title: Codon optimization significantly improves the expression level of a keratinase gene in Pichia pastoris.
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.
authors.affiliation: Institute of Animal Nutrition, Sichuan Agricultural University, Ya'an, Sichuan, PR China.
authors.forename: Hong
authors.initials: H
authors.lastname: Hu
authors.forename: Jie
authors.initials: J
authors.lastname: Gao
authors.forename: Jun
authors.initials: J
authors.lastname: He
authors.forename: Bing
authors.initials: B
authors.lastname: Yu
authors.forename: Ping
authors.initials: P
authors.lastname: Zheng
authors.forename: Zhiqing
authors.initials: Z
authors.lastname: Huang
authors.forename: Xiangbing
authors.initials: X
authors.lastname: Mao
authors.forename: Jie
authors.initials: J
authors.lastname: Yu
authors.forename: Guoquan
authors.initials: G
authors.lastname: Han
authors.forename: Daiwen
authors.initials: D
authors.lastname: Chen
mesh_terms: D001407:Bacillus / Q000201:enzymology; D003001:Cloning, Molecular; D003062:Codon; D018628:Gene Dosage; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007633:Keratins / Q000378:metabolism; D009154:Mutation; D010447:Peptide Hydrolases / Q000096:biosynthesis / Q000235:genetics*; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D003062:Codon; D011994:Recombinant Proteins; D007633:Keratins; D010447:Peptide Hydrolases; C035855:keratinase