Controlling heterologous gene expression inyeast cell factories on different carbon substrates and across the diauxic shift: acomparison of yeast promoter activities

[摘要] BackgroundPredictable control of gene expression is necessary for the rationaldesign and optimization of cell factories. In the yeast Saccharomyces cerevisiae, the promoter is one of the mostimportant tools available for controlling gene expression. However, the complexexpression patterns of yeast promoters have not been fully characterised andcompared on different carbon sources (glucose, sucrose, galactose and ethanol)and across the diauxic shift in glucose batch cultivation. These conditions areof importance to yeast cell factory design because they are commonly used andencountered in industrial processes. Here, the activities of a series of“constitutive” and inducible promoters were characterised in single cellsthroughout the fermentation using green fluorescent protein (GFP) as areporter.ResultsThe “constitutive” promoters, including glycolytic promoters,transcription elongation factor promoters and ribosomal promoters, differed intheir response patterns to different carbon sources; however, in glucose batchcultivation, expression driven by these promoters decreased sharply as glucosewas depleted and cells moved towards the diauxic shift. Promoters induced atlow-glucose levels (PHXT7, PSSA1 and PADH2) varied in induction strength on non-glucose carbon sources(sucrose, galactose and ethanol); in contrast to the “constitutive” promoters,GFP expression increased as glucose decreased and cells moved towards thediauxic shift. While lower than several “constitutive” promoters during theexponential phase, expression from the SSA1promoter was higher in the post-diauxic phase than the commonly-used TEF1 promoter. The galactose-inducible GAL1 promoter provided the highest GFP expressionon galactose, and the copper-inducible CUP1promoter provided the highest induced GFP expression following the diauxicshift.ConclusionsThe data provides a foundation for predictable and optimised controlof gene expression levels on different carbon sources and throughout batchfermentation, including during and after the diauxic shift. This information canbe applied for designing expression approaches to improve yields, rates andtitres in yeast cell factories.

[发布日期] 2015-06-26

[关键词] Promoter;Overexpression;Metabolic engineering;Batch cultivation;Fermentation;Transcription regulation;Flow cytometry