Effect of DR1558, a Deinococcus radiodurans response regulator, on the production of GABA in the recombinant Escherichia coli under low pH conditions

Abstract

Background: Gamma aminobutyric acid (GABA) is an important platform chemical, which has been used as a food additive and drug. Additionally, GABA is a precursor of 2-pyrrolidone, which is used in nylon synthesis. GABA is usually synthesized from glutamate in a reaction catalyzed by glutamate decarboxylase (GAD). Currently, there are several reports on GABA production from monosodium glutamate (MSG) or glucose using engineered microbes. However, the optimal pH for GAD activity is 4, which is the limiting factor for the efficient microbial fermentative production of GABA as fermentations are performed at pH 7. Recently, DR1558, a response regulator in the two-component signal transduction system was identified in Deinococcus radiodurans. DR1558 is reported to confer cellular robustness to cells by binding the promoter regions of genes via DNA-binding domains or by binding to the effector molecules, which enable the microorganisms to survive in various environmental stress conditions, such as oxidative stress, high osmotic shock, and low pH. Results: In this study, the effect of DR1558 in enhancing GABA production was examined using two different strategies: whole-cell bioconversion of GABA from MSG and direct fermentative production of GABA from glucose under acidic culture conditions. In the whole-cell bioconversion, GABA produced by E. coli expressing GadBC and DR1558 (6.52 g/L GABA from 13 g/L MSG•H 2 O) in shake flask culture at pH 4.5 was 2.2-fold higher than that by E. coli expressing only GadBC (2.97 g/L of GABA from 13 g/L MSG•H 2 O). In direct fermentative production of GABA from glucose, E. coli ∆gabT expressing isocitrate dehydrogenase (IcdA), glutamate dehydrogenase (GdhA), GadBC, and DR1558 produced 1.7-fold higher GABA (2.8 g/L of GABA from 30 g/L glucose) than E. coli ∆gabT expressing IcdA, GdhA, and GadBC (1.6 g/L of GABA from 30 g/L glucose) in shake flask culture at an initial pH 7.0. The transcriptional analysis of E. coli revealed that DR1558 conferred acid resistance to E. coli during GABA production. The fed-batch fermentation of E. coli expressing IcdA, GdhA, GadBC, and DR1558 performed at pH 5.0 resulted in the final GABA titer of 6.16 g/L by consuming 116.82 g/L of glucose in 38 h. Conclusion: This is the first report to demonstrate GABA production by acidic fermentation and to provide an engineering strategy for conferring acid resistance to the recombinant E. coli for GABA production.

Improve gamma-aminobutyric acid production in Corynebacterium glutamicum by optimizing the metabolic flux

“…The highest GABA production (70.6 g/L) was obtained from an industrial l-Glu producer which produced 100-110 g/L l-Glu [29]. The highest GABA productions obtained in E. coli and wild-type C. glutamicum ATCC 13,032 were 6.16 g/L [25] and 38.6 g/L [30], respectively. These studies used plasmid expression systems, which need to supplement antibiotics in media, resulting in high production cost and environmental pollution [16].…”

“…The proper pH for l-Glu synthesis is 6.8-7.5. To date, the highest GABA titer was 6.16 g/L by E. coli fermentation [25].…”

Intracellular Flux Prediction of Recombinant Escherichia coli Producing Gamma-Aminobutyric Acid

“…In this study, E. coli DGB303 was used as a GABA-producing strain constructed by transforming pH3BN and pGB103 into E. coli GB300 (BL21(DE3) D gabT D sucA ) [ 19 ]. pH3BN expressing Neurospora crassa gadB and E. coli gadC converted glutamate into GABA.…”

“…More recently, DR1558, a response regulator from Deinococcus radiodurans , was successfully introduced into E. coli , improving cellular tolerance to external stresses, including low pH. This reaction is because GAD’s optimal pH is around 4.5, while E. coli fermentation is conducted near 7.0 [ 19 ]. In particular, DR1558 expression in recombinant E. coli was interesting because of the potentially high GABA productivity during cell growth.…”

Chromosomal editing of Corynebacterium glutamicum ATCC 13032 to produce gamma‐aminobutyric acid

“…The recent studies were focused on direct fermentation from cheap, sustainable, and safe food raw materials. Production of GABA by E. coli fermentation from glucose 38,39 and xylose 40 has been reported; unfortunately, the GABA titer was less than 6.5 g/L.…”