Type I polyketide synthase (PKS) genes consist of modules approximately 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.

To exploit the huge potential of whole-genome sequence information, the ability to efficiently synthesize long, accurate DNA sequences is becoming increasingly important. An approach proposed toward this end involves the synthesis of Ϸ5-kb segments of DNA, followed by their assembly into longer sequences by conventional cloning methods [Smith, H. O., Hutchinson, C. A., III, Pfannkoch, C. & Venter, J. C. (2003) Proc. Natl. Acad. Sci. USA 100, 15440 -15445]. The major current impediment to the success of this tactic is the difficulty of building the Ϸ5-kb components accurately, efficiently, and rapidly from short synthetic oligonucleotide building blocks. We have developed and implemented a strategy for the high-throughput synthesis of long, accurate DNA sequences. Unpurified 40-base synthetic oligonucleotides are built into 500-to 800-bp ''synthons'' with low error frequency by automated PCRbased gene synthesis. By parallel processing, these synthons are efficiently joined into multisynthon Ϸ5-kb segments by using only three endonucleases and ''ligation by selection.'' These large segments can be subsequently assembled into very long sequences by conventional cloning. We validated the approach by building a synthetic 31,656-bp polyketide synthase gene cluster whose functionality was demonstrated by its ability to produce the megaenzyme and its polyketide product in Escherichia coli. The chemical synthesis of genes and genomes has received considerable attention for several decades and is becoming increasingly important in the exploitation of whole-genome sequence information. The field was pioneered by Khorana and coworkers with the then-heroic total synthesis of tRNA structural genes (1, 2) and by Itakura et al. (3) with the synthesis and expression of the somatostatin gene. Since then, DNA synthesis methodology has made steady progress, with current approaches relying on the enzyme-catalyzed assembly of short, chemically synthesized oligonucleotides. Of the various methods, polymerase cycling assembly (PCA) (4) is the most widely used because of its inherent simplicity. Overlapping, complementary oligonucleotides are annealed and recursively elongated with a heat-stable DNA polymerase to ultimately yield a full-length sequence, which is amplified by conventional PCR. PCA, first reported for synthesis of the 303-bp HIV-2 Rev gene (5), has since evolved (6-8) into a widely used general method for synthesis of genes of up to Ϸ1 kb.The 1-kb size barrier was broken in 1990 by Mandecki et al. (9), who synthesized a 2.1-kb plasmid by ligation of 30 fragments, and again in 1995 when Stemmer et al. (7) reported the one-step PCA synthesis of a 2.7-kb plasmid that was purified by antibiotic selection. Smith et al. (4) assembled the 5,386 X174 bacteriophage genome from a single pool of chemically synthesized oligonucleotides by using a combination of ligation and PCA methods, but purification of the product again required biological selection. In 2002, Cello et al. (10) described a stepwise synthesis of a 7,558-bp poliov...

Assembly of DNA parts into DNA constructs is a foundational technology in the emerging field of synthetic biology. An efficient DNA assembly method is particularly important for high-throughput, automated DNA assembly in biofabrication facilities and therefore we investigated one-step, scarless DNA assembly via ligase cycling reaction (LCR). LCR assembly uses single-stranded bridging oligos complementary to the ends of neighboring DNA parts, a thermostable ligase to join DNA backbones, and multiple denaturation-annealing-ligation temperature cycles to assemble complex DNA constructs. The efficiency of LCR assembly was improved ca. 4-fold using designed optimization experiments and response surface methodology. Under these optimized conditions, LCR enabled one-step assembly of up to 20 DNA parts and up to 20 kb DNA constructs with very few single-nucleotide polymorphisms (<1 per 25 kb) and insertions/deletions (<1 per 50 kb). Experimental comparison of various sequence-independent DNA assembly methods showed that circular polymerase extension cloning (CPEC) and Gibson isothermal assembly did not enable assembly of more than four DNA parts with more than 50% of clones being correct. Yeast homologous recombination and LCR both enabled reliable assembly of up to 12 DNA parts with 60-100% of individual clones being correct, but LCR assembly provides a much faster and easier workflow than yeast homologous recombination. LCR combines reliable assembly of many DNA parts via a cheap, rapid, and convenient workflow and thereby outperforms existing DNA assembly methods. LCR assembly is expected to become the method of choice for both manual and automated high-throughput assembly of DNA parts into DNA constructs.

We present a cell-free protein synthesis (CFPS) platform and a one-step, direct conjugation scheme for producing virus-like particle (VLP) assemblies that display multiple ligands including proteins, nucleic acids, and other molecules. Using a global methionine replacement approach, we produced bacteriophage MS2 and bacteriophage Qβ VLPs with surface-exposed methionine analogues (azidohomoalanine and homopropargylglycine) containing azide and alkyne side chains. CFPS enabled the production of VLPs with yields of ~300 μg/mL and with 85% incorporation of methionine analogues without requiring a methionine auxotrophic production host. We then directly conjugated azide- and alkyne-containing proteins (including an antibody fragment and the granulocyte-macrophage colony stimulating factor, or GM-CSF), nucleic acids and poly(ethylene glycol) chains to the VLP surface using Cu(I) catalyzed click chemistry. The GM-CSF protein, after conjugation to VLPs, was shown to partially retain its ability to stimulate the proliferation of cells. Conjugation of GM-CSF to VLPs resulted in a 3–5-fold reduction in its bioactivity. The direct attachment scheme facilitated conjugation of three different ligands to the VLPs in a single step, and enabled control of the relative ratios and surface abundance of the attached species. This platform can be used for the production of novel VLP bioconjugates for use as drug delivery vehicles, diagnostics, and vaccines.

No abstract