Abstract
Type I polyketide synthase (PKS) genes consist of modules ∼3–6 kb long, which encode the structures of 2-carbon units in polyketide products. Alteration or replacement of individual PKS modules can lead to the biosynthesis of 'unnatural' natural products but existing techniques for this are time consuming. Here we describe a generic approach to the design of synthetic PKS genes where facile cassette assembly and interchange of modules and domains are facilitated by a repeated set of flanking restriction sites. To test the feasibility of this approach, we synthesized 14 modules from eight PKS clusters and associated them in 154 bimodular combinations spanning over 1.5-million bp of novel PKS gene sequences. Nearly half the combinations successfully mediated the biosynthesis of a polyketide in Escherichia coli, and all individual modules participated in productive bimodular combinations. This work provides a truly combinatorial approach for the production of polyketides.
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Acknowledgements
We thank Jim Kealey, Sumati Murli and Jonathan Kennedy for providing strains, vectors and procedures for polyketide production in E. coli and Gary Ashley and Leonard Katz for the critical review of the manuscript. This work was supported in part by National Institute of Standards and Technology Advanced Program Grant 70NANB2H3014.
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Supplementary information
Supplementary Fig. 1
Product ion scan of unlabeled (a) and 13C labeled (b) TKLs. (PDF 25 kb)
Supplementary Table 1
Sources of 140 modules analyzed. (PDF 24 kb)
Supplementary Table 2
Restriction sites introduced in PKS building blocks. (PDF 18 kb)
Supplementary Table 3
Substrate specificities of PKS modules used in this study. (PDF 24 kb)
Supplementary Table 4
Expression plasmids used in the bimodular tests. (PDF 47 kb)
Supplementary Table 5
Characterization of triketide lactones obtained in this study by mass spectrometry. (PDF 22 kb)
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Menzella, H., Reid, R., Carney, J. et al. Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes. Nat Biotechnol 23, 1171–1176 (2005). https://doi.org/10.1038/nbt1128
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DOI: https://doi.org/10.1038/nbt1128
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