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Biofouling, caused by the attachment of micro- and macroorganisms, increases the fuel consumption of ships and compromises aquatic infrastructure, especially by microbially induced corrosion. Conventional anti-fouling strategies often rely on the release of toxic substances. Liquid-infused coatings offer an alternative strategy, which has shown promising anti-fouling performance in maritime settings. In this study, we focus on freshwater systems and investigate the performance of a set of recently developed liquid-infused coatings with low environmental impact based on sustainable or non-toxic materials and energy-efficient fabrication. We combine field studies at two freshwater bodies in Bavaria, Germany with laboratory experiments focusing on corrosion and microbial attachment under controlled conditions. Our results indicate that liquid-infused coatings with low environmental impact can show a reduction in general biofouling, mussel attachment, and corrosion rates, as well as the attachment of Thiobacillus thioparus and Pseudomonas fluorescens, organisms known to cause microbially induced corrosion. [ABSTRACT FROM AUTHOR]

Opioids are important analgesics, and their pharmaceutical application is increasing worldwide. Many opioids are based on benzylisoquinoline alkaloids (BIA) and are still industrially produced from Papaver somniferum (opium poppy). (S)-norlaudanosoline ((S)-NLS) is a complex BIA and an advanced intermediate for diverse pharmaceuticals. The efficient synthesis of this scaffold could pave the way for a plant-independent synthesis platform. Although a promising biocatalytic route to (S)-NLS using norcoclaurine synthase (NCS) and ω-transaminase (TAm) has already been explored, the cost-effectiveness of this process still needs much improvement. Therefore, we investigated whether the synthesis could also be performed using whole cells to avoid the use of (partially) purified enzymes. With an optimized mixing ratio of TAm- and NCS-containing cells in batch biotransformations, 50 mM substrate was converted within 3 h with more than 90% yield and a high enantiomeric excess of the product (95%). To further increase the space–time yield, the cells were immobilized to enable their retainment in fixed-bed reactors. A comparison of glass beads, Diaion HP-2MG and alginate revealed that the addition of Diaion during bacterial growth led to the most active immobilisates. To facilitate sustained production of (S)-NLS, a fixed-bed setup was constructed based on lithographically printed columns from biocompatible PRO-BLK 10 plastic. The continuous production at two scales (5 mL and 50 mL columns) revealed insufficient system stability originating from biocatalyst leaching and inactivation. Thus, while the use of whole cells in batch biotransformations represents an immediate process improvement, the transfer to flow catalysis needs further optimization. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In conventional molecular communication (MC) systems, the signaling molecules used for information transmission are stored, released, and then replenished by a transmitter (TX). However, the replenishment of signaling molecules at the TX is challenging in practice. Furthermore, in most envisioned MC applications, e.g., in the medical field, it is not desirable to insert the TX into the MC system, as this might impair natural biological processes. In this paper, we propose the concept of media modulation based MC where the TX is placed outside the channel and utilizes signaling molecules already present inside the system. The signaling molecules can assume different states which can be switched by external stimuli. Hence, in media modulation based MC, the TX modulates information into the state of the signaling molecules. In particular, we exploit the group of photochromic molecules, which undergo light-induced reversible state transitions, for media modulation. We study the usage of these molecules for information transmission in a three-dimensional duct system, which contains an eraser, a TX, and a receiver for erasing, writing, and reading of information via external light, respectively. We develop a statistical model for the received signal which accounts for the distribution of the signaling molecules in the system, the initial states of the signaling molecules, the reliability of the state control mechanism, the randomness of irrepressible, spontaneous state switching, and the randomness of molecule propagation. We adopt a maximum likelihood detector and show that it can be reduced to a threshold based detector. Furthermore, we derive analytical expressions for the optimal threshold value and the resulting bit error rate (BER), respectively. Both the statistical model and BER results are verified by computer simulations. Our results reveal that media modulation enables reliable information transmission, validating it as a promising alternative to MC based on molecule emitting TXs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Most commonly small outer membrane proteins, possessing between 8 and 12 β-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 β-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The asymmetric reduction of alkenes is a widely used transformation in industry. Ene reductases (ERs) are (βα)8‐barrel folded enzymes capable of catalyzing this hydrogenation reaction. At the expense of nicotinamide coenzymes, ERs can reduce a wide range of electron‐deficient alkenes in an anti‐specific manner and with high regio‐ and stereoselectivities. However, a cost‐effective industrial use of these enzymes is hampered, since most ERs prefer nicotinamide adenine dinucleotide phosphate (NADPH) to the more stable and less expensive non‐phosphorylated nicotinamide adenine dinucleotide (NADH) as coenzyme. Here, we demonstrate an approach to both modify the biocatalysts coenzyme selectivity and strongly increase the activity and affinity with NADH. By swapping loop regions of the cyanobacterial NostocER1 for the corresponding regions of two NADH‐favoring ERs, a strong alteration of the biocatalyst's coenzyme binding was achieved. This made possible a transfer of the respective donor‐ER kinetic parameters to NostocER1. Additionally, outperformance of both donors in terms of activity was achieved through combinatorial swapping of loops of both species. These findings demonstrate the high potential of loop swapping as protein engineering approach to selectively optimize the coenzyme binding of ERs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

ABSTRACT Uniform polymersomes (polymer vesicles) made of poly(2-methyloxazoline)15- b-poly(dimethylsiloxane)68- b-poly(2-methyloxazoline)15 (PMOXA15-PDMS68-PMOXA15) can be formed in miniaturized-stirred tank reactors by the aid of a recently published process. In this study, the occurring self-assembly mechanism was elucidated by using transmission electron microscopy. Subsequent to the initial formation of small spherical micelles and the following fusion to worm-like micelles, two simultaneously occurring pathways, describing the transformation of further intermediate structures to the desired vesicles, were found. The resulting particle increase was followed by dynamic light scattering. Thus, the vesicle formation rate was judged by the linear increase of the particle diameter over time. While temperature showed no influence, higher initial polymer concentrations and lower final solvent concentrations accelerated the polymersome formation. Besides, the process was crucially dependent on the agitation speed. While spherical micelles did not transform into polymersomes when no stirring or too slow stirring is applied, the self-assembly process was accelerated by increasing the agitation speed. Uniform polymeric vesicles can be formed under vigorous stirring in stirred-tank reactors in short process times. In this study, the underlying mechanisms of vesicle formation were elucidated, showing that the polymer forms small micellar structures before undergoing two separate pathways to form the desired vesicular structures. The formation rate of the polymer vesicles was mainly dependent on the agitation speed but also on the polymer and solvent concentrations, highlighting the need for controlled formation conditions. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018, 135, 46077. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Abstract: The increased membrane stability of polymersomes compared to their liposomal counterparts is one of their most important advantages. Due to this benefit, polymer vesicles are intended to be used not only as carrier systems for drug delivery purposes but also as nanoreactors for biotechnological applications. Within this work, the stability of polymersomes made of the triblock copolymer poly(2‐methyloxazoline)15‐poly(dimethylsiloxane)68‐poly(2‐methyloxazoline)15 (PMOXA15‐PDMS68‐PMOXA15) toward mechanical stress, typically prevailing in stirred‐tank reactors being the most often used reactor type in the biotechnological industry, was characterized. Dynamic light scattering and turbidity measurements showed that stirrer rotation causing a maximum local energy dissipation of up to 1.23 W/kg−1 did not result in any loss of vesicle quality or quantity. Nevertheless, most probably due to local membrane defects, 6.6% release of the previously encapsulated model dye calcein was recognized at 25°C within 48 h. Moreover, increased temperature, leading to decreased membrane viscosity and increased membrane fluidity, respectively, led to a higher molecule leakage. Besides, the stability of polymersomes in two‐phase systems was investigated. Although alkanes and ionic liquids were shown not to lead to complete vesicle damage, no efficient calcein retention was achieved in either case. [ABSTRACT FROM AUTHOR]

Polymersomes have some fundamental advantages compared to their liposomal counterparts. Due to the increased stability of the polymeric membrane, polymersomes are intended to be reasonably applicable as carrier-systems and universal reaction compartments for diverse medical and biotechnological applications. Regardless of the application area, suitable methods to produce large vesicle quantities in a controlled and cost-effective manner have to be developed to put polymersome technology into action at the industrial scale. In this work, the amphiphilic triblock copolymer poly(2-methyloxazoline)15-poly(dimethylsiloxane)68-poly(2-methyloxazoline)15 was formed into uniform polymersomes. A recently established production process, based on the use of miniaturized stirred-tank reactors at the milliliter-scale (12 mL), was successfully scaled-up to the liter-scale (1.5 L) based on solid process engineering parameters. Dynamic light scattering measurements show that using standard propeller stirrers with a dimensionless diameter [ABSTRACT FROM AUTHOR]

Background: Hollow vesicles formed from block copolymers, so-called polymersomes, have been extensively studied in the last decade for their various applications in drug delivery, in diagnostics and as nanoreactors. The immobilization of proteins on the polymersomes' surface can aid in cell targeting, lead to functional biosensors or add an additional reaction space for multistep syntheses. In almost all surface functionalization strategies to date, a chemical pre-conjugation of the polymer with a reactive group or ligand and the functionalization of the protein are required. To avoid chemical pre-conjugation, we investigated the simple and quick functionalization of preformed poly(2-methyloxazoline)-poly(dimethylsiloxane)-poly(2-methyloxazoline) (PMOXA-PDMS-PMOXA) polymersomes through the spontaneous insertion of four hydrophobic, non-antibacterial peptide anchors into the membrane to display enhanced green fluorescent protein (eGFP) on the polymersomes' surface. Results: Three of the four hydrophobic peptides, the transmembrane domains of a eukaryotic cytochrome b5, of the viral lysis protein L and of the yeast syntaxin VAM3 could be recombinantly expressed as soluble eGFP-fusion proteins and spontaneously inserted into the polymeric membrane. Characterization of the surface functionalization revealed that peptide insertion was linearly dependent on the protein concentration and possible at a broad temperature range of 4–42 °C. Up to 2320 ± 280 eGFP molecules were immobilized on a single polymersome, which is in agreement with the calculated maximum loading capacity. The peptide insertion was stable without disrupting membrane integrity as shown in calcein leakage experiments and the functionalized polymersomes remained stable for at least 6 weeks. Conclusion: The surface functionalization of polymersomes with hydrophilic proteins can be mediated by several peptide anchors in a spontaneous process at extremely mild insertion conditions and without the need of pre-conjugating polymers. [ABSTRACT FROM AUTHOR]

ABSTRACT Polymer vesicles, so-called polymersomes, gain more and more attention as potential carriers for medical and biotechnological applications. To put the production of these nanocompartments into action at an industrial scale, an efficient and scalable process has to be established. Moreover, being able to control the resulting particle size distribution (PSD) is vital. In this work, the amphiphilic triblock copolymer poly(2-methyloxazoline)15-poly(dimethylsiloxane)68-poly(2-methyloxazoline)15 is formed into polymersomes in miniaturized stirred-tank reactors. Varying flow conditions have a huge impact on the resulting PSD. Dynamic light scattering measurements show that driving a S-shaped stirrer at 4000 rpm in unbaffled reactors leads to a monomodal PSD with a low polydispersity index (PDI<0.2). Vesicles with a mean diameter of 200 nm are achieved within less than 1 h in a single production step. The robustness of the established process is shown by producing uniform polymersomes at different temperatures and varying pH and buffer molarities. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 43274. [ABSTRACT FROM AUTHOR]

Background: Whole cell biocatalysts and isolated enzymes are considered as state of the art in biocatalytic preparations for industrial applications. Whole cells as biocatalysts are disadvantageous if substrate or products are toxic to the cells or undesired byproducts are formed due to the cellular metabolism. The use of isolated enzymes in comparison is more expensive due to the required downstream processing. Immobilization of enzymes after purification increases preparation costs for biocatalysts significantly, but allows for the efficient reuse of the enzymes in the biocatalytic process. For a more rapid processing one-step expression and immobilization is desirable. Results: This study focused on the development of a new one-step expression and immobilization technique for enzymes on the example of the β-galactosidase from Escherichia coli K12. The enzyme was expressed in E. coli with a C-terminal membrane anchor originating from cytochrome b5 from rabbit liver and was thus in situ immobilized to the inner surface of the cytosolic membrane. Then, the expression of a lytic phage protein (gene E from PhiX174) caused the formation of a pore in the cell wall of E. coli, which resulted in release of the cytosol. The cellular envelopes with immobilized enzymes were retained. Batch and fed-batch processes were developed for efficient production of these biocatalysts. It was possible to obtain cellular envelopes with up to 27,200 ± 10,460 immobilized enzyme molecules per cellular envelope (753 ± 190 U/gdry weight). A thorough characterization of the effects of membrane immobilization was performed. Comparison to whole cells showed that mass transfer limitation was reduced in cellular envelopes due to the pore formation. Conclusion: In this study the feasibility of a new one-step expression and immobilization technique for the generation of biocatalytic preparations was demonstrated. The technique could be a useful tool especially for enzyme systems, which are not suitable for whole-cell biocatalysts due to severe mass transfer limitations or undesired side reactions mediated by cytosolic enzymes. [ABSTRACT FROM AUTHOR]

Ursodeoxycholic acid (UDCA) is a bile acid which is used as pharmaceutical for the treatment of several diseases, such as cholesterol gallstones, primary sclerosing cholangitis or primary biliary cirrhosis. A potential chemoenzymatic synthesis route of UDCA comprises the two-step reduction of dehydrocholic acid to 12-keto-ursodeoxycholic acid (12-keto-UDCA), which can be conducted in a multienzymatic one-pot process using 3α-hydroxysteroid dehydrogenase (3α-HSDH), 7β-hydroxysteroid dehydrogenase (7β-HSDH), and glucose dehydrogenase (GDH) with glucose as cosubstrate for the regeneration of cofactor. Here, we present a dynamic mechanistic model of this one-pot reduction which involves three enzymes, four different bile acids, and two different cofactors, each with different oxidation states. In addition, every enzyme faces two competing substrates, whereas each bile acid and cofactor is formed or converted by two different enzymes. First, the kinetic mechanisms of both HSDH were identified to follow an ordered bi-bi mechanism with EBQ-type uncompetitive substrate inhibition. Rate equations were then derived for this mechanism and for mechanisms describing competing substrates. After the estimation of the model parameters of each enzyme independently by progress curve analyses, the full process model of a simple batch-process was established by coupling rate equations and mass balances. Validation experiments of the one-pot multienzymatic batch process revealed high prediction accuracy of the process model and a model analysis offered important insight to the identification of optimum reaction conditions. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:375-386, 2015 [ABSTRACT FROM AUTHOR]

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. [Display omitted] • mtDNA in B cells is required for humoral immunity and plasma cell maturation • mtDNA maintains OxPhos in LPS and antigen receptor-stimulated B cells • OxPhos drives the TCA cycle in LPS-activated B cells • OxPhos preserves phosphatidic acid and mTOR activity in activated B cells Urbanczyk et al. report that dominant-negative Twinkle prevents mtDNA replication in B cells. Intact mtDNA fosters OxPhos and restricts glycolysis in LPS- and BCR-activated B cells, enabling the competition of developing plasma cells. OxPhos secures the flux of the TCA cycle and mTOR activity, which is required for plasmablast proliferation and differentiation. [ABSTRACT FROM AUTHOR]

The growing importance of biocatalysis in the syntheses of enantiopure molecules results from the benefits of enzymes regarding selectivity and specificity of the reaction and ecological issues of the process. Ene-reductases (ERs) from the old yellow enzyme family have received much attention in the last years. These flavo-enzymes catalyze the trans-specific reduction of activated CC bonds, which is an important reaction in asymmetric synthesis, because up to two stereogenic centers can be created in one reaction. However, limitations of ERs described in the literature such as their moderate catalytic activity and their strong preference for NADPH promote the search for novel ERs with improved properties. In this study, we characterized nine novel ERs from cyanobacterial strains belonging to different taxonomic orders and habitats. ERs were identified with activities towards a broad spectrum of alkenes. The reduction of maleimide was catalyzed with activities of up to 35.5 U mg−1 using NADPH. Ketoisophorone and (R)-carvone, which were converted to the highly valuable compounds (R)-levodione and (2R,5R)-dihydrocarvone, were reduced with reaction rates of up to 2.2 U mg−1 with NADPH. In contrast to other homologous ERs from the literature, NADH was accepted at moderate to high rates as well: Enzyme activities of up to 16.7 U mg−1 were obtained for maleimide and up to 1.3 U mg−1 for ketoisophorone and (R)-carvone. Additionally, excellent stereoselectivities were achieved in the reduction of (R)-carvone (97-99% de). In particular, AnabaenaER3 from Anabaena variabilis ATCC 29413 and AcaryoER1 from Acaryochloris marina MBIC 11017 were identified as useful biocatalysts. Therefore, novel ERs from cyanobacteria with high catalytic efficiency were added to the toolbox for the asymmetric reduction of alkenes. Biotechnol. Bioeng. 2013; 110: 1293-1301. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Ursodeoxycholic acid (UDCA) is a bile acid of industrial interest as it is used as an agent for the treatment of primary sclerosing cholangitis and the medicamentous, non-surgical dissolution of gallstones. Currently, it is prepared industrially from cholic acid following a seven-step chemical procedure with an overall yield of <30%. In this study, we investigated the key enzymatic steps in the chemo-enzymatic preparation of UDCA-the two-step reduction of dehydrocholic acid (DHCA) to 12-keto-ursodeoxycholic acid using a mutant of 7β-hydroxysteroid dehydrogenase (7β-HSDH) from Collinsella aerofaciens and 3α-hydroxysteroid dehydrogenase (3α-HSDH) from Comamonas testosteroni. Three different one-pot reaction approaches were investigated using whole-cell biocatalysts in simple batch processes. We applied one-biocatalyst systems, where 3α-HSDH, 7β-HSDH, and either a mutant of formate dehydrogenase (FDH) from Mycobacterium vaccae N10 or a glucose dehydrogenase (GDH) from Bacillus subtilis were expressed in a Escherichia coli BL21(DE3) based host strain. We also investigated two-biocatalyst systems, where 3α-HSDH and 7β-HSDH were expressed separately together with FDH enzymes for cofactor regeneration in two distinct E. coli hosts that were simultaneously applied in the one-pot reaction. The best result was achieved by the one-biocatalyst system with GDH for cofactor regeneration, which was able to completely convert 100 mM DHCA to >99.5 mM 12-keto-UDCA within 4.5 h in a simple batch process on a liter scale. Biotechnol. Bioeng. 2013; 110: 68-77. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Industrial biotechnology relies on the availability of highly efficient enzymes and production strains for the development of economically viable processes. Recent advances in the field of genome editing will greatly speed up the implementation of desired metabolic pathways and their optimization, thereby facilitating fast and cost-effective production strain engineering. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

(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. [ABSTRACT FROM AUTHOR]

The utilization of light energy to power organic-chemical transformations is a fundamental strategy of the terrestrial energy cycle. Inspired by the elegance of natural photosynthesis, much interdisciplinary research effort has been devoted to the construction of simplified cell mimics based on artificial vesicles to provide a novel tool for biocatalytic cascade reactions with energy-demanding steps. By inserting natural or even artificial photosynthetic systems into liposomes or polymersomes, the light-driven proton translocation and the resulting formation of electrochemical gradients have become possible. This is the basis for the conversion of photonic into chemical energy in form of energy-rich molecules such as adenosine triphosphate (ATP), which can be further utilized by energy-dependent biocatalytic reactions, e.g., carbon fixation. This review compares liposomes and polymersomes as artificial compartments and summarizes the types of light-driven proton pumps that have been employed in artificial photosynthesis so far. We give an overview over the methods affecting the orientation of the photosystems within the membranes to ensure a unidirectional transport of molecules and highlight recent examples of light-driven biocatalysis in artificial vesicles. Finally, we summarize the current achievements and discuss the next steps needed for the transition of this technology from the proof-of-concept status to preparative applications. [ABSTRACT FROM AUTHOR]