Improved method for the extraction of high-quality DNA from lignocellulosic compost samples for metagenomic studies

Costa, Ângela A.; Santos, Andréia O.; Sousa, Joana; Rodrigues, Joana Lúcia Lima Correia; Gudiña, Eduardo J.; Silvério, Sara C.; Rodrigues, L. R.

doi:10.1007/s00253-021-11647-7

Cited by 14 publications

(12 citation statements)

References 58 publications

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“…Until DNA extraction, the sample was stored at 4 °C. Metagenomic DNA was extracted from 1 g of compost sample following a protocol developed by our research group, already described by Costa and colleagues (Costa et al 2021 ).…”

Section: Methodsmentioning

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: Methodsmentioning

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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“…The compost samples were characterized regarding their temperature, moisture, pH, elemental content, and humic acid concentration, as previously described by Costa et al ( 2021 ). Temperature was determined on site with a thermometer.…”

Section: Methodsmentioning

confidence: 99%

“…The elemental analysis (total organic carbon, nitrogen, hydrogen, and sulfur) was performed by automated dry combustion (Requimte/LAQV, Faculty of Sciences and Technology, Nova University of Lisbon, Portugal). The concentration of humic acids was determined by absorbance measurement at 340 nm, using a standard curve (0–500 ng/μl) previously prepared with commercial humic acids, as described by Costa et al ( 2021 ). Briefly, 1 g of compost samples was added to a 0.1 M NaOH solution (9 ml), stirred for 3 h at room temperature, and then centrifuged at 2500 × g for 10 min.…”

Section: Methodsmentioning

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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“…In the past decades, engineered microorganisms have been demonstrated to be a suitable option to produce numerous compounds with industrially relevant interest [12][13][14][15][16][17] as the production processes are more environmentally friendly and have the potential to become more cost-effective. The use of synthetic biology and metabolic engineering approaches has allowed to construct more efficient strains able to produce heterologous compounds with higher concentrations and yields [18][19][20][21][22][23][24]. Tools such as clustered, regularly interspaced, short palindromic repeats-associated Cas9 (CRISPR-Cas9) have revolutionized the way microorganisms are designed as they make it possible to reconstruct complex metabolic pathways in a short period [25,26].…”

Section: Introductionmentioning

confidence: 99%

Heterologous Production of Acrylic Acid: Current Challenges and Perspectives

Rodrigues

2022

SynBio

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Acrylic acid (AA) is a chemical with high market value used in industry to produce diapers, paints, adhesives and coatings, among others. AA available worldwide is chemically produced mostly from petroleum derivatives. Due to its economic relevance, there is presently a need for innovative and sustainable ways to synthesize AA. In the past decade, several semi-biological methods have been developed and consist in the bio-based synthesis of 3-hydroxypropionic acid (3-HP) and its chemical conversion to AA. However, more recently, engineered Escherichia coli was demonstrated to be able to convert glucose or glycerol to AA. Several pathways have been developed that use as precursors glycerol, malonyl-CoA or β-alanine. Some of these pathways produce 3-HP as an intermediate. Nevertheless, the heterologous production of AA is still in its early stages compared, for example, to 3-HP production. So far, only up to 237 mg/L of AA have been produced from glucose using β-alanine as a precursor in fed-batch fermentation. In this review, the advances in the production of AA by engineered microbes, as well as the hurdles hindering high-level production, are discussed. In addition, synthetic biology and metabolic engineering approaches to improving the production of AA in industrial settings are presented.

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Improved method for the extraction of high-quality DNA from lignocellulosic compost samples for metagenomic studies

Cited by 14 publications

References 58 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

Heterologous Production of Acrylic Acid: Current Challenges and Perspectives

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