A review on Millepachine and its derivatives as potential multitarget anticancer agents.

Chalcones have a long history of being used for many medical purposes. These are the most prestigious scaffolds in medicine. The potential of Millepachine and its derivatives to treat various malignancies has been demonstrated in this review. The anticancer effects of Millepachine and its derivatives on ovarian cancer, hepatocellular carcinoma, breast, liver, colon, cervical, prostate, stomach, and gliomas are highlighted in the current review. Several genes that are crucial in reducing the severity of the disease have been altered by these substances. They mainly work by preventing tubulin polymerizing. They also exhibit apoptosis and cell cycle arrest at the G2/M phase. Additionally, these compounds inhibit invasion and migration and have antiproliferative effects. Preclinical studies have shown that Millepachine and its derivatives offer exceptional potential for treating a number of cancers. These results need to be confirmed in clinical research in order to develop viable cancer therapies.

An overview on monkeypox virus: Pathogenesis, transmission, host interaction and therapeutics.

Orthopoxvirus is one of the most notorious genus amongst the Poxviridae family. Monkeypox (MP) is a zoonotic disease that has been spreading throughout Africa. The spread is global, and incidence rates are increasing daily. The spread of the virus is rapid due to human-to-human and animals-to-human transmission. World Health Organization (WHO) has declared monkeypox virus (MPV) as a global health emergency. Since treatment options are limited, it is essential to know the modes of transmission and symptoms to stop disease spread. The information from host-virus interactions revealed significantly expressed genes that are important for the progression of the MP infection. In this review, we highlighted the MP virus structure, transmission modes, and available therapeutic options. Furthermore, this review provides insights for the scientific community to extend their research work in this field.

Identification of Activated Cdc42-Associated Kinase Inhibitors as Potential Anticancer Agents Using Pharmacoinformatic Approaches.

BACKGROUND: Activated Cdc42-associated kinase (ACK1) is essential for numerous cellular functions, such as growth, proliferation, and migration. ACK1 signaling occurs through multiple receptor tyrosine kinases; therefore, its inhibition can provide effective antiproliferative effects against multiple human cancers. A number of ACK1-specific inhibitors were designed and discovered in the previous decade, but none have reached the clinic. Potent and selective ACK1 inhibitors are urgently needed. METHODS: In the present investigation, the pharmacophore model (PM) was rationally built utilizing two distinct inhibitors coupled with ACK1 crystal structures. The generated PM was utilized to screen the drug-like database generated from the four chemical databases. The binding mode of pharmacophore-mapped compounds was predicted using a molecular docking (MD) study. The selected hit-protein complexes from MD were studied under all-atom molecular dynamics simulations (MDS) for 500 ns. The obtained trajectories were ranked using binding free energy calculations (ΔG kJ/mol) and Gibb's free energy landscape. RESULTS: Our results indicate that the three hit compounds displayed higher binding affinity toward ACK1 when compared with the known multi-kinase inhibitor dasatinib. The inter-molecular interactions of Hit1 and Hit3 reveal that compounds form desirable hydrogen bond interactions with gatekeeper T205, hinge region A208, and DFG motif D270. As a result, we anticipate that the proposed scaffolds might help in the design of promising selective ACK1 inhibitors.

Developing methylotrophic microbial platforms for a methanol-based bioindustry.

Methanol, a relatively cheap and renewable single-carbon feedstock, has gained considerable attention as a substrate for the bio-production of commodity chemicals. Conventionally produced from syngas, along with emerging possibilities of generation from methane and CO2, this C1 substrate can serve as a pool for sequestering greenhouse gases while supporting a sustainable bio-economy. Methylotrophic organisms, with the inherent ability to use methanol as the sole carbon and energy source, are competent candidates as platform organisms. Accordingly, methanol bioconversion pathways have been an attractive target for biotechnological and bioengineering interventions in developing microbial cell factories. This review summarizes the recent advances in methanol-based production of various bulk and value-added chemicals exploiting the native and synthetic methylotrophic organisms. Finally, the current challenges and prospects of streamlining these methylotrophic platforms are discussed.

Reassessing acetyl-CoA supply and NADPH availability for mevalonate biosynthesis from glycerol in Escherichia coli.

Mevalonate is an important platform compound for the biosynthesis of isoprenoids. It can be synthesized from acetyl-CoA in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) by the introduced mvaES operon in Escherichia coli. The influences of E. coli hosts, acetyl-CoA supply, and NADPH availability were assessed and engineered to improve the production titer and yield of mevalonate from glycerol. As a result, E. coli DH5α was found to be the best host with high specific capability and titer of mevalonate from glycerol. Through the engineering of phosphoketolase-phosphotransacetylase (xPK-PTA) bypass and NADPH availability, a final titer of 7.21 g/L with a specific capability of 1.36 g/g dry cell weight was gained in flask culture. Our work could offer new information to metabolically engineer the mevalonate pathway for the efficient production of isoprenoids.

Current Trends in Acetins Production: Green versus Non-Green Synthesis.

To utilize excess glycerol produced from the biodiesel industry, researchers are developing innovative methods of transforming glycerol into value-added chemicals. One strategy adopted is the conversion of glycerol into acetins, which are esters of glycerol that have wide applications in cosmetics, pharmaceuticals, food and fuel additives, and plasticizers and serve as precursors for other chemical compounds. Acetins are synthesized either by traditional chemical methods or by biological processes. Although the chemical methods are efficient, productive, and commercialized, they are "non-green", meaning that they are unsafe for the environment and consumers. On the other hand, the biological process is "green" in the sense that it protects both the environment and consumers. It is, however, less productive and requires further effort to achieve commercialization. Thus, both methodologies have benefits and drawbacks, and this study aims to present and discuss these. In addition, we briefly discuss general strategies for optimizing biological processes that could apply to acetins production on an industrial scale.

Strategies to Mitigate Enteric Methane Emissions from Ruminant Animals.

Human activities account for approximately two-thirds of global methane emissions, wherein the livestock sector is the single massive methane emitter. Methane is a potent greenhouse gas of over 21 times the warming effect of carbon dioxide. In the rumen, methanogens produce methane as a by-product of anaerobic fermentation. Methane released from ruminants is considered as a loss of feed energy that could otherwise be used for productivity. Economic progress and growing population will inflate meat and milk product demands, causing elevated methane emissions from this sector. In this review, diverse approaches from feed manipulation to the supplementation of organic and inorganic feed additives and direct-fed microbial in mitigating enteric methane emissions from ruminant livestock are summarized. These approaches directly or indirectly alter the rumen microbial structure thereby reducing rumen methanogenesis. Though many inorganic feed additives have remarkably reduced methane emissions from ruminants, their usage as feed additives remains unappealing because of health and safety concerns. Hence, feed additives sourced from biological materials such as direct-fed microbials have emerged as a promising technique in mitigating enteric methane emissions.

Metagenomic analysis of gut microbiome reveals a dynamic change in Alistipes onderdonkii in the preclinical model of pancreatic cancer, suppressing its proliferation.

Pancreatic cancer is a lethal cancer with aggressive and invasive characteristics. By the time it is diagnosed, patients already have tumors extended to other organs and show extremely low survival rates. The gut microbiome is known to be associated with many diseases and its imbalance affects the pathogenesis of pancreatic cancer. In this study, we established an orthotopic, patient-derived xenograft model to identify how the gut microbiome is linked to pancreatic ductal adenocarcinoma (PDAC). Using the 16S rDNA metagenomic sequencing, we revealed that the levels of Alistipes onderdonkii and Roseburia hominis decreased in the gut microbiome of the PDAC model. To explore the crosstalk between the two bacteria and PDAC cells, we collected the supernatant of the bacteria or cancer cell culture medium and treated it in a cross manner. While the cancer cell medium did not affect bacterial growth, we observed that the A. onderdonkii medium suppressed the growth of the pancreatic primary cancer cells. Using the bromodeoxyuridine/7-amino-actinomycin D (BrdU/7-AAD) staining assay, we confirmed that the A. onderdonkii medium inhibited the proliferation of the pancreatic primary cancer cells. Furthermore, RNA-seq analysis revealed that the A. onderdonkii medium induced unique transcriptomic alterations in the PDAC cells, compared to the normal pancreatic cells. Altogether, our data suggest that the reduction in the A. onderdonkii in the gut microbiome provides a proliferation advantage to the pancreatic cancer cells. KEY POINTS: • Metagenome analysis of pancreatic cancer model reveals A. onderdonkii downregulation. • A. onderdonkii culture supernatant suppressed the proliferation of pancreatic cancer cells. • RNA seq data reveals typical gene expression changes induced by A. onderdonkii.

Metabolic Engineering of Escherichia coli for Production of α-Santalene, a Precursor of Sandalwood Oil.

α-Santalene belongs to a class of natural compounds with many physiological functions and medical applications. Advances in metabolic engineering enable non-native hosts (e.g., Escherichia coli) to produce α-santalene, the precursor of sandalwood oil. However, imbalances in enzymatic activity often result in a metabolic burden on hosts and repress the synthetic capacity of the desired product. In this work, we manipulated ribosome binding sites (RBSs) to optimize an α-santalene synthetic operon in E. coli, and the best engineered E. coli NA-IS3D strain could produce α-santalene at a titer of 412 mg·L-1. Concerning the observation of the inverse correlation between indole synthesis and α-santalene production, this study speculated that indole-associated amino acid metabolism would be competitive to the synthesis of α-santalene rather than indole toxicity itself. The deletion of tnaA could lead to a 1.5-fold increase in α-santalene production to a titer of 599 mg·L-1 in E. coli tnaA- NA-IS3D. Our results suggested that the optimization of RBS sets of the synthetic module and attenuation of the competitive pathway are promising approaches for improving the production of terpenoids including α-santalene.

The promoter sequences of lettuce cis-prenyltransferase and its binding protein specify gene expression in laticifers.

MAIN CONCLUSION: Promoters of lettuce cis-prenyltransferase 3 (LsCPT3) and CPT-binding protein 2 (LsCBP2) specify gene expression in laticifers, as supported by in situ ß-glucuronidase stains and microsection analysis. Lettuce (Lactuca sativa) has articulated laticifers alongside vascular bundles. In the cytoplasm of laticifers, natural rubber (cis-1,4-polyisoprene) is synthesized by cis-prenyltransferase (LsCPT3) and CPT-binding protein (LsCBP2), both of which form an enzyme complex. Here we determined the gene structures of LsCPT3 and LsCBP2 and characterized their promoter activities using ß-glucuronidase (GUS) reporter assays in stable transgenic lines of lettuce. LsCPT3 has a single 7.4-kb intron while LsCBP2 has seven introns including a 940-bp intron in the 5'-untranslated region (UTR). Serially truncated LsCPT3 promoters (2.3 kb, 1.6 kb, and 1.1 kb) and the LsCBP2 promoter with (1.7 kb) or without (0.8 kb) the 940-bp introns were fused to GUS to examine their promoter activities. In situ GUS stains of the transgenic plants revealed that the 1.1-kb LsCPT3 and 0.8-kb LsCBP2 promoter without the 5'-UTR intron are sufficient to express GUS exclusively in laticifers. Fluorometric assays showed that the LsCBP2 promoter was several-fold stronger than the CaMV35S promoter and was ~ 400 times stronger than the LsCPT3 promoter in latex. Histochemical analyses confirmed that both promoters express GUS exclusively in laticifers, recognized by characteristic fused multicellular structures. We concluded that both the LsCPT3 and LsCBP2 promoters specify gene expression in laticifers, and the LsCBP2 promoter displays stronger expression than the CaMV35S promoter in laticifers. For the LsCPT3 promoter, it appears that unknown cis-elements outside of the currently examined LsCPT3 promoter are required to enhance LsCPT3 expression.

Association of pericardial adipose tissue with left ventricular structure and function: a region-specific effect?

BACKGROUND: The independent role of pericardial adipose tissue (PAT) as an ectopic fat associated with cardiovascular disease (CVD) remains controversial. This study aimed to determine whether PAT is associated with left ventricular (LV) structure and function independent of other markers of general obesity. METHODS: We studied 2471 participants (50.9 % women) without known CVD from the Korean Genome Epidemiology Study, who underwent 2D-echocardiography with tissue Doppler imaging (TDI) and computed tomography measurement for PAT. RESULTS: Study participants with more PAT were more likely to be men and had higher cardiometabolic indices, including blood pressure, glucose, and cholesterol levels (all P < 0.001). Greater pericardial fat levels across quartiles of PAT were associated with increased LV mass index and left atrial volume index (all P < 0.001) and decreased systolic (P = 0.015) and early diastolic (P < 0.001) TDI velocities, except for LV ejection fraction. These associations remained after a multivariable-adjusted model for traditional CV risk factors and persisted even after additional adjustment for general adiposity measures, such as waist circumference and body mass index. PAT was also the only obesity index independently associated with systolic TDI velocity (P < 0.001). CONCLUSIONS: PAT was associated with subclinical LV structural and functional deterioration, and these associations were independent of and stronger than with general and abdominal obesity measures.

Regulatory molecule cAMP changes cell fitness of the engineered Escherichia coli for terpenoids production.

Terpenoids are a class of natural compounds with many important functions and applications. They are synthesized from a long synthetic pathway of isoprenyl unit coupling with the myriads of terpene synthases. Owing to the catalytic divergence of terpenoids synthesis, microbial production of terpenoids is compromised to the complexity of pathway engineering and suffers from the metabolic engineering burden. In this work, the adaptive Escherichia coli HP variant exhibited a general cell fitness in terpenoid synthesis. Especially, it could yield taxadiene of 193.2 mg/L in a test tube culture, which is a five-fold increase over the production in the wild type E. coli DH5α. Mutational analyses indicated that IS10 insertion in adenylate cyclase CyaA (CyaAHP) resulted in lowering intracellular cyclic AMP (cAMP), which could regulate its receptor protein CRP to rewire cell metabolism and contributed to the improved cell fitness. Our results suggested a way to manipulate cell fitness for terpenoids production and other products.

Extension of cell membrane boosting squalene production in the engineered Escherichia coli.

Squalene is a lipophilic and non-volatile triterpene with many industrial applications for food, pharmaceuticals, and cosmetics. Metabolic engineering focused on optimization of the production pathway suffer from little success in improving titers because of a limited space of the cell membrane accommodating the lipophilic product. Extension of cell membrane would be a promising approach to overcome the storage limitation for successful production of squalene. In this study, Escherichia coli was engineered for squalene production by overexpression of some membrane proteins. The highest production of 612 mg/L was observed in the engineered E. coli with overexpression of Tsr, a serine chemoreceptor protein, which induced invagination of inner membrane to form multilayered structure. It was also observed an increase in unsaturated fatty acid in membrane lipids composition, suggesting cellular response to maintain membrane fluidity against squalene accumulation in the engineered strain. This study potentiates the capability of E. coli for squalene production and provides an effective strategy for the enhanced production of such compounds.

Crystal structure of geranylgeranyl pyrophosphate synthase (crtE) from Nonlabens dokdonensis DSW-6.

Isoprenoids comprise a diverse group of natural products with a broad range of metabolic functions. Isoprenoids are synthesized from prenyl pyrophosphates by prenyltransferases that catalyze the isoprenoid chain-elongation process to different chain lengths. We hereby present the crystal structure of geranylgeranyl pyrophosphate synthase from the marine flavobacterium Nonlabens dokdonensis DSW-6 (NdGGPPS). NdGGPPS forms a hexamer composed of homodimeric trimer, and the monomeric structure is composed of 15 α-helices (α1-α15). In this structure, we observed the binding of one pyrophosphate molecule and two glycerol molecules that mimicked substrate binding to the enzyme. The substrate binding site of NdGGPPS contains large hydrophobic residues such as Phe, His and Tyr, and structural and amino acids sequence analyses thereof suggest that the protein belongs to the short-chain prenyltransferase family.

Challenges and tackles in metabolic engineering for microbial production of carotenoids.

Naturally occurring carotenoids have been isolated and used as colorants, antioxidants, nutrients, etc. in many fields. There is an ever-growing demand for carotenoids production. To comfort this, microbial production of carotenoids is an attractive alternative to current extraction from natural sources. This review summarizes the biosynthetic pathway of carotenoids and progresses in metabolic engineering of various microorganisms for carotenoid production. The advances in synthetic pathway and systems biology lead to many versatile engineering tools available to manipulate microorganisms. In this context, challenges and possible directions are also discussed to provide an insight of microbial engineering for improved production of carotenoids in the future.

Development of novel on-line capillary gas chromatography-based analysis method for volatile organic compounds produced by aerobic fermentation.

Many volatile compounds, such as isoprene, a precursor used in the synthesis of natural rubber, have been produced through fermentation using genetically engineered microorganisms. Despite this biotechnological success, measuring the concentrations of volatile compounds during fermentation is difficult because of their high volatility. In current systems, off-line analytical methods usually lead to product loss, whereas on-line methods raise the production cost due to the requirement of complex devices. Here, we developed a novel on-line gas chromatography (GC)-based system for analyzing the concentration of isoprene with the aim to minimize the cost and requirement for devices as compared to current strategies. In this system, a programmable logic controller is used to combine conventional GC with a syringe pump module (SPM) directly connected to the exhaust pipe of the fermentor, and isoprene-containing samples are continuously pumped from the SPM into the GC using an air cylinder recycle stream. We showed that this novel system enables isoprene analysis during fermentation with convenient equipment and without the requirement of an expensive desorption tube. Furthermore, this system may be extended to the detection of other volatile organic compounds in fermentation or chemical processes.

Efficient production of lycopene in Saccharomyces cerevisiae by enzyme engineering and increasing membrane flexibility and NAPDH production.

Lycopene is a red carotenoid pigment with strong antioxidant activity. Saccharomyces cerevisiae is considered a promising host to produce lycopene, but lycopene toxicity is one of the limiting factors for high-level production. In this study, we used heterologous lycopene biosynthesis genes crtE and crtI from Xanthophyllomyces dendrorhous and crtB from Pantoea agglomerans for lycopene production in S. cerevisiae. The crtE, crtB, and crtI genes were integrated into the genome of S. cerevisiae CEN.PK2-1C strain, while deleting DPP1 and LPP1 genes to inhibit a competing pathway producing farnesol. Lycopene production was further improved by inhibiting ergosterol production via downregulation of ERG9 expression and by deleting ROX1 or MOT3 genes encoding transcriptional repressors for mevalonate and sterol biosynthetic pathways. To further increase lycopene production, CrtE and CrtB mutants with improved activities were isolated by directed evolution, and subsequently, the mutated genes were randomly integrated into the engineered lycopene-producing strains via delta-integration. To relieve lycopene toxicity by increasing unsaturated fatty acid content in cell membranes, the OLE1 gene encoding stearoyl-CoA 9-desaturase was overexpressed. In combination with the overexpression of STB5 gene encoding a transcription factor involved in NADPH production, the final strain produced up to 41.8 mg/gDCW of lycopene, which is approximately 74.6-fold higher than that produced in the initial strain.

Microbial Platform for Terpenoid Production: Escherichia coli and Yeast.

Terpenoids, also called isoprenoids, are a large and highly diverse family of natural products with important medical and industrial properties. However, a limited production of terpenoids from natural resources constrains their use of either bulk commodity products or high valuable products. Microbial production of terpenoids from Escherichia coli and yeasts provides a promising alternative owing to available genetic tools in pathway engineering and genome editing, and a comprehensive understanding of their metabolisms. This review summarizes recent progresses in engineering of industrial model strains, E. coli and yeasts, for terpenoids production. With advances of synthetic biology and systems biology, both strains are expected to present the great potential as a platform of terpenoid synthesis.

A Genetically Encoded Biosensor for Monitoring Isoprene Production in Engineered Escherichia coli.

Isoprene is a valuable precursor for synthetic rubber and a signature product of terpenoid pathways. Here, we developed an isoprene biosensor by employing a TbuT transcriptional regulator of Ralstonia pickettii to express a fluorescent reporter gene in response to intracellular isoprene in engineered Escherichia coli. The TbuT regulator recognizes isoprene as its less-preferred effector molecule; thus, we amplified the reporter gene expression using a T7 RNA polymerase-mediated transcriptional cascade and iteratively tuned the promoter transcribing tbuT to improve the sensitivity for detecting isoprene. When the engineered E. coli cells expressed heterologous genes for isoprene biosynthesis, the intracellular isoprene was expelled and the tbuT transcription factor was subsequently activated, leading to gfp expression. The chromosomal isoprene biosensor showed a linear correlation between GFP fluorescence and intracellular isoprene concentration. Using this chromosomal isoprene biosensor, we successfully identified the highest isoprene producer among four different E. coli strains producing different amounts of isoprene. The isoprene biosensor presented here can enable high-throughput screening of isoprene synthases and metabolic pathways for efficient and sustainable production of bioisoprene in engineered microbes.

Metabolic engineering of Escherichia coli for production of mixed isoprenoid alcohols and their derivatives.

BACKGROUND: Current petroleum-derived fuels such as gasoline (C5-C12) and diesel (C15-C22) are complex mixtures of hydrocarbons with different chain lengths and chemical structures. Isoprenoids are hydrocarbon-based compounds with different carbon chain lengths and diverse chemical structures, similar to petroleum. Thus, isoprenoid alcohols such as isopentenol (C5), geraniol (C10), and farnesol (C15) have been considered to be ideal biofuel candidates. NudB, a native phosphatase of Escherichia coli, is reported to dephosphorylate isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) into isopentenol. However, no attention has been paid to its promiscuous activity toward longer chain length (C10-C15) prenyl diphosphates. RESULTS: In this study, the promiscuous activity of NudB toward geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) was applied for the production of isoprenoid alcohol mixtures, including isopentenol, geraniol, and farnesol, and their derivatives. E. coli was engineered to produce a mixture of C5 and C15 alcohols by overexpressing NudB (dihydroneopterin triphosphate diphosphohydrolase) and IspA (FPP synthase) along with a heterologous MVA pathway, which resulted in a total of up to 1652 mg/L mixture of C5 and C15 alcohols and their derivatives. The production was further increased to 2027 mg/L by overexpression of another endogenous phosphatase, AphA, in addition to NudB. Production of DMAPP- and FPP-derived alcohols and their derivatives was significantly increased with an increase in the gene dosage of idi, encoding IPP isomerase (IDI), indicating a potential modulation of the composition of the alcohols mixture according to the expression level of IDI. When IspA was replaced with its mutant IspA*, generating GPP in the production strain, a total of 1418 mg/L of the isoprenoid mixture was obtained containing C10 alcohols as a main component. CONCLUSIONS: The promiscuous activity of NudB was newly identified and successfully used for production of isoprenoid-based alcohol mixtures, which are suitable as next-generation biofuels or commodity chemicals. This is the first successful report on high-titer production of an isoprenoid alcohol-based mixture. The engineering approaches can provide a valuable platform for production of other isoprenoid mixtures via a proportional modulation of IPP, DMAPP, GPP, and FPP syntheses.