11 research outputs found

    Schematic representation of the constructions used for production of free and anchored xylanase in <i>Y. lipolytica</i>.

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    <p>Constructions used for production of free TxXYN A) TxXYN fused with YlCWP110 C-terminal amino acids B), YlPir 100 C-terminal amino acids C) and YlCBM 87 C-terminal amino acids D). ppLIP2, preproLIP2 used as secretion signal peptide; H, 6 histidines tag; LK, 10 amino acids linker peptide; relevant restriction sites in the encoding genes are indicated above the resulting proteins, while relevant amino acids are indicated below (diamonds for the 4 N-Glycosylation sites and stars for the 2 catalytic glutamic acids in TxXYN, 4 cysteines in Pir100 and the asparagine as acceptor of the GPI anchor in CWP110).</p

    Immobilisation efficiencies of different anchoring systems depending on the carbon source.

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    <p>Total xylanase activities obtained for strain <i>Y. lipolytica</i> JMY1212 transformed with JMP62-TEF-ppLIP2-Pir100-TxXYN, JMP62-TEF-ppLIP2-CBM87-TxXYN, JMP62-TEF-ppLIP2-TxXYN-CWP110 cultivated 4 days with 3 different carbon sources; strain JMY1212 transformed with JMP61-POX2-ppLIP2-TxXYN was used as control. Xylanase units per gram dried cells in culture supernatant (25 mL) and lyophilised cell pellets (0.7, 0.89 and 1 g in glycerol, glucose and oleic acid, respectively) are displayed in grey and spotted bars, respectively. Units of bound xylanase per gram dried cells and corresponding percentages are indicated for each construction. Mean and standard deviation of four experiments are presented.</p

    Xylanase activities as determined in cell walls (lyophilised cell pellets) and growth medium when producing the three fusion proteins in <i>Y. lipolytica</i> JMY1212.

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    <p>Xylanase activities determined for strain <i>Y. lipolytica</i> JMY1212 transformed with JMP62-TEF-ppLIP2-CBM87-TxXYN, JMP62-TEF-ppLIP2-Pir100-TxXYN, JMP62-TEF-ppLIP2-TxXYN-CWP110 and cultivated overnight in 10 g/L oleic acid. Xylanase units per gram dried cells in culture supernatant and lyophilised cell pellets are displayed in grey and spotted bars, respectively. Units of bound xylanase per gram dried cells and corresponding percentages are indicated for each construction. The % activity anchored on cells is the ratio between total units in cell pellet and total units in the whole culture (94 mg cell pellet and 10 mL supernatant). Mean and standard deviation of three experiments are presented.</p

    Western blot analysis of xylanase fusion proteins found in supernatant fractions.

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    <p>Proteins released in supernatant were revealed with non position-specific antiHis primary antibodies. Supernatants were concentrated 10 times and deglycosylated with endoglycosidase H prior to western blot analysis. A schematic representation of proteins detected in SDS-PAGE analysis is added.</p

    MOESM1 of Expressing accessory proteins in cellulolytic Yarrowia lipolytica to improve the conversion yield of recalcitrant cellulose

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    Additional file 1: Figure S1. PCR verification of Y. lipolytica transformants expressing multiple cellulases and accessory proteins (A) YLC7, Lane 1 to 6: BGL1, BGL2, 4UASTrEGI, TrEGII, 4UASNcCBHI, 4UASTrCBHII; (B) YLC8, Lane 1 to 6: BGL1, BGL2, 4UASTrEGI, TrEGII, 4UASNcCBHI, 4UASTrCBHII, TrXYNII; (C) YLC9, Lane 1 to 7: BGL1, BGL2, 4UASTrEGI, TrEGII, 4UASNcCBHI, 4UASTrCBHII, TrLPMOA; (D) YLC10, Lane 1 to 7: BGL1, BGL2, 4UASTrEGI, TrEGII, 4UASNcCBHI, 4UASTrCBHII, TrSWO1; (D) YLC11, Lane 1 to 8: BGL1, BGL2, 4UASTrEGI, TrEGII, 4UASNcCBHI, 4UASTrCBHII, TrXYNII, TrLPMOA. Figure S2. Western blot analysis of the heterologous rhTrEGI protein secreted by the engineered Y. lipolytica strains: lane 1, Endo H-treated secretome of YLC6b (20 μL); lane 2, Endo H-treated secretome of YLC7b (20 μL). Figure S3. Characterization of the recombinant XYNII expressed in Y. lipolytica. (a) Effect of pH on the activity of rhXYNII; (B) Effect of temperature on the activity of rhXYNII. Figure S4. Screening of Y. lipolytica expressing cellulases and accessory enzymes on YNB indication plate containing supplemented with 0.2% w/vAzo-CM-Cellulose. Lane 1, Y. lipolytica control; Lane 2 to 4, YLC8, YLC9 and YLC10. Figure S5. The growth of Y. lipolytica in defined medium containing 10 g/L gluconic acid or 10 g/L glucose. Table S1. The sequences of the oligonucleotide primers used for PCR verification of Y. lipolytica-transformants. Table S2. Comparison of cellulose utilization and biomass yield of cellulolytic Y. lipolytica grown on different cellulosic substrates for 120 h in aerobic cultivation without the addition of ascorbic acid

    Additional file 2: of Development of cellobiose-degrading ability in Yarrowia lipolytica strain by overexpression of endogenous genes

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    Figure S1. Multiple alignments of putative conserved domains of the family 3 glycosyl hydrolases of S. fibuligera (Bgl1, GenBank Accession numbers: AAA34314.1) against Yarrowia genome. Figure S2. Screening of Y. lipolytica expressing the 6 putative β-glucosidases on (a) indication plate containing YNBcasa medium supplemented with 1 mM p-nitrophenyl-β-D-glucoside (pNP-βGlc), and (b, c) YNBC plate with cellobiose as sole carbon source. Figure S3. Transcriptional analysis of the expression of the six putative BGLs in wild type strain on glucose (A) and cellobiose (B), and recombinant strain overexpression of BGL1 and BGL2 (C). Figure S4. N-terminal amino acid sequences of Y. lipolytica Bgl1 (A) and Bgl2 (B). The first 50 N-terminal amino acid sequences are indicated with the predicted signal sequence determined with signal P (underlined), the cleavage site predicted with a*, and the N-terminal AA sequence of the purified protein determined by direct sequencing (in bold). Figure S5. Optimal pH (a) and temperature (b) of Bgl1 (square) and Bgl2 (diamond) from Y. lipolytica JMY1212. Each data point represents the mean of three independent experiments and the error bar indicates the standard deviation. Figure S6. Stability of Bgl1 (a) and Bgl2 (b) from Y. lipolytica JMY1212 at pH from 2.0–8.0 as a function of time at 30ºC, and stability of Bgl1 (c) and Bgl2 (d) at temperature from 30ºC to 60ºC as a function of time at pH 5. Each data point represents the mean of three independent experiments and the error bar indicates the standard deviation. Please note that only one curve is given to represent the stability of Bgl2 at pH 4.0, 5.0 and 6.0 (b) and at 30ºC and 40ºC (d) as 100% of enzyme activity remained for these conditions. Figure S7. The hydrolytic activity of Bgl2 on pNP-βGlc (a) and the stability of Bgl2 at 40ºC as a function of time at pH5.0 before and after deglycosylation. Each data point represents the mean of three independent experiments and the error bar indicates the standard deviation

    Reaction media of 44I12 cytoplasmic extract incubated with Impranil at 30°C and pH 7.0 during 24h.

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    <p>Negative control: <i>E</i>. <i>coli</i> host strain Epi100 transformed with the empty pCC1FOS fosmid. For 44I12, the reaction medium, in and out of the tube, is showed, since a solid aggregate could be obtained.</p
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