Red同源重组敲除nagE和manX对大肠杆菌发酵生产氨基葡萄糖的影响

摘要

氨基葡萄糖(GlcN)又称氨基葡糖或葡糖胺,是葡萄糖的一个羟基被氨基取代后的化合物,在医药和保健领域具有广泛应用.在前期研究中,我们构建了一株可高效合成GlcN和其乙酰化衍生物N-乙酰氨基葡萄糖(GlcNAc)的重组大肠肝菌Escherichia coli-glms-gnal(在下游提取过程中用弱酸进行脱乙酰化即可将GlcNAc转化为GlcN).但研究发现,发酵过程中GlcN和GlcNAc能被转运至胞内作为碳源利用,导致胞外产量显著减少.为阻断胞外GlcN和GlcNAc向胞内的转运,利用Red同源重组技术将E.coli-glms-gnal的乙酰氨基葡萄糖磷酸转运子编码基因nagE和甘露糖磷酸转运子编码基因manX敲除,获得nagE基因敲除的工程菌E.coli-glms-gnal-△nagE和nagE/manX基因双敲除的工程菌E.coli-glms-gnal-△nagE-△manX,并在7L发酵罐上利用构建的工程菌进行GlcN和GlcNAc的发酵生产.实验结果表明:培养对照菌株E.coli-glms-gnal 至12h时GlcN产量达到最大值4.06 g/L,GlcNAc产量达到最大值41.46 g/L；而培养单基因敲除菌株E.coli-glms-gnal-△nagE至12h时GlcN产量达到最大值4.38 g/L(是对照菌株的1.08倍),GlcNAc产量达到最大值71.80 g/L(是对照菌株的1.7倍)；培养双基因敲除菌株E.coli-glms-gnal-△nagE-△manX至10h时GlcN产量达到最大值4.82 g/L(是对照菌株的1.2倍),GlcNAc产量达到最大值118.78 g/L(是对照菌株的2.86倍).这表明nagE和manX基因的敲除可显著降低GlcN和GlcNAc向胞内的转运,进而提高其在胞外的积累.研究结果对最终实现GlcN的工业化生产具有一定的指导意义.%Glucosamine (GlcN), also called amino sugar, is a compound derived from the substitution of a hydroxyl group of glucose molecule with an amino group. GlcN finds a wide-range of applications in health food and pharmaceutical industries. In our previous research, a recombinant Escherichia coli-glms-gnal was constructed for the efficient production of GlcN and N-acetylglucosamine (GlcNAc), the latter can be readily deacetylated to GlcN under mild acidic conditions. However, the results indicated that the titer of GlcN and GlcNAc decreased significantly due to the transportation of GlcN and GlcNAc from the culture broth to the inside of cells. To alleviate or block the transportation process, nagE gene (encoding for the GlcNAc-specific transporter) and manX gene (encoding for the mannose transporter) were knocked out with the Red homologous recombination method, and two engineered strains, E. Coli-glms-gnal-AnagE (with nagE gene deletion) and E. Coli-glms-gnal-AnagE-AmanX (with nagE and manX genes deletion), were successfully constructed. The two strains were cultured in a 7-L fermentor for the production of GlcN and GlcNAc. The maximal GlcN concentration of control strain E. Coli-glms-gnal reached 4.06 g/L, and the maximal GlcNAc concentration reached 41.46 g/L. The maximal GlcN and GlcNAc concentration of E. Coli-glms-gnal-AnagE reached 4.38 g/L and 71.80 g/L, respectively, which were 1.08-fold and 1.70-fold of those of E. Coli-glms-gnal, respectively. The maximal GlcN and GlcNAc concentration of E. Coli-glms-gnal-AnagE-AmanX reached 4.82 g/L and 118.78 g/L, respectively, which were 1.20-fold and 2.86-fold of those of E. Coli-glms-gnal, respectively. These results suggested that the deletion of nagE and manX could significantly increase the extracellular accumulation of GlcN and GlcNAc. The results obtained here maybe useful for the microbial GlcN production in an industrial scale.