Biology subjects

Wentzel, A.

Publications and source records attributed to Wentzel, A..

6 recordsLinked to original sources

bioRxiv2024

Xolography for Biomedical Applications: Dual-color Light-sheet Printing of Hydrogels with Local Control over Shape and Stiffness

AbstractCurrent challenges in tissue engineering include creation of extracellular environments that support and interact with cells using biochemical, mechanical, and structural cues. Spatial control over these cues is currently limited due to a lack of suitable fabrication techniques. This study introduces Xolography, an emerging dual-color light-sheet volumetric printing technology, to achieve control over structural and mechanical features for hydrogel-based photoresins at micro-to macroscale while printing within minutes. We propose a water-soluble photoswitch photoinitiator system and are the first to demonstrate Xolography with a library of naturally-derived, synthetic, and thermoresponsive hydrogels. Centimeter-scale, three-dimensional constructs with positive features of 20 {micro}m and negative features of [~] 100 {micro}m are fabricated with control over mechanical properties (compressive moduli 0.2 kPa - 6.5 MPa). Notably, switching from binary to grayscaled light projection enables spatial control over stiffness (0.2 - 16 kPa). As a proof of concept, grayscaled Xolography is leveraged with thermoresponsive hydrogels to introduce reversible anisotropic shape changes beyond isometric shrinkage. We finally demonstrate Xolography of viable cell aggregates, laying the foundation for cell-laden printing of dynamic, cell-instructive environments with tunable structural and mechanical cues in a fast one-step process. Overall, these innovations unlock unique possibilities of Xolography across multiple biomedical applications.

bioengineering↗

bioRxiv2023

Reprogramming Yarrowia lipolytica metabolism for efficient synthesis of itaconic acid from flask to semi-pilot scale

Itaconic acid is an emerging platform chemical with extensive applications. It is currently produced by Aspergillus terreus through biological fermentation. However, A. terreus is a fungal pathogen and needs additional morphology controls, and therefore the production remains problematic. Here, we reprogrammed the GRAS yeast Yarrowia lipolytica metabolism for competitive itaconic acid production. After redirecting the flux of lipid accumulation as carbon sink, we evaluated itaconic acid production both inside and outside the mitochondria, and fine modulated its synthetic pathway. We then mimicked the regulation of nitrogen limitation in nitrogen replete conditions through down regulation of IDH by weak promoter changing, RNAi, or CRISPRi. Ultimately, we optimized fermentation parameters for fed-batch cultivations, and produced itaconic acid with titres of 130.1 g/L in 1L bioreactors and 94.8 g/L in a 50L bioreactor on semi-pilot scale. Our finds provide effective approaches for harnessing GRAS microorganism for competitive industrial itaconic acid production.

bioengineering↗

bioRxiv2020

Automatic reconstruction of metabolic pathways from identified biosynthetic gene clusters

BackgroundA wide range of bioactive compounds are produced by enzymes and enzymatic complexes encoded in biosynthetic gene clusters (BGCs). These BGCs can be identified and functionally annotated based on their DNA sequence. Candidates for further research and development may be prioritized based on properties such as their functional annotation, (dis)similarity to known BGCs, and bioactivity assays. Production of the target compound in the native strain is often not achievable, rendering heterologous expression in an optimized host strain as a promising alternative. Genome-scale metabolic models are frequently used to guide strain development, but large-scale incorporation and testing of heterologous production of complex natural products in this framework is hampered by the amount of manual work required to translate annotated BGCs to metabolic pathways. To this end, we have developed a pipeline for an automated reconstruction of BGC associated metabolic pathways responsible for the synthesis of non-ribosomal peptides and polyketides, two of the dominant classes of bioactive compounds. ResultsThe developed pipeline correctly predicts 72.8% of the metabolic reactions in a detailed evaluation of 8 different BGCs comprising 228 functional domains. By introducing the reconstructed pathways into a genome-scale metabolic model we demonstrate that this level of accuracy is sufficient to make reliable in silico predictions with respect to production rate and gene knockout targets. Furthermore, we apply the pipeline to a large BGC database and reconstruct 943 metabolic pathways. We identify 17 enzymatic reactions using high-throughput assessment of potential knockout targets for increasing the production of any of the associated compounds. However, the targets only provide a relative increase of up to 6% compared to wild-type production rates. ConclusionsWith this pipeline we pave the way for an extended use of genome-scale metabolic models in strain design of heterologous expression hosts. In this context, we identified generic knockout targets for the increased production of heterologous compounds. However, as the predicted increase is minor for any of the single-reaction knockout targets, these results indicate that more sophisticated strain-engineering strategies are necessary for the development of efficient BGC expression hosts.

bioinformatics↗

bioRxiv2020

Microbial-induced calcium carbonate precipitation: An experimental toolbox for in situ and real-time investigation of micro-scale pH evolution

Concrete is the second most consumed product by humans, after water. However, the production of cement, which is used as a binding material in concrete, causes more than 5% of anthropogenic CO2 emissions and has therefore a significant contribution to climate change and global warming. Due to increasing environmental awareness and international climate goals, there is a need for emission-reduced materials, that can replace conventional concrete in certain applications. One path to produce a solid, concrete-like construction material is microbial-induced calcium carbonate precipitation (MICP). As a calcium source in MICP, crushed limestone, which mainly consists out of CaCO3, can be dissolved with acids, for example lactic acid. The pH evolution during crystallization and dissolution processes provides important information about kinetics of the reactions. However, previous research on MICP has mainly been focused on macro-scale pH evolution and on characterization of the finished material. To get a better understanding of MICP it is important to be able to follow also local pH changes in a sample. In this work we present a new method to study processes of MICP at micro-scale in situ and in real time. We present two different methods to monitor the pH changes during the precipitation process of CaCO3. In the first method, the average pHs of small sample volumes are measured in real time, and pH changes are subsequently correlated with processes in the sample by comparing to optical microscope results. The second method is introduced to follow local pH changes at a grain scale in situ and in real time. Furthermore, local pH changes during the dissolution of CaCO3 crystals are monitored. We demonstrate that these two methods are powerful tools to investigate pH changes for both MICP precipitation and CaCO3 dissolution for knowledge-based improvement of MICP-based material properties. Graphical TOC Entry O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/042168v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@dad9bcorg.highwire.dtl.DTLVardef@ce9fborg.highwire.dtl.DTLVardef@1d583b8org.highwire.dtl.DTLVardef@bab03a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2019

Genome-scale Model Constrained by Proteomics Reveals Metabolic Rearrangements in the Heterologous Host Streptomyces coelicolor

Many biosynthetic gene clusters (BGCs) require heterologous expression to realize their genetic potential, including silent and metagenomic BGCs. Although the engineered Streptomyces coelicolor M1152 is a widely used host for heterologous expression of BGCs, a systemic understanding of how its genetic modifications affect the metabolism is lacking and limiting further development. We performed a comparative analysis of M1152 and its ancestor M145, connecting information from proteomics, transcriptomics, and cultivation data into a comprehensive picture of the metabolic differences between these strains. Instrumental to this comparison was the application of an improved consensus genome-scale metabolic model (GEM) of S. coelicolor. Although many metabolic patterns are retained in M1152, we find that this strain suffers from oxidative stress, possibly caused by increased oxidative metabolism. Furthermore, precursor availability is likely not limiting polyketide production, implying that other strategies could be beneficial for further development of S. coelicolor for heterologous production of novel compounds.

systems biology↗

bioRxiv2019

Towards a low CO2 emission building material employing bacterial metabolism in a two-step process of limestone dissolution and recrystallization: The bacterial system and prototype production

The production of concrete for construction purposes is a major source of anthropogenic CO2 emissions. One promising avenue towards a more sustainable construction industry is to make use of naturally occurring mineral-microbe interactions, such as microbial-induced carbonate precipitation (MICP), to produce solid materials. In this paper, we present a new process where calcium carbonate in the form of powdered limestone is transformed to a binder material (termed BioZEment) through microbial dissolution and recrystallization. For the dissolution step, a suitable bacterial strain, closely related to Bacillus pumilus, was isolated from soil near a limestone quarry. We show that this strain produces organic acids from glucose, inducing the dissolution of calcium carbonate in an aqueous slurry of powdered limestone. In the second step, the dissolved limestone solution is used as the calcium source for MICP in sand packed syringe moulds. The amounts of acid produced and calcium carbonate dissolved are shown to depend on the amount of available oxygen as well as the degree of mixing. Precipitation is induced through the pH increase caused by the hydrolysis of urea, mediated by the enzyme urease, which is produced in situ by the bacterium Sporosarcina pasteurii DSM33. The degree of successful consolidation of sand by BioZEment was found to depend on both the amount of urea and the amount of glucose available in the dissolution reaction.

microbiology↗