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

Chair

Biocatalytic synthesis of heterocycles using condensing amidohydrolases

Biotechnology

Heterocycles are essential structural motifs of many pharmaceuticals and agrochemicals. As chemical synthesis of heterocycles typically requires harsh reaction conditions and the use of hazardous agents, the synthetic utility of heterocycle-forming enzymes is increasingly investigated. In recent years, several unusual members of the amidohydrolase superfamily were described that catalyze intramolecular condensation reactions to form heterocycles in natural product biosynthesis pathways. The first such enzyme that was characterized in detail was the condensing amidohydrolase MxcM. MxcM was discovered in the biosynthetic pathway of the siderophore pseudochelin A, where it generates an imidazoline moiety via intramolecular condensation of a β-aminoethyl amide group.[1,2,3] In Streptomyces sp. Tü 6176, a homolog of this condensing amidohydrolase produces a benzoxazole during the biosynthesis of nataxazole.[4] A small enzyme cascade consisting of an AMP ligase and the amidohydrolase is sufficient to synthesize benzoxazoles from aryl carboxylic acids. The AMP ligase first catalyzes the dimerization of two building blocks via ester bond formation. Then, the condensing amidohydrolase catalyzes the biosynthesis of the benzoxazole. Heterologous expression of these two enzymes in M. xanthus led to the production of various benzoxazole derivatives in a combinatorial precursor-directed biosynthesis approach.[5] A phylogenetic analysis based on already described condensing amidohydrolases revealed a large variety of homologous enzymes in different bacterial phyla (unpublished data). This finding suggests that other condensing amidohydrolases involved in heterocycle formation exist and that the synthetic potential of this enzyme group has not yet been fully exploited. In this study, a new condensing amidohydrolase originating from a myxobacterium was investigated. Interestingly, our data indicate that the reaction mechanism of this enzyme differs from the one previously described.[1] Korp J, Winand L, Sester A, Nett M. Environ. Microbiol. 2018, 84(22), e01789-18 [2] Winand L, Vollmann DJ, Hentschel J, Nett M. Catalysts 2021, 11(8), 892. [3] Winand L, Theisen S, Lütz S, Rosenthal K, Nett M. Catalysts 2023, 13(2), 229. [4] Song H, Rao C, Deng Z, Yu Y, Naismith JH. Chem. Int. Ed. Engl. 2020, 59(15), 6054–6061. [5] Winand L, Lernoud L, Meyners SA, Kuhr K, Hiller W, Nett M. ChemBioChem 2023, 24, e202200635.

Short lecture

Prof.

Process developement for heterologous Aurachin production

IntroductionAurachins are a family of prenylated quinoline antibiotics [1], which possess potent antiprotozoal properties [2]. In the natural producer Stigmatella aurantiaca Sg a15 a farnesyl moiety is added to 2-methyl-4-quinolone (MQO) by the farnesyltransferase AuaA to produce aurachin D. The latter is then N-hydroxylated to aurachin C by the Rieske type oxygenase AuaF [3]. Recently, a method for the biocatalytic production of aurachin D from MQO using recombinant E. coli cells was described [4].GoalsDue to their biological properties, the aurachins have gained attention as biochemical tool compounds and as drug candidates. Therefore, there is some interest in their cost-efficient production. The aim of this work was to intensify the biosynthesis of aurachin D in the heterologous host E. coli and to endow this bacterium with the auaF gene so that it can produce aurachin C.ResultsTo simplify the downstream processing we switched from a complex fermentation medium to a defined minimal medium, which initially decreased the aurachin D titer in E. coli from 63.6 mg L-1 to 30.5 mg L‑1. By increasing the glucose concentration in the minimal medium to 15 g L-1 and the NH4Cl concentration to 2 g L‑1, aurachin D titers comparable to the complex medium were reached. With the increase of the substrate concentration from 90 mg L‑1 to 180 mg L‑1 an aurachin D titer of 104.2 mg L-1 could be achieved. Another rise of the product titer to 117.2 3 mg L-1 was possible after extending the cultivation time from 24 h to 48 h.Plasmid-based co-expression of AuaA and AuaF enabled the production of aurachin C (6.6 mg L-1). Moreover, by lowering the precursor concentrations and auaF expression levels, an aurachin C titer of 23.5 mg L-1 was achieved.SummaryThe intensification of aurachin D production was successful and the product titer was almost doubled. Moreover, a system for aurachin C production was established. In the future aurachin C production can be optimized and new aurachin C derivatives can be produced by precursor-directed biosynthesis and screened for their antiprotozoal properties.References[1] Kunze B, Höfle G, Reichenbach H, J. Antibiot. 1987; 40(3): 258.[2] Kruth S, Zimmermann CJ-M, Kuhr K, Hiller W, Lütz S, Pietruszka J, Kaiser M, Nett M, Molecules 2023; 28(3): 1066.[3] Pistorius D, Li Y, Sandmann A, Müller R, Mol. Biosyst. 2011; 7(12): 3308.[4] Kruth S, Schibajew L, Nett M, AMB Expr. 2022; 12(1): 138.

Development of Bacillus subtilis as a production platform for bioactive secondary metabolites

Bacillus subtilis is considered a workhorse in biotechnology. In this context, the Bacillus family demonstrates a remarkable biosynthetic potential for the production of bioactive metabolites. Here, bacteriocins and lipopeptides are important representatives involved in intercellular interactions as well as showing promising application potentials due to their structural properties. However, in order to use the potential of bioactive metabolites in B. subtilis, molecular regulatory circuits in production strains have to be studied and adapted by molecular strain engineering and tailored bioproduction processes. In this way, surfactin as a representative of the lipopeptide family and mersacidin as a member of the bacteriocin class have already been successfully introduced into adapted B. subtilis production strains by understanding the associated regulatory networks, followed by rational strain engineering.In order to understand molecular regulatory networks within the biosynthetic pathways of surfactin and mersacidin, both the domesticated laboratory B. subtilis strain 168 and the sporulation-deficient strain 3NA were selected. Using reporter strains together with the integration of combinatorial gene knockouts, the expression of biosynthetic machineries could be evaluated and the influence of certain regulatory circuits could be studied. In this context, the cell differentiation associated with Spo0A-derived sporulation initiation and the regulation of the global transcriptional regulator AbrB were found to have significant relevance. This regulatory network plays a crucial role in the productivity of engineered B. subtilis production strains, which has been applied in adapted high cell-density fed-batch bioreactor cultivation processes. Targeted strain engineering allowed an increase in the production of surfactin to about 40 g/L, which is a record in scientific literature, whereas a mersacidin titer of about 100 mg/L could be reached, a 10-fold increase compared to the wild-type production strain.In sum, molecular strain engineering based on understanding molecular regulatory networks provides the opportunity to realize the biosynthetic potential of B. subtilis as a production platform for bioactive metabolites. In addition, the use of tailored fed-batch bioreactor production processes with high cell-density enables the production of the target products mentioned in high quantities to enable future applications.

MyxoTech: harnessing the power of microbial evolutionary diversity

Myxobacteria (phylum Myxococcota) are environmentally distributed, Gram-negative bacteria described as prolific producers of bioactive secondary ("special") metabolites. These potently engage a variety of cellular mechanisms-of-action in pro- and eukaryotes. MyxoTech re-purposes the myxobacterial drug discovery platform technology developed at HZI-HIPS for commercial application in indication areas outside the institute"s core mission, infectious disease. Under license we access collections of myxobacterial isolates and associated know-how. Our first collection follows the conceptual framework established by Hoffmann et al. (2018) to maximize chemical diversity, hence discovery potential, as a function of taxonomic diversity. The collection comprises isolates from 21 genera, 119 operational taxonomic units (OTU) of myxobacteria, with 196 isolates genome-sequenced, cultivated, extracted and prefractionated for biological screening at third party labs. MyxoTech provides this collection preplated and assay-ready for convenient screening. Downstream activities such as activity-directed dereplication, preparation of neat compound samples and structure elucidation can be accessed as fee-for-service or collaboration. We are committed to contributing to discoveries in a number of application and indication areas including hematology/oncology, immunity and inflammation. We plan assay applications for cell aging, mitochondrial function as well as payloads with novel, differentiated mechanism-of-action. We also plan to venture into non-pharmaceutical applications and seek scientific dialogue on these topics with the VAAM conference attendants.

Prof. Dr.

Optimization of biofilm-based production of platform chemicals using microscale membrane reactors

1. IntroductionIn the transition to a circular economy, alternatives to conventional chemical production routes must be developed based on renewable carbon or atmospheric CO2 to restore the balance of the global carbon cycle. We propose the production of platform chemicals using a genetically engineered Cupriavidus necator H16 strain thriving as biofilm on gas diffusion membranes. Due to its metabolic versatility, C. necator is able to utilize organic acids resulting from dark fermentations of biogenic waste and, as Knallgasbacterium, can fix CO2 using H2 as energy source. The targeted products can be entered into synthesis routes, e.g., for sustainable jet fuel.2. GoalsThe multiphase substrates of the bioconversion demand a suitable setup for efficient mass transfer and biomass retention, which can be realized in a membrane biofilm reactor (MBR). However, insights into biofilm formation, productivity per biomass, and influence of process parameters remain limited in such a setup. We established a microfluidic flow cell with an incorporated membrane that allows in-depth characterization and serves as a screening platform for a lab-scale MBR and robust process conditions. 3. MethodsC. necator was optimized for acetoin and 2,3-BDO production under availability of inorganic and organic carbon. The engineered strain was characterized regarding carbon efficiency and cultivation conditions. Cultivation as biofilm under autotrophic and heterotrophic conditions was performed in microfluidic reactors. Biofilm growth was monitored using optical coherence tomography and limitations were elucidated. 4. ResultsThe engineered strain reaches a carbon efficiency of 86% for the production of acetoin and 2,3-BDO from organic acids and CO2 (ratio 2:1). Cultivation as a membrane-bound biofilm allows a substantially higher cell density compared to planktonic systems, while downstream processing is simplified through cell retention. By fine-tuning the gas flow rate, growth can be limited to the membrane surface and the biofilm height controlled.5. SummaryGenetically engineered C. necator is employed as biofilm on gas diffusion membranes to produce valuable chemicals from renewable carbon and CO2. A microfluidic flow cell was developed for detailed analysis, serving as a screening platform to optimize conditions in a MBR. This approach demonstrates high carbon efficiency and cell densities, facilitating robust production suitable for large-scale applications.

Contaminant transformation and greywater treatment in redox-differentiated green roof model systems

Introduction: Green roofs have great potential to treat domestic greywater on-site while improving the urban microclimate and biodiversity. Preliminary experiments have shown that wetland roof systems are able to efficiently treat greywater which can be used as service water after appropriate hygienization (Rahman, 2023). However, further research is needed to understand underlying removal mechanisms to optimize green roof systems for wastewater treatment.Objectives: We are investigating which redox conditions are beneficial for the treatment of greywater and the microbial transformation of poorly degradable contaminants.Methods: Transformation of four wastewater chemicals of concern (namely benzotriazole, diclofenac, sulfamethoxazole and perfluorooctanoic acid) as well as the treatment of synthetic greywater were investigated. For this, two polypropylene boxes (each 37 cm × 26 cm) equipped with helophyte mats planted with Carex acutiformis served as down-scaled model systems under defined conditions. Redox-conditions were changed in the systems via permeation of nitrogen gas or compressed air and monitored using sensitive dissolved-oxygen sensors. Treatment of synthetic greywater was evaluated by measuring standard parameters for wastewater analysis. Transformation of the model chemicals was investigated by using LC-MS/MS technology. De novo proteomics were used to study the microbial community.Results: We were able to establish different oxic and anoxic subzones within the model systems. Despite the small model size and low amount of plants (three plants per system), the synthetic greywater was treated efficiently with high removal of BOD, COD, TOC, total nitrogen and ammonium – all parameters showing higher removal efficiencies under aeration with compressed air. The transformation of the four recalcitrant model chemicals was highly redox-dependent. Especially benzotriazole and diclofenac showed significantly higher removal when introducing oxic zones, while sulfamethoxazole also showed high removal efficiencies under very anoxic conditions. Identification of transformation products and de novo proteomics gave further insights, highlighting once more the importance of redox-conditions in microbial processes.Conclusion: This study shows the great potential of redox-dependent microbial transformation for the treatment of greywater in green roof systems.Rahman, K.Z. et al., Water 2023, 15, 3375. doi: 10.3390/w15193375

Dipl. Ing

Towards high atom economy in whole-cell redox biocatalysis: Up-scaling light-driven cyanobacterial ene-reductions in a flat panel photobioreactor

Light-driven biotransformations in recombinant cyanobacteria benefit from the atom-efficient regeneration of reaction equivalents, such as NADPH, from water and light via oxygenic photosynthesis. However, effective light distribution is hindered by self-shading effects within photosynthetic cells and extended light paths, significantly limiting scalability.We introduced a flat-panel photobioreactor with a 1-cm optical path length as a scalable system to provide efficient light distribution at high cell densities. Genes encoding five distinct classes of ene-reductases were heterologously expressed in Synechocystis sp. PCC 6803. The resulting strains were evaluated for their efficacy in light-driven ene-reduction reactions across a range of prochiral substrates.Under standard small-scale reaction conditions, the recombinant strains expressing the ene-reductases OYE3 and mutated TsOYE demonstrated specific activities of up to 150 U gCDW⁻¹, marking the highest activities recorded for photobiotransformations to date. These strains were chosen for scale-up in a 120-mL flat-panel photobioreactor. The strains achieved volumetric productivities up to 1 g L⁻¹ h⁻¹, surpassing current photosynthesis-driven processes. This setup enabled the conversion of 50 mM 2-methyl maleimide in a fed-batch within approximately 8 hours. The atom economy of this process reached 88% outperforming traditional processes relying on sacrificial cosubstrates such as glucose and formic acid. An E-Factor of 203 indicates that volumetric yield and water consumption for cell cultivation are critical parameters impacting process sustainability.In summary, we highlight essential factors influencing the sustainability of light-driven whole-cell biotransformations, establishing a robust foundation for future optimization and scale-up efforts in photosynthesis-driven bioproduction

Computationally assisted design of in situ activatable recombinant microbial transglutaminase variants

Microbial transglutaminase (MTG, EC: 2.3.2.13) from Streptomyces mobaraense catalyzes the formation of isopeptide bonds between proteinaceous glutamine and lysine residues. In addition, MTG catalyzes the conjugation of various amines to glutamine residues. These characteristics make it a valuable tool for usage in different industrial applications from medicine to food production. Native MTG is secreted as an inactive zymogen by Streptomyces mobaraense and proteolytically activated in the extracellular space. The zymogen consists of the mature polypeptide and an N-terminal pro-peptide, which is crucial for correct folding and keeping MTG inactive until its activity is needed. The expression as an inactive zymogen presents some difficulties for the recombinant production of highly active MTG, as the removal of the pro-peptide to activate MTG is a neccessary processing step. This extra step complicates purification, increases costs and can introduce unwanted side-effects, such as overdigestion of MTG by non-native proteases. To overcome these challenges, various approaches, e.g., the introduction of self-cleaving pro-peptide variants or the introduction of destabilizing mutations in the pro-peptide have been published. To simplify the production of active recombinant MTG, we systematically searched for mutations in the pro-peptide sequence, that would lead to protease-independent MTG-activation. By in silico site-saturation mutagenesis of selected pro-peptide residues and comparison of Rosetta ΔΔG predictions, we obtained destabilizing and stabilizing mutations. Several of these pro-peptide mutations were selected and the pro-peptide sequence was combined with the highly active and thermostable mature MTG variants TG16 and TG2A via Golden Gate Cloning. The resulting twelve single and two double mutants per MTG variant were expressed, purified and tested for protein stability, activity at a broad range of temperatures and ability to crosslink β-casein. Moreover, we present several MTG variants, that can be readily produced, easily purified and activated, as well as deactivated in situ.

Microbial biotechnology

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