Codon optimization significantly improves the expression level of a keratinase gene in Pichia pastoris

PLoS One. 2013;8(3):e58393. doi: 10.1371/journal.pone.0058393. Epub 2013 Mar 5.

Abstract

The main keratinase (kerA) gene from the Bacillus licheniformis S90 was optimized by two codon optimization strategies and expressed in Pichia pastoris in order to improve the enzyme production compared to the preparations with the native kerA gene. The results showed that the corresponding mutations (synonymous codons) according to the codon bias in Pichia pastoris were successfully introduced into keratinase gene. The highest keratinase activity produced by P. pastoris pPICZαA-kerAwt, pPICZαA-kerAopti1 and pPICZαA-kerAopti2 was 195 U/ml, 324 U/ml and 293 U/ml respectively. In addition, there was no significant difference in biomass concentration, target gene copy numbers and relative mRNA expression levels of every positive strain. The molecular weight of keratinase secreted by recombinant P. pastori was approx. 39 kDa. It was optimally active at pH 7.5 and 50°C. The recombinant keratinase could efficiently degrade both α-keratin (keratin azure) and β-keratin (chicken feather meal). These properties make the P. pastoris pPICZαA-kerAopti1 a suitable candidate for industrial production of keratinases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacillus / enzymology
  • Cloning, Molecular
  • Codon*
  • Gene Dosage
  • Gene Expression Regulation, Enzymologic*
  • Gene Expression Regulation, Fungal*
  • Glycosylation
  • Hydrogen-Ion Concentration
  • Hydrolysis
  • Keratins / metabolism
  • Mutation
  • Peptide Hydrolases / biosynthesis
  • Peptide Hydrolases / genetics*
  • Pichia / metabolism*
  • Plasmids / metabolism
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Substrate Specificity
  • Temperature

Substances

  • Codon
  • Recombinant Proteins
  • Keratins
  • Peptide Hydrolases
  • keratinase

Grants and funding

This research was granted by Feed Biotechnology Project of Sichuan Province of China with grant number 2010GZ0193. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.