Abstract
BACKGROUND: Gene manipulation has a wide array of applications in microorganisms. We can construct multifunctional bacterial strains by gene manipulation and gene editing in order to produce several industrial biomaterials including enzymes at the same time. OBJECTIVE: According to the importance of cellulase in various industries, including food industry, the purpose of this study was aimed to produce cellulase in an indigenous Bacillus cereus EG296 strain through gene manipulation. MATERIALS AND METHODS: The Bacillus subtilis 168 cellulase gene, located between the regulatory upstream and downstream regions of Bacillus cereus protease gene (aprE), was amplified by SOEing PCR and transformed into the Bacillus cereus EG296 by natural transformation. After selection of the strains with cellulase activity, the scoC gene (Negative transcriptional regulator of aprE gene) was also deleted from the genome of the transformant by homologous recombination in order to increase the cellulase and protease activities simultaneously. RESULTS: The Bacillus cereus cells were acquired the cellulase gene into their genome with cellulase activity of about 0.61 u.mL(-1). By scoC gene deletion, the protease activity reached to 363.14 u.mL(-1) from 230 u.mL(-1). Meanwhile, the cellulase activity under the control of the protease promoter was also increased to 0.78 u.mL(-1) from 0.61 u.mL(-1). The cellulase and protease expressed in B. cereus have an instability index of 26.16 and 20.18 respectively, which is much lower than threshold of 40. Accordingly, it can be concluded that both enzymes are considered to be stable. CONCLUSION: As a result, we obtained a genetically engineered strain that had the ability to produce and secrete two important industrial extracellular enzymes (cellulase and protease), with easy downstream purification processes.