State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China.
School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
Microb Cell Fact. 2019 Apr 11;18(1):69. doi: 10.1186/s12934-019-1119-8.
Our laboratory has constructed a Bacillus stearothermophilus α-amylase (AmyS) derivative with excellent enzymatic properties. Bacillus subtilis is generally regarded as safe and has excellent protein secretory capability, but heterologous extracellular production level of B. stearothermophilus α-amylase in B. subtilis is very low.
In this study, the extracellular production level of B. stearothermophilus α-amylase in B. subtilis was enhanced by signal peptide optimization, chaperone overexpression and α-amylase mutant selection. The α-amylase optimal signal peptide (SP) was obtained by screening 173 B. subtilis signal peptides. Although the extracellular α-amylase activity that was produced by the resulting recombinant strain was 3.5-fold greater than that of the control, significant quantities of inclusion bodies were detected. Overexpressing intracellular molecular chaperones significantly reduced inclusion body formation and further increased α-amylase activity. Error-prone PCR produced an amylase mutant K82E/S405R (AmySA) with enzymatic activity superior to that of AmyS. Expression of the amySA gene with the SP while overexpressing molecular chaperones resulted in a 7.1-fold improvement in α-amylase activity. When the final expression strain (WHS11YSA) was cultivated in a 3-L fermenter for 92 h, the α-amylase activity of the culture supernatant was 9201.1 U mL, which is the highest level that has been reported to date.
This is the first report that describes an improvement of B. stearothermophilus α-amylase extracellular production levels in B. subtilis using these strategies, and this represents the highest extracellular production level ever reported for α-amylase from B. stearothermophilus in B. subtilis. This high-level production provides a basis for enhanced industrial production of α-amylase. These extracellular production level improvement approaches are also expected to be valuable in the expression of other enzymes in B. subtilis.
本实验室构建了一种具有优良酶学性质的嗜热脂肪芽孢杆菌α-淀粉酶（AmyS）衍生物。枯草芽孢杆菌通常被认为是安全的，并且具有出色的蛋白质分泌能力，但在枯草芽孢杆菌中外源表达嗜热脂肪芽孢杆菌α-淀粉酶的水平非常低。
本研究通过优化信号肽、过表达伴侣蛋白和α-淀粉酶突变体筛选的方法，提高了枯草芽孢杆菌中嗜热脂肪芽孢杆菌α-淀粉酶的胞外表达水平。通过筛选 173 种枯草芽孢杆菌信号肽，获得了α-淀粉酶最优信号肽（SP）。虽然所得重组菌的胞外α-淀粉酶活性比对照提高了 3.5 倍，但检测到大量的包涵体。过表达细胞内分子伴侣显著减少了包涵体的形成，并进一步提高了α-淀粉酶的活性。易错 PCR 产生了酶活优于 AmyS 的突变体 K82E/S405R（AmySA）。在过表达分子伴侣的同时表达 amySA 基因和 SP，α-淀粉酶活性提高了 7.1 倍。当最终表达菌株（WHS11YSA）在 3-L 发酵罐中培养 92 h 时，培养上清液中的α-淀粉酶活性达到 9201.1 U/mL，这是迄今为止报道的最高水平。
这是首次描述了使用这些策略提高枯草芽孢杆菌中嗜热脂肪芽孢杆菌α-淀粉酶胞外表达水平的研究，这代表了迄今为止枯草芽孢杆菌中嗜热脂肪芽孢杆菌α-淀粉酶胞外表达的最高水平。这种高水平的表达为α-淀粉酶的工业生产提供了基础。这些胞外表达水平提高的方法也有望在枯草芽孢杆菌中表达其他酶中具有应用价值。
