Ribosomal binding site sequences and promoters for expressing glutamate decarboxylase and producing \u03b3-aminobutyrate in Corynebacterium glutamicum

Feng Shi,Mingyue Luan,Yongfu Li

doi:10.1186/s13568-018-0595-2

Feng Shi, Mingyue Luan + Show 1 more

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https://doi.org/10.1186/s13568-018-0595-2

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Abstract

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.

Highlights

Introduction γAminobutyric acid (GABA) is a non-protein amino acid widely synthesized by diverse microorganisms, plants and animals (Li and Cao 2010; Shi et al 2016)
γ-aminobutyric acid (GABA) is mainly converted from l-glutamate (Glu) by lactic acid bacteria (LAB) and Escherichia coli strains with high Glu decarboxylase (GAD) activity (Li and Cao 2010; Shi et al 2016)
Optimization of ribosomal binding site (RBS) sequence for gadB2 expression and GABA production RBS is a pivotal region for controlling translation initiation and protein expression

Summary

Introduction

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 antiobesity (Wong et al 2003; Mohler 2012; Shi et al 2016). Glu must be added as the precursor during GABA production by LAB

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