School of Biotechnology, Jiangnan Universitygrid.258151.a, Wuxi, Jiangsu, China.
Microbiol Spectr. 2022 Oct 26;10(5):e0132222. doi: 10.1128/spectrum.01322-22. Epub 2022 Aug 29.
The development of efficient, low-cost, and robust expression systems is important for the mass production of proteins and natural products in large amounts using cell factories. Glycerol is an ideal carbon source for large-scale fermentation due to its low cost and favorable maintenance of the fermentation process. Here, we used the antiterminator protein GlpP and its target promoter P to construct a highly efficient glycerol-inducible expression system (GIES) in Bacillus subtilis. This system was able to express heterologous genes in an autoinducible manner based on the sequential utilization of glucose and glycerol under the regulation of carbon catabolite repression. In such a system, the concentration of glycerol regulated the strength of gene expression, and the concentration of glucose affected both the timing of induction and the strength of gene expression. By enhancing GlpP, the GIES was further strengthened for high-level intracellular expression of aspartase and secretory expression of nattokinase. High yields of nattokinase in a 5-L fermenter through batch and fed-batch fermentation demonstrated the potential to apply the GIES for large-scale enzyme production. Through the evolution of the -10 box of P, mutants with gradient activities were obtained. In addition, hybrid glycerol-inducible promoters were successfully constructed by combining the constitutive promoters and the 5' untranslated region of P. Collectively, this study developed a GIES to obtain high-value products from inexpensive glycerol. More importantly, the great potential of the pair of inherent terminator and antiterminator protein as a portable biological tool for various purposes in synthetic biology is proposed. In this study, a GIES was constructed in B. subtilis by employing the antiterminator protein GlpP and the GlpP-regulated promoter P. Based on the sequential utilization of glucose and glycerol by B. subtilis, the GIES was able to express genes in an autoinducible manner. The amounts and ratio of glucose and glycerol can regulate the gene induction timing and expression strength. The GIES was further applied for high yields of nattokinase, and its robustness in production scale-up was confirmed in a 5-L fermenter. The high-level expression of heterologous proteins demonstrated the huge application potential of the GIES. Furthermore, mutants of P with gradient activities and hybrid glycerol-inducible promoters were obtained through the evolution of the -10 box of P and the combination of the constitutive promoters and the 5' untranslated region of P, respectively. These results demonstrated the use of the antiterminator protein as a regulator for various purposes in synthetic biology.
高效、低成本、稳健的表达系统的开发对于使用细胞工厂大量生产蛋白质和天然产物至关重要。由于成本低廉且有利于维持发酵过程，甘油是大规模发酵的理想碳源。在这里，我们使用抗终止蛋白 GlpP 及其靶启动子 P 在枯草芽孢杆菌中构建了一种高效的甘油诱导表达系统 (GIES)。该系统能够根据葡萄糖和甘油的顺序利用，在碳分解代谢物阻遏的调控下，以自动诱导的方式表达异源基因。在这样的系统中，甘油的浓度调节基因表达的强度，葡萄糖的浓度影响诱导的时间和基因表达的强度。通过增强 GlpP，GIES 进一步增强了天冬氨酸酶的细胞内高水平表达和纳豆激酶的分泌表达。通过分批和补料分批发酵在 5-L 发酵罐中获得了纳豆激酶的高产，证明了 GIES 具有大规模生产酶的潜力。通过对 P 的 -10 框的进化，获得了具有梯度活性的突变体。此外，通过组合组成型启动子和 P 的 5'非翻译区，成功构建了混合甘油诱导启动子。总之，本研究开发了一种 GIES，以从廉价的甘油中获得高价值的产品。更重要的是，提出了固有终止子和抗终止子蛋白作为一种用于合成生物学中各种目的的便携式生物工具的巨大潜力。在这项研究中，通过使用抗终止蛋白 GlpP 和 GlpP 调节的启动子 P，在枯草芽孢杆菌中构建了 GIES。基于枯草芽孢杆菌对葡萄糖和甘油的顺序利用，GIES 能够以自动诱导的方式表达基因。葡萄糖和甘油的量和比例可以调节基因诱导的时间和表达强度。GIES 进一步应用于纳豆激酶的高产，并在 5-L 发酵罐中证实了其在生产规模扩大方面的稳健性。异源蛋白的高水平表达证明了 GIES 的巨大应用潜力。此外，通过对 P 的 -10 框的进化和组成型启动子与 P 的 5'非翻译区的组合，分别获得了具有梯度活性的 P 突变体和混合甘油诱导启动子。这些结果表明，抗终止蛋白可作为合成生物学中各种目的的调节剂。
