Acetyl-CoA and Oliver Kayser - TU Dortmund Japan Duboisia Laura Kohnen.… · Principal Studies on...
-
Upload
phungkhuong -
Category
Documents
-
view
230 -
download
3
Transcript of Acetyl-CoA and Oliver Kayser - TU Dortmund Japan Duboisia Laura Kohnen.… · Principal Studies on...
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]
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