Long-term continuous adaptation of Escherichia coli to high succinate stress and transcriptome analysis of the tolerant strain.

To understand the responses of Escherichia coli to high succinate stress and to determine the roles of upregulated genes in high succinate tolerance, a continuous culture of wild-type E. coli W3110 was performed for 268 days in a gradually increasing concentration of succinate. Growth of the final adapted strain, designated DST160, proceeded growth rate of 0.20 h(-1) without a lag phase in medium containing 0.592 M succinate, while the wild-type strain showed 0.02 h(-1) in 38 h. The growth rates of DST160 in media containing either 0.61 M NaCl, 0.61 M KCl, or at pH 4.5 were 25% higher, 18% lower, and 57% higher than those of wild-type, respectively, implying DST160 acquired salt tolerance and pH shock tolerance as well as succinate tolerance. DNA microarray and real-time PCR results indicated that genes controlling active transport and biosynthesis of osmoprotectants were upregulated in DST160 compared to W3110. When ygjE, encoding a putative tartrate/succinate antiporter, and betA, encoding betaine biosynthesis, were expressed in a wild-type E. coli as represent genes for active transport and osmoprotectant synthesis, respectively, greater growth rates were achieved under 0.592 M succinate stress conditions (seven times higher due to ygjE expression and six times higher due to betA expression) than wild-type. The potential to design a metabolic engineering for microbial succinate production is suggested based on the transcriptional regulation of the long-term adapted DST160.