The suitability of bacteria as microbial cell factories is dependent on several factors such as price of feedstock, product range, production yield and ease of downstream processing. The facultative methylotroph Bacillus methanolicus is gaining interest as a thermophilic cell factory for production of value-added products from methanol. The aim of this study was to expand the capabilities of B. methanolicus as a microbial cell factory by establishing a system for secretion of recombinant proteins. Native and heterologous signal peptides were tested for secretion of α-amylases and proteases, and we have established the use of the thermostable superfolder green fluorescent protein (sfGFP) as a valuable reporter protein in B. methanolicus. We demonstrated functional production and secretion of recombinant proteases, α-amylases and sfGFP in B. methanolicus MGA3 at 50 °C and showed that the choice of signal peptide for optimal secretion efficiency varies between proteins. In addition, we showed that heterologous production and secretion of α-amylase from Geobacillus stearothermophilus enables B. methanolicus to grow in minimal medium with starch as the sole carbon source. An in silico signal peptide library consisting of 169 predicted peptides from B. methanolicus was generated and will be useful for future studies, but was not experimentally investigated any further here. A functional system for recombinant production of secreted proteins at 50 °C has been established in the thermophilic B. methanolicus. In addition, an in silico signal peptide library has been generated, that together with the tools and knowledge presented in this work will be useful for further development of B. methanolicus as a host for recombinant protein production and secretion at 50 °C.

中文翻译：

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建立在嗜热芽孢杆菌中于50°C重组生产分泌蛋白的功能系统。

细菌作为微生物细胞工厂的适用性取决于几个因素，例如原料价格，产品范围，产量和下游加工的难易程度。兼性的甲基营养型甲醇芽孢杆菌作为嗜热的细胞工厂越来越受到关注，可以从甲醇生产增值产品。这项研究的目的是通过建立分泌重组蛋白的系统来扩大甲醇芽孢杆菌作为微生物细胞工厂的能力。测试了天然和异源信号肽的α-淀粉酶和蛋白酶的分泌，我们已经建立了将热稳定的超文件夹绿色荧光蛋白（sfGFP）用作甲醇芽孢杆菌中有价值的报告蛋白的用途。我们证明了B中重组蛋白酶，α-淀粉酶和sfGFP的功能性生产和分泌。MGA3在50°C的温度下燃烧，表明蛋白质之间最佳分泌效率的信号肽选择有所不同。此外，我们显示异源生产和嗜热脂肪芽孢杆菌分泌α-淀粉酶使甲醇芽孢杆菌能够在以淀粉为唯一碳源的基本培养基中生长。生成了由甲醇双歧杆菌预测的169个肽组成的计算机内信号肽文库，该文库可用于将来的研究，但此处未进行任何实验研究。在嗜热芽孢杆菌中已经建立了在50℃下重组生产分泌蛋白的功能系统。此外，已经生成了计算机信号肽文库，该文库与本工作中介绍的工具和知识一起将对B的进一步开发有用。