代谢改造黑曲霉合成乙酰氨基葡萄糖

Metabolic Engineering of Aspergillus niger to Produce N-acetylglucosamine

该研究以黑曲霉为底盘细胞,首先敲除N-乙酰氨基葡萄糖(N-acetylglucosamine,GlcNAc)摄取相关转运蛋白ngtA编码基因,获得GlcNAc的吸收利用缺陷型菌株,有利于胞外GlcNAc的积累。在此基础上,通过表达大肠杆菌来源的卤酸脱卤酶样磷酸酶编码基因yqaB,构建黑曲霉GlcNAc的完整合成途径,实现GlcNAc的合成,产量为1.78 g/L。通过共过表达氨基葡萄糖-6-磷酸乙酰转移酶基因gnaA和氨基葡萄糖-6-磷酸合成酶基因gfaA强化GlcNAc合成途径,GlcNAc的合成水平提升至3.64 g/L。为增加GlcNAc合成前体GlcNAc6P的胞内供给,利用RNA干扰技术对乙酰氨基葡萄糖-6-磷酸(GlcNAc6P)分解代谢途径中氨基葡萄糖-6-磷酸脱氨基酶基因nagB和乙酰氨基葡萄糖-6-磷酸脱乙酰酶基因nagA基因表达进行弱化表达,GlcNAc产量进一步提高至4.03 g/L。为减弱黑曲霉糖酵解途径与GlcNAc合成途径竞争共同前体物质果糖-6-磷酸,对磷酸果糖激酶基因pfkA进行弱化表达,GlcNAc的最终产量为4.61 g/L。

In this study,Aspergillus niger was used as the chassis cell,and the uptake transporter ngtA,the encoding gene of N-acetylglucosamine(GlcNAc),was firstly knocked out to obtain a defective strain for absorbing and utilizing GlcNAc,which was beneficial to the accumulation of extracellular GlcNAc.On this basis,a complete synthesis pathway of GlcNAc in A.niger was constructed by expressing the encoding gene yqaB of haloacid dehalogenase-like phosphatase from Escherichia coli,and the synthesis of GlcNAc was successfully achieved in A.niger with a yield of 1.78 g/L.By co-overexpressing the glucosamine-6-phosphate acetyltransferase gene gnaA and the GlcN6P synthase gene gfaA,the GlcNAc biosynthesis pathway was enhanced,and the synthesis level of GlcNAc increased to 3.64 g/L.To increase the intracellular supply of GlcNAc6P,the synthesis precursor of GlcNAc,attenuated expressions were performed to the glucosamine-6-phosphate deaminase gene nagB and acetylglucosamine-6-phosphate deacetylase gene nagA in GlcNAc6P catabolic pathway by RNA interference.The yield of GlcNAc further increased to 4.03 g/L.To reduce the competition of fructose 6-phosphate between the glycolytic pathway and GlcNAc synthesis pathway of A.niger,the phosphofructokinase gene pfkA was weakened,and the final yield of GlcNAc increased to 4.61 g/L.