A New Expression System Based on Psychrotolerant Debaryomyces macquariensis Yeast and Its Application to the Production of Cold-Active ß-d-Galactosidase from Paracoccus sp. 32d.

Yeasts provide attractive host/vector systems for heterologous gene expression. The currently used yeast-based expression platforms include mesophilic and thermotolerant species. A eukaryotic expression system working at low temperatures could be particularly useful for the production of thermolabile proteins and proteins that tend to form insoluble aggregates. For this purpose, an expression system based on an Antarctic psychrotolerant yeast Debaryomyces macquariensis strain D50 that is capable of growing at temperatures ranging from 0 to 30 °C has been developed. The optimal physical culture conditions for D. macquariensis D50 in a fermenter are as follows: temperature 20 °C, pH 5.5, aeration rate of 1.5 vvm, and a stirring speed of 300 rpm. Four integrative plasmid vectors equipped with an expression cassette containing the constitutive GAP promoter and CYC1 transcriptional terminator from D. macquariensis D50 were constructed and used to clone and express a gene-encoding cold-active ß-d-galactosidase of Paracoccus sp. 32d. The yield was 1150 U/L of recombinant yeast culture. Recombinant D. macquariensis D50 strains were mitotically stable under both selective and non-selective conditions. The D. macquariensis D50 host/vector system has been successfully utilized for the synthesis of heterologous thermolabile protein, and it can be an alternative to other microbial expression systems.

The Natural Product Elegaphenone Potentiates Antibiotic Effects against Pseudomonas aeruginosa.

Natural products represent a rich source of antibiotics that address versatile cellular targets. The deconvolution of their targets via chemical proteomics is often challenged by the introduction of large photocrosslinkers. Here we applied elegaphenone, a largely uncharacterized natural product antibiotic bearing a native benzophenone core scaffold, for affinity-based protein profiling (AfBPP) in Gram-positive and Gram-negative bacteria. This study utilizes the alkynylated natural product scaffold as a probe to uncover intriguing biological interactions with the transcriptional regulator AlgP. Furthermore, proteome profiling of a Pseudomonas aeruginosa AlgP transposon mutant provided unique insights into the mode of action. Elegaphenone enhanced the elimination of intracellular P. aeruginosa in macrophages exposed to sub-inhibitory concentrations of the fluoroquinolone antibiotic norfloxacin.

The psychrotrophic yeast Sporobolomyces roseus LOCK 1119 as a source of a highly active aspartic protease for the in vitro production of antioxidant peptides.

A psychrotrophic yeast strain producing a cold-adapted protease at low temperature was classified as Sporobolomyces roseus. In standard YPG medium, S. roseus LOCK 1119 synthesized an extracellular protease with an activity of approximately 560 U/L. Optimization of medium composition and process temperature considerably enhanced enzyme biosynthesis; an approximate 70% increase in activity (2060 U/L). The native enzyme was purified to homogeneity by cation exchange chromatography followed by a size exclusion step, resulting in a 103-fold increase in specific activity (660 U/mg) with 25% recovery. The enzyme displayed 10%-30% of its maximum activity at 0-25 °C, with the optimum temperature being 50°C. Protease G8 was strongly inactivated by pepstatin A, an aspartic protease inhibitor. The enzyme was used to hydrolyze four natural substrates, and their antioxidant activities were evaluated against 1,1-diphenyl-2-picrylhydrazyl. The highest antioxidant activity (69%) was recorded for beef casein.

Quantitative Map of ß-Lactone-Induced Virulence Regulation.

ß-Lactones have recently been introduced as the first selective ClpP inhibitors that attenuate virulence of both sensitive Staphylococcus aureus and multiresistant strains (MRSA). Although previous knockout studies showed that ClpP is essential for S. aureus alpha-toxin production, a link between ß-lactone inhibition and molecular virulence mechanisms has been lacking so far. We here perform a chemical-proteomic approach to elucidate antivirulence pathways. First, we demonstrate by gel-free activity-based protein profiling that ClpP is the predominant target of ß-lactones. Only a few off-targets were discovered, which, unlike ClpP, were not involved in the reduction of alpha-toxin expression. Second, in-depth mechanistic insight was provided by a full proteomic comparison between lactone treated and untreated S. aureus cells. Quantitative mass-spectrometric analysis revealed increased repressor of toxin (Rot) levels and a corresponding down-regulation of α-toxin, providing the first direct connection between the lactone-dependent phenotype and a corresponding cellular mechanism. By building up a quantitative virulence regulation network, we visualize the impact of ClpP inhibition in a systems biology context. Interestingly, a lack of in vitro Rot degradation by either ClpXP or ClpCP calls either for a proteolysis mechanism with yet unknown adaptor proteins or for an indirect mode of action that may involve ClpX-mediated RNA signaling and feedback circuits.

A strategy for dual inhibition of the proteasome and fatty acid synthase with belactosin C-orlistat hybrids.

The proteasome, a validated cellular target for cancer, is central for maintaining cellular homeostasis, while fatty acid synthase (FAS), a novel target for numerous cancers, is responsible for palmitic acid biosynthesis. Perturbation of either enzymatic machine results in decreased proliferation and ultimately cellular apoptosis. Based on structural similarities, we hypothesized that hybrid molecules of belactosin C, a known proteasome inhibitor, and orlistat, a known inhibitor of the thioesterase domain of FAS, could inhibit both enzymes. Herein, we describe proof-of-principle studies leading to the design, synthesis and enzymatic activity of several novel, ß-lactone-based, dual inhibitors of these two enzymes. Validation of dual enzyme targeting through activity-based proteome profiling with an alkyne probe modeled after the most potent inhibitor, and preliminary serum stability studies of selected derivatives are also described. These results provide proof of concept for dual targeting of the proteasome and fatty acid synthase-thioesterase (FAS-TE) enabling a new approach for the development of drug-candidates with potential to overcome resistance.

Activity-Based Protein Profiling in Bacteria.

Understanding the molecular mechanisms of bacterial pathogenesis and virulence is of great importance from both an academic and clinical perspective, especially in view of an alarming increase in bacterial resistance to existing antibiotics and antibacterial agents. Use of small molecules to dissect the basis of these dynamic processes is a very attractive approach due to their ability for rapid spatiotemporal control of specific biochemical functions. Activity-based protein profiling (ABPP), employing small molecule probes to interrogate enzyme activities in complex proteomes, has emerged as a powerful tool to study bacterial pathogenesis. In this chapter, we present a set of ABPP methods to identify and analyze enzymes essential for growth, metabolism and virulence of different pathogens including S. aureus and L. monocytogenes using natural product-inspired activity-based probes.

Comparative Activity-Based Flavin-Dependent Oxidase Profiling.

Activity-based protein profiling (ABPP) has become a powerful chemoproteomic technology allowing for the dissection of complex ligand-protein interactions in their native cellular environment. One of the biggest challenges for ABPP is the extension of the proteome coverage. In this chapter a new ABPP strategy dedicated to monoamine oxidases (MAO) is presented. These enzymes are representative examples of flavin-dependent oxidases, playing a crucial role in the regulation of nervous system signaling.

Target identification of covalently binding drugs by activity-based protein profiling (ABPP).

The characterization of the target proteins of drug molecules has become an important goal in understanding its mode of action and origin of side effects due to off-target binding. This is especially important for covalently binding drugs usually containing electrophilic moieties, which potentially can react with nucleophilic residues found in many proteins. This review gives a comprehensive overview of the use of activity-based protein profiling (ABPP) as an efficient tool for the target identification of covalently binding drugs.

An Aromatic Hydroxyamide Attenuates Multiresistant Staphylococcus aureus Toxin Expression.

Methicillin-resistant Staphylococcus aureus (MRSA) causes severe infections with only few effective antibiotic therapies currently available. To approach this challenge, chemical entities with a novel and resistance-free mode of action are desperately needed. Here, we introduce a new hydroxyamide compound that effectively reduces the expression of devastating toxins in various S. aureus and MRSA strains. The molecular mechanism was investigated by transcriptome analysis as well as by affinity-based protein profiling. Down-regulation of several pathogenesis associated genes suggested the inhibition of a central virulence-related pathway. Mass spectrometry-based chemical proteomics revealed putative molecular targets. Systemic treatment with the hydroxyamide showed significant reduction of abscess sizes in a MRSA mouse skin infection model. The absence of resistance development in vitro further underlines the finding that targeting virulence could lead to prolonged therapeutic options in comparison to antibiotics that directly address bacterial survival.

Effects of genetic modifications and fermentation conditions on 2,3-butanediol production by alkaliphilic Bacillus subtilis.

Two recombinants of alkaliphilic Bacillus subtilis LOCK 1086, constructed via different strategies such as cloning the gene encoding bacterial hemoglobin from Vitreoscilla stercoraria (vhb) and overexpression of the gene encoding acetoin reductase/2,3-butanediol dehydrogenase (bdhA) from B. subtilis LOCK 1086, did not produce more 2,3-butanediol (2,3-BD) than the parental strain. In batch fermentations, this strain synthesized 9.46 g/L in 24 h and 12.80 g/L 2,3-BD in 46 h from sugar beet molasses and an apple pomace hydrolysate, respectively. 2,3-BD production by B. subtilis LOCK 1086 was significantly enhanced in fed-batch fermentations. The highest 2,3-BD concentration (75.73 g/L in 114 h, productivity of 0.66 g/L × h) was obtained in the sugar beet molasses-based medium with four feedings with glucose. In a medium based on the apple pomace hydrolysate with three feedings with sucrose, B. subtilis LOCK 1086 produced up to 51.53 g/L 2,3-BD (in 120 h, productivity of 0.43 g/L × h).

Application of enzymatic apple pomace hydrolysate to production of 2,3-butanediol by alkaliphilic Bacillus licheniformis NCIMB 8059.

2,3-Butanediol (2,3-BD) synthesis by a nonpathogenic bacterium Bacillus licheniformis NCIMB 8059 from enzymatic hydrolysate of depectinized apple pomace and its blend with glucose was studied. In shake flasks, the maximum diol concentration in fed-batch fermentations was 113 g/L (in 163 h, from the hydrolysate, feedings with glucose) while in batch processes it was around 27 g/L (in 32 h, from the hydrolysate and glucose blend). Fed-batch fermentations in the 0.75 and 30 L fermenters yielded 87.71 g/L 2,3-BD in 160 h, and 72.39 g/L 2,3-BD in 94 h, respectively (from the hydrolysate and glucose blend, feedings with glucose). The hydrolysate of apple pomace, which was for the first time used for microbial 2,3-BD production is not only a source of sugars but also essential minerals.

Synthesis of (±)-spongiolactone enabling discovery of a more potent derivative.

An eleven-step synthesis of (±)-spongiolactone from 1,3-cyclohexanedione is reported that relies on a diastereoselective, nucleophile-catalyzed aldol lactonization (NCAL) process with an advanced ketoacid intermediate that installed the anticipated ß-lactone pharmacophore of the natural product. In addition, a stereoselective cyclohexenyl zinc addition to a substituted cyclohexanone simultaneously installed two fully substituted vicinal stereocenters. The reported synthesis enabled preliminary structure-activity studies that revealed a regio- and stereoisomeric derivative of spongiolactone with greater antiproliferative activity towards a leukemia (K562) cell line. Furthermore, unusual antiproliferative selectivity of these spongiolactone derivatives toward the K562 cell line was observed with no inhibition of the breast, liver, and lung cancer cell lines tested.

Activity-based probes for studying the activity of flavin-dependent oxidases and for the protein target profiling of monoamine oxidase inhibitors.

High profile: new activity-based protein profiling (ABPP) probes have been designed that target exclusively monoamine oxidases A and B within living cells (see picture; FAD=flavin adenine dinucleotide, FMN=flavin monodinucleotide). With these probes it could be shown that the MAO inhibitor deprenyl, which is in clinical use against Parkinson's disease, shows unique protein specificity despite its covalent mechanism of action.

Activity-based protein profiling for natural product target discovery.

Natural products represent an important treasure box of biologically active molecules, from which many drug candidates have been sourced. The identification of the target proteins addressed by these natural products is a foremost goal for which new techniques are required. Activity-based protein profiling (ABPP), exploiting protein-reactive functional groups present in many natural products, offers unseen opportunities in this respect. This review article describes the current status of this field. Many examples are given for the annotation of biological target proteins of natural products containing epoxides, lactones, lactams, Michael acceptors, and other electrophilic groups. In addition, the development of probe molecules identified from biomimetic natural product libraries is discussed.