Study On The High-efficiency Expression And Designation Of Chymosin Gene Of Bos Taurus

Chymosin is a general term for a group of enzymes that can coagulate emulsions and is a key type of enzyme in cheese processing.With the increasing demand for chymosin,traditional animal-derived chymosin can no longer satisfy milk Development of the product industry.The rennet producing strain obtained by genetic engineering technology has the advantages of high efficiency,high yield and economy,and is widely used in the production of chymosin,and it is expected to solve the problem of rennet shortage.In this paper,bovine taurus prorennin gene is used to improve the expression standard of recombinant bovine chymosin in P.pastoris by strategies such as codon optimization,artificial targeted modification,and increasing gene dosage.High-yield recombinant engineering bacteria,laying the foundation for the industrialization of rennet.There are main contents of this study:(1)Based on the codon preference of Pichia pastoris,codon optimization of the bovine rennet gene pchy was performed to obtain the synthetic gene pchyO.Heterologous expression of pchyO in Pichia X-33 was achieved;In order to explore the effect of the prorennin leading peptide on its protein function,this study obtained the gene chyO without the leader peptide sequence by PCR technology,and introduced the renin gene chyO into yeast.Result:The prorennin gene pchyO was expressed and the enzyme activity was detected in Pichia pastoris,the enzyme activity was 1.5 SU/mL.The chymosin gene chyO gene lacking the leader peptide was not expressed in Pichia pastoris,which indicated that the lead peptide is essential to the expression of its protein in P.pastoris.(2)This study further carried out site-directed mutagenesis and codon optimization of three amino acids in the original rennet through molecular modification.The designed gene pchy2aO was obtained by artificial synthesis,which realized the high efficient expression of gene pchy2aO in Pichia pastoris.(3)This study successfully constructed a multicopy tandem expression cassette of gene pchy2aO.The results showed that the single copy strain P.pastoris/pchy2aO-6 had an enzyme activity of 2000 SU/mL at the shake flask level;this study successfully constructed For three or four copies of the engineered bacteria of the pchy2aO gene,the enzyme activity of the four copy strain is twice that of the single copy enzyme activity,and the enzyme activity of the four copy strain P.pastoris/pchy2aO-Q2 is 4000 SU/mL.(4)In this study,the production intensity of recombinant strain P.pastoris/pchy2aO-Q2 was evaluated at the level of 50-L bioreactor.The results showed that in the bioreactor,the enzyme activity reached 40,000 SU/mL,which was the level of shake flask fermentation about 10 times.In this study,the artificial design of the calf rennet gene was successfully realized in yeast and its high expression in yeast was successfully achieved.The recombinant strain P.pastoris/pchy2aO-Q2 with high yield and high activity was screened and its production strength meets the industrial production requirements of enzyme preparations,which has the potential for large-scale industrial production.