Extreme dependence of Chloroflexus aggregans LOV domain thermo- and photostability on the bound flavin species.

Light-oxygen-voltage (LOV) domains are common photosensory modules that found many applications in fluorescence microscopy and optogenetics. Here, we show that the Chloroflexus aggregans LOV domain can bind different flavin species (lumichrome, LC; riboflavin, RF; flavin mononucleotide, FMN; flavin adenine dinucleotide, FAD) during heterologous expression and that its physicochemical properties depend strongly on the nature of the bound flavin. We show that whereas the dissociation constants for different chromophores are similar, the melting temperature of the protein reconstituted with single flavin species varies from ~ 60 °C for LC to ~ 81 °C for FMN, and photobleaching half-times vary almost 100-fold.

Optochemical Control of Bacterial Gene Expression: Novel Photocaged Compounds for Different Promoter Systems.

Photocaged compounds are applied for implementing precise, optochemical control of gene expression in bacteria. To broaden the scope of UV-light-responsive inducer molecules, six photocaged carbohydrates were synthesized and photochemically characterized, with the absorption exhibiting a red-shift. Their differing linkage through ether, carbonate, and carbamate bonds revealed that carbonate and carbamate bonds are convenient.

Substrate Access Mechanism in a Novel Membrane-Bound Phospholipase A of Concordant with Specificity and Regioselectivity.

PlaF is a cytoplasmic membrane-bound phospholipase A from that alters the membrane glycerophospholipid (GPL) composition and fosters the virulence of this human pathogen. PlaF activity is regulated by a dimer-to-monomer transition followed by tilting of the monomer in the membrane. However, how substrates reach the active site and how the characteristics of the active site tunnels determine the activity, specificity, and regioselectivity of PlaF for natural GPL substrates have remained elusive.

The bacterial phylum Planctomycetes as novel source for bioactive small molecules.

Extensive knowledge and methodological expertise on the bacterial cell biology have been accumulated over the last decades and bacterial cells have now become an integral part of several (bio-)technological processes. While it appears reasonable to focus on a relatively small number of fast-growing and genetically easily manipulable model bacteria as biotechnological workhorses, the for the most part untapped diversity of bacteria needs to be explored when it comes to bioprospecting for natural product discovery. Members of the underexplored and evolutionarily deep-branching phylum Planctomycetes have only recently gained increased attention with respect to the production of small molecules with biomedical activities, e.

Pseudomonas putida KT2440 endures temporary oxygen limitations.

The obligate aerobic nature of Pseudomonas putida, one of the most prominent whole-cell biocatalysts emerging for industrial bioprocesses, questions its ability to be cultivated in large-scale bioreactors, which exhibit zones of low dissolved oxygen tension. P. putida KT2440 was repeatedly subjected to temporary oxygen limitations in scale-down approaches to assess the effect on growth and an exemplary production of rhamnolipids.

Exploiting unconventional prokaryotic hosts for industrial biotechnology.

Developing cost-efficient biotechnological processes is a major challenge in replacing fossil-based industrial production processes. The remarkable progress in genetic engineering ensures efficient and fast tailoring of microbial metabolism for a wide range of bioconversions. However, improving intrinsic properties such as tolerance, handling, growth, and substrate consumption rates is still challenging.

MIXed plastics biodegradation and UPcycling using microbial communities: EU Horizon 2020 project MIX-UP started January 2020.

This article introduces the EU Horizon 2020 research project MIX-UP, "Mixed plastics biodegradation and upcycling using microbial communities". The project focuses on changing the traditional linear value chain of plastics to a sustainable, biodegradable based one. Plastic mixtures contain five of the top six fossil-based recalcitrant plastics [polyethylene (PE), polyurethane (PUR), polypropylene (PP), polyethylene terephthalate (PET), polystyrene (PS)], along with upcoming bioplastics polyhydroxyalkanoate (PHA) and polylactate (PLA) will be used as feedstock for microbial transformations.

Aqueous ionic liquids redistribute local enzyme stability via long-range perturbation pathways.

Ionic liquids (IL) and aqueous ionic liquids (aIL) are attractive (co-)solvents for biocatalysis due to their unique properties. On the other hand, the incubation of enzymes in IL or aIL often reduces enzyme activity. Recent studies proposed various aIL-induced effects to explain the reduction, classified as direct effects, e.

Real-Time Tracking of Proton Transfer from the Reactive Cysteine to the Flavin Chromophore of a Photosensing Light Oxygen Voltage Protein.

LOV (light oxygen voltage) proteins are photosensors ubiquitous to all domains of life. A variant of the short LOV protein from (DsLOV) exhibits an exceptionally fast photocycle. We performed time-resolved molecular spectroscopy on DsLOV-M49S and characterized the formation of the thio-adduct state with a covalent bond between the reactive cysteine (C72) and C of the FMN.

Metabolic and process engineering for microbial production of protocatechuate with Corynebacterium glutamicum.

3,4-Dihydroxybenzoate (protocatechuate, PCA) is a phenolic compound naturally found in edible vegetables and medicinal herbs. PCA is of high interest in the chemical industry and has wide potential for pharmaceutical applications. We designed and constructed a novel Corynebacterium glutamicum strain to enable the efficient utilization of d-xylose for microbial production of PCA.

Production of C20, C30 and C40 terpenes in the engineered phototrophic bacterium Rhodobacter capsulatus.

Terpenes constitute one of the largest groups of secondary metabolites that are used, for example, as food-additives, fragrances or pharmaceuticals. Due to the formation of an intracytoplasmic membrane system and an efficient intrinsic tetraterpene pathway, the phototrophic α-proteobacterium Rhodobacter capsulatus offers favorable properties for the production of hydrophobic terpenes. However, research efforts have largely focused on sesquiterpene production.

Towards robust Pseudomonas cell factories to harbour novel biosynthetic pathways.

Biotechnological production in bacteria enables access to numerous valuable chemical compounds. Nowadays, advanced molecular genetic toolsets, enzyme engineering as well as the combinatorial use of biocatalysts, pathways, and circuits even bring new-to-nature compounds within reach. However, the associated substrates and biosynthetic products often cause severe chemical stress to the bacterial hosts.

Applied biocatalysis beyond just buffers - from aqueous to unconventional media. Options and guidelines.

In nature, enzymes conventionally operate under aqueous conditions. Because of this, aqueous buffers are often the choice for reaction media when enzymes are applied in chemical synthesis. However, to meet the demands of an industrial application, due to the poor water solubility of many industrially relevant compounds, an aqueous reaction system will often not be able to provide sufficient substrate loadings.

Engineering adipic acid metabolism in Pseudomonas putida.

Bio-upcycling of plastics is an upcoming alternative approach for the valorization of diverse polymer waste streams that are too contaminated for traditional recycling technologies. Adipic acid and other medium-chain-length dicarboxylates are key components of many plastics including polyamides, polyesters, and polyurethanes. This study endows Pseudomonas putida KT2440 with efficient metabolism of these dicarboxylates.

Promiscuous Esterases Counterintuitively Are Less Flexible than Specific Ones.

Understanding mechanisms of promiscuity is increasingly important from a fundamental and application point of view. As to enzyme structural dynamics, more promiscuous enzymes generally have been recognized to also be more flexible. However, examples for the opposite received much less attention.

Towards bio-upcycling of polyethylene terephthalate.

Over 359 million tons of plastics were produced worldwide in 2018, with significant growth expected in the near future, resulting in the global challenge of end-of-life management. The recent identification of enzymes that degrade plastics previously considered non-biodegradable opens up opportunities to steer the plastic recycling industry into the realm of biotechnology. Here, the sequential conversion of post-consumer polyethylene terephthalate (PET) into two types of bioplastics is presented: a medium chain-length polyhydroxyalkanoate (PHA) and a novel bio-based poly(amide urethane) (bio-PU).

pyFOOMB: Python framework for object oriented modeling of bioprocesses.

Quantitative characterization of biotechnological production processes requires the determination of different key performance indicators (KPIs) such as titer, rate and yield. Classically, these KPIs can be derived by combining black-box bioprocess modeling with non-linear regression for model parameter estimation. The presented pyFOOMB package enables a guided and flexible implementation of bioprocess models in the form of ordinary differential equation systems (ODEs).

Less Unfavorable Salt Bridges on the Enzyme Surface Result in More Organic Cosolvent Resistance.

Biocatalysis for the synthesis of fine chemicals is highly attractive but usually requires organic (co-)solvents (OSs). However, native enzymes often have low activity and resistance in OSs and at elevated temperatures. Herein, we report a smart salt bridge design strategy for simultaneously improving OS resistance and thermostability of the model enzyme, Bacillus subtilits Lipase A (BSLA).

Construction and comprehensive characterization of an EcLDCc-CatIB set-varying linkers and aggregation inducing tags.

Background: In recent years, the production of inclusion bodies that retained substantial catalytic activity was demonstrated. These catalytically active inclusion bodies (CatIBs) were formed by genetic fusion of an aggregation inducing tag to a gene of interest via short linker polypeptides and overproduction of the resulting gene fusion in Escherichia coli. The resulting CatIBs are known for their high stability, easy and cost efficient production, and recyclability and thus provide an interesting alternative to conventionally immobilized enzymes.

Understanding substrate binding and the role of gatekeeping residues in PigC access tunnels.

Semi-rational redesign of the substrate binding pocket and access tunnels of prodigiosin ligase PigC enhanced the catalytic efficiency in the synthesis of pyrrolic anti-cancer agents more than 45 times. A molecular understanding was gained on residues V333 and T334 relevant to substrate binding and translocation of small pyrroles through PigC access tunnels.

Automated Rational Strain Construction Based on High-Throughput Conjugation.

Molecular cloning is the core of synthetic biology, as it comprises the assembly of DNA and its expression in target hosts. At present, however, cloning is most often a manual, time-consuming, and repetitive process that highly benefits from automation. The automation of a complete rational cloning procedure, , from DNA creation to expression in the target host, involves the integration of different operations and machines.