Dynamic control over feedback regulatory mechanisms improves NADPH fluxes and xylitol biosynthesis in engineered E. coli

We report improved NADPH flux and xylitol biosynthesis in engineered E. coli. Xylitol is produced from xylose via an NADPH dependent reductase. We utilize two-stage dynamic metabolic control to compare two approaches to optimize xylitol biosynthesis, a stoichiometric approach, wherein competitive fluxes are decreased, and a regulatory approach wherein the levels of key regulatory metabolites are reduced. The stoichiometric and regulatory approaches lead to a 16 fold and 100 fold improvement in xylitol production, respectively. Strains with reduced levels of enoyl-ACP reductase and glucose-6-phosphate dehydrogenase, led to altered metabolite pools resulting in the activation of the membrane bound transhydrogenase and a new NADPH generation pathway, namely pyruvate ferredoxin oxidoreductase coupled with NADPH dependent ferredoxin reductase, leading to increased NADPH fluxes, despite a reduction in NADPH pools. These strains produced titers of 200 g/L of xylitol from xylose at 86% of theoretical yield in instrumented bioreactors. We expect dynamic control over enoyl-ACP reductase and glucose-6-phosphate dehydrogenase to broadly enable improved NADPH dependent bioconversions.HighlightsDecreases in NADPH pools lead to increased NADPH fluxesPyruvate ferredoxin oxidoreductase coupled with NADPH-ferredoxin reductase improves NADPH production in vivo.Dynamic reduction in acyl-ACP/CoA pools alleviate inhibition of membrane bound transhydrogenase and improve NADPH fluxXylitol titers > 200g/L in fed batch fermentations with xylose as a sole feedstock.

Brevifoliol and its Analogs: A New Class of Antitubercular Agents

: Brevifoliol is an abeo-taxane isolated from the Taxus wallichiana needles, eighteen semi-synthetic esters derivatives of brevifoliol were prepared by Steglich esterification and screened for their anti-tubercular potential against Mycobacterium tuberculosis H37Ra avirulent strain. The 3-[chloro (7)] and 3, 5-[dinitro (8)] benzoic acid ester derivatives were most active (MIC 25 ug/ml) against the pathogen. Further, in silico docking studies of the active derivative 7 with mycobacterium enzyme inhA (enoyl-ACP reductase) gave a LibDock score of 152.68 and binding energy of -208.62 and formed three hydrogen bonds with SER94, MET98, and SER94. Similarly, when derivative 8 docked with inhA, it gave a LibDock score of 113.55 and binding energy of -175.46 and formed a single hydrogen bond with GLN100 and Pi-interaction with PHE97. On the other hand the known standard drug isoniazid (INH) gave a LibDock score of 61.63, binding energy of -81.25 and formed one hydrogen bond with ASP148. These molecular docking results and the way of binding pattern indicated that compound 7 and 8 bound well within the binding pocket of inhA and showed a higher binding affinity than the known drug Isoniazid. Additionally, both the derivatives (7 and 8) showed no cytotoxicity towards the healthy liver cell lines CHANG.