Laboratory of Biotransformation and Biocatalysis, School of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou, Dongfeng Road 5, Henan, 450000, People's Republic of China.
School of Food and Bioengineering/Collaborative Innovation Center for Food Production and Safety, Zhengzhou University of Light Industry, Dongfeng Road 5, Zhengzhou, Henan, 450001, People's Republic of China.
Biotechnol Lett. 2022 Sep;44(9):1051-1061. doi: 10.1007/s10529-022-03281-8. Epub 2022 Aug 3.
Glycerol kinase is the key enzyme in glycerol metabolism, and its catalytic efficiency has an important effect on glycerol utilization. Based on an analysis of the glycerol utilization pathway and regulation mechanism in B. subtilis, we conducted site-directed mutagenesis of the key glycerol kinase gene (glpK) on the chromosome to improve the glycerol utilization efficiency of Bacillus subtilis. Recombinant wild-type Bacillus subtilis glycerol kinase (BsuGlpK) and two mutants (BsuGlpK and BsuGlpK) were successfully overexpressed in Escherichia coli BL21(DE3) and purified by Ni-IDA metal chelate chromatography. The specific activity of the BsuGlpK mutant (62.6 U/mg) was significantly higher (296.2%) than that of wild-type BsuGlpK (15.8 U/mg). By contrast, the mutant BsuGlpK (4.89 U/mg) exhibited lower (69.1%) activity than BsuGlpK, which suggested that variant S71V exhibited reduced catalytic efficiency for the substrate. Furthermore, the mutant strain B. subtilis M270I was constructed using a markerless delivery system, and exhibited a higher specific growth rate (improved by 11.3%, from 0.453 ± 0.012 to 0.511 ± 0.017 h) and higher maximal biomass (cell dry weight increased by 16%, from 0.577 ± 0.033 to 0.721 ± 0.015 g/L) than the parental strain with a shortened lag phase (2 ~ 4 h shorter) in M9 minimal medium with glycerol. These results indicate that the mutated glpK resulted in improved glycerol utilization, which has broad application prospects.
甘油激酶是甘油代谢中的关键酶，其催化效率对甘油的利用有重要影响。基于对枯草芽孢杆菌甘油利用途径和调控机制的分析，我们对染色体上的关键甘油激酶基因 (glpK) 进行了定点突变，以提高枯草芽孢杆菌的甘油利用效率。重组野生型枯草芽孢杆菌甘油激酶 (BsuGlpK) 和两种突变体 (BsuGlpK 和 BsuGlpK) 在大肠杆菌 BL21(DE3) 中成功过表达，并通过 Ni-IDA 金属螯合层析进行纯化。突变体 BsuGlpK 的比活性 (62.6 U/mg) 显著高于野生型 BsuGlpK (15.8 U/mg) (296.2%)。相比之下，突变体 BsuGlpK 的活性较低 (69.1%)，表明变体 S71V 对底物的催化效率降低。此外，使用无标记递送系统构建了枯草芽孢杆菌 M270I 突变株，其比生长速率更高 (提高了 11.3%，从 0.453 ± 0.012 到 0.511 ± 0.017 h)，最大生物量更高 (细胞干重增加了 16%，从 0.577 ± 0.033 到 0.721 ± 0.015 g/L)，在以甘油为碳源的 M9 基本培养基中比原始菌株的延滞期更短 (短 2 到 4 h)。这些结果表明突变的 glpK 导致甘油利用得到改善，具有广阔的应用前景。
