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Characterisation of p-hydroxycinnamate catabolism in a soil Actinobacterum

ABSTRACT: p-Hydroxycinnamates, such as p-coumarate and ferulate, are components of plant cell walls and have a number of commercial applications. Previously, we had shown that the soil Actinobacterium Rhodococcus jostii RHA1 (RHA1) grows on ferulate, catabolizing it via vanillate and the β-ketoadipate pathway. We used transcriptomics to identify genes in RHA1 that were specifically up-regulated during growth on ferulate. These include three operons predicted to encode the uptake and β-oxidative deacetylation of ferulate and p-coumarate: couHLT, couMNO and couR. A couL mutant did not grow on p-coumarate, ferulate or their dihydro derivatives, but grew on vanillate. Purified CouL catalyzed the thioesterification of several p-hydroxycinnamates. Among the tested substrates, the best were p-coumarate and caffeate (kcat/KM ~400 mM-1s-1), and sinapate was not transformed. Of these, p-coumarate was also RHA1’s preferred growth substrate. Although the data indicate that p-hydroxycinnamates are catabolized via β-oxidation, the pathway lacks a typical β-ketothiolase. The data further suggest the involvement of two formaldehyde detoxification pathways in vanillate catabolism. This study augments our understanding of the bacterial catabolism of biomass and facilitates the production of aromatics from renewable feedstocks.

ORGANISM(S): Rhodococcus jostii

PROVIDER: GSE56355 | GEO | 2014/09/28

SECONDARY ACCESSION(S): PRJNA243026

REPOSITORIES: GEO

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Project description:Strains of Pseudomonas putida have emerged as promising biocatalysts for the conversion of sugars and aromatics obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimizing the conversion of biomass to fuels and chemicals. The functioning of CCR in a P. putida strain, P. putida M2, capable of growing on both hexose and pentose sugars as well as aromatics, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 was able to co-utilize sugars and aromatics simultaneously; however, during co-cultivation with glucose and phenylpropanoid aromatics (p-coumarate and ferulate), intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis of these co- cultivations revealed that glucose exerted stronger repression compared to xylose on the proteins involved in aromatic catabolism. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during co-cultivation with aromatics implying simultaneous catabolite repression by sugars and aromatics. Downregulation of crc mediated by CRISPRi led to faster growth and uptake of both glucose and p-coumarate in the CRISPRi strains compared to the control while no difference was observed on xylose + p-coumarate. The increased abundance of the Eda protein and amino acids biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that the levels of CrcY and CrcZ homologs in M2, previously identified as sRNAs involved in CCR in P. putida strains, were lower under strong CCR (glucose + p-coumarate) condition compared to when repression was absent (p-coumarate or glucose only).

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Characterisation of p-hydroxycinnamate catabolism in a soil Actinobacterum

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