National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.
Science Center for Future Foods, Jiangnan University, Wuxi, 214122, China.
Microb Cell Fact. 2022 Mar 19;21(1):42. doi: 10.1186/s12934-022-01772-x.
Lipoxygenase (LOX) is a non-heme iron containing dioxygenase that is widely used to improve food quality and produce active drug intermediates and biodiesel. Escherichia coli is one of the most widely used host microorganisms for recombinant protein expression; however, its weak extracellular secretion ability precludes its effective production of recombinant proteins into the extracellular environment. To facilitate subsequent characterization and application of LOX, improving its secretion efficiency from E. coli is a major challenge that needs to be solved.
Several strategies were adopted to improve the extracellular secretion of LOX based on the signal peptides and cell wall permeability of E. coli. Here, we studied the effect of signal peptides on LOX secretion, which increased the secretory capacity for LOX marginally. Although surfactants could increase the permeability of the cell membrane to promote LOX secretion, the extracellular LOX yield could not meet the requirements of industrialization production. Subsequently, an autolysis system was constructed in E. coli based on the bacteriophage lysis gene ΦX174-E to enhance the production of extracellular proteins. Thus, the extracellular production of LOX was achieved and the content of inclusion bodies in the cell was reduced by optimizing cell lysis conditions. The extracellular LOX yield reached 368 ± 1.4 U mL in a 5-L bioreactor under optimized lysis conditions that is, an induction time and temperature, and arabinose concentration of 5 h, 25 °C, and 0.6 mM, respectively.
In this study, the different signal peptides and cell autolysis system were developed and characterized for extracellular LOX production in E. coli. Finally, the cell autolysis system presented a slight advantage on extracellular LOX yield, which also provides reference for other protein extracellular production.
脂氧合酶（LOX）是一种广泛用于改善食品质量和生产活性药物中间体和生物柴油的非血红素铁含双氧酶。大肠杆菌是最常用于重组蛋白表达的宿主微生物之一；然而，其弱的细胞外分泌能力使其无法将重组蛋白有效分泌到细胞外环境中。为了方便随后对 LOX 的表征和应用，提高其从大肠杆菌中的分泌效率是需要解决的主要挑战。
基于大肠杆菌的信号肽和细胞壁通透性，采用了几种策略来提高 LOX 的细胞外分泌。在这里，我们研究了信号肽对 LOX 分泌的影响，这略微提高了 LOX 的分泌能力。尽管表面活性剂可以增加细胞膜的通透性以促进 LOX 分泌，但细胞外 LOX 的产量仍不能满足工业化生产的要求。随后，在大肠杆菌中构建了基于噬菌体 lysis 基因 ΦX174-E 的自溶系统，以增强细胞外蛋白质的生产。因此，实现了 LOX 的细胞外生产，并通过优化细胞裂解条件减少了细胞内包涵体的含量。在优化的裂解条件下，即在诱导时间和温度分别为 5 小时、25°C 和 0.6mM 阿拉伯糖的条件下，在 5L 生物反应器中 LOX 的细胞外产量达到 368±1.4 U mL。
本研究开发并表征了不同的信号肽和细胞自溶系统用于大肠杆菌中 LOX 的细胞外生产。最后，细胞自溶系统在细胞外 LOX 产量方面略有优势，这也为其他蛋白质的细胞外生产提供了参考。
