Elsevier

Metabolic Engineering

Volume 13, Issue 2, March 2011, Pages 234-240
Metabolic Engineering

The expression of 1-deoxy-d-xylulose synthase and geraniol-10-hydroxylase or anthranilate synthase increases terpenoid indole alkaloid accumulation in Catharanthus roseus hairy roots

https://doi.org/10.1016/j.ymben.2010.11.005Get rights and content

Abstract

The terpenoid indole alkaloid (TIA) pathway in Catharanthus roseus produces two important anticancer drugs, vinblastine and vincristine, in very low yields. This study focuses on overexpressing several key genes in the upper part of the TIA pathway in order to increase flux toward downstream metabolites within hairy root cultures. Specifically, we constructed hairy root lines with inducible overexpression of 1-deoxy-D-xylulose synthase (DXS) or geraniol-10-hydroxylase (G10H). We also constructed hairy root lines with inducible expression of DXS and anthranilate synthase α subunit (ASA) or DXS and G10H. DXS overexpression resulted in a significant increase in ajmalicine by 67%, serpentine by 26% and lochnericine by 49% and a significant decrease in tabersonine by 66% and hörhammericine by 54%. Co-overexpression of DXS and G10H caused a significant increase in ajmalicine by 16%, lochnericine by 31% and tabersonine by 13%. Likewise, DXS and ASA overexpression displayed a significant increase in hörhammericine by 30%, lochnericine by 27% and tabersonine by 34%. These results point to the need for overexpressing multiple genes within the pathway to increase the flux toward vinblastine and vincristine.

Introduction

Since their discovery in the late 1950s (Pearce and Miller, 2005), vinblastine and vincristine have been used as powerful anticancer drugs for a variety of cancer treatments. These compounds are classified as terpenoid indole alkaloids and were discovered during a screening for novel anti-diabetic drugs in Catharanthus roseus (van der Heijden et al., 2004). Vinblastine and vincristine are produced in small quantities within the leaves of the plant. Approximately 500 kg of dried leaves are needed to produce 1 g of vinblastine (Noble, 1990). For commercial production, a semi-synthetic route has been used to couple vindoline and catharanthine; both compounds are present in higher concentrations within the leaves (van der Heijden et al., 2004). The importance of these anticancer drugs has lead researchers to study the biosynthesis and regulation of the terpenoid indole alkaloid pathway and to explore ways to engineer the increased production of these metabolites.

The terpenoid indole alkaloids (TIAs) are formed by the condensation of the indole moiety tryptamine and the monoterpenoid secologanin by strictosidine synthase (STR) to form strictosidine, the precursor to a wide variety of TIAs (Fig. 1). Throughout the TIA pathway there are a number of key enzymes that regulate the flux through the pathway. Anthranilate synthase (AS) catalyzes the first committed step in the synthesis of tryptophan and is subjected to feedback inhibition by tryptophan (Li and Last, 1996). AS is a tetramer composed of two α subunits (ASα) and two β subunits (ASβ). The α subunit catalyzes the aromatization of chorismate while the β subunit donates the amino subunit. The inhibition by tryptophan can be overcome by expressing a feedback-insensitive ASα. This technique has been successfully used to increase tryptophan content in rice, soybean, and C. roseus (Dubouzet et al., 2007, Hong et al., 2006b, Hughes et al., 2004a, Inaba et al., 2007). In C. roseus hairy roots, the overexpression of a feedback-insensitive ASα caused huge increases in tryptophan and tryptamine concentrations (>300 fold and 10 fold, respectively), but only led to the modest increase (2 fold) of one TIA, lochnericine (Hughes et al., 2004a). The conversion of tryptophan to tryptamine is another branch point in the pathway and a potential control step. When tryptophan decarboxylase (TDC) was overexpressed in C. roseus hairy roots, tryptamine did not increase but serpentine showed a significant increase (Hughes et al., 2004b). The overexpression of both ASα and TDC in C. roseus hairy roots caused a 6-fold increase in tryptamine levels while the TIAs levels did not increase (Hughes et al., 2004b).

1-deoxy-d-xylulose 5-phosphate synthase (DXS) catalyzes the first step in the methyl-erythritol phosphate (MEP) pathway and is hypothesized to be an important control step within the MEP pathway. The following examples highlight the role of the DXS pathway in plants. In Arabidopsis the overexpression of DXS led to an increase in terpenoid concentrations while the repression of DXS decreased terpenoid concentrations (Estevez et al., 2001). DXS expression pattern followed the accumulation pattern of carotenoids during fruit development in tomato (Lois et al., 2000). Deoxyxylulose feeding caused an increase of 30–60% in the concentration of two indole alkaloids, lochnericine and tabersonine, in C. roseus hairy roots (Hong et al., 2003). In C. roseus cell suspension cultures, geraniol-10-hydroxylase (G10H) is considered to be an important limiting enzyme in the production of TIAs (Collu et al., 2002, Collu et al., 2001, Dagnino et al., 1995, Schiel et al., 1987). In native roots high levels of G10H activity have been reported (Meijer et al., 1993) highlighting a potential difference between root and cell culture. DXS and G10H mRNA levels are up-regulated by a number of signaling molecules including jasmonic acid, ethylene, and cytokinin (Collu et al., 2001, Papon et al., 2005, van der Fits and Memelink, 2000).

The use of feeding studies can help us to determine potential limiting steps within a pathway. For the TIA pathway, there are conflicting reports as to whether the terpenoid or indole pathway is the limiting pathway in the production of TIAs. In cell suspension cultures, it has been shown that tryptophan feeding (indole pathway) has resulted in increased serpentine levels in one study, in reduced alkaloid levels in another, and in no effect in another (Kargi and Ganapathi, 1991, Knobloch and Berlin, 1980, Zenk et al., 1977). Cell cultures that overexpressed TDC exhibited an increase in tryptophan accumulation but no change in TIA accumulation (Canel et al., 1998). Loganin or secologanin feeding (terpenoid pathway) increases TIAs in cell suspension cultures (Mérillon et al., 1989, Moreno et al., 1993) and increases TIAs in cells overexpressing STR or TDC (Whitmer et al., 2002a, Whitmer et al., 2002b). The timing of precursor feeding may affect which pathway is limiting in hairy roots. In the exponential growth phase, tryptophan feeding significantly increased the production of TIAs but the feeding of geraniol, 10-hydroxygeraniol, and loganin had no significant effect. In early stationary growth phase, feeding geraniol, 10-hydroxygeraniol, or loganin increased the amount of tabersonine that accumulated, while the addition of tryptophan had no effect (Morgan and Shanks, 2000). These reports suggest that both pathways may be limiting—when the limitation of one pathway is overcome, likely the other pathway becomes limiting.

This paper explores the effect of overexpressing several important TIA pathway genes alone and in concert with other genes in hairy root cultures of C. roseus. Hairy roots were chosen due to the fact that they produce significantly higher concentrations of TIAs than cell suspension cultures (Montiel et al., 2007). Specifically, the single effects of overexpressing DXS, G10H, and previously published ASA (Hughes et al., 2004a) are investigated. The mixed results of single gene overexpression lead us to explore the effect of overexpressing two genes simultaneously. The effect of co-expressing DXS and G10H or DXS and ASA resulted in an increase in several TIA metabolites. These results suggest that expression of multiple key enzymes maybe necessary to overcome the native regulation and increase TIA metabolite accumulation.

Section snippets

Plasmid construction

The Arabidopsis DXS gene, ClaI, was provided as a cDNA clone in pBluescript by Dr. Patricia Leon. G10H was amplified from cDNA prepared from RNA purified from C. roseus hairy roots using polyT primers and M-MLV reverse transcriptase according to manufacturer's instructions (Promega). The primers used for G10H amplification were 5′-ACTTCCATTCCATGGATTACCTTACCA-3′ and 5′-TTAAAGGGTGCTTGGTACAGCAC-3′. The sequence from the resulting gene matched the published sequence for G10H. G10H was moved from

Results

Hairy root cultures were generated by infecting C. roseus seedlings with A. rhizogenes strain ATCC 15834 carrying the Ri plasmid and p7002DXS, p7002G10H, or p7002ASA/DXS or A. tumefaciens strain GV3101 carrying the plasmid pPZPROL containing the rolABC genes (Hong et al., 2006a) and p7002G10H/DXS. The resulting hairy roots were screened on hygromycin selection plates and adapted to liquid media for several generations. pTA7002 contains a glucocorticoid-inducible promoter system which uses the

Discussion

DXS overexpression in hairy roots showed mixed results in TIA profiles. Three metabolites, ajmalicine, serpentine, and lochnericine, exhibited an increase in concentration while two others, tabersonine and hörhammericine, showed a decrease in concentration upon the overexpression of DXS (Fig. 3). These differences make it hard to generalize the overall effect of DXS on TIAs. Another puzzling result is the difference seen in TIA metabolites between DXS overexpression and deoxyxylulose feeding in

Conclusion

This paper demonstrates some of the potential problems associated with the overexpression of single genes within an organism such as C. roseus. Simultaneously overexpression of several multiple enzymes within the TIA pathway are needed to see significant increases in the metabolites of interest. In addition, optimization of the expression of each enzyme in the pathway may be necessary in order to maximize metabolite production.

Acknowledgments

The authors would like to thank Dr. Nam-Hai Chua at the Rockefeller University for providing the inducible promoter plasmid (pTA7002), Dr. Eugene Nester at the University of Washington for providing the A. rhizogenes 15834 strain used, Dr. Johan Memelink at the University of Leiden for providing us with the geraniol-10-hydroxylase sequence prior to publication, and Dr. Patricia Leon from the Universidad Nacional Autonoma de Mexico for providing the cDNA clone of Arabidopsis DXS. This work was

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