Metabolic engineering of high-salinity-induced biosynthesis of γ-aminobutyric acid improves salt-stress tolerance in a glutamic acid-overproducing mutant of an ectoine-deficient .

A moderately halophilic eubacterium, , has been used as cell factory to produce fine chemical 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine), which functions as a major osmolyte protecting the cells from high-salinity stress. To explore the possibility of using to biosynthesize other valuable osmolytes, an ectoine-deficient salt-sensitive deletion mutant strain KA1 (Δ), which only grows well in minimal medium containing up to 3% NaCl, was subjected to an adaptive mutagenesis screening in search of mutants with restored salt tolerance. Consequently, we obtained a mutant, which tolerates 6% NaCl in minimal medium by overproducing L-glutamic acid (Glu). However, this Glu-overproducing (GOP) strain has a lower tolerance level than the wild-type , possibly because the acidity of Glu interferes with the pH homeostasis of the cell and hinders its own cellular accumulation. Enzymatic decarboxylation of Glu to γ-aminobutyric acid (GABA) by a Glu decarboxylase (GAD) could restore cellular pH homeostasis; therefore, we introduced an engineered salt-inducible gene, which encodes a wide pH-range GAD mutant, into the genome of the GOP strain. We found that the resulting GOP-Gad strain exhibits higher salt tolerance than the GOP strain by accumulating high concentration of GABA as an osmolyte in the cell (176.94 µmol/g cell dry weight in minimal medium containing 7% NaCl). With OUT30018 genetic background, GOP-Gad strain can utilize biomass-derived carbon and nitrogen compounds as its sole carbon and nitrogen sources, making it a good candidate for the development of GABA-producing cell factories.IMPORTANCEWhile the wild-type moderately halophilic can synthesize ectoine as a high-value osmolyte via the aspartic acid metabolic pathway, a mutant GOP strain identified in this work opens doors for the biosynthesis of alternative valuable osmolytes via glutamic acid metabolic pathway. Further metabolic engineering to install a GAD system into the GOP strain successfully created a GOP-Gad strain, which acquired higher tolerance to salt stress by accumulating GABA as a major osmolyte. With the ability to assimilate biomass-derived carbon and nitrogen sources and thrive in high-salinity environment, the GOP-Gad strain can be used in the development of sustainable GABA-producing cell factories.