Biology subjects

Schada von Borzyskowski, L.

Publications and source records attributed to Schada von Borzyskowski, L..

6 recordsLinked to original sources

bioRxiv2024

A new pathway in central metabolism mediates nutrient control of development and antibiotic production by Streptomyces

The amino sugar N-acetylglucosamine (GlcNAc) plays a central role in primary metabolism and is a key signaling molecule for the onset of morphological and chemical differentiation of Streptomyces. The global nutrient-sensory regulator DasR acts as the gatekeeper of development in streptomycetes, and its activity is modulated by aminosugar phosphates. Here, we report the discovery of a novel pathway in aminosugar metabolism that governs GlcNAc sensing. GlcNAc-6P is converted into a toxic metabolite via two new enzyme functions, namely dehydration of N-acetylglucosamine-6-phosphate by NagS to form 6P-Chromogen I, a reaction that has not yet been described in the textbooks, and its subsequent deacetylation by NagA producing a cytotoxic structural analogue of ribose. The latter reveals an unexpected promiscuous activity for GlcNAc-6P deacetylase NagA. The crystal structures of NagS apoenzyme and NagS in complex with its substrate GlcNAc-6P or its inhibitor 6-phosphogluconate were resolved at 2.3 [A], 2.6 [A], and 1.7 [A] resolution, respectively. Detailed in vivo and in vitro studies resolved the key residues of the NagS catalytic site. Thus, we have uncovered a novel pathway in aminosugar metabolism that sheds new light on nutrient-mediated control of development and antibiotic production in Streptomyces.

microbiology↗

bioRxiv2024

Promiscuous NAD-dependent dehydrogenases enable efficient bacterial growth on the PET monomer ethylene glycol

Ethylene glycol is widely used as antifreeze agent and monomer of the ubiquitous plastic PET (polyethylene terephthalate). Its global production amounts to more than 50 million tons per year, and it constitutes an environmental pollutant of increasing concern. Although it is generally accepted that bacteria oxidize ethylene glycol to use it as growth substrate, the enzymes involved in this process are not well understood. Here we show that the soil bacterium Paracoccus denitrificans is able to assimilate ethylene glycol efficiently via NAD-dependent alcohol and aldehyde dehydrogenases. Using comparative proteome analysis, we identify a previously unknown gene cluster that is strongly expressed in the presence of ethylene glycol. We report the kinetic parameters and cryo-EM structures of EtgB and EtgA, the key enzymes encoded by this etg gene cluster. These novel biocatalysts pave the way for more efficient biotechnological conversion of ethylene glycol. We furthermore show that the transcriptional activator EtgR controls expression of the etg gene cluster. Directed evolution of P. denitrificans on ethylene glycol results in faster growing strains, which is enabled by increased activities of EtgB and EtgA. Bioinformatic analysis reveals that the etg gene cluster and variants thereof are widely distributed among Proteobacteria, suggesting a previously underappreciated role of NAD-dependent dehydrogenases in microbial ethylene glycol assimilation.

microbiology↗

bioRxiv2024

Multiple levels of transcriptional regulation control glycolate metabolism in Paracoccus denitrificans

The hydroxyacid glycolate is a highly abundant carbon source in the environment. Glycolate is produced by unicellular photosynthetic organisms and excreted at petagram scales to the environment, where it serves as growth substrate for heterotrophic bacteria. In microbial metabolism, glycolate is first oxidized to glyoxylate by the enzyme glycolate oxidase. The recently described {beta}-hydroxyaspartate cycle (BHAC) subsequently mediates the carbon-neutral assimilation of glyoxylate into central metabolism in ubiquitous Alpha- and Gammaproteobacteria. While the reaction sequence of the BHAC was elucidated in Paracoccus denitrificans, little is known about the regulation of glycolate and glyoxylate assimilation in this relevant alphaproteobacterial model organism. Here, we show that regulation of glycolate metabolism in P. denitrificans is surprisingly complex, involving two regulators, the IclR-type transcription factor BhcR that acts as an activator for the BHAC gene cluster, as well as the GntR-type transcriptional regulator GlcR, a previously unidentified repressor that controls the production of glycolate oxidase. Furthermore, an additional layer of regulation is exerted at the global level, which involves the transcriptional regulator CceR that controls the switch between glycolysis and gluconeogenesis in P. denitrificans. Together, these regulators control glycolate metabolism in P. denitrificans, allowing the organism to assimilate glycolate together with other carbon substrates in a simultaneous fashion, rather than sequentially. Our results show that the metabolic network of Alphaproteobacteria shows a high degree of flexibility to react to the availability of multiple substrates in the environment.

microbiology↗

bioRxiv2022

Implementation of the β-hydroxyaspartate cycle increases growth performance of Pseudomonas putida on the PET monomer ethylene glycol

Ethylene glycol (EG) is a promising next generation feedstock for bioprocesses. It is a key component of the ubiquitous plastic polyethylene terephthalate (PET) and other polyester fibers and plastics, used in antifreeze formulations, and can also be generated by electrochemical conversion of syngas, which makes EG a key compound in a circular bioeconomy. The majority of biotechnologically relevant bacteria assimilate EG via the glycerate pathway, a wasteful metabolic route that releases CO2 and requires reducing equivalents as well as ATP. In contrast, the recently characterized {beta}-hydroxyaspartate cycle (BHAC) provides a more efficient, carbon-conserving route for C2 assimilation. Here we aimed at overcoming the natural limitations of EG metabolism in the industrially relevant strain Pseudomonas putida KT2440 by replacing the native glycerate pathway with the BHAC. We first prototyped the core reaction sequence of the BHAC in Escherichia coli before establishing the complete four-enzyme BHAC in Pseudomonas putida. Directed evolution on EG resulted in an improved strain that exhibits 35% faster growth and 20% increased biomass yield compared to a recently reported P. putida strain that was evolved to grow on EG via the glycerate pathway. Genome sequencing and proteomics highlight plastic adaptations of the genetic and metabolic networks in response to the introduction of the BHAC into P. putida and identify key mutations for its further integration during evolution. Taken together, our study shows that the BHAC can be utilized as plug-and-play module for the metabolic engineering of two important microbial platform organisms, paving the way for multiple applications for a more efficient and carbon-conserving upcycling of EG in the future.

synthetic biology↗

bioRxiv2022

On the flexibility of the cellular amination network in E. coli

Ammonium (NH4+) is essential to generate the nitrogenous building blocks of life. It gets assimilated via the canonical biosynthetic routes to glutamate and is further distributed throughout metabolism via a network of transaminases. To study the flexibility of this network, we constructed an Escherichia coli glutamate auxotrophic strain. This strain allowed us to systematically study which amino acids serve as amine source and found that several amino acids complement the auxotrophy, either by producing glutamate via transamination reactions or by their conversion to glutamate. In this network, we identified aspartate transaminase AspC as a major connector between many amino acids and glutamate. Additionally, we extended the transaminase network by the amino acids {beta}-alanine, alanine, glycine and serine as new amine sources and identified D-amino acid dehydrogenase (DadA) as an intracellular amino acid sink removing substrates from transaminase reactions. Finally, ammonium assimilation routes producing aspartate or leucine were introduced. Our study reveals the high flexibility of the cellular amination network, both in terms of transaminase promiscuity and adaptability to new connections and ammonium entry points.

systems biology↗

bioRxiv2019

Sulfur-oxidizing symbionts without canonical genes for autotrophic CO2 fixation

Since the discovery of symbioses between sulfur-oxidizing (thiotrophic) bacteria and invertebrates at hydrothermal vents over 40 years ago, it has been assumed that autotrophic fixation of CO2 by the symbionts drives these nutritional associations. In this study, we investigated Candidatus Kentron, the clade of symbionts hosted by Kentrophoros, a diverse genus of ciliates which are found in marine coastal sediments around the world. Despite being the main food source for their hosts, Kentron lack the key canonical genes for any of the known pathways for autotrophic fixation, and have a carbon stable isotope fingerprint unlike other thiotrophic symbionts from similar habitats. Our genomic and transcriptomic analyses instead found metabolic features consistent with growth on organic carbon, especially organic and amino acids, for which they have abundant uptake transporters. All known thiotrophic symbionts have converged on using reduced sulfur to generate energy lithotrophically, but they are diverse in their carbon sources. Some clades are obligate autotrophs, while many are mixotrophs that can supplement autotrophic carbon fixation with heterotrophic capabilities similar to those in Kentron. We have shown that Kentron are the only thiotrophic symbionts that appear to be entirely heterotrophic, unlike all other thiotrophic symbionts studied to date, which possess either the Calvin-Benson-Bassham or reverse tricarboxylic acid cycles for autotrophy. Significance StatementMany animals and protists depend on symbiotic sulfur-oxidizing bacteria as their main food source. These bacteria use energy from oxidizing inorganic sulfur compounds to make biomass autotrophically from CO2, serving as primary producers for their hosts. Here we describe apparently non-autotrophic sulfur symbionts called Kentron, associated with marine ciliates. They lack genes for known autotrophic pathways, and have a carbon stable isotope fingerprint heavier than other symbionts from similar habitats. Instead they have the potential to oxidize sulfur to fuel the uptake of organic compounds for heterotrophic growth, a metabolic mode called chemolithoheterotrophy that is not found in other symbioses. Although several symbionts have heterotrophic features to supplement primary production, in Kentron they appear to supplant it entirely.

microbiology↗