A promiscuous ancestral enzyme´s structure unveils protein variable regions of the highly diverse metallo-β-lactamase family.

The metallo-β-lactamase fold is an ancient protein structure present in numerous enzyme families responsible for diverse biological processes. The crystal structure of the hyperthermostable crenarchaeal enzyme Igni18 from Ignicoccus hospitalis was solved at 2.3 Å and could resemble a possible first archetype of a multifunctional metallo-β-lactamase.

Heterologous Production of β-Caryophyllene and Evaluation of Its Activity against Plant Pathogenic Fungi.

Terpenoids constitute one of the largest and most diverse groups within the class of secondary metabolites, comprising over 80,000 compounds. They not only exhibit important functions in plant physiology but also have commercial potential in the biotechnological, pharmaceutical, and agricultural sectors due to their promising properties, including various bioactivities against pathogens, inflammations, and cancer. In this work, we therefore aimed to implement the plant sesquiterpenoid pathway leading to β-caryophyllene in the heterologous host and achieved a maximum production of 139 ± 31 mg L culture.

An Optimized for Itaconic Acid Production at Maximal Theoretical Yield.

, a member of the Ustilaginaceae family, is a promising host for the production of several metabolites including itaconic acid. This dicarboxylate has great potential as a bio-based building block in the polymer industry, and is of special interest for pharmaceutical applications. Several itaconate overproducing strains have been generated by metabolic and morphology engineering.

The Plant Sesquiterpene Nootkatone Efficiently Reduces Parasitism by Activating Plant Defense.

Plant parasitic nematodes, including the beet cyst nematode , constitute a devastating problem for crops worldwide. The limited availability of sustainable management options illustrates the need for new eco-friendly control means. Plant metabolites represent an invaluable source of active compounds for the discovery of such novel antagonistic agents.

Consolidated bioprocessing of cellulose to itaconic acid by a co-culture of Trichoderma reesei and Ustilago maydis.

Background: Itaconic acid is a bio-derived platform chemical with uses ranging from polymer synthesis to biofuel production. The efficient conversion of cellulosic waste streams into itaconic acid could thus enable the sustainable production of a variety of substitutes for fossil oil based products. However, the realization of such a process is currently hindered by an expensive conversion of cellulose into fermentable sugars.

CompassR Yields Highly Organic-Solvent-Tolerant Enzymes through Recombination of Compatible Substitutions.

The CompassR (computer-assisted recombination) rule enables, among beneficial substitutions, the identification of those that can be recombined in directed evolution. Herein, a recombination strategy is systematically investigated to minimize experimental efforts and maximize possible improvements. In total, 15 beneficial substitutions from Bacillus subtilis lipase A (BSLA), which improves resistance to the organic cosolvent 1,4-dioxane (DOX), were studied to compare two recombination strategies, the two-gene recombination process (2GenReP) and the in silico guided recombination process (InSiReP), employing CompassR.

Azulitox-A P28-Derived Cancer-Cell-Specific Protein Photosensitizer.

Azulitox as a new fusion polypeptide with cancer cell specificity and phototoxicity was generated and is composed of a photosensitizer domain and the cell-penetrating peptide P28. The photosensitizer domain (EcFbFP) was derived from a bacterial blue-light receptor, which belongs to the family of light-oxygen-voltage proteins and produces reactive oxygen species (ROS) upon excitation. P28 is derived from the cupredoxin protein azurin that is known to specifically penetrate cancer cells and bind to the tumor suppressor protein p53.

Adaptive laboratory evolution of and to enhance anthranilate tolerance.

Microbial bioproduction of the aromatic acid anthranilate (-aminobenzoate) has the potential to replace its current, environmentally demanding production process. The host organism employed for such a process needs to fulfil certain demands to achieve industrially relevant product levels. As anthranilate is toxic for microorganisms, the use of particularly robust production hosts can overcome issues from product inhibition.

Integration of Genetic and Process Engineering for Optimized Rhamnolipid Production Using .

Rhamnolipids are biosurfactants produced by microorganisms with the potential to replace synthetic compounds with petrochemical origin. To promote industrial use of rhamnolipids, recombinant rhamnolipid production from sugars needs to be intensified. Since this remains challenging, the aim of the presented research is to utilize a multidisciplinary approach to take a step toward developing a sustainable rhamnolipid production process.

A Straightforward Assay for Screening and Quantification of Biosurfactants in Microbial Culture Supernatants.

A large variety of microorganisms produces biosurfactants with the potential for a number of diverse industrial applications. To identify suitable wild-type or engineered production strains, efficient screening methods are needed, allowing for rapid and reliable quantification of biosurfactants in multiple cultures, preferably at high throughput. To this end, we have established a novel and sensitive assay for the quantification of biosurfactants based on the dye Victoria Pure Blue BO (VPBO).

Complex Evolution of Light-Dependent Protochlorophyllide Oxidoreductases in Aerobic Anoxygenic Phototrophs: Origin, Phylogeny, and Function.

Light-dependent protochlorophyllide oxidoreductase (LPOR) and dark-operative protochlorophyllide oxidoreductase are evolutionary and structurally distinct enzymes that are essential for the synthesis of (bacterio)chlorophyll, the primary pigment needed for both anoxygenic and oxygenic photosynthesis. In contrast to the long-held hypothesis that LPORs are only present in oxygenic phototrophs, we recently identified a functional LPOR in the aerobic anoxygenic phototrophic bacterium (AAPB) Dinoroseobacter shibae and attributed its presence to a single horizontal gene transfer event from cyanobacteria. Here, we provide evidence for the more widespread presence of genuine LPOR enzymes in AAPBs.

Effect of Photocaged Isopropyl β-d-1-thiogalactopyranoside Solubility on the Light Responsiveness of LacI-controlled Expression Systems in Different Bacteria.

Photolabile protecting groups play a significant role in controlling biological functions and cellular processes in living cells and tissues, as light offers high spatiotemporal control, is non-invasive as well as easily tuneable. In the recent past, photo-responsive inducer molecules such as 6-nitropiperonyl-caged IPTG (NP-cIPTG) have been used as optochemical tools for Lac repressor-controlled microbial expression systems. To further expand the applicability of the versatile optochemical on-switch, we have investigated whether the modulation of cIPTG water solubility can improve the light responsiveness of appropriate expression systems in bacteria.

Serves as a Novel Production Host for the Synthesis of Plant and Fungal Sesquiterpenoids.

Sesquiterpenoids are important secondary metabolites with various pharma- and nutraceutical properties. In particular, higher basidiomycetes possess a versatile biosynthetic repertoire for these bioactive compounds. To date, only a few microbial production systems for fungal sesquiterpenoids have been established.

The length of ribosomal binding site spacer sequence controls the production yield for intracellular and secreted proteins by Bacillus subtilis.

Background: Bacillus subtilis is widely used for the industrial production of recombinant proteins, mainly due to its high secretion capacity, but higher production yields can be achieved only if bottlenecks are removed. To this end, a crucial process is translation initiation which takes place at the ribosome binding site enclosing the Shine Dalgarno sequence, the start codon of the target gene and a short spacer sequence in between. Here, we have studied the effects of varying spacer sequence lengths in vivo on the production yield of different intra- and extracellular proteins.

Benzoate Synthesis from Glucose or Glycerol Using Engineered Pseudomonas taiwanensis.

Benzoic acid is one of the most commonly used food preservatives, but currently exclusively produced in petrochemical processes. In this study, a bio-based production pathway using an engineered strain of Pseudomonas taiwanensis is described. In a phenylalanine-overproducing strain, bacterial and plant genes are heterologously expressed to achieve production of benzoate via a β-oxidation pathway.

Catalytically-active inclusion bodies for biotechnology-general concepts, optimization, and application.

Bacterial inclusion bodies (IBs) have long been considered as inactive, unfolded waste material produced by heterologous overexpression of recombinant genes. In industrial applications, they are occasionally used as an alternative in cases where a protein cannot be expressed in soluble form and in high enough amounts. Then, however, refolding approaches are needed to transform inactive IBs into active soluble protein.

Rapid and comprehensive evaluation of microalgal fatty acids via untargeted gas chromatography and time-of-flight mass spectrometry.

Due to their high triacylglyceride content, microalgae are intensively investigated for bio-economy and food applications. However, lipid analysis is a laborious task incorporating extraction, transesterification and typically gas chromatographic measurement. Co-elution induces a significant risk of fatty acid misidentification and thus, additional purification steps like thin layer chromatography are needed.

Characterization of Context-Dependent Effects on Synthetic Promoters.

Understanding the composability of genetic elements is central to synthetic biology. Even for seemingly well-known elements such as a sigma 70 promoter the genetic context-dependent variability of promoter activity remains poorly understood. The lack of understanding of sequence to function results in highly limited design of novel genetic element combinations.

Unraveling 1,4-Butanediol Metabolism in KT2440.

Plastics, in all forms, are a ubiquitous cornerstone of modern civilization. Although humanity undoubtedly benefits from the versatility and durability of plastics, they also cause a tremendous burden for the environment. Bio-upcycling is a promising approach to reduce this burden, especially for polymers that are currently not amenable to mechanical recycling.

Detailed small-scale characterization and scale-up of active YFP inclusion body production with Escherichia coli induced by a tetrameric coiled coil domain.

During heterologous protein production with Escherichia coli, the formation of inclusion bodies (IBs) is often a major drawback as these aggregated proteins are usually inactive. However, different strategies for the generation of IBs consisting of catalytically active proteins have recently been described. In this study, the archaeal tetrameric coiled-coil domain of the cell-surface protein tetrabrachion was fused to a target reporter protein to produce fluorescent IBs (FIBs).

A Novel Polyester Hydrolase From the Marine Bacterium Structural and Functional Insights.

Biodegradation of synthetic polymers, in particular polyethylene terephthalate (PET), is of great importance, since environmental pollution with PET and other plastics has become a severe global problem. Here, we report on the polyester degrading ability of a novel carboxylic ester hydrolase identified in the genome of the marine hydrocarbonoclastic bacterium VGXO14 . The enzyme, designated PE-H, belongs to the type IIa family of PET hydrolytic enzymes as indicated by amino acid sequence homology.

Structural and dynamic insights revealing how lipase binding domain MD1 of Pseudomonas aeruginosa foldase affects lipase activation.

Folding and cellular localization of many proteins of Gram-negative bacteria rely on a network of chaperones and secretion systems. Among them is the lipase-specific foldase Lif, a membrane-bound steric chaperone that tightly binds (K = 29 nM) and mediates folding of the lipase LipA, a virulence factor of the pathogenic bacterium P. aeruginosa.

Systematically Scrutinizing the Impact of Substitution Sites on Thermostability and Detergent Tolerance for Lipase A.

Improving an enzyme's (thermo-)stability or tolerance against solvents and detergents is highly relevant in protein engineering and biotechnology. Recent developments have tended toward data-driven approaches, where available knowledge about the protein is used to identify substitution sites with high potential to yield protein variants with improved stability, and subsequently, substitutions are engineered by site-directed or site-saturation (SSM) mutagenesis. However, the development and validation of algorithms for data-driven approaches have been hampered by the lack of availability of large-scale data measured in a uniform way and being unbiased with respect to substitution types and locations.

An Ustilago maydis chassis for itaconic acid production without by-products.

Ustilago maydis is a promising yeast for the production of a range of valuable metabolites, including itaconate, malate, glycolipids and triacylglycerols. However, wild-type strains generally produce a potpourri of all of these metabolites, which hinders efficient production of single target chemicals. In this study, the diverse by-product spectrum of U.

Process engineering of pH tolerant Ustilago cynodontis for efficient itaconic acid production.

Background: Ustilago cynodontis ranks among the relatively unknown itaconate production organisms. In comparison to the well-known and established organisms like Aspergillus terreus and Ustilago maydis, genetic engineering and first optimizations for itaconate production were only recently developed for U. cynodontis, enabling metabolic and morphological engineering of this acid-tolerant organism for efficient itaconate production.