Recombinant Expression And Fermentation Process Of Recombinant Human Hyaluronidase In Pichia Pastoris

Hyaluronidase（HAase）is a kind of glycosaminoglycan degrading enzyme,which mainly degrades hyaluronic acid（HA）and is widely used in medicine,chemical industry and other fields.Recombinant human hyaluronidase PH-20（rh PH-20）has been widely studied in recent years and is thought to be a safer alternative to animal tissue derived ones.In medicine,commercialized rh PH-20 have been used for the delivery of a variety of injectable drugs to facilitate drug diffusion in the body by degrading HA in the extracellular matrix.At present,all commercially available rh PH-20 preparations are produced by animal cells such as CHO cells,whose cost is higher than that of microbial fermentation.In recent years,rh PH-20 has been expressed in Pichia pastoris,but no optimization of rh PH-20 secretion has been reported.Therefore,in this study,the expression of rh PH-20 in P.pastoris was systematically optimized through the construction of expression vectors,the optimization of expression elements,and the enhancement of secreted expression pathway,and the optimization of culture conditions and the scale-up fermentation of the optimized recombinant strain were explored.The main research results of this paper are as follows:（1）Recombinant expression and cassette optimization of rh PH-20.The Homo sapiens PH-20 spermatoacrosomal hyaluronidase was analyzed using SMART database,Signal P5.0 and TMHMM2.0 webserver.rh PH-20-encoding gene with removed signal peptide and membrane anchoring region was synthesized.The recombinant strain Pichia pastoris GS115/p PICZα-rh PH20 was constructed.The secreted enzyme activity was 1.02 U·m L^-1.On this basis,the recombinant expression cassette was modified by promoter adaptation,and the constitutive promoter P_GCW14 was selected.The enzyme activity of the corresponding recombinant strain Pichia pastoris GS115/p GCZα-rh PH20 was increased to 1.91 U·m L^-1.（2）Optimization and engineering of protein translocation.Firstly,the modification of rh PH-20 was performed by replacing MF-αsignal peptide with ost1-proαsignal peptide,which mediated protein into the cotranslational translocation pathway,and the secretion of rh PH-20was increased by 20%.The cotranslational translocation pathway of recombinant P.pastoris was further enhanced.By separately overexpressing key proteins in this pathway,it was found that single overexpression of Srp54p subunits of signal recognition particles increased secreted expression enzyme activity by 48%.The secreted expression enzyme activity of recombinant strain OE54 increased to 3.14 U·m L^-1 after the two-step modification.（3）Overexpression of Hac1p,the transcription factor of unfolded protein response.On the basis of the previous modification,we further overexpressed the transcription factor of unfolded protein response,HAC1 from Pichia pastoris,Saccharomyces cerevisiae and human to enhance the post-translational processing of the recombinant protein in the endoplasmic reticulum,and found that the overexpression of Sc HAC1 from saccharomyces cerevisiae significantly improved the protein secretion and expression.The secretory efficiency of recombinant strain OE54-Sc HAC1 increased by 32%,reaching 4.06 U·m L^-1.The recombinant bacteria were characterized before and after overexpression of Sc HAC1,and it was found that several molecular chaperone genes were significantly up-regulated,and genes related to endoplasmic reticulum stress,cell stress and oxidative stress were down-regulated.At the same time,the down-regulation of intracellular oxygen free radical（ROS）levels after overexpression of Sc HAC1 was found,and the negative effect of ROS on the active secretion and expression of rh PH-20 was demonstrated by the backfilling experiment.（4）Fermentation optimization and scale-up fermentation.Through the optimization of carbon source concentration,it was found that the accumulation and secreted expression enzyme activity of strain increased with the glycerol concentration increasing in the range of10-40 g·L^-1.By optimizing initial p H,it was found that the enzyme production efficiency of recombinant strain was the highest at p H 6.0.By optimizing the culture temperature,it was found that reducing the culture temperature of recombinant bacteria to 20°C was beneficial to the secretion of recombinant protein.At the same time,the optimal filling volume of the medium was determined to be 30 m L and the inoculation volume was 10%by shaking flask optimization.The optimized medium was used for batch fermentation of the recombinant bacteria on the tank,with the highest enzyme activity of 8.42 U·m L^-1.Finally,the yield of rh PH-20 was increased to 19.82 U·m L^-1 by fed-batch fermentation.