title: Evaluation of Mut(S) and Mut⁺ Pichia pastoris strains for membrane-bound catechol-O-methyltransferase biosynthesis.
abstract: Catechol-O-methyltransferase (COMT, EC 2.1.1.6) is an enzyme that catalyzes the methylation of catechol substrates, and while structural and functional studies of its membrane-bound isoform (MBCOMT) are still hampered by low recombinant production, Pichia pastoris has been described as an attractive host for the production of correctly folded and inserted membrane proteins. Hence, in this work, MBCOMT biosynthesis was developed using P. pastoris X33 and KM71H cells in shake flasks containing a semidefined medium with different methanol concentrations. Moreover, after P. pastoris glass beads lysis, biologically and immunologically active hMBCOMT was found mainly in the solubilized membrane fraction whose kinetic parameters were identical to its correspondent native enzyme. In addition, mixed feeds of methanol and glycerol or sorbitol were also employed, and its levels quantified using liquid chromatography coupled to refractive index detection. Overall, for the first time, two P. pastoris strains with opposite phenotypes were applied for MBCOMT biosynthesis under the control of the strongly methanol-inducible alcohol oxidase (AOX) promoter. Moreover, this eukaryotic system seems to be a promising approach to deliver MBCOMT in high quantities from fermentor cultures with a lower cost-benefit due to the cheaper cultivation media coupled with the higher titers tipically achieved in biorreactors, when compared with previously reported mammallian cell cultures.
authors.affiliation: CICS-UBI-Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6201-001, Covilhã, Portugal.
authors.forename: A Q
authors.initials: AQ
authors.lastname: Pedro
authors.forename: D
authors.initials: D
authors.lastname: Oppolzer
authors.forename: M J
authors.initials: MJ
authors.lastname: Bonifácio
authors.forename: C J
authors.initials: CJ
authors.lastname: Maia
authors.forename: J A
authors.initials: JA
authors.lastname: Queiroz
authors.forename: L A
authors.initials: LA
authors.lastname: Passarinha
mesh_terms: D000429:Alcohol Oxidoreductases / Q000378:metabolism; D002394:Catechol O-Methyltransferase / Q000096:biosynthesis* / Q000235:genetics; D018929:Cell Culture Techniques; D005285:Fermentation; D005990:Glycerol / Q000737:chemistry; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000378:metabolism; D000432:Methanol / Q000378:metabolism; D010641:Phenotype; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D008565:Membrane Proteins; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C029293:methanol oxidase; D002394:Catechol O-Methyltransferase; D005990:Glycerol; D000432:Methanol