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Evaluation of MutS and Mut+ Pichia pastoris Strains for Membrane-Bound Catechol-O-Methyltransferase Biosynthesis

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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.

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Abbreviations

AOX:

Alcohol oxidase

COMT:

Catechol-O-methyltransferase

E. coli :

Escherichia coli

DNA:

Deoxyribonucleic acid

gDNA:

Genomic DNA

HPLC:

High-peformance liquid chromatography

LLOQ:

Lower limit of quantification

MBCOMT:

Membrane-bound catechol-O-methyltransferase

MP:

Membrane protein

Mut:

Methanol utilization

MutS :

Methanol utilization slow

Mut+ :

Methanol utilization plus

OD600 :

Optical density 600 nm

PCR:

Polymerase chain reaction

P. pastoris :

Pichia pastoris

RID:

Refractive index detection

SAM:

S-adenosyl-L-methionine

SCOMT:

Soluble catechol-O-methyltransferase

SDS-PAGE:

Reducing sodium dodecyl sulfate polyacrylamide

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Acknowledgments

A.Q. Pedro acknowledges a doctoral fellowship (SFRH/BD/81222/2011) from Fundação para a Ciência e Tecnologia and D. Oppolzer acknowledges a fellowship (CENTRO-07-ST24_FEDER-002014-TPCR-2-004) from Programa “Mais Centro” within the scope of QREN–POPH–Advanced Formation programs cofunded by Fundo Social Europeu and MEC. This work was partially funded by Fundação para a Ciência e Tecnologia I.P. (PIDDAC) and Fundo Europeu de Desenvolvimento Regional-FEDER funds through Programa Operacional Factores de Competitividade (POFC)–COMPETE: FCOMP-01-0124-FEDER-027563 and by National Funds through FCT–Fundação para a Ciência e Tecnologia within the scope of Project “EXPL/BBB478/BQB/0960/2012.”

Conflict of Interest

All authors declare they do not have any conflict of interest.

Compliance with Ethical Standards

In this work, no studies involving human participants or animals were carried out.

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Authors and Affiliations

  1. CICS-UBI–Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6201-001, Covilhã, Portugal

    A. Q. Pedro, D. Oppolzer, C. J. Maia, J. A. Queiroz & L. A. Passarinha

  2. Bial–Departamento de Investigação e Desenvolvimento, 4745-457, São Mamede do Coronado, Portugal

    M. J. Bonifácio

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  1. A. Q. Pedro
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  2. D. Oppolzer
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Correspondence to L. A. Passarinha.

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Pedro, A.Q., Oppolzer, D., Bonifácio, M.J. et al. Evaluation of MutS and Mut+ Pichia pastoris Strains for Membrane-Bound Catechol-O-Methyltransferase Biosynthesis. Appl Biochem Biotechnol 175, 3840–3855 (2015). https://doi.org/10.1007/s12010-015-1551-0

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