MAMMUT: Metabolic burden in Pseudomonas putida: Optimizing amino acid cost during expression of synthetic pathways for assimilation and bioproduction

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 introduction of heterologous or synthetic pathways comes along with a higher demand for cellular resources, which 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, ribosomes, and amino acids, but also chemical and redox energy. If any of these resources becomes limiting, usually the growth rate decreases and heterologous production ceases. This phenomenon is referred to as metabolic burden.

The general aim of this proposal is to develop a comprehensive, system-level understanding of the physiology and metabolism of heterologous protein overproduction using Pseudomonas putida as a host and apply this knowledge to a biotechnological process. We will perform a systematic investigation of the metabolic response to an introduced metabolic task in three different setups: the production of a fluorescently labelled dummy protein, the expression of a foreign heterologous pathway, and the production of novel metabolites from synthetic pathways by employing endogenous pathways of the cell. The collected quantitative data will enter a mathematical model providing a detailed description of resource allocation and metabolic burden in P. putida, which will lay the basis for tuning the performance of heterologous pathways in future applications.

Project supervisor: M.Sc. Carina Meiners

Start of the project: 01.11.2022