Graduate Students Mini-Symposium II 2025

13:15 h Dennis Wiens - MPRG Hochberg

Evolution of Rubisco’s chaperone dependence

Molecular chaperones assist the folding and assembly of proteins, but little is known about their evolutionary origins. Rubisco, the enzyme responsible for carbon fixation in photosynthesis, is a heteromeric protein complex and serves as a model system to study chaperone evolution. Despite high sequence similarity, Rubisco from higher plants requires much more extensive chaperone assistance during assembly of the complex than cyanobacterial Rubisco. We seek to answer why and how this complexity arose using ancestral sequence reconstruction. We could previously show that cyanobacterial chaperones increase assembly efficiency. Through biochemical characterization and comparison of ancestral Rubisco proteins we were able to show that dependence on additional chaperones emerged early in the evolution of plants. We verified this result by the first reconstitution of a basal Streptophyte Rubisco. We further show that dependence can be reversed by a few historical substitutions. These substitutions can also influence chaperone dependence in extant Rubisco proteins. With these insights we can possibly engineer chaperone-independent Rubisco and gain a fundamental understanding about the evolutionary and structural mechanisms governing chaperone dependence.

13:45 Adrian Podolski - AG Bode

Development of a Golden Gate cloning Toolkit for NRPS engineering

Natural products (NP) have contributed significantly to treat infectious diseases, cancer and other diseases. These NPs are complex chemical molecules that often need further structural modifications for pharmacological use. Many of them are biosynthesised by mega-enzymes such as non-ribosomal peptide synthetases (NRPS). Modifying those enzymes requires engineering at specific breakpoints, allowing the exchange of NRPS components in a modular fashion⁹. The active exchange of frequent building blocks in an NPRS system can quickly become tedious and time-consuming in library-like approaches. Due to specific fusion sites described in the “eXchange Unit between Thiolation domains” (XUT) approach, efficient assembly methods such as Golden Gate assembly have become feasible. In this way, it is possible to use the NRPS of interest as an acceptor, while modules of a pre-made NRPS library (i.e. XUTs) are used as donors. Specifically designed overhangs for the NRPS fusion sites facilitate a high degree of compatibility between the donor and acceptor systems. Therefore, it is possible to engineer specific NRPS modules and, consequently, modify specific chemical regions of NPs in a high-throughput manner. This enables direct high-throughput screening using NRPS-produced NP derivatives rather than chemically synthesised compounds.

14:15 Leonie Schenk - AG Bode

Development of a colorimetric assay for detecting functional engineered NRPS

Non-ribosomal peptide synthetases (NRPS) synthesize a wide range of natural products (NPs) with diverse biological activities, including potent drugs like cyclosporin and daptomycin. Due to the rise of antimicrobial resistance, the creation of new de novo peptides is of high interest, especially for the pharmaceutical industry. The modular composition of NRPS allows engineering of the mega synthetases by exchanging modules and catalytic subunits. During the past years, a multitude of engineering strategies and high throughput methods were established enhancing the generation of de novo NRPS. However, the screening for functional engineered NRPS and the respective NPs remains a bottleneck, including time-consuming steps like heterologous expression, NP extraction and HPLC-MS analysis. Establishing a screening assay identifying functional NRPS through a visual readout connected to the production of NPs would revolutionize NRPS engineering. To achieve this, we focused on the fluorescent siderophore pyoverdine, synthetized by an NRPS from Pseudomonas entomophila for NRPS engineering. Leveraging its intrinsic fluorescence, we successfully created first engineered fluorescent de novo NPs, which are easily screenable using a plate reader.