Corynebacterium glutamicum is a traditional food-grade industrial microorganism, in which an efficient endotoxin-free recombinant protein expression factory is under developing in recent years. However, the intrinsic disadvantage of low recombinant protein expression level is still difficult to be solved. Here, according to the bacteria-specific polycistronic feature that multiple proteins can be translated in one mRNA, efforts have been made to insert a leading peptide gene upstream of target genes as an expression enhancer, and it is found that this can remarkably improve the expression level of proteins under the control of inducible tac promoter in C. glutamicum. In this research, the Escherichia coli (E. coli) tac promoter combined with 24 different fore-cistron sequences were constructed in a bicistronic manner in C. glutamicum. Three strong bicistronic expression vectors were isolated and exhibited high efficiency under different culture conditions. The compatibility of these bicistronic vectors was further validated using six model proteins- aldehyde dehydrogenase (ALDH), alcohol dehydrogenase (ADH), RamA (regulator of acetate metabolism), Bovine interferon-α (BoIFN-α), glycoprotein D protein (gD) of infectious bovine rhinotracheitis virus (IBRV) and procollagen type Ι N-terminal peptide (PΙNP). All examined proteins were highly expressed compared with the original vector with tac promoter. Large-scale production of PΙNP was also performed in fed-batch cultivation, and the highest PΙNP production level was 1.2 g/L. In this study, the strength of the inducible tac promoter for C. glutamicum was improved by screening and inserting fore-cistron sequences in front of the target genes. Those vectors with bicistronic expression patterns have strong compatibility for expressing various heterogeneous proteins in high yield. This new strategy could be used to further improve the performance of inducible promoters, achieving double competence of inducible control and high yield.

中文翻译：

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通过构建双顺反子基因表达系统，增强了谷氨酸棒杆菌中重组蛋白的生产。

谷氨酸棒杆菌是一种传统的食品级工业微生物，近年来，正在开发一种高效的无内毒素的重组蛋白表达工厂。然而，重组蛋白表达水平低的内在缺点仍然难以解决。在此，根据细菌特异性的多顺反子特征，即可以在一个mRNA中翻译多种蛋白质，已经进行了努力，将前导肽基因插入靶基因的上游作为表达增强剂，发现这可以显着改善表达。谷氨酸棒杆菌中可诱导的tac启动子控制下蛋白质的水平。在这项研究中，以双顺反子方式在谷氨酸棒杆菌中构建了与24个不同的顺反子正反序列相结合的大肠杆菌tac启动子。分离了三种强双顺反子表达载体，并在不同培养条件下显示出高效率。这些双顺反子载体的兼容性使用六种模型蛋白进一步验证：醛脱氢酶（ALDH），醇脱氢酶（ADH），RamA（乙酸酯代谢调节剂），牛干扰素-α（BoIFN-α），糖蛋白D蛋白（gD）传染性牛鼻气管炎病毒（IBRV）和胶原蛋白原型N末端肽（P1NP）的鉴定。与带有tac启动子的原始载体相比，所有检测的蛋白质均高表达。在补料分批培养中也进行了大规模的P1NP生产，最高的P1NP生产水平为1.2 g / L。在这项研究中，诱导型tac启动子对C的强度。通过筛选和在目标基因前面插入反顺反子序列来改善谷氨酸。那些具有双顺反子表达模式的载体对于以高产量表达各种异质蛋白具有很强的相容性。该新策略可用于进一步改善诱导型启动子的性能，实现诱导型控制和高产量的双重能力。