CHEAP

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 the proposed 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 the proposed 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.