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

2. Kosan Business Technology platform High value pharmaceuticals
polyketide
alteration & production
Link to biz description, add value and topspin to Point B
You know of BT companies with a strong tech platform; and you know of BT companies that have a strong product portfolio. Kosan has both. This provides Kosan and its investors with a unique opportunity to share in the short and long-term benefits of both.
We have a strong technology platform that permits alteration and production of a class of compounds known as polyketides. We are also translating the platform to very high value pharmaceuticals.

3. What Are Polyketides? Many more where they came from
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
Many more where they came from
This is a list of polyketides that have been in man/animals
Used in every important therapeutic area
Sales of almost $15 billion a year; equivalent to all protein pharmaceuticals
Reasonable to expect that more high value polyketides will be added to the list, and it is obvious why we at Kosan are so excited about the opportunity we have
Mevacor (Lovastatin)
Merck
Cholesterol-lowering
Mevastatin (Compactin)
Sankyo
Pravastatin
Sankyo, Bristol-Myers Squibb
Zocor
Merck
Zearalenone
Schering
Ascomycin (Immunomycin)
Merck
Immunosuppressant
FK506
Fujisawa
Sirolimus (Rapamycin)
Wyeth-Ayerst
Spinosad
Dow Elanco
Insecticide
Avermectin
Merck
Veterinary Med
Lasalocid A
Hoffman LaRoche
Milbemycin
Sankyo
Monensin
Lilly
Tylosin
Lilly

4. Polyketides Defined ~ 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
Pluck, paraphrase and add value

5. Polypeptide - Polyketide Analogy
Protein AA
DNA sequence
(3 bp codon)
Anti-codon
DNA sequence
(~5,000 bp module)
enzyme module
10,000 - about 100 or 1% found way into man; today, maybe 1/million necessary by HTS
Kosan has developed novel and very proprietary technology that allows us to change the structure of the polyketide product, by changing the genes--
In the past, such technology was only applicable to protein therapeutics, vaccines or targets; now, for the first time, we can use gene-altering technologies to produce organic, orally active drugs.
When you understand how polyketides are made, you will understand Kosan’s technology
Let me start with the polyketide gene
PK 2-carbon unit
Change DNA sequence  Change PK structure

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

7. Change Module to Change Structure
PKS
Gene
Cluster
PKS
Polyketide
2-carbon building blocks

8. Change Module to Change Structure
PKS
Gene
Cluster
PKS
Polyketide
Novel 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  

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

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

28. Parallel 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
4,000 bp
Module

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