Construction Of Engineering Yeast Strain Expressing Mn14-sod Gene And Research Of The Expression Condition

The metalloenzyme superoxide dismutases (SODs, EC1.15.1.1) are classified as the copper-zinc type (CuZn-SOD), the manganese type (Mn-SOD), the iron type (Fe-SOD) and the most recently described nickel type (Ni-SOD), iron-zinc type (FeZn-SOD) according to the metal ion cofactor required for their activity. Superoxide dismutases, which catalyze the conversion of superoxide radical ion into hydrogen peroxide and oxygen, play a central role in protecting organisms against oxidative stress.Bacillus cereus M22, a biocontrol agent isolated from the plant, could produce a high yield of SOD. It is hypothesized that SOD may be involved in the biocontrol process and facilitate the colonization of B. cereus M22.According to the sequence of previously cloned Mnl4-sod gene, a pair of specific primer had been designed to amplify Mn14-sod gene open reading frame. A recombinant plasmid pPICZαA-sodMn14 was constructed by inserting the encoding sequence into a yeast expression vector pPICZαA and then transformed into the yeast cell by electroporation. Positive transformants containing Mn14-sod gene Were identified by Zeocin^TM-resistance screening and PCR with specific primers. Three strains obtained, named P_MSA, P_MSB, P_MSC, respectively, showed significant activities of SOD in their fermentative supernatants by PAGE analysis.The optimum culture conditions of genetically engineered strain P_MSA in shake-flask cultivation were investigated. The maximum SOD activity of 211 U·mL^-1 in supernatant at the 72th hour was obtained when the culture suspension (OD₆₀₀=10) was induced by 1% (V/V) methanol.