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 Biochem Biotechnol. 2022 Mar;194(3):1206-1220. doi: 10.1007/s12010-021-03617-6. Epub 2021 Oct 15.
Bacillus subtilis has many attributes that make it a popular host for recombinant protein production. Although its protein production ability has been enhanced through protease gene disruption, residual proteases like quality control HtrA and HtrB can limit protein yield by degrading inadequately folded proteins present during overexpression. In this study, two strategies were employed to increase production of industrial enzyme pullulanase: enhancing extracellular pullulanase folding and optimizing its signal peptide. The hypothesis was that disruption of dltB gene of expression host B. subtilis WS9 would enhance recombinant extracellular folding by increasing cation binding to the cell's outer envelope. Consistent with this hypothesis, disrupting dltB enhanced pullulanase production by 49% in shake-flask cultures. The disruption also enhanced extracellular α-CGTase and β-CGTase production by 25% and 35%, respectively. Then, more effective signal peptide for pullulanase production was identified through high-throughput screening of 173 unique B. subtilis signal peptides. Replacing the native signal peptide of pullulanase with that encoded by ywtF increased extracellular pullulanase activity by an additional 12%. Three-liter fermenter scale-up production yielded the highest extracellular pullulanase activity reported to date: 8037.91 U·mL. This study highlights the usefulness of dltB deletion and signal peptide optimization in enhancing extracellular protein production.
枯草芽孢杆菌具有许多特性，使其成为重组蛋白生产的理想宿主。尽管通过蛋白酶基因敲除提高了其蛋白质生产能力，但像质量控制 HtrA 和 HtrB 这样的残留蛋白酶仍然可以通过降解表达过程中存在的折叠不完全的蛋白质来限制蛋白质产量。在这项研究中，采用了两种策略来提高工业酶普鲁兰酶的产量：增强细胞外普鲁兰酶的折叠和优化其信号肽。假设通过表达宿主枯草芽孢杆菌 WS9 中 dltB 基因的敲除，增加阳离子与细胞外膜的结合，从而增强重组细胞外折叠。与该假设一致，dltB 的敲除使摇瓶培养中的普鲁兰酶产量提高了 49%。该敲除还分别使细胞外 α-CGTase 和 β-CGTase 的产量提高了 25%和 35%。然后，通过对 173 种独特的枯草芽孢杆菌信号肽进行高通量筛选，确定了更有效的普鲁兰酶生产用信号肽。用 ywtF 编码的信号肽替代普鲁兰酶的天然信号肽，使细胞外普鲁兰酶活性额外提高了 12%。在 3 升发酵罐规模放大生产中，获得了迄今为止报道的最高细胞外普鲁兰酶活性：8037.91 U·mL。本研究强调了 dltB 缺失和信号肽优化在增强细胞外蛋白质生产中的作用。
