谷氨酸棒杆菌中基于5'UTR及其下游序列的增强型表达载体构建

谷氨酸棒杆菌中基于5'UTR及其下游序列的增强型表达载体构建

Identify the 5'UTR (5'-untranslated region) of the gene with the highest protein abundance and its downstream sequence from the genome of Corynebacterium glutamicum CGMCC1.15647, and use these 5'UTRs and their downstream sequences to combine with two high-strength promoters, PH36 and Ptac, respectively, to construct a series of monocistronic and bicistronic expression vectors. The 5'UTR and its downstream sequence significantly enhance the expression intensity of the promoter, and the fluorescence intensity of pTac - B2826 - EGFP with the highest expression intensity is 3.6 times that of the positive control pTac - Positive. The selected enhanced expression vectors were used to successfully express the VHH protein (variable domain of heavy chain of heavy - chain antibody) in Corynebacterium glutamicum, and at the shake flask fermentation level, the secreted expression amount of VHH reached 85.4 mg/L. The selected bicistronic vector can become a favorable tool for overexpression of endogenous genes and production of recombinant proteins in Corynebacterium glutamicum.

从谷氨酸棒杆菌CGMCC1.15647基因组中鉴定蛋白丰度最高基因的5'UTR(5'-untranslated region)及其下游序列,并利用这些5'UTR及其下游序列与两种高强度启动子PH36和Ptac组合分别构建一系列单顺反子和双顺反子表达载体。5'UTR及其下游序列显著提升启动子的表达强度,其中表达强度最高的pTac-B2826-EGFP荧光强度是阳性对照pTac-Positive的3.6倍。使用筛选的增强型表达载体在谷氨酸棒杆菌中成功地表达VHH蛋白(variable domain of heavy chain of heavy-chain antibody),在摇瓶发酵水平,VHH的分泌表达量达到85.4 mg/L。筛选的双顺反子载体可成为谷氨酸棒杆菌内源基因过表达和重组蛋白生产的有利工具。