TY - JOUR
T1 - Evolved Peroxygenase-Aryl Alcohol Oxidase Fusions for Self-Sufficient Oxyfunctionalization Reactions
AU - Gomez De Santos, Patricia
AU - Lazaro, Sofia
AU - Vinã-Gonzalez, Javier
AU - Hoang, Manh Dat
AU - Sánchez-Moreno, Israel
AU - Glieder, Anton
AU - Hollmann, Frank
AU - Alcalde, Miguel
PY - 2020/11/20
Y1 - 2020/11/20
N2 - Fungal peroxygenases are deemed emergent biocatalysts for selective C-H bond oxyfunctionalization reactions. In this study, we have engineered a functional and stable self-sufficient chimeric peroxygenase-oxidase fusion. The bifunctional biocatalyst carried a laboratory-evolved version of the fungal peroxygenase fused to an evolved fungal aryl-alcohol oxidase that supplies H2O2 in situ. Enzyme fusion libraries with peptide linkers of different sizes and amino acid compositions were designed, while attached leader sequences favored secretion in yeast. The most promising functional enzyme fusions were characterized biochemically and further tested for the synthesis of dextrorphan, a metabolite of the antitussive drug dextromethorphan. This reaction system was optimized to control the aromatic alcohol transformation rate, and therefore the H2O2 supply, to achieve total turnover numbers of 62,000, the highest value reported for the biocatalytic synthesis of dextrorphan to date. Accordingly, our study opens an avenue for the use of peroxygenase-aryl alcohol oxidase fusions in the pharmaceutical and chemical sectors.
AB - Fungal peroxygenases are deemed emergent biocatalysts for selective C-H bond oxyfunctionalization reactions. In this study, we have engineered a functional and stable self-sufficient chimeric peroxygenase-oxidase fusion. The bifunctional biocatalyst carried a laboratory-evolved version of the fungal peroxygenase fused to an evolved fungal aryl-alcohol oxidase that supplies H2O2 in situ. Enzyme fusion libraries with peptide linkers of different sizes and amino acid compositions were designed, while attached leader sequences favored secretion in yeast. The most promising functional enzyme fusions were characterized biochemically and further tested for the synthesis of dextrorphan, a metabolite of the antitussive drug dextromethorphan. This reaction system was optimized to control the aromatic alcohol transformation rate, and therefore the H2O2 supply, to achieve total turnover numbers of 62,000, the highest value reported for the biocatalytic synthesis of dextrorphan to date. Accordingly, our study opens an avenue for the use of peroxygenase-aryl alcohol oxidase fusions in the pharmaceutical and chemical sectors.
KW - aryl alcohol oxidase
KW - enzyme fusion
KW - human drug metabolites
KW - in situ HOgeneration
KW - unspecific peroxygenase
UR - http://www.scopus.com/inward/record.url?scp=85096917214&partnerID=8YFLogxK
U2 - 10.1021/acscatal.0c03029
DO - 10.1021/acscatal.0c03029
M3 - Article
AN - SCOPUS:85096917214
SN - 2155-5435
VL - 10
SP - 13524
EP - 13534
JO - ACS Catalysis
JF - ACS Catalysis
IS - 22
ER -