State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
Appl Microbiol Biotechnol. 2018 Jun;102(12):5089-5103. doi: 10.1007/s00253-018-8965-x. Epub 2018 Apr 20.
To study the effect of proteases on pullulanase production, six protease-encoding genes (nprB, bpr, mpr, epr, vpr, and wprA) in the genome of Bacillus subtilis strain WS5, which already lacks the protease-encoding genes nprE and aprE, were sequentially disrupted using a CRISPR/Cas9 system. This created strains WS6-WS11, respectively. The strains WS3 (none) and WS4 (ΔnprE) were constructed earlier. After addition of expression plasmid pHYPULd4 into the strains WS3-WS11, the pullulanase production levels of the resulting strains (WS3PUL-WS11PUL, respectively) were investigated in shake-flask cultivations and recombinant strain WS5PUL produced the highest pullulanase activity (148.2 U/mL). Then, the scale-up pullulanase production levels of four recombinant strains WS5PUL, WS9PUL, WS10PUL, and WS11PUL were investigated in the 3-L fermenter cultivations. Strain WS9PUL produced the highest pullulanase activity (2449.6 U/mL) when fed an inorganic nitrogen source. However, the specific activity of the pullulanase obtained in a 3-L fermenter generally decreased as the number of protease deletions increased. Meanwhile, using pullulanase, α-cyclodextrin glucosyltransferase and β-cyclodextrin glucosyltransferase as reporter proteins, the protein production differences among strains WS3, WS9, and the widely used WB600 were investigated. Finally, the carbon to organic nitrogen source ratio of the feeding solution used in the 3-L fermenter was optimized. Recombinant strain WS9PUL fed with carbon and organic nitrogen sources in a ratio of 4:1 achieved a pullulanase activity of 5951.8 U/mL, the highest activity reported to date.
为了研究蛋白酶对普鲁兰酶生产的影响，利用 CRISPR/Cas9 系统依次敲除了枯草芽孢杆菌 WS5 基因组中已缺失蛋白酶编码基因 nprE 和 aprE 的 6 个蛋白酶编码基因（nprB、bpr、mpr、epr、vpr 和 wprA），分别构建了 WS6-WS11 菌株。此前构建了 WS3（无）和 WS4（ΔnprE）菌株。将表达质粒 pHYPULd4 添加到 WS3-WS11 菌株中后，研究了所得菌株（分别为 WS3PUL-WS11PUL）在摇瓶培养中的普鲁兰酶生产水平，重组菌株 WS5PUL 产生的普鲁兰酶活性最高（148.2 U/mL）。然后，在 3-L 发酵罐培养中研究了四个重组菌株 WS5PUL、WS9PUL、WS10PUL 和 WS11PUL 的放大普鲁兰酶生产水平。当以无机氮源为底物时，菌株 WS9PUL 产生的普鲁兰酶活性最高（2449.6 U/mL）。然而，在 3-L 发酵罐中获得的普鲁兰酶的比活通常随着蛋白酶缺失数目的增加而降低。同时，利用普鲁兰酶、α-环糊精葡萄糖基转移酶和β-环糊精葡萄糖基转移酶作为报告蛋白，研究了 WS3、WS9 和广泛使用的 WB600 菌株之间的蛋白产量差异。最后，优化了 3-L 发酵罐中补料液的碳源与有机氮源的比例。重组菌株 WS9PUL 以碳源与有机氮源 4:1 的比例进料，可获得 5951.8 U/mL 的普鲁兰酶活性，这是迄今为止报道的最高活性。
