Development of Yarrowia lipolytica expression systems for recombinant proteins
Bulani, Siyavuya Ishmael
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The first aim of this study was to evaluate Yarrowia lipoytica as a host for heterologous protein production of functionally active histidine (His) tagged and non-tagged Fusarium solani pisi cutinase (FSCut) directed by the pre-pro Lip2 secretion signal (Lip2ss) and the native cutinase secretion signal (Nss). Single copy plasmid, pKOV323, directed to the pBR322 docking platform was used for expression of FSCut. The expression levels of FSCut in the P01g strain were highly variable, with the 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. In addition, 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 of Thermomyces lenuginosus lipase (TLL). Similarly to FSCut, the effect of the TLL Nss and the Lip2ss were evaluated for directing the secretion of His-tagged and non-tagged TLL. The plasmid pKOV323 and the multi-copy plasmid, pKOV410, were used for expression of TLL in the strains Po1g and Po1f strains, respectively. Both secretion signals directed expression of functionally active TLL. The His-tag affected TLL expression levels, its activity, and thermostability. The expressed TLL showed differences in band patterns on the SDS gel. This study demonstrated that secretion signals 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 industrial production of FScut and TLL. Characterization of all purified proteins expressed using different approaches showed the highest hydrolytic activity at 60°C and pH 9.0. High-level protein production of the His-tagged and non-tagged TLL was obtained when the enzyme was directed by the pre-pro Lip2ss for secretion into the extracellular medium, with the non-tagged showing the highest levels of total protein production. The results reported in this study indicate that Y. lipolytica is prolific producer of TLL with potential large-scale industrial production of the enzyme. For the third aim of this study, a novel rDNA based plasmid was developed for display of heterologous proteins on the cell surface of Y. Iipolytica using the Cterminal end of the glycosylphosphatidylinositol (GPI) anchored Y. lipolytica cell wall protein 1 (YICWP1). This system employed mCherry as a model protein. High-level display of mCherry on the yeast cell was shown by changes in the phenotypic colour of the cells and culture medium. The displayed mCherry was cleaved from the yeast cells using enterokinase. The developed cell surface displaying plasmid was used to display 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 Histagged cell free enzymes expressed in Y. lipolytica. The display of active hydrolytic enzymes on the cells of Y. lipolytica demonstrated that this yeast have a potential industrial application as whole-cell catalyst. The last aim of this study takes advantage of the plasmid used for over-expressing mCherry displayed on Y. Iipolytica cell surface. Using the novel constructed system, RANTES fused to mCherry on the cell surface of Y. lipolytica cell surface was successfully expressed. The developed expression system offers a simplified process for downstream processing of fusion proteins.