Establishment of a functional system for recombinant production of secreted proteins at 50 °C in the thermophilic Bacillus methanolicus
Establishment of a functional system for recombinant production of secreted proteins at 50 °C in the thermophilic Bacillus methanolicus
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DOI：
10.1186/s12934-020-01409-x
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发表时间：
2020-07-28
影响因子：
6.4
通讯作者：
Hakvag, Sigrid
中科院分区：
文献类型：
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作者：
Irla, Marta;Drejer, Eivind B.;Hakvag, Sigrid
细菌作为微生物细胞工厂的适宜性取决于几个因素，如原料价格、产品范围、产量和下游加工的简便性。兼性甲基芽孢杆菌作为一种从甲醇中生产附加值产品的嗜热细胞工厂正受到越来越多的关注。结果对天然和异源信号肽进行了α-淀粉酶和蛋白水解酶的分泌试验，确定了耐热超折叠绿色荧光蛋白(SfGFP)作为报告蛋白在甲醇芽孢杆菌中的应用。我们展示了在50℃下在甲醇假单胞菌MGA3中产生和分泌重组蛋白酶、α-淀粉酶和sfGFP的功能，并表明最佳分泌效率的信号肽的选择在不同的蛋白质中是不同的。此外，我们还发现，嗜热脂肪芽孢杆菌异源生产和分泌α-淀粉酶使其能够在以淀粉为唯一碳源的最小培养基中生长。构建了一个由169个预测的甲烷假单胞菌多肽组成的电子信号多肽文库，该文库可用于未来的研究，但尚未进行进一步的实验研究。结论已在嗜热甲烷假单胞菌中建立了在50℃条件下重组分泌蛋白的功能系统。此外，还建立了一个电子信号多肽文库，该文库与本工作中提供的工具和知识相结合，将有助于进一步发展甲醇假单胞菌作为50℃下重组蛋白生产和分泌的宿主。
BackgroundThe 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.ResultsNative and heterologous signal peptides were tested for secretion of alpha -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, alpha -amylases and sfGFP in B. methanolicus MGA3 at 50 degrees 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 alpha -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.ConclusionA functional system for recombinant production of secreted proteins at 50 degrees 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 degrees C.