Enhanced Expression Of Glr Gene Encoding Glutamate Racemase And Its Regulatory Effects On Synthesis Of Poly-γ-Glutamic Acid

The poly-y-glutamic acid (γ-PGA) is a viscous slime biopolymer containing D-and L-glutamate residues produced mainly by microbes. It has broad applications in fields of cosmetics, food, agriculture, medicinal industries and many others. However, y-PGA with different stereochemical composition has different applications.The glr gene from a newly isolated strain Bacillus licheniformis WX-02, encoding glutamate racemase involved in the conversion of L-glutamic acid to the D-isomer was cloned and expressed in B. licheniformis WX-02. Shake flask fermentation experiments preliminarily showed that:overexpression of the glr gene in B. licheniformis WX-02 not only increased the yield of y-PGA by 22.5%, but also increased the proportion of D-glutamate in y-PGA from 77% to 85%. The activity of glutamate racemase in expressing strain was higher than in the original strain throughout cultivation time. This is the first report that overexpression of the glr gene could participate in the L-and D-glutamate conversion in B. Licheniformis and increase the proportion of D-glutamate in y-PGA and the yield of y-PGA.In this study, we constructed a glutamate racemase knockout vector pDG780-194orits-Kan-AB and transformed it into B. licheniformis WX-02. PCR analysis indicated that pDG780-194orits-Kan-AB has been successfully single exchanged into the chromosome. However, no mutant strain was isolated from our screen.Furthermore, in this study, the gdh gene, which encodes glutamate dehydrogenase involved in the conversion of a-ketoglutarate to L-glutamic acid, was cloned and expressed in B. licheniformis WX-02. Shake flask fermentation experiments preliminarily showed that:overexpression of the gdh gene in B. licheniformis WX-02 did not increase the yield of y-PGA.