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High throughput gene synthesis and cloning of polyketide synthase modules

High throughput gene synthesis and cloning of polyketide synthase modules. Kosan Biosciences Sarah Reisinger . Kosan Business. High value pharmaceuticals. Technology platform. polyketide alteration & production. What Are Polyketides?. Product. Company. Therapeutic Area. Azithromycin.

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High throughput gene synthesis and cloning of polyketide synthase modules

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  1. High throughput gene synthesis and cloning of polyketide synthase modules Kosan Biosciences Sarah Reisinger

  2. Kosan Business High value pharmaceuticals Technology platform polyketide alteration & production

  3. What Are Polyketides? Product Company Therapeutic Area Azithromycin Pfizer Antibacterial Clarithromycin Abbott Erythromycin Abbott, others Josamycin Yamanouchi Minocycline (Dynacil) Wyeth-Ayerst Miokamycin Meiji Seika Mycinamicin Asahi Oleandomycin Pfizer Pseudomonic acid SmithKline Beecham Rifamycins (Rifampin) Novartis, Lepetit Rokitamycin (Ricamycin) Asahi Tetracyclines Pfizer, Wyeth-Ayerst Aclarubicin (aclacinomycin) Bristol-Myers Squibb Anticancer Adriamycin (Doxorubicin) Pharmacia-Upjohn Chromomycin Takeda Daunorubicin Astra, Chiron Enediynes Wyeth-Ayerst Idarubicin (Idamycin) Pharmacia-Upjohn Amphotericin B Bristol-Myers Squibb Antifungal Candicidin Hoechst Marion Roussel Griseofulvin Schering, Wyeth-Ayerst, Ortho Nystatin/Mycostatin Bristol-Myers Squibb, others Spiramycin Rhône-Poulenc Mevacor (Lovastatin) Merck Cholesterol-lowering Mevastatin (Compactin) Sankyo Pravastatin Sankyo, Bristol-Myers Squibb Zocor Merck Schering Zearalenone Ascomycin (Immunomycin) Merck Immunosuppressant FK506 Fujisawa Sirolimus (Rapamycin) Wyeth-Ayerst Insecticide Spinosad Dow Elanco Avermectin Merck Veterinary Med Lasalocid A Hoffman LaRoche Milbemycin Sankyo Monensin Lilly Tylosin Lilly

  4. ~ 10,000 known polyketides Produced by soil micro-organisms (actinomycetes & myxobacterial) Diverse, complex structures Produced by modular enzymes Similar precursors, similar mechanisms Each 2 carbon atoms encoded by DNA sequence Polyketides Defined

  5. Polypeptide - Polyketide Analogy DNA sequence (3 bp codon) Anti-codon Protein AA DNA sequence (~5,000 bp module) enzyme module PK 2-carbon unit Change DNA sequence  Change PK structure

  6. Polyketide Synthesis 2-carbon unit building blocks module 1 module 2 module 3 module 4 PKS Gene Cluster Assembly-line blueprint PolyKetide Synthase (PKS) The assembly-line The raw materials The polyketide product

  7. Change Module to Change Structure module 1 module 2 module 3 module 4 module 3 PKS Gene Cluster PKS Polyketide 2-carbon building blocks

  8. Change Module to Change Structure module 1 module 2 module 3 module 4 module 3 PKS Gene Cluster PKS Novel Polyketide Polyketide 2-carbon building blocks

  9. Morphing • In theory, could sew PKS modules together to make any or many polyketides • In practice, difficult to obtain functional PKS module interactions

  10. Morphing Objectives • Learn how to connect PKS modules from different PKS gene clusters to make any or many polyketides

  11. Morphing Toolbox Objectives: • Develop a library of modules to express in genetic host • Connect modules in all permutations • Determine which module sets produce products • Learn how to correct inefficient module sets

  12. Develop a Library of Modules Possibilities: • Natural modules • Pros • Already exist • Cons • Requires isolated genes • High G+C content; possible expression problems • No convenient restriction sites • Synthetic genes • Pros • Control of G+C content; fewer expression • problems • Designer restriction sites; simple to • mobilize module/domains • Cons • Huge effort to create synthetic genes • (100 modules = 500 kbp)

  13. High Throughput Gene Synthesis

  14. Objective To develop a fully automated process to quickly and efficiently synthesize and engineer large PKS. Output: Synthetic Gene of Interest Input: Gene Sequence Gene Design Synthesis

  15. Module Gene Design Develop a system for generating synthetic PKS modules that allows for: • Codon optimization for expression in E. coli • Common restriction sites at module and domain edges • Additional restriction sites within modules to facilitate partial domain or module swaps/replacements

  16. Module Gene Design Generic design for ~200 known modules identified conserved regions for engineering restriction sites between domains within modules

  17. Software Automation • Developed suite of tools for gene synthesis design and analysis • Synthetic gene design • Split gene into smaller parts, codon optimize, restriction sites • Oligo design/specificity testing/order • Automation input information • Sequence analysis • Database

  18. Gene Morphing System (GeMS) User selected: Restriction enzymes, Distance between sites, Fragment size Input: Protein/DNA sequence Codon optimization Restriction site insertion/deletion Oligo design and testing Design validation Output: Oligo ordering file Automation files for oligo mixing and cloning http://software.kosan.com/GeMS

  19. Gene Synthesis: Fragment Generation Input: Oligo components of 500 bp synthons • Distribution of individual oligos to gene synthesis wells • Gene synthesis • Clone into vector • Transformation into E. coli • Isolation of colonies • DNA sequencing Output: 500 bp synthons in plasmids with correct sequence

  20. Flow Chart of Synthesis

  21. Gene Synthesis Assemble, amplify Clone ~500 bp Synthon A B Plasmids containing synthons B A synthon 40mer oligos

  22. Generation of Synthetic Fragment U-U-U

  23. HTP Cloning • Criteria • Purification of PCR products unnecessary • High efficiency • Amenable to HTP automation

  24. HTP Cloning: UDG Cloning 5’-UXUXUX UXUXUX-5’ PCR AXAXAX 5’-UXUXUX AXAXAX UXUXUX-5’ UDG AXAXAX AXAXAX No purification necessary! transform Synthon in vector Vector with long 5’ ends Annealed insert-vector

  25. Generation of Synthetic DNA • > 500 synthetic DNA fragments generated • 100% success rate • GC content from 44-69% • Size between 129 and 1400 bp • Over 250,000 bp synthesized • Average error rate around 1.5 errors/kb • Fully automated most steps in process

  26. Gene Synthesis: Module Assembly Input: 500 bp synthons in plasmids with correct sequence • Digestion • Ligation • Transformation • Isolation of colonies • Verification of correct clone • Repeat until full-length gene assembled Output: Complete module (>5kb) in plasmid with correct sequence

  27. Gene Assembly (“Synthon Stitching“) ~10 plasmids containing 500 bp synthons B A synthon Synthon 1 Synthon 2 Synthon 3 Synthon 10 • Criteria: • Accurate • Amenable to HT 5,000 bp module

  28. Parallel Ligations to Assemble Modules 500 bp 1,000 bp 2,000 bp 4,000 bp 1 1-2 2 1-2-3-4 3 3-4 1-2-3-4-5-6-7-8 4 5 5-6 Module 6 5-6-7-8 7 7-8 8

  29. Synthon Stitching Method • Utilize Type IIs restriction enzymes • Cut DNA outside of recognition site • Use different Type IIs enzymes to create compatible overhangs • Same enzymes can be used for all synthon pairs to facilitate automation Bsa I: 5´ ... G G T C T C (N)1^ ... 3´ 3´ ... C C A G A G (N)5^ ... 5´

  30. Stitching Method: Use of Type IIs RE

  31. Synthon Stitching Method • Unique selectable markers on two sister plasmids eliminates need for purification of fragments

  32. Alternation of vector pairings allows for unique selection at each round of stitching

  33. Results of Synthon Stitching • 26 complete modules constructed • > 250 successful ligations • Selection scheme works extremely well • Majority of ligations performed gave only correct product • Use of Type IIs enzymes makes method amenable to automation

  34. Improvements of Gene Synthesis: Designer Vectors • 3-plasmid system for synthon stitching • Counter-selectable markers • Allows 4-piece ligations of unpurified digests

  35. Synthetic Vector Family:Multiple-synthon Ligations Use of counter-selection allows for stitching of multiple fragments without purification

  36. Second Round Stitching Can combine 8 fragments in 2 steps with no fragment purification!

  37. Testing of Modules

  38. Proof of Concept • Expressed synthetic 6-module DEBS gene cluster in E. coli • Protein subunits observed on SDS-PAGE in the soluble fraction • Product (6-dEB) identified by LC-MS

  39. Results of Module Testing • Tested 14 synthetic modules in 154 bimodular combinations • 72 of the 154 combinations tested produced measurable triketide lactone • All modules tested worked

  40. Summary • Successfully developed method for high throughput gene synthesis • High-throughput method for assembly of DNA fragments into larger genes (modules) developed • Populated module library and tested in bimodular cases

  41. Acknowledgements Kosan Biosciences – Morphing Group • Dan Santi • Ralph Reid • Kedar Patel • Sebastian Jayaraj • Hugo Menzella • Sunil Chandran

  42. Summary of Major Synthesis Efforts aEach experiment represents the parallel processed synthesis of the DNA indicated. bAssuming Poisson distribution of errors cAny specific error was counted only once

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