Insights into the functionality of endophytic actinobacteria with a focus on their biosynthetic potential and secondary metabolites production

Abstract

Endophytic actinobacteria play an important role in growth promotion and development of host plant by producing enormous quantities of novel bioactive natural products. In the present investigation, 169 endophytic actinobacteria were isolated from endospheric tissues of Rhynchotoechum ellipticum. Based on their antimicrobial potential, 81 strains were identified by 16rRNA gene analysis, which were taxonomically grouped into 15 genera. All identified strains were screened for their plant growth promoting attributes and, for the presence of modular polyketide synthases (PKSI, PKSII and nonribosomal peptide synthetase (NRPS) gene clusters to correlate the biosynthetic genes with their functional properties. Expression studies and antioxidant potential for four representative strains were evaluated using qRT-PCR and DPPH assay respectively. Additionally, six antibiotics (erythromycin, ketoconazole, fluconazole, chloramphenicol, rifampicin and miconazole) and nine phenolic compounds (catechin, kaempferol, chebulagic acid, chlorogenic acid, Asiatic acid, ferulic acid, arjunic acid, gallic acid and boswellic acid) were detected and quantified using UHPLC-QqQ_LIT-MS/MS. Furthermore, three strains (BPSAC77, 121 and 101) showed the presence of the anticancerous compound paclitaxel which was reported for the first time from endophytic actinobacteria. This study provides a holistic picture, that endophytic actinobacteria are rich bacterial resource for bioactive natural products, which has a great prospective in agriculture and pharmaceutical industries.

Figure 1

Maximum likelihood phylogenetic tree using Kimura-2 parameter model based on 16S rRNA gene sequences of endophytic actinobacterial isolates showing the phylogenetic relationship between the endophytes sequences with closest type strain sequences. Numbers at branches indicate bootstrap values in 1,000 replicates.

Figure 2

Relative gene expression of PKS type I gene and NRPS gene on five alternate days across strains BPSAC77, BPSAC101, BPSAC121 and BPSAC147. The expression level calculated by the formula 2^−ΔΔCt represents the x-fold difference from the calibrator and 16S rRNA gene as endogenous control. Threshold cycle (C_T) was compared with log₁₀ relative copy number of the sample from a dilution series. Error bars indicate standard error. Sample after 12^th day of incubation shows highest expression level.

Figure 3

Scavenging activity (%) on DPPH radicals (IC₅₀) of methanolic extract of potential four endophytic actinobacterial strain (Streptomyces olivaceus strain BPSAC77; Streptomyces sp. strain BPSAC101; Streptomyces sp. strain BPSAC121 & Streptomyces thermocarboxydus strain BPSAC147) at different concentration (µg/mL). Ascorbic acid was used as a positive control in DPPH radical scavenging test and the experiment was performed in triplicate for all the samples.

Figure 4

MS/MS spectra of reference analytes of standard six antibiotics; Fluconazole, Chloramphenicol, Erythromycin, Ketoconazole, Rifampicin, Miconazole.

Figure 5

MRM-extracted ion chromatogram (XICs) of six pair of antibiotics obtained by UPLC-ESI–MS/MS.

Figure 6

MS/MS spectra of reference analytes of standard nine phenolic compound; Catechin, Kaempferol, Chebulagic acid, Chlorogenic acid, Asiatic acid, Ferulic acid, Arjunic acid, Gallic acid, Boswellic acid and an anticancerous compound Paclitaxel.

Figure 7

MRM-extracted ion chromatogram (XICs) of nine pair of phenolic and an anticancer compound obtained by UPLC-ESI–MS/MS.