Over-Expression of Salt-Tolerant Glutaminase by Integration of Mglu Genes in Bacillus subtilis 168

Abstract:

Heterologous over-expression of glutaminase in microorganisms has been extensively studied and its application in industrial production has been limited by its poor separation stability under nonresistant conditions with free plasmids. To obtain a strain used in industrial glutaminase production, glutaminase（from Micrococcus luteus K-3） was successfully over-expressed by integration of the Mglu gene in chromosome of GRAS strain Bacillus subtilis 168. Two genes encoding specific protease were blocked by integration of Mglu gene, and the zeo resistance marker was deleted by Cre recombinase catalyzing the recombination of DNA between lox sequences. After continuous cell culture without adding any antibiotics, the enzyme activity was basically unchanged when the recombinant strain was transmitted for 42 generations. By using 5 L bioreactor for fed-batch fermentation of recombinant glutaminase producing strain, the highest enzyme activity was about 41.5 U/mL. This study provides a reference for improving the genetic stability of glutaminase recombinant strain.