Fetherolf, Morgan M.;
Levy-Booth, David J.;
Navas, Laura E.;
Liu, Jie;
Grigg, Jason C.;
Wilson, Andrew;
Katahira, Rui;
Beckham, Gregg T.;
Mohn, William W.;
Eltis, Lindsay D.
Characterization of alkylguaiacol-degrading cytochromes P450 for the biocatalytic valorization of lignin
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Medientyp:
E-Artikel
Titel:
Characterization of alkylguaiacol-degrading cytochromes P450 for the biocatalytic valorization of lignin
Beteiligte:
Fetherolf, Morgan M.;
Levy-Booth, David J.;
Navas, Laura E.;
Liu, Jie;
Grigg, Jason C.;
Wilson, Andrew;
Katahira, Rui;
Beckham, Gregg T.;
Mohn, William W.;
Eltis, Lindsay D.
Erschienen:
National Academy of Sciences, 2020
Erschienen in:Proceedings of the National Academy of Sciences of the United States of America
Sprache:
Englisch
ISSN:
0027-8424;
1091-6490
Entstehung:
Anmerkungen:
Beschreibung:
<p>Cytochrome P450 enzymes have tremendous potential as industrial biocatalysts, including in biological lignin valorization. Here, we describe P450s that catalyze the <italic>O</italic>-demethylation of lignin-derived guaiacols with different ring substitution patterns. Bacterial strains <italic>Rhodococcus rhodochrous</italic> EP4 and <italic>Rhodococcus jostii</italic> RHA1 both utilized alkylguaiacols as sole growth substrates. Transcriptomics of EP4 grown on 4-propylguaiacol (4PG) revealed the up-regulation of <italic>agcA</italic>, encoding a CYP255A1 family P450, and the <italic>aph</italic> genes, previously shown to encode a <italic>meta</italic>-cleavage pathway responsible for 4-alkylphenol catabolism. The function of the homologous pathway in RHA1 was confirmed: Deletion mutants of <italic>agcA</italic> and <italic>aphC</italic>, encoding the <italic>meta</italic>-cleavage alkylcatechol dioxygenase, grew on guaiacol but not 4PG. By contrast, deletion mutants of <italic>gcoA</italic> and <italic>pcaL</italic>, encoding a CYP255A2 family P450 and an <italic>ortho</italic>-cleavage pathway enzyme, respectively, grew on 4-propylguaiacol but not guaiacol. CYP255A1 from EP4 catalyzed the <italic>O</italic>-demethylation of 4-alkylguaiacols to 4-alkylcatechols with the following apparent specificities (<italic>k</italic>
<sub>cat</sub>/<italic>K</italic>
<sub>M</sub>): propyl > ethyl > methyl > guaiacol. This order largely reflected AgcA’s binding affinities for the different guaiacols and was the inverse of GcoA<sub>EP4</sub>’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.</p>