Discovery of highly potent HDAC8 PROTACs with anti-tumor activity.

Various diseases are deeply associated with aberrations in HDAC8 functions. These aberrations can be assigned to either structural functions or catalytic functions of HDAC8. Therefore, development of HDAC8 degradation inducers might be more promising than HDAC8 inhibitors. We employed the proteolysis targeting chimera (PROTAC) strategy to develop a selective and potent HDAC8 degradation inducer CT-4 with single-digit nanomolar DC50 values and over 95% Dmax in both triple-negative breast cancer MDA-MB-231 cells and T-cell leukemia cells. Notably, CT-4 demonstrated potent anti-migration activity and limited anti-proliferative activity in MDA-MB-231 cells. In contrast, CT-4 effectively induced apototic cell death in Jurkat cells, as assessed by a caspase 3/7 activity assay and flow cytometry. Our findings suggest that the development of HDAC8 degradation inducers holds great potential for the treatment of HDAC8-related diseases.

Development and application of CRISPR-based genetic tools in Bacillus species and Bacillus phages.

Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed into a precise and efficient genome editing tool. Since its discovery as an adaptive immune system in prokaryotes, it has been applied in many different research fields including biotechnology and medical sciences. The high demand for rapid, highly efficient and versatile genetic tools to thrive in bacteria-based cell factories accelerates this process. This review mainly focuses on significant advancements of the CRISPR system in Bacillus subtilis, including the achievements in gene editing, and on problems still remaining. Next, we comprehensively summarize this genetic tool's up-to-date development and utilization in other Bacillus species, including B. licheniformis, B. methanolicus, B. anthracis, B. cereus, B. smithii and B. thuringiensis. Furthermore, we describe the current application of CRISPR tools in phages to increase Bacillus hosts' resistance to virulent phages and phage genetic modification. Finally, we suggest potential strategies to further improve this advanced technique and provide insights into future directions of CRISPR technologies for rendering Bacillus species cell factories more effective and more powerful.

Identification of a Bromodomain-like Region in 15-Lipoxygenase-1 Explains Its Nuclear Localization.

Lipoxygenase (LOX) activity provides oxidative lipid metabolites, which are involved in inflammatory disorders and tumorigenesis. Activity-based probes to detect the activity of LOX enzymes in their cellular context provide opportunities to explore LOX biology and LOX inhibition. Here, we developed Labelox B as a potent covalent LOX inhibitor for one-step activity-based labeling of proteins with LOX activity. Labelox B was used to establish an ELISA-based assay for affinity capture and antibody-based detection of specific LOX isoenzymes. Moreover, Labelox B enabled efficient activity-based labeling of endogenous LOXs in living cells. LOX proved to localize in the nucleus, which was rationalized by identification of a functional bromodomain-like consensus motif in 15-LOX-1. This indicates that 15-LOX-1 is not only involved in oxidative lipid metabolism, but also in chromatin binding, which suggests a potential role in chromatin modifications.

A regulated synthetic operon facilitates stable overexpression of multigene terpenoid pathway in Bacillus subtilis.

The creation of microbial cell factories for sustainable production of natural products is important for medical and industrial applications. This requires stable expression of biosynthetic pathways in a host organism with favorable fermentation properties such as Bacillus subtilis. The aim of this study is to construct B. subtilis strains that produce valuable terpenoid compounds by overexpressing the innate methylerythritol phosphate (MEP) pathway. A synthetic operon allowing the concerted and regulated expression of multiple genes was developed. Up to 8 genes have been combined in this operon and a stably inherited plasmid-based vector was constructed resulting in a high production of C30 carotenoids. For this, two vectors were examined, one with rolling circle replication and another with theta replication. Theta-replication constructs were clearly superior in structural and segregational stability compared to rolling circle constructs. A strain overexpressing all eight genes of the MEP pathway on a theta-replicating plasmid clearly produced the highest level of carotenoids. The level of transcription for each gene in the operon was similar as RT-qPCR analysis indicated. Hence, that corresponding strain can be used as a stable cell factory for production of terpenoids. This is the first report of merging and stably expressing this large-size operon (eight genes) from a plasmid-based system in B. subtilis enabling high C30 carotenoid production.

Exoproteome Heterogeneity among Closely Related Staphylococcus aureus t437 Isolates and Possible Implications for Virulence.

Staphylococcus aureus with spa-type t437 has been identified as a predominant community-associated methicillin-resistant S. aureus clone from Asia, which is also encountered in Europe. Molecular typing has previously shown that t437 isolates are highly similar regardless of geographical regions or host environments. The present study was aimed at assessing to what extent this high similarity is actually reflected in the production of secreted virulence factors. We therefore profiled the extracellular proteome, representing the main reservoir of virulence factors, of 20 representative clinical isolates by mass spectrometry. The results show that these isolates can be divided into three groups and nine subgroups based on exoproteome abundance signatures. This implies that S. aureus t437 isolates show substantial exoproteome heterogeneity. Nonetheless, 30 highly conserved extracellular proteins, of which about 50% have a predicted role in pathogenesis, were dominantly identified. To approximate the virulence of the 20 investigated isolates, we employed infection models based on Galleria mellonella and HeLa cells. The results show that the grouping of clinical isolates based on their exoproteome profile can be related to virulence. We consider this outcome important as our approach provides a tool to pinpoint differences in virulence among seemingly highly similar clinical isolates of S. aureus.

Cytotoxic Deoxypodophyllotoxin Can Be Extracted in High Purity from Anthriscus sylvestris Roots by Supercritical Carbon Dioxide.

Deoxypodophyllotoxin is present in the roots of Anthriscus sylvestris. This compound is cytotoxic on its own, but it can also be converted into podophyllotoxin, which is in high demand as a precursor for the important anticancer drugs etoposide and teniposide. In this study, deoxypodophyllotoxin is extracted from A. sylvestris roots by supercritical carbon dioxide extraction. The process is simple and scalable. The supercritical carbon dioxide method extracts 75 - 80% of the total deoxypodophyllotoxin content, which is comparable to a single extraction by traditional Soxhlet. However, less polar components are extracted. The activity of the supercritical carbon dioxide extract containing deoxypodophyllotoxin was assessed by demonstrating that the extract arrests A549 and HeLa cells in the G2/M phase of the cell cycle. We conclude that biologically active deoxypodophyllotoxin can be extracted from A. sylvestris by supercritical carbon dioxide extraction. The method is solvent free and more sustainable compared to traditional methods.

Metabolic engineering of Bacillus subtilis for terpenoid production.

Terpenoids are the largest group of small-molecule natural products, with more than 60,000 compounds made from isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). As the most diverse group of small-molecule natural products, terpenoids play an important role in the pharmaceutical, food, and cosmetic industries. For decades, Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae) were extensively studied to biosynthesize terpenoids, because they are both fully amenable to genetic modifications and have vast molecular resources. On the other hand, our literature survey (20 years) revealed that terpenoids are naturally more widespread in Bacillales. In the mid-1990s, an inherent methylerythritol phosphate (MEP) pathway was discovered in Bacillus subtilis (B. subtilis). Since B. subtilis is a generally recognized as safe (GRAS) organism and has long been used for the industrial production of proteins, attempts to biosynthesize terpenoids in this bacterium have aroused much interest in the scientific community. This review discusses metabolic engineering of B. subtilis for terpenoid production, and encountered challenges will be discussed. We will summarize some major advances and outline future directions for exploiting the potential of B. subtilis as a desired "cell factory" to produce terpenoids.

The Promiscuity of Squalene Synthase-Like Enzyme: Dehydrosqualene Synthase, a Natural Squalene Hyperproducer?

Dehydrosqualene synthase (CrtM), as a squalene synthase-like enzyme from Staphylococcus aureus, can naturally utilize farnesyl diphosphate to produce dehydrosqualene (C30H48). However, no study has documented the natural production of squalene (C30H50) by CrtM. Here, based on an HPLC-Q-Orbitrap-MS/MS study, we report that the expression of crtM in vitro or in Bacillus subtilis 168 both results in the output of squalene, dehydrosqualene, and phytoene (C40H64). Notably, wild-type CrtM exhibits a significantly higher squalene yield compared to squalene synthase (SQS) from Bacillus megaterium with an approximately 2.4-fold increase. Moreover, the examination of presqualene diphosphate's stereostructures in both CrtM and SQS enzymes provides further understanding into the presence of multiple identified terpenoids. In summary, this study not only provides insights into the promiscuity demonstrated by squalene synthase-like enzymes but also highlights a new strategy of utilizing CrtM as a potential replacement for SQS in cell factories, thereby enhancing squalene production.

Production of α-cuprenene in Xanthophyllomyces dendrorhous: a step closer to a potent terpene biofactory.

BACKGROUND: The red yeast Xanthophyllomyces dendrorhous is a natural producer of the carotenoid astaxanthin. Because of its high flux, the native terpene pathway leading to the production of the tetraterpene is of particular interest as it can be redirected toward the production of other terpene compounds. The genetic tools for the transformation of the yeast with the concurrent knock-out of genes involved in the astaxanthin biosynthesis are made available and here we show that the production of the sesquiterpene α-cuprenene is possible in mutant strains of X. dendrorhous transformed with the Cop6 gene originating from the fungus Coprinus cinereus. For the evaluation of the production levels, we chose to express the same gene and analyze the accumulation of α-cuprenene in Escherichia coli and Saccharomyces cerevisiae, as well. Here we propose that X. dendrorhous is a candidate in the search for the potential platform organism for the production of terpenes. RESULTS: All three X. dendrorhous mutants functionally express the Cop6 gene and accumulate α-cuprenene. The production of α-cuprenene in the red yeast reached 80 mg/L, which represents a far higher concentration compared to the levels obtained in the E. coli and S. cerevisiae mutants. At this expression levels the pool of terpene precursors has not become a limiting factor in the X. dendrorhous mutants since the expression of the Cop6 gene in the genomic rDNA of the yeast allows production of both α-cuprenene and astaxanthin without affecting the growth or the accumulation levels of both compounds. CONCLUSIONS: We have shown that X. dendrorhous can produce α-cuprenene, and the results here presented, next to the capability of accumulating at least two more non-native sesquiterpenes, demonstrates the high potential of this yeast to become an interesting terpene-based drugs producer.

Receptor Specificity Engineering of TNF Superfamily Ligands.

The tumor necrosis factor (TNF) ligand family has nine ligands that show promiscuity in binding multiple receptors. As different receptors transduce into diverse pathways, the study on the functional role of natural ligands is very complex. In this review, we discuss the TNF ligands engineering for receptor specificity and summarize the performance of the ligand variants in vivo and in vitro. Those variants have an increased binding affinity to specific receptors to enhance the cell signal conduction and have reduced side effects due to a lowered binding to untargeted receptors. Refining receptor specificity is a promising research strategy for improving the application of multi-receptor ligands. Further, the settled variants also provide experimental guidance for engineering receptor specificity on other proteins with multiple receptors.

Fighting Acinetobacter baumannii infections with the acylase PvdQ.

Acinetobacter baumannii is an opportunistic Gram-negative bacterial pathogen that poses a threat for frail patients worldwide. The high ability to withstand environmental stresses as well as its resistance towards a broad range of antibiotics make A. baumannii an effective hard-to-eradicate pathogen. One of the key mechanisms mediating tolerance against antibiotic treatment is the formation of biofilms, a process that is controlled by a multitude of different regulatory mechanisms. A key factor with major impact on biofilm formation is cell-to-cell communication by quorum-sensing, which in A. baumannii is mediated by acyl homoserine lactone signaling molecules. Here we show that the Ntn-Hydrolase PvdQ from Pseudomonas aeruginosa can reduce biofilm formation by the A. baumannii ATCC 17978 type strain and several clinical isolates on abiotic surfaces. Further, our study shows that a combination treatment of PvdQ-mediated quorum-quenching with the antibiotic gentamicin has a synergistic effect on the clearance of A. baumannii biofilms and possible biofilm dispersal. Moreover, we demonstrate in a Galleria mellonella larval infection model that PvdQ administration significantly prolongs survival of the larvae. Altogether, we conclude that the acylase-mediated irreversible cleavage of quorum-sensing signaling molecules as exemplified with PvdQ can set a profound limit to the progression of A. baumannii infections.

Discovery of chromene compounds as inhibitors of PvdQ acylase of Pseudomonas aeruginosa.

The acquisition of iron is a crucial mechanism for the survival of pathogenic bacteria such as Pseudomonas aeruginosa in eukaryotic hosts. The key iron chelator in this organism is the siderophore pyoverdine, which was shown to be crucial for iron homeostasis. Pyoverdine is a non-ribosomal peptide with several maturation steps in the cytoplasm and others in the periplasmatic space. A key enzyme for its maturation is the acylase PvdQ. The inhibition of PvdQ stops the maturation of pyoverdine causing a significant imbalance in the iron homeostasis and hence can negatively influence the survival of P. aeruginosa. In this work, we successfully synthesized chromene-derived inhibitory molecules targeting PvdQ in a low micromolar range. In silico modeling as well as kinetic evaluations of the inhibitors suggest a competitive inhibition of the PvdQ function. Further, we evaluated the inhibitor in vivo on P. aeruginosa cells and report a dose-dependent reduction of pyoverdine formation. The compound also showed a protecting effect in a Galleria mellonella infection model.

Dihydroartemisinin-Transferrin Adducts Enhance TRAIL-Induced Apoptosis in Triple-Negative Breast Cancer in a P53-Independent and ROS-Dependent Manner.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype independent of estrogen receptor, progesterone receptor, or human epidermal growth factor receptor 2. It has a poor prognosis and high recurrence. Due to its limited treatment options in the clinic, novel therapies are urgently needed. Single treatment with the death receptor ligand TRAIL was shown to be poorly effective. Recently, we have shown that artemisinin derivatives enhance TRAIL-induced apoptosis in colon cancer cells. Here, we utilized transferrin (TF) to enhance the effectiveness of dihydroartemisinin (DHA) in inducing cell death in TNBC cell lines (MDA-MB-231, MDA-MB-436, MDA-MB-468 and BT549). We found that the combination of DHA-TF and the death receptor 5-specific TRAIL variant DHER leads to an increase in DR5 expression in all four TNBC cell lines, while higher cytotoxicity was observed in MDA-MB-231, and MDA-MB-436. All the data point to the finding that DHA-TF stimulates cell death in TNBC cells, while the combination of DHA-TF with TRAIL variants will trigger more cell death in TRAIL-sensitive cells. Overall, DHA-TF in combination with TRAIL variants represents a potential novel combination therapy for triple-negative breast cancer.

High level production of amorphadiene using Bacillus subtilis as an optimized terpenoid cell factory.

The anti-malarial drug artemisinin, produced naturally in the plant Artemisia annua, experiences unstable and insufficient supply as its production relies heavily on the plant source. To meet the massive demand for this compound, metabolic engineering of microbes has been studied extensively. In this study, we focus on improving the production of amorphadiene, a crucial artemisinin precursor, in Bacillus subtilis. The expression level of the plant-derived amorphadiene synthase (ADS) was upregulated by fusion with green fluorescent protein (GFP). Furthermore, a co-expression system of ADS and a synthetic operon carrying the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway genes was established. Subsequently, farnesyl pyrophosphate synthase (FPPS), a key enzyme in formation of the sesquiterpene precursor farnesyl pyrophosphate (FPP), was expressed to supply sufficient substrate for ADS. The consecutive combination of these features yielded a B. subtilis strain expressing chromosomally integrated GFP-ADS followed by FPPS and a plasmid encoded synthetic operon showing a stepwise increased production of amorphadiene. An experimental design-aided systematic medium optimization was used to maximize the production level for the most promising engineered B. subtilis strain, resulting in an amorphadiene yield of 416 ± 15 mg/L, which is 20-fold higher than that previously reported in B. subtilis and more than double the production in Escherichia coli or Saccharomyces cerevisiae on a shake flask fermentation level.

Engineering of Multiple Modules to Improve Amorphadiene Production in Bacillus subtilis Using CRISPR-Cas9.

Engineering strategies to improve terpenoids' production in Bacillus subtilis mainly focus on 2C-methyl-d-erythritol-4-phosphate (MEP) pathway overexpression. To systematically engineer the chassis strain for higher amorphadiene (precursor of artemisinin) production, a clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system was established in B. subtilis to facilitate precise and efficient genome editing. Then, this system was employed to engineer three more modules to improve amorphadiene production, including the terpene synthase module, the branch pathway module, and the central metabolic pathway module. Finally, our combination of all of the useful strategies within one strain significantly increased extracellular amorphadiene production from 81 to 116 mg/L after 48 h flask fermentation without medium optimization. For the first time, we attenuated the FPP-derived competing pathway to improve amorphadiene biosynthesis and investigated how the TCA cycle affects amorphadiene production in B. subtilis. Overall, this study provides a universal strategy for further increasing terpenoids' production in B. subtilis by comprehensive and systematic metabolic engineering.

Immobilized Acylase PvdQ Reduces Pseudomonas aeruginosa Biofilm Formation on PDMS Silicone.

The bacterial biofilm plays a key role in nosocomial infections, especially those related to medical devices in sustained contact with patients. The active dispersion of bacterial cells out of biofilms acts as a reservoir for infectious diseases. The formation of such biofilms is a highly complex process, which is coordinated by many regulatory mechanisms of the pathogen including quorum sensing (QS). Many bacteria coordinate the expression of key virulence factors dependent on their population density through QS. The inhibition of this system is called quorum quenching (QQ). Thus, preventing the development of biofilms is considered a promising approach to prevent the development of hard to treat infections. Enzymatic QQ is the concept of interfering with the QS system of bacteria outside the cell. PvdQ is an acylase with an N-terminal nucleophile (Ntn-hydrolase) that is a part of the pyoverdine gene cluster (pvd). It is able to cleave irreversibly the amide bond of long chain N-acyl homoserine lactones (AHL) rendering them inactive. Long chain AHLs are the main signaling molecule in the QS system of the gram-negative pathogen Pseudomonas aeruginosa PA01, which is known for surface-associated biofilms on indwelling catheters and is also the cause of catheter-associated urinary tract infections. Furthermore, PA01 is a well characterized pathogen with respect to QS as well as QQ. In this study, we immobilized the acylase PvdQ on polydimethylsiloxane silicone (PDMS), creating a surface with quorum quenching properties. The goal is to control infections by minimizing the colonization of indwelling medical devices such as urinary catheters or intravascular catheters. The enzyme activity was confirmed by testing the degradation of the main auto-inducer that mediates QS in P. aeruginosa. In this article we report for the first time a successful immobilization of the quorum quenching acylase PvdQ on PDMS silicone. We could show that immobilized PvdQ retained its activity after the coating procedure and showed a 6-fold reduction of the auto-inducer 3-oxo-C12 in a biosensor setup. Further we report significant reduction of a P. aeruginosa PA01 biofilm on a coated PDMS surface compared to the same untreated material.

Death Receptor 5 Displayed on Extracellular Vesicles Decreases TRAIL Sensitivity of Colon Cancer Cells.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is considered to be a promising antitumor drug because of its selective proapoptotic properties on tumor cells. However, the clinical application of TRAIL is until now limited because of the resistance of several cancer cells, which can occur at various levels in the TRAIL signaling pathway. The role of decoy receptors that can side-track TRAIL, thereby preventing the formation of an activated death receptor, has been extensively studied. In this study, we have focused on extracellular vesicles (EVs) that are known to play a role in cell-to-cell communication and that can be released by donor cells into the medium transferring their components to recipient cells. TRAIL-induced apoptotic signaling is triggered upon the binding of two death receptors, DR4 and DR5. Here, we found that DR5 but not DR4 is present in the conditioned medium (CM)-derived from various cancer cells. Moreover, we observed that DR5 was exposed on EVs and can act as "decoy receptor" for binding to TRAIL. This results in a strongly reduced number of apoptotic cells upon treatment with DR5-specific TRAIL variant DHER in CM. This reduction happened with EVs containing either the long or short isoform of DR5. Taken together, we demonstrated that colon rectal tumor cells can secrete DR5-coated EVs, and this can cause TRAIL resistance. This is to our knowledge a novel finding and provides new insights into understanding TRAIL sensitivity.

Artemisinin Derivatives Stimulate DR5-Specific TRAIL-Induced Apoptosis by Regulating Wildtype P53.

Artemisinin derivatives, widely known as commercial anti-malaria drugs, may also have huge potential in treating cancer cells. It has been reported that artemisinin derivatives can overcome resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in liver and cervical cancer cells. In our study, we demonstrated that artesunate (ATS) and dihydroartemisinin (DHA) are more efficient in killing colon cancer cells compared to artemisinin (ART). ATS/DHA induces the expression of DR5 in a P53 dependent manner in HCT116 and DLD-1 cells. Both ATS and DHA overcome the resistance to DHER-induced apoptosis in HCT116, mainly through upregulating death receptor 5 (DR5). We also demonstrate that DHA sensitizes HCT116 cells to DHER-induced apoptosis via P53 regulated DR5 expression in P53 knockdown assays. Nevertheless, a lower effect was observed in DLD-1 cells, which has a single Ser241Phe mutation in the P53 DNA binding domain. Thus, the status of P53 could be one of the determinants of TRAIL resistance in some cancer cells. Finally, the combination treatment of DHA and the TRAIL variant DHER increases cell death in 3D colon cancer spheroid models, which shows its potential as a novel therapy.

Production of Squalene in Bacillus subtilis by Squalene Synthase Screening and Metabolic Engineering.

Squalene synthase (SQS) catalyzes the conversion of two farnesyl pyrophosphates to squalene, an important intermediate in between isoprene and valuable triterpenoids. In this study, we have constructed a novel biosynthesis pathway for squalene in Bacillus subtilis and performed metabolic engineering aiming at facilitating further exploitation and production of squalene-derived triterpenoids. Therefore, systematic studies and analysis were performed including selection of multiple SQS candidates from various organisms, comparison of expression vectors, optimization of cultivation temperatures, and examination of rate-limiting factors within the synthetic pathway. We were, for the first time, able to obtain squalene synthesis in B. subtilis. Furthermore, we achieved a 29-fold increase of squalene yield (0.26-7.5 mg/L) by expressing SQS from Bacillus megaterium and eliminating bottlenecks within the upstream methylerythritol-phosphate pathway. Moreover, our findings showed that also ispA could positively affect the production of squalene.

Quorum-quenching acylase reduces the virulence of Pseudomonas aeruginosa in a Caenorhabditis elegans infection model.

The Pseudomonas aeruginosa PAO1 gene pvdQ encodes an acyl-homoserine lactone (AHL) acylase capable of degrading N-(3-oxododecanoyl)-L-homoserine lactone by cleaving the AHL amide. PvdQ has been proven to function as a quorum quencher in vitro in a number of phenotypic assays. To address the question of whether PvdQ also shows quorum-quenching properties in vivo, an infection model based on the nematode Caenorhabditis elegans was explored. In a fast-acting paralysis assay, strain PAO1(pMEpvdQ), which overproduces PvdQ, was shown to be less virulent than the wild-type strain. More than 75% of the nematodes exposed to PAO1(pMEpvdQ) survived and continued to grow when using this strain as a food source. Interestingly, in a slow-killing assay monitoring the survival of the nematodes throughout a 4-day course, strain PAO1-Delta pvdQ was shown to be more virulent than the wild-type strain, confirming the role of PvdQ as a virulence-reducing agent. It was observed that larval stage 1 (L1) to L3-stage larvae benefit much more from protection by PvdQ than L4 worms. Finally, purified PvdQ protein was added to C. elegans worms infected with wild-type PAO1, and this resulted in reduced pathogenicity and increased the life span of the nematodes. From our observations we can conclude that PvdQ might be a strong candidate for antibacterial therapy against Pseudomonas infections.