| Gamma-aminobutyric acid (GABA) is a nonprotein amino acid known to be a majorinhibitory neurotransmitter in the mammalian nervous systems, and it has many physiologicalproperties. GABA is mainly synthesized through the decarboxylation of L-glutamate byglutamate decarboxylase (GAD). In this process of GABA preparation, the precursorL-glutamate or monosodium glutamate (MSG) must be added. In this work, microbialfermentation of GABA from cheap raw materials such as glucose was achieved.Firstly, the ΔargB fragment that lacked491bp of N-acetylglutamate kinase (NAGK)gene which encodes the key enzyme of L-arginine metabolic pathway in C. crenatum SYPA5-5was obtained by overlap PCR. The homologous integrative vector pK18-ΔargB::tacgadwas constructed by linking ΔargB to pK18mobsacB. The argB knock-out strain crenatum E01was obtained through two homologous recombination. Fermentation was carried out withglucose as substrate, after36hours,34g/L L-Glu was detected, however, no L-Arg wasdetectd.To construct a recombinant C. crenatum that could direct convert glucose into GABA,the GAD encoding gene lpgad from Lactobacillus plantarum GB01-21was amplified andcloned in the E. coli/C. crenatum shuttle vector pJC-tac. The recombinant plasmid pGAD wastransformed into C. crenatum E01. As a result, GAD was successfully expressed inrecombinant strain E01/pGAD. The fermentation was carried out with glucose as substrate for96hours, and the final yield of GABA was13.66g/L.Considering the safety and stability, GAD gene was integrated onto the C. crenatumSYPA5-5genome to construct an integrative recombinant strain. Firstly, the GAD geneattched tac promoter was inserted into the ΔargB fragment. Using the upstream anddownstream of ΔargB as homologous arms, we constructed pK18-ΔargB::tacgad which wasused for the integration of tacgad gene onto the C. crenatum SYPA5-5genome. Through twohomologous recombination, we got an argB blocked strain C. crenatum ΔargB::tacgad whichcould successfully express GAD. Fermentation was carried out with glucose as substrate for96hours, and8.58g/L GABA was detected.The stability tests showed that the recombinant strain C. crenatum ΔargB::tacgaddisplayed better stability than E01/pGAD. After six times of inoculations, plasmid retentionrate of E01/pGAD was more than88%, which demonstrated good stability but also a certaindegree of degradation. Each C. crenatum ΔargB::tacgad culture was identified by colony PCRusing primers of argB gene, and the products of six C. crenatum ΔargB::tacgad cultures wereΔargB::tacgad gene fragments, which showed the good stability of C. crenatumΔargB::tacgad. However, the GAD enzyme activity of C. crenatum ΔargB::tacgad was lowerthan E01/pGAD. The highest GAD enzyme activities in C. crenatum ΔargB::tacgad andE01/pGAD were2.85U/mL and4.07U/mL respectively, which were6.8%and9.7%of L.plantarum GB01-21.The preliminary fermentation optimization results were as follows: addition time of ureawas28h, glucose concentration was130g/L, and added content of PLP was0.1mmol/L. Through the optimization, GABA productions of E01/pGAD and C. crenatum ΔargB::tacgadwere both promoted significantly. There were19.92g/L and14.89g/L GABA accumulated inE01/pGAD and C. crenatum ΔargB::tacgad fermentation broth respectively, which increasedby45.8%and73.5%compared with preliminary fermentation results.The fermentation of recombinant strains in5L fermentor were carried out. Thefermentation conditions were as follows: ventilation volume was1.5L/(L min), stirringspeed was600r/min, and culture temperature was30℃. During the initial stage offermentation, the pH was controlled at7.2by adding ammonia until28h. During the laterperiod of fermentation, GABA was accumulated, and the final GABA yields of E01/pGADand C. crenatum ΔargB::tacgad were26.25g/L and19.44g/L respectively. In this study,GAD was successfully expressed in C. crenatum. This research constructed an high safty andgood stability recombinant strain, and provided a new approach for GABA production. |
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