Dataset Information

Cloning and characterization of the pnb genes, encoding enzymes for 4-nitrobenzoate catabolism in Pseudomonas putida TW3.

ABSTRACT: Pseudomonas putida strain TW3 is able to metabolize 4-nitrotoluene via 4-nitrobenzoate (4NBen) and 3, 4-dihydroxybenzoic acid (protocatechuate [PCA]) to central metabolites. We have cloned, sequenced, and characterized a 6-kbp fragment of TW3 DNA which contains five genes, two of which encode the enzymes involved in the catabolism of 4NBen to PCA. In order, they encode a 4NBen reductase (PnbA) which is responsible for catalyzing the direct reduction of 4NBen to 4-hydroxylaminobenzoate with the oxidation of 2 mol of NADH per mol of 4NBen, a reductase-like enzyme (Orf1) which appears to have no function in the pathway, a regulator protein (PnbR) of the LysR family, a 4-hydroxylaminobenzoate lyase (PnbB) which catalyzes the conversion of 4-hydroxylaminobenzoate to PCA and ammonium, and a second lyase-like enzyme (Orf2) which is closely associated with pnbB but appears to have no function in the pathway. The central pnbR gene is transcribed in the opposite direction to the other four genes. These genes complete the characterization of the whole pathway of 4-nitrotoluene catabolism to the ring cleavage substrate PCA in P. putida strain TW3.

SUBMITTER: Hughes MA

PROVIDER: S-EPMC94995 | biostudies-literature | 2001 Feb

REPOSITORIES: biostudies-literature

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Cloning and characterization of the pnb genes, encoding enzymes for 4-nitrobenzoate catabolism in Pseudomonas putida TW3.

Hughes M A MA Williams P A PA

Journal of bacteriology 20010201 4

Pseudomonas putida strain TW3 is able to metabolize 4-nitrotoluene via 4-nitrobenzoate (4NBen) and 3, 4-dihydroxybenzoic acid (protocatechuate [PCA]) to central metabolites. We have cloned, sequenced, and characterized a 6-kbp fragment of TW3 DNA which contains five genes, two of which encode the enzymes involved in the catabolism of 4NBen to PCA. In order, they encode a 4NBen reductase (PnbA) which is responsible for catalyzing the direct reduction of 4NBen to 4-hydroxylaminobenzoate with the o ...[more]

PMID: 11157934

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Project description:Pseudomonas alloputida KT2440 (previously misclassified as P. putida KT2440 based on 16S rRNA gene homology) has emerged as an ideal host strain for plan t biomass valorization. However, P. alloputida KT2440 is unable to natively utilize abundant pentose sugars (e.g., xylose and arabinose) in hydrolysate streams, which may account for up to 25% of lignocellulosic biomass. In the last decades, microbes have been engineered to utilize the pentose sugars. However, most of the engineered strains were either slow-growing or displayed phenotypes that could not be replicated. In this work, we successfully isolated five Pseudomonas species with the native capability to utilize glucose, xylose and p-coumarate as a sole carbon source. These isolates were in two clusters; one set of isolates (M2 and M5) and the second set of isolates (BP6 and BP7) showed 85.6% and 96.2% ANI, respectively, to P. alloputida KT24440. BP8 showed 84.6% ANI to P. putida KT2440 and does not belong to any neighboring type strains indicating a new species. Notably, the isolates showed robust growth solely on xylose and higher growth rates (m, 0.36-0.49 h-1) when compared to only known xylose-utilizing Pseudomonas taiwanenesis VLB120 (m, 0.28 h-1) as a control. Unexpectedly, among five isolates, M2 and M5 grew solely on arabinose as well. Comprehensive analysis of genomics, transcriptomics and proteomics revealed the isolates utilize xylose and arabinose via Weimberg pathway (xylD-xylX-xylA) and oxidative pathway (araD-araX-araA), respectively. Furthermore, a preliminary result demonstrated the production of flaviolin solely on xylose and arabinose in the isolate, showing noteworthy potential to be an alternative host for lignocellulosic feedstocks into valuable products. This is the first report on isolating Pseudomonas strains natively capable of utilizing all of the major carbon sources in lignocellulosic biomass, and leading to higher consumption of available substrates and therefore maximizing the product yield.

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Dataset Information

Cloning and characterization of the pnb genes, encoding enzymes for 4-nitrobenzoate catabolism in Pseudomonas putida TW3.

Publications

Cloning and characterization of the pnb genes, encoding enzymes for 4-nitrobenzoate catabolism in Pseudomonas putida TW3.

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