热葡糖苷酶地芽胞杆菌NCIMB 11955高效电转感受态细胞电转体系的建立

Development of highly efficient electrocompetent cells for electroporation of Geobacillus thermoglucosidasius NCIMB 11955

摘要:

热葡糖苷酶地芽胞杆菌(Geobacillus thermoglucosidasius)是一种革兰氏阳性兼性厌氧菌，因其高温生长速度快、不易染菌的特性，作为下一代工业生物技术的候选底盘，已被广泛用于生产生物燃料和高附加值的化学品。然而，相比于经典的模式菌株大肠杆菌(Escherichia coli)，热葡糖苷酶地芽胞杆菌因其转化效率低，限制了其作为底盘菌株的高效应用。本研究的目的是开发一种能够提高该菌株电转化效率的方法。本研究通过敲除该菌株中预测的限制性内切酶基因，添加细胞壁弱化剂和细胞膜抑制剂，优化目标菌株高效感受态细胞的制备，从而提高转化效率。热葡糖苷酶地芽胞杆菌的4个限制性内切酶基因的敲除可以使感受态细胞电转化效率提高至1.2×10⁴ CFU/(μg DNA)；在敲除株的基础上，分别通过添加吐温80、dl-苏氨酸和甘氨酸进一步优化感受态细胞转化效率，其中吐温80的添加使感受态细胞电转化效率提高22.5倍左右，dl-苏氨酸的添加可以使感受态细胞电转化效率增加44倍，甘氨酸的添加可以使感受态细胞电转化效率进一步提高334倍左右，优化整合添加各类试剂后感受态细胞电转化效率在限制性内切酶缺失菌株的基础上提高至4.6×10⁶ CFU/(μg DNA)，相对野生型菌株提高了3个数量级。这为热葡糖苷酶地芽胞杆菌的高效遗传操作和代谢工程奠定了基础。

Abstract:

Geobacillus thermoglucosidasius is a kind of Gram-positive facultative anaerobic bacteria. The fast growth rate under high temperature and less susceptibility to microbial contamination enable G. thermoglucosidasius to be a desirable producer of biofuels and high-value-added chemicals for the next-generation industrial biotechnology. However, compared with the classical model strain Escherichia coli, the applications of G. thermoglucosidasius are hampered by its low transformation efficiency. This study aimed at obtaining competent cells with high transformation efficiency through inactivating restriction enzymes, adding cell membrane inhibitors and cell wall weakening agents. The results showed that the electro-transformation efficiency achieved 1.2×10⁴ CFU/(μg DNA) by knocking out four genes encoding restriction enzymes. Adding a certain amount of tween 80, dl-threonine and glycine further increased the competent efficiency about 22.5, 44, and 334 times, respectively. The electro-transformation efficiency was enhanced to 4.6×10⁶ CFU/(μg DNA) under the optimized conditions, laying a foundation for genetic manipulation and metabolic engineering of G. thermoglucosidasius.