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.

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Aims:  To immobilize the purified Aspergillus flavipesl‐methioninase on solid carriers for continuous production of methanethiol with high purity, by the enzymatic methods. Methods and Results:  The purified l‐methioninase was immobilized using different methods, and physicochemical and kinetic studies for the potent immobilized enzyme were conducted parallel to the soluble one. The activity of the purified extracellular enzyme was 1·8‐fold higher than intracellular one from submerged cultures of A. flavipes. Among the tested methods, polyacrylamide (42·2%), Ca‐alginate (40·9%) and chitin (40·8%) displayed the highest immobilization efficiency. The thermal inactivation rate was strongly decreased for chitin‐immobilized enzyme (0·222 s−1) comparing to soluble enzyme (0·51 s−1). Enzyme immobilization efficiency was greatly improved using 4·0% glutaraldehyde and 41·6/6·3 (T/C) as spacers for chitin and polyacrylamide‐enzyme conjugates, comparing to their controls. Also the incorporation of lysine, glutathione, cysteine and dithiothreitol as active site protectants significantly enhance the catalytic efficiency of immobilized enzyme. The activity of enzyme was increased by 4·5‐ and 3·5‐fold using glutathione plus DDT and glutathione plus methionine, for chitin and polyacrylamide enzyme, respectively. Conclusion:  Chitin enzyme gave a plausible stability till fourth cycle for production of methanethiol under controlled system. Applying GC and HNMR analysis, methanethiol has identical chemical structure to the standard compound. Significance and Impact of the Study:  Technically, a new method for continuous production of pure methanethiol, with broad applications, was developed using a simple low expenses method.

Endophytic fungi have been considered as a repertoire for bioactive secondary metabolites with potential application in medicine, agriculture and food industry. The biosynthetic pathways by fungal endophytes raise the argument of acquisition of these machineries of such complex metabolites from the plant host. Diterpenoids “Taxol” is the most effective anticancer drug with highest annual sale, since its discovery in 1970 from the Pacific yew tree, Taxus brevifolia. However, the lower yield of Taxol from this natural source (bark of T. brevifolia), availability and vulnerability of this plant to unpredicted fluctuation with the ecological and environmental conditions are the challenges. Endophytic fungi from Taxus spp. opened a new avenue for industrial Taxol production due to their fast growth, cost effectiveness, independence on climatic changes, feasibility of genetic manipulation. However, the anticipation of endophytic fungi for industrial Taxol production has been challenged by the loss of its productivity, due to the metabolic reprograming of cells, downregulating the expression of its encoding genes with subculturing and storage. Thus, the objectives of this review were to (1) Nominate the endophytic fungal isolates with the Taxol producing potency from Taxaceae and Podocarpaceae; (2) Emphasize the different approaches such as molecular manipulation, cultural optimization, co-cultivation for enhancing the Taxol productivities; (3) Accentuate the genome mining of the rate-limiting enzymes for rapid screening the Taxol biosynthetic machinery; (4) Triggering the silenced rate-limiting genes and transcriptional factors to activates the biosynthetic gene cluster of Taxol.

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