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David Hopwood Lecture 2 (DH2). Part 1 Aspects of the programming of Type II PKSs Chain length control Tang, Tsai & Khosla (2003) JACS 125: 12708 Keatings-Clay, A. T. et al . (2004) Nature Struct. Biol. 11: 888. Chain length control by Type II ketosynthases. ACP. tcm KS -act CLF. ACP. - PowerPoint PPT Presentation

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  • David Hopwood

    Lecture 2

    (DH2)

  • Part 1

    Aspects of the programming of Type II PKSs

    Chain length control

    Tang, Tsai & Khosla (2003) JACS 125: 12708

    Keatings-Clay, A. T. et al. (2004) Nature Struct. Biol. 11: 888

  • Chain length control by Type II ketosynthasesACPACPtcm KS-act CLFact KS-tcm CLFno product!

  • McDaniel, Ebert-Khosla, Hopwood, Khosla (1993) Science 262: 1546Engineered Biosynthesis of Novel PolyketidesThe CLF (perhaps in conjunction with the KS) could provide a water-excluding pocket with appropriate molecular dimensions for the nascent polyketide chain

  • Role of the chain length factorCLFKS18 channellid

  • Role of the chain length factor

  • Role of the chain length factor in chain length controlCritical residues in the channel are smaller for longer carbon chains

  • (b) Unimodular and bimodular Type II PKSs

    Tang, Y. et al. (2003) Biochemistry 42: 6588 Tang, Y. et al. (2004) Public Library of Science Biology 2:227

    Tang, Y. et al. (2004) Biochemistry 43: 9546

  • Unimodular and bimodular PKSsKSKSElongation moduleInitiation module

  • Unimodular and bimodular PKSsKSKSElongation moduleInitiation moduleXInitiation moduleX

  • KSActinorhodin biosynthesis bya unimodular polyketide synthaseDMACActinorhodin

  • 11111222212 R1128 biosynthesis bya bimodular PKSInitiation moduleElongation module

  • Initiation ketosynthases prefer initiation ACPs

  • Elongation ketosynthases prefer elongation ACPs

  • 1 2 Recombining initiation and elongation modulesR1128 initiation module + octaketide synthase

  • Recombining initiation and elongation modulesR1128 initiation module + decaketide synthase

  • Part 2

    PKS gene synthesis and morphingof modular Type I PKSsKOSAN Biosciences

  • Requirements for PKS gene synthesis and morphing

    E. coli as expression host

    Pfeifer, B. A. et al. (2001) Microbiol. Mol. Biol. Rev. 65: 106

    Rapid gene synthesis, e.g. ~32 kb DEBS cluster

    Kodumal, S. J. (2004) PNAS 101: 15573

    Synthetic PKS building blocks

    Combinatorial biosynthesis of novel polyketides

    Menzella, H. G. et al. (2005) Nat. Biotech. 23: 1171

  • E. coli as host for polyketide biosynthesis

  • One letter code for 2-C extensions added by modules in database

  • The code for erythromycin

  • Input: PKS module sequence

    Optimize and randomize codon usageAutomated restriction site assignmentAvoid secondary structures in RNAOptimized oligo overlap specificity

    Output: overlapping oligos 40mersGEMS softwareJayaraj, S. et al. (2005) Nucleic Acids Res. 33: 3011

  • Fast and accurate gene synthesis

  • Generic module designAlignment of 150 modules revealed conserved sequences at bordersGeneric module design

  • Synthetic PKS building blocksCurrent collectionLM = loading module 4LI = intrapeptide linker40LN = N-terminal linker module40LC = C-terminal linker40TE = thioesterase 3

  • Bimodular test system17 donor X 17 acceptor modules = 289 bimodules47% gave TKL product

  • Bimodular test systemLI: Intrapeptide linkerLC: C- terminal Interpeptide linkerLN: N- terminal interpeptide linker

  • 6x6=36 polyketides expected from the 289 bimodular PKSsTKLs from bimodular tests

  • 264 unnatural PKSs tested, 118 active (45%)TKLs from bimodular tests

  • Rescuing inactive bimodules

    Chandran, S. S. et al. (2006) Chemistry & Biology 13:469

  • Rescuing inactive bimodules

  • Rescuing inactive bimodules

  • Rational design and assemblyof synthetic trimodular PKSs

    Menzella, H. G. et al. (2007) Chemistry & Biology 14: 143

  • If modA - modB makes a product,

    and modB - modC makes a product,

    will modA - modB - modC make a product ?Rational assembly of trimodular PKSs

  • Rational assembly of trimodular PKSs

  • 54 A-B-C trimodular PKSs assembled , with A-B and B-C active as bimodulese.g.: pairs sor6-ery5 and ery5-rap3 are active, so sor6-ery5-rap3 is testedRational assembly of trimodular PKSs

  • Expected tetraketide products from 54 trimodular PKSs assembledRational assembly of trimodular PKSs

  • 52 out of 54 trimodular PKSs active (96%)Rational assembly of trimodular PKSs

  • Searching for the discodermolidePKS genes

    Schirmer, A. et al. (2005) Appl. Env. Microbiol. 71: 4840

  • DiscodermiadissolutaDiscodermolide

  • An abundant, simple PKS

  • A multimodular PKS gene cluster from Discodermia

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