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Expression of Hyoscyamine-6β-Hydroxylase in genetically modified E. coli and S. cerevisiae
for the conversion of Hyoscyamine to Scopolamine
Nils Averesch *, Marcello Siena, Oliver Kayser
Laboratory of Technical Biochemistry, Department of Biochemical and Chemical Engineering, TU Dortmund
* [email protected]
Abstract
Tropane alkaloids are a class of plant secondary metabolites that exclusively exist in Solanaceae. The tropane alkaloid scopolamine and its precursor hyoscyamine, are widely used as
pharmaceuticals due to their anticholinergic activity.[5,6] Scopolamine is the more valuable alkaloid, with a 10x higher commercial demand than that of hyoscyamine because of the fewer side
effects and higher physiological activity.[5] Unfortunately hyoscyamine is the more abundant alkaloid produced in plants.[6] Hashimoto et al., first identified a 2-oxoglutarate-dependent dioxygenase
from Hyoscyamus niger that hydroxylates hyoscyamine at the 6β-position and further epoxidates it to scopolamine: Hyoscyamine-6β-Hydroxylase (H6H, EC 1.14.11.11).[1,2,3] This project is focused
on a production system for Hyoscyamine-6β-Hydroxylase with genetically modified microorganisms to convert abundant hyoscyamine to scopolamine. As has been reported for several natural h6hgenes, a synthetic h6h-gene originating from H. niger was introduced into Escherichia coli[3,7,8] and Saccharomyces cerevisiae[5] host strains to express an active enzyme. Outgoing from pET15b and
pYES2/CT, vectors were constructed for E. coli and S. cerevisiae respectively, by cloning a synthetic h6h-gene into the designated vectors restriction sites. The E. coli constructs were designed with
and without an N-terminal 6xHis-Tag. For S. cerevisiae four different constructs were designed: With and without C-terminal V5-epitope + 6xHis-Tag each with two different consensus sequences
for initiation of translation. The E. coli strain Rosetta Gami 2 DE3, transformed with the pET15b-h6h vector, expressed the His-tagged protein, validated by SDS-PAGE after IMAC utilizing a column
with a Ni2+ charged resin. For the pYES2/CT-h6h transformed protease knock out strain YPL154c (derived from BY4742), expression could proven by detecting the V5-epitope on a Western Blot.
Flow Chart
Hyoscyamine-6β-Hydroxylase
Optimization of gene
 Oxidoreductase acting on paired donors, found in the roots of Solanaceae [1]
 Cofactors: Fe2+, ascorbate[1]
 Highly stereo-selective[1]
 Average mass: 39 - 41 kDa[1]
Isolation of gene
Native gene
Synthetic gene
 Synthetic gene from Hyoscyamus niger
 Codon usage optimized for eukaryotes
Hyoscyamus niger
Cloning
Bioactivity
Vector
Host: E. coli
Transformation
Scale Up
Expression
Enzyme
in vivo / in vitro
catalysis
Host: S. cerevisiae
Industrial Application
Extent of current work
Strains & Expression Conditions
YPL154c (PEP4)
 Alleviated codon bias
 trxB/gor mutant
 Lysogen of λDE3
 Hydroxylation of l-hyoscyamine at the 6β-position to 6β-hydroxyhyoscyamine[1,2]
 Epoxidation to scopolamine by dehydrogenation of the 6β-hydrogen[2,3]
 Epoxidase activity is only 1-10% of the hydroxylase activity (bottleneck in the respective pathway)[3,4]
Vectors & Cloning Strategy
h6h-gene: Codon usage optimized for yeast → reasons for choice of strains
Strain for expression in E. coli:
Strain for expression in S. cerevisiae:
Rosetta Gami 2 DE3
Reactions catalysed by
Hyoscyamine-6β-Hydroxylase
 Derived from BY4742
 Genotype: MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0
 Vacuolar aspartyl protease (proteinase A) k. o.
 Chloramphenicol-resistant pRARE2 plasmid supplies seven rare tRNAs
 Enhance disulfide bond formation selectable on tetracycline
 Carries chromosomal copy of T7 RNA polymerase gene under control of lacUV5 promoter
pET15b Vector
 Cloning in E. coli
 Cloning & expression of proteins in E. coli
 Expression of proteins
in S. cerevisiae
 N-terminal His-Tag
 Thrombin cleavage site
 C-terminal V5 epitope
& 6xHis-Tag
 Outgoing from a synthetic h6h-gene on a pMA-SK vector the inserts for the vectors were build by PCR:
Cultivation conditions:
 LB medium pH 7 in baffled shake flasks
 1 mM IPTG for induction of expression
 Antibiotics: Amp, Cam, Tet
 Cultivation: 30°C, 200 rpm, 50 mm orbit, 22h
 Minimal medium pH 6 in baffled shake flasks
 2% glucose (precultures) 2% galactose (expression
cultures), + amino acids & vitamines, no uracil
 Cultivation: 30°C, 200 rpm, 50 mm orbit, 23h
pYES2/CT Vector
PCR
Inserts for constructs expressing H6H without Tag:
Nco-No: For construct with untagged H6H
STOP
PCR
TCT STOP
Inserts for constructs with V5 epitope & 6xHis-Tag:
NdeHis: For construct with 6xHis-Tag
V5
TCT V5
 Inserts and vectors were prepared by digestion with the respective restriction enzymes:
Expression Results, Discussion & Outlook
BamHI &
NcoI/NdeI
pET15b
 Protein extraction methods were the same for E. coli and S. cerevisiae: French pressing three times in
protease inhibiting buffer (10 mL / g cell wet weight) followed by centrifugation (10 min, RCF = 25000 x g)
 In case of E. coli the supernatant was used for immobilized metal affinity chromatography (IMAC) followed
by SDS-PAGE of the fractions  Protein band around 40 kDa (H6H + His-Tag) in the elution fractions
 For S. cerevisiae a Western Blot was performed directly with the crude protein extract. V5 epitope
detection by chemiluminescence revealed a band at 41 kDa (H6H + V5 epitope + 6xHis-Tag) but also
several bands at lower masses. Conceivable might be splicing of the mRNA
Which is quite unlikely as the
gene is optimized for expression
in yeast, also regarding exon /
intron recognition sites. Most
conceivable might be posttranslational degradation of the
protein caused by cleavage of
another protease besides the
knocked out PEP4.
1
2
3
4 Marker
SDS-PAGE of fractions eluted from the
nickel column of protein extract from a
NdeHis Rosetta Gami 2 DE3 transformant
Western Blot of protein extract from YPL154c transformants,
1 & 2: V5 constructs, 3 & 4: TCT V5 constructs
Fakultät Bio- und Chemieingenieurwesen
Prof. Dr. Oliver Kayser
Lehrstuhl Technische Biochemie
Emil-Figge-Str. 66
D-44227 Dortmund
Tel.: +49 (0)231 / 755 - 7487
Fax: +49 (0)231 / 755 - 7489
[email protected]
http://www.tb.tu-dortmund.de/
pYES2/CT
insert
+
insert
 The inserts were ligated into the respective vectors restriction sites:
Cloning
Cloning
Nco-No
NdeHis
For expression of
an untagged H6H
For expression of
H6H with 6xHis-Tag
STOP
V5
&
&
TCT STOP
TCT V5
For expression of
untagged H6H
For expression of
H6H with V5 epitope
& 6xHis-Tag
Cloning Results & Discussion
All of a ligation reactions products were used to transform DH5α E. colis by heat shock. Cloning of the h6hgene was successful for more than 60% of all transformants. This was proven by PCR from colonies with gene
specific primers and gel electrophoresis of the products on a 1% agarose gel:
pET15b-h6h constructs
pYES2/CT-h6h constructs
As these are preliminary results, reproducibility should be verified, and diverse parameters customized:
 Solubility of the protein was already reviewed by excluding the production of inclusion bodies in E. coli
 Temperature, time course (& IPTG conc. in case of E. coli) should be adjusted for optimal expression level
 Transcript level might be checked by mRNA extraction and analysis
 The nature of the protein has to be confirmed, either by specific antibodies or by sequencing of the protein
 The activity of the enzyme from E. coli has to be tested in vivo and in vitro and collated with the yeast’s
 Other genes could be used, e.g. a natural gene, or a gene that promotes an outward transfer of the enzyme
+
HindIII
& XhoI
3000 bp
1000 bp
3 kbp
3 kbp
3 kbp
3 kbp
1 kbp
1 kbp
1 kbp
1 kbp
Nco-No
3000 bp
1000 bp
NdeHis
STOP
TCT STOP
V5
TCT V5
References
[1] Hyoscyamine-6β-Hydroxylase, a 2-oxoglutarate-dependent dioxygenase, in alkaloid-producing root cultures; T. Hashimoto, Y. Yamada; Plant Physiol. 1986, 81:619-625
[2] Purification and characterization of Hyoscyamine-6β-Hydroxylase from root cultures of Hyoscyamus niger L.; T. Hashimoto, Y. Yamada; Eur. J. Biochem. 1987, 164:277-285
[3] Two-step epoxidation of Hyoscyamine to Scopolamine is catalyzed by bifunctional Hyoscyamine-6β-Hydroxylase; T. Hashimoto, J. Matsuda, Y. Yamada; FEBS Lett. 1993, 329, 1-2:35-39
[4] Alkaloid Biogenesis: Molecular Aspects; T. Hashimoto, Y. Yamada; Annu. Rev. Plant Physiol. Plant Mol. BioI. 1994, 45:257-85
[5] Expression of Brugmansia candida Hyoscyamine-6β-Hydroxylase gene in Saccharomyces cerevisiae and its potential use as biocatalyst; A. Cardillo, J. Rodríguez Talou, A. Giulietti; Microb. Cell Fact. 2008, 7:17
[6] Application of Metabolic Engineering to the Production of Scopolamine; J. Palazón, A. Navarro-Ocaña, L. Hernandez-Vazquez, M. H. Mirjalili; Molecules 2008, 13:1722-1742
[7] Biochemical and structural characterization of recombinant Hyoscyamine-6β-Hydroxylase from Datura metel L.; K.K. Pramod, S. Singh, C. Jayabaskaran; Plant Physiol. Biochem. 2010, 48, 12:966-970
[8] Functional identification of Hyoscyamine-6β-Hydroxylase from Anisodus acutangulus and overproduction of Scopolamine in genetically-engineered Escherichia coli; G. Kai, Y. Liu, X. Wang, S. Yang, X. Fu, X. Luo, P. Liao; Biotechnol. Lett. 2011, 33, 7:1361-1365