High-quality genome-scale metabolic network reconstruction of probiotic bacterium Escherichia coli Nissle 1917.

BACKGROUND: Escherichia coli Nissle 1917 (EcN) is a probiotic bacterium used to treat various gastrointestinal diseases. EcN is increasingly being used as a chassis for the engineering of advanced microbiome therapeutics. To aid in future engineering efforts, our aim was to construct an updated metabolic model of EcN with extended secondary metabolite representation. RESULTS: An updated high-quality genome-scale metabolic model of EcN, iHM1533, was developed based on comparison with 55 E. coli/Shigella reference GEMs and manual curation, including expanded secondary metabolite pathways (enterobactin, salmochelins, aerobactin, yersiniabactin, and colibactin). The model was validated and improved using phenotype microarray data, resulting in an 82.3% accuracy in predicting growth phenotypes on various nutrition sources. Flux variability analysis with previously published 13C fluxomics data validated prediction of the internal central carbon fluxes. A standardised test suite called Memote assessed the quality of iHM1533 to have an overall score of 89%. The model was applied by using constraint-based flux analysis to predict targets for optimisation of secondary metabolite production. Modelling predicted design targets from across amino acid metabolism, carbon metabolism, and other subsystems that are common or unique for influencing the production of various secondary metabolites. CONCLUSION: iHM1533 represents a well-annotated metabolic model of EcN with extended secondary metabolite representation. Phenotype characterisation and the iHM1533 model provide a better understanding of the metabolic capabilities of EcN and will help future metabolic engineering efforts.

Galacturonate Metabolism in Anaerobic Chemostat Enrichment Cultures: Combined Fermentation and Acetogenesis by the Dominant sp. nov. "Candidatus Galacturonibacter soehngenii".

Agricultural residues such as sugar beet pulp and citrus peel are rich in pectin, which contains galacturonic acid as a main monomer. Pectin-rich residues are underexploited as feedstocks for production of bulk chemicals or biofuels. The anaerobic, fermentative conversion of d-galacturonate in anaerobic chemostat enrichment cultures provides valuable information toward valorization of these pectin-rich feedstocks. Replicate anaerobic chemostat enrichments, with d-galacturonate as the sole limiting carbon source and inoculum from cow rumen content and rotting orange peels, yielded stable microbial communities, which were dominated by a novel Lachnospiraceae species, for which the name "Candidatus Galacturonibacter soehngenii" was proposed. Acetate was the dominant catabolic product, with formate and H2 as coproducts. The observed molar ratio of acetate and the combined amounts of H2 and formate deviated significantly from 1, which suggested that some of the hydrogen and CO2 formed during d-galacturonate fermentation was converted into acetate via the Wood-Ljungdahl acetogenesis pathway. Indeed, metagenomic analysis of the enrichment cultures indicated that the genome of "Candidatus G. soehngenii" encoded enzymes of the adapted Entner-Doudoroff pathway for d-galacturonate metabolism as well as enzymes of the Wood-Ljungdahl pathway. The simultaneous operation of these pathways may provide a selective advantage under d-galacturonate-limited conditions by enabling a higher specific ATP production rate and lower residual d-galacturonate concentration than would be possible with a strictly fermentative metabolism of this carbon and energy source.IMPORTANCE This study on d-galacturonate metabolism by open, mixed-culture enrichments under anaerobic, d-galacturonate-limited chemostat conditions shows a stable and efficient fermentation of d-galacturonate into acetate as the dominant organic fermentation product. This fermentation stoichiometry and population analyses provide a valuable baseline for interpretation of the conversion of pectin-rich agricultural feedstocks by mixed microbial cultures. Moreover, the results of this study provide a reference for studies on the microbial metabolism of d-galacturonate under different cultivation regimes.