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 the O-demethylation of lignin-derived guaiacols with different ring substitution patterns. Bacterial strains Rhodococcus rhodochrous EP4 and Rhodococcus jostii RHA1 both utilized alkylguaiacols as sole growth substrates. Transcriptomics of EP4 grown on 4-propylguaiacol (4PG) revealed the up-regulation of agcA, encoding a CYP255A1 family P450, and the aph genes, previously shown to encode a meta-cleavage pathway responsible for 4-alkylphenol catabolism. The function of the homologous pathway in RHA1 was confirmed: Deletion mutants of agcA and aphC, encoding the meta-cleavage alkylcatechol dioxygenase, grew on guaiacol but not 4PG. By contrast, deletion mutants of gcoA and pcaL, encoding a CYP255A2 family P450 and an ortho-cleavage pathway enzyme, respectively, grew on 4-propylguaiacol but not guaiacol. CYP255A1 from EP4 catalyzed the O-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.

Engineering a Cytochrome P450 for Demethylation of Lignin-Derived Aromatic Aldehydes

“…The GcoA active site has been described as a tight-fitting hydrophobic pocket with a series of hydrophobic amino acids responsible for positioning the substrate’s aromatic ring. 26 Binding of several different aromatic molecules with various ring substituents have shown accommodation with only subtle reorganization of the surrounding enzyme side chains, with varying results on turnover by GcoA. 26 Here, multiple crystal structures were successfully solved for engineered complexes of T296A/G/S:guaiacol ( Table S2 ), T296A/G/S: p -vanillin ( Table S3 ), and F169A/S/V: o -vanillin ( Table S4 ).…”

“… 26 Binding of several different aromatic molecules with various ring substituents have shown accommodation with only subtle reorganization of the surrounding enzyme side chains, with varying results on turnover by GcoA. 26 Here, multiple crystal structures were successfully solved for engineered complexes of T296A/G/S:guaiacol ( Table S2 ), T296A/G/S: p -vanillin ( Table S3 ), and F169A/S/V: o -vanillin ( Table S4 ). The resolution of these nine structures, ranging from 1.60 to 1.88 Å, allowed robust refinement of the bound ligand orientations and surrounding active site residue positions ( Figure S6 ).…”

“…At least three enzyme classes, Rieske nonheme iron monooxygenases, tetrahydrofolate-dependent O- demethylases, and cytochrome P450 enzymes (hereafter P450s), are known to catalyze aromatic O- demethylation. 20 − 26 P450s are powerful redox catalysts that have served as exceptionally useful scaffolds for biological transformations. 27 The P450 system at the heart of the current study was first identified by Eltis and colleagues, who demonstrated O- demethylation of guaiacol (2-methoxyphenol) ( Scheme 1 A), the simplest compound with a G-type substitution pattern on the aromatic ring, and reported the N-terminal sequence of the P450 oxygenase.…”

Editorial: CAZymes in Biorefinery: From Genes to Application

“…Especially, CAZymes capable of degrading polysaccharide fraction of plant biomass into simple sugars have proven significant, as the resulting monomers can either be used a carbon source for fermentation-based production processes or be biotransformed into bioactive compounds, such as bioactive oligosaccharides like alginate oligosaccharides (Falkeborg et al, 2014 ; Rakotoarivonina et al, 2016 ; Oliveira et al, 2019 ). The non-carbohydrate fraction of plant biomass, like lignin, can also be converted into relevant products, such as ferulic acid and vanillin, by lignin-modifying enzymes (Sainsbury et al, 2013 ; Tian et al, 2017 ; Fetherolf et al, 2020 ).…”

Bacterial Transformation of Aromatic Monomers in Softwood Black Liquor

“…SYK-6 (Kamimura et al, 2017), Pseudomonas putida KT2440 (Weimer et al, 2020), Novosphingobium aromaticivorans DSM 12444 (Perez et al, 2021), Rhodococcus opacus PD630 (Kosa and Ragauskas, 2012), and Rhodococcus jostii RHA1 (Eltis and Singh, 2018). More recently, we isolated Rhodococcus rhodochrous EP4 for its ability to grow on alkylated guaiacols generated in the reductive catalytic fractionation (RCF) of hardwood lignin (Levy-Booth et al, 2019;Fetherolf et al, 2020). These strains not only have exceptional abilities to aerobically degrade LDACs, but also have a high natural resistance to toxic compounds (Krell et al, 2012;Xu et al, 2019).…”