Enhancing Tropane Alkaloid Production Based on the Functional Identification of Tropine-Forming Reductase in Scopolia lurida, a Tibetan Medicinal Plant.

Tengfei Zhao,Chunxian Yang,Xiaoqiang Liu,Kaihui Zhao,Min Chen,Junlan Zeng,Lingjiang Zeng,Xiaozhong Lan,Zhihua Liao,Fei Qiu,Haoxing Zhang

doi:10.3389/fpls.2017.01745

Abstract

Scopolia lurida, a native herbal plant species in Tibet, is one of the most effective producers of tropane alkaloids. However, the tropane alkaloid biosynthesis in this plant species of interest has yet to be studied at the molecular, biochemical, and biotechnological level. Here, we report on the isolation and characterization of a putative short chain dehydrogenase (SDR) gene. Sequence analysis showed that SlTRI belonged to the SDR family. Phylogenetic analysis revealed that SlTRI was clustered with the tropine-forming reductases. SlTRI and the other TA-biosynthesis genes, including putrescine N-methyltransferase (SlPMT) and hyoscyamine 6β-hydroxylase (SlH6H), were preferably or exclusively expressed in the S. lurida roots. The tissue profile of SlTRI suggested that this gene might be involved in tropane alkaloid biosynthesis. By using GC-MS, SlTRI was shown to catalyze the tropinone reduction to yield tropine, the key intermediate of tropane alkaloids. With the purified recombinant SlTRI from Escherichia coli, an enzymatic assay was carried out; its result indicated that SlTRI was a tropine-forming reductase. Finally, the role of SlTRI in promoting the tropane alkaloid biosynthesis was confirmed through metabolic engineering in S. lurida. Specifically, hairy root cultures of S. lurida were established to investigate the effects of SlTRI overexpression on tropane alkaloid accumulation. In the SlTRI-overexpressing root cultures, the hyoscyamine contents were 1.7- to 2.9-fold higher than those in control while their corresponding scopolamine contents were likewise elevated. In summary, this functional identification of SlTRI has provided for a better understanding of tropane alkaloid biosynthesis. It also provides a candidate gene for enhancing tropane alkaloid biosynthesis in S. lurida via metabolic engineering.

Highlights

Scopolia lurida, known as Himalayan Scopolia (Mills and Jackson, 1972) is a perennial herb of the family Solanaceae and indigenous to the Himalayas of the Qinghai-Tibet Plateau (Qin et al, 2014)
The BLASTP analysis showed that SlTRI belonged to the family of SDRs, the NADPH-dependent short chain dehydrogenases/reductases (Dräger, 2006)
The SDR-specific motifs were found in SlTRI (Figure 3)

Summary

Introduction

Known as Himalayan Scopolia (Mills and Jackson, 1972) is a perennial herb of the family Solanaceae and indigenous to the Himalayas of the Qinghai-Tibet Plateau (Qin et al, 2014). Some Solanaceae species including Hyoscyamus niger (Yun et al, 1992), Datura species (Jakabová et al, 2012), Atropa belladonna (Wang et al, 2011), and S. lurida (Wang et al, 2010) are widely used as anticholinergic agents, especially the pharmaceutical tropane alkaloids (TAs), such as hyoscyamine and scopolamine that are produced exclusively by the medicinal plant family Among these TA-producing plant species, S. lurida is one of the most effective producers of TAs (Jovanovicet al., 1991) because of its high biomass and content of hyoscyamine which was reported as up to 1.5% dry weight in the aerial parts (Mills and Jackson, 1972). Biosynthesis and regulation of TAs in S. lurida is largely unknown at the molecular, biochemical and biotechnological level

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Conclusion