Efficient cell surface display of Lip2 lipase using C-domains of glycosylphosphatidylinositol-anchored cell wall proteins of Yarrowia lipolytica

Abstract

The cell surface display of enzymes is of great interest because of its simplified purification stage and the possibility for recycling in industrial processes. In this study, we have focused on the cell wall immobilization of Yarrowia lipolytica Lip2 protein--an enzyme that has a wide technological application. By genome analysis of Y. lipolytica in addition to already characterized Ylcwp1, we identified five putative open reading frames encoding glycosylphosphatidylinositol-anchored proteins. Lip2 translation fusion with the carboxyl termini of these proteins revealed that all proteins were capable of immobilizing lipase in active form on the cell surface. The highest level of cell-bound lipase activity was achieved using C-domains encoded by YlCWP1, YlCWP3 (YALI0D27214g) and YlCWP6 (YALI0F18282g) comprising 16,173 ± 1,800, 18,785 ± 1,130 and 17,700 ± 2,101 U/g dry cells, respectively. To the best of our knowledge, these results significantly exceed the highest cell-bound lipase activity previously reported for engineered Saccharomyces cerevisiae and Pichia pastoris strains. Furthermore, the lyophilized biomass retained the activity and was robust to collecting/resuspending procedures. Nevertheless, in most cases, a substantial amount of lipase activity was also found in the growth medium. Further work will be necessary to better understand the nature of this phenomenon.

Cell Surface Display of Yarrowia lipolytica Lipase Lip2p Using the Cell Wall Protein YlPir1p, Its Characterization, and Application as a Whole-Cell Biocatalyst

“…In our previous study, Lip2p lipase was displayed on Y. lipolytica cells using the C-domains of six putative GPI-anchored cell wall proteins [11]. The active site of lipase is located near the Cterminus; therefore, to prevent the activity, loosing the insertion of a linker peptide between Lip2p and C-domains has been performed.…”

“…The resulting plasmid was named php4d-YlPIR1-LIP2. Plasmid php4d-YlPIR1-LIP2 was digested with NheI and XmaJI to cleave the hp4d-YlPIR1-LIP2 cassette, which was then inserted into the NheI and XmaJI sites of pY-exp [11]. The obtained plasmid, pY-YlPIR1-LIP2, was used for cell surface display of the lipase (Fig.…”

“…The Y. lipolytica strain Po1f was purchased from American Type Culture Collection (ATCC MYA-2613). Po1f (Leu + ) and Po1f (Leu + Ura + ) strains were generated as described previously [11]. The yeast minimal growth medium used was YNBD (0.67 % yeast nitrogen base, Difco, USA) with glucose (2.0 %).…”

“…This procedure was performed to eliminate the Eco31I site inside the gene. The amplified YlPIR1 fragment was digested with Eco31I and XmaJI and introduced into the BpiI and XmaJI sites of the plasmid pUC-hp4d [11]. The synthetic hybrid hp4d promoter was constructed as described by Madzak et al [36].…”

Bioadsorption Strategies with Yeast Molecular Display Technology

“…Several kinds of cell wall proteins in Y. lipolytica have been investigated for use in molecular display technology. YlCWP1 and YlPIR1 from Y. lipolytica, which are GPI-anchored cell wall proteins, allow N-and C-terminus-free display, respectively Bulani et al, 2012;Yuzbasheva et al 2011. Shibasaki et al, 2001bShibasaki et al, 2006, vaccines Shibasaki et al, 2013 and a combinatorial peptide library for use in screening for bioactive molecules Matsui et al, 2009 .…”