Comparison of elicitor-based effects on metabolic responses of Taxus\u2009\xd7\u2009media hairy roots in perfluorodecalin-supported two-phase culture system

K Sykłowska-Baranek,M Grech-Baran,W Rymaszewski,M Pilarek,A Pietrosiuk,P Rokicki,J Hennig,M Gaweł

doi:10.1007/s00299-018-2351-0

Abstract

Key messageTwo lines of Taxus × media hairy roots harbouring or not the TXS transgene demonstrated diverse gene expression and taxane yield during cultivation in PFD-supported two liquid-phase culture system.Two lines of Taxus × media hairy roots were subjected to single or twice-repeated supplementation with methyl jasmonate, sodium nitroprusside, l-phenylalanine, and sucrose feeding. One line harboured transgene of taxadiene synthase (ATMA), while the second (KT) did not. Both hairy root lines were cultured in two-phase culture systems containing perfluorodecalin (PFD) in aerated or degassed form. The relationship between TXS (taxadiene synthase), BAPT (baccatin III: 3-amino, 3-phenylpropanoyltransferase), and DBTNBT (3′-N-debenzoyl-2-deoxytaxol-N-benzoyltransferase) genes and taxane production was analysed. The ATMA and KT lines differed in their potential for taxane accumulation, secretion, and taxane profile. In ATMA biomass, both paclitaxel and baccatin III were detected, while in KT roots only paclitaxel. The most suitable conditions for taxane production for ATMA roots were found in single-elicited supported with PFD-degassed cultures (2 473.29 ± 263.85 µg/g DW), whereas in KT roots in single-elicited cultures with PFD-aerated (470.08 ± 25.15 µg/g DW). The extracellular levels of paclitaxel never exceeded 10% for ATMA roots, while for KT increased up to 76%. The gene expression profile was determined in single-elicited cultures supported with PFD-degassed, where in ATMA roots, the highest taxane yield was obtained, while in KT the lowest one. The gene expression pattern in both investigated root lines differed substantially what resulted in taxane yield characterized particular lines. The highest co-expression of TXS, BAPT and DBTNBT genes noted for ATMA roots harvested 48 h after elicitation corresponded with their higher ability for taxane production in comparison with the effects observed for KT roots.

Highlights

Taxane-type compounds are well-known drug agents used in anti-cancer therapies against tumors of ovaries, breast, prostate and lung cancers, Kaposi’s sarcoma, squamous cell carcinoma of head and neck, and many others (Yared and Tkaczuk 2012)
The profiling of transcripts after methyl jasmonate (MJ) elicitation of cells in suspension cultures of various Taxus species, demonstrated upregulation of genes involved in taxane biosynthesis, and genes engaged in the stimulation of plant hormone and phenylpropanoid biosynthesis, MJ-signaling, taxane transport, degradation, as well as transcriptional regulation (Lenka et al 2012; Li et al 2012; Sun et al 2013)
The most influencing and statistically significant factor determinative for the taxane production output seems to be the duration of elicitation, because the maximal value of taxane accumulation was obtained after 2 weeks of elicitation

Summary

Introduction

Taxane-type compounds are well-known drug agents used in anti-cancer therapies against tumors of ovaries, breast, prostate and lung cancers, Kaposi’s sarcoma, squamous cell carcinoma of head and neck, and many others (Yared and Tkaczuk 2012). An immense effort was undertaken to elucidate MJ molecular mechanism of action in Taxus cell suspension cultures, which led to revealing up-regulation of the majority of genes engaged in metabolic pathways leading to taxane biosynthesis (Nims et al 2006; Exposito et al 2010; Onrubia et al 2010, 2013b; Patil et al 2012; SabaterJara et al 2014) These investigations unraveled the 1–7-day delay between a time course for mRNA accumulation of the known taxane biosynthetic genes and taxane production (Nims et al 2006; Lenka et al 2012). Despite the enormous efforts undertaken for a better understanding of taxane metabolism in plant biomass from in vitro cultures, the taxane biosynthesis rate-limiting steps remained still unidentified, the late pathway steps seems to be potentially and significantly rate-influencing steps in paclitaxel production (Nims et al 2006; Vongpaseuth and Roberts 2007; Sabater-Jara et al 2014; Cusido et al 2014)

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