Abstract
The bacterial catabolism of aromatic compounds has considerable promise to convert lignin depolymerization products to commercial chemicals. Alkylphenols are a key class of depolymerization products whose catabolism is not well-elucidated. We isolated Rhodococcus rhodochrous EP4 on 4-ethylphenol and applied genomic and transcriptomic approaches to elucidate alkylphenol catabolism in EP4 and Rhodococcus jostii RHA1. RNA-Seq and RT-qPCR revealed a pathway encoded by the aphABCDEFGHIQRS genes that degrades 4-ethylphenol via the meta-cleavage of 4-ethylcatechol. This process was initiated by a two-component alkylphenol hydroxylase, encoded by the aphAB genes, which were upregulated ~3,000-fold. Purified AphAB from EP4 had highest specific activity for 4-ethylphenol and 4-propylphenol (~2,000 U/mg) but did not detectably transform phenol. Nevertheless, a ΔaphA mutant in RHA1 grew on 4-ethylphenol by compensatory upregulation of phenol hydroxylase genes (pheA1-3). Deletion of aphC, encoding an extradiol dioxygenase, prevented growth on 4-alkylphenols but not phenol. Disruption of pcaL in the β-ketoadipate pathway prevented growth on phenol but not 4-alkylphenols. Thus, 4-alkylphenols are catabolized exclusively via meta-cleavage in rhodococci while phenol is subject to ortho-cleavage. A putative genomic island encoding aph genes was identified in EP4 and several other rhodococci. Overall, this study identifies a 4-alkylphenol pathway in rhodococci, demonstrates key enzymes involved, and presents evidence that the pathway is encoded in a genomic island. These advances are of particular importance for wide-ranging industrial applications of rhodococci, including upgrading of lignocellulose biomass.
Highlights
Lignin, a heterogeneous aromatic polymer, accounts for up to 40% dry weight of terrestrial plant biomass (Ragauskas et al, 2014)
Bacterial biocatalysts provide a means of transforming mixtures of aromatic compounds to single compounds due to the convergent nature of their catabolic pathways, whereby substrates are transformed to central metabolites via shared intermediates, such as catechols (Linger et al, 2014; Eltis and Singh, 2018)
The lack of Pacific Biosciences (PacBio) long reads overlapping the 5′ and 3′ genome regions indicated that the EP4 genome is linear
Summary
A heterogeneous aromatic polymer, accounts for up to 40% dry weight of terrestrial plant biomass (Ragauskas et al, 2014). Rhodococcus is a genus of mycolic acid-producing Actinobacteria that catabolize a wide variety of aromatic compounds (Yam et al, 2010), including phenols (Kolomytseva et al, 2007; Gröning et al, 2014) These bacteria have considerable potential as biocatalysts for the industrial production of compounds ranging from nitriles to steroids and high-value lipids (Alvarez et al, 1996; Round et al, 2017; Shields-Menard et al, 2017; Sengupta et al, 2019). Knowledge gained by this study will facilitate the efficient valorization of lignin following its depolymerization
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