Annual Conference 2025 of the Association for General and Applied Microbiology - Jahrestagung 2025 der Vereinigung für Allgemeine und Angewandte Mikrobiologie

Prof.

Chair

New tricks and tools for efficient enzyme production and engineering employing P. pastoris

Invited lecture

From Host Selection to Process Innovation: Aspergillus niger and Its Alternatives in Enzyme and Protein Production

FG Biotransformation

The selection of an optimal host organism for heterologous protein production is a critical and challenging aspect of industrial biotechnology. Each host offers unique advantages and limitations that must be carefully considered for efficient enzyme or protein production. To address this challenge, BRAIN Biotech has developed a suite of producer strains, including Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Komagataella phaffii, and Aspergillus niger. Among these, A. niger has emerged as one of the most productive hosts in terms of final product titers, making it an attractive candidate for industrial applications.This presentation will focus on the strain and process optimization strategies employed to enhance A. niger as a production host. Key advancements include the use of proprietary CRISPR nucleases for targeted genome editing to facilitate the insertion of expression cassettes and the deletion of protease-encoding genes to reduce protein degradation. Additional modifications, such as the deletion of genes influencing viscosity in the fermentation broth, have been implemented to improve process efficiency. Furthermore, process optimization and downstream processing enhancements have been explored to maximize yields and reduce production costs.By combining cutting-edge genetic engineering techniques with process engineering innovations, we have significantly improved the utility of A. niger as a robust and efficient production platform for industrial enzymes and proteins. This talk will give insights into how BRAIN Biotech utilizes A. niger for the biotechnological production of enzymes and other proteins.

Short lecture

Accelerating the discovery of biocatalysts through cell-free protein synthesis: Advances and applications

Cell-free protein synthesis (CFPS) is a well-established but constantly evolving method that enables protein synthesis in vitro without the need for living cells. This method has proven its effectiveness as a robust platform for screening biocatalysts and enables the rapid discovery and optimization of enzymes for various biotechnological applications. By separating protein synthesis from cell growth, CFPS has significant advantages compared to conventional heterologous protein synthesis in vivo, e.g. shorter production times and the ability to synthesize proteins that are toxic to cells.In recent years, we have developed and utilized various CFPS systems for the screening of a wide range of enzymes. Mainly based on Escherichia coli extracts, a total of seven proprietary CFPS systems have been developed and more than 30 different enzymes have been successfully synthesized across five enzyme classes. The presentation will provide insights into the application of CFPS systems based on E. coli extracts as a screening tool using various examples. These applications encompass the screening of enzyme homologs and variants, evaluation of various expression hosts, investigation of (co-)substrate ranges, and assessment of gene expressibility, such as genes from higher organisms in standard prokaryotic systems [1-3]. In addition, protein synthesis under non-natural conditions [4] and the synthesis of complex proteins such as membrane proteins using CFPS are discussed. In addition to the fast reaction, the openness of the system, which means that it can be easily modified, if necessary, is a major advantage in order to create highly defined environmental conditions for protein synthesis.Overall, CFPS has been demonstrated to serve as a flexible platform for biocatalyst development, with promising applications spanning areas such as industrial biotechnology and drug discovery.[1] Rolf J., Siedentop R., Lütz S., Rosenthal K. Int. J. Mol. Sci 2019, 21(1), 105[2] Rolf J., Nerke P., Britner A., Krick S., Lütz S., Rosenthal K. Catalysts 2021, 11(9), 1038[3] Rolf J., Ngo A., Lütz S., Tischler D., Rosenthal K. ChemBioChem 2022, 23(15), e202200121[4] Bartsch T., Lütz S., Rosenthal K. Beilstein J. Org. Chem. 2024, 20, 2242-2253

Heterologous expression in the eukaryotic micro alga Chlamydomonas reinhardtii

Green microalgae harbour great potential as sustainable light-driven hosts for engineered bioproduction of high-value products. Their capacity for efficient photosynthesis fuels a strong metabolic flux towards various hydrocarbons, which can be tapped for the production of valuable hydrocarbon based chemicals such as Terpenoids and Carotenoids.The microalga Chlamydomonas reinhardtii stands out as a well-developed model organism: established transformation methods allow feasible transformation and straightforward mutant screening is provided by a large set of reporter genes. However, advanced genetic and metabolic engineering driven from the nuclear genome is generally hindered by low transgene transcription rates and limited knowledge of underlying eukaryotic regulation.In the last decade, a great molecular toolkit has been developed, which is still expanding. Here we present some of the most striking strategies to enhance gene expression in the microalga including promotor engineering, intron mediated enhancement and targeted integration. These and further strategies allow a more efficient use of the microalgas potential for protein expression and bioproduction.

BPP Bioportides – How to standardize a protein based platform for gene delivery

Biotechnology

Introduction: Gene delivery is a topic of utmost importance that we are constantly confronted with in all fields of microbiology. Genetic modifications may take all kinds of forms. At times, it is necessary to insert a gene for recombinant expression and sometimes we want to silence certain pathways. BPP Bioportides promise to implement one tool for various such applications in a broad variety of different organisms. The nucleic acid binding domain of the BPP Bioportide has been observed to show affinity for different types of nucleic acids. The cell penetrating domain can be adapted to the target membrane. This capability was successfully employed in projects involving the transfection of plants but also the transformation of filamentous microorganisms.Project Goals: BPP Bioportides are a novel tool. Hence, it is necessary to establish a standard procedure to assess the quality of a certain batch but also to investigate the suitability of a certain Bioportide with respect to a certain nucleic acid and organsism. E. coli is used as a standard organism that is transformed with standard plasmids using different Bioportides and heat shock as a reference method in order to establish a relative scale and method to compare BPP Bioportide suitability for different organisms.Materials and methods: A series of different Bioportides is used for transformation in E.coli and compared with heat shock transformation. The read out is assessed using two methods. The classical approach is two count CFUs on selection agar plates and evaluate transformation efficiency and frequency. The second method employs analysis of culture growth in a fluid selection medium. The culture growth is investigated using light scattering and oxygen concentration as readouts.Results: The data of both methods are in line and show that the investigated Bioportides lead to higher transformation efficiencies than the heat shock method in log phase E. coli cells. The data collected in fluid culture add information regarding the vitality of the cells. Both methods are also useful to highlight differences in performance between the BPP Bioportides in use.Summary: BPP Bioportides are novel platform for gene delivery in various microorganisms. The study at hand provides a tool for the comparison and standardization of Bioportide utilization in unknown organisms. This provides new opportunities to improve and optimize gene delivery.

FG Biotransformation

Appointment

Chair

Programme