从枯草芽孢杆菌1A 747菌株基因组克隆Am yX基因信号肽序列,构建枯草芽孢杆菌分泌表达载体pYGAm yX,转化枯草杆菌W B 700得W B 700(pYGAm yX)。采用重叠PCR技术,将Am yX基因信号肽突变,使其N端正电荷增加,H区疏水性降低,利用突变后信号肽序列构建枯草杆菌分泌表达载体pYGMUT,转化枯草杆菌W B 700得W B 700(pYGMUT)。对W B 700(pYGAm yX)和W B 700(pYGMUT)的表达产物进行检测,研究Am yX基因信号肽突变对枯草杆菌蛋白分泌途径的影响。结果表明,W B 700(pYGAm yX)和W B 700(pYGMUT均能表达β-半乳糖苷酶,W B 700(pYGAm yX)的最高胞外酶活性达2 300 U,W B 700(pYGMUT)的最高酶活性达5 200 U。结果表明Am yX基因信号肽突变后构建的表达载体pYGMUT分泌表达β-半乳糖苷酶明显提高,说明β-半乳糖苷酶通过枯草杆菌T at途径分泌表达。
An integrated expression vector pGT9GH was constructed by using guaC gene of receptor strain as guide sequence for homologous recombination.And then,by using the method of double crossover recombination between plasmid and chromosome,the linearized plasmid pGT9GH were integrated into B.subtilis GJ06 to get B.subtilis GJ07 with guaC gene mutant,in which,one copy of riboflavin operon was inserted between 5' and 3' end of guaC gene.The homologous recombination events were confirmed by PCR methods,and then,the riboflavin yield of mutant strain GJ07 was measured.The results of shake flask culture showed that the production of riboflavin of GJ07 was 24.5% higher than that of GJ06 in 60 h.
