Molecular Cloning And Expression Of The Glutamic Acid Decarboxylase Gene From Saccharomyces Cerevisiae For Biosynthesis Of γ-Amino Butyric Acid

γ-Amino Butyric Acid(GABA),a non-protein amino acid,is a natural active constituent in some organisms.GABA can lower blood pressure,function as an anti-anxiety substance,cure epilepsy,tranquilize the mind,control asthma,regulate hormone secretion,promoting the reproduction and activate hepatorenal,etc. Preparation and application of GABA were received by people's concern and attention. The Glutamic Acid Decarboxylase(GAD,EC4.1.1.15) catalyze L-glutamate byα-decarboxylation reaction to generate GABA.It is rate-limiting enzyme for biocatalysis GABA.GAD is the key gene to regulate the production of GABA.In this study,screening a yeast strain of high-yielding GABA was used as the GAD sources.A full-length GAD of Saccharomyces cerevisiae was cloned by PCR. The gene was sequenced and analyzed by homology and function.Besides,the similarity of GAD between S.cerevisiae and other microorganisms was explored. Recombinant expression vectors which contained S.cerevisiae GAD were constructed and transformed into Escherichia coli BL21(DE3) prokaryotic expression host cells and Pichia pastoris GS115 yeast eukaryotic expression system.The strains of efficient expression were selected and the expression products of S.cerevisiae GAD were analyzed by SDS-PAGE.The major experiment results of dissertation were as follows:1.Screening of yeast for biosynthesisγ-amino butyric acid56 strains of yeast which could produceγ-amino butyric acid were isolated from the fruit surface of pears,apples and kiwi,etc.A strain named MJ2 was comparatively high-yielding GABA(4.727 g/L).MJ2 strain was identified as S.cerevisiae, according to morphology and biochemistry.The culture substrate and fermentation conditions of MJ2 were optimized by ways of single factor experiment and L₉(3⁴) orthogonal test.The results showed that when soluble starch as carbon source,beef paste as nitrogen source,beginning pH 5.5,culture temperature at 31℃,shaking speed 180 rpm,the higher yield ofγ-Amino butyric acid could be reached.The content of GABA was 7.539 g/L in the optimum culture conditions.2.Cloning and sequence analysis of S.cerevisiae GADMJ2 yeast strain was used as the GAD sources.Yeast genomic DNA was extracted using improved liquid nitrogen-CTAB method.A pair of specific primers were designed bases both ends of S.cerevisiae GAD sequences region.A fragment of 1758 bp-length named Scgad was cloned by PCR.Identity analysis of full-length Scgad sequence shows 98%,69%and 67%similarity with S.cerevisiae,Candida glabrata and Kluyveromyces lactis GAD sequence,respectively.The obtained full length Scgad(1758 bp) contained an open reading frame(1755 bp) encoding 585 amino acids,the molecular weight of its deduced polypeptide was 65.897 kDa.The deduced amino acids sequence of Scgad shows 100%,65%and 62%similarity with S. cerevisiae,C.glabrata and K.lactis GAD in the polypeptide level,respectively.3.Prokaryotic Expression of Saccharomyces cerevisiae GADThe obtained gene encoding the nucleotide sequence of Scgad was inserted into the prokaryotic expression vector pET30a(+).The recombinant prokaryotic expression vector pET30gad was constructed,identified both by PCR and dual-enzyme digestion,in order to check it had been transformed into E.coli BL21 (DE3) prokaryotic expression host cells.The exogenous recombinant protein induced expression by IPTG,were analyzed by SDS-PAGE.4.Eukaryotic Expression of Saccharomyces cerevisiae GADThe obtained gene encoding the nucleotide sequence of Scgad was inserted into the Pichia pastoris yeast expression vector pPIC9K.The recombinant eukaryotic expression vector pPIC9Kgad was constructed and integrated into the eukaryotic expression host cells' chromosome genomes of P.pastoris GS115.The exogenous recombinant protein were induced expression by methanol.The result of SDS-PAGE analysis on fermentation broth shows that Scgad was expressed and secreted in P. pastoris prokaryotic expression system finally.