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Identification of the Gene Responsible for Lignin-Derived Low-Molecular-Weight Compound Catabolism in Pseudomonas sp. Strain LLC-1.

ABSTRACT: Pseudomonas sp. strain LLC-1 (NBRC 111237) is capable of degrading lignin-derived low-molecular-weight compounds (LLCs). The genes responsible for the catabolism of LLCs were characterized in this study using whole-genome sequencing. Despite the close phylogenetic relationship with Pseudomonas putida, strain LLC-1 lacked the genes usually found in the P. putida genome, which included fer, encoding an enzyme for ferulic acid catabolism, and vdh encoding an NAD⁺-dependent aldehyde dehydrogenase specific for its catabolic intermediate, vanillin. Cloning and expression of the 8.5 kb locus adjacent to the van operon involved in vanillic acid catabolism revealed the bzf gene cluster, which is involved in benzoylformic acid catabolism. One of the structural genes identified, bzfC, expresses the enzyme (BzfC) having the ability to transform vanillin and syringaldehyde to corresponding acids, indicating that BzfC is a multifunctional enzyme that initiates oxidization of LLCs in strain LLC-1. Benzoylformic acid is a catabolic intermediate of (R,S)-mandelic acid in P. putida. Strain LLC-1 did not possess the genes for mandelic acid racemization and oxidation, suggesting that the function of benzoylformic acid catabolic enzymes is different from that in P. putida. Genome-wide characterization identified the bzf gene responsible for benzoylformate and vanillin catabolism in strain LLC-1, exhibiting a unique mode of dissimilation for biomass-derived aromatic compounds by this strain.

SUBMITTER: Hirose J

PROVIDER: S-EPMC7760541 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Identification of the Gene Responsible for Lignin-Derived Low-Molecular-Weight Compound Catabolism in Pseudomonas sp. Strain LLC-1.

Hirose Jun J Tsukimata Ryusei R Miyatake Munetoshi M Yokoi Haruhiko H

Genes 20201127 12

Pseudomonas sp. strain LLC-1 (NBRC 111237) is capable of degrading lignin-derived low-molecular-weight compounds (LLCs). The genes responsible for the catabolism of LLCs were characterized in this study using whole-genome sequencing. Despite the close phylogenetic relationship with Pseudomonas putida, strain LLC-1 lacked the genes usually found in the P. putida genome, which included fer, encoding an enzyme for ferulic acid catabolism, and vdh encoding an NAD<s ...[more]

PMID: 33260964

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Project description:The gene loci fcs, encoding feruloyl coenzyme A (feruloyl-CoA) synthetase, ech, encoding enoyl-CoA hydratase/aldolase, and aat, encoding beta-ketothiolase, which are involved in the catabolism of ferulic acid and eugenol in Pseudomonas sp. strain HR199 (DSM7063), were localized on a DNA region covered by two EcoRI fragments (E230 and E94), which were recently cloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The nucleotide sequences of parts of fragments E230 and E94 were determined, revealing the arrangement of the aforementioned genes. To confirm the function of the structural genes fcs and ech, they were cloned and expressed in Escherichia coli. Recombinant strains harboring both genes were able to transform ferulic acid to vanillin. The feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase activities of the fcs and ech gene products, respectively, were confirmed by photometric assays and by high-pressure liquid chromatography analysis. To prove the essential involvement of the fcs, ech, and aat genes in the catabolism of ferulic acid and eugenol in Pseudomonas sp. strain HR199, these genes were inactivated separately by the insertion of omega elements. The corresponding mutants Pseudomonas sp. strain HRfcsOmegaGm and Pseudomonas sp. strain HRechOmegaKm were not able to grow on ferulic acid or on eugenol, whereas the mutant Pseudomonas sp. strain HRaatOmegaKm exhibited a ferulic acid- and eugenol-positive phenotype like the wild type. In conclusion, the degradation pathway of eugenol via ferulic acid and the necessity of the activation of ferulic acid to the corresponding CoA ester was confirmed. The aat gene product was shown not to be involved in this catabolism, thus excluding a beta-oxidation analogous degradation pathway for ferulic acid. Moreover, the function of the ech gene product as an enoyl-CoA hydratase/aldolase suggests that ferulic acid degradation in Pseudomonas sp. strain HR199 proceeds via a similar pathway to that recently described for Pseudomonas fluorescens AN103.

Dataset Information

Identification of the Gene Responsible for Lignin-Derived Low-Molecular-Weight Compound Catabolism in Pseudomonas sp. Strain LLC-1.

Publications

Identification of the Gene Responsible for Lignin-Derived Low-Molecular-Weight Compound Catabolism in <i>Pseudomonas</i> sp. Strain LLC-1.

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