Systems metabolic engineering in the halophilic bacteria Chromohalobacter salexigens for overproduction of ectoines

Abstract

The halophilic broad salt-gi owing bacterium Nfiromofio/o#octer solezipens is a natui al producer of ectoines, compatible solutes with piomising biotechnological applications as power ful biostabilizei s in Biomedicine, kJolecular Biology, and Dermopharmacy, among other s. These solutes are synthesized and intracellularly accumulated by this bacterium, in iesponse to increasing salinity in the external medium, and thus adapting its metabolism to support this biosynthetic route. Due to the complex regulation of this metabolic adaptation, novel metabolic engineering appi oaches based on Systems Biology (omics analysis and genome-scale metabolic models) that allows the rational rewiring of cell metabolism to inci ease bioproduction have been applied for ectoines overproduction. To accomplish this purpose, in this Doctoral Thesis a piuned genome-scale metabolic model of this bacterium, obtained from a pievious one (iFP764), was iefined and used to piedict potential genetic targets by using differ ent algorithms. As a iesult, they suggested a set of genes to knockout and/or to be up/down-regulated to obtain overpioducing str ains at high and low salinities. In order to 3n s3/ico analyze the effect of these peitui Nations in the distribution of metabolic fluxes, additional computational methods were successfully applied to simulate the cellular metabolic iesponse at different salinities in mutant str ains of N. solezipens. kJoi eover, an integrative analysis of multiomics data derived from ti anscriptomic, pioteomic and metabolomics, obtained at differ ent conditions of salinities and temperatui e, was also car ried out to find metabolic cori elations that could complement these analysis. Furthermore, a number of new genetic tools have been impi oved and developed in this work to assist in the /u r/ro implementation of some of the genetic modification str ategies predicted by the metabolic model. Finally, multiple knockout mutants of U. sa/ez/peus were obtained and phenotypically char acterised by deter mining their growth and intracellular ectoines accumulation profiles at differ ent salinities. A significant increase in ectoines production (measui ed as yield and specific pioduction i ates) was found in some of the single and multiple mutant str ains indicating that the metabolic model is capable to efficiently simulate 3n si/ico the metabolism of N. soleziqens, and thus being able to piedict ectoine-over producing strains. These findings would be of great value for the improvement of the biotechnological exploitation of ectoines.