Taxomyces andreanae: A Presumed Paclitaxel Producer Demystified?

Abstract

The 1990s brought an abundance of reports on paclitaxel-producing endophytes, initially heralded as a discovery having tremendous implications for cancer therapy. As the vision of large-scale fermentation tanks producing vast quantities of relatively inexpensive paclitaxel and novel taxanes has faded and has been replaced by controversial silence, we carried out an in-depth investigation of Taxomyces andreanae – the very first presumed endophytic synthesizer of the diterpenoid. On one hand, metabolic profiling by means of chromatographic, spectroscopic and immunoenzymatic techniques predominant in literature was taken up. On the other, the experimental procedure was brought to an alternative, previously unattempted level aiming at revealing the genetic background of paclitaxel biosynthesis in the endophyte. The profound PCR-based screening for taxadiene synthase (txs) – a gene unique to the formation of the primary taxane-skeleton, as well as phenylpropanoyl transferase (bapt) encoding for the catalyst of the final acylation of the core structure rendering the ultimate efficacy of the drug, confirmed the molecular blueprint for paclitaxel biosynthesis to be an inherent genetic trait of the endophyte. However, as the thorough metabolic analysis of Taxomyces andreanae commercial isolate brought no confirmation of endophytic paclitaxel production even after considerable up-scaling endeavors, we postulate that proclaiming the strain “a fungus factory for Taxol” might have been premature.

Key words

paclitaxel - Taxomyces andreanae - hyphomyces - endophyte - taxadiene synthase gene - phenylpropanoyl transferase gene - PCR

References

• 1 Croteau R. Taxol biosynthesis and molecular genetics. Wageningen, the Netherlands; Terpnet 2005
• 2 Stierle A, Strobel G A, Stierle D B, Grothaus P, Bignami G. The search for a taxol-producing microorganism among the endophytic fungi of the Pacific yew, Taxus brevifolia.  J Nat Prod. 1995;  58 1315-1324
• 3 Stierle A, Strobel G A, Stierle D B. Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of Pacific yew.  Science. 1993;  260 214-216
• 4 Stone R. Surprise! A fungus factory for taxol?.  Science. 1993;  260 154-155
• 5 Walker K, Croteau R. Taxol biosynthetic genes.  Phytochemistry. 2001;  58 1-7
• 6 Wildung M R, Croteau R. A cDNA clone for taxadiene synthase, the diterpene cyclase that catalyzes the committed step of taxol biosynthesis.  J Biol Chem. 1996;  271 9201-9204
• 7 Walker K, Long R, Croteau R. The final acylation step in taxol biosynthesis: cloning of the taxoid C13-side chain N-benzoyltransferase from Taxus.  PNAS. 2002;  99 9166-9171
• 8 Kurland C G, Canback B, Berg O G. Horizontal gene transfer: a critical view.  PNAS. 2003;  100 9658-9662
• 9 Rosewich U L, Kistler H C. Role of horizontal gene transfer in the evolution of fungi.  Annu Rev Phytopathol. 2000;  38 325-363
• 10 Grothaus P G, Bignami G S, O'Malley S, Harada K E, Byrnes J B, Waller D F, Raybould T J, McGuire M T, Alvarado B. Taxane-specific monoclonal antibodies: measurement of taxol, baccatin III, and “total taxanes" in Taxus brevifolia extracts by enzyme immunoassay.  J Nat Prod. 1995;  58 1003-1014
• 11 Strobel G A, Stierle A, Stierle D B. Taxol production by Taxomyces andreanae. 1994 US Patent 5322779
• 12 Ballero M, Loi M C, van Rozendaal E L, van Beek T A, van de Haar C, Poli F, Appendino G. Analysis of pharmaceutically relevant taxoids in wild yew trees from Sardinia.  Fitoterapia. 2003;  74 34-39
• 13 Grothaus P G, Raybould T J, Bignami G S, Lazo C B, Byrnes J B. An enzyme immunoassay for the determination of taxol and taxanes in Taxus sp. tissues and human plasma.  J Immunol Methods. 1993;  158 5-15
• 14 Besumbes O, Sauret-Güeto S, Phillips M A, Imperial S, Rodriguez-Concepcion M, Boronat A. Metabolic engineering of isoprenoid biosynthesis in Arabidopsis for the production of taxadiene, the first committed precursor of taxol.  Biotechnol Bioeng. 2004;  88 168-175
• 15 Zhou X, Wang Z, Jiang K, Wei Y, Lin J, Sun X, Tang K. Screening of taxol-producing endophytic fungi from Taxus chinensis var. mairei.  Prikl Biokhim Mikrobiol. 2007;  43 490-494
• 16 Zhang P, Zhou P, Jiang C, Yu H, Yu L J. Screening of taxol-producing fungi based on PCR amplification from Taxus.  Biotechnol Lett. 2008;  30 2119-2123
• 17 Bohlmann J, Meyer-Gauen G, Croteau R. Plant terpenoid synthases: molecular biology and phylogenetic analysis.  PNAS. 1998;  95 4126-4133
• 18 Lesburg C A, Zhai G, Cane D E, Christianson D W. Crystal structure of pentalenene synthase: mechanistic insights on terpenoid cyclization reactions in biology.  Science. 1997;  277 1820-1824
• 19 Trapp S C, Croteau R. Genomic organization of plant terpene synthases and molecular evolutionary implications.  Genetics. 2001;  158 811-832
• 20 NCBI .Available at. www.ncbi.nlm.nih.gov/BLAST Accessed May 3, 2007
• 21 NCBI .Available at. www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=cd00868&version=v2.06 Accessed May 17, 2007
• 22 Tarshis L C, Yan M, Poulter C D, Sacchettini J C. Crystal structure of recombinant farnesyl diphosphate synthase at 2.6-A resolution.  Biochemistry. 1994;  33 10871-10877
• 23 Staniek A, Woerdenbag H J, Kayser O. Endophytes: exploiting biodiversity for the improvement of natural product-based drug discovery.  J Plant Interact. 2008;  3 75-93
• 24 Ketchum R E, Gibson D M. Paclitaxel production in suspension cell cultures of Taxus.  Plant Cell Tiss Organ Cult. 1996;  46 9-16
• 25 Lawrence J G, Roth J R. Selfish operons: horizontal transfer may drive the evolution of gene clusters.  Genetics. 1996;  143 1843-1860
• 26 Hines P J, Zahn L M. What's bugging plants? Introduction to special issue.  Science. 2009;  324 741
• 27 Chakravarthi B V S K, Das P, Surendranath K, Karande A A, Jayabaskaran C. Production of paclitaxel by Fusarium solani isolated from Taxus celebica.  J Biosci. 2008;  33 259-267
• 28 Strobel G A, Hess W M, Ford E, Sidhu R S, Yang X. Taxol from fungal endophytes and the issue of biodiversity.  J Indian Microbiol Biotechnol. 1996;  17 417-423
• 29 Miller K, Neilan B, Sze D M Y. Development of taxol and other endophyte produced anti-cancer agents.  Recent Pat Anticancer Drug Discov. 2008;  3 14-19