Restoring the Taxol biosynthetic machinery of Aspergillus terreus by Podocarpus gracilior Pilger microbiome, with retrieving the ribosome biogenesis proteins of WD40 superfamily

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 10 th 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.

Conjugation of Aspergillus flavipes Taxol with Porphyrin Increases the Anticancer Activity of Taxol and Ameliorates Its Cytotoxic Effects

“…Fungal endophytes elevated the hope for mass production of Taxol due to their fast growth, feasibility of fermentation, genetic manipulation, and independence of climatic changes [20]. Nevertheless, the anticipation of fungi for commercial Taxol production has been challenged by their lower yield and drastic loss of Taxol yield with sub-culturing and storage [5,6,8,13,21]. Thus, exploring novel fungal isolates with Taxol-producing potency was the objective.…”

“…Four fungal isolates, namely, Aspergillus flavipes [1], A. terreus, A. flavus, and A. parasiticus [6], were selected based on their higher Taxol productivity. The isolates were grown on potato dextrose broth (PDB) and incubated, and then Taxol was extracted and quantified according to [1,6,8]. The yield of Taxol was emphasized by the TLC and HPLC analyses ( Figure 1).…”

“…Taxol is now being commercially produced by a semi-synthetic method, starting with 10-deacetylbaccatin III [7] Molecules 2020, 25, 263 2 of 13 extracted from the needles of T. baccata and T. walichiana growing in Europe and Asia. However, this method is challenged with lower yield, climatic dependence, and seasonal variations (reviewed by [5,8]. Chemical synthesis of Taxol has been emphasized [9,10]; however, the technical complexity and higher expenses are the main challenges stopping this technology from becoming an economically affordable approach [11,12].…”

“…Deciphering the potency of endophytic fungi for Taxol production opened a new avenue for commercial Taxol production due to their rapid growth, independence on climatic changes, and feasibility of genetic manipulation [5,13]. Taxomyces andreanae was the first recognized Taxol-producing endophyte of Taxus spp [14], after which an unlimited number of studies became motivated to isolate endophytic fungi with Taxol metabolic-producing potency [5][6][7][8]. Nevertheless, the implementation of these fungi for commercial production of Taxol has been confronted by the loss of Taxol productivity with subculturing and storage [1,6].…”

Plant Endophytes and Epiphytes: Burgeoning Sources of Known and “Unknown” Cytotoxic and Antibiotic Agents?

“…microbes on the leaf surface. Following on from this discovery, in 2019, the group published a second paper [29] in which they identified three proteins that were exponentially suppressed during subculturing. On the subsequent addition of sterilized leaves of the tree, as mentioned in the preceding paper, production of taxol resumed.…”