Biology subjects

Potocki-Veronese, G.

Publications and source records attributed to Potocki-Veronese, G..

4 recordsLinked to original sources

bioRxiv2026

Metabolic engineering of Escherichia coli strains for the in vivo synthesis of GP-mediated oligosaccharides

Enzymatic synthesis of rare disaccharides by reverse-phosphorolysis is a potentially sustainable route to produce high-value glycosides for human health and nutrition. We report the metabolic engineering of Escherichia coli for in vivo production of {beta}-mannobiose with different osidic linkages from hexose sugars. We demonstrate production of {beta}-1,2-mannobiose with this approach as proof of concept. Phosphotransferase system (PTS) inactivation enables import of non-phosphorylated mannose via heterologous permease GalP, restoring growth on mannose in a PTS- background and allowing mannose into the reverse biosynthetic pathway. Deletion of pfkA, which promotes intracellular accumulation of key sugar phosphates (G1P, M1P), establishes a favorable metabolic chassis for oligosaccharide production using glycoside-phosphorylases. Using this chassis, we expressed two {beta}-mannoside phosphorylases to enable the direct production of {beta}-1,2- and {beta}-1,4-mannobiose from mannose. The same chassis was also employed for laminaribiose production through the expression of a laminaribiose-phosphorylase. pfkA deletion significantly increased product titer (> 0.6 g{middle dot}L-1) and yield (up to 9% g/g mannose), highlighting a favorable redistribution of carbon fluxes toward disaccharide formation. Moreover, a combination of mixed-substrate cultures using glycerol as carbon and energy source and further metabolic engineering enabled partial growth-production decoupling, redirecting mannose utilization primarily toward product synthesis, with a yield of 60%. These results demonstrate the modularity and efficiency of the proposed platform for fermentative production of non-conventional oligosaccharides and expand the scope of metabolic engineering strategies for glycoside biosynthesis.

synthetic biology↗

bioRxiv2026

Metagenomic insights into microbial mediation of Caulerpa prolifera invasion in a coastal lagoon

Opportunistic seaweeds of the genus Caulerpa have benefited from human-driven environmental change to expand within coastal lagoons, frequently displacing native seagrasses. In the Mar Menor lagoon (southeastern Spain), Caulerpa prolifera has progressively expanded since the 1970s, coinciding with the decline of the native seagrass Cymodocea nodosa. Sediments colonized by Ca. prolifera are characterized by elevated sulfide concentrations, which are toxic to seagrasses. Because sulfide is microbially produced, these sediment communities likely shape the competition between Ca. prolifera and Cy. nodosa. However, their metabolic potential and activity remain poorly understood. Here, we examined the taxonomic composition and functional capabilities of sediment microbial communities using 12 paired metagenomes and metatranscriptomes from areas dominated by either Ca. prolifera or Cy. nodosa. Caulerpa-associated sediments displayed a higher potential for organic matter degradation and sulfate reduction. Several metagenome-assembled genomes (MAGs) encoded enzymes putatively involved in the degradation of pectin-like polysaccharides characteristic of the Caulerpa cell wall. Comparison of metagenomic and metatranscriptomic data further revealed that the most abundant MAGs were not necessarily the most transcriptionally active. This finding challenges the common assumption that ecological importance is primarily determined by abundance and highlights the potential contribution of less abundant microorganisms to biogeochemical cycling. Further, 97 of the 100 most highly expressed genes lacked functional annotation. Recombinant production and functional characterization of the protein encoded by the most highly expressed gene in the analyzed sediments revealed that it is an enzyme able to breakdown endogenous bacterial peptidoglycan. Collectively, these results underscore the importance of integrating metagenomics and metatranscriptomics with experimental validation to achieve a more comprehensive understanding of microbial functional capabilities, an integration that is still uncommon in the microbial ecology field.

microbiology↗

bioRxiv2024

Functional exploration of in vivo and in vitro lignocellulose-fed rumen bacterial microbiomes reveals novel enzymes involved in polysaccharide breakdown

BackgroundPlant cell walls are the main carbon sources for ruminal bacteria, which have evolved to produce sophisticated multi-functional enzyme cocktails in response to the structural diversity of lignocelulloses. Since a large proportion of ruminal bacteria are not yet cultured, we developed a high-throughput activity-based metagenomic approach to gain insight into this enzymatic diversity. ResultsA multi-step screening methodology was implemented to identify metagenomic clones acting on polysaccharides and polyaromatic compounds. This approach was used to explore the functional potential of two different microbial consortia derived from in vivo and in vitro enrichments of the bovine rumen microbiome on wheat straw. One hundred and sixty-eight fosmid clones were isolated from libraries. Five to seven times more {beta}-mannanase and {beta}-glucanase clones, and seven times less xylanase clones were obtained from the in vitro enrichment compared to the in vivo one. The sequencing of 51 fosmids, covering in total 1.4 Gb of metagenomic DNA, enabled the identification of various novel glycoside-hydrolases, esterases and oxidoreductases mostly encoded by unknown bacterial genera. Functional analysis showed that most of the identified xylanases belonged to Firmicutes members that were not enriched in the fermenter, while most cellulases and mannanases originate from Bacteroidetes. ConclusionThese enzymes, that, for most of them, had not been previously identified by in depth-metagenome sequencing, present a high potential for biotechnological applications, as they could be used alone or in cocktails to break down plant cell walls. The relationships established between enzyme function and taxonomy highlight the complementary roles played by ruminal Firmicutes and Bacteroidetes in plant cell wall degradation.

biochemistry↗

bioRxiv2018

A catalog of microbial genes from the bovine rumen reveals the determinants of herbivory

BackgroundThe rumen microbiota provides essential services to its host and, through its role in ruminant production, contributes to human nutrition and food security. A thorough knowledge of the genetic potential of rumen microbes will provide opportunities for improving the sustainability of ruminant production systems. The availability of gene reference catalogs from gut microbiomes has advanced the understanding of the role of the microbiota in health and disease in humans and other mammals. In this work, we established a catalog of reference prokaryote genes from the bovine rumen.\n\nResultsUsing deep metagenome sequencing we identified 13,825,880 non-redundant prokaryote genes from the bovine rumen. Compared to human, pig and mouse gut metagenome catalogs, the rumen is larger and richer in functions and microbial species associated with the degradation of plant cell wall material and production of methane. Genes encoding enzymes catalyzing the breakdown of plant polysaccharides showed a particularly high richness that is otherwise impossible to infer from available genomes or shallow metagenomics sequencing. The catalog expands by several folds the dataset of carbohydrate-degrading enzymes described in the rumen. Using an independent dataset from a group of 77 cattle fed 4 common dietary regimes, we found that only <0.1% of genes were shared by all animals, which contrast with a large overlap for functions, i.e. 63% for KEGG functions. Different diets induced differences in the relative abundance rather than the presence or absence of genes explaining the great adaptability of cattle to rapidly adjust to dietary changes.\n\nConclusionsThese data bring new insights into functions, carbohydrate-degrading enzymes and microbes of the rumen that is complementing the available information on microbial genomes. The catalog is a significant biological resource enabling deeper understanding of phenotypes and biological processes and will be expanded as new data is made available.

microbiology↗