Construction And Application Of A Surface Display System For Candida Tropicalis

To evaluate the potential application of cell surface display technology in Candida tropicalis,we used C.tropicalis as host cells to construct a new surface display system using exogenous anchoring protein genes.The new display system was used to display exogenous amylase as well as β-glucosidase and verifying the viability.The main elements are as follows:(1)The gene sequences of five anchor protein SED1,AGα1,EGT2,CWP2 and DAN4 derived from Saccharomyces cerevisiae were obtained via NCBI.Five recombinant strains with different anchoring proteins displaying ye GFP were constructed.The effect of ye GFP displayed in C.tropicalis was assessed by laser confocal microscopy(LSCM),immunofluorescence and flow cytometry assays.It showed that the anchoring domains SED1,CWP2 and AGα1 successfully displayed ye GFP on the cell wall,the display efficiency of the anchoring domains SED1 and CWP2 was higher than that of AGα1.(2)Six β-glucosidase gene sequences of C.tropicalis were obtained by NCBI,namely bgl I-0,bgl I-1,BGL2,SIM1-0,SIM1-1 and SUN41.The cell growth of strains that knocked out of six β-glucosidase genes showed that the most important gene affecting the growth was BGL2.The recombinant strains for displaying BGL2 showed a lower enzyme activity,which indicated the weak ability of C.tropicalis to hydrolyze cellobiose.(3)Recombinant strains displaying the β-glucosidase(BGL1)derived from Aspergillus aculeatus or the α-amylase(ROA1)from Rhizopus oryzae were constructed.It was found that the strains GBSE1 using anchor protein SED1 to display BGL1 or GRSE1 using anchor protein SED1 to display ROA1 had the highest enzyme activity,7.93 U/g CDW and 6.37 U /g CDW,respectively.The enzyme activity was 8 and 6 times higher than that of strains GBAG and GRAG using anchor protein AGα1 to display BGL1 and ROA1,respectively.Real-time fluorescence quantitative PCR analysis showed that strains GBSE1 or GRSE1 had the highest transcript levels.In addition,the strain GBSE3,which displayed BGL1 using signal peptide of SED1 and anchoring protein SED1,as well as the strain GRSE2,which displayed ROA1 using signal peptide of CWP2 and anchoring protein SED1,had the highest enzymatic activity of 8.18 U/g CDW and 8.86 U/g CDW,respectively.(4)Investigation of the enzymatic properties for recombinant strains GBSE1 or GRSE1.It was discovered that the Optimum reaction temperature for surface-displayed β-glucosidase was 55°C;the Optimum reaction p H was 4.0;and the enzymatic activity remained above 85%when kept at 55°C and p H 4.0 for 1 h.The Optimum reaction temperature displayedα-amylase was 60°C;the Optimum reaction p H was 5.0,and the enzymatic activity ofα-amylase remained above 80% when maintained at 60°C and p H 5.0 for 1 h.(5)The recombinant strains DRPB2 and DRPB3 were constructed by integrating fragments containing the SED1 anchoring domain into strain DRPB1.Then the β-carotene content of the recombinant strains was investigated after 120 h fermentation.The results showed that the β-carotene content of DRPB2 and DRPB3 were 2.72 mg/L and 4.54 mg/L,which were 1.58 and 2.34 folds higher compared to the control strains,respectively.