Experimental analysis and genetic modifications of the bacterial metabolism (Team Bettenbrock)
Biological systems are complex by nature. Even a simple model organism such as the bacterium Escherichia coli are made up of a large number of molecules ranging from simple metabolites to proteins, RNA and DNA. The molecules interact in complex ways to ensure survival and replication under varying conditions. The team of Katja Bettenbrock aims to achieve a holistic and quantitative understanding of these interactions by combining experimental biological research with mathematical modeling and computational methods.
Experimental techniques routinely used in our lab include:
Genetic modifications: Defined genetic modifications are introduced into our model organisms using various genetic and molecular methods. We mainly use recombineering to delete or insert genes into the genome. In addtion, CRISPR-based methods are used e.g. for the introduction of (point) mutations. Heterologous genes are introduced either by plasmid vectors or by integratiion into the genome of the respective host. To achieve controlled gene expression, we use a variety of different sytems, ranging from the well-established lac system to optogenetic tools and CRISPRi.
Cultivation: To characterize strains we perform well-controlled growth assays ranging from shake flasks to batch, fed-batch or continuous cultication in bioreactors. One focus is to vary between aerobic, microaerobic and anaerobic growth conditions. Medium composition is varied according to the specific project.
Analytics: To obtain a holistic view of the physiology of investigated cells, we combine a variety of different analytical methods. We use HPLC, HPLC-MS or enzymatic assays to quantify the uptake of growth substrates, to quantify the different products and to analyze the concentrations of selected intracellular metabolites. In addition, we use real-time PCR or NGS methods to analyse RNA levels in our strains or use reporter genes to analyse gene expression of selected genes. Where appropiate, we also analyze protein levels, activity or modifications.
We apply these techniques in different research projects including:
1) Understanding bacterial metabolism and regulation
Due to their small size and their lifestyle bacteria are subjected to rapid and drastic changes in their environment. To cope with this, each cell must monitor its environment and respond in an appropriate way. This is achieved through a complex network of sensors and regulatory systems. Regulatory systems control gene expression and thereby tune catabolic and anabolic metabolic pathways in catabolism as well as in anabolism. In addition, metabolism is influenced by the control of enzyme activities through modification or allosteric control. A deep understanding of bacterial metabolism and its regulation is vital for engineering bacteria for biotechnological applications. We are therefore investigating the influence of regulatory systems on metabolism under different external conditions in the model bacterium E. coli. A major goal is to use this understanding for the targeted modification of metabolism and regulation in the construction of production strains.
2) Engineering Zymomonas mobilis as workhorse for biotechnological applications
The bacterium Zymomonas mobilis is characterised by a particularly high glucose uptake rate and a high glycolytic flux compared to most other microorganisms. In addition, the glucose taken up by Z. mobilis is almost completely converted into its main fermentation product, ethanol. These characteristics make Z. mobilis a promising workhorse for biotechnology applications. Genetic engineering for targeted modification of the Z. mobilis genome is possible but efficient tools are not yet available. We are developing a genetic toolbox for efficient genetic engineering of Z. mobilis. This toolbox includes various plasmid vectors, different promoter elements for controlled gene expression, a set of ribosome binding sites, as well as tools for genome integration and gene knock out. We use this toolbox to engineer Z. mobilis for the production of other products than ethanol. Model-based analysis predicts promising modifications which are tested in our lab. In addtion, we are aiming to a better understanding of more general aspects of Z. mobilis physiology such as the function and role of its respiratory chain, the mechanisms underlying the uncoupled growth phenotype and ATP metabolism.
3) Dynamic Process Optimization in Biotechnology
The application of biological processes in the production of building blocks is becoming increasingly important. In order to replace petrochemicals, the biological production processes must become more efficient and cheaper. Often the synthesis of the desired product is hampered by slow growth of the cells, reduced substrate uptake rates and/or by the use of the compounds as building blocks for the cells themselves. In order to overcome this obstacle, the application of dynamic process control strategies is promising. In addition to the control of external parameters, also the control of intracellular parameters such as gene expression and protein activity is required. In this project, different targets and strategies for the control of intracellular parameters is evaluated. Most importantly, gene expression systems need to be designed, to allow fine-tuning of gene expression during the production process. Optogenetic tools are suitable for these applications but other tools are also being investigated in our group.