谷氨酸棒杆菌(Corynebacterium glutamicum)作为应用日益广泛的蛋白表达宿主菌,其全基因组虽然已经被测序,但是一些影响蛋白表达的关键基因仍未被研究。基因NCgl2632被预测可能是影响谷氨酸棒杆菌能量代谢的关键基因之一。因此该文首先分别构建了NCgl2632敲除菌株、NCgl2632过表达菌株,测定了生长曲线和胞内ATP水平的变化,并用增强绿色荧光蛋白(enhanced green fluorescent protein, eGFP)和重链抗体可变区蛋白(variable domain of heavy chain antibody, VHH)验证该基因表达水平的变化对外源蛋白表达的影响,发现敲除该基因对外源蛋白表达量提高有利。同时,由于前期工作中发现NCgl0909的敲除表型比较有利于外源蛋白表达水平的提高,因此在其基础上构建了C.g 15647ΔNCgl2632ΔNCgl0909双敲除菌株,并表达VHH蛋白进行了验证。此外,该文还选取了一种有潜力的蛋白human MutT homolog 1(hMTH1)进行了表达验证和活性检测,由于其分子质量较小且较稳定易于表达,未来可以作为新的模式蛋白验证菌株产外源蛋白的能力。最终对双敲除菌株表达该蛋白的发酵培养基配比进行了优化,得到了较优的培养基配比。
Corynebacterium glutamicum is an excellent protein expression host. Although the entire genome of C. glutamicum has been sequenced, some key genes that affect protein expression have not yet been researched. The gene NCgl2632 is predicted to be one of the key genes affecting the energy metabolism of C. glutamicum. Therefore, the NCgl2632 knockout strain and NCgl2632 overexpression strain were constructed, and the growth curve and changes of intracellular ATP levels were measured. Further, enhanced green fluorescent protein (eGFP) and variable domain of heavy chain antibody (VHH) were used to verify the effect of changes in the gene expression level on the expression of exogenous proteins, which indicated that knocking out the gene improved the expression of exogenous proteins. At the same time, since the knockout phenotype of NCgl0909 was proved to increase the expression of exogenous proteins in previous work, the C.g 15647ΔNCgl2632ΔNCgl0909 co-knockout strain was constructed on this basis. The model protein VHH was used to validate the ability of expressing exogenous proteins in the co-knockout strain. It was proved that the co-knockout strain was beneficial to the expression of VHH. In addition, a potential protein human MutT homolog 1(hMTH1) was selected for expression verification and activity detection, in which case the co-knockout strain also increased the expression of hMTH1. Owing to its small molecular weight and stability, hMTH1 can be used as a new model protein to verify the ability of strains to produce exogenous proteins in the future. Finally, to obtain a better medium ratio for protein expression, the fermentation medium of the co-knockout strain was optimized.
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