Functional assembly of camphor converting two-component Baeyer–Villiger monooxygenases with a flavin reductase from E. coli

The major limitation in the synthetic application of two-component Baeyer–Villiger monooxygenases was addressed by identifying the 28-kDa flavin-reductase Fre from Escherichia coli as a suitable supplier of reduced FMN for these enzymes. Coexpression of Fre with either 2,5- or 3,6-diketocamphane mon...

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Published in:Applied microbiology and biotechnology 2014-05, Vol.98 (9), p.3975-3986
Main Authors: Kadow, Maria, Balke, Kathleen, Willetts, Andrew, Bornscheuer, Uwe T, Bäckvall, J.-E
Format: Article
Language:English
Subjects:
Analysis
Baeyer-Villiger monooxygenases
Biocatalysts
Biochemistry
Biomedical and Life Sciences
Biosynthesis
Biotechnologically Relevant Enzymes and Proteins
Biotechnology
Camphor
Camphor - metabolism
Catalysis
Coenzymes - metabolism
E coli
Enzymes
Escherichia coli
Escherichia coli - enzymology
Escherichia coli - metabolism
Escherichia coli Proteins - genetics
Escherichia coli Proteins - metabolism
Flavin Mononucleotide - metabolism
Flavin reductase
FMN Reductase - genetics
FMN Reductase - metabolism
Fre
Fusion protein
Gene Expression
Genes
ketones
Life Sciences
Microbial Genetics and Genomics
Microbiology
Mixed Function Oxygenases - genetics
Mixed Function Oxygenases - metabolism
Multi-component flavin-dependent monooxygenases
open reading frames
Organic chemistry
Oxidases
Physiological aspects
Polypeptides
Proteins
Pseudomonas putida
Pseudomonas putida - enzymology
Pseudomonas putida - metabolism
Pseudomonas putida NCIMB10007
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
Studies
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description The major limitation in the synthetic application of two-component Baeyer–Villiger monooxygenases was addressed by identifying the 28-kDa flavin-reductase Fre from Escherichia coli as a suitable supplier of reduced FMN for these enzymes. Coexpression of Fre with either 2,5- or 3,6-diketocamphane monooxygenase from Pseudomonas putida NCIMB 10007 significantly enhanced the conversion of camphor and norcamphor serving as representative ketones. With purified enzymes, full conversion was achieved, while only slight amounts of product were formed in the absence of this flavin reductase. Fusion of the genes of Fre and DKCMOs into single open reading frame constructs resulted in unstable proteins exhibiting flavin reducing, but poor oxygenating activity, which led to overall decreased conversion of camphor.
doi_str_mv 10.1007/s00253-013-5338-3
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Coexpression of Fre with either 2,5- or 3,6-diketocamphane monooxygenase from Pseudomonas putida NCIMB 10007 significantly enhanced the conversion of camphor and norcamphor serving as representative ketones. With purified enzymes, full conversion was achieved, while only slight amounts of product were formed in the absence of this flavin reductase. 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Fusion of the genes of Fre and DKCMOs into single open reading frame constructs resulted in unstable proteins exhibiting flavin reducing, but poor oxygenating activity, which led to overall decreased conversion of camphor.</description><subject>Analysis</subject><subject>Baeyer-Villiger monooxygenases</subject><subject>Biocatalysts</subject><subject>Biochemistry</subject><subject>Biomedical and Life Sciences</subject><subject>Biosynthesis</subject><subject>Biotechnologically Relevant Enzymes and Proteins</subject><subject>Biotechnology</subject><subject>Camphor</subject><subject>Camphor - metabolism</subject><subject>Catalysis</subject><subject>Coenzymes - metabolism</subject><subject>E coli</subject><subject>Enzymes</subject><subject>Escherichia coli</subject><subject>Escherichia coli - enzymology</subject><subject>Escherichia coli - metabolism</subject><subject>Escherichia coli Proteins - genetics</subject><subject>Escherichia coli Proteins - metabolism</subject><subject>Flavin Mononucleotide - metabolism</subject><subject>Flavin reductase</subject><subject>FMN Reductase - genetics</subject><subject>FMN Reductase - metabolism</subject><subject>Fre</subject><subject>Fusion protein</subject><subject>Gene Expression</subject><subject>Genes</subject><subject>ketones</subject><subject>Life Sciences</subject><subject>Microbial Genetics and Genomics</subject><subject>Microbiology</subject><subject>Mixed Function Oxygenases - genetics</subject><subject>Mixed Function Oxygenases - metabolism</subject><subject>Multi-component flavin-dependent monooxygenases</subject><subject>open reading frames</subject><subject>Organic chemistry</subject><subject>Oxidases</subject><subject>Physiological aspects</subject><subject>Polypeptides</subject><subject>Proteins</subject><subject>Pseudomonas putida</subject><subject>Pseudomonas putida - enzymology</subject><subject>Pseudomonas putida - metabolism</subject><subject>Pseudomonas putida NCIMB10007</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Studies</subject><issn>0175-7598</issn><issn>1432-0614</issn><issn>1432-0614</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2014</creationdate><recordtype>article</recordtype><sourceid>M0C</sourceid><recordid>eNqNkstu1DAUhiMEokPhAdiAJTYgkeH4kouXQy9QqRISpd1aTmKnrhJ7aiedzqIS78Ab8iR4lKEwCCHkhSWf7__lc86fJM8xzDFA8S4AkIymgGmaUVqm9EEyw4ySFHLMHiYzwEWWFhkv95InIVwBYFLm-eNkjzDMgfFyltwdj7YejLOyQzIE1VfdGjmNatkvL51HtbM3yg_GtmhYubR2_dJZZQf0Xqq18t-_frswXWda5VHvrHO361ZZGVRAKzNcIol0J2-MRV41Yz3EAtLe9ehoHp078zR5pGUX1LPtvZ-cHx99OfiYnn76cHKwOE3rnBVDyutM8rJpNJSMAqVFXTQ4qwCDxpUmFa140_CSZ1KxBuMmb4jOteacZ3mZlZzuJ28n37BSy7ESS2966dfCSSMOzcVCON-KMAoMjGGI-OsJX3p3PaowiN6EWnWdtMqNQeCMAC14Qcr_QHFeEJrzjeurP9ArN_o4-IkCRhktflGt7JQwVrvBy3pjKha0iP-jPNtQ879Q8TSqN3FnSpv4viN4syOIzKBuh1aOIYiTs8-7LJ7Y2rsQvNL388IgNsETU_BEDJ7YBE_QqHmxbW6setXcK34mLQJku4JYsjEvv3X_D9eXk0hLJ2TrTRDnZwQwA4C4WcLpD_YL61c</recordid><startdate>20140501</startdate><enddate>20140501</enddate><creator>Kadow, Maria</creator><creator>Balke, Kathleen</creator><creator>Willetts, Andrew</creator><creator>Bornscheuer, Uwe T</creator><creator>Bäckvall, J.-E</creator><general>Springer-Verlag</general><general>Springer Berlin Heidelberg</general><general>Springer</general><general>Springer Nature B.V</general><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>ISR</scope><scope>3V.</scope><scope>7QL</scope><scope>7T7</scope><scope>7WY</scope><scope>7WZ</scope><scope>7X7</scope><scope>7XB</scope><scope>87Z</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8FL</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BEZIV</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FRNLG</scope><scope>FYUFA</scope><scope>F~G</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K60</scope><scope>K6~</scope><scope>K9.</scope><scope>L.-</scope><scope>LK8</scope><scope>M0C</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PQBIZ</scope><scope>PQBZA</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>7X8</scope><scope>7QO</scope><scope>ABAVF</scope><scope>ADTPV</scope><scope>AOWAS</scope><scope>D8T</scope><scope>DG7</scope><scope>ZZAVC</scope></search><sort><creationdate>20140501</creationdate><title>Functional assembly of camphor converting two-component Baeyer–Villiger monooxygenases with a flavin reductase from E. coli</title><author>Kadow, Maria ; 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subjects Analysis
Baeyer-Villiger monooxygenases
Biocatalysts
Biochemistry
Biomedical and Life Sciences
Biosynthesis
Biotechnologically Relevant Enzymes and Proteins
Biotechnology
Camphor
Camphor - metabolism
Catalysis
Coenzymes - metabolism
E coli
Enzymes
Escherichia coli
Escherichia coli - enzymology
Escherichia coli - metabolism
Escherichia coli Proteins - genetics
Escherichia coli Proteins - metabolism
Flavin Mononucleotide - metabolism
Flavin reductase
FMN Reductase - genetics
FMN Reductase - metabolism
Fre
Fusion protein
Gene Expression
Genes
ketones
Life Sciences
Microbial Genetics and Genomics
Microbiology
Mixed Function Oxygenases - genetics
Mixed Function Oxygenases - metabolism
Multi-component flavin-dependent monooxygenases
open reading frames
Organic chemistry
Oxidases
Physiological aspects
Polypeptides
Proteins
Pseudomonas putida
Pseudomonas putida - enzymology
Pseudomonas putida - metabolism
Pseudomonas putida NCIMB10007
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
Studies
title Functional assembly of camphor converting two-component Baeyer–Villiger monooxygenases with a flavin reductase from E. coli
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