Abstract
BackgroundThe oleaginous yeast Yarrowia lipolytica is increasingly used as an alternative cell factory for the production of recombinant proteins. Recently, regulated promoters from genes EYK1 and EYD1, encoding an erythrulose kinase and an erythritol dehydrogenase, respectively, have been identified and characterized in this yeast. Hybrid promoters up-regulated by polyols such as erythritol and erythrulose have been developed based on tandem copies of upstream activating sequences from EYK1 (UAS1EYK1) and XPR2 (encoding extracellular protease, UAS1XPR2) promoters.ResultsThe strength of native (pEYD1) and engineered promoters (pEYK1-3AB and pHU8EYK) was compared using the extracellular lipase CalB from Candida antarctica as a model protein and a novel dedicated host strain. This latter is engineered in polyol metabolism and allows targeted chromosomal integration. In process conditions, engineered promoters pEYK1-3AB and pHU8EYK yielded 2.8 and 2.5-fold higher protein productivity, respectively, as compared to the reference pTEF promoter. We also demonstrated the possibility of multicopy integration in the newly developed host strain. In batch bioreactor, the CalB multi-copy strain RIY406 led to a 1.6 fold increased lipase productivity (45,125 U mL−1) within 24 h as compared to the mono-copy strain.ConclusionsThe expression system described herein appears promising for recombinant extracellular protein production in Y. lipolytica.
Highlights
The oleaginous yeast Yarrowia lipolytica is increasingly used as an alternative cell factory for the produc‐ tion of recombinant proteins
Construction of the host strain JMY7126 for erythritol‐inducible based expression system Yarrowia lipolytica strain JMY1212 was previously developed to target integration of an expression cassette at a zeta docking platform located at the LEU2 locus [27]
Despite strain JMY1212 has been used successfully for heterologous gene expression [27, 29,30,31], it suffers from several drawbacks: (i) it possesses only a single auxotrophy based on uracil metabolism, impairing multiple genome editions; (ii) it is able to metabolize erythritol and erythrulose, preventing their utilization as free inducer
Summary
The oleaginous yeast Yarrowia lipolytica is increasingly used as an alternative cell factory for the produc‐ tion of recombinant proteins. Results: The strength of native (pEYD1) and engineered promoters (pEYK1-3AB and pHU8EYK) was compared using the extracellular lipase CalB from Candida antarctica as a model protein and a novel dedicated host strain. This latter is engineered in polyol metabolism and allows targeted chromosomal integration. Regulated promoters derived from the LIP2 and POX2 genes encoding an extracellular lipase and an acyl-CoA oxidase, respectively, have been developed for recombinant gene expression [17]. These regulated promoters delivered strong gene expression, their utilization at industrial scale is impeded by the hydrophobic nature of their inducers (i.e. triglycerides and fatty acids)
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