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

Laura.Kohnen@bci.tu-dortmund.de, Friederike.Ullrich@bci.tu-dortmund.de, nils.averesch@uq.net.au, Oliver.Kayser@bci.tu-dortmund.de

Acknowledgements

This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement No 613513.

References

[1] Ziegler J, Facchini PJ (2008) Alkaloid Biosynthesis: Metabolism and Trafficking. Annu Rev Plant Biol 59:735–769. doi: 10.1146/annurev.arplant.59.032607.092730 [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

Tropane alkaloids

- Tropane alkaloids (TA), including scopolamine and hyoscyamine, are secondary plant components mainly occurring in the family of Solanaceae

- Scopolamine is an important bulk compound in the semi-synthesis of drugs for clinical medicines like Buscopan® or Spiriva®

- TA are mainly obtained via extraction from field-grown Duboisia hybrids - Demand for scopolamine based drugs is expected to increase in the future - A biotechnological process may help to compensate fluctuations in crop yield

of the medicinal plants

Objectives

- Reconstruction of a heterologous pathway requires fundamental understanding of the merging pathways, the respective biosynthetic genes, their transcription and regulation - here we focus on:

I) Analysis of the principal pathway and most important enzymes for their expression and biochemical profile II) Construction of a cDNA library from cytosolic root cells of Duboisia species III) Analysis of metabolic profiles of Duboisia species IV) In silico analysis of a heterologous production system utilizing metabolic network modelling

Intr

od

uct

ion

B

iosy

nth

esis

an

d lo

caliz

atio

n A

nalytics an

d m

etabo

lom

ics M

etab

olic

net

wo

rk a

nal

ysis

0

5

10

15

20

25

30

A B C D E

Alk

alo

id c

on

cen

trat

ion

[m

g/g]

Genotype

Scopolamine

Hyoscyamine

6β-OH-hyoscyamine

Littorine

Norscopolamine

Norhyoscyamine

HPLC-MS-based tropane alkaloid quantitation

- 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 highest scopolamine levels

- Wild types B and C predominantly produce hyoscyamine

- Method potentially applicable for monitor-ing alkaloid bio-synthesis in a hetero-logous production system

NMR-based metabolomics

- Global analysis of leaf and root extracts - Detection of primary (amino acids, sugars) as well as secondary metabolites

(flavonoids, TA) - Glucose, sucrose and

myo-inositol positively correlated with plant growth

- Environmental and genetic factors strongly affect scopolamine production in Duboisia plants

Fig. 1: Section of late tropane alkaloid pathway

Tropane alkaloids

Sugars

Amino acids

Flavonoids

Biosynthetic pathway of TA

- Biosynthesis of TA is located in the roots[1]

- TA are transported to the aerial parts of the plants - Storage and accumulation of hyoscyamine, 6-OH-hyoscyamine and

scopolamine in the leaves (cf. HPLC-MS based quantitation)

Dehydro-

genase

H6H H6H Littorine

Synthase

CYP80F1

MALDI imaging-MS of Duboisia myoporoides roots

- Investigation of three different growth stages (6 weeks, 3 months, and 6 months) of the roots

- Spatial distribution of the TA tropine, hyoscyamine, and scopolamine

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 hyoscyamine ([M+H]+; m/z 290.18), localization of scopolamine ([M+H]+; m/z 304.15). Scale bar 1 mm.

- Different spatial distribution over time; biosynthesis of young plants is located within the central cylinder, whereas the localization of TA in older plants is found in the inner cortex and outer central cylinder

6 weeks 3 months 6 months

Tropine

Hyoscyamine

Scopolamine

Elementary flux mode and thermodynamic analysis of recombinant scopolamine production

- The obtained array of flux distributions (Fig. 7) shows that de novo production of scopolamine from glucose is theoretically possible in a microbial system

- Maximum theoretical carbon yields range from 62 – 70% depending on model (E. coli vs. S. cerevisiae) and alternative biochemical reactions / routes In particular three pathway-variations were differentiated:

I. Co-factor utilisation of hydroxyphenylpyruvate reductase: NADH dependency benefits yield, NADPH utilisation favours thermodynamics of phenyllactate formation - can impact the titer

II. Biosynthetic route for putrescine formation: outgoing from ornithine the maximum theoretical carbon yield is higher than via arginine - thermodynamically both are favoured

III. SAM / methyl-THF regeneration mechanism: Utilizing the glycine cleavage system (GCS) the achievable carbon yields were restricted - could be improved when using formate to regenerate methyl-THF, provided by a pyruvate formate lyase (natural in E. coli, heterologous in case of S. cerevisiae)

- Assuming a tropine-feed to allow glucose + tropine co-utilisation significantly higher maximum theoretical carbon yields (82 – 86%) are possible

- Tropine-feed also avoids the metabolic bottleneck of methylation co-factor regeneration - avoiding thermodynamic restrictions may allow higher titers

Fig. 3: Characteristic NMR spectrum of a Duboisia leaf extract.

Fig. 4: Alkaloid profile of different genotypes of Duboisia quantified via HPLC-MS.

Fig. 5: Pathways to scopolamine and tropine. In the pathway to scopolamine (a) the reduction of phenylpyruvate to phenyllactate can be NADH or NADPH dependent. Formation of putrescine towards biosynthesis of tropine (b) can proceed outgoing from ornithine or arginine.

NADPH

NADP+

SAM

SAH

PutrescineCarbamoylputrescine

Agmatine

OrnithineArginine

Methylputresine

Methylpyrrolinium

Methylpyrrolinium-acetoacetyl-CoA

Tropinone

Tropine

CO2

CO2

NH3

CO2 NH3

CO2 H2O2

O2

CO2

Acetoacetyl-CoA

H-CoA

b

Erythrose 4-phosphate Phosphoenolpyruvate

3-Dehydroquinate

3-Dehydroshikimate

Shikimate

Shikimate 3-phosphate

5-Enolpyruvylshikimate 3-phosphate

Chorismate

Phenylalanine

Tyrosine

2-Dehydro-3-deoxy-D-arabino-heptonate 7-phosphate

NADPH

NADP+

ATP

ADP

Phosphoenolpyruvate

H2O

H2O

Phosphate

Phosphate

Phosphate

Phosphate

Tryptophan

Prephenate

Folate

Phenylpyruvate

CO2

Phenyllactate

NAD(P)H

NAD(P)+

Acetyl-CoA

Acetate

Phenyllactyl-CoATropine

H-CoA

Littorine

Hyoscyamine

6β-Hydroxyhyoscyamine

Scopolamine

2-Oxoglutarate

Succinate

O2

CO2

NADH

NAD+

a Figure 6: Different options for regeneration of THF – (c) glycine cleavage system (GCS) and (d) using formate as methyl group donor. Methyl-THF is then used to regenerate SAM.

5-Methyl-THF

THF

5,10-Methenyl-THF

5,10-Methylene-THF

10-Formyl-THF

FormateATP

ADP

NADPH

NADP+

NADPH

NADP+d

NADH

NAD+

THF

5-Methyl-THF

5,10-Methylene-THFNADPH

NADP+

SER

CO2

NH3

GLYTHF

c

Fig. 7: Product carbon vs. biomass yield plots of elementary flux modes for E. coli (I) and S. cerevisiae (II) metabolic networks enabling production of scopolamine, de novo from glucose (a) or utilizing a glucose & tropine co-feed (b).

0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100

Product carbon yield [%]

Bio

mas

s yi

eld

[%

]

100 90 80 70 60 50 40 30 20 10

0 100

90 80 70 60 50 40 30 20 10

0

Scopolamine de novo from glucose

in E. coli

aI

Scopolamine from glucose & tropine

co-feed in E. coli

bI

Scopolamine de novo from glucose

in S. cerevisiae

aII

Scopolamine from glucose & tropine

co-feed in S. cerevisiae

bII

- Spatial metabolite distribution is age dependent - Alkaloid and metabolite profile largely depends on genotype - Robust LC-MS method for alkaloid quantitation was developed and validated - Heterologous de novo production of TA in microbes theoretically possible - External tropine supply significantly improves theoretical maximum yield - Circumventing one-carbon metabolism may warrant significant titers Su

mm

ary O

utlo

ok

- Refine metabolic models, develop strain construction strategies - Stepwise heterologous reconstruction[2] of the alkaloid pathway - in vivo validation of the in silico model:

I. E. coli vs. S. cerevisiae II. de novo production vs. glucose & tropine co-feed

- Establish sustainable scopolamine production

Tropine

Phenyllactate

Littorine Hyoscyamine

aldehyde

Hyoscyamine 6-OH-Hyoscyamine Scopolamine