nimmoniana (with maximum CPT yield reported up to 0.3% dry weight (DW)) (Padmanabha et al., 2006). Owing to extensive harvesting and habitat loss, N. nimmoniana population has declined by 50–80% in India (Khan, 2010). In case of C. acuminata, over-harvesting by the pharmaceutical industry has decimated the population of the endemic trees in China with less than 4000 of the trees remaining in the wild (Balasubramanian and Narayanan, 2012). Considering these facts, search for alternative sources of the drug has become inevitable. Plant cell cultures (Fulzele et al., 2001, Pi et al., 2010, Saito et al., 2001) and total chemical synthesis (Chavan et al., 2004, Yu et al., 2011) have already been explored but have not been extensively exploited at industrial scale due to limitations like complex and uneconomical production, difficulty in scale-up, slow growth and genetic/biochemical instability of plant cells in culture, etc.

Under these circumstances, the discovery of endophytic fungi capable of producing CPT has offered microbial fermentation route as an exciting and potential alternative for production of CPT at commercial level (Zhao et al., 2010). Despite the extensive documentation of various endophytes capable of producing CPT (Table 1), very few studies have been undertaken on bioprocess development and subsequent optimization for yield and productivity enhancement of CPT from endophytic fungi (Amna et al., 2012, Amna et al., 2006, Musavi et al., 2014, Pu et al., 2013, Rehman et al., 2009). Hence, there is an immense scope for exploring bioprocess optimization strategies and developing an optimized microbial fermentation based production platform for CPT. However, the potential of endophytes as sustainable alternative production platforms has been severely hampered due to the widely reported problem of subculture based attenuation in product yield, resulting in inconsistent and low production. Lack of host stimuli in culture medium and genomic instability or silencing of the biosynthetic genes in axenic cultures have been hypothesized as possible reasons for the attenuation (Sachin et al., 2013). Strategies to overcome the attenuation problem including re-inoculation into the host followed by re-isolation, supplementing the axenic culture with host plant extracts, have been reported to be successful in some cases (Soliman and Raizada, 2013, Zhao et al., 2013) and not so in other cases (Sachin et al., 2013, Kusari et al., 2011). Thus, in order to improve CPT production in potential endophytes, exogenous addition of possible elicitors including small molecules and non-CPT producing plant extracts were explored as yield enhancement strategies. In the present study, the effect of addition of Catharanthus roseus leaf extract and a small molecule elicitor (ethanol) in the growth medium was investigated on biomass and CPT production in the suspension culture of the endophyte, Fusarium solani MTCC 9668.

Section snippets

Fungal culture used

The endophytic fungal strain used in the present study was, F. solani MTCC 9668 (Shweta et al., 2010), procured from Microbial Type Culture Collection and GenBank, Chandigarh, India.

Development of the suspension culture of F. solani MTCC 9668

The CPT yield in the biomass obtained under control conditions was very low (0.5 μg/g DW) and inconsistent. Similar to the observations reported in another endophytic strain of F. solani (Kusari et al., 2011), subsequent attenuation in the CPT yield was observed in the suspension culture of F. solani upon sub-culture in fresh liquid medium. Hence, the inoculum used in all the experiments was freshly prepared each time from the glycerol stocks originally prepared from the lyophilized culture

Conclusions

Addition of a non-CPT producing plant (C. roseus) extract and a small molecule elicitor (ethanol) in the medium enhanced the CPT production in the suspension culture of F. solani MTCC 9668. The study reported for the first time that ethanol can possibly act both as an elicitor and as a preferred carbon/energy source for enhanced CPT production by F. solani suspension culture. The results obtained in the study establish that the manipulation of the culture environment via bioprocess optimization

References (35)

  • D.P. Fulzele et al.

    Growth and production of camptothecin by cell suspension cultures of Nothapodytes foetida

    Planta Med.

    (2001)
  • Khan, N., 2010. Quantitative estimation of camptothecin in Nothapodytes nimmoniana by HPLTC method collected from...
  • S. Kusari et al.

    An endophytic fungus from Camptotheca acuminata that produces camptothecin and analogues

    J. Nat. Prod.

    (2009)
  • S. Kusari et al.

    Effect of artificial reconstitution of the interaction between the plant Camptotheca acuminata and the fungal endophyte Fusarium solani on camptothecin biosynthesis

    J. Nat. Prod.

    (2011)
  • L.K. Kvols

    Radiation sensitizers: a selective review of molecules targeting DNA and non-DNA targets

    J. Nucl. Med.

    (2005)
  • Y.Q. Liu et al.

    Anti-HSV activity of camptothecin analogues

    Nat. Prod. Res.

    (2010)
  • A. Marcoleta et al.

    Glucose and ethanol-dependent transcriptional regulation of the astaxanthin biosynthesis pathway in Xanthophyllomyces dendrorhous

    BMC Microbiol.

    (2011)
  • Cited by (45)

    • Current advances of endophytes as a platform for production of anti-cancer drug camptothecin

      2021, Food and Chemical Toxicology
      Citation Excerpt :

      Consequently, the production of camptothecin was increased by 2.5 times when the endophytic fungus was cultured in Sabouraud medium with tryptophan and leucine (Amna et al., 2012). Venugopalan and Srivastava tried to increase the production of camptothecin in F. solani MTCC 9668 by using C. roseus leaves extract (strictosidine rich) as a precursor for camptothecin biosynthesis (Venugopalan and Srivastava, 2015). The production of camptothecin increased up to 5-fold when ethanolic extract of C. roseus leaves was added in the fermentation medium (Venugopalan and Srivastava, 2015).

    View all citing articles on Scopus
    View full text