Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes

• Published in: Nature biotechnology Vol. 23; no. 9; pp. 1171 - 1176
• Main Authors: Santi, Daniel V, Menzella, Hugo G, Reid, Ralph, Carney, John R, Chandran, Sunil S, Reisinger, Sarah J, Patel, Kedar G, Hopwood, David A
• Format: Journal Article
• Language: English
• Published: New York, NY Nature 01.09.2005; Nature Publishing Group
• Subjects: Amino Acid Sequence; Biological and medical sciences; Biotechnology; Biotechnology - methods; Combinatorial Chemistry Techniques; Escherichia coli; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic Engineering - methods; Genetic technics; Lactones - chemistry; Macrolides - chemistry; Methods. Procedures. Technologies; Models, Chemical; Modification of gene expression level; Molecular Sequence Data; Plasmids - metabolism; Polyketide Synthases - biosynthesis; Polyketide Synthases - chemistry; Protein Engineering - methods; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Polyketide; Escherichia coli; Production; Bacteria; Biosynthesis; Gene expression; Hybrid gene; Enterobacteriaceae
• ISSN: 1087-0156; 1546-1696
• DOI: 10.1038/nbt1128

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.