Biology subjects

Molzahn, C.

Publications and source records attributed to Molzahn, C..

3 recordsLinked to original sources

bioRxiv2024

Mining Yeast Diversity Unveils Novel Targets for Improved Heterologous Laccase Production in Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae is a widely utilized host cell for recombinant protein production due to its well studied and annotated genome, its ability to secrete large and post-translationally modified proteins, fast growth and cost-effective culturing. However, recombinant protein yields from S. cerevisiae often fall behind that of other host systems. To address this, we developed a high throughput screen of wild, industrial and laboratory S. cerevisiae isolates to identify strains with a natural propensity for greater recombinant protein production, specifically focussing on laccase multicopper oxidases from the fungi Trametes trogii and Myceliophthora thermophila. Using this method, we identified 20 non-laboratory strains with higher capacity to produce active laccase. Interestingly, lower levels of laccase mRNA were measured in most cases, indicating that the drivers of elevated protein production capacity lie beyond the regulation of recombinant gene expression. We characterized the identified strains using complementary genomic and proteomic approaches to reveal several potential pathways driving the improved expression phenotype. Gene ontology analysis suggests broad changes in cellular metabolism, specifically in genes/proteins involved in carbohydrate catabolism, thiamine biosynthesis, transmembrane transport and vacuolar degradation. Targeted deletions of the hexose transporter HXT11 and the Coat protein complex II interacting paralogs PRM8 and 9, involved in ER to Golgi transport, resulted in significantly improved laccase production from the S288C laboratory strain. Whereas the deletion of the Hsp110 SSE1 gene, guided by our proteomic analysis, also led to higher laccase activity, we did not observe major changes of the protein homeostasis network within the strains with higher laccase activity. This study opens new avenues to leverage the vast diversity of Saccharomyces cerevisiae for recombinant protein production, as well as offers new strategies and insights to enhance recombinant protein yields of current strains.

molecular biology↗

bioRxiv2024

Analysis of protein levels and solubility in distinct brain regions reveals several elements of the protein homeostasis network that are impacted by aging

The onset of protein conformation diseases is inextricably linked to aging. During aging, cellular protein quality control declines which results in diminished protein homeostasis (proteostasis). In model organisms, such as C. elegans and killifish, proteostatic decline with age has been linked to the onset of aggregation of proteins in wild-type animals, observed through detergent-insoluble fractionation. Analysis of studies applying detergent-insoluble fractionation in mice revealed that the composition of detergent-insoluble proteins changes with age. However, these individual fractionation studies have generally been limited to small numbers of mice. Herein, we expand on our previous analysis by extending the experiments to a larger cohort of mice and to two brain regions implicated in neurodegenerative diseases, the cortex and hippocampus. These experiments unveil insights into alterations in the abundance and solubility of proteins involved in protein quality control and in inflammation. For example, ribosomal proteins and many chaperone proteins are downregulated with age. Consistent enrichment of subunits of the extracellular C1q complex was also observed in both brain regions alongside an increase in immunoglobulin signal indicating that markers of increased inflammation may also become insoluble during aging. More generally, insoluble proteins share features observed in datasets of impaired protein degradation indicating that the loss of activity of cellular protein degradation machinery may contribute to the specific aggregation of these proteins.

cell biology↗

bioRxiv2020

Kinetic-Mechanistic Evidence for Which E. coli RNA Polymerase-{lambda}PR Open Promoter Complex Initiates and for Stepwise Disruption of Contacts in Bubble Collapse

In transcription initiation, specific contacts between RNA polymerase (RNAP) and promoter DNA are disrupted as the RNA-DNA hybrid advances into the cleft, resulting in escape of RNAP. From the pattern of large and small rate constants for steps of initiation at {lambda}PR promoter at 19{degrees}C, we proposed that in-cleft interactions are disrupted in extending 3-mer to 5-mer RNA, -10 interactions are disrupted in extending 6-mer to 9-mer, and -35 interactions are disrupted in extending 10-mer to 11-mer, allowing RNAP to escape. Here we test this mechanism and determine enthalpic and entropic activation barriers of all steps from kinetic measurements at 25{degrees}C and 37{degrees}C. Initiation at 37{degrees}C differs significantly from expectations based on lower-temperature results. At low concentration of the second iNTP (UTP), synthesis of full-length RNA at 37{degrees}C is slower than at 25{degrees}C and no transient short RNA intermediates are observed, indicating a UTP-dependent bottleneck step early in the 37{degrees}C mechanism. Analysis reveals that the 37{degrees}C {lambda}PR OC (RPO) cannot initiate and must change conformation to a less-stable initiation complex (IC) capable of binding the iNTP. We find that IC is the primary {lambda}PR OC species below 25{degrees}C, and therefore conclude that IC must be the I3 intermediate in RPO formation. Surprisingly, Arrhenius activation energy barriers to five steps where RNAP-promoter in-cleft and -10 contacts are disrupted are much smaller than for other steps, including a negative barrier for the last of these steps. We interpret these striking effects as enthalpically-favorable, entropically-unfavorable, stepwise bubble collapse accompanying disruption of RNAP contacts. SignificanceTranscription initiation is highly regulated. To understand regulation, mechanisms of initiation and escape of RNA polymerase (RNAP) from the promoter must be understood. RNAP forms a highly-stable open complex (RPO) with {lambda}PR promoter at 37{degrees}C. From experiments determining effects of temperature on rate constants for each step of RNA synthesis, we find that RPO cannot bind the initiating nucleotides, that the I3 intermediate and not RPO is the initiation complex, and that contacts of RNAP with single-stranded DNA of the discriminator and -10 region and with -35 duplex DNA are disrupted stepwise as the RNA-DNA hybrid moves into the cleft. Evidence is obtained for stepwise bubble collapse and base stacking accompanying disruption of interactions of the single-stranded discriminator and -10 regions with RNAP.

biochemistry↗