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

Chair

Prof. Dr.

Engineering growth-coupled biosynthesis of animal pigments in bacteria

FG Synthetic microbiology

The mining of genomes across life has unearthed a bounty of biosynthetic potential for diverse molecules essential to build a bio-based future. While the heterologous expression of metabolic pathways has been adopted with broad success, most approaches face a common challenge: low initial production levels that require extensive, resource-intensive iterative strain engineering refinement. In this talk, I will discuss a growth-coupled biosynthesis strategy that links microbial growth with the production of specialized compounds—some of which, such as animal pigments, have not been produced in microbes before. The plug-and-play versatility of this strategy is showcased by producing the structurally-complex animal biopigment xanthommatin, a color-shifting ommochrome with promising material and cosmetic applications. In this framework, xanthommatin biosynthesis drives the growth of an engineered Pseudomonas putida 5,10-methylenetetrahydrofolate auxotroph (PUMA). With the support of genome-scale metabolic modeling, PUMA was engineered to be regulated by endogenous formate, a co-product generated in the multi-step conversion of tryptophan to xanthommatin. Adaptive laboratory evolution further streamlined gram-scale xanthommatin bioproduction through growth-rate selection, positioning this strategy as a solid approach for initiating and optimizing microbial production of high-value molecules.

Invited lecture

Rhodococcus for bioeconomy through synthetic biology

Environmental pollution has been the biggest trade-off caused by modernization and industrial revolution. Several efforts are now being channelled to circumvent this issue on environmental pollution and its repercussions such as global warming. The use of biological agents like exploiting the machineries of microorganisms has now emerged as a promising option. Microorganisms such as bacteria can adapt and evolve given the right amount of selective pressure. Therefore, it can be promising to exploit their inherent tools and machineries.Actinobacteria such as Streptomyces, Gordonia, and Rhodococcus are considered metabolic powerhouses that can degrade and transform different compounds such as styrene, rubber, phenols, and nitroaromatics. Several studies have shown and explored the enzymes responsible in degrading these pollutants. But unlike model organisms such as Pseudomonas putida and Escherichia coli, we have yet to understand on how to use an actinobacteria such as Rhodococcus for the purpose of a microbial cell factory through synthetic approaches. Nonetheless, Rhodococcus presents a more robust system as it can withstand harsher conditions compared to other model organisms. It is also easier to cultivate than other actinobacteria. Here, we present available information for Rhodococcus such as a tailored genetic toolbox and how to utilize them for protein production. Moreover, the current progress of Rhodococcus towards the route of bioeconomy will be discussed such as its potential for upcycling. This includes conversion of lignin-derived compounds like vanillin or polyethylene for industrially valuable products. This highlights how engineering Rhodococcus for bioeconomy can be promising.

Short lecture

Automating the DBTL-cycle for E. coli: Integration of modular cloning, CRISPRCas9 and Proteomics for advanced strain engineering

Introduction: The current process for developing microbial strains is both time-consuming and costly, mainly due to the many manual steps involved. Through miniaturization, parallelization, automation and digitalization, there is great potential for a significant acceleration of strain engineering.Methods: Here, we present automated workflows tailored to E. coli based on the seamless integration of modular cloning techniques (MoClo), genome editing tools (CRISPR-Cas9) and quantitative omics methods (proteomics).Results:MoClo enables the rapid screening of libraries with various DNA building blocks such as promoters or ribosomal binding sites. The plasmid-based workflow includes the entire Golden Gate reaction up to the transformation and plating of 96 assemblies on one plate. The additional integration of an automated CRISPR-Cas9 module enables the precise and targeted modification of the E. coli genome, which is crucial for the further metabolic engineering of producer strains. The latter covers the entire process from the generation of single guide RNA plasmids to the actual knock-out and curing step. Finally, to accelerate the subsequent phenotypic characterization of the genetically modified strains, an automated high-throughput module for untargeted proteomics was set up. The entire workflow, spanning from cultivation through bead mill processing to the Bradford assay, can be represented on the platform.Conclusion: In summary, the automated MoClo, CRISPR-Cas9 and proteomics workflows provide a comprehensive toolset for the rapid engineering of E. coli strains. Each module can be operated with minimal human interaction and high robustness, also facilitating the exploration of the genetic diversity of this important model organism.References: AutoBioTech─A Versatile Biofoundry for Automated Strain EngineeringTobias Michael Rosch, Julia Tenhaef, Tim Stoltmann, Till Redeker, Dominic Kösters, Niels Hollmann, Karin Krumbach, Wolfgang Wiechert, Michael Bott, Susana Matamouros, Jan Marienhagen, and Stephan NoackACS Synthetic Biology 2024 13 (7), 2227-2237DOI: 10.1021/acssynbio.4c00298

Analysis of the homeostatic control of glutamate in Bacillus subtilis

Glutamate plays an essential role in cellular metabolism, representing a crucial metabolic intersection linking carbon and nitrogen metabolism1. Therefore, this study aimed to analyze the export and consumption of glutamate in strains of Bacillus subtilis. To this end, B. subtilis was used as a chassis for deleting the pbuE gene, whose deletion can increase the intracellular concentrations of NAD+/NADH2. The engineered and parental strains were cultivated in 500 mL of mineral medium enriched with 60 g/L glucose for 50 h. During cultivation, samples were taken to evaluate cell growth spectrophotometrically and the concentration of glucose and glutamate in the medium by HPLC. B. subtilis wt exported glutamate to the medium during the exponential growth phase, reaching the peak between 20h and 30h. B. subtilis has been described to maintain high levels of intracellular glutamate during this phase, which may lead to the export of excess glutamate to sustain homeostatic levels3. B. subtilis ΔpbuE exported 0.15 g/L more glutamate than the parental strain. This indicates that increased concentrations of NAD+/NADH may be a determining factor in the availability of the amino acid in the cell. In B. subtilis, the only reaction that directly connects glutamate to NAD+/NADH is the conversion of glutamate into 2-ketoglutarate catalyzed by the RocG, which requires NAD+4. Once the glucose concentration dropped and became limiting to growth, cells started to consume the glutamate from the medium. B. subtilis has a complex regulatory system related to glutamate, which can assess the availability of carbon and nitrogen in the medium and thus adjust glutamate homeostasis accordingly3. Therefore, the data provided in this study contribute to understanding the complex dynamics of glutamate synthesis and export in different metabolic contexts in B. subtilis during the fermentation process.1Commichau, FM et al. (2006) Regulatory links between carbon and nitrogen metabolism. Curr Opin Microbiol 9: 167–172.2Michon, C et al. (2020) A bacterial cell factory converting glucose into scyllo-inositol, a therapeutic agent for Alzheimer"s disease. Commun Biol. 3(93).3Dormeyer M et al. (2019) Variants of the Bacillus subtilis LysR-type regulator GltC with altered activator and repressor function. Front Microbiol. 10:2321.4Belitsky BR, Sonenshein AL (1998) Role and Regulation of Bacillus subtilis Glutamate Dehydrogenase Genes. J Bacteriol 180

Mining of Vibrionacea genomes for plasmid origins as to obtain stable single-copy backbones for applications in Vibrio natriegens

Plasmids are extrachromosomal replicons which are ubiquitous in many families of bacteria archaea. This is also the case for the Vibrionacea, a family with many famous members, e.g. the human pathogens Vibrio cholerae and Vibrio parahaemolytics. Previous studies have shown that approximately a third of Vibrionacea isolates harbor plasmids and it is likely that these plasmids carry genes for niche adaptation and pathogenicity. Additionally, plasmids have a long history in biotechnological applications, like the production of insulin, and more recently, the synthesis of mRNA vaccines.Plasmids can be present in copy numbers from a single copy per cell, up to several hundreds. Especially single- and low-copy plasmids carry dedicated plasmid segregation systems. One such example is the ParABS machinery, consisting of the parS DNA sequence, typically located close to the origin of replication. parS is recognized by the CTPase ParB. The third component is ParA, a Walker-type ATPase, which binds unspecifically to DNA and interacts with ParB. Through a Brownian-ratchet mechanism caused by unbinding of ParA from the nucleoid, both copies of the ParB-bound plasmids are translocated to opposite cell poles, thereby ensuring stable inheritance of the plasmids to both daughter cells.We are interested in identifying ParABS systems present in Vibrionacea to investigate the diversity of these segregation mechanisms and to harness them as novel tools.Through a Hidden-Markov-Model-based genome mining approach, we identified almost 50 ParABS systems on plasmids from Vibrionacea isolates, with a replication protein in close proximity. Through this bioinformatic analysis, we identified sequences, which are likely responsible for replication and segregation of these plasmids. We proceeded by cloning these sequences and evaluated their performance in the fast-growing bacterium Vibrio natriegens. Our results indicate that the resulting plasmids have a single or low-copy number and varying degrees of stability. In some cases, the resulting plasmids are highly stable and persist in the population even in absence of antibiotic selection.In summary, we performed genome mining of Vibrionacea genomes to identify ParABS systems with accompanying replication proteins as plasmid origins. We experimentally characterized these sequences in V. natriegens to obtain stable single-copy origins which might allow the construction of synthetic chromosome in future projects.

FG Synthetic microbiology

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