title: High level expression of organophosphorus hydrolase in Pichia pastoris by multicopy ophcM assembly.
abstract: The residues of organophosphorus pesticides bring serious impact on the environmental safety and people's health. Biodegradation of organophosphorus pesticides is recognized as an ideal method. An organophosphorus hydrolase (OPHCM) from Pseudomonas pseudoalcaligenes was synthesized and expressed in Pichia pastoris. The yield reached approximately 470 mg/l after a 6-d induction in shake flasks. To improve the enzyme production, we describe a novel approach to express OPHCM efficiently with a biobrick assembly method in vitro. Four recombinant plasmids containing 1-4 copies of ophcM-expressing cassettes were constructed and transformed into P. pastoris. Increasing the copy number of ophcM gene enhanced the expression level of OPHCM. The maximum yield and specific activity in P. pastoris harboring two-copy tandem ophcM-expressing cassettes reached 610 mg/l after a 6-d induction in shake flasks and 7.8 g/l in high-density fermentation with specific activity of 13.7 U/mg. The optimum pH and temperature of the recombinant OPHCM activity were 11.0 and 50 °C, respectively. In addition, the enzyme activity of recombinant OPHCM enhanced 57.6% and 30.1% in the presence of 1 mM Cd(2+) and 5% glycerol, respectively. The high expression and good properties of recombinant OPHCM provide an effective solution to solve the pollution of organophosphorus pesticides in the environment. Moreover, the approach for generating multicopy gene expressing vectors here will benefit the study for enhancing the expression level of genes of interest.
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Shen
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, PR China.
authors.forename: Min
authors.initials: M
authors.lastname: Shu
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, PR China.
authors.forename: Lixin
authors.initials: L
authors.lastname: Ma
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, PR China.
authors.forename: Hong
authors.initials: H
authors.lastname: Ni
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, PR China. Electronic address: hon9yan@yeah.net.
authors.forename: Hong
authors.initials: H
authors.lastname: Yan
mesh_terms: D043303:Aryldialkylphosphatase / Q000096:biosynthesis* / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli; D018628:Gene Dosage; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D010843:Pichia; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D043303:Aryldialkylphosphatase
keywords: Biobrick assembly; Multicopy gene expression; Organophosphorus hydrolase; Pichia pastoris