Date Published: April 18, 2018
Publisher: Springer Berlin Heidelberg
Author(s): Feng Shi, Mingyue Luan, Yongfu Li.
Glutamate decarboxylase (GAD) converts l-glutamate (Glu) into γ-aminobutyric acid (GABA). Corynebacterium glutamicum that expresses exogenous GAD gene, gadB2 or gadB1, can synthesize GABA from its own produced Glu. To enhance GABA production in C. glutamicum, ribosomal binding site (RBS) sequence and promoter were searched and optimized for increasing the expression efficiency of gadB2. R4 exhibited the highest strength among RBS sequences tested, with 6 nt the optimal aligned spacing (AS) between RBS and start codon. This combination of RBS sequence and AS contributed to gadB2 expression, increased GAD activity by 156% and GABA production by 82% compared to normal strong RBS and AS combination. Then, a series of native promoters were selected for transcribing gadB2 under optimal RBS and AS combination. PdnaK, PdtsR, PodhI and PclgR expressed gadB2 and produced GABA as effectively as widely applied Ptuf and PcspB promoters and more effectively than Psod promoter. However, each native promoter did not work as well as the synthetic strong promoter PtacM, which produced 20.2 ± 0.3 g/L GABA. Even with prolonged length and bicistronic architecture, the strength of PdnaK did not enhance. Finally, gadB2 and mutant gadB1 were co-expressed under the optimal promoter and RBS combination, thus converted Glu into GABA completely and improved GABA production to more than 25 g/L. This study provides useful promoters and RBS sequences for gene expression in C. glutamicum.
γ-Aminobutyric acid (GABA) is a non-protein amino acid widely synthesized by diverse microorganisms, plants and animals (Li and Cao 2010; Shi et al. 2016). It functions as the major inhibitory neurotransmitter for the mammalian central nervous system and has several therapeutic abilities including hypotension, anti-anxiety, anti-depression, anti-schizophrenia, diuresis and anti-obesity (Wong et al. 2003; Mohler 2012; Shi et al. 2016). Therefore, GABA has been used as a bioactive component in functional foods and feeds. In addition, GABA is a crucial building block for the synthesis of bio-plastics, such as the biodegradable polyamide nylon 4 (Park et al. 2013).
To improve GAD expression and GABA production in recombinant C. glutamicum SH, the RBS sequences and promoters for expressing GAD gene were explored here. As expression of gadB2 produced more GABA in C. glutamicum than expression of gadB1 (Shi and Li 2011), gadB2 was selected for engineering of RBS sequence and promoter.
This study aims to ascertain an optimal combination of promoter and RBS sequence applying for the expression of GAD gene(s) and production of GABA in C. glutamicum. The expression level of heterologous genes has been illustrated to be influenced by multiple factors, including gene dosage, promoter strength, secondary structure of mRNA and RBS sequence (Stenstrom et al. 2001; Salis et al. 2009). To obtain the optimal expression element, successive investigation of RBS sequences and promoters was conducted.