The Study On High-level Expression Of XYL1in Pichia Pastoris Based On Industrial Production

Xylanase could specific hydrolysis xylan to produce xylo-oligosaccharides and xylose,which showed upstanding application prospect in field of pulping and papermaking, feedindustry, food industry, and Brewing industry, and also provided newly solving strategy toenergy shortage and environmental pollution. Xylanase was widely derived from prokaryoticmicroorganisms including Bacillus subtilis, class of Bacillus, Bacillus pumilus, and fungus suchas Aspergillus niger, Trichodermareesei, Penicillium, Streptomyces. There existed a bottleneckthat the productions of xylanase in parent microorganisms were too low to satisfy therequirement of industry, thus limiting the application of xylanase. According to the Pichiapastoris favored codons, this study designed and artificially synthesized the cDNA fragmentencoding the mature peptide of XYL1（Xlyanase1） from Streotomyces sp.S38. The host X33andthe GAP promoter were selected to construct the constitutive expression strain of XYL1. Therecombinant plasmid pGAPZ A-XYL1was integrated into X33genome DNA byelectroporation. The recombinant strain was transfered of culture stably without obvioussignificant gene loss in10generation, thus satisfied the demand of fed-batch fermentation. Theproduction of XYL1was215±10IU/mLmL in flask fermentation.The recombianat XYL1was purified through5kDa packge concentration, ion exehnagechromatography Q-Sepharose FF, and molecular sieve Sephadex G-25, with an estimating puritygreater than90%by SDS-PAGE. The specific activity of xylanase was improved from796.80IU/mg to1328.69IU/mg after purification. PMF analysis shown that the purified XYL1matchedto the β^-1,4endo xylanase1（No. X98518.1） from Streptomyces sp. S38. The recombinant XYL1presented satisfactory enzymatic properties including good thermal stability and wide pHadaptability, which was better than its parental molecule. The optimal pH and temperature forrecombinant XYL1were6.0and55oC, respectively. The catalysis reaction of recombinantXYL1was accorded with the Michaelis-Menton equation, and the Km and Vmax values ofXYL1were2.0mg/mL and5000μmol min^-1mg^-1, respectively.Glucose was selected as the carbon source for the fermentation of X33/pGAPZ A-XYL1,with an optimal initial concentration of30g/L. The continuous fermentation of X33/pGAPZ A-XYL1was performed in5L fermentor. The fermentation of recombinant XYL1get into stable state in the fifth day,with the steady wet cell weight （WCW） of400g/L and the volumetric cellproductivity （DCx） was8.14g/L.h. The expression level of XYL1was maintained at1100IU/mL, and the volumetric product productivity （DCp） was23028IU/L.h. The concentration ofresidual sugar in fermentation borth was kept constantly at a low level of2.3g/L in thecontinuous fermentation process. The continuous fermentation lasted for17days, with thedilution rate （D）of0.48D^-1. Finally, the production yield of continuous fermentation was1.66fold of fed-batch fermentation.The engineering strain X33/pGAPZ A-XYL1displayed genetic stability in thefermentation, as no obvious gene lost was detected. Meanwhile, significant degration of XYL1did not observed in fermentation, resulting from short-term retention of the dosage liquor.Relevant kinetics parameters of fermentation were obtained in the chemostatic culture of X33/pGAPZ A-XYL1in5L fermentor with glucose as the restrictive substrate. The relationshipbetween specific cell growth rate （μ） and substrate concentration （Cs） was according with theMonod equation. The yeast growth kinetics was coincide with the equation, μ=0.35Cs/（0.38+Cs）, hence μmax=0.35h^-1and Ks=0.38g·L^-1. The specific cell growth rate （μ） and specificproduct formation rate （qp） was according with the equation qp=-0.0529μ²+0.0168μ-0.0003（0.024≤μ≤0.212）. The substrate consumption kinetics model equation was qs=1.6025μ+0.0277and the theoretical maximum biomass yield coefficient （YG） was0.624g/g. The theoreticaloptimal dilution rate was0.316h^-1, which was from the study on the relationship of specificgrowth rate and volumetric cell productivity （DCx）, and the estimating the maximum volumetriccell productivity （DCx）mwas7.509g/L·h. The relationship of specific growth rate and productformation was also studied, which was similar to the relationship of specific cell growth rate （μ）and specific product formation rate （qp）, and the peak value was μ=0.156h^-1.We also compaired the influence of different fed-batch mode, including intermissionfed-batch, constant velocity fed-batch and exponential fed-batch in50L fermentor, on thegrowth of Pichia pastoris X33/pGAPZ A-XYL1and the production of recombinant XYL1,which was evaluated by kinetic parameters such as products yield coefficient （Yp/s）, biomassyield coefficient（YX/S）, specific product formation rate （q_p）. Compaired with intermissionfed-batch and constant velocity fed-batch, the exponential fed-batch was the effective fed-batchmode for the high density fermentation of X33/pGAPZ A-XYL1. Based on the exponential fed-batch, we also used the fed-batch controlled in stages strategy in the50-L fermentation.According to the combination fermentation strategy above, the wet cell weight was415gram perliter borth, and the activity of xylanase was2788IU/mL. Finally, three batch of10m³scalefermentation were carried out, using the fed-batch controlled in stages strategy. Compared to the50L scale fermentation, the yield of XYL1in10m³scale fermentation was increased by23%.The fermentation process was stable and the highest wet cell weight and xylanase activity in thefermentation borth was486g/L and3420IU/mL respectively.