Principal Studies on Scopolamine Biosynthesis in Duboisia spec. for Heterologous Reconstruction of Tropane Alkaloid Biosynthesis Laura Kohnen1, Friederike Ullrich1, Nils J. H. Averesch2 and Oliver Kayser1 (1) Technical Biochemistry, Technical University Dortmund, Dortmund, Germany (2) Centre for Microbial Electrochemical Systems (CEMES), The University of Queensland, Brisbane, Australia [email protected], [email protected], [email protected], [email protected] Tropane alkaloids Objectives - Tropane alkaloids (TA), including scopolamine and hyoscyamine, are - Reconstruction of a heterologous pathway requires fundamental secondary plant components mainly occurring in the family of Solanaceae understanding of the merging pathways, the respective biosynthetic genes, - Scopolamine is an important bulk compound in the semi-synthesis of drugs their transcription and regulation - here we focus on: for clinical medicines like Buscopan® or Spiriva® I) Analysis of the principal pathway and most important enzymes for their - TA are mainly obtained via extraction from field-grown Duboisia hybrids expression and biochemical profile - Demand for scopolamine based drugs is expected to increase in the future II) Construction of a cDNA library from cytosolic root cells of Duboisia - A biotechnological process may help to compensate fluctuations in crop yield species of the medicinal plants III) Analysis of metabolic profiles of Duboisia species Introduction IV) In silico analysis of a heterologous production system utilizing metabolic network modelling Biosynthetic pathway of TA NMR-based metabolomics - Biosynthesis of TA is located in the roots[1] - Global analysis of leaf and root extracts - TA are transported to the aerial parts of the plants - Detection of primary (amino acids, sugars) as well as secondary metabolites - Storage and accumulation of hyoscyamine, 6-OH-hyoscyamine and (flavonoids, TA) scopolamine in the leaves (cf. HPLC-MS based quantitation) - Glucose, sucrose and Tropane alkaloids myo-inositol positively Sugars Tropine Littorine CYP80F1 Dehydro- H6H H6H correlated with plant Synthase genase growth Littorine Hyoscyamine Hyoscyamine 6-OH-Hyoscyamine Scopolamine - Environmental and Amino acids Analytics and metabolomics Phenyllactate aldehyde Fig. 1: Section of late tropane alkaloid pathway genetic factors strongly MALDI imaging-MS of Duboisia myoporoides roots affect scopolamine Flavonoids production in Duboisia - Investigation of three different growth stages (6 weeks, 3 months, and 6 plants months) of the roots - Spatial distribution of the TA tropine, hyoscyamine, and scopolamine Fig. 3: Characteristic NMR spectrum of a Duboisia leaf extract. 6 weeks 3 months 6 months HPLC-MS-based tropane alkaloid quantitation Tropine - Quantitation of scopolamine itself and of its direct precursors and degradation products in different genotypes of Duboisia (genotype A = D. myoporoides; genotypes B, C = D. leichhardtii; genotypes D, E = D. hybrids) - Hybrids D and E show 30 highest scopolamine Hyoscyamine levels 25 - Wild types B and C predominantly 20 produce hyoscyamine Scopolamine Biosynthesis and localization Biosynthesis 15 - Method potentially Hyoscyamine Scopolamine 6β-OH-hyoscyamine applicable for monitor- 10 Littorine ing alkaloid bio- Norscopolamine Norhyoscyamine Fig. 2: Ion images showing the spatial distribution of tropane alkaloids in Duboisia myoporoides root. Localization of tropine ([M+H]+; m/z 142.12, localization of synthesis in a hetero- [mg/g] concentration Alkaloid 5 hyoscyamine ([M+H]+; m/z 290.18), localization of scopolamine ([M+H]+; m/z 304.15). Scale bar 1 mm. logous production 0 - Different spatial distribution over time; biosynthesis of young plants is located system A B C D E within the central cylinder, whereas the localization of TA in older plants is Genotype found in the inner cortex and outer central cylinder Fig. 4: Alkaloid profile of different genotypes of Duboisia quantified via HPLC-MS. Erythrose 4-phosphate Phosphoenolpyruvate THF H2O THF Elementary flux mode and thermodynamic analysis SER ATP Formate Phosphate Arginine Ornithine THF 2-Dehydro-3-deoxy-D-arabino-heptonate 7-phosphate + ADP CO2 GLY NAD Agmatine Phosphate CO2 10-Formyl-THF of recombinant scopolamine production CO2 3-Dehydroquinate CO2 NH3 NH NADH NH3 3 Carbamoylputrescine Putrescine 5,10-Methylene-THF H2O 5,10-Methenyl-THF 3-Dehydroshikimate SAM NADPH - The obtained array of flux distributions (Fig. 7) shows that de novo production NADPH NADPH SAH NADP+ c d NADP+ Methylputresine NADP+ of scopolamine from glucose is theoretically possible in a microbial system O2 5-Methyl-THF Shikimate a b 5,10-Methylene-THF ATP CO2 H2O2 NADPH - Maximum theoretical carbon yields range from 62 – 70% depending on Methylpyrrolinium Figure 6: Different options for regeneration of ADP Acetoacetyl-CoA THF – (c) glycine cleavage system (GCS) and + Shikimate 3-phosphate NADP Phosphoenolpyruvate (d) using formate as methyl group donor. model (E. coli vs. S. cerevisiae) and alternative biochemical reactions / routes Methylpyrrolinium 5-Methyl-THF Methyl-THF is then used to regenerate SAM. Phosphate -acetoacetyl-CoA In particular three pathway-variations were differentiated: 5-Enolpyruvylshikimate 3-phosphate CO2 H-CoA Phosphate Tropinone Folate Chorismate Tryptophan NADPH 100 I. Co-factor utilisation of hydroxyphenylpyruvate reductase: NADH dependency benefits yield, NADPH 90 Scopolamine de novo from glucose Scopolamine from glucose & tropine + NADP 80 in E. coli co-feed in E. coli utilisation favours thermodynamics of phenyllactate formation - can impact the titer Prephenate Tyrosine Tropine 70 60 II. Biosynthetic route for putrescine formation: outgoing from ornithine the maximum theoretical carbon CO2 50 aI bI Phenylpyruvate Phenylalanine 40 yield is higher than via arginine - thermodynamically both are favoured NAD(P)H 30 + 20 III. SAM / methyl-THF regeneration mechanism: Utilizing the glycine cleavage system (GCS) the achievable NAD(P) Fig. 5: Pathways to 10 Phenyllactate scopolamine and tropine. In 0 carbon yields were restricted - could be improved when using formate to regenerate methyl-THF, Acetyl-CoA 100 the pathway to scopolamine 90 Scopolamine de novo from glucose Scopolamine from glucose & tropine provided by a pyruvate formate lyase (natural in E. coli, heterologous in case of S. cerevisiae) Acetate (a) the reduction of 80 in S. cerevisiae co-feed in S. cerevisiae Phenyllactyl-CoA 70 phenylpyruvate to yield [%] Biomass Tropine 60 aII bII - Assuming a tropine-feed to allow glucose + tropine co-utilisation significantly phenyllactate can be NADH 50 H-CoA or NADPH dependent. 40 Littorine 30 higher maximum theoretical carbon yields (82 – 86%) are possible Formation of putrescine 20 Hyoscyamine towards biosynthesis of 10 0 - Tropine-feed also avoids the metabolic bottleneck of methylation co-factor 2-Oxoglutarate O2 tropine (b) can proceed 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 outgoing from ornithine or Product carbon yield [%] Succinate CO2 6β-Hydroxyhyoscyamine arginine. Fig. 7: Product carbon vs. biomass yield plots of elementary flux modes for E. coli (I) Metabolic network analysis network Metabolic regeneration - avoiding thermodynamic restrictions may allow higher titers NAD+ and S. cerevisiae (II) metabolic networks enabling production of scopolamine, NADH de novo from glucose (a) or utilizing a glucose & tropine co-feed (b). Scopolamine - Spatial metabolite distribution is age dependent - Refine metabolic models, develop strain construction strategies O - Alkaloid and metabolite profile largely depends on genotype - Stepwise heterologous reconstruction[2] of the alkaloid pathway utlook - Robust LC-MS method for alkaloid quantitation was developed and validated - in vivo validation of the in silico model: - Heterologous de novo production of TA in microbes theoretically possible I. E. coli vs. S. cerevisiae - External tropine supply significantly improves theoretical maximum yield II. de novo production vs. glucose & tropine co-feed - Circumventing one-carbon metabolism may warrant significant titers - Establish sustainable scopolamine production Summary References Acknowledgements [1] Ziegler J, Facchini PJ (2008) Alkaloid Biosynthesis: Metabolism and Trafficking. Annu Rev Plant Biol 59:735–769. doi: This project has received funding from the European Union’s Seventh Framework Programme for research, 10.1146/annurev.arplant.59.032607.092730 technological development and demonstration under grant agreement No 613513. [2] Averesch NJH, Kayser O (2014) Assessing Heterologous Expression of Hyoscyamine 6β-Hydroxylase – A Feasibility Study. Procedia Chem 13:69–78. doi: http://dx.doi.org/10.1016/j.proche.2014.12.008 .