Reiterative Recombination for the in vivo assembly of libraries of multigene pathways

Abstract

The increasing sophistication of synthetic biology is creating a demand for robust, broadly accessible methodology for constructing multigene pathways inside of the cell. Due to the difficulty of rationally designing pathways that function as desired in vivo, there is a further need to assemble libraries of pathways in parallel, in order to facilitate the combinatorial optimization of performance. While some in vitro DNA assembly methods can theoretically make libraries of pathways, these techniques are resource intensive and inherently require additional techniques to move the DNA back into cells. All previously reported in vivo assembly techniques have been low yielding, generating only tens to hundreds of constructs at a time. Here, we develop "Reiterative Recombination," a robust method for building multigene pathways directly in the yeast chromosome. Due to its use of endonuclease-induced homologous recombination in conjunction with recyclable markers, Reiterative Recombination provides a highly efficient, technically simple strategy for sequentially assembling an indefinite number of DNA constructs at a defined locus. In this work, we describe the design and construction of the first Reiterative Recombination system in Saccharomyces cerevisiae, and we show that it can be used to assemble multigene constructs. We further demonstrate that Reiterative Recombination can construct large mock libraries of at least 10(4) biosynthetic pathways. We anticipate that our system's simplicity and high efficiency will make it a broadly accessible technology for pathway construction and render it a valuable tool for optimizing pathways in vivo.

Fig. 1.

General scheme of Reiterative Recombination, showing two rounds of elongation. Each round of elongation is achieved by recombination between an acceptor module (shown here in the linear chromosome) and a donor module (in the circular plasmid). The two modules contain orthogonal homing endonuclease cleavage sites (triangles) adjacent to different selectable markers (purple and green). Both markers are downstream of a homology region (gray), but only the acceptor module contains a promoter (white) driving marker expression. Endonuclease cleavage of the acceptor module stimulates recombination, joining the fragments being assembled (orange) and replacing the acceptor module’s endonuclease site and expressed selectable marker with those of the donor module. Repeating this procedure with a donor module of the opposite polarity returns the acceptor module to its original state, allowing the assembly to be elongated indefinitely.

Fig. 2.

Reiterative Recombination reporter proof-of-principle system. (A) Details of the assembly process for the proof-of-principle system, in which the three reporter genes lacZ, gusA, and MET15 were sequentially integrated into the chromosome. (B) Construction of donor plasmids by plasmid gap repair, in which a digested universal donor plasmid and PCR fragments with appropriate homology regions were cotransformed into the Reiterative Recombination strain and assembled via homologous recombination. (C) Results of the round 2 induction step are shown as a representative example. As negative controls, cells containing identical donor plasmids lacking the SceI endonuclease gene and/or the gusA fragment with lacZ homology were induced in parallel. A calculated 6 × 10⁶ cells were plated on SC(-Histidine) media to assay for selective marker conversion after a 12-h galactose induction. (D) Phenotypes of 12 unique cured colonies from each round of assembly. In columns, recombinants are assayed for the HIS3 [SC(-Histidine)] and LEU2 [SC(-Leucine)] markers. In rows, recombinants are assayed for lacZ (Magenta-Gal), gusA (X-Gluc), and MET15 [SC(-Methionine)].

Fig. 3.

Assembly of the lycopene biosynthesis pathway using Reiterative Recombination. (A) Order of crt gene insertion. (B–E) Phenotypes of cured round 3 colonies containing wild-type crtE and (B) crtB-stop + crtI-stop, (C) crtB-stop + crtI-silent, (D) crtB-silent + crtI-stop, and (E) crtB-silent + crtI-silent. For (E), 315 out of 317 colonies had an orange phenotype; none of the other plates contained any orange colonies. (F, G) Restriction analysis of the three orange cured recombinants recovered from the 10⁴∶1 (100∶1 crtB stop∶silent + 100∶1 crtI stop∶slient) lycopene library screen. Regions of the crtB (F) and crtI (G) alleles containing the diagnostic mutations were amplified by colony PCR and digested with EcoRV and BsmBI, respectively. Only alleles containing the silent mutations are cut by these enzymes. The plasmids with the B-stop, B-silent, I-stop, and I-silent alleles that served as PCR templates for the subfragments were PCR amplified and digested in parallel as controls. The ladder is a 100 bp DNA ladder from New England Biolabs.