Development of Yarrowia lipolytica expression systems for recombinant proteins

[摘要] The first aim of this study was to evaluate Yarrowia lipoytica as a host forheterologous protein production of functionally active histidine (His) tagged and non-taggedFusarium solani pisi cutinase (FSCut) directed by the pre-pro Lip2 secretionsignal (Lip2ss) and the native cutinase secretion signal (Nss). Single copy plasmid,pKOV323, directed to the pBR322 docking platform was used for expression ofFSCut. The expression levels of FSCut in the P01g strain were highly variable, withthe purified non-tagged FSCut higher and more active that the His-tagged FSCut.The His-tag affected the expression levels and activity of the recombinant FScut. Inaddition, the His-tag influenced the enzyme activity when assayed at its optimal pH.The second aim of the study was to evaluate Y. lipolytica as a host for expression ofThermomyces lenuginosus lipase (TLL). Similarly to FSCut, the effect of the TLL Nssand the Lip2ss were evaluated for directing the secretion of His-tagged and non-taggedTLL. The plasmid pKOV323 and the multi-copy plasmid, pKOV410, wereused for expression of TLL in the strains Po1g and Po1f strains, respectively. Bothsecretion signals directed expression of functionally active TLL. The His-tag affectedTLL expression levels, its activity, and thermostability. The expressed TLL showeddifferences in band patterns on the SDS gel. This study demonstrated that secretionsignals play an important role in both secretion and activity of recombinant proteins.In addition, Y. lipolytica has a potential to be used for large-scale industrialproduction of FScut and TLL.Characterization of all purified proteins expressed using different approaches showedthe highest hydrolytic activity at 60°C and pH 9.0. High-level protein production of theHis-tagged and non-tagged TLL was obtained when the enzyme was directed by thepre-pro Lip2ss for secretion into the extracellular medium, with the non-taggedshowing the highest levels of total protein production. The results reported in thisstudy indicate that Y. lipolytica is prolific producer of TLL with potential large-scaleindustrial production of the enzyme.For the third aim of this study, a novel rDNA based plasmid was developed fordisplay of heterologous proteins on the cell surface of Y. Iipolytica using the Cterminalend of the glycosylphosphatidylinositol (GPI) anchored Y. lipolytica cell wallprotein 1 (YICWP1). This system employed mCherry as a model protein. High-leveldisplay of mCherry on the yeast cell was shown by changes in the phenotypic colourof the cells and culture medium. The displayed mCherry was cleaved from the yeastcells using enterokinase. The developed cell surface displaying plasmid was used todisplay functionally active His-tagged FSCut and TLL on Y. Iipolytica cell surface.The FSCut and TLL displayed on Y. Iipolytica cells were more active that the Histaggedcell free enzymes expressed in Y. lipolytica. The display of active hydrolyticenzymes on the cells of Y. lipolytica demonstrated that this yeast have a potentialindustrial application as whole-cell catalyst.The last aim of this study takes advantage of the plasmid used for over-expressingmCherry displayed on Y. Iipolytica cell surface. Using the novel constructed system,RANTES fused to mCherry on the cell surface of Y. lipolytica cell surface wassuccessfully expressed. The developed expression system offers a simplifiedprocess for downstream processing of fusion proteins.

[发布机构] University of the Free State