High throughput gene synthesis High throughput gene synthesis and cloning of polyketide and cloning of polyketide

synthase modulessynthase modules

High throughput gene synthesis High throughput gene synthesis and cloning of polyketide and cloning of polyketide

synthase modulessynthase modules

Kosan BiosciencesKosan Biosciences

Sarah Reisinger Sarah Reisinger

High valueHigh valuepharmaceuticalspharmaceuticals

Technology platformTechnology platform

polyketide polyketide alteration & productionalteration & production

Kosan BusinessKosan BusinessKosan BusinessKosan Business

What Are Polyketides?What Are Polyketides?What Are Polyketides?What Are Polyketides?AzithromycinClarithromycinErythromycinJosamycinMinocycline (Dynacil)MiokamycinMycinamicinOleandomycinPseudomonic acidRifamycins (Rifampin)Rokitamycin (Ricamycin)TetracyclinesAclarubicin (aclacinomycin)Adriamycin (Doxorubicin)ChromomycinDaunorubicinEnediynesIdarubicin (Idamycin)Amphotericin BCandicidinGriseofulvinNystatin/MycostatinSpiramycinMevacor (Lovastatin)Mevastatin (Compactin)PravastatinZocorZearalenoneAscomycin (Immunomycin)FK506Sirolimus (Rapamycin)SpinosadAvermectinLasalocid AMilbemycinMonensinTylosin

ProductPfizerAbbottAbbott, othersYamanouchiWyeth-AyerstMeiji SeikaAsahiPfizerSmithKline BeechamNovartis, LepetitAsahiPfizer, Wyeth-AyerstBristol-Myers SquibbPharmacia-UpjohnTakedaAstra, ChironWyeth-AyerstPharmacia-UpjohnBristol-Myers SquibbHoechst Marion RousselSchering, Wyeth-Ayerst, OrthoBristol-Myers Squibb, othersRhône-PoulencMerckSankyoSankyo, Bristol-Myers SquibbMerck

MerckFujisawaWyeth-AyerstDow ElancoMerckHoffman LaRocheSankyoLillyLilly

CompanyAntibacterial

Anticancer

Antifungal

Cholesterol-lowering

Immunosuppressant

InsecticideVeterinary Med

Therapeutic Area

Schering

Polyketides DefinedPolyketides DefinedPolyketides DefinedPolyketides Defined

• ~ 10,000 known polyketides~ 10,000 known polyketides

• Produced by soil micro-organisms Produced by soil micro-organisms

(actinomycetes & myxobacterial)(actinomycetes & myxobacterial)

• Diverse, complex structuresDiverse, complex structures

• Produced by modular enzymesProduced by modular enzymes

• Similar precursors, similar mechanismsSimilar precursors, similar mechanisms

• Each 2 carbon atoms encoded by DNA sequence Each 2 carbon atoms encoded by DNA sequence

O

OH

OHO

O

OH

Polypeptide - Polyketide AnalogyPolypeptide - Polyketide AnalogyPolypeptide - Polyketide AnalogyPolypeptide - Polyketide Analogy

Protein AA

DNA sequence(3 bp codon)

Anti-codon

PK 2-carbon unit

enzyme module

DNA sequence(~5,000 bp

module)

Change DNA sequence Change DNA sequence Change PK structure Change PK structure

PKSGene Cluster

module 3 module 4module 1 module 2

Assembly-line blueprint

The assembly-line

The raw materials

The polyketide product

2-carbon unit building blocks

PolyKetide

Synthase(PKS)

Polyketide SynthesisPolyketide SynthesisPolyketide SynthesisPolyketide Synthesis

2-carbon building blocks

PKS

Polyketide

Change Change ModuleModule to Change Structure to Change StructureChange Change ModuleModule to Change Structure to Change Structure

PKSGene Cluster

module 3 module 4module 1 module 2module 3

2-carbon building blocks

PKS

PolyketideNovel Polyketide

Change Change ModuleModule to Change Structure to Change StructureChange Change ModuleModule to Change Structure to Change Structure

PKSGene Cluster

module 3 module 4module 1 module 2module 3

MorphingMorphingMorphingMorphing

• In In theorytheory, could sew PKS modules together to make , could sew PKS modules together to make

any or many polyketidesany or many polyketides

• In In practicepractice, difficult to obtain functional PKS module , difficult to obtain functional PKS module

interactionsinteractions

Morphing ObjectivesMorphing ObjectivesMorphing ObjectivesMorphing Objectives

• Learn how to connect PKS modules from different Learn how to connect PKS modules from different PKS gene clusters to make any or many polyketidesPKS gene clusters to make any or many polyketides

Morphing ToolboxMorphing ToolboxMorphing ToolboxMorphing Toolbox

Objectives:Objectives:

• Develop a library of modules to express in genetic Develop a library of modules to express in genetic

host host

• Connect modules in all permutations Connect modules in all permutations

• Determine which module sets produce productsDetermine which module sets produce products

• Learn how to correct inefficient module setsLearn how to correct inefficient module sets

Develop a Library of ModulesDevelop a Library of ModulesDevelop a Library of ModulesDevelop a Library of Modules

PossibilitiesPossibilities::

• Natural modulesNatural modules– ProsPros

• Already existAlready exist

– ConsCons• Requires isolated genesRequires isolated genes

• High G+C content; possible High G+C content; possible expression problemsexpression problems

• No convenient restriction No convenient restriction sitessites

• Synthetic genesSynthetic genes– ProsPros

• Control of G+C content; fewer expressionControl of G+C content; fewer expression

problemsproblems

• Designer restriction sites; simple toDesigner restriction sites; simple to

mobilize module/domainsmobilize module/domains

– ConsCons• Huge effort to create synthetic genes Huge effort to create synthetic genes

(100 modules = 500 kbp)(100 modules = 500 kbp)

High Throughput Gene SynthesisHigh Throughput Gene SynthesisHigh Throughput Gene SynthesisHigh Throughput Gene Synthesis

ObjectiveObjectiveObjectiveObjective

To develop a fully automated process to To develop a fully automated process to

quickly and efficiently synthesize and quickly and efficiently synthesize and

engineer large PKS.engineer large PKS.

Input:

Gene SequenceGene Design Synthesis

Output:

Synthetic Gene

of Interest

Module Gene DesignModule Gene DesignModule Gene DesignModule Gene Design

Develop a system for generating synthetic PKS Develop a system for generating synthetic PKS

modules that allows for:modules that allows for:– Codon optimization for expression in Codon optimization for expression in E. coliE. coli

– Common restriction sites at module and domain edgesCommon restriction sites at module and domain edges

– Additional restriction sites within modules to facilitate partial Additional restriction sites within modules to facilitate partial

domain or module swaps/replacementsdomain or module swaps/replacements

Module Gene DesignModule Gene DesignModule Gene DesignModule Gene Design Generic design for ~200 known modules identified conserved regions for engineering restriction

sites between domains within modules

Software AutomationSoftware 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

Protein/DNA sequenceUser selected: Restriction enzymes,Distance between sites,Fragment size

1. Codon optimization 2. Restriction site insertion/deletion 3. Oligo design and testing

Design validation

Output:

Input:

Oligo ordering fileAutomation files for oligo mixing and cloning

Gene Morphing System (GeMS)Gene Morphing System (GeMS)Gene Morphing System (GeMS)Gene Morphing System (GeMS)

  http://software.kosan.com/GeMS

Gene Synthesis: Fragment GenerationGene Synthesis: Fragment GenerationGene Synthesis: Fragment GenerationGene Synthesis: Fragment Generation

• Distribution of individual oligos to gene synthesis wellsDistribution of individual oligos to gene synthesis wells

• Gene synthesisGene synthesis

• Clone into vectorClone into vector

• Transformation into Transformation into E. coliE. coli

• Isolation of coloniesIsolation of colonies

• DNA sequencingDNA sequencing

InputInput: Oligo components of 500 bp synthons: Oligo components of 500 bp synthons

OutputOutput: 500 bp synthons in plasmids with correct sequence: 500 bp synthons in plasmids with correct sequence

Flow Chart of Synthesis Receive Oligos

Generate Synthetic Fragments (2-Step PCR)

Clone (UDG-LIC)

Transform

Isolate Colonies

Isolate Plamid DNA

Sequence Clones

Assemble Genes from Correct Synthons

Completed Synthetic Gene

Gene SynthesisGene SynthesisGene SynthesisGene Synthesis

Assemble, amplify

40mer oligos

A B~500 bp

Synthon

synthon

A B

Plasmids containing synthons

Clone

PCR step 2: Products areamplified with primerscontaining dU flanking region

PCR step 1: Assemblyof 40-mers into full-length fragment

U-U-UU-U-U

Generation of Synthetic FragmentGeneration of Synthetic FragmentGeneration of Synthetic FragmentGeneration of Synthetic Fragment

HTP CloningHTP CloningHTP CloningHTP Cloning

CriteriaCriteria

• Purification of PCR products unnecessaryPurification of PCR products unnecessary

• High efficiencyHigh efficiency

• Amenable to HTP automationAmenable to HTP automation

HTP Cloning: UDG CloningHTP Cloning: UDG CloningHTP Cloning: UDG CloningHTP Cloning: UDG Cloning

5’-UXUXUX

UXUXUX-5’

5’-UXUXUXUXUXUX-5’

AXAXAXAXAXAX

AXAXAXAXAXAX

PCR

UDG

Vector with long 5’ ends Annealedinsert-vector

Synthon in vector

transform

No purification necessary!

Generation of Synthetic DNAGeneration of Synthetic DNAGeneration of Synthetic DNAGeneration of Synthetic DNA

• > 500 synthetic DNA fragments generated> 500 synthetic DNA fragments generated– 100% success rate100% success rate

– GC content from 44-69%GC content from 44-69%

– Size between 129 and 1400 bpSize between 129 and 1400 bp

• Over 250,000 bp synthesizedOver 250,000 bp synthesized

• Average error rate around 1.5 errors/kbAverage error rate around 1.5 errors/kb

• Fully automated most steps in processFully automated most steps in process

Gene Synthesis: Module AssemblyGene Synthesis: Module AssemblyGene Synthesis: Module AssemblyGene Synthesis: Module Assembly

• DigestionDigestion

• LigationLigation

• TransformationTransformation

• Isolation of coloniesIsolation of colonies

• Verification of correct cloneVerification of correct clone

• Repeat until full-length gene assembledRepeat until full-length gene assembled

InputInput: 500 bp synthons in plasmids with correct sequence: 500 bp synthons in plasmids with correct sequence

OutputOutput: Complete module (>5kb) in plasmid with correct sequence: Complete module (>5kb) in plasmid with correct sequence

Gene Assembly (“Synthon Stitching“)Gene Assembly (“Synthon Stitching“)Gene Assembly (“Synthon Stitching“)Gene Assembly (“Synthon Stitching“)

synthon

A B

~10 plasmids containing

500 bp synthons

Synthon 1 Synthon 2 Synthon 3 Synthon 10

5,000 bp module

Criteria:AccurateAmenable to HT

Parallel Ligations to Assemble ModulesParallel Ligations to Assemble ModulesParallel Ligations to Assemble ModulesParallel Ligations to Assemble Modules

500 bp

1

2

3

4

5

6

7

8

1,000 bp

1-2

3-4

5-6

7-8

2,000 bp

1-2-3-4

5-6-7-8

4,000 bp

Module

1-2-3-4-5-6-7-8

Synthon Stitching MethodSynthon Stitching MethodSynthon Stitching MethodSynthon Stitching Method

• Utilize Type IIs restriction enzymesUtilize Type IIs restriction enzymes– Cut DNA outside of recognition siteCut DNA outside of recognition site

– Use different Type IIs enzymes to create compatible Use different Type IIs enzymes to create compatible

overhangsoverhangs

– Same enzymes can be used for all synthon pairs to facilitate Same enzymes can be used for all synthon pairs to facilitate

automationautomation

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 REStitching Method: Use of Type IIs REStitching Method: Use of Type IIs REStitching Method: Use of Type IIs RE

5'N N N G G T C T C N N N N N N N N N N N N N N N N N G A T C G N G T C T T C N N N N 3' 5'N N N C T C T T C N G A T C G N N N N N N N N N N N N N N N N N G T C T T C N N N N 3'3'N N N C C A G A G N N N N N N N N N N N N N N N N N C T A G C N C A G A A G N N N N 5' 3'N N N G A G A A G N C T G G C N N N N N N N N N N N N N N N N N C A G A A G N N N N 5'

5'G A T C G N N N N N N N N N N N N N N N N N G T C T T C N N N N N 3'5'N N N G G T C T C N N N N N N N N N N N N N N N N N 3' 3'C N N N N N N N N N N N N N N N N N C A G A A G N N N N N 5'3'N N N C C A G A G N N N N N N N N N N N N N N N N N C T A G 5'

5'N N N G G T C T C N N N N N N N N N N N N N N N N N G A T C G N N N N N N N N N N N N N N N N N G T C T T C N N N N N 3'3'N N N C C A G A G N N N N N N N N N N N N N N N N N C T A G C N N N N N N N N N N N N N N N N N C A G A A G N N N N N 5'

Bsa I

Bbs I

Synthon 1 Sequence Bsa I

Bbs I

Synthon 2 Sequence

Bsa I Synthon 1 Sequence

Digest with Bbs IDigest with Bsa I

Bbs I

Synthon 2 Sequence

Bsa I Synthon 1 Sequence

Bbs I

Synthon 2 Sequence

Ligate

Synthon Stitching MethodSynthon Stitching MethodSynthon Stitching MethodSynthon Stitching Method

• Unique selectable markers on two sister plasmids Unique selectable markers on two sister plasmids

eliminates need for purification of fragmentseliminates need for purification of fragments

• Alternation of Alternation of vector pairings vector pairings allows for unique allows for unique selection at each selection at each round of stitchinground of stitching

Results of Synthon StitchingResults of Synthon StitchingResults of Synthon StitchingResults of Synthon Stitching

• 26 complete modules constructed26 complete modules constructed

• > 250 successful ligations> 250 successful ligations

• Selection scheme works extremely well Selection scheme works extremely well – Majority of ligations performed gave only correct productMajority of ligations performed gave only correct product

• Use of Type IIs enzymes makes method amenable to Use of Type IIs enzymes makes method amenable to automationautomation

Improvements of Gene Synthesis: Improvements of Gene Synthesis: Designer VectorsDesigner VectorsImprovements of Gene Synthesis: Improvements of Gene Synthesis: Designer VectorsDesigner Vectors

• 3-plasmid system for synthon stitching 3-plasmid system for synthon stitching – Counter-selectable markers Counter-selectable markers

– Allows 4-piece ligations of unpurified digestsAllows 4-piece ligations of unpurified digests

Synthetic Vector Family:Synthetic Vector Family:Multiple-synthon LigationsMultiple-synthon LigationsSynthetic Vector Family:Synthetic Vector Family:Multiple-synthon LigationsMultiple-synthon Ligations

Use of counter-selection allows for stitching of multiple Use of counter-selection allows for stitching of multiple fragments without purificationfragments without purification

ApR

XhoI

SacB

Syn 1

KmR

Unique BbsI

ApR

SacB

Syn 2

BsaI BbsI

ApR

SacB

Syn 3

BsaI BbsI

ApR

XhoI SacB

Syn 4

TetR

BsaI BbsI

Digest BbsI, BsaI and XhoI.

Ligate

Select TetR, KmR, Sucrose

XhoI

KmR

TetR

Syn 14

Unique BbsI

ApR

Second Round StitchingSecond Round StitchingSecond Round StitchingSecond Round Stitching

XhoI

KmR

TetR

Syn 14

Unique BbsI

XhoI

CmR

StrR

Syn 58

BsaI Unique

ApR ApR

XhoI

KmR

ApR StrR

Syn 18

Unique Unique

Digest BbsI, BsaI, XhoI

Ligate

Select KmR, StrR

Can combine 8 fragments in 2 steps with no fragment purification!

Testing of ModulesTesting of ModulesTesting of ModulesTesting of Modules

Proof of ConceptProof of ConceptProof of ConceptProof of Concept

• Expressed synthetic 6-module DEBS gene cluster in Expressed synthetic 6-module DEBS gene cluster in

E. coliE. coli

• Protein subunits observed on SDS-PAGE in the Protein subunits observed on SDS-PAGE in the

soluble fractionsoluble fraction

• Product (6-dEB) identified by LC-MSProduct (6-dEB) identified by LC-MS

Results of Module TestingResults of Module TestingResults of Module TestingResults of Module Testing

• Tested 14 synthetic modules in 154 bimodular Tested 14 synthetic modules in 154 bimodular

combinationscombinations

• 72 of the 154 combinations tested produced 72 of the 154 combinations tested produced

measurable triketide lactonemeasurable triketide lactone

• All modules tested workedAll modules tested worked

SummarySummarySummarySummary

• Successfully developed method for high throughput Successfully developed method for high throughput

gene synthesis gene synthesis

• High-throughput method for assembly of DNA High-throughput method for assembly of DNA

fragments into larger genes (modules) developedfragments into larger genes (modules) developed

• Populated module library and tested in bimodular Populated module library and tested in bimodular

casescases

AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgements

Kosan Biosciences – Morphing GroupKosan Biosciences – Morphing Group

• Dan SantiDan Santi

• Ralph ReidRalph Reid

• Kedar PatelKedar Patel

• Sebastian JayarajSebastian Jayaraj

• Hugo MenzellaHugo Menzella

• Sunil ChandranSunil Chandran

Summary of Major Synthesis EffortsSummary of Major Synthesis EffortsSummary of Major Synthesis EffortsSummary of Major Synthesis Efforts

Synthon size (bp) Correct clones (%) EFExperiment No. ofsynthons

Range Average

Total bpsynthesized a

Totalclonessequenced

SequencedDNA (bp)

Predictedb Foundc Mismatch Deletion Insertion Total

1 102 229-541 496 50,634 647 308,938 43 39 0.12 0.047 0.004 0.1762 118 129-781 510 59,884 797 409,267 30 32 0.17 0.070 0.004 0.243 44 286-748 519 27,004 706 346,222 20 20 0.27 0.06 0.006 0.344 85 112-650 502 42,743 589 317,382 19 16 0.24 0.078 0.02 0.33

Ave. orTotal

349 112-748 505 180,265 2,739 1,381,809 28 27 0.20 0.064 0.007 0.27

aEach experiment represents the parallel processed synthesis of the DNA indicated. bAssuming Poisson distribution of errorscAny specific error was counted only once