Functional genomics analysis reveals two novel genes required for littorine biosynthesis

Abstract

Some medicinal plants of the Solanaceae produce pharmaceutical tropane alkaloids (TAs), such as hyoscyamine and scopolamine. Littorine is a key biosynthetic intermediate in the hyoscyamine and scopolamine biosynthetic pathways. However, the mechanism underlying littorine formation from the precursors phenyllactate and tropine is not completely understood.Here, we report the elucidation of littorine biosynthesis through a functional genomics approach and functional identification of two novel biosynthesis genes that encode phenyllactate UDP-glycosyltransferase (UGT1) and littorine synthase (LS).UGT1 and LS are highly and specifically expressed in Atropa belladonna secondary roots. Suppression of either UGT1 or LS disrupted the biosynthesis of littorine and its TA derivatives (hyoscyamine and scopolamine). Purified His-tagged UGT1 catalysed phenyllactate glycosylation to form phenyllactylglucose. UGT1 and LS co-expression in tobacco leaves led to littorine synthesis if tropine and phenyllactate were added.This identification of UGT1 and LS provides the missing link in littorine biosynthesis. The results pave the way for producing hyoscyamine and scopolamine for medical use by metabolic engineering or synthetic biology.

Biosynthesis of medicinal tropane alkaloids in yeast

“…A tropine biosynthesis module (II) incorporated (i) seven enzymes: A. belladonna and Datura stramonium putrescine N-methyltransferases (AbPMT1 and DsPMT1), Datura metel N-methylputrescine oxidase engineered for improved peroxisomal activity (DmMPO1 ΔC-PTS1 ), A. belladonna pyrrolidine ketide synthase (AbPYKS) and tropinone synthase (AbCYP82M3), Arabidopsis thaliana cytochrome P450 reductase (AtATR1) and D. stramonium tropinone reductase 1 (DsTR1); and (ii) disruptions to five aldehyde dehydrogenases (Hfd1, Ald2, Ald3, Ald4 and Ald5) to reduce loss of pathway intermediates. We designed a third module (III) for production of the acyl donor 1-O-β-phenyllactoylglucose (PLA glucoside) from phenylalanine via aromatic aminotransferases Aro8 and Aro9, phenylpyruvate reductase (PPR) and PLA UDP-glucosyltransferase (UGT84A27) 16 . Yeast produce 3-phenylpyruvate from phenylalanine via Aro8 and Aro9 21 , and wild-type yeast and CSY1251 produce trace levels of PLA, potentially via nonspecific activity of a lactate dehydrogenase (LDH) acting on 3-phenylpyruvate 22 .…”

“…The identification of a type III polyketide synthase (PYKS) and cytochrome P450 (CYP82M3) catalyzing the cyclization of N-methylpyrrolinium to tropinone in Atropa belladonna 13 enabled us and others to engineer yeast strains for de novo production of tropine 14 , 15 . The recent report of a UDP-glucosyltransferase (UGT84A27) and serine carboxypeptidase-like (SCPL) acyltransferase (littorine synthase, LS) catalyzing the condensation of tropine and phenyllactate to littorine 16 resolved a debate about the nature of the acyl transfer reaction 9 . However, functional expression of plant SCPL acyltransferases (SCPL-ATs) in non-plant hosts has not been reported.…”

Development of Atropa belladonna L. Plants with High-Yield Hyoscyamine and without Its Derivatives Using the CRISPR/Cas9 System

“…Generally, substrates are able to accumulate at higher levels when the biosynthesis genes downstream from them are suppressed or disrupted. The suppression or silencing of TA biosynthesis genes, such as ornithine decarboxylase [26], tropinone synthase [27], phenylpyruvic acid reductase [28], and littorine synthase [29], dramatically reduced the corresponding alkaloid production, while markedly increasing the accumulation of related substrates and precursors in A. belladonna. Because anisodamine and scopolamine are the products of H6H catalysis, the CRISPR/Cas9-mediated loss of H6H function halted the conversion of hyoscyamine into its products, and, thus, the hyoscyamine accumulation level was increased.…”

“…The sample solution was injected at 20 µL each time. The method for the analysis of the TAs content was the same as that described in our recent work [26,28,29,35,36].…”

Cross-Species Comparison of Fruit-Metabolomics to Elucidate Metabolic Regulation of Fruit Polyphenolics Among Solanaceous Crops

“…[21], and Atropa spp. [22]. However, developing similar resources for related crops is still a goal of the scientific community [23].…”