for the conversion of Hyoscyamine to Scopolamine › Poster ›...
1
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] 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 Hyoscyamine-6β-Hydroxylase Oxidoreductase acting on paired donors, found in the roots of Solanaceae [1] Cofactors: Fe 2+ , ascorbate [1] 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] Flow Chart Strains & Expression Conditions h6h-gene: Codon usage optimized for yeast → reasons for choice of strains 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 h6h- genes, 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 Ni 2+ 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. 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/ Highly stereo-selective [1] Average mass: 39 - 41 kDa [1] Reactions catalysed by Hyoscyamine-6β-Hydroxylase Strain for expression in E. coli: Rosetta Gami 2 DE3 Alleviated codon bias trxB/gor mutant Lysogen of λDE3 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 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 Strain for expression in S. cerevisiae: YPL154c (PEP4) Derived from BY4742 Genotype: MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 Vacuolar aspartyl protease (proteinase A) k. o. 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 Cloning Results & Discussion All of a ligation reactions products were used to transform DH5α E. colis by heat shock. Cloning of the h6h- gene 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: Nco-No NdeHis STOP TCT STOP V5 TCT V5 pET15b-h6h constructs pYES2/CT-h6h constructs Vectors & Cloning Strategy Inserts and vectors were prepared by digestion with the respective restriction enzymes: The inserts were ligated into the respective vectors restriction sites: Outgoing from a synthetic h6h-gene on a pMA-SK vector the inserts for the vectors were build by PCR: PCR Nco-No: For construct with untagged H6H NdeHis: For construct with 6xHis-Tag STOP V5 TCT STOP TCT V5 Inserts for constructs with V5 epitope & 6xHis-Tag: Inserts for constructs expressing H6H without Tag: PCR BamHI & NcoI/NdeI HindIII & XhoI pET15b + pYES2/CT + insert insert pET15b Vector Cloning & expression of proteins in E. coli N-terminal His-Tag Thrombin cleavage site pYES2/CT Vector Cloning in E. coli Expression of proteins in S. cerevisiae C-terminal V5 epitope & 6xHis-Tag Nco-No For expression of an untagged H6H NdeHis For expression of H6H with 6xHis-Tag STOP & TCT STOP V5 & TCT V5 Cloning Cloning For expression of untagged H6H For expression of H6H with V5 epitope & 6xHis-Tag Western Blot of protein extract from YPL154c transformants, 1 & 2: V5 constructs, 3 & 4: TCT V5 constructs SDS-PAGE of fractions eluted from the nickel column of protein extract from a NdeHis Rosetta Gami 2 DE3 transformant 1 2 3 4 Marker Bioactivity Native gene in vivo / in vitro catalysis Isolation of gene Scale Up Expression Results, Discussion & Outlook 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 post- translational degradation of the protein caused by cleavage of another protease besides the knocked out PEP4. Optimization of gene Hyoscyamus niger Synthetic gene from Hyoscyamus niger Codon usage optimized for eukaryotes Extent of current work Industrial Application Synthetic gene Cloning Vector Transformation Host: E. coli Host: S. cerevisiae Expression Enzyme 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 3000 bp 1000 bp 3000 bp 1000 bp 3 kbp 1 kbp 3 kbp 1 kbp 3 kbp 1 kbp 3 kbp 1 kbp
Transcript of for the conversion of Hyoscyamine to Scopolamine › Poster ›...
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
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
Hyoscyamine-6β-Hydroxylase
Oxidoreductase acting on paired donors, found in the roots of Solanaceae [1]
Cofactors: Fe2+, ascorbate[1]
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]
Flow Chart
Strains & Expression Conditions
h6h-gene: Codon usage optimized for yeast → reasons for choice of strains
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 h6h-genes, 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.
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/
Highly stereo-selective[1] Average mass: 39 - 41 kDa[1]
Reactions catalysed by Hyoscyamine-6β-Hydroxylase
Strain for expression in E. coli:
Rosetta Gami 2 DE3
Alleviated codon bias trxB/gor mutant Lysogen of λDE3
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
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
Strain for expression in S. cerevisiae:
YPL154c (PEP4)
Derived from BY4742 Genotype: MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 Vacuolar aspartyl protease (proteinase A) k. o.
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
Cloning Results & Discussion
All of a ligation reactions products were used to transform DH5α E. colis by heat shock. Cloning of the h6h-gene 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:
Nco-No
NdeHis STOP TCT STOP V5 TCT V5
pET15b-h6h constructs
pYES2/CT-h6h constructs
Vectors & Cloning Strategy
Inserts and vectors were prepared by digestion with the respective restriction enzymes:
The inserts were ligated into the respective vectors restriction sites:
Outgoing from a synthetic h6h-gene on a pMA-SK vector the inserts for the vectors were build by PCR:
PCR
Nco-No: For construct with untagged H6H
NdeHis: For construct with 6xHis-Tag
STOP
V5
TCT STOP
TCT V5
Inserts for constructs with V5 epitope & 6xHis-Tag:
Inserts for constructs expressing H6H without Tag:
PCR
BamHI & NcoI/NdeI
HindIII & XhoI
pET15b + pYES2/CT + insert insert
pET15b Vector Cloning & expression of proteins in E. coli N-terminal His-Tag Thrombin cleavage site
pYES2/CT Vector
Cloning in E. coli
Expression of proteins in S. cerevisiae
C-terminal V5 epitope & 6xHis-Tag
Nco-No For expression of an untagged H6H
NdeHis For expression of
H6H with 6xHis-Tag
STOP & TCT STOP
V5 & TCT V5
Cloning Cloning For expression of
untagged H6H
For expression of H6H with V5 epitope
& 6xHis-Tag
Western Blot of protein extract from YPL154c transformants, 1 & 2: V5 constructs, 3 & 4: TCT V5 constructs
SDS-PAGE of fractions eluted from the nickel column of protein extract from a NdeHis Rosetta Gami 2 DE3 transformant
1 2 3 4 Marker
Bioactivity
Native gene
in vivo / in vitro catalysis
Isolation of gene
Scale Up
Expression Results, Discussion & Outlook
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 post-translational degradation of the protein caused by cleavage of another protease besides the knocked out PEP4.
Optimization of gene
Hyoscyamus niger
Synthetic gene from Hyoscyamus niger Codon usage optimized for eukaryotes
Extent of current work Industrial Application
Synthetic gene
Cloning
Vector
Transformation
Host: E. coli
Host: S. cerevisiae
Expression
Enzyme
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
3000 bp
1000 bp
3000 bp
1000 bp
3 kbp
1 kbp
3 kbp
1 kbp
3 kbp
1 kbp
3 kbp
1 kbp
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