Establishment, in silico analysis, and experimental verification of a large-scale metabolic network of the xanthan producing Xanthomonas campestris pv. campestris strain B100

The γ-proteobacterium Xanthomonas campestris pv. campestris (Xcc) B100 synthesizes the polysaccharide xanthan, a commercially important viscosifier. Since the complete genome of Xcc B100 is available, systems biology tools were applied to obtain a deeper understanding of the metabolism involved in xanthan biosynthesis. A large-scale metabolic network was reconstructed and manually curated. The reconstructed network included 352 genes, 437 biochemical reactions, 10 transport reactions, and 338 internal metabolites. To use this network for flux balance analysis, the biomass composition of Xcc B100 was determined. The comprehensive model obtained was applied for in silico analyses to predict biomass generation and gene essentiality. Predictions were extensively validated by analyzing batch culture performance and by carbon balancing including xanthan production. Single gene deletion mutants causing deficiencies in the central carbohydrate metabolism were constructed to enforce major flux redistributions. The impact of xanthan production was studied in vivo and in silico, comparing the physiology of a gumD mutant, negative in xanthan production, with the original strain. The results indicate a redistribution of resources from xanthan to biomass, rather than a reduction in carbon uptake. With this high quality metabolic model, both systems biology analyses and synthetic biology reengineering of Xcc gained an important tool.

► A large-scale metabolic network of Xcc was reconstructed with 437 reactions and 338 metabolites.
► The comprehensive model has no dead ends and allows quantitative analysis of metabolic processes.
► The enzyme-encoding genes edd, tkt, pgi, pfkA, and gumD were deleted and functionally analyzed.
► Mutants deficient in metabolic key reactions enabled verification of simulation results.
► Parameters relevant for xanthan biosynthesis were analyzed in a bioreactor.