枯草芽孢杆菌L-脯氨酸合成途径中glnA、proB、proA基因功能探究

Function of glnA, proB and proA genes in L-proline anabolic pathway of Bacillus subtilis

【目的】从基因水平探究枯草芽孢杆菌渗透压调节因子L-脯氨酸合成途径中glnA、proB、proA基因的功能,通过分子改造实现对代谢途径的人工扰动。【方法】从枯草芽孢杆菌WB600出发,通过向胞内引入一系列基因敲除或过表达,分别构建了proB和proA基因过表达的重组菌WB601和WB602、glnA基因缺失的重组菌WB603以及在此基础之上过表达proB基因的重组菌WB604。借助菌株胞外和胞内游离脯氨酸积累的表型分析影响途径的关键节点。【结果】在非胁迫条件下,重组菌WB601和WB602胞外脯氨酸含量分别是原始菌的2.21倍和2.82倍,单位细胞胞外脯氨酸得率分别是原始菌的4.09倍和9.80倍,胞内游离脯氨酸含量分别是原始菌的1.91倍和3.34倍;重组菌WB603胞外脯氨酸含量上升至1221.43 mg/L,是原始菌的6.28倍,单位细胞胞外和胞内游离脯氨酸得率分别为原始菌的9.13倍和3.66倍;而重组菌WB604胞外脯氨酸含量最高达1391.65 mg/L,相比菌株WB603,其胞外脯氨酸含量及单位细胞得率分别提高了13.94%和14.10%,且胞内游离脯氨酸含量提高了32.60%。在5%Na Cl胁迫条件下,重组菌WB601和WB602的胞外脯氨酸含量分别是原始菌的1.94倍和1.54倍,单位细胞胞外脯氨酸得率分别是原始菌的2.15倍和2.19倍;重组菌WB603胞外脯氨酸含量及其单位细胞得率分别是原始菌的4.16倍和7.29倍;相同条件下,相比于重组菌WB603,重组菌WB604的胞外脯氨酸含量及其单位细胞得率分别提高了32.61%和5.54%。此外,实验组菌株的胞内游离脯氨酸含量均高于非胁迫时,并达到相对平衡状态。【结论】proB和proA基因的过表达均能显著提升细胞合成脯氨酸的能力,并且能增强细胞的耐盐性;glnA基因的缺失能增强脯氨酸合成途径,提高脯氨酸的积累;两种效应的正向叠加可进一步提升细胞脯氨酸合成能力。

[Objective] We studied the function of glnA, proB and proA genes in the pathway of proline biosynthesis in Bacillus subtilis. [Methods] B. subtilis WB600 was used as the original strain. Al^er a series of intracellular gene deletion and overexpression, we constructed recombinant strain WB601 with proB gene overexpression and WB602 with proA gene overexpression; WB603 with glnA gene deletion. Then, WB604 with proB gene overexpression was constructed. The anabolic key nodes were analyzed by phenotypes of extracellular and intracellular free proline accumulation. [Results] Under non-stress conditions, the extracellular proline content of the recombinant strains WB601 and WB602 were 2.21 times and 2.82 times of that of the original strain, the unit cell extracellular proline yield were 4.09 times and 9.80 times of that of the original strain, and the intracellular free proline content were 1.91 times and 3.34 times of that of the original strain, respectively. The extracellular proline content of the recombinant strain WB603 increased to 1221.43 mg/L, 6.28 times of that of the original strain, and the unit cell extracellular and intracellular free proline yield were 9.13 times and 3.66 times of that of the original strain, respectively. The extracellular proline content of the recombinant strain WB604 up to 1391.65 mg/L, compared with strain WB603, the extracellular proline content and unit cell yield were increased by 13.94% and 14.10%, respectively, and the intracellular free proline content increased by 32.60%. Under 5% NaC1 stress, the extracellular proline content of recombinant strains WB601 and WB602 were 1.94 times and 1.54 times of the original strain, and the unit cell yield were 2.15 times and 2.19 times of the original strain, respectively; the extracellular proline content and unit cell yield of recombinant strain WB603 were 4.16 times and 7.29 times of the original strain, respectively; under the same conditions, compared with the recombinant strain WB603, the extracellular proline content and unit cell yield of the recombinant strain WB604 were increased by 32.61% and 5.54%, respectively. In addition, the intracellular free proline content of the experimental groups were higher than that of non stress conditions, and reached a relative equilibrium state. [Conclusion] Overexpression ofproB and proA gene can significantly enhance the ability of cells to synthesize proline and enhance the salt tolerance of cells; deletion of glnA could enhance proline de novo synthesis pathway and increase the accumulation of proline; moreover, the positive accumulation of the two effects could further improve the ability ofproline synthesis.