A novel toggle switch under acid-base regulation for tunable gamma-aminobutyric acid (GABA) production in Escherichia coli

Background: The efficient utilization of natural regulatory networks offers a practical strategy for optimizing the production of crucial bio-chemicals. In recent years, acid resistance has emerged as a prominent regulatory network in Escherichia coli. Method: A tunable interplay between glutamate decarboxylase (GadA/B) and tryptophan operons as a novel regulatory tool in E. coli is developed. The protein of native Gad operon and tryptophanase A (TnaA) are analyzed under varying pH in four E. coli strains. Moreover, the influence of 4-aminobutyric acid (GABA) and indole on the expression of tnaA or Gad operon is individually explored under acid, neutral and base conditions. Afterwards, the regulation is further applied to realize the production of valuable chemical GABA. Significant findings: The activities of GadA/B correlate with indole formation induced by TnaA, displaying a notable relationship under varying pH conditions. The GadA promoter triggered by tryptophan, facilitates the conversion of glutamate to GABA. Conversely, TnaA activity, indicated by mRNA levels is inhibited by GABA. Therefore, tnaA-deficient mutants were used for tunable GABA fabrication via in vitro approaches, especially for base -favorable W3110 and acid-benign Nissle 1917 (EcN). Finally, GABA production was increased from 12 g/L to 59 g/L in the deficient strain E3. The results demonstrated the potential of acid/base regulation mediated by gad and tna operons as a novel toggle switch and control element for enhancing GABA production.