Abstract:

Glutamic acid decarboxylase (GAD) is a rate-limiting enzyme for the biocatalysis of γ-aminobutyric acid
(GABA). Therefore, GAD gene is the key gene to regulate the production of GABA. In order to improve the productive
capacity of GABA in yeasts, in the present study, we used the CTAB method to purify S. cerevisiae 28 genomic DNA as the
template. A 1 758 bp gene fragment was amplified and sequenced as the GAD1 gene that had 98.58% homology with the
S. cerevisiae S288c gene. The primers were designed for amplifying the 3 490 bp sequence of GAD1 full-length gene,
including GAD1 gene upstream promoter, regulatory sequence and downstream terminator. The full-length gene sequence
was cloned into plasmid pUG6, to construct the recombinant plasmid p28. The strain 28 was used as the starting strain
for the conversion, and the transformant was gained by G418 resistance screening, and further verified by PCR, plasmid
extraction and double digest identification as the recombinant DL28. By exploring the characteristics of the recombinant
plasmid p28, we found that it had good genetic stability, and the recombinant plasmid was still stable after subculture for 10
generations without resistance training. The enzyme activity was determined after transformation, and the GAD activity of
recombinant DL28 was improved by 73.8% when compared with the strain 28. Therefore, GAD gene is highly expressed in
recombinant DL28, which also has higher G418 resistance.

Key words: S. cerevisiae, γ-aminobutyric acid, glutamic acid decarboxylase, cloning, expression