Red Biotechnology Potential of Taxol-Producing Endophytic Fungus Lasodiplodia Theobromea Isolated from a Non-Taxol-Producing Plant

Taxol^®(generic name Paclitaxel) a plant-derived anticancer drug, widely used against breast,ovarian and lung cancer,was originally isolated from the Pacific Yew,Taxus brevifolia.The limited supply of the drug prompted research to find alternative sources,such as chemical synthesis,tissue and cell culture of the Taxus species both of which are expensive and yield low levels.Thus,a sustainable, economical and unconventional alternative source of Taxol such as endophytic fungi has been actively researched.

A rich variety of endophytic fungi from Taxol- producing and non-Taxol-producing plants are known to produce Taxol in axenic cultures. Lasodiplodia theobromea species are among the most important endophytic fungi with biotechnological potential. In search of Taxol-producing endophyitc fungi from non-Taxol producing plant such as Piper nigrum, Zizypus sp and Mesura ferrae we isolated total of eight L.theobromea, three of which were found to produce Taxol.One of these three L.theobromea isolated from non-Taxol-producing Piper nigrum was selected in this study. Its identity was confirmed by morphological characteristics and ITS rDNA sequence analyses. Taxol produced by L.theobromea was shown to be identical to the authentic Taxol as analysed by TLC, HPLC, LC-MS and MS/MS analyses. The quantity of Taxol produced by the fungus was estimated to be 247µg/L.

As an evidence to support the independent production of Taxol by the L.theobromea,the gene encoding the 10-deacetylbaccatinIII-10-0-acetyltransferasse(DBAT), which catalyzing the penultimate acetylation in the Taxol biosynthesis pathway, was cloned and sequenced from L.theobromea. Additionally the same gene was cloned from the Taxol- producing Taxus celebica plant. The predicted amino sequence of the L.theobromea and the plant DBAT shows high homology to DBATs fromTaxol- producing Taxus celebica and Taxus cuspidate plants as well as the endophytic fungus Cladosporium. Further, the ORFs of L.theobromea and T.celebica DBAT were heterologously expressed in E.coli and the purified recombinant DBAT enzymes were shown to be capable of converting 10-deacetylbaccatinIII to baccatinIII. Therefore, Taxol biosynthetic DBAT gene in L.theobromea seems to have evolved independently and thus rule out horizontal gene transfer between the host plant and its endophytes.

This study can provide a road map to improve the production level and minimise production cost of Taxol at industrial level by implementing techniques of synthetic biology.