Characterization of p -Hydroxycinnamate Catabolism in a Soil Actinobacterium

Abstract

p-Hydroxycinnamates, such as ferulate and p-coumarate, are components of plant cell walls and have a number of commercial applications. Rhodococcus jostii RHA1 (RHA1) catabolizes ferulate via vanillate and the ␤-ketoadipate pathway. Here, we used transcriptomics to identify genes in RHA1 that are upregulated during growth on ferulate versus benzoate. The upregulated genes included three transcriptional units predicted to encode the uptake and ␤-oxidative deacetylation of p-hydroxycinnamates: couHTL, couNOM, and couR. Neither ⌬couL mutants nor ⌬couO mutants grew on p-hydroxycinnamates, but they did grow on vanillate. Among several p-hydroxycinnamates, CouL catalyzed the thioesterification of p-coumarate and caffeate most efficiently (k cat /K m ‫؍‬ ϳ400 mM ؊1 s ؊1 ). p-Coumarate was also RHA1's preferred growth substrate, suggesting that CouL is a determinant of the pathway's specificity. CouL did not catalyze the activation of sinapate, in similarity to two p-coumaric acid:coenzyme A (CoA) ligases from plants, and contains the same bulged loop that helps determine substrate specificity in the plant homologues. The couO mutant accumulated 4-hydroxy-3-methoxyphenyl-␤-ketopropionate in the culture supernatant when incubated with ferulate, supporting ␤-oxidative deacetylation. This phenotype was not complemented with a D257N variant of CouO, consistent with the predicted role of Asp257 as a metal ligand in this amidohydrolase superfamily member. These data suggest that CouO functionally replaces the ␤-ketothiolase and acyl-CoA thioesterase that occur in canonical ␤-oxidative pathways. Finally, the transcriptomics data suggest the involvement of two distinct formaldehyde detoxification pathways in vanillate catabolism and identify a eugenol catabolic pathway. The results of this study augment our understanding of the bacterial catabolism of aromatics from renewable feedstocks.

Investigation of the Chemocatalytic and Biocatalytic Valorization of a Range of Different Lignin Preparations: The Importance of β-O-4 Content

“…The appearance of vanillic acid as a bio-product is unexpected and puzzling, since the enzymatic oxidation of vanillin to vanillic acid has been disrupted by gene knockout. In these cases it is possible that vanillic acid is formed from -oxidation of ferulic acid via the pathway elucidated by Otani et al, 41 or from another unidentified pathway, but one would expect that the vanillic acid would be further metabolised via demethylation and subsequently via the -ketoadipate pathway, as shown in Figure 7. One possible rationalisation is that the vanillate demethylase gene, which is normally induced by the presence of vanillin, 42 is not induced by syringaldehyde, which would result in a shortage of vanillate demethylase enzyme in the host organism, thereby accumulating the substrate vanillic acid.…”

Chemical intervention in bacterial lignin degradation pathways: Development of selective inhibitors for intradiol and extradiol catechol dioxygenases

“…Small amounts of protocatechuic acid were observed upon treatment of R. jostii with inhibitor D13, but only when the carbon source was ferulic acid, rather than lignocellulose, and no substrate accumulation was observed for inhibitor D3 in E. coli. We have observed previously that ferulic acid is converted via b-oxidation into vanillic acid in R. jostii RHA1 [8], and a cluster of genes responsible for metabolism of ferulic acid in R. jostii has been identified [19]. In the case of protocatechuic acid, it is possible that, upon accumulation of this metabolite, R. jostii can convert this compound via decarboxylation into catechol [24], preventing the accumulation of higher concentrations of protocatechuic acid.…”

“…It is known that ferulic acid (4) is converted into vanillic acid (2) in R. jostii RHA1 via b-oxidation [19]. Using media containing wheat straw lignocellulose as carbon source, protocatechuic acid was not detected, implying that its concentration in this case is too low to detect by LC-MS.…”

Rapid, parallel identification of pathways for catabolism of lignin-derived aromatic compounds

“…SYK-6, with FerA and FerB performing the deacetylation 16 , followed by oxidation by LigV or DesV 17,18 . Likewise, β-oxidative routes have been described for ferulate and coumarate in Rhodococcus jostii RHA1 19 and for ferulate, coumarate, and caffeate in Agrobacterium fabrum 20 . However, no β-oxidative pathway has previously been demonstrated for conversion of sinapate to syringate.…”