Metagenomic Approaches as a Tool to Unravel Promising Biocatalysts from Natural Resources: Soil and Water

Sousa, Joana; Silvério, Sara C.; Costa, Ângela A.; Rodrigues, L. R.

doi:10.3390/catal12040385

Cited by 11 publications

(5 citation statements)

References 215 publications

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“…The demand for efficient glycosyl hydrolases with outstanding catalytic activity, namely hemicellulases, is crucial to facilitate the bioconversion of hemicellulose into several value-added products (Periyasamy et al 2022 ; Nargotra et al 2023 ). Xylanases with interesting catalytic features have been searched from extreme environments using metagenomic approaches (Sousa et al 2022 ).…”

Section: Discussionmentioning

confidence: 99%

Heterologous expression and structure prediction of a xylanase identified from a compost metagenomic library

Sousa,

Santos-Pereira,

Gomes

et al. 2024

Appl Microbiol Biotechnol

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Xylanases are key biocatalysts in the degradation of the β‐1,4‐glycosidic linkages in the xylan backbone of hemicellulose. These enzymes are potentially applied in a wide range of bioprocessing industries under harsh conditions. Metagenomics has emerged as powerful tools for the bioprospection and discovery of interesting bioactive molecules from extreme ecosystems with unique features, such as high temperatures. In this study, an innovative combination of function-driven screening of a compost metagenomic library and automatic extraction of halo areas with in-house MATLAB functions resulted in the identification of a promising clone with xylanase activity (LP4). The LP4 clone proved to be an effective xylanase producer under submerged fermentation conditions. Sequence and phylogenetic analyses revealed that the xylanase, Xyl4, corresponded to an endo-1,4-β-xylanase belonging to glycosyl hydrolase family 10 (GH10). When xyl4 was expressed in Escherichia coli BL21(DE3), the enzyme activity increased about 2-fold compared to the LP4 clone. To get insight on the interaction of the enzyme with the substrate and establish possible strategies to improve its activity, the structure of Xyl4 was predicted, refined, and docked with xylohexaose. Our data unveiled, for the first time, the relevance of the amino acids Glu133 and Glu238 for catalysis, and a close inspection of the catalytic site suggested that the replacement of Phe316 by a bulkier Trp may improve Xyl4 activity. Our current findings contribute to enhancing the catalytic performance of Xyl4 towards industrial applications. Key points • A GH10 endo-1,4-β-xylanase (Xyl4) was isolated from a compost metagenomic library • MATLAB’s in-house functions were developed to identify the xylanase-producing clones • Computational analysis showed that Glu133 and Glu238 are crucial residues for catalysis Graphical abstract

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Section: Discussionmentioning

confidence: 99%

Heterologous expression and structure prediction of a xylanase identified from a compost metagenomic library

Sousa,

Santos-Pereira,

Gomes

et al. 2024

Appl Microbiol Biotechnol

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“…Environmental DNA from soil, water, animal gut, industrial sewages, and extreme environments, such as composting, has been shown to have a great potential to uncover promising enzymes for biotechnological applications (Datta et al 2020 ; Sousa et al 2022 ). Due to the huge variety of microorganisms able to degrade lignocellulosic biomass involved in composting, it is considered one of the most important (bio)reactors that contribute to the renewable bioenergy on the planet.…”

Section: Introductionmentioning

confidence: 99%

Functional and sequence-based metagenomics to uncover carbohydrate-degrading enzymes from composting samples

Santos-Pereira

Sousa

Costa

et al. 2023

Appl Microbiol Biotechnol

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The renewable, abundant , and low-cost nature of lignocellulosic biomass can play an important role in the sustainable production of bioenergy and several added-value bioproducts, thus providing alternative solutions to counteract the global energetic and industrial demands. The efficient conversion of lignocellulosic biomass greatly relies on the catalytic activity of carbohydrate-active enzymes (CAZymes). Finding novel and robust biocatalysts, capable of being active under harsh industrial conditions, is thus imperative to achieve an economically feasible process. In this study, thermophilic compost samples from three Portuguese companies were collected, and their metagenomic DNA was extracted and sequenced through shotgun sequencing. A novel multi-step bioinformatic pipeline was developed to find CAZymes and characterize the taxonomic and functional profiles of the microbial communities, using both reads and metagenome-assembled genomes (MAGs) as input. The samples’ microbiome was dominated by bacteria, where the classes Gammaproteobacteria, Alphaproteobacteria, and Balneolia stood out for their higher abundance, indicating that the degradation of compost biomass is mainly driven by bacterial enzymatic activity. Furthermore, the functional studies revealed that our samples are a rich reservoir of glycoside hydrolases (GH), particularly of GH5 and GH9 cellulases, and GH3 oligosaccharide-degrading enzymes. We further constructed metagenomic fosmid libraries with the compost DNA and demonstrated that a great number of clones exhibited β-glucosidase activity. The comparison of our samples with others from the literature showed that, independently of the composition and process conditions, composting is an excellent source of lignocellulose-degrading enzymes. To the best of our knowledge, this is the first comparative study on the CAZyme abundance and taxonomic/functional profiles of Portuguese compost samples. Key points • Sequence- and function-based metagenomics were used to find CAZymes in compost samples. • Thermophilic composts proved to be rich in bacterial GH3, GH5, and GH9 enzymes. • Compost-derived fosmid libraries are enriched in clones with β-glucosidase activity.

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“…Recent investigations have revealed that over 99% of prokaryotic microorganisms remain elusive through existing pure culture isolation techniques ( Tiwari et al, 2021 ). Metagenomic sequencing technology has emerged as a potent avenue to directly access functional gene sequences from environmental DNA ( Kaushal et al, 2021 ; Robinson et al, 2021 ; Sousa et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

Characterization of a thermophilic and glucose-tolerant GH1 β-glucosidase from hot springs and its prospective application in corn stover degradation

Huang,

Lv,

Zheng

et al. 2023

Front. Microbiol.

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Introductionβ-Glucosidase serves as the pivotal rate-limiting enzyme in the cellulose degradation process, facilitating the hydrolysis of cellobiose and cellooligosaccharides into glucose. However, the widespread application of numerous β-glucosidases is hindered by their limited thermostability and low glucose tolerance, particularly in elevated-temperature and high-glucose environments.MethodsThis study presents an analysis of a β-glucosidase gene belonging to the GH1 family, denoted lqbg8, which was isolated from the metagenomic repository of Hehua hot spring located in Tengchong, China. Subsequently, the gene was cloned and heterologously expressed in Escherichia coli BL21(DE3). Post expression, the recombinant β-glucosidase (LQBG8) underwent purification through a Ni affinity chromatography column, thereby enabling the in-depth exploration of its enzymatic properties.ResultsLQBG8 had an optimal temperature of 70°C and an optimum pH of 5.6. LQBG8 retained 100 and 70% of its maximum activity after 2-h incubation periods at 65°C and 70°C, respectively. Moreover, even following exposure to pH ranges of 3.0–10.0 for 24 h, LQBG8 retained approximately 80% of its initial activity. Notably, the enzymatic prowess of LQBG8 remained substantial at glucose concentrations of up to 3 M, with a retention of over 60% relative activity. The kinetic parameters of LQBG8 were characterized using cellobiose as substrate, with Km and Vmax values of 28 ± 1.9 mg/mL and 55 ± 3.2 μmol/min/mg, respectively. Furthermore, the introduction of LQBG8 (at a concentration of 0.03 mg/mL) into a conventional cellulase reaction system led to an impressive 43.7% augmentation in glucose yield from corn stover over a 24-h period. Molecular dynamics simulations offered valuable insights into LQBG8’s thermophilic nature, attributing its robust stability to reduced fluctuations, conformational changes, and heightened structural rigidity in comparison to mesophilic β-glucosidases.DiscussionIn summation, its thermophilic, thermostable, and glucose-tolerant attributes, render LQBG8 ripe for potential applications across diverse domains encompassing food, feed, and the production of lignocellulosic ethanol.

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Metagenomic Approaches as a Tool to Unravel Promising Biocatalysts from Natural Resources: Soil and Water

Cited by 11 publications

References 215 publications

Heterologous expression and structure prediction of a xylanase identified from a compost metagenomic library

Heterologous expression and structure prediction of a xylanase identified from a compost metagenomic library

Functional and sequence-based metagenomics to uncover carbohydrate-degrading enzymes from composting samples

Characterization of a thermophilic and glucose-tolerant GH1 β-glucosidase from hot springs and its prospective application in corn stover degradation

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