Identification of TFG- and Autophagy-Regulated Proteins and Glycerophospholipids in B Cells.

Autophagy supervises the proteostasis and survival of B lymphocytic cells. Trk-fused gene (TFG) promotes autophagosome-lysosome flux in murine CH12 B cells, as well as their survival. Hence, quantitative proteomics of CH12tfgKO and WT B cells in combination with lysosomal inhibition should identify proteins that are prone to lysosomal degradation and contribute to autophagy and B cell survival. Lysosome inhibition via NH4Cl unexpectedly reduced a number of proteins but increased a large cluster of translational, ribosomal, and mitochondrial proteins, independent of TFG. Hence, we propose a role for lysosomes in ribophagy in B cells. TFG-regulated proteins include CD74, BCL10, or the immunoglobulin JCHAIN. Gene ontology (GO) analysis reveals that proteins regulated by TFG alone, or in concert with lysosomes, localize to mitochondria and membrane-bound organelles. Likewise, TFG regulates the abundance of metabolic enzymes, such as ALDOC and the fatty acid-activating enzyme ACOT9. To test consequently for a function of TFG in lipid metabolism, we performed shotgun lipidomics of glycerophospholipids. Total phosphatidylglycerol is more abundant in CH12tfgKO B cells. Several glycerophospholipid species with similar acyl side chains, such as 36:2 phosphatidylethanolamine and 36:2 phosphatidylinositol, show a dysequilibrium. We suggest a role for TFG in lipid homeostasis, mitochondrial functions, translation, and metabolism in B cells.

Dimer Stabilization by SpyTag/SpyCatcher Coupling of the Reductase Domains of a Chimeric P450 BM3 Monooxygenase from Bacillus spp. Improves its Stability, Activity, and Purification.

The vast majority of known enzymes exist as oligomers, which often gives them high catalytic performance but at the same time imposes constraints on structural conformations and environmental conditions. An example of an enzyme with a complex architecture is the P450 BM3 monooxygenase CYP102A1 from Bacillus megaterium. Only active as a dimer, it is highly sensitive to dilution or common immobilization techniques. In this study, we engineered a thermostable P450BM3 chimera consisting of the heme domain of a CYP102A1 variant and the reductase domain of the homologous CYP102A3. The dimerization of the hybrid was even weaker compared to the corresponding CYP102A1 variant. To create a stable dimer, we covalently coupled the C-termini of two monomers of the chimera via SpyTag003/SpyCatcher003 interaction. As a result, purification, thermostability, pH stability, and catalytic activity were improved. Via a bioorthogonal two-step affinity purification, we obtained high purity (94 %) of the dimer-stabilized variant being robust against heme depletion. Long-term stability was increased with a half-life of over 2 months at 20 °C and 80-90 % residual activity after 2 months at 5 °C. Most catalytic features were retained with even an enhancement of the overall activity by ~2-fold compared to the P450BM3 chimera without SpyTag003/SpyCatcher003.

Enhancing the activity of a monomeric alcohol dehydrogenase for site-specific applications by site-directed mutagenesis.

Gene fusion or co-immobilization are key tools to optimize enzymatic reaction cascades by modulating catalytic features, stability and applicability. Achieving a defined spatial organization between biocatalysts by site-specific applications is complicated by the involvement of oligomeric enzymes. It can lead to activity losses due to disturbances of the quaternary structures and difficulties in stoichiometric control. Thus, a toolkit of active and robust monomeric enzymes is desirable for such applications. In this study, we engineered one of the rare examples of monomeric alcohol dehydrogenases for improved catalytic characteristics by site-directed mutagenesis. The enzyme from the hyperthermophilic archaeon Thermococcus kodakarensis naturally exhibits high thermostability and a broad substrate spectrum, but only low activity at moderate temperatures. The best enzyme variants showed an ~5-fold (2-heptanol) and 9-fold (3-heptanol) higher activity while preserving enantioselectivity and good thermodynamic stability. These variants also exhibited modified kinetic characteristics regarding regioselectivity, pH dependence and activation by NaCl.

Measurement of Minute Liquid Volumes of Chiral Molecules Using In-Fiber Polarimetry.

We report an optofluidic method that enables to efficiently measure the enantiomeric excess of chiral molecules at low concentrations. The approach is to monitor the optical activity induced by a Kagome-lattice hollow-core photonic crystal fiber filled with a sub-µL volume of chiral compounds. The technique also allows monitoring the enzymatic racemization of R-mandelic acid.

Mitochondrial respiration in B lymphocytes is essential for humoral immunity by controlling the flux of the TCA cycle.

To elucidate the function of oxidative phosphorylation (OxPhos) during B cell differentiation, we employ CD23Cre-driven expression of the dominant-negative K320E mutant of the mitochondrial helicase Twinkle (DNT). DNT-expression depletes mitochondrial DNA during B cell maturation, reduces the abundance of respiratory chain protein subunits encoded by mitochondrial DNA, and, consequently, respiratory chain super-complexes in activated B cells. Whereas B cell development in DNT mice is normal, B cell proliferation, germinal centers, class switch to IgG, plasma cell maturation, and T cell-dependent as well as T cell-independent humoral immunity are diminished. DNT expression dampens OxPhos but increases glycolysis in lipopolysaccharide and B cell receptor-activated cells. Lipopolysaccharide-activated DNT-B cells exhibit altered metabolites of glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle and a lower amount of phosphatidic acid. Consequently, mTORC1 activity and BLIMP1 induction are curtailed, whereas HIF1α is stabilized. Hence, mitochondrial DNA controls the metabolism of activated B cells via OxPhos to foster humoral immunity.

Balance of carbon species combined with stable isotope ratios show critical switch towards bicarbonate uptake during cyanobacteria blooms.

Next to water quality deterioration, cyanobacteria blooms can affect turnover of aqueous carbon, including dissolved inorganic carbon (DIC), dissolved organic carbon (DOC), and particulate organic carbon (POC). We investigated interactions of these three phases and their stable isotopes in a freshwater pond with periodic cyanobacterial blooms over a period of 23 months. This helped to map turnover and sources of aqueous carbon before, during, and after bloom events. During bloom events POC isotope values (δ13CPOC) increased up to -17.4‰, after aqueous CO2 (CO2(aq)) fell below an atmospheric equilibration value of 412 µatm. Additionally, carbon isotope enrichment between CO2(aq) and POC (εCO2-phyto) ranged from 2.0 to 21.5‰ with lowest fractionations observed at pH values above 8.9. The increase of δ13CPOC and decrease of εCO2-phyto values at low pCO2 and high pH was most likely caused by the activation of the carbon concentrating mechanism (CCM). This mechanism correlated with prevalent assimilation of 13C-enriched HCO3-. Surprisingly, CO2(aq) still contributed more than 50% to the POC pool down to pCO2 values of around 150 µatm. Only after this threshold the reduced εCO2-phyto suggested incorporation of 13C-enriched HCO3-.

Inhibitory properties of crude microalgal extracts on the in vitro replication of cyprinid herpesvirus 3.

Microalgae are possible sources of antiviral substances, e.g. against cyprinid herpesvirus 3 (CyHV-3). Although this virus leads to high mortalities in aquacultures, there is no treatment available yet. Hence, ethanolic extracts produced with accelerated solvent extraction from six microalgal species (Arthrospira platensis, Chlamydomonas reinhardtii, Chlorella kessleri, Haematococcus pluvialis, Nostoc punctiforme and Scenedesmus obliquus) were examined in this study. An inhibition of the in vitro replication of CyHV-3 could be confirmed for all six species, with the greatest effect for the C. reinhardtii and H. pluvialis crude extracts. At still non-cytotoxic concentrations, viral DNA replication was reduced by over 3 orders of magnitude each compared to the untreated replication controls, while the virus titers were even below the limit of detection (reduction of 4 orders of magnitude). When pre-incubating both cells and virus with C. reinhardtii and H. pluvialis extracts before inoculation, the reduction of viral DNA was even stronger (> 4 orders of magnitude) and no infectious viral particles were detected. Thus, the results of this study indicate that microalgae and cyanobacteria are a promising source of natural bioactive substances against CyHV-3. However, further studies regarding the isolation and identification of the active components of the extracts are needed.

Three Steps, Two Enzymes, One Pot, but a Multitude of Nanocompartments: Combined Cycles of Kinetic Resolutions and Re-racemization with Incompatible Biocatalysts.

Deracemizations are clearly preferable to kinetic resolutions in the production of chiral molecules from racemates, as they allow up to 100% chemical and optical yield. Here we present a new process route for multienzymatic deracemizations that is relevant for reaction systems with incompatible reaction conditions of the biocatalysts. This often applies to combinations of lipases used for stereoselective acylation and solvent-sensitive racemases. By encapsulating a model racemase in polymeric vesicles, it was protected from inactivation by the organic solvent up to phase proportions of 99%. As high yields in the lipase reaction required either water proportions well below 1% or racemase-denaturating acyl donor concentrations, a one-pot reaction was implemented through the sequential use of lipase and racemase-containing nanocompartments. This strategy allowed us to perform two kinetic resolutions with intermittent re-racemization in one pot yielding 72% (0.72 mM after 120 h) of an enantiopure product.

Enzymatic Synthesis of Sialic Acids in Microfluidics to Overcome Cross-Inhibitions and Substrate Supply Limitations.

Multienzymatic cascade reactions are a powerful strategy for straightforward and highly specific synthesis of complex materials, such as active substances in drugs. Cross-inhibitions and incompatible reaction steps, however, often limit enzymatic activity and thus the conversion. Such limitations occur, e.g., in the enzymatic synthesis of the biologically active sialic acid cytidine monophosphate N-acetylneuraminic acid (CMP-Neu5Ac). We addressed this challenge by developing a confinement and compartmentalization concept of hydrogel-immobilized enzymes for improving the efficiency of the enzyme cascade reaction. The three enzymes required for the synthesis of CMP-Neu5Ac, namely, N-acyl-d-glucosamine 2-epimerase (AGE), N-acetylneuraminate lyase (NAL), and CMP-sialic acid synthetase (CSS), were immobilized into bulk hydrogels and microstructured hydrogel-enzyme-dot arrays, which were then integrated into microfluidic devices. To overcome the cytidine triphosphate (CTP) cross-inhibition of AGE and NAL, only a low CTP concentration was applied and continuously conveyed through the device. In a second approach, the enzymes were compartmentalized in separate reaction chambers of the microfluidic device to completely avoid cross-inhibitions and enable the use of higher substrate concentrations. Immobilization efficiencies of up to 25% and pronounced long-term activity of the immobilized enzymes for several weeks were realized. Moreover, immobilized enzymes were less sensitive to inhibition and the substrate-channeling effect between immobilized enzymes promoted the overall conversion in the trienzymatic cascade reaction. Based on this, CMP-Neu5Ac was successfully synthesized by immobilized enzymes in noncompartmentalized and compartmentalized microfluidic devices. This study demonstrates the high potential of immobilizing enzymes in (compartmentalized) microfluidic devices to perform multienzymatic cascade reactions despite cross-inhibitions under continuous flow conditions. Due to the ease of enzyme immobilization in hydrogels, this concept is likely applicable for many cascade reactions with or without cross-inhibition characteristics.

Increased Protein Encapsulation in Polymersomes with Hydrophobic Membrane Anchoring Peptides in a Scalable Process.

Hollow vesicles made from a single or double layer of block-copolymer molecules, called polymersomes, represent an important technological platform for new developments in nano-medicine and nano-biotechnology. A central aspect in creating functional polymersomes is their combination with proteins, especially through encapsulation in the inner cavity of the vesicles. When producing polymersomes by techniques such as film rehydration, significant proportions of the proteins used are trapped in the vesicle lumen, resulting in high encapsulation efficiencies. However, because of the difficulty of scaling up, such methods are limited to laboratory experiments and are not suitable for industrial scale production. Recently, we developed a scalable polymersome production process in stirred-tank reactors, but the statistical encapsulation of proteins resulted in fairly low encapsulation efficiencies of around 0.5%. To increase encapsulation in this process, proteins were genetically fused with hydrophobic membrane anchoring peptides. This resulted in encapsulation efficiencies of up to 25.68%. Since proteins are deposited on the outside and inside of the polymer membrane in this process, two methods for the targeted removal of protein domains by proteolysis with tobacco etch virus protease and intein splicing were evaluated. This study demonstrates the proof-of-principle for production of protein-functionalized polymersomes in a scalable process.

Internal Illumination to Overcome the Cell Density Limitation in the Scale-up of Whole-Cell Photobiocatalysis.

Cyanobacteria have the capacity to use photosynthesis to fuel their metabolism, which makes them highly promising production systems for the sustainable production of chemicals. Yet, their dependency on visible light limits the cell-density, which is a challenge for the scale-up. Here, it was shown with the example of a light-dependent biotransformation that internal illumination in a bubble column reactor equipped with wireless light emitters (WLEs) could overcome this limitation. Cells of the cyanobacterium Synechocystis sp. PCC 6803 expressing the gene of the ene-reductase YqjM were used for the reduction of 2-methylmaleimide to (R)-2-methylsuccinimide with high optical purity (>99 % ee). Compared to external source of light, illumination by floating wireless light emitters allowed a more than two-fold rate increase. Under optimized conditions, product formation rates up to 3.7 mm h-1 and specific activities of up to 65.5 U gDCW -1 were obtained, allowing the reduction of 40 mm 2-methylmaleimide with 650 mg isolated enantiopure product (73 % yield). The results demonstrate the principle of internal illumination as a means to overcome the intrinsic cell density limitation of cyanobacterial biotransformations, obtaining high reaction rates in a scalable photobioreactor.

Fast and effective chromatographic separation of polymersomes from proteins by multimodal chromatography.

Artificial vesicles made of block copolymers, so-called polymersomes, represent a versatile chassis for the creation of functionalized nanocompartments with a wide range of biotechnological applications. The specific application depends on the biomolecules - usually proteins - that are positioned in the interior, in the membrane or on the surface of the vesicles. However, not all added proteins are integrated into the vesicles during the usual manufacturing processes of polymersomes. Excess proteins must therefore be removed. The separation techniques currently used for this, however, are associated with decisive disadvantages, such as damaged vesicles, long process times, or small sample volumes that can be processed. To overcome these drawbacks, we investigated the applicability of Capto™ Core 700 resin for polymersome purification. Polymersomes were not damaged or otherwise affected by passage through the column verified by hollow fiber flow field flow fractionation technique. Using three proteins with divergent physico-chemical properties as examples, it was demonstrated that different types of unentrapped proteins were efficiently removed from polymersome dispersions. The dynamic binding capacities in the presence of polymersomes varied between 9.5 and 16.5 mg per mL resin for the proteins applied. The technique can be used for small and large sample volumes alike. In addition, it can be used without special laboratory equipment. This adds a new and easy-to-use purification method for polymer vesicles to the repertoire that will also facilitate the large-scale production of functionalized polymersomes.

A ß-barrel for oil transport through lipid membranes: Dynamic NMR structures of AlkL.

The protein AlkL is known to increase permeability of the outer membrane of bacteria for hydrophobic molecules, yet the mechanism of transport has not been determined. Differing crystal and NMR structures of homologous proteins resulted in a controversy regarding the degree of structure and the role of long extracellular loops. Here we solve this controversy by determining the de novo NMR structure in near-native lipid bilayers, and by accessing structural dynamics relevant to hydrophobic substrate permeation through molecular-dynamics simulations and by characteristic NMR relaxation parameters. Dynamic lateral exit sites large enough to accommodate substrates such as carvone or octane occur through restructuring of a barrel extension formed by the extracellular loops.

Polymersomes as Nanoreactors Enabling the Application of Solvent-Sensitive Enzymes in Different Biphasic Reaction Setups.

There is an increasing interest in biocatalysis to perform chemical reactions in biphasic systems, consisting of an aqueous phase and a water-immiscible organic solvent or ionic liquid. In most cases, the hydrophobic phase is used as reservoir for poorly water-soluble substrates or for in situ product removal. However, many enzymes are solvent-sensitive and cannot be used in such systems. In this study, the solvent-sensitive enzyme mandelate racemase is exemplarily protected from the organic phase by its entrapment in (crosslinked) polymersomes. The covalent crosslinking of the individual chains of the block copolymer poly(2-methyloxazoline)15 -poly(dimethylsiloxane)68 -poly(2-methyloxazoline)15 via terminal methacrylates leads to enhanced membrane stability. This effect is especially pronounced for long-time incubation in the presence of organic solvents and ionic liquids. By using a gentle polymerization initiator at its minimal necessary concentration, the prior encapsulated enzymes remain intact during crosslinking. Although the insertion of natural channel proteins into the membrane improves the mass transport into the vesicles, it is non-essential. Mandelate racemase in (crosslinked) polymersomes remains active in different highly dispersed biphasic systems for more than 24 h. The free enzyme, on the other hand, gets completely inactivated within 1 h, thus illustrating the potential of polymersomes as nanoreactors in biphasic reaction setups.

Resonance assignment of the outer membrane protein AlkL in lipid bilayers by proton-detected solid-state NMR.

Most commonly small outer membrane proteins, possessing between 8 and 12 ß-strands, are not involved in transport but fulfill diverse functions such as cell adhesion or binding of ligands. An intriguing exception are the 8-stranded ß-barrel proteins of the OmpW family, which are implicated in the transport of small molecules. A representative example is AlkL from Pseudomonas putida GPoI, which functions as a passive importer of hydrophobic molecules. This role is of high interest with respect to both fundamental biological understanding and industrial applications in biocatalysis, since this protein is frequently utilized in biotransformation of alkanes. While the transport function of AlkL is generally accepted, a controversy in the transport mechanism still exists. In order to address this, we are pursuing a structural study of recombinantly produced AlkL reconstituted in lipid bilayers using solid-state NMR spectroscopy. In this manuscript we present 1H, 13C and 15N chemical shift assignments obtained via a suite of 3D experiments employing high magnetic fields (1 GHz and 800 MHz) and the latest magic-angle spinning (MAS) approaches at fast (60-111) kHz rates. We additionally analyze the secondary structure prediction in comparison with those of published structures of homologous proteins.

Asymmetric Whole-Cell Bio-Reductions of (R)-Carvone Using Optimized Ene Reductases.

(2R,5R)-dihydrocarvone is an industrially applied building block that can be synthesized by site-selective and stereo-selective C=C bond bio-reduction of (R)-carvone. Escherichia coli (E. coli) cells overexpressing an ene reductase from Nostoc sp. PCC7120 (NostocER1) in combination with a cosubstrate regeneration system proved to be very effective biocatalysts for this reaction. However, the industrial applicability of biocatalysts is strongly linked to the catalysts' activity. Since the cell-internal NADH concentrations are around 20-fold higher than the NADPH concentrations, we produced E. coli cells where the NADPH-preferring NostocER1 was exchanged with three different NADH-accepting NostocER1 mutants. These E. coli whole-cell biocatalysts were used in batch operated stirred-tank reactors on a 0.7 l-scale for the reduction of 300 mM (R)-carvone. 287 mM (2R,5R)-dihydrocarvone were formed within 5 h with a diasteromeric excess of 95.4% and a yield of 95.6%. Thus, the whole-cell biocatalysts were strongly improved by using NADH-accepting enzymes, resulting in an up to 2.1-fold increased initial product formation rate leading to a 1.8-fold increased space-time yield when compared to literature.

Probing Membrane Protein Insertion into Lipid Bilayers by Solid-State NMR.

Determination of the environment surrounding a protein is often key to understanding its function and can also be used to infer the structural properties of the protein. By using proton-detected solid-state NMR, we show that reduced spin diffusion within the protein under conditions of fast magic-angle spinning, high magnetic field, and sample deuteration allows the efficient measurement of site-specific exposure to mobile water and lipids. We demonstrate this site specificity on two membrane proteins, the human voltage dependent anion channel, and the alkane transporter AlkL from Pseudomonas putida. Transfer from lipids is observed selectively in the membrane spanning region, and an average lipid-protein transfer rate of 6 s-1 was determined for residues protected from exchange. Transfer within the protein, as tracked in the 15 N-1 H 2D plane, was estimated from initial rates and found to be in a similar range of about 8 to 15 s-1 for several resolved residues, explaining the site specificity.

Membrane functionalization of polymersomes: alleviating mass transport limitations by integrating multiple selective membrane transporters for the diffusion of chemically diverse molecules.

Recently, the interest in polymersomes as nanoreactors for synthetic applications has increased due to interesting proof-of-concept studies, indicating a versatile use of polymeric vesicles to compartmentalize complex reaction cascades. However, the low permeability of polymeric membranes and the requirement for a controlled mass transport across the compartment boundaries have posed a major limitation to the broad applicability of polymersomes for synthetic reactions. Current advances in the functional integration of membrane proteins (MPs) into poly(2-dimethylsiloxane)-based membranes have allowed the selective increase of the permeability for a controlled mass transport of the desired compounds across the membrane. Herein we demonstrate that polymer membranes are capable of harboring different MPs to alleviate the mass transport limitations of chemically diverse molecules, thereby enabling complex cascade reactions to be performed within the nanoreactors. The ability to functionalize the polymer membrane with multiple, highly selective MPs allows a reduction in mass transport limitations without abandoning compartmentalization of the reaction space on a low molecular mass level. As the model reaction, a two enzyme system consisting of a ketoreductase (KR) and a formate dehydrogenase was studied. For the transport of the hydrophobic substrate and product of the KR, the MPs AlkL, OmpW, OprG and TodX were investigated. For the transport of formate, OmpF, PhoE and FocA were used. AlkL showed the highest integration efficiency (39%) and a maximum of 120 AlkL molecules were successfully inserted into each polymersome. The highest channel-specific effects on the mass transfer were achieved using TodX and PhoE, respectively. The combination of both proteins led to an improvement of the space-time yield of the product (S)-pentafluorophenyl ethanol by 2.32-fold compared to nanoreactors without MPs.

Polymersomes as nanoreactors for preparative biocatalytic applications: current challenges and future perspectives.

Polymersomes are hollow, spherical vesicles that are surrounded by a polymer membrane. The applied polymer must be amphiphilic to promote self-assembly in aqueous solution. At the same time, the polymer composition is highly versatile, which leads to diverse properties in terms of chemical and mechanical stability, membrane permeability and the ability to functionalize the membrane. By encapsulating chemical or biological substances within the polymersomes, drug delivery systems, cell mimetics or catalytic nanoreactors can be assembled. Whereas drug delivery systems and cell mimetics based on polymersomes have been reviewed excessively, we lay focus on the current challenges and perspectives of polymersomes as nanoreactors for preparative biocatalytic applications. We discuss the importance of membrane properties for the use of polymersomes for synthetic applications and highlight advances in polymersome production and membrane functionalization. Finally, we summarize recent applications of polymersomes as nanoreactors, discuss the associated challenges and disclose future requirements and perspectives for the industrial use of polymersomes as nanoreactors.

Overcoming the Incompatibility Challenge in Chemoenzymatic and Multi-Catalytic Cascade Reactions.

Multi-catalytic cascade reactions bear a great potential to minimize downstream and purification steps, leading to a drastic reduction of the produced waste. In many examples, the compatibility of chemo- and biocatalytic steps could be easily achieved. Problems associated with the incompatibility of the catalysts and their reactions, however, are very frequent. Cascade-like reactions can hardly occur in this way. One possible solution to combine, in principle, incompatible chemo- and biocatalytic reactions is the defined control of the microenvironment by compartmentalization or scaffolding. Current methods for the control of the microenvironment of biocatalysts go far beyond classical enzyme immobilization and are thus believed to be very promising tools to overcome incompatibility issues and to facilitate the synthetic application of cascade reactions. In this Minireview, we will summarize recent synthetic examples of (chemo)enzymatic cascade reactions and outline promising methods for their spatial control either by using bio-derived or synthetic systems.