| Xylan is a main anti-nutritional factor in cereals. Xylanases (EC3.2.1.8) are glycosidases that break long sugar chain of xylan into xylooligosaccharides (XOs), the synergic effects of both endo-xylanase and exo-xylosidase (EC3.2.1.37) catalyze xylan into D-xylose. In the past decade, xylanases have been widely used in paper pulp, food and feed industries. Xylose has been of tremendeous interests to produce bio-ethanol. Therefore, it is desirable for xylanase to exhibit high catalytic activity and thermostability to handle industrial tasks. The current study was designed to clone four xylanase genes from family11and one xylosidase gene, modify the gene with high catalytic activity and good properties, and construct recombinant yeast containing high-copy xyalanse genes. After that, xylanase and xylosidase were heterologously co-expressed in flask medium. The secreted proteins were further subjected to analyze substrate morphology and hydrolytic products of birchwood xylan. The main results were as follow:1Cloning and expression of endo-xylanse and xylosidase in E. coliFour family11xylanase genes (Aspergillus niger xylanase, anx; Bacillus subtilis xylanase, bsx; Thermomonospora fusca xylanase, tfx; hybrid xylanase, atx) and B. subtilis xylosidase (xylo) gene were cloned and expressed in E. coli. All xylanases were stable under acidy conditions. Among them, both ATx and Tfx showed high acitivity and good thermostability. Furthermore, Tfx was observed to be capable to endure xylanase inhibior and contain a carbohydrate binding module (CBM), which was seldom found in family11xylanses. With the context, xylanase genes atx and tfx were employed for further protein enginnering.2Molecular modification of atx and construction of the recombinant yeastA directed evolution strategy comprising of error-prone PCR (EP-PCR) and high-throughput screening based on micro-plate was employed to improve the catalytic activity of atx, according to the non-rational degisn. A total of1530clones were screened to obtain the mutant FSI-A124with most improved xylanase activity. The wild type gene atx and the mutant FSI-A124were transformed into Pichia pastoris GS115to target the PGAP locus of yeast genome by electroporation. The recombinant xylanases driven by the Saccharomyces cerevisiae a-mating factor were secreted into culture medium. After incubation in YPD medium for96h, the specific activities of YATx and YFSI-A124reached954and1556U/mg, respectively. The Km of YFSI-A124was4.25mg/ml,4.7%lower than that of YATx, while the turnover number kcat was44%higher. Nevertheless, YFSI-A124was less thermally stable than YATx. Amino acid alignment and three-dimensional strucute revealed that a single substitution L49P was formed within the sequence of YFSI-A124. The mutagenesis possibly made the catalytic module of YFSI-A124more flexible than wild type. Therfore, the pocket cavity was supposed to have good affinity to substrate and catalytic efficiency. The decrease of thermostability was attributed to decline of hydrogen bond, van der Waals force and hydrophobic interactions with other residues nearby the catalytic center.3Molecular modification of tfxBoth directed evolution and site-directed mutagenesis were employed to improve the catalytic activity and thermostability of tfx. Two round EP-PCR and high-throughput screening based on micro-plate were initially used to screen7000clones for six mutants with improved activity. Four mutation sites possibly relative to catalytic acticvity and enzymatic properties were optimized using site-saturation mutagenesis. After that, all mutants obtained were subjected to DNA shuffling and StEP. A total of3000clones were screened to obtain three mutants with improved activity. A single substitution S144C was further introduced into G3DS2to obtain the mutant G4SM1(S62T/S144C/N198D/A217V) with most improved activity and thermostability. The specific activity of G4SM1was2036±45.8U/mg,2.12times wild type, while the Km value (1.84mg/ml) was27.8%lower (P<0.05). Furthermore, the kcat/Km ratio of G4SM1was improved by76%, indicating that the affinity to substrate and catalytic effiency were dramatically enhanced. Three-dimensional structure analysis revealed that the substitutions S62T and S144C, located at the catalytic domain, were responsible for the improved activity and thermostability.4Construction of recombinant yeast containg high-copy G4SM1 The construction of recombinant yeast containg high-copy genes were achieved using two different methods. The YPD plates with high concentrations of zeocin were used to obtain high-zeocin resistant transformants. After electroporation, the transformed cells were spread onto YPD plates supplemented with500,1000,1500and2000μg/ml zeocin. The positive clones of1500and2000μg/ml zeocin plates were picked and subjected to evaluation of gene copy number. Southern blot and qPCR showed that the No.28strain (designated as33-1) was a recombinant yeast containing two-copy integrated G4SM1. It was suggested by both previous study and our result, CBM2of Tfx was non-essential for catalysis. With the context, CBM2was removed from Tfx and the catalytic domain was used for construction of high-copy yeast. A pair of isocaudarners (BamHⅠ and BglⅡ) was employed to establish a shuttle vector pGAPZαA/G4SM1_CBM (4), which contains four tandemly arranged expression cassettes. The resultant plasmid was transformed into P. pastoris SMD1168and the gene copy number of the strain (also named as33-2) was confirmed by Southern blot and qPCR.5Construction of a recombinant yeast co-expressing endo-xylanase and xylosidase, fermentation in flask medium, and characterization of xylan morphology and its hydrolytic productsThe co-expression pladmid was constructed by ligating pGAPZaA/G4SM1_CBM(4) and pPICZaA/xylo using BamHⅠ and BglⅡ. The resultant plasmid was transformed into P. pastoris SMD1168to obtain the co-expression strain33-3. Both33-1and33-2were cultured in YPD medium for120h. The recombinant protein concentrations secredted into supernatant were0.49and0.65g/l. The co-expression strain33-3could either secrete endo-xylanase only in YPD medium or secrete both endo-xylansae and xylosidasae when methanol was supplemented. After incubation for120h, total protein concentration in culture supernatant was1.22g/1/. The kurtosis surface of xylan substrate was observed to be noticeably altered. The untreated birchwood xylan exhibited well-arranged polysaccharide molecules and a sharp kurtosis surface, and polysaccharides were observed to be viscous. After hydrolysis of xylan by endo-xylanase, endo-xylanase broke the ligation between polysaccharides into small particles, the concentrations of xylobiose and xylotriose were0.954±0.093and0.360±0.022mg/ml, or62.6%and23.6%of total XOs. Furthermore, the synergic effects of endo-xylanase and β-xylosidase were capable to convert xylan into xylose. After hydrolysis for24h, the concentration of xylose was1.198±0.016mg/ml,69.1%of total XOs. In summary, endo-xylanase gene tfx was modified using rational and non-rational design. The mutant gene was subjected to construct two high-copy yeasts33-1and33-2, and a co-expression yeast33-3. The proteins concentrations in culture medium of these strains were high, and could be further improved by high-density fermentation in fermenter. The strains are potential candidates for application in feed and biofuel industries. |
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