Flexible and Versatile Strategy for the Construction of Large Biochemical Pathways

Yongbo Yuan, Erik Andersen, Huimin Zhao

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)46-52
Number of pages7
JournalACS synthetic biology
Volume5
Issue number1
DOIs
StatePublished - Jan 15 2016

Keywords

  • DNA assembler
  • ligase cycling reaction
  • pathway engineering
  • synthetic biology
  • yeast homologous recombination

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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