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A modular Golden Gate toolkit for Yarrowia lipolytica synthetic biology.

Macarena LarroudeYoung-Kyoung ParkPaul SoudierMonika KubiakJean-Marc NicaudTristan Rossignol
Published in: Microbial biotechnology (2019)
The oleaginous yeast Yarrowia lipolytica is an established host for the bio-based production of valuable compounds and an organism for which many genetic tools have been developed. However, to properly engineer Y. lipolytica and take full advantage of its potential, we need efficient, versatile, standardized and modular cloning tools. Here, we present a new modular Golden Gate toolkit for the one-step assembly of three transcription units that includes a selective marker and sequences for genome integration. Perfectly suited to a combinatorial approach, it contains nine different validated promoters, including inducible promoters, which allows expression to be fine-tuned. Moreover, this toolbox incorporates six different markers (three auxotrophic markers, two antibiotic-resistance markers and one metabolic marker), which allows the fast sequential construction and transformation of multiple elements. In total, the toolbox contains 64 bricks, and it has been validated and characterized using three different fluorescent reporter proteins. Additionally, it was successfully used to assemble and integrate a three-gene pathway allowing xylose utilization by Y. lipolytica. This toolbox provides a powerful new tool for rapidly engineering Y. lipolytica strains and is available to the community through Addgene.
Keyphrases
  • genome wide
  • poor prognosis
  • escherichia coli
  • copy number
  • healthcare
  • saccharomyces cerevisiae
  • air pollution
  • crispr cas
  • binding protein
  • long non coding rna
  • high density
  • single molecule