Abstract
Attenuating the Taxol yield of Aspergillus terreus with the subculturing and storage were the technical challenges that prevent this fungus to be a novel platform for industrial Taxol production. Thus, the objective of this study was to unravel the metabolic machineries of A. terreus associated with attenuation of Taxol productivity, and their restoring potency upon cocultivation with the Podocarpus gracilior microbiome. The Taxol yield of A. terreus was drastically reduced with the fungal subculturing. At the 10th subculture, the yield of Taxol was reduced by four folds (78.2 µg/l) comparing to the original culture (268 µg/l), as authenticated from silencing of molecular expression of the Taxol-rate limiting enzymes (GGPPS, TDS, DBAT and BAPT) by qPCR analyses. The visual fading of A. terreus conidial pigmentation with the subculturing, revealing the biosynthetic correlation of melanin and Taxol. The level of intracellular acetyl-CoA influx was reduced sequentially with the fungal subculturing, rationalizing the decreasing on Taxol and melanin yields. Fascinatingly, the Taxol biosynthetic machinery and cellular acetyl-CoA of A. terreus have been completely restored upon addition of 3% surface sterilized leaves of P. gracilior, suggesting the implantation of plant microbiome on re-triggering the molecular machinery of Taxol biosynthesis, their transcriptional factors, and/or increasing the influx of Acetyl-CoA. The expression of the proteins of 74.4, 68.2, 37.1 kDa were exponentially suppressed with A. terreus subculturing, and strongly restored upon addition of P. gracilior leaves, ensuring their profoundly correlation with the molecular expression of Taxol biosynthetic genes. From the proteomic analysis, the restored proteins 74.4 kDa of A. terreus upon addition of P. gracilior leaves were annotated as ribosome biogenesis proteins YTM and microtubule-assembly proteins that belong to WD40 superfamily. Thus, further ongoing studies for molecular cloning and expression of these genes with strong promotors in A. terreus, have been initiated, to construct a novel platform of metabolically stable A. terreus for sustainable Taxol production. Attenuating the Taxol yield of A. terreus with the multiple-culturing and storage might be due to the reduction on main influx of acetyl-CoA, or downregulation of ribosome biogenesis proteins that belong to WD40 protein superfamily.
Highlights
Taxol is a diterpenoid natural product, had been originally isolated and chemically identified from the bark of Pacific yew Taxus brevifolia[1]
The putative Taxol biosynthetic pathway in fungi requires about 19 enzymatic steps starting from the precursor geranylgeranyl diphosphate (GGPP) that cyclized into taxa-4(5),11(12)-diene by taxadiene synthase followed by hydroxylation of taxadiene nucleus by cytochrome P450-monooxygenases[3,8,9]
The Taxol yield for the original culture of A. terreus grown on modified MID media was 268 μg/l, while their yields for the 5th, 7th and 10th generations were 133.4, 94.7 and 78.2 μg/L, respectively, as revealed from the thin layer chromatography (TLC) and HPLC chromatograms
Summary
Taxol is a diterpenoid natural product, had been originally isolated and chemically identified from the bark of Pacific yew Taxus brevifolia[1]. It has been approved by FDA in 1992 as a broad-spectrum drug for treatment of www.nature.com/scientificreports/. Twenty-four endophytic fungal isolates were recovered from Podocarpus gracilior and screened for Taxol biosynthetic potency, among them, Aspergillus terreus EFB108 was reported as a potent Taxol producer[7]. The objective of this work was to evaluate the Taxol yield of A. terreus “endophyte of Podocarpus gracilior” in response to storage, multiple subculturing and potency to restore their Taxol biosynthetic machinery upon addition of P. gracilior leaves. To unravel the molecular and metabolic identity of Taxol biosynthetic machineries of A. terreus associated with the attenuation and restoration of Taxol biosynthesis upon addition of P. gracilior leaves
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