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

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.

Metabolic Characterization of Hyoscyamus niger Ornithine Decarboxylase

“…The detection methods used here were similar to those applied in the enzymatic assays described below. Tropane alkaloid content was quantified by adhering to previously described methodologies (Qiang et al, 2016; Zhao et al, 2017; Geng et al, 2018). For this, 200 mg of dry powder from each plant part was accurately weighed for alkaloid extraction and detection, with at least three independent plants used for metabolite detections.…”

“…These enzymes include putrescine N -methyltransferase (Liu et al, 2005; Kai et al, 2009b; Geng et al, 2018), tropinone reductase I (Nakajima et al, 1993b; Kai et al, 2009a; Qiang et al, 2016), tropinone reductase II (Hashimoto et al, 1992; Nakajima et al, 1993a,b), CYP80F1 (Li et al, 2006), and hyoscyamine 6β-hydroxylase (Matsuda et al, 1991; Li et al, 2012). Since their identification, the associated TA-biosynthesis genes have been applied to engineer TA biosynthesis in plants via the overexpression method (Zhang et al, 2004; Wang et al, 2011; Zhao et al, 2017). In this context, it is thus very important to distinguish those enzymes with higher catalytic activities to better facilitate the metabolic engineering of metabolite biosynthesis.…”

Chemistry of Secondary Metabolites

“…Scopolialurida, otherwise called Himalayan Scopolia, a local home grown plant species in Tibet, is a standout amongst the best makers of tropane alkaloids. Some Solanaceae species including Hyoscyamus niger, Datura species, Atropa belladonna and S. lurida are generally utilized as anticholinergic specialists, particularly the pharmaceutical tropane alkaloids, for example, hyoscyamine and scopolamine that are created only by the medicinal plant family (Figure 33) [85].…”

Differential expression and functional analysis of two short-chain alcohol dehydrogenases/reductases in Hedychium coronarium

“…Furthermore, short-chain dehydrogenase/reductases (SDRs) are a gene family involved in various secondary metabolic pathways. At present, the research on cloning of SDR genes has mainly been focused on dicots, such as genus Solanum (Sonawane et al, 2018), Catharanthus roseus (Stavrinides et al, 2018), Scopolia lurida (Zhao et al, 2017), Hevea brasiliensis (Loh et al, 2016), Camellia sinensis (Zhou et al, 2015), Medicago truncatula (Pan et al, 2014), Papaver somniferum (Chen and Facchini, 2013;, Lycopersicon esculentum Mill. (Moummou et al, 2012), Gossypium spp.…”