Affiliation:
1. Department of Microbiology and Immunology, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada
Abstract
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 β-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 β-oxidative deacetylation of
p
-hydroxycinnamates:
couHTL
,
couNOM
, and
couR
. Neither Δ
couL
mutants nor Δ
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
= ∼400 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-β-ketopropionate in the culture supernatant when incubated with ferulate, supporting β-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 β-ketothiolase and acyl-CoA thioesterase that occur in canonical β-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.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
50 articles.
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