| A total of 78 marine yeast strains from seawater, sediments, mud of salterns, guts of the marine fish and marine algae were obtained. After crude protein of the yeasts was estimated, we found that seven strains of the marine yeasts (hcx-2, L2-2, Wl-6, Qme, SWJ-1b, W2B and Z172 1-2) contained more than 41.0 g protein per 100 g of cell dry weight. Among them, strain SWJ-1b isolated from fish gut contained the highest crude protein. The protein content (w/w) and cell mass (g/L) of the strain SWJ-1b were 47.6% and 6.7, respectively. Therefore, it had potential use as single cell protein. The results of routine identification, Biolog identification and molecular methods further confirmed that it belonged to a strain of Yarrowia lipolytica.The marine yeast Y. lipolytica was genetically modified using the surface display system. When the gene encoding enhanced green fluorescent protein (EGFP) was inserted into the surface display plasmid pINA1317-YlCWP110 and expressed in uracil mutant of Y. lipolytica SWJ-1b. The protein EGFP was successfully displayed on the cell surface, and 100% of the yeast cells exhibited the anchored target proteins. The protein content, cell mass, amino acid composition, and fatty acids compositions of the marine-derived yeast Y. lipolytica SWJ-1b, its uracil mutant 22a-2 and the selected transformant (strain 106) displaying EGFP were analyzed and it was found that crude protein contents (w/w) of the yeast Y. lipolytica SWJ-1b, its uracilmutant 22a-2 and the selected transformant (strain 106) were 54.9±1.3% ,54.9±1.6% and 51.1±0.8%, respectively, while cell mass (g/L) of the yeast Y. lipolytica SWJ-1b, its uracil mutant 22a-2 and the selected transformant (strain 106) were12.0±0. 1, 12.0±0.2 and 16.0±0.1, respectively. There were no big changes in amino acid compositions of the marine yeast Y. lipolytica SWJ-1b, its uracil mutant 22a-2 and the selected transformant (strain 106), especially the essential amino acids, such as lysine, methionine, and leucine. The selected transformant (strain 106) synthesized higher amount of C18:1 and C18:3 fatty acids, but lower amount of C16:0 and C16:1 fatty acids than its wild-type Y. lipolytica SWJ-1b. The results suggest that the marine-derived Y. lipolytica SWJ-1b can be armed with the heterologous protein by the genetic modification and further used as single cell protein.The DNA fragment encoding the N-terminus of scallop H2A was inserted into the expression vector pINA1317 and expressed in Y. lipolytica SWJ-1b. After cultivation in PBB medium for 120 h, the transformant Yl-H2A-29a produced high level of the antibacterial peptide and the supernatant obtained had the killing activity against Vibrio harveyi, Vibrio anguillarum and Vibrio parahaemolyticus. The minimum inhibition concentrations (MIC) for V. harveyi, V. anguillarum and V. parahaemolyticus was 4.47±0.4μg of protein/mL, 8.95±1.4μg of protein/mL and 1.12±0.2μg of protein/mL, respectively. The diameters of the inhibition zone were 14.0 mm, 11.0 mm and24.0 mm, respectively. However, our results show that the supernatant had no activity against Escherichia coli, Staphyloccocus aureus and Bacillus subtilis. The recombinant antibacterial peptide with 6×His tag was purified by Ni2+ affinity chromatography. The molecular mass of the recombinant antibacterial peptide was 45.0 kDa. It could be clearly seen from the photograph of transmission electron microscope that after the whole cells and protoplast cells of the V. parahaemolyticus were exposed to the purified antibacterial peptide, the antibacterial peptide could cause leakage of intracellular components in both the whole cells and protoplast cells and cell death, suggesting that the antibacterial peptide can pass different barriers such as the extracellular matrix, or the bacterial lipopolysaccharides, outer membrane and/or peptidoglycan layers and form pores on cell membrane. After the SWISS model of the antibacterial peptide used in this study was made, we found that the antibacterial peptide indeed had theα-helical structure . The hydropathy profile of amino acids of the antibacterial peptide indicates that the antibacterial peptide had many hydrophobic amino acids. Theα-helical structure and hydrophobic amino acids may be related to the antibacterial activity of the peptide. After the yeast transformant Yl-H2A-29a was grown in YPD medium, PBB medium and the hydrolysate of soybean meal with ammonium sulfate, the protein content of the cells and cell growth were determined, respectively. The yeast transformant Yl-H2A-29a grown in YPD medium, PBB medium and the hydrolysate of soybean meal with ammonium sulfate still contained 48.9±0.3%, 45.3±0.8% and 46.1±1.4% (w/w) protein in their cells, respectively and cell dry weights( g/L)were 12.0±0.9, 11.5±0.7 and 9.9±0.6, respectively. Y. lipolytica SWJ-1b which was able to secrete the antibacterial peptide still could be used as single cell protein. This is the first time to report that the marine-derived Y. lipolytica with high content of protein was used to produce the recombinant antibacterial peptide.The two DNA fragments encoding the N-terminus of scallop H2A and the inulinase gene INU1 were inserted into the expression vector pINA1317, respectively. Then, the two DNA fragments were co-expressed in Y. lipolytica SWJ-1b. The recombinant products obtained from the transformant carrying the two DNA fragments had both the killing activity against V. harveyi, V. parahaemolyticus and V. anguillarum and inulinase activity. The diameters of the clear zones for V. harveyi, V. anguillarum, V. parahaemolyticus and were 12.0 mm, 8.0 mm and 15.0 mm, respectively. The MICs were 6.28±0.2μg of protein/mL, 2.51±0.7μg of protein/mLand 13.63±0.4μg of protein/mL. respectively. However, our results show that the recombinant products had no activity against E. coli, S. aureus and B. subtilis. The inulinase activity of the recombinant products was 32.7±0.3 U/mL. During the 2-L fermentation, we found that 38.6±0.4 U/mL of the maximum inulinase activity was produced within 72 h of fermentation. The crude protein contents (w/w) and cell mass (g/L) were 46.3±0.2% and 19.2±0.3 at this time. The fermentation broth had the hightest killing activity against V. harveyi, V. parahaemolyticus. and V. anguillarum and the diameters of the clear zones for V. harveyi, V. anguillarum and V. parahaemolyticus were 13.0 mm, 10.0 mm and 16.0 mm after 120 h of the fermentation.
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