Flexible and Versatile Strategy for the Construction of Large Biochemical Pathways.
Synthetic pathways and circuits have been increasingly used for microbial production of fuels and chemicals. Here, we report a flexible and versatile DNA assembly strategy that allows rapid, modular, and reliable construction of biological pathways and circuits from basic genetic parts. This strategy combines the automation-friendly ligase cycling reaction (LCR) method and the high-fidelity in vivo yeast-based DNA assembly method, DNA assembler. Briefly, LCR is used to preassemble basic genetic parts into gene expression cassettes or to preassemble small parts into larger parts to reduce the number of parts, in which many basic genetic parts can be reused. With the help of specially designed unique linkers, all preassembled parts will then be directly assembled using DNA assembler to build the target constructs. As proof of concept, three plasmids with varying sizes of 13.4, 24, and 44 kb were rapidly constructed with fidelities of 100, 88, and 71%, respectively. The yeast strain harboring the constructed 44 kb plasmid was confirmed to be capable of utilizing xylose, cellobiose, and glucose to produce zeaxanthin. This strategy should be generally applicable to any custom-designed pathways, circuits, or plasmids.
Department of Chemical and Biomolecular Engineering, Institute for Genomic Biology, ‡Departments of Chemistry, Biochemistry, and Bioengineering, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Yongbo
Y
Yuan
Department of Chemical and Biomolecular Engineering, Institute for Genomic Biology, ‡Departments of Chemistry, Biochemistry, and Bioengineering, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Erik
E
Andersen
Department of Chemical and Biomolecular Engineering, Institute for Genomic Biology, ‡Departments of Chemistry, Biochemistry, and Bioengineering, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Huimin
H
Zhao
D020029:Base Pairing; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008025:Ligases / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D010957:Plasmids / Q000378:metabolism; D058615:Synthetic Biology / Q000379:methods*
D004247:DNA; D008025:Ligases
DNA assembler; ligase cycling reaction; pathway engineering; synthetic biology; yeast homologous recombination