A versatile, efficient strategy for assembly of multi-fragment expression vectors in Saccharomyces cerevisiae using 60 bp synthetic recombination sequences

Abstract

BackgroundIn vivo recombination of overlapping DNA fragments for assembly of large DNA constructs in the yeast Saccharomyces cerevisiae holds great potential for pathway engineering on a small laboratory scale as well as for automated high-throughput strain construction. However, the current in vivo assembly methods are not consistent with respect to yields of correctly assembled constructs and standardization of parts required for routine laboratory implementation has not been explored. Here, we present and evaluate an optimized and robust method for in vivo assembly of plasmids from overlapping DNA fragments in S. cerevisiae.ResultsTo minimize occurrence of misassembled plasmids and increase the versatility of the assembly platform, two main improvements were introduced; i) the essential elements of the vector backbone (yeast episome and selection marker) were disconnected and ii) standardized 60 bp synthetic recombination sequences non-homologous with the yeast genome were introduced at each flank of the assembly fragments. These modifications led to a 100 fold decrease in false positive transformants originating from the backbone as compared to previous methods. Implementation of the 60 bp synthetic recombination sequences enabled high flexibility in the design of complex expression constructs and allowed for fast and easy construction of all assembly fragments by PCR. The functionality of the method was demonstrated by the assembly of a 21 kb plasmid out of nine overlapping fragments carrying six glycolytic genes with a correct assembly yield of 95%. The assembled plasmid was shown to be a high fidelity replica of the in silico design and all glycolytic genes carried by the plasmid were proven to be functional.ConclusionThe presented method delivers a substantial improvement for assembly of multi-fragment expression vectors in S. cerevisiae. Not only does it improve the efficiency of in vivo assembly, but it also offers a versatile platform for easy and rapid design and assembly of synthetic constructs. The presented method is therefore ideally suited for the construction of complex pathways and for high throughput strain construction programs for metabolic engineering purposes. In addition its robustness and ease of use facilitate the construction of any plasmid carrying two or more genes.

Laboratory Evolution of a Biotin-Requiring Saccharomyces cerevisiae Strain for Full Biotin Prototrophy and Identification of Causal Mutations

“…The amplified BIO gene sequences included 0.5-kb terminator sequences. The 3= and 5= primers for amplification of promoter and terminator fragments, respectively, contained 60-bp synthetic extensions designed for efficient in vivo assembly of DNA fragments (56). Promoters and coding regions were fused by fusion PCR (57) and subsequently assembled into pJET1.2/blunt vectors with a CloneJet PCR cloning kit (Thermo Scientific), resulting in the vector constructs pUD416 and pUD418 (Table 5).…”

Pathway swapping: Toward modular engineering of essential cellular processes

“…In practice, unintended genome rearrangements caused by HR, either between the engineered genetic elements themselves or between engineered genetic elements and the native yeast genome, were not observed. These results highlight the amazing efficiency and versatility of in vivo assembly and CRISPR/Cas9-facilitated genome editing in S. cerevisiae (6,24).…”

“…1 and SI Appendix, Fig. S1), 13 DNA cassettes each consisting of a S. cerevisiae glycolytic gene, including its native promoter and terminator, flanked by 60-bp synthetic homologous recombination (SHR) sequences (6). SHR sequences share no homology with the S. cerevisiae genome and can be used for efficient in vivo assembly and integration, by homologous recombination (HR), of the glycoblocks.…”

High molecular weight DNA assembly in vivo for synthetic biology applications

“…[33] An improved method for multi-fragment DNA assembly in S. cerevisiae utilizes 60 bp synthetic recombination sequences that are non-homologous with the yeast genome to enhance reliability, flexibility and accuracy of the yeast homologous recombination for assembly of plasmids. [46] The synthetic recombination sequences are non-homologous with the yeast genome to avoid interference and recombination with the host cell genomic DNA. Separation of the survival elements of the plasmid backbone (selection marker and yeast episome) into two DNA fragments flanked by 60 bp synthetic recombination sequences led to a 100 fold decrease in the number of false positive transformants as compared to previous methods (such as using linearized plasmid backbone).…”

“…Using this approach, nine parts were assembled into a 21 kb plasmid with an accuracy of 95%. [46] An improved DNA assembly method, dubbed RADOM (rapid assembly of DNA overlapping multifragments), has been developed recently [47] (Figure 1). RADOM combines yeast homologous recombination with blue/white screening in E. coli, to reduce the time and labor required for screening for correctly assembled DNA fragments.…”