Genetic tools for streamlined and accelerated pathway engineering in Yarrowia lipolytica

Lynn Wong, Benjamin Holdridge, Jake Engel, Peng Xu*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

10 Scopus citations

Abstract

Yarrowia lipolytica is an industrial oleaginous yeast that has many attractive physiological and metabolic characteristics for various biotechnological applications. Although it has a long history of industrial applications, the number of genetic tools available to effectively and efficiently engineer Y. lipolytica still falls behind the vast number of tools available for common organisms such as Escherichia coli and Saccharomyces cerevisiae. In this protocol, we have developed a complete and versatile genetic toolkit tailored for facile genetic manipulation in Y. lipolytica. We created a versatile DNA assembly platform YaliBrick, which can streamline the cloning of large multigene pathways with reused genetic parts. We established a sensitive luciferase reporter assay to characterize a set of 12 native promoters. In addition, we used YaliBrick to generate different gene configurations in multigene constructs. The five-gene biosynthetic pathway of the anticancer, antimicrobial pigment violacein was rapidly assembled in 1 week to demonstrate the simplicity and effectiveness of integrating pathway-balancing strategies with our YaliBrick vectors. In the end, we incorporated CRISPR-Cas9 into our YaliBrick vectors and achieved indel mutation and frameshift gene deletion at the CAN1 (arginine permease) genomic loci of Yarrowia lipolytica. The reported protocol provides a standard procedure to streamline and accelerate metabolic pathway engineering in Yarrowia lipolytica.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages155-177
Number of pages23
DOIs
StatePublished - 2019
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume1927
ISSN (Print)1064-3745

Keywords

  • Combinatorial pathway library
  • Gene assembly
  • Genome editing
  • Metabolic engineering
  • Promoter engineering
  • Reporter
  • Synthetic biology
  • YaliBrick

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