【摘要】 在核黄素操纵子过表达的枯草芽孢杆菌中,采用无标记基因敲除方法,分别敲除GMP还原酶基因(guaC)、腺苷琥珀酸合成酶基因(purA)、嘌呤操纵子(pur operon)阻遏蛋白编码基因簇(purR-yabJ),并且用cryⅢA mRNA稳定子替换嘌呤操纵子的mRNA前导区,构建了菌株LX35.采用实时定量PCR方法,测定其胞内嘌呤操纵子mRNA的相对水平.摇瓶发酵,测定其发酵液中的核黄素含量.结果显示,菌株LX35嘌呤操纵子的mRNA水平相对提高了53.33倍,核黄素产量提高了126.6%.使用cryⅢA mRNA稳定子,能够有效提高嘌呤操纵子的表达水平,有助于核黄素过量合成.更多还原

【Abstract】 Bacillus subtilis with over-expressed riboperon was engineered to be LX35,which the genes for GMP reductase(guaC),adenylosuccinate synthetase(purA),pur operon repressor(purR-yabJ)were inactivated by marker-free deletions,and the mRNA leader region of pur operon was replaced with the cryⅢA mRNA stabilizer.RT-PCR analysis indicated that the transcriptional level of pur operon was 53.33-fold higher in LX35 than the original strain.At the same time,shake flask results showed that the yield of riboflavin in LX35 was also increased by 126.6%in the comparison to the control strain.It suggested that the cryⅢA mRNA stabilizer could effectively improve the transcriptional level of pur operon,which made a contribution to the riboflavin synthesis.更多还原