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

恶臭假单胞菌是当代生物技术应用的重要工具，因为它具有易于处理和快速生长，低营养需求以及对代谢和生理应激的内在抗性。本提案的总体目标是开发一个全面的，系统水平的理解的生理和代谢的异种蛋白过量生产利用恶臭假单胞菌作为宿主。事实上，诱导蛋白质过量生产导致资源分配从正常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，还包括化学和氧化还原能量。为了面对这种新的资源分配，新陈代谢作为一个整体必须适应，以确保细胞的生长或至少存活。如果这一方法失败，意味着这些资源中的任何一种变得有限，通常生长速度会下降，异种生产也会停止。这种现象被称为“代谢负担”。在这个项目中，我们将对p.p putida对异源蛋白生产的代谢反应进行广泛的系统水平的研究。与来自法国和德国两个实验室的跨学科科学家团队一起，我们将系统地收集表达代谢负荷的恶臭杆菌细胞的定量数据。因此，将创建一个质粒收集（目标#1），使我们能够具体地改变不同的输入，提供有关细胞容量的信息。此外，我们将开发遗传结构来量化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），然后使用适用于恶臭假单胞菌（目标#2）的方法对其进行深入分析（目标#4）。这些方法的结合将导致在不同水平上的异源蛋白生产的细胞过程的透彻理解。所有这些数据将进入一个数学模型，该模型考虑了菌株的特定特征和特征，目的是能够为异源蛋白分配特定的成本项，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？异种蛋白生产的瓶颈在哪里？这些信息是否可以一般化并最终被预测？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，也可以预测一个小途径，以实现最佳性能。

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.