Browsing by Person "Seitl, Ines"

Now showing 1 - 6 of 6

Functional and structural studies of a C-terminally extended YidC
(2015) Seitl, Ines; Kuhn, Andreas
Members of the YidC/Oxa1/Alb3 protein family catalyze the insertion of integral membrane proteins into the lipid bilayer of the bacterial plasma membrane (YidC), the inner mitochondrial membrane (Oxa1), and the chloroplast thylakoid membrane (Alb3) (Saller et al., 2012; Dalbey et al., 2014). The insertase homologs are comprised of a conserved core region of 5 transmembrane domains, but are provided with additionally flanking N- and C-terminal regions of variable lengths and functions. The Gram-negative YidC is characterized by an additional N-terminal domain, while Gram-positive bacteria, mitochondria and plastids developed C-terminally extended insertase-domains. These domains are involved e.g. in direct interaction with ribosomes and facilitate a functional overlap with the co-translational SRP-targeting pathway. An extended C-terminal highly positively charged tail region was also found in the YidC homologs of the Gram-negative marine bacteria Rhodopirellula baltica and Oceanicaulis alexandrii, but not in Escherichia coli. The primary subject of this work was to characterize and analyze in detail the C-terminally extended YidC chimera, composed of the E. coli YidC and the C-terminally extended domains of the marine YidC homologs. Biochemical binding assays with the purified YidC proteins and isolated, vacant E. coli 70S ribosomes showed that the C-tails mediate specific binding to ribosomes independently of the translational state of the ribosome. Furthermore, a ribosome-bound insertase complex was visualized by cryo-electron microscopy. The enhanced affinity of the C-terminally extended YidC was used to isolate stable complexes with stalled ribosomes, carrying a nascent polypeptide chain of a YidC substrate protein (MscL). The cryo-EM structure of a YidC-ribosome nascent chain complex (RNC) was solved to a 8,6 Å resolution and allowed the visualization of the nascent chain from the peptidyl transferase center through the ribosomal exit tunnel into the YidC density. The structure revealed the helix H59 of the 23S rRNA and the two ribosomal proteins L24 and L29 as the major contacts sites of YidC at the ribosomal tunnel exit. Pull down assays confirmed a significantly interaction of the C-terminal ribosome binding domain and the ribosomal protein L29, while L24 seems to be a universal contact site for the YidC-insertase core domain. Strikingly, the cryo-EM structure clearly showed a single monomer of YidC bound to the translating ribosome. This suggests that monomeric YidC might be the minimal functional unit for YidC-dependent, co-translational insertion of inner membrane proteins. In addition to the in vitro tests, a possible role of the C-terminal YidC extensions in co-translational protein targeting was tested in vivo in E. coli. For that purpose the targeting and localization of the SRP-dependent YidC-substrate protein MscL (Facey et al., 2007) was investigated as a GFP fusion protein via fluorescence microscopy. In addition, the proper membrane insertion of MscL was analyzed in radioactive pulse chase experiments via AMS gel shift assays, either in the absence of a functional SRP or SRP receptor (FtsY). Both in vivo assays clearly showed that the C-terminal ribosome binding domain of the R. baltica YidC homolog can partially substitute for the SRP receptor function in E. coli, while the cytosolic signal recognition particle is still required for correct insertion of the MscL protein. Therefore, a new co-translational targeting and insertion model of YidC-only substrates was proposed. This works also highlights evolutionary aspects of the accessory YidC domains and indicates that the C-terminal extended tail of YidC in the planctomycete group may be an ancestral remnant of a primordial translocation system operating without a typical SRP receptor. The second part focuses on the interaction of the signal recognition particle with SRP signal sequences. Isolated mutant signal sequence peptides were used to determine the specificity of SRP recognition in proteins. The interaction studies were established in an in vitro system and binding affinities of purified SRP to the isolated signal sequence peptides were determined via microscale thermophoresis (MST). A short sequence of 27 amino acid residues at the very N-terminal tail of the large cytoplasmic domain of KdpD was identified as a SRP signal sequence. Furthermore, a direct influence of the amino acid composition in the signal peptide on its SRP binding affinity in vitro was demonstrated. This confirms a low influence of an altered charge in the N-terminal region while mutations in the hydrophobic core region causes significantly reduced binding affinities to SRP. Taken together, this study contributes to the understanding of the molecular mechanisms of co-translational membrane protein biogenesis in bacteria.
Importance of the 5’ untranslated region for recombinant enzyme production in isolated Bacillus subtilis 007
(2025) Senger, Jana; Schulz, Adriana; Seitl, Ines; Heider, Martin; Fischer, Lutz
The production of industrial enzymes requires an efficient expression system with a suitable host. This study investigated the isolated Bacillus subtilis 007 as a host for expressing three enzymes with potential application in the food industry. Firstly, testing the PaprE and P43 promoters and the corresponding 5’ untranslated regions revealed great differences in the production of the recently discovered β-galactosidase from Paenibacillus wnnyii. Expression controlled by the PaprE promoter yielded a significantly higher activity of 2515 µkat/L, compared to 56 µkat/L with the P43 promoter. Modifications on the PaprE core promoter region or the spacer, the sequence between the Shine-Dalgarno sequence and the start codon, did not improve β-galactosidase production. Since the aprE 5’ untranslated region contributes to a high mRNA stability, it was incorporated into the P43 construct to determine whether mRNA stability is responsible for the differences observed in β-galactosidase production. Interestingly, mRNA stability was significantly improved and led to a nearly 50-fold higher β-galactosidase production of 2756 µkat/L. This strategy was successfully validated by the expression of two other enzymes: the cellobiose-2-epimerase from Caldicellulosiruptor saccharolyticus and the β-glucosidase from Pyrococcus furiosus. These findings underscored the crucial role of post-transcriptional regulation and emphasized mRNA stability as a key role in recombinant enzyme production in B. subtilis 007.
Orotic acid production by Yarrowia lipolytica under conditions of limited pyrimidine
(2021) Swietalski, Paul; Hetzel, Frank; Klaiber, Iris; Pross, Eva; Seitl, Ines; Fischer, Lutz
Orotic acid (OA) is an intermediate of the pyrimidine biosynthesis with high industrial relevance due to its use as precursor for production of biochemical pyrimidines or its use as carrier molecule in drug formulations. It can be produced by fermentation of microorganisms with engineered pyrimidine metabolism. In this study, we surprisingly discovered the yeast Yarrowia lipolytica as a powerful producer of OA. The overproduction of OA in the Y. lipolytica strain PO1f was found to be caused by the deletion of the URA3 gene which prevents the irreversible decarboxylation of OA to uridine monophosphate. It was shown that the lack of orotidine‐5′‐phosphate decarboxylase was the reason for the accumulation of OA inside the cell since a rescue mutant of the URA3 deletion in Y. lipolytica PO1f completely prevented the OA secretion into the medium. In addition, pyrimidine limitation in the cell massively enhanced the OA accumulation followed by secretion due to intense overflow metabolism during bioreactor cultivations. Accordingly, supplementation of the medium with 200 mg/L uracil drastically decreased the OA overproduction by 91%. OA productivity was further enhanced in fed‐batch cultivation with glucose and ammonium sulfate feed to a maximal yield of 9.62 ± 0.21 g/L. Y. lipolytica is one of three OA overproducing yeasts described in the literature so far, and in this study, the highest productivity was shown. This work demonstrates the potential of Y. lipolytica as a possible production organism for OA and provides a basis for further metabolic pathway engineering to optimize OA productivity.
Recombinant production of Paenibacillus wynnii β-galactosidase with Komagataella phaffii
(2024) Bechtel, Anna; Seitl, Ines; Pross, Eva; Hetzel, Frank; Keutgen, Mario; Fischer, Lutz
The β-galactosidase from Paenibacillus wynnii (β-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low KM value) compared to industrially used β-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, β-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce β-gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the β-gal-Pw in K. phaffii either extracellularly or intracellularly.ResultsFirstly, 11 different signal peptides were tested for extracellular production of β-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of β-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular β-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric β-galactosidase activity of 7537 ± 66 µkatoNPGal/Lculture was achieved by the K. phaffii clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14 ± 0.05 µkatoNPGal/gDCW/h was achieved when using the GAP promoter for β-gal-Pw production compared to the AOX1 promoter. After partial purification, a β-gal-Pw enzyme preparation with a total β-galactosidase activity of 3082 ± 98 µkatoNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter).ConclusionThis study showed that the β-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular β-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme.
Secretion of the cytoplasmic and high molecular weight β-galactosidase of Paenibacillus wynnii with Bacillus subtilis
(2024) Senger, Jana; Seitl, Ines; Pross, Eva; Fischer, Lutz
Background: The gram-positive bacterium Bacillus subtilis is widely used for industrial enzyme production. Its ability to secrete a wide range of enzymes into the extracellular medium especially facilitates downstream processing since cell disruption is avoided. Although various heterologous enzymes have been successfully secreted with B. subtilis, the secretion of cytoplasmic enzymes with high molecular weight is challenging. Only a few studies report on the secretion of cytoplasmic enzymes with a molecular weight > 100 kDa. Results: In this study, the cytoplasmic and 120 kDa β-galactosidase of Paenibacillus wynnii (β-gal-Pw) was expressed and secreted with B. subtilis SCK6. Different strategies were focused on to identify the best secretion conditions. Tailormade codon-optimization of the β-gal-Pw gene led to an increase in extracellular β-gal-Pw production. Consequently, the optimized gene was used to test four signal peptides and two promoters in different combinations. Differences in extracellular β-gal-Pw activity between the recombinant B. subtilis strains were observed with the successful secretion being highly dependent on the specific combination of promoter and signal peptide used. Interestingly, signal peptides of both the general secretory- and the twin-arginine translocation pathway mediated secretion. The highest extracellular activity of 55.2 ± 6 µkat/Lculture was reached when secretion was mediated by the PhoD signal peptide and expression was controlled by the PAprE promoter. Production of extracellular β-gal-Pw was further enhanced 1.4-fold in a bioreactor cultivation to 77.5 ± 10 µkat/Lculture with secretion efficiencies of more than 80%. Conclusion: For the first time, the β-gal-Pw was efficiently secreted with B. subtilis SCK6, demonstrating the potential of this strain for secretory production of cytoplasmic, high molecular weight enzymes.
Toward food-grade production of the Bacteroides helcogenes protein-glutamine glutaminase with an optimized Bacillus subtilis strain
(2026) Senger, Jana; Keutgen, Mario; Roth, Nicole; Seitl, Ines; Fischer, Lutz; Senger, Jana; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr 25, 70599, Stuttgart, Germany; Keutgen, Mario; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr 25, 70599, Stuttgart, Germany; Roth, Nicole; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr 25, 70599, Stuttgart, Germany; Seitl, Ines; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr 25, 70599, Stuttgart, Germany; Fischer, Lutz; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr 25, 70599, Stuttgart, Germany
Protein-glutamine glutaminases (PGs; EC 3.5.1.44) have gained attention in the food industry due to their application in plant protein products. The recently discovered PG from Bacteroides helcogenes (PGB) has especially been shown to provide promising characteristics for improving the techno-functional properties of plant proteins. A prerequisite for food enzymes, such as the PG, is their production with an expression host that meets food safety and yield requirements. The antibiotic-free and secretory production of the PGB was targeted in this study using the undomesticated Bacillus subtilis 007. The CRISPR/Cas9-mediated approach enabled specific genomic PGB integrations, while simultaneously deleting unwanted B. subtilis traits. Firstly, the PGB expression cassette was integrated into the sigF gene, leading to an asporogenic strain and extracellular activity of 4.1 µkat/Lculture in bioreactor cultivations. However, excessive foaming hampered the production process tremendously. Consequently, a second PGB copy was integrated into the sfp locus, which is involved in the production of lipopeptides, such as surfactin. As a result, the PGB activity was increased to 5.4 µkat/Lculture, and foaming during cultivation was reduced significantly. The introduction of a third PGB copy for preventing cell motility did not increase production; however, the integration into the well-established amyE locus improved the PGB yield during reactor cultivations. A final extracellular activity of 9.5 µkat/Lculture was reached. The multiple genomic integrations of the PGB gene enabled the efficient PGB secretion in an optimized B. subtilis host without the need for antibiotics.Key points• Site-specific PGB integration enabled by genome sequencing of B. subtilis 007.• Antibiotic-free and secretory PGB production with an optimized B. subtilis host.• Increased PGB production reaching 9.5 µkat/Lculture.