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

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.

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

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.

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

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

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.

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)

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)

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

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.

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

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.

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