Total synthesis of long DNA sequences: synthesis of a contiguous 32-kb polyketide synthase gene cluster

Abstract

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 approximately 5-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 approximately 5-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 PCR-based gene synthesis. By parallel processing, these synthons are efficiently joined into multisynthon approximately 5-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.

Fig. 1.

LBS with type IIS restriction enzymes and double antibiotic selection of ligated products. The procedure is as described in Results and Discussion.P and P′ represent the PCR primer sites incorporated at the insert ends.

Fig. 2.

Dendrogram of the plan for a three-cycle, eight-fragment LBS synthesis of a 3,408-bp PKS module. Assigned synthon numbers, insert length in bp, and resistance markers used for LBS are indicated. Other sequences were planned and prepared in a similar fashion.

Fig. 3.

Construction of synthetic DEBS ORFs, TUs, and gene cluster. (Top) Components of the DEBS ORFs were excised from their LBS vectors and assembled in a pUC derivative to give pKOS 422-33-1 (DEBS 1), pKOS 422-51-1 (DEBS 2), and pKOS 422-31-2 (DEBS 3). (Middle) The TU cloning vector, pKOS 422-174-3, was created by cloning a 270-bp synthetic fragment containing, from 5′ to 3′, a BglII site, a T7 promoter (Pr), a lac operator (Op), a ribosome binding site (RBS), NdeI/EcoRI cloning sites, a T7 transcriptional terminator (TT), and a MfeI restriction site into the BglII/EcoRI sites of pET22b. The DEBS's ORFs were excised as NdeIT7/EcoRI fragments and cloned into the NdeI/EcoRI sites of pKOS 422-74-3 to generate the TUs pKOS 422-80-1 (DEBS 1), pKOS 422-80-2 (DEBS 1), and pKOS 422-80-3 (DEBS 1). (Bottom) The XbaI/PacI fragment of pKOS 422-80-3 containing the DEBS 3 TU was cloned into the SpeI/PacI sites of pKOS 422-80-2, adjacent to the DEBS 2 TU to give pKOS 422-81-1. The XbaI/PacI fragment of this plasmid was inserted into the SpeI/PacI sites of pKOS 422-80-1 containing the DEBS 1 TU to obtain the three-ORF gene cluster, pDE1.