Abstract
Engineering and evaluation of synthetic routes for generating valuable compounds require accurate and cost-effective de novo synthesis of genetic pathways. Here, we present an economical and streamlined de novo DNA synthesis approach for engineering a synthetic pathway with microchip-synthesized oligonucleotides (oligo). The process integrates entire oligo pool amplification, error-removal, and assembly of long DNA molecules. We utilized this method to construct a functional lycopene biosynthetic pathway (11.9 kb encoding 10 genes) in Escherichia coli using a highly error-prone microchip-synthesized oligo pool (479 oligos) without pre-purification, and the error-frequency was reduced from 14.25/kb to 0.53/kb. This low-equipment-dependent and cost-effective method can be widely applied for rapid synthesis of biosynthetic pathways in general molecular biology laboratories.
Highlights
The development of high-throughput DNA sequencing and bioinformatics has generated massive amount of information about metabolic pathways of important compounds[1]
The ribosome-binding site (RBS) sequences were flanked by the ends of the contiguous genes
All the 36 target subpools were separately re-amplified from the MP-polymerase chain reaction (PCR) amplicons (Fig. 4a) and were assembled into target fragments (Fig. 4b). These results showed that 36 subpools (479 oligos) could be amplified simultaneously in one MP-PCR reaction without missing target oligos, and that the concentration of each oligo was sufficient for fragment assembly
Summary
The development of high-throughput DNA sequencing and bioinformatics has generated massive amount of information about metabolic pathways of important compounds[1]. We developed a low-equipment-dependent process, in which the throughput of each synthetic reaction (oligo amplification, error-removal, and DNA assembly) was highly improved using the commercial Mcp-oligo pools. Each subpool was selectively amplified from the Mcp-oligo pool, processed for error-removal using MICC, and assembled using LCR.
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