title: Standardized Markerless Gene Integration for Pathway Engineering in Yarrowia lipolytica.
abstract: The yeast Yarrowia lipolytica is a promising microbial host due to its native capacity to produce lipid-based chemicals. Engineering stable production strains requires genomic integration of modified genes, avoiding episomal expression that requires specialized media to maintain selective pressures. Here, we develop a CRISPR-Cas9-based tool for targeted, markerless gene integration into the Y. lipolytica genome. A set of genomic loci was screened to identify sites that were accepting of gene integrations without impacting cell growth. Five sites were found to meet these criteria. Expression levels from a GFP expression cassette were consistent when inserted into AXP, XPR2, A08, and D17, with reduced expression from MFE1. The standardized tool is comprised of five pairs of plasmids (one homologous donor plasmid and a CRISPR-Cas9 expression plasmid), with each pair targeting gene integration into one of the characterized sites. To demonstrate the utility of the tool we rapidly engineered a semisynthetic lycopene biosynthesis pathway by integrating four different genes at different loci. The capability to integrate multiple genes without the need for marker recovery and into sites with known expression levels will enable more rapid and reliable pathway engineering in Y. lipolytica.
authors.affiliation: Chemical and Environmental Engineering, University of California , Riverside, California 92521, United States.
authors.forename: Cory
authors.initials: C
authors.lastname: Schwartz
authors.affiliation: Chemical and Biomolecular Engineering, Clemson University , Clemson, South Carolina 29634, United States.
authors.forename: Murtaza
authors.initials: M
authors.lastname: Shabbir-Hussain
authors.affiliation: Chemical and Environmental Engineering, University of California , Riverside, California 92521, United States.
authors.forename: Keith
authors.initials: K
authors.lastname: Frogue
authors.affiliation: Chemical and Biomolecular Engineering, Clemson University , Clemson, South Carolina 29634, United States.
authors.forename: Mark
authors.initials: M
authors.lastname: Blenner
authors.affiliation: Chemical and Environmental Engineering, University of California , Riverside, California 92521, United States.
authors.forename: Ian
authors.identifier: 0000-0002-3492-7539
authors.initials: I
authors.lastname: Wheeldon
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics; D002338:Carotenoids / Q000096:biosynthesis; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D056426:Genetic Loci / Q000235:genetics; D005819:Genetic Markers / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D000077276:Lycopene; D060847:Metabolic Engineering / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D025062:Yarrowia / Q000235:genetics*
chemical_list: D005819:Genetic Markers; D002338:Carotenoids; D000077276:Lycopene
keywords: CRISPR-Cas9; carotenoids; genome editing; metabolic engineering; standardized genetic tool; synthetic biology