Characterization of alkylguaiacol-degrading cytochromes P450 for the biocatalytic valorization of lignin

Abstract

Cytochrome P450 enzymes have tremendous potential as industrial biocatalysts, including in biological lignin valorization. Here, we describe P450s that catalyze theO-demethylation of lignin-derived guaiacols with different ring substitution patterns. Bacterial strainsRhodococcus rhodochrousEP4 andRhodococcus jostiiRHA1 both utilized alkylguaiacols as sole growth substrates. Transcriptomics of EP4 grown on 4-propylguaiacol (4PG) revealed the up-regulation ofagcA, encoding a CYP255A1 family P450, and theaphgenes, previously shown to encode ameta-cleavage pathway responsible for 4-alkylphenol catabolism. The function of the homologous pathway in RHA1 was confirmed: Deletion mutants ofagcAandaphC, encoding themeta-cleavage alkylcatechol dioxygenase, grew on guaiacol but not 4PG. By contrast, deletion mutants ofgcoAandpcaL, encoding a CYP255A2 family P450 and anortho-cleavage pathway enzyme, respectively, grew on 4-propylguaiacol but not guaiacol. CYP255A1 from EP4 catalyzed theO-demethylation of 4-alkylguaiacols to 4-alkylcatechols with the following apparent specificities (kcat/KM): propyl > ethyl > methyl > guaiacol. This order largely reflected AgcA’s binding affinities for the different guaiacols and was the inverse of GcoAEP4’s specificities. The biocatalytic potential of AgcA was demonstrated by the ability of EP4 to grow on lignin-derived products obtained from the reductive catalytic fractionation of corn stover, depleting alkylguaiacols and alkylphenols. By identifying related P450s with complementary specificities for lignin-relevant guaiacols, this study facilitates the design of these enzymes for biocatalytic applications. We further demonstrated that the metabolic fate of the guaiacol depends on its substitution pattern, a finding that has significant implications for engineering biocatalysts to valorize lignin.