Expression Platforms for Functional Metagenomics: Emerging Technology Options Beyond Escherichia coli

Lewin, Anna; Lale, Rahmi; Wentzel, Alexander

doi:10.1007/978-3-319-61510-3_2

Cited by 8 publications

(7 citation statements)

References 194 publications

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“…Likewise, bacterial artificial chromosomes (BACs) or plasmids with low DNA copy number should also become amenable to droplet screening. If growth can be spatially adjusted, an extension of this work flow to any expression host [64] is also conceivable. Finally, in addition to single enzyme screening, ultra-high-throughput discovery and combinatorial engineering of complex metabolic pathways is now practically possible.…”

Section: Ultra-high Throughput Sorting Of Intracellular Enzymesmentioning

confidence: 99%

Investigating host-microbiome interactions by droplet based microfluidics

et al. 2020

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Background Despite the importance of the mucosal interface between microbiota and the host in gut homeostasis, little is known about the mechanisms of bacterial gut colonization, involving foraging for glycans produced by epithelial cells. The slow pace of progress toward understanding the underlying molecular mechanisms is largely due to the lack of efficient discovery tools, especially those targeting the uncultured fraction of the microbiota. Results Here, we introduce an ultra-high-throughput metagenomic approach based on droplet microfluidics, to screen fosmid libraries. Thousands of bacterial genomes can be covered in 1 h of work, with less than ten micrograms of substrate. Applied to the screening of the mucosal microbiota for β-N-acetylgalactosaminidase activity, this approach allowed the identification of pathways involved in the degradation of human gangliosides and milk oligosaccharides, the structural homologs of intestinal mucin glycans. These pathways, whose prevalence is associated with inflammatory bowel diseases, could be the result of horizontal gene transfers with Bacteroides species. Such pathways represent novel targets to study the microbiota-host interactions in the context of inflammatory bowel diseases, in which the integrity of the mucosal barrier is impaired. Conclusion By compartmentalizing experiments inside microfluidic droplets, this method speeds up and miniaturizes by several orders of magnitude the screening process compared to conventional approaches, to capture entire metabolic pathways from metagenomic libraries. The method is compatible with all types of (meta)genomic libraries, and employs a commercially available flow cytometer instead of a custom-made sorting system to detect intracellular or extracellular enzyme activities. This versatile and generic workflow will accelerate experimental exploration campaigns in functional metagenomics and holobiomics studies, to further decipher host-microbiota relationships.

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Section: Ultra-high Throughput Sorting Of Intracellular Enzymesmentioning

confidence: 99%

Investigating host-microbiome interactions by droplet based microfluidics

et al. 2020

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Section: Introductionmentioning

confidence: 99%

“…All of these systems involve the use of mesophilic hosts, which when expressing extremozymes have even greater limitations as these enzymes are usually active in conditions that mimic their original environments. 16 To our knowledge, there is only one publication where an extremophile host was used for the construction of functional metagenomic libraries. 17 In this work, the authors reported that the use of Thermus thermophilus as host to find hydrolytic enzymes from hot environments retrieved more positive clones than when using E. coli.…”

Section: Introductionmentioning

confidence: 99%

Synthetic Biology Toolbox for Antarctic Pseudomonas sp. Strains: Toward a Psychrophilic Nonmodel Chassis for Function-Driven Metagenomics

et al. 2023

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One major limitation of function-driven metagenomics is the ability of the host to express the metagenomic DNA correctly. Differences in the transcriptional, translational, and posttranslational machinery between the organism to which the DNA belongs and the host strain are all factors that influence the success of a functional screening. For this reason, the use of alternative hosts is an appropriate approach to favor the identification of enzymatic activities in function-driven metagenomics. To be implemented, appropriate tools should be designed to build the metagenomic libraries in those hosts. Moreover, discovery of new chassis and characterization of synthetic biology toolbox in nonmodel bacteria is an active field of research to expand the potential of these organisms in processes of industrial interest. Here, we assessed the suitability of two Antarctic psychrotolerant Pseudomonas strains as putative alternative hosts for functiondriven metagenomics using pSEVA modular vectors as scaffold. We determined a set of synthetic biology tools suitable for these hosts and, as a proof of concept, we demonstrated their fitness for heterologous protein expression. These hosts represent a step forward for the prospection and identification of psychrophilic enzymes of biotechnological interest.

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“…To circumvent these problems, other host strains and vectors have been developed [ 288 , 289 , 290 ]. In activity-based metagenomics, the positive hit rate greatly depends on the metagenomic DNA source, its abundance in the metagenomic sample, the chosen cloning vector and expression host, the variability of heterologous gene expression, and the sensitivity and inherent bias of the screening assay.…”

Section: Glycoside-phosphorylasesmentioning

confidence: 99%

Discovery and Biotechnological Exploitation of Glycoside-Phosphorylases

Benkoulouche

Ladevèze

et al. 2022

IJMS

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Among carbohydrate active enzymes, glycoside phosphorylases (GPs) are valuable catalysts for white biotechnologies, due to their exquisite capacity to efficiently re-modulate oligo- and poly-saccharides, without the need for costly activated sugars as substrates. The reversibility of the phosphorolysis reaction, indeed, makes them attractive tools for glycodiversification. However, discovery of new GP functions is hindered by the difficulty in identifying them in sequence databases, and, rather, relies on extensive and tedious biochemical characterization studies. Nevertheless, recent advances in automated tools have led to major improvements in GP mining, activity predictions, and functional screening. Implementation of GPs into innovative in vitro and in cellulo bioproduction strategies has also made substantial advances. Herein, we propose to discuss the latest developments in the strategies employed to efficiently discover GPs and make the best use of their exceptional catalytic properties for glycoside bioproduction.

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Expression Platforms for Functional Metagenomics: Emerging Technology Options Beyond Escherichia coli

Cited by 8 publications

References 194 publications

Investigating host-microbiome interactions by droplet based microfluidics

Investigating host-microbiome interactions by droplet based microfluidics

Synthetic Biology Toolbox for Antarctic Pseudomonas sp. Strains: Toward a Psychrophilic Nonmodel Chassis for Function-Driven Metagenomics

Discovery and Biotechnological Exploitation of Glycoside-Phosphorylases

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