A versatile in situ cofactor enhancing system for meeting cellular demands for engineered metabolic pathways.

Cofactor imbalance obstructs the productivities of metabolically engineered cells. Herein, we employed a minimally perturbing system, xylose reductase and lactose (XR/lactose), to increase the levels of a pool of sugar phosphates which are connected to the biosynthesis of NAD(P)H, FAD, FMN, and ATP in Escherichia coli. The XR/lactose system could increase the amounts of the precursors of these cofactors and was tested with three different metabolically engineered cell systems (fatty alcohol biosynthesis, bioluminescence light generation, and alkane biosynthesis) with different cofactor demands. Productivities of these cells were increased 2-4-fold by the XR/lactose system. Untargeted metabolomic analysis revealed different metabolite patterns among these cells, demonstrating that only metabolites involved in relevant cofactor biosynthesis were altered. The results were also confirmed by transcriptomic analysis. Another sugar reducing system (glucose dehydrogenase) could also be used to increase fatty alcohol production but resulted in less yield enhancement than XR. This work demonstrates that the approach of increasing cellular sugar phosphates can be a generic tool to increase in vivo cofactor generation upon cellular demand for synthetic biology.

Protein Modification via Nitrile Oxide-Dehydroalanine Cycloaddition: Formation of Isoxazoline Ring on the Protein Backbone.

Here we describe a novel catalyst-free 1,3-dipolar cycloaddition bioconjugation approach for chemical modification of proteins. The dehydroalanine (Dha)-containing protein reacts with nitrile oxides generated in situ through 1,3-dipolar cycloaddition in fully aqueous-buffered systems. This leads to the formation of a new isoxazoline ring at a pre-defined site (Dha) of the protein. Furthermore, the 1-pyrene isoxazoline-installed annexin V acts as a fluorescent probe, which successfully labels the outer cellular membranes of human cholangiocarcinoma (HuCCA-1) cells for detection of apoptosis.

Enzymes in riboflavin biosynthesis: Potential antibiotic drug targets.

The rapid resistance of pathogens to antibiotics has emerged as a major threat to global health. Identification of new antibiotic targets is thus needed for developing alternative drugs. Genes encoding enzymes involved in the biosynthesis of riboflavin and flavin cofactors (FMN/FAD) are attractive targets because these enzymatic reactions are necessary for most bacteria to synthesize flavin cofactors for use in their central metabolic reactions. Moreover, humans lack most of these enzymes because we uptake riboflavin from our diet. This review discusses the current knowledge of enzymes involved in bacterial biosynthesis of riboflavin and other flavin cofactors, as well as the functions of the FMN riboswitch. Here, we highlight recent progress in the structural and mechanistic characterization, and inhibition of GTP cyclohydrolase II (GCH II), lumazine synthase (LS), riboflavin synthase (RFS), FAD synthetase (FADS), and FMN riboswitch, which have been identified as plausible antibiotic targets. As the structures and functions of these enzymes and regulatory systems are not completely understood, they are attractive as subjects for future in-depth biochemical and biophysical analysis.

Luciferin Synthesis and Pesticide Detection by Luminescence Enzymatic Cascades.

D-Luciferin (D-LH2 ), a substrate of firefly luciferase (Fluc), is important for a wide range of bioluminescence applications. This work reports a new and green method using enzymatic reactions (HELP, HadA Enzyme for Luciferin Preparation) to convert 19 phenolic derivatives to 8 D-LH2 analogues with ≈51 % yield. The method can synthesize the novel 5'-methyl-D-LH2 and 4',5'-dimethyl-D-LH2 , which have never been synthesized or found in nature. 5'-Methyl-D-LH2 emits brighter and longer wavelength light than the D-LH2 . Using HELP, we further developed LUMOS (Luminescence Measurement of Organophosphate and Derivatives) technology for in situ detection of organophosphate pesticides (OPs) including parathion, methyl parathion, EPN, profenofos, and fenitrothion by coupling the reactions of OPs hydrolase and Fluc. The LUMOS technology can detect these OPs at parts per trillion (ppt) levels. The method can directly detect OPs in food and biological samples without requiring sample pretreatment.

Naphthalene Derivatives and Quinones from Ventilago denticulata and Their Nitric Oxide Radical Scavenging, Antioxidant, Cytotoxic, Antibacterial, and Phosphodiesterase Inhibitory Activities.

New naphthalene derivatives (1 and 2) and a new isomer (3) of ventilagolin, together with known anthraquinones, chrysophanol (4), physcion or emodin 3-methyl ether (5), and emodin (6), were isolated from vines of Ventilago denticulata. The isolated compounds exhibited cytotoxic activity with IC50 values of 1.15 - 40.54 µg/ml. Compounds 1 - 3 selectively exhibited weak antibacterial activity (MIC values of 200.0 - 400.0 µg/ml), while emodin (6) displayed moderate antibacterial activity with MIC value of 25.0 µg/ml. The isolated compounds showed nitric oxide and DPPH radical scavenging activities. Compounds 1 - 3 and 6 exhibited weak xanthine oxidase inhibitory activity, while emodin (6) acted as an aromatase inhibitor with the IC50 value of 10.1 µm. Compounds 1 and 2 exhibited phosphodiesterase 5 inhibitory activity with IC50 values of 8.28 µm and 6.48 µm, respectively.

Water-Assisted Nitrile Oxide Cycloadditions: Synthesis of Isoxazoles and Stereoselective Syntheses of Isoxazolines and 1,2,4-Oxadiazoles.

Conventional methods generate nitrile oxides from oxime halides in organic solvents under basic conditions. However, the present work revealed that water-assisted generation of nitrile oxides proceeds under mild acidic conditions (pH 4-5). Cycloadditions of nitrile oxides with alkynes and alkenes easily occurred in water without using catalysts, thus yielding isoxazoles and isoxazolines, respectively, with excellent stereoselectivity toward five- and six-membered cyclic alkenes. A double stereoselective cycloaddition of two units of a nitrile oxide with cyclohexene was also achieved, thus yielding 1,2,4-oxadiazole derivatives having a unique hybrid isoxazoline-oxadiazole skeleton. Enantiomerically pure isoxazolines were prepared from monoterpenes with a ring strain. In one case, the isoxazoline with a butterfly-like structure was simply prepared, and it might be used as a ligand in asymmetric catalysis.

Altenusin, a fungal metabolite, alleviates TGF-ß1-induced EMT in renal proximal tubular cells and renal fibrosis in unilateral ureteral obstruction.

Renal fibrosis is a pathological feature of chronic kidney disease (CKD), progressing toward end-stage kidney disease (ESKD). The aim of this study is to investigate the therapeutic potential of altenusin, a farnesoid X receptor (FXR) agonist derived from fungi, on renal fibrosis. The effect of altenusin was determined (i) in vitro using the transforming growth factor ß1 (TGF-ß1)-induced epithelial to mesenchymal transition (EMT) of human renal proximal tubular cells and (ii) in vivo using mouse unilateral ureteral obstruction (UUO). The findings revealed that incubation of 10 ng/ml TGF-ß1 promotes morphological change in RPTEC/TERT1 cells, a human renal proximal tubular cell line, from epithelial to fibroblast-like cells. TGF-ß1 markedly increased EMT markers namely α-smooth muscle actin (α-SMA), fibronectin, and matrix metalloproteinase 9 (MMP-9), while decreased the epithelial marker E-cadherin. Co-incubation TGF-ß1 with altenusin preserved the epithelial characteristics of the renal epithelial cells by antagonizing TGF-ß/Smad signaling pathway, specifically a decreased phosphorylation of Smad2/3 with an increased level of Smad7. Interestingly, the antagonizing effect of altenusin does not require FXR activation. Moreover, altenusin could reverse TGF-ß1-induced fibroblast-like cells to epithelial-like cells. Treatment on UUO mice with 30 mg/kg altenusin significantly reduced the expression of α-SMA, fibronectin, and collagen type 1A1 (COL1A1). The reduction in the renal fibrosis markers is correlated with the decreased phosphorylation of Smad2/3 levels but does not improve E-cadherin protein expression. Collectively, altenusin reduces EMT in human renal proximal tubular cells and renal fibrosis by antagonizing the TGF-ß/Smad signaling pathway.

Enzymatic reactions and pathway engineering for the production of renewable hydrocarbons.

Hydrocarbons such as alkanes and alkenes are extensively used as organic compounds for combustion reactions and as building block components for the synthesis of numerous materials. Various synthetic enzymatic cascades and engineered metabolic pathways can be used to produce alkanes and alkenes from bio-based materials. An understanding of the native reactions and pathways used by various organisms to synthesize these compounds together with novel approaches in biocatalysis and synthetic biology have been instrumental in the development of methods to produce alkanes and alkenes with reasonable yield. This article discusses the present state of knowledge regarding hydrocarbon biosynthetic pathways and discusses current mechanistic understanding of relevant enzymatic reactions in cyanobacteria, aerobic bacteria, insects, algae, and plants. Recent advancements in metabolic engineering and process scale up for production of hydrocarbons from fatty acids are also discussed. This technology is important for sustainability, as it provides a clean and eco-friendly method for the future production of fuels and industrial materials. Further development towards whole cell biocatalysts that are able to provide good yield with a low production cost may allow countries without big oil reserves to be capable of producing precursors for the materials industries in the future.