AbstractBackgroundMicrobial engineering strategies that elicit global metabolic perturbations have the capacity to increase organism robustness for targeted metabolite production. In particular, perturbations to regulators of cellular systems that impact glycolysis and amino acid production while simultaneously decreasing fermentation by-products such as acetate and CO2 make ideal targets. Intriguingly, perturbation of the Carbon Storage Regulator (Csr) system has been previously implicated in large changes in central carbon metabolism in E. coli. Therefore, we hypothesized that perturbation of the Csr system through the CsrA-CsrB ribonucleoprotein complex might increase production of biofuels and their intermediates from heterologous pathways.ResultsWe engaged the CsrA-CsrB ribonucleoprotein complex of E. coli via overexpression of CsrB. CsrB is a 350-nucleotide non-coding RNA that antagonizes CsrA, an RNA-binding protein that regulates translation of specific mRNA targets. By using shotgun proteomics and targeted metabolomics we established that elevation of CsrB levels leads to alterations in metabolite and protein levels in glycolysis, the TCA cycle and amino acid levels. Consequently, we show that such changes can be suitably applied to improve the production of desired compounds through the native fatty acid and heterologous n-butanol and isoprenoid pathways by up to two-fold. We also observed concomitant decreases in undesirable fermentation by-products such as acetate and CO2.ConclusionsWe have demonstrated that simple engineering of the RNA-based Csr global regulatory system constitutes a novel approach to obtaining pathway-independent improvements within engineered hosts. Additionally, since Csr is conserved across most prokaryotic species, this approach may also be amenable to a wide variety of production hosts.

The mechanisms of Desulfovibrio vulgaris Hildenborough responses to long-term NaCl exposure were studied by global transcriptional and metabolite analyses. The growth of D. vulgaris was inhibited by high salinity, and salt inhibition could be relieved by an addition of amino acids (e.g., glutamate, alanine) or yeast extract. Salt shock (sudden increase in salt concentration) and salt adaptation (inoculating cells in the medium containing high concentrations of salt) showed a significant difference in respective transcriptomes. Salt adaptation induced expression of genes involved in amino acid biosynthesis and transportation, electron transfer, hydrogen oxidation, and general stress responses (e.g., heat shock proteins, phage shock proteins, and oxidative stress response proteins). Genes involved in energy metabolism, cell motility, and phage structures were repressed. Genes involved in Na+/H+ transport, K+ uptake and transportation, and proline biosynthesis and transportation were not significantly affected. Metabolite assays and external addition of amino acids into the growth medium of D. vulgaris suggest that amino acids, such as glutamate and alanine may accumulate as osmoprotectants in D. vulgaris. A conceptual model is proposed to link our observed results to currently available knowledge for further understanding the mechanisms of adaptation of D. vulgaris to sodium chloride.