Abstract
BACKGROUND: Variations in codon usage between species are one of the major causes affecting recombinant protein expression levels, with a significant impact on the economy of industrial enzyme production processes. The use of codon-optimized genes may overcome this problem. However, designing a gene for optimal expression requires choosing from a vast number of possible DNA sequences and different codon optimization methods have been used in the past decade. Here, a comparative study of the two most common methods is presented using calf prochymosin as a model. RESULTS: Seven sequences encoding calf prochymosin have been designed, two using the "one amino acid-one codon" method and five using a "codon randomization" strategy. When expressed in Escherichia coli, the variants optimized by the codon randomization approach produced significantly more proteins than the native sequence including one gene that produced an increase of 70% in the amount of prochymosin accumulated. On the other hand, no significant improvement in protein expression was observed for the variants designed with the one amino acid-one codon method. The use of codon-optimized sequences did not affect the quality of the recovered inclusion bodies. CONCLUSIONS: The results obtained in this study indicate that the codon randomization method is a superior strategy for codon optimization. A significant improvement in protein expression was obtained for the largely established process of chymosin production, showing the power of this strategy to reduce production costs of industrial enzymes in microbial hosts.