Biology subjects

Kabisch, J.

Publications and source records attributed to Kabisch, J..

8 recordsLinked to original sources

bioRxiv2026

Bacterial Spores as a Scalable, Modular Platform forthe Production of Amyloids for Materials

We present a proof-of-concept platform in which amyloids are displayed on the surface of engineered Bacillus subtilis spores for bioengineered materials. Amyloids possess high tensile strength, elasticity, and tunable assembly, but their use is limited by inaccessible native sources and low-yield or toxic heterologous expression. Here, spores were engineered to display the native amyloid TasA and Humboldt squid suckerins 9 and 10 as fusions to the spore coat protein CotY. Amyloid production and fibril formation were confirmed by Western blot and X-34 staining, and quantitative analysis indicated mg/L-level yields. Atomic force microscopy revealed altered stiffness and surface ultrastructure, and incorporation of amyloid-displaying spores into resin-based 3D printing modified tensile strength. These findings highlight spore-based amyloid display as a scalable, modular platform for materials applications, leveraging established industrial spore production.

bioengineering↗

bioRxiv2021

Functionalizing cell-free systems with CRISPR-associated proteins: Application to RNA-based circuit engineering

Cell-free systems have become a compelling choice for the prototyping of synthetic circuits. Many robust protocols for preparing cell-free systems are now available along with toolboxes designed for a variety of applications. Thus far the production of cell-free extracts has often been decoupled from the production of functionalized proteins. Here, we leveraged the most recently published protocol for E. coli-based cell extracts with the endogenous production of two CRISPR-associated proteins, Csy4 and dCas9. We found pre-expression did not affect the resulting extract performance, and the final concentrations of the endonucleases matched the level required for synthetic circuit prototyping. We demonstrated the benefits and versatility of dCas9 and Csy4 through the use of RNA circuitry based on a combination of single guide RNAs, small transcriptional activator RNAs and toehold switches. For instance, we show that Csy4 processing increased fourfold the dynamic range of a previously published AND-logic gate. Additionally, blending the CRISPR-enhanced extracts enabled us to reduce leakage in a multiple inputs gate, and to extend the type of Boolean functions available for RNA-based circuits, such as NAND-logic. Finally, the use of dual transcriptional and translational reporters for the engineering of RNA-based circuits, allowed us to gain better insight into their underlying mechanisms. We hope this work will facilitate the adoption of advanced processing tools for RNA-based circuit prototyping in a cell-free environment.

synthetic biology↗

bioRxiv2020

Bacillus subtilis spore surface display of photodecarboxylase for the transformation of lipids to hydrocarbons

The display of enzymes on the surface of spores allows the rapid and very simple biotechnological production of immobilized enzymes. Here we describe the development of a Bacillus subtilis spore display platform and its application to produce hydrocarbons from lipids obtained from the oleaginous yeasts Yarrowia lipolytica, Cutaneotrichosporon oleaginosus as well as olive oil. Lipid hydrolysis was examined in a bienzymatic one-pot cascade using a commercially immobilized lipase (RO lipase) as well as spores with and without additional heterologous lipase expression. Decarboxylation of the released fatty acids was achieved displaying a photodecarboxylase (CvFAP) on the spore surface. Differences in composition of the formed hydrocarbons were observed depending on the lipids source. Using 3D printed lighting equipment titers of up to 64.0 {+/-} 5.6 mg/L hydrocarbons were produced.

bioengineering↗

bioRxiv2019

Characterization of three Yarrowia lipolytica strains in respect to different cultivation temperatures and metabolite secretion

Despite the increasing relevance, ranging from academic research to industrial applications, only a limited number of nonconventional, oleaginous Yarrowia lipolytica strains are characterized in detail. Therefore, we analyzed three strains in regard to their metabolic and physiological properties and in respect to important characteristics of a production strains. A flow cytometry method was set up to evaluate their fitness in a rapid manner. By investigating different cultivation conditions and media compositions, similarities and differences between the distinct strain backgrounds could be derived. Especially sugar alcohol production, as well as a agglomeration of cells were found to be connected with growth at high temperatures. In addition, sugar alcohol production was independent of high substrate concentrations under these conditions. To investigate particular traits, including growth characteristics and metabolite concentrations, genomic analysis were performed. We found sequence variations for one third of the annotated proteins but no obvious link to all phenotypic features.

microbiology↗

bioRxiv2019

Quantification and Isolation of Bacillus subtilis Spores using Cell Sorting and automated Gating.

The Gram-positive bacterium Bacillus subtilis is able to form endospores which have a variety of biotechnological applications. Due to this ability, B. subtilis is as well a model organism for cellular differentiation processes. Sporulating cultures of Bacillus subtilis form sub-populations which include vegetative cells, spore forming cells and spores. In order to readily and rapidly quantify spore formation we employed flow cytometric and fluorescence activated cell sorting techniques in combination with nucleic acid fluorescent staining in order to investigate the distribution of sporulating cultures on a single cell level. Moreover we tested different fluorescent dyes as well as different conditions in order to develop a method for optimal separation of distinct populations during sporulation. Automated gating procedures using k-means clustering and thresholding by gaussian mixture modeling were employed to avoid subjective gating and allow for the simultaneous measurement of controls. We utilized the presented method for monitoring sporulation over time in strains harboring different genome modifications. We identified the different subpopulations formed during sporulation by employing sorting and microscopy. Finally, we employed the technique to show that a double knock-out mutant of the phosphatase gene encoding Spo0E and of the spore killing factor SkfA results in faster spore formation.

microbiology↗

bioRxiv2019

Optimization of the experimental parameters of the ligase cycling reaction

The ligase cycling reaction (LCR) is a scarless and efficient method to assemble plasmids from fragments of DNA. This assembly method is based on the hybridization of DNA fragments with complementary oligonucleotides, so-called bridging oligos (BOs), and an experimental procedure of thermal denaturation, annealing and ligation. In this study, we explore the effect of molecular crosstalk of BOs and various experimental parameters on the LCR by utilizing a fluorescence-based screening system. The results indicate an impact of the melting temperatures of BOs on the overall success of the LCR assembly. Secondary structure inhibitors, such as DMSO and betaine, are shown to negatively impact the number of correctly assembled plasmids. Adjustments of the annealing, ligation and BO-melting temperature further improved the LCR. The optimized LCR was confirmed by validation experiments. Based on these findings, a step-by-step protocol is offered within this study to ensure a routine for high efficient LCR assemblies.

bioengineering↗

bioRxiv2018

Drop-In Biofuel production by using fatty acid photodecarboxylase from Chlorella variabilis in the oleaginous yeast Yarrowia lipolytica.

BackgroundOleaginous yeasts are potent hosts for the renewable production of lipids and harbor great potential for derived products, such as biofuels. Several promising processes have been described that produce hydrocarbon drop-in biofuels based on fatty acid decarboxylation and fatty aldehyde decarbonylation. Unfortunately, besides fatty aldehyde toxicity and high reactivity, the most investigated enzyme, aldehyde-deformylating oxygenase, shows unfavorable catalytic properties which hindered high yields in previous metabolic engineering approaches.\n\nResultsTo demonstrate an alternative alkane production pathway for oleaginous yeasts, we describe the production of diesel-like, odd-chain alkanes and alkenes, by heterologously expressing a recently discovered light-driven oxidase from Chlorella variabilis (CvFAP) in Yarrowia lipolytica. Initial experiments showed that only strains engineered to have an increased pool of free fatty acids showed to be susceptible to sufficient decarboxylation. Providing these strains with glucose and light in a synthetic medium resulted in titers of 10.9 mg/L of hydrocarbons. Using custom 3D printed labware for lighting bioreactors, and an automated pulsed glycerol fed-batch strategy, intracellular titers of 58.7 mg/L were achieved.\n\nConclusionsOleaginous yeasts such as Yarrowia lipolytica can transform renewable resources such as glycerol into fatty acids and lipids. By heterologously expressing a fatty acid photodecarboxylase from the algae Chlorella variabilis hydrocarbons were produced in several scales from microwell plate to 400 ml bioreactors. The developed bioprocess shows a route to the renewable production of hydrocarbons for a variety of applications ranging from representing a substrate for further enzymatic or chemical modification or as a drop-in biofuel blend.\n\nShort abstractOleaginous yeasts are potent hosts for the renewable production of lipids, fatty acids and derived products such as biofuels. Here, we describe, the production of odd-numbered alkanes and alkenes with a length of 17 and 15 carbons by expression of a fatty acid photodecarboxylase (CvFAP) from Chlorella variabilis in different Yarrowia lipolytica strains under different regimes of blue light exposure in several scales from microwell plate to 400 ml bioreactors.

bioengineering↗

bioRxiv2018

CopySwitch - in vivo optimization of gene copy numbers for heterologous gene expression in Bacillus subtilis

1.The Gram-positive bacterium Bacillus subtilis has long been used as a host for production and secretion of industrially relevant enzymes like amylases and proteases. It is imperative for optimal efficiency, to balance protein yield and correct folding. Gene copy numbers are an important tuning valve for the optimization of heterologous gene expression. While some genes are best expressed from many gene copies, for other genes, medium or even single copy numbers are the only way to avoid formation of inclusion bodies, toxic gene dosage effects or achieve desired levels for metabolic engineering. In order to provide a simple and robust method to address above-mentioned issues in the Gram-positive bacterium Bacillus subtilis, we have developed an automatable system for the tuning of heterologous gene expression based on the hosts intrinsic natural competence and homologous recombination capabilities. By supplying our reporter strains with a linearized, low copy number plasmid containing homology regions left and right of the reporter genes and an antibiotic resistance marker, we could show an up to 3.6-fold higher gfp (green fluorescent protein) expression and up to 1.3-fold higher mPLC (mature phospholipase C) expression after successful recombination and thus circularization of our plasmid. Furthermore, the plasmid-borne gfp expression seems to be more stable, since over the whole cultivation period the share of fluorescent cells compared to all measured cells is consistently higher.

bioengineering↗