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Improvement in the osmotolerance ofEscherichia coli is essential for the production of high titers of various bioproducts. In this work, acusS mutation that was identified in the previously constructed high-succinate-producingE. coli strain HX024 was investigated for its effect on osmotolerance. CusS is part of the two-component system CusSR that protects cells from Ag(I) and Cu(I) toxicity. ChangingcusS from strain HX024 back to its original sequence led to a 24% decrease in cell mass and succinate titer under osmotic stress (12% glucose). When cultivated with a high initial glucose concentration (12%), introduction of thecusS mutation into parental strain Suc-T110 led to a 21% increase in cell mass and a 40% increase in succinate titer. When the medium was supplemented with 30 g/liter disodium succinate, thecusS mutation led to a 120% increase in cell mass and a 492% increase in succinate titer. Introducing thecusS mutation into the wild-type strain ATCC 8739 led to increases in cell mass of 87% with 20% glucose and 36% using 30 g/liter disodium succinate. ThecusS mutation increased the expression ofcusCFBA , and gene expression levels were found to be positively related to osmotolerance abilities. Because high osmotic stress has been associated with deleterious accumulation of Cu(I) in the periplasm, activation of CusCFBA may alleviate this effect by transporting Cu(I) out of the cells. This hypothesis was confirmed by supplementing sulfur-containing amino acids that can chelate Cu(I). Adding methionine or cysteine to the medium increased the osmotolerance ofE. coli under anaerobic conditions.IMPORTANCE In this work, an activating Cus copper efflux system was found to increase the osmotolerance ofE. coli . In addition, new osmoprotectants were identified. Supplementation with methionine or cysteine led to an increase in osmotolerance ofE. coli under anaerobic conditions. These new strategies for improving osmotolerance will be useful for improving the production of chemicals in industrial bioprocesses.

Osmotolerance in Escherichia coli Is Improved by Activation of Copper Efflux Genes or Supplementation with Sulfur-Containing Amino Acids