title: Potential of Pichia pastoris for the production of industrial penicillin G acylase.
abstract: This study deals with the potential of Pichia pastoris X-33 for the production of penicillin G acylase (PGAA) from Achromobacter sp. CCM 4824. Synthetic gene matching the codon usage of P. pastoris was designed for intracellular and secretion-based production strategies and cloned into vectors pPICZ and pPICZα under the control of AOX1 promoter. The simple method was developed to screen Pichia transformants with the intracellularly produced enzyme. The positive correlation between acylase production and pga gene dosage for both expression systems was demonstrated in small scale experiments. In fed-batch bioreactor cultures of X-33/PENS2, an extracellular expression system, total PGAAexpressed from five copies reached 14,880 U/L of an active enzyme after 142 h; however, 60% of this amount retained in the cytosol. The maximum PGAAproduction of 31,000 U/L was achieved intracellularly from nine integrated gene copies of X-33/PINS2 after 90 h under methanol induction. The results indicate that in both expression systems the production level of PGAAis similar but there is a limitation in secretion efficiency.
authors.affiliation: Institute of Microbiology of the CAS, v.v.i, Vídeňská 1083, 14220, Prague 4, Czech Republic.
authors.forename: Helena
authors.initials: H
authors.lastname: Marešová
authors.affiliation: Institute of Microbiology of the CAS, v.v.i, Vídeňská 1083, 14220, Prague 4, Czech Republic. palyzova@biomed.cas.cz.
authors.forename: Andrea
authors.initials: A
authors.lastname: Palyzová
authors.affiliation: Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Viničná 5, 12840, Prague 2, Czech Republic.
authors.forename: Martina
authors.initials: M
authors.lastname: Plačková
authors.affiliation: Institute of Microbiology of the CAS, v.v.i, Vídeňská 1083, 14220, Prague 4, Czech Republic.
authors.forename: Michal
authors.initials: M
authors.lastname: Grulich
authors.affiliation: Fermenta Biotech Ltd., Thane, India.
authors.forename: Vyasa Williams
authors.initials: VW
authors.lastname: Rajasekar
authors.affiliation: Institute of Microbiology of the CAS, v.v.i, Vídeňská 1083, 14220, Prague 4, Czech Republic.
authors.forename: Václav
authors.initials: V
authors.lastname: Štěpánek
authors.affiliation: Institute of Microbiology of the CAS, v.v.i, Vídeňská 1083, 14220, Prague 4, Czech Republic.
authors.forename: Eva
authors.initials: E
authors.lastname: Kyslíková
authors.affiliation: Institute of Microbiology of the CAS, v.v.i, Vídeňská 1083, 14220, Prague 4, Czech Republic.
authors.forename: Pavel
authors.initials: P
authors.lastname: Kyslík
mesh_terms: D042402:Achromobacter / Q000235:genetics / Q000378:metabolism; D019149:Bioreactors / Q000382:microbiology; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D018628:Gene Dosage; D015870:Gene Expression; D005822:Genetic Vectors; D007218:Industrial Microbiology / Q000379:methods*; D010399:Penicillin Amidase / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic
chemical_list: D003062:Codon; D011994:Recombinant Proteins; D010399:Penicillin Amidase