Biology subjects

Wendisch, V. F.

Publications and source records attributed to Wendisch, V. F..

5 recordsLinked to original sources

bioRxiv2026

Isoprenoid quinone profiling in complex biological samples using a novel semi-quantitative HPLC-MS/MS method

Isoprenoid quinones are ubiquitous redox lipids that mediate electron transfer in various cellular processes across all domains of life. These molecules also serve as taxonomic and metabolic markers, facilitating the characterisation of microbial communities. However, their structural diversity and extreme hydrophobicity are challenging for comprehensive detection and quantification in complex biological matrices. In this study, we present a semi-quantitative HPLC-MS/MS method that enables the sensitive analysis of the widest range of quinones reported to date. Using a 16-quinone standard mixture, we optimised separation within a 14-minute HPLC gradient and achieved femtomole-level sensitivity in targeted analyses. When applied to sewage sludges sampled weekly over three weeks, our method detected 57 distinct quinones, revealing stage-specific quinone profiles that reflect shifts in bacterial communities during wastewater treatment. This rapid and sensitive workflow provides a robust tool for accurate quinone profiling in complex samples, opening avenues for the discovery of novel quinones through untargeted approaches. By pushing the boundaries of quinone profiling, our method holds significant promise for advancing microbial ecology, environmental monitoring, and biotechnological applications. HighlightsO_LIuHPLC-Orbitrap method for the semi-quantitative profiling of isoprenoid quinones C_LIO_LIAnalysis of the widest range of isoprenoid quinones to date C_LIO_LIFemtomole-level sensitivity in just 14 minutes of chromatographic separation C_LIO_LIDetection of 57 quinones in complex wastewater sludge matrices C_LIO_LIMost comprehensive set of quinone standards including purified microbial quinones C_LI

biochemistry↗

bioRxiv2024

Systems-level analysis provides insights on methanol-based production of L-glutamate and its decarboxylation product γ-aminobutyric acid by Bacillus methanolicus

BackgroundBacillus methanolicus is the next workhorse in biotechnology using methanol, an alternative and economical one-carbon feedstock that can be obtained directly from carbon dioxide, as both carbon and energy source for the production of various value-added chemicals. The wild-type strain B. methanolicus MGA3 naturally overproduces O_SCPLOWLC_SCPLOW-glutamate in methanol-based fed-batch fermentations. ResultsHere we generated, by induced mutagenesis, an evolved B. methanolicus strain exhibiting enhanced O_SCPLOWLC_SCPLOW-glutamate production capability (>150%). To showcase the potential of this evolved strain, further metabolic engineering enabled the production of {gamma}-aminobutyric acid (GABA) directly from O_SCPLOWLC_SCPLOW-glutamate, with a yield of 13.2 g/L from methanol during fed-batch fermentations. By using a systems-level analysis, encompassing whole-genome sequencing, RNA sequencing, fluxome analysis and genome-scale metabolic modelling, we were able to elucidate the metabolic and regulatory adaptations that sustain the biosynthesis of these products. The metabolism of the mutant strain evolved to prioritize energy conservation and efficient carbon utilization. Key metabolic shifts include the downregulation of energy-intensive processes such as flagellation and motility and the rerouting of carbon fluxes towards -ketoglutarate and its derivative, O_SCPLOWLC_SCPLOW-glutamate. Moreover, we observed that transformation of the evolved strain with a GABA biosynthesis plasmid had a positive effect on O_SCPLOWLC_SCPLOW-glutamate production, likely due to an upregulation of various transaminases involved in the O_SCPLOWLC_SCPLOW-glutamate biosynthesis from -ketoglutarate. ConclusionsThese results and insights provide a foundation for further rational metabolic engineering and bioprocess optimization, enhancing the industrial viability of B. methanolicus for sustainable production of O_SCPLOWLC_SCPLOW-glutamate and its derivatives.

systems biology↗

bioRxiv2023

COQ4 is required for the oxidative decarboxylation of the C1 carbon of Coenzyme Q in eukaryotic cells

Coenzyme Q (CoQ) is a redox lipid that fulfills critical functions in cellular bioenergetics and homeostasis. CoQ is synthesized by a multi-step pathway that involves several COQ proteins. Two steps of the eukaryotic pathway, the decarboxylation and hydroxylation of position C1, have remained uncharacterized. Here, we provide evidence that these two reactions occur in a single oxidative decarboxylation step catalyzed by COQ4. We demonstrate that COQ4 complements an Escherichia coli strain deficient for C1 decarboxylation and hydroxylation and that COQ4 displays oxidative decarboxylation activity in the non-CoQ producer Corynebacterium glutamicum. Overall, our results substantiate that COQ4 contributes to CoQ biosynthesis, not only via its previously proposed structural role, but also via oxidative decarboxylation of CoQ precursors. These findings fill a major gap in the knowledge of eukaryotic CoQ biosynthesis, and shed new light on the pathophysiology of human primary CoQ deficiency due to COQ4 mutations.

biochemistry↗

bioRxiv2021

Growth response and recovery of Corynebacterium glutamicum colonies on single-cell level upon defined pH stress pulses

Bacteria respond to pH changes in their environment via pH homeostasis to keep the intracellular pH as constant as possible within a small range. A change of the intracellular pH value influences e.g., the enzyme activity, protein stability, solubility of trace elements and the proton motive force. Here, the species Corynebacterium glutamicum has been chosen as a neutralophilic and moderately alkali-tolerant bacterium capable of maintaining an internal pH of 7.5 {+/-} 0.5 in environments with an external pH between 5.5 and 9. In the recent years, the phenotypic response of C. glutamicum to pH changes has been systematically investigated at the bulk population level. A detailed understanding of the C. glutamicum cell responding to defined short-term pH perturbations/ pulses is missing. In this study, dynamic microfluidic single-cell cultivation (dMSCC) was applied to analyse the physiological growth response of C. glutamicum upon precise pH stress pulses at a single-cell level. Analysis of the growth behaviour at the colony level by dMSCC exposed to single pH stress pulses (pH = 4, 5, 10, 11) revealed a decrease in the viability with increasing stress duration. Colony regrowth was possible after increasing lag phases when stress durations were increased from 5 min to 9 h for all tested pH values. Furthermore, the single-cell analysis revealed heterogeneous regrowth of cells after pH stress, which can be distinguished into two distinct behaviours firstly, cells continue to grow without interruption after the pH stress, and secondly, some cells rest for several hours after the pH stress before they start to grow again after this lag phase. This study provides the first insights into the single-cell response to acidic and alkaline pH stress adaptation of C. glutamicum.

microbiology↗

bioRxiv2019

Charting the metabolic landscape of the facultative methylotroph Bacillus methanolicus

Bacillus methanolicus MGA3 is a thermotolerant and relatively fast-growing methylotroph able to secrete large quantities of glutamate and lysine. These natural characteristics make B. methanolicus a good candidate to become a new industrial chassis organism, especially in a methanol-based economy. This has motivated a number of omics studies of B. methanolicus at the genome, transcript, protein and metabolic levels. Intriguingly, the only substrates known to support B. methanolicus growth as sole source of carbon and energy are methanol, mannitol, and to a lesser extent glucose and arabitol. We hypothesized that comparing methylotrophic and non-methylotrophic metabolic states at the flux level would yield new insights into MGA3 metabolism. 13C metabolic flux analysis (13C-MFA) is a powerful computational method to estimate carbon flows from substrate to biomass (i.e. the in vivo reaction rates of the central metabolic pathways) from experimental labeling data. In this study, we designed and performed a 13C-MFA of the facultative methylotroph B. methanolicus MGA3 growing on methanol, mannitol and arabitol to compare the associated metabolic states. The results obtained validate previous findings on the methylotrophy of B. methanolicus, allowed us to characterize the assimilation pathway of one of the studied carbon sources, and provide a better overall understanding of this strain. IMPORTANCEMethanol is cheap, easy to transport and can be produced both from renewable and fossil resources without mobilizing arable lands. As such, it is regarded as a potential carbon source to transition toward a greener industrial chemistry. Metabolic engineering of bacteria and yeast able to efficiently consume methanol is expected to provide cell factories that will transform methanol into higher-value chemicals in the so-called methanol economy. Toward that goal, the study of natural methylotrophs such as B. methanolicus is critical to understand the origin of their efficient methylotrophy. This knowledge will then be leveraged to transform such natural strains into new cell factories, or to design methylotrophic capability in other strains already used by the industry.

systems biology↗