Rational Design of Hyoscyamine 6 β ‐Hydroxylase for the Efficient Production of Scopolamine

Scopolamine is a medicinal tropane alkaloid used for motion sickness, analgesia and anaesthesia and as a precursor to tiotropium for COPD therapy (Srinivasan and Smolke 2020). Industrial production relies on natural plant sources that accumulate low levels of scopolamine, limiting supply. Increasing pathway efficiency through the engineering of key enzymes remains a major objective (Gong et al. 2022). Hyoscyamine 6β-hydroxylase (H6H) catalyses the two-step conversion of hyoscyamine to anisodamine and subsequently to scopolamine (Figure 1a) (Hashimoto and Yamada 1986). H6Hs have been identified in several Solanaceae species, with Hyoscyamus niger H6H (HnH6H) outperforming homologues for metabolic engineering applications (Gong et al. 2022) (Figure 1b). We employed consensus sequence design (Sternke et al. 2019) to engineer HnH6H for enhanced catalytic performance. We retrieved all the sequences of H6Hs from the public databank and used them for consensus sequence analysis. Employing PSI-BLAST, we constructed a position-specific scoring matrix (PSSM) for H6Hs. Then, 19 single-site variants were constructed and evaluated in E. coli supplemented with hyoscyamine (Figure 1c). Seven mutants increased anisodamine accumulation by more than 20%, with HnH6HH43D showing the highest improvement (1.98-fold anisodamine and 4.95-fold scopolamine vs. wild type) (Figure 1d,e). Based on this variant, double-site combinations were produced; H43D + Q247E yielded 5.07-fold anisodamine and 13.68-fold scopolamine (Figure 1d,e). Subsequent triple mutants revealed H43D + Q247E + D324E to be superior, achieving 6.11- and 17.56-fold increases in anisodamine and scopolamine, respectively (Figure 1d,e). Additional mutations did not result in further improvements in product yields. The optimal variant, HnH6HH43D+Q247E+D324E (HnH6H3M), was chosen for biochemical characterisation. His-tagged HnH6H/HnH6H3M were purified from engineered E. coli and used for enzymatic assays. The optimal pH and temperature for catalysis did not differ significantly between HnH6H and HnH6H3M. Both enzymes shared identical optimal conditions: pH 7.8 and 30°C for hyoscyamine-to-anisodamine conversion, and pH 7.8 and 20°C for anisodamine-to-scopolamine conversion. Accordingly, the enzyme kinetics of the two-step reactions were measured under these optimum conditions. The Km value of HnH6H was 0.0557 mM for hyoscyamine and 0.1356 mM for anisodamine, respectively (Figure 1f,h). The Kcat/Km value of HnH6H was 6.8384 s−1·mM−1 for hyoscyamine and 0.0833 s−1·mM−1 for anisodamine (Figure 1f,h). HnH6H preferentially utilises hyoscyamine over anisodamine as a substrate, and catalyses the formation of anisodamine more efficiently than scopolamine. Furthermore, HnH6H3M exhibited Km values of 0.0516 mM for hyoscyamine and 0.1947 mM for anisodamine (Figure 1g,i), showing no significant difference compared to HnH6H. However, the Kcat/Km values of HnH6H3M were 18.2338 s−1·mM−1 for hyoscyamine and 0.1554 s−1·mM−1 for anisodamine, indicating a marked improvement in catalytic efficiency over HnH6H (Figure 1g,i). Although the three amino acid substitutions did not significantly alter HnH6H's affinity for substrate binding, they substantially enhanced the enzyme's efficiency in producing both anisodamine and scopolamine. In addition, HnH6H3M has nearly 10-fold higher catalytic efficiency than the AaH6H S14K/K97A (1.8678 s−1·mM−1) variant in converting hyoscyamine to anisodamine (Cao et al. 2015). To elucidate the molecular basis of enhancement, molecular dynamics simulations were performed (Figure S1a–h). HnH6H3M exhibited lower backbone RMSD values (Figure S1a,c), reduced RMSF in substrate-contact regions (Figure S1b,d), and smaller and more stable Rg (Figure S1e,g), indicating increased structural compaction and reduced conformational flexibility. Hydrogen bond analysis showed that HnH6H3M formed more stable and persistent hydrogen bonds within the catalytic pocket, thereby facilitating substrate stabilisation and efficient catalysis (Figure S1f,h). To study whether the mutations enhanced protein stability, thermal unfolding was monitored by circular dichroism at 208 nm. The ellipticity data were fitted to a two-state sigmoidal model to determine the melting temperature (Tm). HnH6H exhibited a Tm value of 75.55°C, whereas HnH6H3M showed a Tm value of 77.59°C, indicating that the mutations increased the overall structural stability (Figure S2). The spatial orientation (Fe-O-H(C7) approach angle) is critical for the epoxidation reaction catalysed by H6H, and even the modest deviations in this angle can lead to a substantial decrease in scopolamine production (Wenger et al. 2024). These results are consistent with the catalytic mechanism proposed by Wenger, suggesting that the stabilised protein structure may favour the formation of the optimal Fe-O-H(C7) angle, thereby promoting the efficiency of epoxidation. Atropa belladonna, a major industrial source of tropane alkaloids, is an ideal system for studying metabolic engineering using hairy root cultures. Therefore, we compared the AbH6H (from A. belladonna), HnH6H and HnH6H3M on engineering the production of scopolamine in hairy root cultures of A. belladonna. The control group produced hyoscyamine, anisodamine and scopolamine at concentrations of 1.96 mg/g dry weight (DW), 0.29 mg/g DW and 0.18 mg/g DW (Figure 1n–p). When AbH6H was overexpressed, the levels of hyoscyamine, anisodamine and scopolamine reached 1.17, 0.40 and 0.64 mg/g DW (Figure 1n–p). In hairy root cultures overexpressing HnH6H, the corresponding levels were 0.83 mg/g DW for hyoscyamine, 0.60 mg/g DW for anisodamine and 0.90 mg/g DW for scopolamine (Figure 1n–p). Overexpression of HnH6H3M resulted in the highest accumulation of anisodamine (0.94 mg/g DW) and scopolamine (2.02 mg/g DW), along with the lowest level of hyoscyamine (0.49 mg/g DW) (Figure 1n–p). The scopolamine level in hairy root cultures overexpressing HnH6H3M was 2.24 times higher than that in cultures overexpressing HnH6H (Figure 1p). Transgenic results indicate that HnH6H3M outperforms HnH6H and AbH6H in the engineered production of scopolamine. Furthermore, overexpression of HnH6H3M resulted in the highest total alkaloid levels, indicating that its enhanced catalytic activity efficiently drove the metabolic flux (Figure 1q). Overexpression of the terminal enzyme H6Hs drives scopolamine production via a ‘pull effect’, which may relieve upstream feedback inhibition (O'Connor 2015). Y.M., Z.L. and J.Z. designed the experiments and wrote the paper. Y.M., J.Z., Y.W., W.C., J.L., S.L., C.Y., X.L. and M.C. performed the experiments. L.Z. detected metabolites using an Orbitrap Exploris 120 LC–MS. This work was supported by Base and Talent Foundation of Science and Technology Department of Xizang Autonomous Region, XZ202501JD0026; Key project at central government level: The ability establishment of sustainable use for valuable Chinese medicine resources, 2060302; the Forth National Survey of Traditional Chinese Medicine Resources, 20191217-540124, 20200501-542329; the National Natural Science Foundation of China, 32500224; the Chongqing Postdoctoral Research Project Special Support program, 2412013580147856; the scientific funding given by Mianyang Habio Bioengineering Co., Ltd. HnH6H3M is included in an authorised patent (ZL202510695276.4). The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Figure S1: Molecular dynamics (MD) simulations of HnH6H and HnH6H3M. Figure S2: Determination of the melting curve of H6H by circular dichroism. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.