1、High throughput gene synthesis and cloning of polyketide synthase modules,Kosan Biosciences Sarah Reisinger,High value pharmaceuticals,Technology platform,polyketide alteration & production,Kosan Business,What Are Polyketides?,Azithromycin,Clarithromycin,Erythromycin,Josamycin,Minocycline (Dynacil),
2、Miokamycin,Mycinamicin,Oleandomycin,Pseudomonic acid,Rifamycins (Rifampin),Rokitamycin (Ricamycin),Tetracyclines,Aclarubicin (aclacinomycin),Adriamycin (Doxorubicin),Chromomycin,Daunorubicin,Enediynes,Idarubicin (Idamycin),Amphotericin B,Candicidin,Griseofulvin,Nystatin/Mycostatin,Spiramycin,Mevacor
3、 (Lovastatin),Mevastatin (Compactin),Pravastatin,Zocor,Zearalenone,Ascomycin (Immunomycin),FK506,Sirolimus (Rapamycin),Spinosad,Avermectin,Lasalocid A,Milbemycin,Monensin,Tylosin,Product,Pfizer,Abbott,Abbott, others,Yamanouchi,Wyeth-Ayerst,Meiji Seika,Asahi,Pfizer,SmithKline Beecham,Novartis, Lepeti
4、t,Asahi,Pfizer, Wyeth-Ayerst,Bristol-Myers Squibb,Pharmacia-Upjohn,Takeda,Astra, Chiron,Wyeth-Ayerst,Pharmacia-Upjohn,Bristol-Myers Squibb,Hoechst Marion Roussel,Schering, Wyeth-Ayerst, Ortho,Bristol-Myers Squibb, others,Rhne-Poulenc,Merck,Sankyo,Sankyo, Bristol-Myers Squibb,Merck,Merck,Fujisawa,Wye
5、th-Ayerst,Dow Elanco,Merck,Hoffman LaRoche,Sankyo,Lilly,Lilly,Company,Antibacterial,Anticancer,Antifungal,Cholesterol-lowering,Immunosuppressant,Insecticide,Veterinary Med,Therapeutic Area,Schering,Polyketides Defined, 10,000 known polyketides Produced by soil micro-organisms (actinomycetes & myxoba
6、cterial) Diverse, complex structures Produced by modular enzymes Similar precursors, similar mechanisms Each 2 carbon atoms encoded by DNA sequence,Polypeptide - Polyketide Analogy,PK 2-carbon unit,enzyme module,DNA sequence (5,000 bp module),Change DNA sequence Change PK structure,PKS Gene Cluster,
7、module 3,module 4,module 1,module 2,Assembly-line blueprint,The assembly-line,The raw materials,The polyketide product,PolyKetide Synthase (PKS),Polyketide Synthesis,2-carbon building blocks,PKS,Polyketide,Change Module to Change Structure,PKS Gene Cluster,module 3,module 4,module 1,module 2,module
8、3,2-carbon building blocks,PKS,Polyketide,Novel Polyketide,Change Module to Change Structure,PKS Gene Cluster,module 3,module 4,module 1,module 2,module 3,Morphing,In theory, could sew PKS modules together to make any or many polyketidesIn practice, difficult to obtain functional PKS module interact
9、ions,Morphing Objectives,Learn how to connect PKS modules from different PKS gene clusters to make any or many polyketides,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
10、 correct inefficient module sets,Develop a Library of Modules,Possibilities: Natural modules Pros Already existCons Requires isolated genes High G+C content; possible expression problems No convenient restriction sites,Synthetic genesProsControl of G+C content; fewer expressionproblemsDesigner restr
11、iction sites; simple tomobilize module/domainsConsHuge effort to create synthetic genes (100 modules = 500 kbp),High Throughput Gene Synthesis,Objective,To develop a fully automated process to quickly and efficiently synthesize and engineer large PKS.,Input: Gene Sequence,Gene Design,Synthesis,Outpu
12、t: Synthetic Gene of Interest,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
13、swaps/replacements,Module Gene Design,Generic design for 200 known modules identified conserved regions for engineering restriction sites between domains within modules,Software Automation,Developed suite of tools for gene synthesis design and analysis Synthetic gene design Split gene into smaller p
14、arts, codon optimize, restriction sites Oligo design/specificity testing/order Automation input information Sequence analysis Database,Protein/DNA sequence,User selected: Restriction enzymes, Distance between sites, Fragment size,Codon optimization Restriction site insertion/deletion Oligo design an
15、d testing,Design validation,Output:,Input:,Oligo ordering file Automation files for oligo mixing and cloning,Gene Morphing System (GeMS),http:/ Synthesis: Fragment Generation,Distribution of individual oligos to gene synthesis wells Gene synthesis Clone into vector Transformation into E. coli Isolat
16、ion of colonies DNA sequencing,Input: Oligo components of 500 bp synthons,Output: 500 bp synthons in plasmids with correct sequence,Flow Chart of Synthesis,Gene Synthesis,40mer oligos,U-U-U,Generation of Synthetic Fragment,HTP Cloning,Criteria Purification of PCR products unnecessary High efficiency
17、 Amenable to HTP automation,HTP Cloning: UDG Cloning,5-UXUXUX,UXUXUX-5,5-UXUXUX,UXUXUX-5,AXAXAX,AXAXAX,AXAXAX,AXAXAX,PCR,UDG,Vector with long 5 ends,Annealed insert-vector,Synthon in vector,transform,No purification necessary!,Generation of Synthetic DNA, 500 synthetic DNA fragments generated 100% s
18、uccess rate GC content from 44-69% Size between 129 and 1400 bpOver 250,000 bp synthesizedAverage error rate around 1.5 errors/kbFully automated most steps in process,Gene Synthesis: Module Assembly,Digestion Ligation Transformation Isolation of colonies Verification of correct clone Repeat until fu
19、ll-length gene assembled,Input: 500 bp synthons in plasmids with correct sequence,Output: Complete module (5kb) in plasmid with correct sequence,Gene Assembly (“Synthon Stitching“),5,000 bp module,Criteria: Accurate Amenable to HT,Parallel Ligations to Assemble Modules,Synthon Stitching Method,Utili
20、ze Type IIs restriction enzymes Cut DNA outside of recognition siteUse different Type IIs enzymes to create compatible overhangsSame 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,Stitching Method: Use of Type IIs RE,Syntho
21、n Stitching Method,Unique selectable markers on two sister plasmids eliminates need for purification of fragments,Alternation of vector pairings allows for unique selection at each round of stitching,Results of Synthon Stitching,26 complete modules constructed 250 successful ligationsSelection schem
22、e works extremely well Majority of ligations performed gave only correct productUse of Type IIs enzymes makes method amenable to automation,Improvements of Gene Synthesis: Designer Vectors,3-plasmid system for synthon stitching Counter-selectable markers Allows 4-piece ligations of unpurified digest
23、s,Synthetic Vector Family: Multiple-synthon Ligations,Use of counter-selection allows for stitching of multiple fragments without purification,Second Round Stitching,Can combine 8 fragments in 2 steps with no fragment purification!,Testing of Modules,Proof of Concept,Expressed synthetic 6-module DEB
24、S gene cluster in E. coliProtein subunits observed on SDS-PAGE in the soluble fractionProduct (6-dEB) identified by LC-MS,Results of Module Testing,Tested 14 synthetic modules in 154 bimodular combinations72 of the 154 combinations tested produced measurable triketide lactoneAll modules tested worke
25、d,Summary,Successfully developed method for high throughput gene synthesis High-throughput method for assembly of DNA fragments into larger genes (modules) developedPopulated module library and tested in bimodular cases,Acknowledgements,Kosan Biosciences Morphing Group Dan Santi Ralph Reid Kedar Patel Sebastian Jayaraj Hugo Menzella Sunil Chandran,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,
