Cell‐surface display of the active mannanase in Yarrowia lipolytica with a novel surface‐display system

Abstract

A novel surface-display system was constructed using the cell-wall anchor protein Flo1p from Saccharomyces cerevisiae, the mannanase (man1) from Bacillus subtilis fused with the C-terminus of Flo1p and the 6xHis tag was inserted between Flo1p and man1. The fusion protein was displayed on the cell surface of Yarrowia lipolytica successfully, and it was confirmed by immunofluorescence. In succession, the surface-displayed mannanase was characterized. The optimum catalytic conditions for the recombinant mannanase were 55 degrees C at pH 6.0, and it exhibited high stability against pH variation. The highest activity of the recombinant mannanase reached 62.3 IU/g (dry cell weight) after the recombinant was cultivated for 96 h in YPD medium [1% (w/v) yeast extract/2% (w/v) peptone/2% (w/v) glucose]. To our knowledge, the present paper is the first to report that high-activity mannanase is displayed on the cell surface of Y. lipolytica with Flo1p.

Development of a novel rDNA based plasmid for enhanced cell surface display on Yarrowia lipolytica

“…In this study, we constructed a novel rDNA based plasmid for surface display of heterologous proteins on Y. lipolytica . The plasmid of [Yue et al (2007]) and ([Yang et al 2009]) employ ura3d1 and LEU2 selection markers for single copy integration into the zeta targeting sequence and the pBR322 docking platform, respectively [(Madzak et al 2000]; [Nicaud et al 2002]). In addition, vectors carrying both a zeta sequence and ura3d4 are integrated into Y. lipolytica genome with low transformation frequencies [(Juretzek et al 2001]).…”

“…All identified proteins were used successfully to display active Lip2 lipase on Y. lipolytica employing a zeta-based plasmid . The displaying plasmid of ([Yue et al 2007]) has been used to carry FLO1 for cell surface display of an active mannanase on Y. lipolytica cells ([Yang et al 2009]).…”

Metabolic Engineering for Expanding the Substrate Range of Yarrowia lipolytica

“…Important enzymes that release monosaccharides from various substrates have been successfully expressed in this yeast in studies focused on enzyme characterization [60][61][62]. However, no research has been conducted on the ability of these enzymes to allow yeast growth on such substrates when they are the sole carbon source.…”

“…Another is the mannanase found in Bacillus subtilis (encoded by the man1 gene); it is displayed on Y. lipolytica's cell surface and breaks down glucomannan, a major component of lignocellulosic biomass [61].…”

Progress in Bacillus subtilis Spore Surface Display Technology towards Environment, Vaccine Development, and Biocatalysis

“…Genetically engineered yeast has many applications in chemical synthesis, biotechnology productions, environmental pollutants adsorptions, biorefineries, and protein evolution [Tanaka et al, 2012]. Displaying costly enzymes on the surface of yeast make them costeffective and easily available for cultivation and easy recovery with excellent enzymatic characteristics for industrial application, including oil drilling and hydrolysis of coffee [Yang et al, 2009]. However, yeast surface display has certain drawbacks because of its nutritive properties, susceptibility towards heat and other harsh industrial processes, and chances of cell cracking during protein recovery when using different chemicals.…”