Biology subjects

Virant, D.

Publications and source records attributed to Virant, D..

3 recordsLinked to original sources

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↗

bioRxiv2021

Unraveling the kinetochore nanostructure in Schizosaccharomyces pombe using multi-color single-molecule localization microscopy

The key to ensuring proper chromosome segregation during mitosis is the kinetochore complex. This large and tightly regulated multi-protein complex links the centromeric chromatin to the microtubules attached to the spindle pole body and as such leads the segregation process. Understanding the architecture, function and regulation of this vital complex is therefore essential. However, due to its complexity and dynamics, only its individual subcomplexes could be studied in high-resolution structural detail so far. In this study we construct a nanometer-precise in situ map of the human-like regional kinetochore of Schizosaccharomyces pombe (S. pombe) using multi-color single-molecule localization microscopy (SMLM). We measure each kinetochore protein of interest (POI) in conjunction with two reference proteins, cnp1CENP-A at the centromere and sad1 at the spindle pole. This arrangement allows us to determine the cell cycle and in particularly the mitotic plane, and to visualize individual centromere regions separately. From these data, we determine protein distances within the complex using Bayesian inference, establish the stoichiometry of each POI for individual chromosomes and, consequently, build an in situ kinetochore model for S.pombe with so-far unprecedented precision. Being able to quantify the kinetochore proteins within the full in situ kinetochore structure, we provide valuable new insights in the S.pombe kinetochore architecture.

microbiology↗

bioRxiv2020

Post-mitotic expansion of cell nuclei requires ACTN4-mediated nuclear actin filament bundling

The actin cytoskeleton operates in a multitude of cellular processes including cell shape and migration, mechanoregulation, as well as membrane or organelle dynamics. However, its filamentous properties and functions inside the mammalian cell nucleus are less well explored. We previously described transient actin assembly at mitotic exit that promotes nuclear expansion during chromatin decondensation. Here, we identify non-muscle ACTN4 as a critical regulator to facilitate F-actin formation, reorganization and bundling during postmitotic nuclear expansion. ACTN4 binds to nuclear actin filaments and ACTN4 clusters associate with nuclear F-actin in a highly dynamic fashion. ACTN4 but not ACTN1 is required for proper postmitotic nuclear volume expansion, mediated by its actin binding domain. Using super-resolution imaging to quantify actin filament numbers and widths in individual nuclei we find that ACTN4 is necessary for postmitotic nuclear actin assembly and actin filament bundling. Our findings uncover a nuclear cytoskeletal function for ACTN4 to control nuclear size during mitotic cell division.

cell biology↗