Optimization of calf rennin gene and construction of gene construct for the recombinant chymosin production in Pichia pastoris

Animal chymosin due to the high quality of cheese texture and flavor is the most effective enzyme for cheese production. Due to the increasing demand for this enzyme, in addition to the plant and microbial sources, recombinant sources should be considered for chymosin production. In this study, in order to produce recombinant chymosin, as well as improving the expression of bovine chymosin gene in yeast, the sequence of the chymosin A gene was optimized and synthesized based on the codon usage of the Pichia pastoris and was cloned in an appropriate expression vector. The codon adaptation index (CAI) increased from 0.59 to 0.72 after codon optimization. There were 41 yeast rare codons (with frequency of less than 10%) in the bovine chymosin A gene, whereas no rare codons in the codon optimized sequence. In order to transfer the resynthesized chymosin A gene to the yeast expression vector pPIC9, this gene was proliferated using the specific primers and was cloned in the PTG-19 vector. Recombinant (PTG19-chymA) colonies were selected by the screening of white-blue colonies method. The resynthesized chymosin A gene was then cut from PTG19-chymA vector using the restriction enzymes Not I and EcoR I, cloned into the pPIC9 vector and was transformed into DH5α strain of E. coli. Colonies containing recombinant vector (pPIC9-chymA) were identified and selected using colony-PCR technique. The recombinant nature and correct insertion of resynthesized chymosin A gene in the yeast expression vector pPIC9 were confirmed by plasmid extraction and its digestion with BamH I enzyme.