Efficient Expression Of Glutamic Acid Decarboxylase Using RBS Sequence And Promoter Strategy

?-aminobutyric acid?GABA?functions as the major inhibitory neurotransmitter in the mammalian central nervous system and has several therapeutic abilities including hypotension,anti-anxiety,etc.Therefore,GABA can be used in several fields.Previously,two glutamate decarboxylase?GAD?genes?gadB1 and gadB2?derived from Lactobacillus brevis Lb85 were expressed in an L-glutamate producing Corynebacterium glutamicum strain SH and successfully converted the endogenous L-glutamate into GABA.To improve GAD expression and GABA production in C.glutamicum,the ribosomal binding site?RBS?sequence and promoters for expressing GAD were optimized.The main results are as follows.?1?To improve translation efficiency of gadB2,12 strains expressing gadB2under different RBS sequence were constructed.The GAD activity of R4-B2?16.5±0.2 U/g DCW?increased by 52.8%as compared with that of R-B2.Meanwhile,GABA production of R4-B2?13.3±0.5 g/L?was 19.8%higher than that of R-B2.The results suggested that the RBS sequence of R4 was beneficial for gadB2 translation and GABA conversion.?2?Whereafter,the distance of aligned spacing?AS?between RBS and initiation codon was optimized.GAD activity?32.0±2.5 U/g DCW?and GABA production?20.2±0.3 g/L?of R4a-B2 with AS of 6 nt were obviously higher than those of R4-B2with AS of 7 nt and R4b-B2 with AS of 8 nt.The results suggested that the AS of 6 nt with RBS of R4?R4a?was more preferable for translation of GadB2 and production of GABA in C.glutamicum.?3?To improve transcription level of gadB2,three types of native promoters were researched.First,gadB2 was expressed by 7 constitutive promoters separately.However,the transcription level of gadB2 and the activity of GAD?1.1-7.3 U/g DCW?of these strains were low.Among these 7 strains,PdtsR-B2 produced the highest level of GABA?16.4±0.1 g/L?.Then,gadB2 was expressed by 5 SigB-dependent promoters and PsigB.Transcription level of gadB2 was improved during GABA rapid conversion stage in this strains,however,GAD activity was not improved as compared with R4a-B2 under PtacM.The strain PsigB-B2 produced most GABA?9.9±0.8 g/L?among these 6 strains.Finally,6 stress-induced promoters were used to express gadB2.In the GABA conversion stage,the gadB2 transcription level of these6 strains was higher than that of R4a-B2 under PtacM,but the GAD activity was lower during fermentation.The highest production of GABA was obtained by PdnaK-B2?15.8 g/L?among these 6 strains.However,GABA production of all strains expressed by these three types of promoters was lower than that of R4a-B2,indicating that PtacM was the robust promoter for expression of gadB2.?4?Afterwards,the length of the strongest native promoter PdnaK was extended to obtain PdnaK?+1?,PdnaK?-1?,PdnaK?-2?,and PdnaK?-3?,and the dicistronic promoter PdnaK2SD was constructed to express gadB2.Elongation of PdnaK did not increase gadB2transcription level and GAD activity,but PdnaK?+1?-B2 and PdnaK?-1?-B2 produced the same level of GABA as the PdnaK-B2?about 16 g/L?.Although GAD activity of the strain under bicistronic promoter PdnaK2SD increased,its GABA production did not increase.?5?To further improve the GAD activity and yield of GABA,gadB1^mut was co-expressed with gadB2,both under the robust promoter of PtacM and RBS sequence of R4a.GAD activity of co-expressing strain?127±18 U/g DCW?increased by 3-fold and GABA production increased by 24.8%as compared to R4a-B2.Therefore,co-expression of gadB1^mut and gadB2 under PtacM promoter and R4a RBS sequence can improve GAD activity and GABA production in C.glutamicum.