The high level food-grade expression of glutamate decarboxylase in Bacillus subtilis through combination of genomic integration and free plasmid

gamma-Aminobutyric acid (GABA) is a bioactive compound with diverse physiological functions. It has a wide range of applications in food and medicine and mainly biosynthesized through glutamate decarboxylase (GAD) catalysis. Bacillus subtilis is recognized for its robust secretion capabilities and high food safety standards, making it a prevalent choice for recombinant protein expression. In traditional industrial enzyme production in B. subtilis, antibiotics are required to sustain plasmid stability. However, the incorporation of antibiotics fails to align with the criteria applicable to enzymes for use in the food industry. To eliminate the need for antibiotics in the production of GAD preparations, we constructed a marker-free recombinant strain B. subtilis WS9D-GAD. This strain was developed based on antibiotic-free multi-copy gene expression vector pUBDAL-amyL and d-alanine racemase (dal)-deficient B. subtilis WS9D, ultimately enabling the food-grade expression of GAD. To enhance GAD expression levels, we integrated the gadA expression cassette into the genome of B. subtilis using the Cre/lox system method. Additionally, strain WS9C6D-GAD was generated through the co-expression of free plasmid and genome integration, which carried the free plasmid pUBDAL-gadA and featured six copies of the gadA expression cassette within its genome. The enzyme activity during shake flask fermentation reached 28.15 U mL(-1), while the enzyme activity in high-density 3-L fermenter culture reached 199.49 U mL(-1), marking the highest level of food-grade GAD expression with a multitude of potential applications. This study presents an effective strategy for the expression of food-grade industrial enzymes in B. subtilis.