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高度顺从的工业生产宿主，用于生产许多具有商业价值的酶。枯草杆菌在重组蛋白的工业化生产中的突出作用是基于其基因组可以轻松地进行工程(最近包括CRISPR技术)，以及通过直接分泌到细胞外培养基来生产高产率蛋白质的能力，从而促进具有成本效益的纯化。除了是工业生产宿主外，枯草杆菌还是研究最多的细菌模式生物之一。原因之一是其特有的细胞分化过程导致不同的细胞亚群进入不同的细胞分化途径。然而，分化成区域亚群的能力对枯草杆菌作为蛋白质生产宿主的功能产生了负面影响，因为并不是所有产生的细胞类型都对蛋白质生产过程做出贡献。在纽卡斯尔大学和皇家帝斯曼公司以生物技术为主题的联合研究项目中，我们将确定如何根据枯草杆菌在工业类发酵条件下的能量状态来优化其适合性，以提高异源蛋白质的生产。一个特别的重点将是研究细胞自溶过程，这会影响相当大比例的细胞群体，从而降低蛋白质产量。如何优化发酵条件以最大限度地减少自溶，以及如何重新设计生产宿主以抑制自溶过程，将进行研究，最终目标是提高重组蛋白的产量。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。定期与帝斯曼生物技术中心(代尔夫特，荷兰)的工业合作伙伴进行研究是研究计划不可或缺的一部分。

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.