title: Engineering Promoter Architecture in Oleaginous Yeast Yarrowia lipolytica.
abstract: Eukaryotic promoters have a complex architecture to control both the strength and timing of gene transcription spanning up to thousands of bases from the initiation site. This complexity makes rational fine-tuning of promoters in fungi difficult to predict; however, this very same complexity enables multiple possible strategies for engineering promoter strength. Here, we studied promoter architecture in the oleaginous yeast, Yarrowia lipolytica. While recent studies have focused on upstream activating sequences, we systematically examined various components common in fungal promoters. Here, we examine several promoter components including upstream activating sequences, proximal promoter sequences, core promoters, and the TATA box in autonomously replicating expression plasmids and integrated into the genome. Our findings show that promoter strength can be fine-tuned through the engineering of the TATA box sequence, core promoter, and upstream activating sequences. Additionally, we identified a previously unreported oleic acid responsive transcription enhancement in the XPR2 upstream activating sequences, which illustrates the complexity of fungal promoters. The promoters engineered here provide new genetic tools for metabolic engineering in Y. lipolytica and provide promoter engineering strategies that may be useful in engineering other non-model fungal systems.
authors.affiliation: Department of Chemical and Biomolecular Engineering and ‡Department of Genetics and Biochemistry, Clemson University , Clemson, South Carolina 29634, United States.
authors.forename: Murtaza
authors.initials: M
authors.lastname: Shabbir Hussain
authors.affiliation: Department of Chemical and Biomolecular Engineering and ‡Department of Genetics and Biochemistry, Clemson University , Clemson, South Carolina 29634, United States.
authors.forename: Lauren
authors.initials: L
authors.lastname: Gambill
authors.affiliation: Department of Chemical and Biomolecular Engineering and ‡Department of Genetics and Biochemistry, Clemson University , Clemson, South Carolina 29634, United States.
authors.forename: Spencer
authors.initials: S
authors.lastname: Smith
authors.affiliation: Department of Chemical and Biomolecular Engineering and ‡Department of Genetics and Biochemistry, Clemson University , Clemson, South Carolina 29634, United States.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Blenner
mesh_terms: D001483:Base Sequence; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D016385:TATA Box / Q000235:genetics*; D025062:Yarrowia / Q000235:genetics* / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D012319:RNA Polymerase II
keywords: POX2; UAS; Yarrowia lipolytica; enhancers; oleate response elements; promoter