Codon optimization of Saccharomyces cerevisiae mating factor alpha prepro-leader to improve recombinant protein production in Pichia pastoris
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To evaluate different codon optimization parameters on the Saccharomyces cerevisiae-derived mating factor α prepro-leader sequence (MFLS) to improve Candida antarctica lipase B (CAL-B) secretory production in Pichia pastoris.
Codon optimization based on the individual codon usage (ICU) and codon context (CC) design parameters enhanced secretory production of CAL-B to 7 U/ml and 12 U/ml, respectively. Only 3 U/ml was obtained with the wild type sequence while the sequence optimized using both ICU and CC objectives showed intermediate performance of 10 U/ml. These results clearly show that CC is the most relevant parameter for the codon optimization of MFLS in P. pastoris, and there is no synergistic effect achieved by considering both ICU and CC together.
The CC optimized MFLS increased secretory protein production of CAL-B in P. pastoris by fourfold.
KeywordsCodon optimization Codon-pair context Individual codon usage Mating factor α prepro-leader sequence Pichia pastoris Saccharomyces cerevisiae Synthetic biology
This work was supported by the National University of Singapore, Biomedical Research Council of A*STAR (Agency for Science, Technology and Research), Singapore and Grants from the Global R&D project program (N011500017), Ministry of Trade, Industry and Energy (MOTIE), Republic of Korea, the Next-Generation BioGreen 21 Program (SSAC, No. PJ01109405), Rural Development Administration, Republic of Korea and a grant from KRIBB Research Initiative Program. The authors would like to thank Dr. Chung for his help in the in silico design of sequences.
Additional information: Calculation of ICU and CC fitness. Supplementary Table 1: Primers used in this study. Supplementary Figure 1: 1st repeated comparison of the secretory CAL-B production using different codon optimized MFLS. Supplementary Figure 2: 2nd repeated comparison of the secretory CAL-B production using different codon optimized MFLS with lipase activity measured at different temperatures.