CRISPR-Cas9 Mediated Knockout of LacZ Gene in Escherichia Coli for Enhanced Production of Asparaginase

Genome editing with CRISPR-Cas9 offers a powerful approach for enhancing enzyme production in microorganisms. This study aimed to genetically engineer the lacZ gene in Escherichia coli using CRISPR-Cas9 to evaluate its impact on asparaginase production during submerged fermentation with rice bran serving as a glucose source. Both edited and wild-type E. coli strains were cultured at optimal conditions to produce and characterize asparaginase. The edited E. coli formed distinct colonies, displaying a blue phenotype when exposed to Cas9 without sgRNA or arabinose, yielding a total of 96 colonies. No colonies were observed when Cas9 and sgRNA were present without arabinose, while the addition of Cas9 and arabinose without sgRNA resulted in 309 blue colonies. With Cas9, sgRNA, and arabinose present, repair activation produced 114 distinct white colonies. The editing of the lacZ gene was validated through multiplex PCR and gel electrophoresis, with bands at 650 bp indicated lacZ gene editing, while bands at 1,100 bp indicated the wild-type. Asparaginase production was assessed using plate method assay, submerged fermentation using rice bran as a glucose source, and subsequent purification via ammonium sulfate precipitation and ion-exchange chromatography. Ion-exchange chromatography revealed enhanced purity and activity in the edited strain, with peak activity observed at an elution of 80 mL. The CRISPR-Cas9 edited strain exhibiting significantly higher enzyme activity (1.2 ± 0.002 U/ mL) compared to the wild-type (0.8 ± 0.005 U/mL). Both strains demonstrated maximum asparaginase activity at 40 ^o C and pH 7. This study concludes that CRISPR-Cas9 meditated lacZ gene editing in E. coli improves its ability to utilize rice bran as a substrate, significantly enhancing asparaginase production. These findings highlight the potential of genetic engineering and agricultural by-products for sustainable enzyme production.