Endophytic fungi harbored in Camptotheca acuminata, Hypericum perforatum and Juniperus communis plants as promising sources of camptothecin, hypericin and deoxypodophyllotoxin

Abstract

Endophytic microorganisms are a diverse group of microbes that colonize living, internal tissues of plants without causing any immediate, overt negative effects within the hosts. A number of novel endophytic microorganisms are capable of producing host plant-specific secondary metabolites with therapeutic potential. The main objective of this study was isolation, identification, biological and biochemical characterization of endophytic fungi capable of indigenously producing camptothecin (CPT), hypericin and deoxypodophyllotoxin, harbored in Camptotheca acuminata, Hypericum perforatum and Juniperus communis plants, respectively. Secondary metabolites were identified and quantified by highly selective and sensitive LC-ESI-MS/MS and LC-ESI-HRMSn.

C. acuminata plants were sampled from different botanical gardens and tissue culture laboratories across Germany as well as from China. The aerial parts were extracted and analyzed for CPT, 9-methoxycamptothecin and 10-hydroxycamptothecin. Chemometric evaluation revealed CPT to be positively correlated with both the metabolites. Endophytic fungi were isolated and characterized from all plants, only one of which was capable of producing CPT, 9-methoxycamptothecin and 10-hydroxycamptothecin in rich mycological media under axenic submerged shake-flask fermentation. The fungus was identified as Fusarium solani by its morphology and authenticated by ITS-5.8S rDNA analysis. CPT along with both the metabolites were additionally identified by 1H NMR spectroscopy, and confirmed by comparison with authentic standards. A substantial decrease in the production of CPT by the in vitro cultured endophyte over repeated subculturing was observed. The survival strategy of the endophyte against CPT toxicity was evaluated by identifying the typical amino acid residues Asn352, Glu356, Arg488, Gly503, and Gly717 (numbered according to human topoisomerase I) which prevent CPT binding to topoisomerase I, and the point mutation Met370Thr on the CPT-binding and catalytic domain of its topoisomerase I enzyme (encoded by Top1). A cross-species biosynthetic pathway was then deciphered where the fungal endophyte utilizes indigenous G10H (geraniol 10-hydroxylase), SLS (secologanin synthase), and TDC (tryptophan decarboxylase) to biosynthesize CPT precursors. However, to complete CPT biosynthesis, the endophyte requires the host STR (strictosidine synthase). The fungal CPT biosynthetic genes destabilize ex planta over successive subculture generations. The seventh subculture predicted proteins exhibited reduced homologies to the original enzymes proving that such genomic instability leads to dysfunction at the amino acid level. The endophyte with an impaired CPT biosynthetic capability was artificially inoculated into the living host plants and then recovered after colonization. CPT biosynthesis could still not be restored. This demonstrated that the observed phenomenon of genomic instability is irreversible.

Several Hypericum species were sampled from the natural populations of Slovakia and India, extracted, and analyzed for eight pharmacologically important secondary compounds (hypericin, pseudohypericin, emodin, hyperforin, hyperoside, rutin, quercetin, and quercitrin). Chemometric evaluation not only revealed various strong positive and negative correlations among the different phytochemicals but also depicted H. montanum as an alternative source to H. perforatum. Although endophytic fungi were isolated and characterized from all plants, only one endophytic fungus was capable of indigenously producing hypericin and emodin under axenic submerged shake-flask fermentation. The fungus was identified as Thielavia subthermophila by its morphology and authenticated by 28S rDNA and ITS-5.8S rDNA analyses. The growth of the endophyte and production of hypericin remained independent of the illumination conditions and media spiking with emodin. Protohypericin could not be detected, irrespective of either spiking or illumination conditions. The hyp-1 gene, suggested to encode for the Hyp-1 phenolic coupling protein in plant cell cultures, was absent in the genome of the endophyte. Thus, it is proposed that emodin anthrone is the common precursor of both hypericin and emodin in the fungal endophyte, which is governed by a different molecular mechanism than the host plant or host cell suspension cultures. Like the CPT producing endophyte, this endophyte also showed a substantial decrease in the production of hypericin and emodin in vitro over repeated subculturing and on storage.

Juniperus and Podophyllum species were collected from natural populations of India and Germany. Extraction and analyses were performed for four potential pro-drugs (podophyllotoxin, deoxypodophyllotoxin, demethylpodophyllotoxin, and podophyllotoxone). Chemometric evaluation revealed both infraspecific and infrageneric correlations among the different phytochemicals. Endophytic fungi were isolated and characterized from all plants. Only one endophytic fungus was capable of producing deoxypodophyllotoxin under axenic submerged shake-flask fermentation. The fungus was identified as Aspergillus fumigatus Fresenius by its morphology and 28S rDNA analysis. The growth and production kinetics showed the potential of the endophyte in the indigenous production of deoxypodophyllotoxin, but in vitro subculturing showed no production from the third subculture generation.

The results reported in this thesis reveal the immense potential of novel endophytic fungi as a source of important bioactive pro-drugs. Emphasis is also laid on the difficulties ahead if endophytic fungi are to be exploited for industrial production of bioactive secondary metabolites.