Cascade synthesis of uridine-5'-diphosphate glucuronic acid by coupling multiple whole cells expressing hyperthermophilic enzymes.

BACKGROUND: Enzymatic glycan synthesis has leapt forward in recent years and a number of glucuronosyltransferase (EC 2.4.1.17) have been identified and prepared, which provides a guide to an efficient approach to prepare glycans containing glucuronic acid (GlcA) residues. The uridine 5'-diphosphate (UDP) activated form, UDP-GlcA, is the monosaccharide donor for these glucuronidation reactions. RESULTS: To produce UDP-GlcA in a cost-effective way, an efficient three-step cascade route was developed using whole cells expressing hyperthermophilic enzymes to afford UDP-GlcA from starch. By coupling a coenzyme regeneration system with an appropriate expression level with UDP-glucose 6-dehydrogenase in a single strain, the cells were able to meet NAD+ requirements. Without addition of exogenous NAD+, the reaction produced 1.3 g L-1 UDP-GlcA, representing 100% and 46% conversion of UDP-Glc and UTP respectively. Finally, an anion exchange chromatography purification method was developed. UDP-GlcA was successfully obtained from the cascade system. The yield of UDP-GlcA during purification was about 92.0%. CONCLUSIONS: This work built a de novo hyperthermophilic biosynthetic cascade into E. coli host cells, with the cells able to meet NAD+ cofactor requirements and act as microbial factories for UDP-GlcA synthesis, which opens a door to large-scale production of cheaper UDP-GlcA.

Three new compounds from the twigs and leaves of Nageia fleuryi Hickel.

Two new diterpenoids, 12,15-di-O-acetylhypargenin B (1) and taiwanin F-12-O-ß-D-glucopyranoside (2), one new monoterpenoid, (S)-7-methyl-3-methyleneoct-6-ene-1,2-diyl diacetate (3), together with eight known compounds (4-11), were obtained from the twigs and leaves of Nageia fleuryi Hickel. The structures of the new compounds were elucidated by extensive spectroscopic techniques including HR-ESI-MS and 1 D and 2 D NMR experiments. Spectroscopic data of the known compound 4 were provided for the first time. Compounds 1 and 11 exhibited strong inhibitory activity on LPS-stimulated production of NO in RAW 264.7 murine macrophages, while compounds 1, 3, and 5 showed significant quinone reductase inducing activity in Hepa 1c1c7 murine hepatoma cells. Moreover, compounds 7 and 8 showed inhibitory activity against the proliferation of the human prostate carcinoma DU145 cells.

Cell-based and cell-free biocatalysis for the production of D-glucaric acid.

D-Glucaric acid (GA) is a value-added chemical produced from biomass, and has potential applications as a versatile platform chemical, food additive, metal sequestering agent, and therapeutic agent. Marketed GA is currently produced chemically, but increasing demand is driving the search for eco-friendlier and more efficient production approaches. Cell-based production of GA represents an alternative strategy for GA production. A series of synthetic pathways for GA have been ported into Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris, respectively, and these engineered cells show the ability to synthesize GA de novo. Optimization of the GA metabolic pathways in host cells has leapt forward, and the titer and yield have increased rapidly. Meanwhile, cell-free multi-enzyme catalysis, in which the desired pathway is constructed in vitro from enzymes and cofactors involved in GA biosynthesis, has also realized efficient GA bioconversion. This review presents an overview of studies of the development of cell-based GA production, followed by a brief discussion of potential applications of biosensors that respond to GA in these biosynthesis routes.

The second member of the bacterial UDP-N-acetyl-d-glucosamine:heparosan alpha-1, 4-N-acetyl-d-glucosaminyltransferase superfamily: GaKfiA from Gallibacterium anatis.

Bacterial UDP-N-acetyl-d-glucosamine:heparosan alpha-1, 4-N-acetyl-d-glucosaminyltransferases (KfiAs) are in high demand for the development of animal-free heparin (HP) production. Until now, EcKfiA from Escherichia coli O10:K5:H4 was the sole identified member of this family. The lack of known members has limited research into molecular structure and catalytic mechanism of the KfiA superfamily, and restricted its application in enzymatic glycan synthesis. Herein, we report the identification and characterization of Gallibacterium anatis GaKfiA, doubling the number of known members of the KfiA family. GaKfiA is a monofunctional enzyme that transfers N-acetyl-d-glucosamine (GlcNAc) residues from their nucleotide forms to the nonreducing ends of saccharide chains structurally equivalent to the backbone of HP. The catalytic efficiency of GaKfiA is lower than that of EcKfiA. However, a single mutation of GaKfiA, N56D, resulted in a drastic increase in kcat/Km compared with wild-type GaKfiA. These data once again indicate the key role of a complete DXD motif for the catalytic efficiency of glycosyltransferases. This study deepens understanding of the mechanism of KfiA, and will assist in research into animal-free HP production.

Cloning and Characterization of a Chondroitin AC Exolyase from Arthrobacter sp. SD-04.

Glycosaminoglycans (GAGs) and their low-molecular weight derivates have received considerable interest in terms of their potential clinical applications, and display a wide variety of pharmacological and pharmacokinetic properties. Structurally distinct GAG chains can be prepared by enzymatic depolymerization. A variety of bacterial chondroitin sulfate (CS) lyases have been identified, and have been widely used as catalysts in this process. Here, we identified a putative chondroitin AC exolyase gene, AschnAC, from an Arthrobacter sp. strain found in a CS manufacturing workshop. We expressed the enzyme, AsChnAC, recombinantly in Escherichia coli, then purified and characterized it in vitro. The enzyme indeed displayed exolytic cleavage activity toward HA and various CSs. Removing the putative N-terminal secretion signal peptide of AsChnAC improved its expression level in E. coli while maintaining chondroitin AC exolyase activity. This novel catalyst exhibited its optimal activity in the absence of added metal ions. AsChnAC has potential applications in preparation of low-molecular weight GAGs, making it an attractive catalyst for further investigation.