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

Atropa belladonna L. is one of the most important herbal plants that produces hyoscyamine or atropine, and it also produces anisodamine and scopolamine. However, the in planta hyoscyamine content is very low, and it is difficult and expensive to independently separate hyoscyamine from the tropane alkaloids in A. belladonna. Therefore, it is vital to develop A. belladonna plants with high yields of hyoscyamine, and without anisodamine and scopolamine. In this study, we generated A. belladonna plants without anisodamine and scopolamine, via the CRISPR/Cas9-based disruption of hyoscyamine 6ß-hydroxylase (AbH6H), for the first time. Hyoscyamine production was significantly elevated, while neither anisodamine nor scopolamine were produced, in the A. belladonna plants with homozygous mutations in AbH6H. In summary, new varieties of A. belladonna with high yields of hyoscyamine and without anisodamine and scopolamine have great potential applicability in producing hyoscyamine at a low cost.

Engineered Microbes for Producing Anticholinergics.

The tropane alkaloids (TAs) hyoscyamine and scopolamine function as acetylcholine receptor antagonists and are used clinically as parasympatholytics to treat neuromuscular disorders in humans. While TAs are synthesized in a small subset of plant families, these specialized metabolites are only accumulated in limited quantities in plant organs. The complex chemical structures of these compounds make their industrial production by chemical synthesis very challenging, Therefore, the supply of these TAs still relies on intensive farming of Duboisia shrubs in tropical countries. Many adverse factors such as climate fluctuations and pandemics can thus influence annual world production. Based on the landmark microbial production of the antimalarial semi-synthetic artemisinin, the Smolke group recently developed a yeast cell factory capable of de novo synthesizing hyoscyamine and scopolamine, thus paving the way for an alternative production of these compounds.

Biosynthesis of medicinal tropane alkaloids in yeast.

Tropane alkaloids from nightshade plants are neurotransmitter inhibitors that are used for treating neuromuscular disorders and are classified as essential medicines by the World Health Organization1,2. Challenges in global supplies have resulted in frequent shortages of these drugs3,4. Further vulnerabilities in supply chains have been revealed by events such as the Australian wildfires5 and the COVID-19 pandemic6. Rapidly deployable production strategies that are robust to environmental and socioeconomic upheaval7,8 are needed. Here we engineered baker's yeast to produce the medicinal alkaloids hyoscyamine and scopolamine, starting from simple sugars and amino acids. We combined functional genomics to identify a missing pathway enzyme, protein engineering to enable the functional expression of an acyltransferase via trafficking to the vacuole, heterologous transporters to facilitate intracellular routing, and strain optimization to improve titres. Our integrated system positions more than twenty proteins adapted from yeast, bacteria, plants and animals across six sub-cellular locations to recapitulate the spatial organization of tropane alkaloid biosynthesis in plants. Microbial biosynthesis platforms can facilitate the discovery of tropane alkaloid derivatives as new therapeutic agents for neurological disease and, once scaled, enable robust and agile supply of these essential medicines.

[Effects of nitrogen form on accumulation of alkaloids and expression of relative genes in Atropa belladonna].

Hyoscyamine and scopolamine are two main alkaloids in Atropa belladonna with great medicinal value. In this paper, the contents of hyoscyamine and scopolamine, the upstream products in alkaloid synthesis, and the expression levels of key enzyme genes PMT, TRⅠ and H6H in secondary metabolism of A. belladonna seedlings were measured to clarify the mechanism of nitrogen forms regulating alkaloids synthesis.The results showed that the 50/50 (NH⁺4/NO⁻3) treatment was more favorable for the accumulation of alkaloids and the conversion of hyoscyamine to scopolamine. The content of putrescine was almost consistent with the change of key enzymes activities in the synthesis of putrescine, they both increased with the rise of ammonium ratio, reaching the highest at 75/25 (NH⁺4/NO⁻3). The detection of signaling molecule nitric oxide (NO) showed that the NO concentration decreased with the decrease of nitrate proportion. Further detection of gene expression levels of PMT, TRⅠ and H6H in TAs synthesis pathway showed that a certain amount of ammonium promoted the expression of PMT and H6H in roots. When the ratio of ammonium to nitrate was 50/50, PMT, TRⅠ and H6H in leaves and roots had higher expression levels. It can be speculated that the regulation of the formation of hyoscyamine to scopolamine by nitrogen forms mainly through affecting the expression of key enzyme genes. 50/50 (NH⁺4/NO⁻3) treatment increased the gene expression of TRⅠ in both leaves and roots as well as PMT and H6H in roots, promoting the synthesis of putrescine to hyoscyamine and the conversion of hyoscyamine to scopolamine.

Localization and Organization of Scopolamine Biosynthesis in Duboisia myoporoides R. Br.

Tropane alkaloids (TAs), especially hyoscyamine and scopolamine, are important precursors for anticholinergic and antispasmodic drugs. Hyoscyamine and scopolamine are currently obtained at commercial scale from hybrid crosses of Duboisia myoporoides × Duboisia leichhardtii plants. In this study, we present a global investigation of the localization and organization of TA biosynthesis in a Duboisia myoporoides R. Br. wild-type line. The tissue-specific spatial distribution of TAs within D. myoporoides is presented, including quantification of the TAs littorine, 6-hydroxy hyoscyamine, hyoscyamine, scopolamine and, additionally, hyoscyamine aldehyde as well as scopolamine glucoside. Scopolamine (14.77 ± 5.03 mg g-1), and to a lesser extent hyoscyamine (3.01 ± 1.54 mg g-1) as well as 6-hydroxy hyoscyamine (4.35 ± 1.18 mg g-1), are accumulated in leaves during plant development, with the highest concentration of total TAs detected in 6-month-old plants. Littorine, an early precursor in TA biosynthesis, was present only in the roots (0.46 ± 0.07 mg g-1). During development, the spatial distribution of all investigated alkaloids changed due to secondary growth in the roots. Transcripts of pmt, tr-I and cyp80f1 genes, involved in early stages of TA biosynthesis, were found to be most abundant in the roots. In contrast, the transcript encoding hyoscyamine 6ß-hydroxylase (h6h) was highest in the leaves of 3-month-old plants. This investigation presents the spatial distribution of biochemical components as well as gene expression profiles of genetic factors known to participate in TA biosynthesis in D. myoporoides. The results of this investigation may aid in future breeding or genetic enhancement strategies aimed at increasing the yields of TAs in these medicinally valuable plant species.

Optimization of the culture medium composition to improve the production of hyoscyamine in elicited Datura stramonium L. hairy roots using the Response Surface Methodology (RSM).

Traditionally, optimization in biological analyses has been carried out by monitoring the influence of one factor at a time; this technique is called one-variable-at-a-time. The disadvantage of this technique is that it does not include any interactive effects among the variables studied and requires a large number of experiments. Therefore, in recent years, the Response Surface Methodology (RSM) has become the most popular optimization method. It is an effective mathematical and statistical technique which has been widely used in optimization studies with minimal experimental trials where interactive factors may be involved. This present study follows on from our previous work, where RSM was used to optimize the B5 medium composition in [NO(3-)], [Ca(2+)] and sucrose to attain the best production of hyoscyamine (HS) from the hairy roots (HRs) of Datura stramonium elicited by Jasmonic Acid (JA). The present paper focuses on the use of the RSM in biological studies, such as plant material, to establish a predictive model with the planning of experiments, analysis of the model, diagnostics and adjustment for the accuracy of the model. With the RSM, only 20 experiments were necessary to determine optimal concentrations. The model could be employed to carry out interpolations and predict the response to elicitation. Applying this model, the optimization of the HS level was 212.7% for the elicited HRs of Datura stramonium, cultured in B5-OP medium (optimized), in comparison with elicited HRs cultured in B5 medium (control). The optimal concentrations, under experimental conditions, were determined to be: 79.1 mM [NO(3-)], 11.4 mM [Ca(2+)] and 42.9 mg/L of sucrose.