1. Epoxomicin: Assembly Line Engineering for Pharmaceutical Drug Production Using Natural Product Gene Clusters
Anna Klavins, Haley Hoffman
August 13, 2015
California Polytechnic State University

2. Epoxomicin  Carfilzomib
Epoxomicin is a natural product peptide, and we are substituting amino acids in order to make YU-101
Epoxomicin inhibits the 20S proteasome in humans
Discovered at BMS in early 1990s. Craig Crews at Yale showed that this molecule inhibited the catalytic subunit of the 20S proteosome. In this lab and Proteolix, a spin off company, a more selective version, YU-101 was developed. Had issues with solubility,
Epoxomicin isolated yield ~ 2 mg/L
Many drugs are based on Epoxomicin’s structure including cancer drug Carfilzomib
Kim, K. B.; Crews, C. M. Nat. Prod. Rep. 2013, 30, 600–4.

3. 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

4. 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 R OR
P450
NRPS
PKS
ACAD
Grey arrows represent tailoring enzymes and regulators of the pathway
Schorn, M.; Zettler, J.; Noel, J. P.; Dorrestein, P. C.; Moore, B. S.; Kaysser, L. ACS Chem. Biol. Ahead of print. Oct. 29, 2013.

5. Adenylation Domains
NRPS
Module 1
Module 2
Module 3
Module 4
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

6. 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.
Find a picture

7. Changes will be made to adenylation domains using site directed mutagenesis
- A4 does not need modification
Module 1
Module 2
Module 3
Module 4
Epoxomicin Isoleucine Isoleucine Threonine Leucine
Carfilzomib Homophenylalanine Leucine Phenylalanine Leucine
Key
A – adenylation domain
C – condensation domain
Add table and combine with slide 8
Marahiel, A.; Mootz, D. Chem. Biol. 1999, 6, 493–505.

8. Methods
Isolated Epoxomicin gene cluster in a plasmid (Dr. Leonard Kayseer)
Each module is placed in its own DNA construct
Amplify 3 modules of NRPS
pET28
Module 3
pET28
pET28
Module 1
Module 2
Site-directed mutagenesis of adenylation domains
Analyze metabolites
Isolate genomic DNA -> Isolate NRPS sequence -> Assemble onto DNA Construct with pET28 -> Modify adenylation domains in NRPS -> Express in Heterologous Host -> Analyze Metabolites
Express in a heterologous host
Assemble all 3 modules into gene cluster (Gibson Assembly)

9. Methods
Isolated Epoxomicin gene cluster in a plasmid (Dr. Leonard Kayseer)
Each module is placed in its own DNA construct
Amplify 3 modules of NRPS
pET28 ✔ ✔
Module 3
pET28
pET28
Module 1
Module 2 ✔
Site-directed mutagenesis of adenylation domains
Analyze metabolites
Isolate genomic DNA -> Isolate NRPS sequence -> Assemble onto DNA Construct with pET28 -> Modify adenylation domains in NRPS -> Express in Heterologous Host -> Analyze Metabolites
Express in a heterologous host
Assemble all 3 modules into gene cluster (Gibson Assembly)

10. Primer Problems Module 2: ~ 4000 base pairs
First set of primers, only 12nts long
Second set of primers, 20nts long, more specific

11. Results Module 1: ~4300 base pairs Module 2: ~4000 base pairs
10 uL reactions using Epx MS01 DNA Template to determine primer specificity and success.

12. Isolation of Module DNA
Gel electrophoresis using Epx MS01 DNA as template DNA
Gel extraction (GeneJET kit used) to extract each module band to make Module DNA
10 uL reaction using Module DNA Template for better isolation of each module

13. 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.
Module 1
Module 2
Module 3
Low molecular weight bands, trying to change
PCR procedure in order to rid of these bands
and do PCR extraction instead of gel extraction.
This could help improve our DNA yield.

14. 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