Optimising protein production from high density Bacillus subtilis cultures by supressing autolysis

革兰氏阳性细菌枯草芽孢杆菌是一种非常适合工业生产的宿主，用于生产许多有商业价值的酶。B的突出作用。枯草芽孢杆菌在重组蛋白的工业生产中的应用是基于其基因组可以被工程化的容易性（最近包括CRISPR技术），以及通过直接分泌到细胞外培养基来产生高产量蛋白质的能力，从而促进成本有效的纯化。除了作为工业生产主机，B。枯草芽孢杆菌是研究得最好的细菌模式生物之一。其原因之一是其特征性的细胞分化过程，其导致进入不同细胞分化途径的不同细胞亚群。然而，分化为地区亚人口的能力对B的功能具有负面影响。枯草芽孢杆菌作为蛋白质生产宿主的优势在于并非所有所得细胞类型都有助于蛋白质生产过程。在纽卡斯尔大学和皇家帝斯曼之间的这个以生物技术为主题的联合研究项目中，我们将确定B。在类似工业发酵条件期间，就其能量状态而言，可以优化枯草芽孢杆菌以改善异源蛋白质生产。一个具体的重点将是研究细胞自溶过程，这会影响很大比例的细胞群，从而降低蛋白质的产量。如何优化发酵条件以减少自溶，以及如何重新设计生产宿主以抑制自溶过程，最终目标是提高重组蛋白的生产。该跨学科项目结合了分子和细胞生物学的最新技术，包括分子克隆和高端显微镜，以及生物技术方法，包括生产宿主优化，发酵，和重组蛋白生产。博士课程在细菌细胞生物学中心（纽卡斯尔大学）进行，这是一个专注于微生物学的世界级研究中心。与帝斯曼生物技术中心（德尔夫特，荷兰）的工业合作伙伴进行定期研究是研究计划的一个组成部分。

The Gram-positive bacterium Bacillus subtilis is a highly amenable industrial production host used to produce numerous commercially valuable enzymes. The prominent role of B. subtilis in industrial production of recombinant proteins is based on the ease with which its genome can be engineered (more recently including the CRISPR technology), and the ability to produce high yields of protein by secretion directly to the extracellular medium thereby facilitating cost-effective purification. In addition to being an industrial production host, B. subtilis is among the best-studied bacterial model organism. One of the reasons for this is its characteristic cell differentiation processes that result in distinct sub-populations of cells that enter distinct cellular differentiation pathways. However, the ability to differentiate into district sub-population has negative consequences for the function of B. subtilis as a protein production host as not all of the resulting cell types contribute to the protein production process. In this joint biotechnology-themed research project between Newcastle University and Royal DSM, we will determine how the fitness of B. subtilis, in terms of its energy status during industry-like fermentation conditions, can be optimised for improved heterologous protein production. A specific focus will be on studying the cell autolytic process, which affects a significant proportion of the cell population and thus reduces the protein production yield. How the fermentation conditions can be optimised to minimise autolysis, and how the production host can be re-engineered to suppress the autolytic process will be studied with the ultimate goal to improve recombinant protein production.The interdisciplinary project combines state-of-the-art techniques in molecular and cellular biology including molecular cloning and high-end microscopy, and biotechnological methods including production host optimisation, fermentation, and recombinant protein production. The PhD programme is carried out at the Centre for Bacterial Cell Biology (Newcastle University), a world-class research centre with focus on microbiology. Regular research stays with the industrial partner at the DSM Biotechnology Center (Delft, the Netherlands) form an integral part of the research programme.