Dissection of the cellular processes during heterologous protein production in Pseudomomas putida – or how much is a protein?

恶臭假单胞菌异源蛋白质生产过程中细胞过程的剖析 â 或蛋白质有多少？

• 批准号: 406709163
• 负责人: Professor Dr.-Ing. Andreas Kremling
• 金额: --
• 依托单位: LISBP - Laboratoire dIngénierie des Systèmes Biologiques et des Procédés
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/406709163?language=en
• 关键词: Dissection; cellular; processes; during; heterologous

Pseudomonas putida is an important workhorse for contemporary biotechnological applications, as it combines easy handling and fast growth with a low nutrient demand and an intrinsic resistance to metabolic and physiological stresses. The general aim of this proposal is to develop a comprehensive, system-level understanding of the physiology and metabolism of heterologous protein overproduction using P. putida as a host. Indeed, the induction of protein overproduction leads to a switch in resource allocation from the normal cellular functions towards the additionally implemented task. These resources comprise not only the transcriptional and translational machinery, including RNA polymerase and ribosomes, but also chemical and redox energy. To face this new resource distribution, the metabolism as a whole has to adapt to ensure growth or at least the survival of the cells. If this fails, meaning that any of these resources becomes limiting, usually the growth rate decreases and heterologous production ceases. This phenomenon is referred to as `metabolic burden´.In this project, we will perform an extensive, system-level investigation of the metabolic response to heterologous protein production in P. putida. With an interdisciplinary team of scientists from two laboratories in France and Germany, we will systematically collect quantitative data from P. putida cells, which are expressing a metabolic load. Therefore, a plasmid collection will be created (Objective #1) that allows us to specifically vary different inputs that provide information about the cellular capacity. Additionally, we will develop genetic constructs to quantify the RNA polymerase and ribosomes. We will compare the metabolic response of the wild type strain with the one of a streamlined cell-factory P. putida. In a screening process we will select the most interesting and promising conditions (Objective #3), which will then be analysed in depth (Objective #4) with a methodology adapted for P. putida (Objective #2). The combination of these approaches will lead to a thorough understanding of the cellular processes during heterologous protein production on different levels. All these data will enter a mathematical model which takes into account the strain specific traits and features with the aim of being able to assign a specific cost term to the heterologous protein and even to predict the cost for other proteins (Objective #5).In this project the following questions will be tackled: What is the cost for the production of a specific protein? Where are the bottlenecks during heterologous protein production? Can this information be generalized and eventually predicted? Our vision is to develop a toolbox that allows us to predict the optimal environment for a specific gene or, eventually, also for a small pathway in order to achieve the best performance possible.

恶臭假单胞菌是当代生物技术应用的重要主力，因为它结合了易于处理和快速生长，营养需求低，对代谢和生理应激具有内在抗性。本提案的总体目标是发展一个全面的，系统水平的了解异源蛋白质过量生产的生理和代谢，使用恶臭假单胞菌作为宿主。事实上，蛋白质过量生产的诱导导致资源分配从正常的细胞功能转向额外执行的任务。这些资源不仅包括转录和翻译机制，包括RNA聚合酶和核糖体，而且还包括化学和氧化还原能。面对这种新的资源分配，新陈代谢作为一个整体必须适应，以确保生长或至少细胞的生存。如果这失败了，意味着这些资源中的任何一个变得有限，通常生长速率降低，异源生产停止。这种现象被称为“代谢负担”。在这个项目中，我们将进行广泛的，系统级的调查的代谢反应，异源蛋白生产在恶臭假单胞菌。与来自法国和德国两个实验室的跨学科科学家团队一起，我们将系统地收集来自表达代谢负荷的恶臭假单胞菌细胞的定量数据。因此，将创建质粒集合（目标#1），其允许我们特异性地改变提供关于细胞能力的信息的不同输入。此外，我们将开发基因结构来定量RNA聚合酶和核糖体。我们将比较野生型菌株与流线型细胞工厂恶臭假单胞菌的代谢反应。在筛选过程中，我们将选择最有趣和最有希望的条件（目标#3），然后将采用适用于恶臭假单胞菌的方法（目标#2）对其进行深入分析（目标#4）。这些方法的组合将导致在不同水平上的异源蛋白质生产过程中的细胞过程的透彻理解。所有这些数据都将进入一个数学模型，该模型考虑到菌株的具体性状和特征，目的是能够为异源蛋白质分配一个具体的成本项，甚至预测其他蛋白质的成本（目标#5）。在这个项目中，将解决以下问题：生产特定蛋白质的成本是多少？异源蛋白生产的瓶颈在哪里？这些信息能否被概括并最终预测？我们的愿景是开发一个工具箱，使我们能够预测特定基因的最佳环境，或者最终预测一个小途径的最佳环境，以实现最佳性能。