DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways

Abstract

The assembly of large recombinant DNA encoding a whole biochemical pathway or genome represents a significant challenge. Here, we report a new method, DNA assembler, which allows the assembly of an entire biochemical pathway in a single step via in vivo homologous recombination in Saccharomyces cerevisiae. We show that DNA assembler can rapidly assemble a functional D-xylose utilization pathway (approximately 9 kb DNA consisting of three genes), a functional zeaxanthin biosynthesis pathway (approximately 11 kb DNA consisting of five genes) and a functional combined D-xylose utilization and zeaxanthin biosynthesis pathway (approximately 19 kb consisting of eight genes) with high efficiencies (70-100%) either on a plasmid or on a yeast chromosome. As this new method only requires simple DNA preparation and one-step yeast transformation, it represents a powerful tool in the construction of biochemical pathways for synthetic biology, metabolic engineering and functional genomics studies.

Figure 1.

Construction of the d-xylose utilization pathway in Saccharomyces cerevisiae. (a) The assembled d-xylose utilization pathway with its corresponding verification primers; (b) PCR analysis of the assembled d-xylose utilization pathway on a plasmid [d-xylose utilization (p), using pRS426m as a backbone] and on a chromosome [d-xylose utilization (c), integrated to the δ site(s)]. M represents 1 kb DNA ladder from Invitrogen; (c) Growth of S. cerevisiae strains carrying d-xylose utilization pathway using d-xylose as the sole carbon source. Cells carrying the empty vector pRS426 were used as the negative control.

Figure 2.

Construction of the zeaxanthin biosynthesis pathway in Saccharomyces cerevisiae. (a) The assembled zeaxanthin biosynthesis pathway with its corresponding verification primers; (b) PCR analysis of the assembled zeaxanthin biosynthesis pathway on a plasmid [zeaxanthin biosynthesis (p), using pRS426m as a backbone] and on a chromosome [zeaxanthin biosynthesis (c), integrated to the δ site(s)]. M represents 1 kb DNA ladder from Invitrogen; (c) HPLC analysis of the cell extracts from S. cerevisiae carrying the zeaxanthin biosynthesis pathway. Cells carrying the empty vector pRS426 were used as the negative control.

Figure 3.

Construction of the combined d-xylose utilization and zeaxanthin biosynthesis pathway in Saccharomyces cerevisiae. (a) The assembled combined d-xylose utilization and zeaxanthin biosynthesis pathway with its corresponding verification primers; (b) PCR analysis of the assembled combined d-xylose utilization and zeaxanthin biosynthesis pathway on a plasmid [combined pathway (p), using pRS426m as a backbone] and on a chromosome [combined pathway (c), integrated to the δ site(s)]. M represents 1 kb DNA ladder from Invitrogen; (c) Growth of S. cerevisiae strains carrying the combined pathway using d-xylose as the sole carbon source; (d) HPLC analysis of the cell extracts from S. cerevisiae carrying the combined pathway. Cells carrying the empty vector pRS426 were used as the negative control.

Figure 4.

Physical characterization of the recombinant clones using pRS416m as a backbone through restriction digestion. Plasmids corresponding to the clones from the eight-gene pathway with ∼50 bp, ∼125 bp and 270–430 bp overlaps were digested by BamHI and EcoRI. The correct clones should exhibit five bands with sizes of 2233, 2551, 3218, 4454 and 11 386 bp.

Scheme 1.

A one-step method for assembly and integration of a biochemical pathway using in vivo homologous recombination in S. cerevisiae. (a) One-step assembly into a vector. (b) One-step integration to a δ site on a S. cerevisiae chromosome. n represents the number of DNA fragments.