Codon optimization of the Bos taurus Chymosin gene (CYM) for its expression in Pichia pastoris was performed in this study. A synthetic CYM gene was designed in silico by replacing codons rarely used by P. pastoris with equivalent nucleotide combinations that codify for the same amino acid but that are more frequently encountered in the genome of P. pastoris. A total of 332 nucleotides were modified to optimize 289 codons. The synthetic CYM gene was cloned into the expression vector pPICZαA and transformed into P. pastoris. The transformed strains were grown in artificial media supplemented with glycerol as a carbon source to increase biomass and then cultured in a similar medium replacing glycerol with methanol as a carbon source to initiate gene induction. Raw extracts of the growth media exhibited milk-clotting activity of 146.11 SU/mL. Produced recombinant chymosin showed coagulant activity from 25 to 50 °C, and within a pH range of 5-6.9, having optimum activity at 35-40 °C, and pH 5.0. These results show that codon optimization is a viable strategy to improve CYM gene expression levels in P. pastoris for the production of recombinant chymosin.
Keywords: Chymosin; Codon optimization; Pichia pastoris; Prochymosin; Recombinant protein.