N-乙酰氨基葡萄糖(N-acetylglucosamine,GlcNAc)在食品、保健品以及药品领域有着广泛的应用。该课题组前期构建了一株具有较高GlcNAc合成效率的枯草芽孢杆菌底盘细胞FMIK,由于GlcNAc前体的过量积累引起了磷酸糖胁迫,限制了FMIK的 GlcNAc产量。因此,该文首先通过实施荧光定量PCR验证磷酸糖胁迫对于glcR-ywpJ操纵子转录水平影响,并通过凝胶迁移率确定GlcR与PglcR的结合能力;然后,利用易错PCR构建PglcR-ep突变体库,设计并使用基于荧光检测的高通量筛选系统,鉴定得到PglcR与GlcR结合的关键区域;最后,在FMIK菌株的基础上对筛选得到的PglcR与GlcR的结合关键区域进行敲除,得到工程菌株FMIK-m4。该菌株有效降低了GlcNAc前体浓度,解除了磷酸糖胁迫导致的二次生长现象,且摇瓶内GlcNAc产量提高至18.21 g/L。
N-acetylglucosamine (GlcNAc) is widely used in the fields of food, nutrition and drugs. In previous studies, our team constructed a chassis cell of Bacillus subtilis with high synthesis efficiency of GlcNAc through metabolic regulation. However, the excessive accumulation of GlcNAc precursor caused phosphosugar stress, which limited the yield of GlcNAc. In this study, the effect of phosphate stress on the transcription level of glcR-ywpJ operon was verified by qRT-PCR; the binding ability between GlcR and PglcR was determined by EMSA. Then, a PglcR-ep mutant library was constructed by error prone PCR, and a high-throughput screening system based on fluorescence detection was designed to identify the key regions of PglcR binding to GlcR. Finally, on the basis of FMIK strain, the key binding regions between PglcR and GlcR were knocked out, and the engineering strain FMIK-m4 was obtained. This strain effectively reduced the concentration of GlcNAc precursor, relieved the secondary growth phenomenon caused by phosphate sugar stress, and increased the yield of GlcNAc in the shake flask to 18.21 g/L.
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