Engineering Promoter Architecture in Oleaginous Yeast Yarrowia lipolytica

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...

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Bibliographic Details
Published in:ACS synthetic biology 2016-03, Vol.5 (3), p.213-223
Main Authors: Shabbir Hussain, Murtaza, Gambill, Lauren, Smith, Spencer, Blenner, Mark A
Format: Article
Language:English
Subjects:
Base Sequence
Enhancer Elements, Genetic - genetics
Fungal Proteins - genetics
Fungal Proteins - metabolism
Genetic Engineering
Plasmids - genetics
Plasmids - metabolism
Real-Time Polymerase Chain Reaction
RNA Polymerase II - genetics
RNA Polymerase II - metabolism
TATA Box - genetics
Yarrowia - genetics
Yarrowia - metabolism
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Summary: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.
ISSN:2161-5063
2161-5063
DOI:10.1021/acssynbio.5b00100