Characterization of the Furfural and 5-Hydroxymethylfurfural (HMF) Metabolic Pathway in the Novel Isolate Pseudomonas putida ALS1267

Crigler, Jacob; Eiteman, Mark A.; Altman, Elliot

doi:10.1007/s12010-019-03130-x

Cited by 19 publications

(24 citation statements)

References 43 publications

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“…HMF-oxidizing activity was first identified in Cupriavidus basilensis strain HMF14 ( 3 , 4 ) and more recently in Pseudomonas putida strain ALS1267 ( 5 ), with their metabolic pathways being characterized. The hmfH gene of C. basilensis encodes an HMFO (UniProt D5KB61) that contributes to the conversion of HMF into FDCA, while the lack of this gene in P. putida suggests that, in this species, the conversion of HMF is catalyzed by different enzymes.…”

Section: Introductionmentioning

confidence: 99%

Screening and Evaluation of New Hydroxymethylfurfural Oxidases for Furandicarboxylic Acid Production

Viñambres

Espada

Martínez

et al. 2020

Appl Environ Microbiol

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The enzymatic production of 2,5-furandicarboxylic acid (FDCA) from 5-hydroxy-methylfurfural (HMF) has gained interest in recent years, as the renewable precursor of poly(ethylene-2,5-furandicarboxylate) (PEF). 5-Hydroxymethylfurfural oxidases (HMFOs) form a flavoenzyme family with genes annotated in a dozen bacterial species, but only one enzyme purified and characterized to date (after heterologous expression of a Methylovorus sp hmfo gene). This oxidase acts on both furfuryl alcohols and aldehydes being, therefore, able to catalyze the conversion of HMF into FDCA through 2,5-diformylfuran (DFF) and 2,5-formylfurancarboxylic acid (FFCA), with the only need of oxygen as cosubstrate. To enlarge the repertoire of HMFO enzymes available, genetic databases were screened for putative hmfo genes, followed by heterologous expression in Escherichia coli. After unsuccessful trials with other bacterial hmfo genes, HMFOs from two Pseudomonas species were produced as active soluble enzymes, purified and characterized. The Methylovorus sp enzyme was also produced and purified in parallel for comparison. Enzyme stability against temperature, pH and hydrogen peroxide, three key aspects for application, were evaluated (together with optimal conditions for activity) revealing differences between the three HMFOs. Also the kinetic parameters for HMF, DFF and FFCA oxidation were determined, having the new HMFOs higher efficiencies for the oxidation of FFCA, which constitutes the bottleneck in the enzymatic route for FDCA production. These results were used to set up the best conditions for FDCA production by each enzyme, attaining a compromise between optimal activity and half-life under different operation conditions. IMPORTANCE: HMFO is the only enzyme described to date catalyzing by itself the three consecutive oxidation steps to produce FDCA from HMF. Unfortunately, only one HMFO enzyme is currently available for biotechnological application. This availability is enlarged here by the identification, heterologous production, purification and characterization of two new HMFOs from Pseudomonas nitroreducens and an unidentified Pseudomonas species. Compared to the previously known Methylovorus HMFO, the new enzyme from P. nitroreducens exhibits better performance for FDCA production in wider pH and temperature ranges, with higher tolerance to the hydrogen peroxide formed, longer half-life during oxidation, and higher yield and total turnover number in long-term conversions under optimized conditions. All these features are relevant properties for the industrial production of FDCA. In summary, gene screening and heterologous expression can facilitate the selection and improvement of HMFO enzymes as biocatalysts for the enzymatic synthesis of renewable building blocks in the production of bioplastics.

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Section: Introductionmentioning

confidence: 99%

Screening and Evaluation of New Hydroxymethylfurfural Oxidases for Furandicarboxylic Acid Production

Viñambres

Espada

Martínez

et al. 2020

Appl Environ Microbiol

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“…A limited number of bacterial species owning the ability to use furfural or HMF as sole carbon sources have been characterized at genetic level, with Pseudomonas putida Fu1 [19] , Cupriavidus basilensis HMF14 [20] , and Pseudomonas putida ALS1267 [21] being the best characterized examples. The pioneering work of Nichols and Mertens provided the first pieces of evidence about furans biodegradation in P. putida strain Fu1 by identification of catabolic and regulatory psf genes [19] .…”

Section: Introductionmentioning

confidence: 99%

“…No specific aldehyde dehydrogenases have been identified for initial HMF oxidation into HMFCA, suggesting that such activity is related to broad-specificity dehydrogenases protecting cells against toxic aldehydes [18] , [20] , [22] . Recently, the genes involved in HMF catabolism have been characterized in P. putida ALS1267, which carries all hmf genes previously described in strain HMF14 except for hmfH , suggesting that another gene would replace this FAD-dependent oxidoreductase [21] . Interestingly, two additional genes are present in hmf operon from P. putida ALS1267, which were also previously found in P. putida Fu1 and termed psfG and psfA, encoding a putative short-chain alcohol dehydrogenase and a putative NAD-dependent aldehyde dehydrogenase, respectively [19] .…”

Section: Introductionmentioning

confidence: 99%

“…Interestingly, two additional genes are present in hmf operon from P. putida ALS1267, which were also previously found in P. putida Fu1 and termed psfG and psfA, encoding a putative short-chain alcohol dehydrogenase and a putative NAD-dependent aldehyde dehydrogenase, respectively [19] . These enzymatic functions have been suggested to be related to the oxidation of furfuryl alcohol/furfural and HMF alcohol/HMF [19] , [21] , but additional support has not been provided.

Fig.…”

Section: Introductionmentioning

confidence: 99%

“…1 Predicted metabolic pathways of HMF and furfural in Cupriavidus and Pseudomonas species converged toward FA as central intermediate. The diagram of reconstructed pathways has been adapted and integrated from previous reports [19] , [20] , [21] . FA conversion into α-ketoglutaric acid is accomplished by hmfABCDE -encoded enzymes constituting the lower pathway.…”

Section: Introductionmentioning

confidence: 99%

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