epoxomicin: assembly line engineering for pharmaceutical drug production using natural product gene...
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Epoxomicin:Assembly Line Engineering for Pharmaceutical Drug Production Using Natural Product Gene Clusters
Anna Klavins, Haley HoffmanAugust 13, 2015
California Polytechnic State University
NNH
HN
O
NH
O O
OHO
O
O
epoxomicinNatural Product
ATCC 53904
Epoxomicin Carfilzomib
NNH
HN
O
NH
O O
OHO
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epoxomicinNatural Product
ATCC 53904N
NH
HN
O
NH
O O
O
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YU-101SyntheticProteolix
NNH
HN
O
NH
O O
O
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O
N
O
CarfilzomibSynthetic
Onyx PharmaceuticalsKim, K. B.; Crews, C. M. Nat. Prod. Rep. 2013, 30, 600–4.
• Epoxomicin is a natural product peptide, and we are substituting amino acids in order to make YU-101
• Many drugs are based on Epoxomicin’s structure including cancer drug Carfilzomib
• Epoxomicin inhibits the 20S proteasome in humans
Introduction
• Carfilzomib costs $10,000 for one 28 day treatment when chemically synthesized
• Use of natural product Epoxomicin for biosynthesis could potentially decrease costs for this cancer treatment
NNH
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NH
O O
OHO
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epoxomicinNatural Product
ATCC 53904
NNH
HN
O
NH
O O
O
O
O
N
O
CarfilzomibSynthetic
Onyx Pharmaceuticals
NRPS-PKS Sequence
• This gene cluster consists of a non-ribosomal polypeptide synthase and polyketide synthase
• Entire gene cluster ~ 28 kb• NRPS is ~ 15 kb
ACADPKSNRPSP450ORR
Schorn, M.; Zettler, J.; Noel, J. P.; Dorrestein, P. C.; Moore, B. S.; Kaysser, L. ACS Chem. Biol. Ahead of print. Oct. 29, 2013.
Adenylation Domains
• Each module of the NRPS catalyzes the incorporation of one amino acid into the growing peptide
• Adenylation domains are responsible for what amino acid is incorporated (the “gate-keeper”)- Found boundaries of the domains using the known sequence
from NCBI, and using antiSMASH and Serial Cloner software
Module 2 Module 3
NRPS
Module 1 Module 4
Goals
• Demonstrate metabolic engineering as a strategy for production of a therapeutically relevant molecule
• Changing specificity of adenylation domains in the natural gene cluster through site-directed mutagenesis (based on past experiments done by two other research groups)
• Thirlway, J.; Lewis, R.Nunns, L.; Al Nakeeb, M.; Styles, M.; Struck, A.-W.; Smith, C. P.; Micklefield, J. Angew. Chem. Int. Ed. 2012, 51, 7181–4.
• Zhang, K.; Nelson, K. M.; Bhuripanyo, K.; Grimes, K. D.; Zhao, B.; Aldrich, C. C.; Yin, J. Chem. Biol. 2013, 20, 92–101.
• Marahiel, A.; Mootz, D. Chem. Biol. 1999, 6, 493–505.
Module 2 Module 3Module 1
Epoxomicin Isoleucine IsoleucineThreonine LeucineCarfilzomib Homophenylalanine LeucinePhenylalanine Leucine
Module 4
• Changes will be made to adenylation domains using site directed mutagenesis
KeyA – adenylation domainC – condensation domain
- A4 does not need modification
235 236 239 278 299 301 322 330 331 517Thr D F W N I G M V H KPhe D A W T I A A V C KIle D G F F L G V V Y K
Leu D A W F L G N V V KHph D L G A I G C V I K
Position number and amino acids of adenylation domains linked to specificity
Incorporated Amino Acid
Marahiel, A.; Mootz, D. Chem. Biol. 1999, 6, 493–505.
Methods
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NH
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Isolated Epoxomicin gene cluster in a plasmid (Dr. Leonard Kayseer)
Amplify 3 modules of NRPS
Assemble all 3 modules into gene cluster (Gibson Assembly)
Express in a heterologous host
Analyze metabolites
Module 1Module 2
Module 3
Each module is placed in its own DNA construct
Site-directed mutagenesis of adenylation domains
pET28
pET28 pET28
Methods
NNH
HN
O
NH
O O
O
O
O
Isolated Epoxomicin gene cluster in a plasmid (Dr. Leonard Kayseer)
Amplify 3 modules of NRPS
Assemble all 3 modules into gene cluster (Gibson Assembly)
Express in a heterologous host
Analyze metabolites
Module 1Module 2
Module 3
Each module is placed in its own DNA construct
Site-directed mutagenesis of adenylation domains
pET28
pET28 pET28
✔
✔✔
Primer Problems
First set of primers, only 12nts long
Second set of primers, 20nts long, more specific
Module 1: ~ 4300 base pairs
Module 3: ~ 3100 base pairs Module 1
Module 2: ~ 4000 base pairs
Module 3
10 uL reaction using Module DNA Template for better isolation of each module
Gel electrophoresis using Epx MS01 DNA as template DNA
Gel extraction (GeneJET kit used) to extract each module band to make Module DNA
Isolation of Module DNA
Results
Pictured: 20 uL reactions using Module DNA as the template, scaling the reaction up from 10 uL to 20 uL appears to be successful. This will help us increase module DNA yield.
Low molecular weight bands, trying to changePCR procedure in order to rid of these bandsand do PCR extraction instead of gel extraction.This could help improve our DNA yield.
Module 1 Module 2 Module 3
Conclusion/Future Outlook
• What will you do next?– Insert module DNA fragments into pET 28– Use site directed mutagenesis to change
adenylation domains to code for the amino acids needed to biosynthesize the base of Carfilzomib
• Funding: CBF/Frost Funds, CSUperb• Acknowledgements: Dr. Leonard Kaysser,
University of Tuebingen, Germany