A synthetic biochemistry platform for cell free production of monoterpenes from glucose.

Cell-free systems designed to perform complex chemical conversions of biomass to biofuels or commodity chemicals are emerging as promising alternatives to the metabolic engineering of living cells. Here we design a system comprises 27 enzymes for the conversion of glucose into monoterpenes that generates both NAD(P)H and ATP in a modified glucose breakdown module and utilizes both cofactors for building terpenes. Different monoterpenes are produced in our system by changing the terpene synthase enzyme. The system is stable for the production of limonene, pinene and sabinene, and can operate continuously for at least 5 days from a single addition of glucose. We obtain conversion yields >95% and titres >15 g l-1. The titres are an order of magnitude over cellular toxicity limits and thus difficult to achieve using cell-based systems. Overall, these results highlight the potential of synthetic biochemistry approaches for producing bio-based chemicals

Production of FAME biodiesel in E. coli by direct methylation with an insect enzyme.

Most biodiesel currently in use consists of fatty acid methyl esters (FAMEs) produced by transesterification of plant oils with methanol. To reduce competition with food supplies, it would be desirable to directly produce biodiesel in microorganisms. To date, the most effective pathway for the production of biodiesel in bacteria yields fatty acid ethyl esters (FAEEs) at up to ~1.5 g/L. A much simpler route to biodiesel produces FAMEs by direct S-adenosyl-L-methionine (SAM) dependent methylation of free fatty acids, but FAME production by this route has been limited to only ~16 mg/L. Here we employ an alternative, broad spectrum methyltransferase, Drosophila melanogaster Juvenile Hormone Acid O-Methyltransferase (DmJHAMT). By introducing DmJHAMT in E. coli engineered to produce medium chain fatty acids and overproduce SAM, we obtain medium chain FAMEs at titers of 0.56 g/L, a 35-fold increase over titers previously achieved. Although considerable improvements will be needed for viable bacterial production of FAMEs and FAEEs for biofuels, it may be easier to optimize and transport the FAME production pathway to other microorganisms because it involves fewer enzymes

Bacteriorhodopsin folds through a poorly organized transition state.

The folding mechanisms of helical membrane proteins remain largely uncharted. Here we characterize the kinetics of bacteriorhodopsin folding and employ φ-value analysis to explore the folding transition state. First, we developed and confirmed a kinetic model that allowed us to assess the rate of folding from SDS-denatured bacteriorhodopsin (bRU) and provides accurate thermodynamic information even under influence of retinal hydrolysis. Next, we obtained reliable φ-values for 16 mutants of bacteriorhodopsin with good coverage across the protein. Every φ-value was less than 0.4, indicating the transition state is not uniquely structured. We suggest that the transition state is a loosely organized ensemble of conformations

An Adaptation To Life In Acid Through A Novel Mevalonate Pathway.

Extreme acidophiles are capable of growth at pH values near zero. Sustaining life in acidic environments requires extensive adaptations of membranes, proton pumps, and DNA repair mechanisms. Here we describe an adaptation of a core biochemical pathway, the mevalonate pathway, in extreme acidophiles. Two previously known mevalonate pathways involve ATP dependent decarboxylation of either mevalonate 5-phosphate or mevalonate 5-pyrophosphate, in which a single enzyme carries out two essential steps: (1) phosphorylation of the mevalonate moiety at the 3-OH position and (2) subsequent decarboxylation. We now demonstrate that in extreme acidophiles, decarboxylation is carried out by two separate steps: previously identified enzymes generate mevalonate 3,5-bisphosphate and a new decarboxylase we describe here, mevalonate 3,5-bisphosphate decarboxylase, produces isopentenyl phosphate. Why use two enzymes in acidophiles when one enzyme provides both functionalities in all other organisms examined to date? We find that at low pH, the dual function enzyme, mevalonate 5-phosphate decarboxylase is unable to carry out the first phosphorylation step, yet retains its ability to perform decarboxylation. We therefore propose that extreme acidophiles had to replace the dual-purpose enzyme with two specialized enzymes to efficiently produce isoprenoids in extremely acidic environments

Electron g-2 in Light-Front Quantization

Basis Light-front Quantization has been proposed as a nonperturbative framework for solving quantum field theory. We apply this approach to Quantum Electrodynamics and explicitly solve for the light-front wave function of a physical electron. Based on the resulting light-front wave function, we evaluate the electron anomalous magnetic moment. Nonperturbative mass renormalization is performed. Upon extrapolation to the infinite basis limit our numerical results agree with the Schwinger result obtained in perturbation theory to an accuracy of 0.06%.Comment: 6 pages, 4 figure

Shielding superconductors with thin films

Determining the optimal arrangement of superconducting layers to withstand large amplitude AC magnetic fields is important for certain applications such as superconducting radiofrequency cavities. In this paper, we evaluate the shielding potential of the superconducting film/insulating film/superconductor (SIS') structure, a configuration that could provide benefits in screening large AC magnetic fields. After establishing that for high frequency magnetic fields, flux penetration must be avoided, the superheating field of the structure is calculated in the London limit both numerically and, for thin films, analytically. For intermediate film thicknesses and realistic material parameters we also solve numerically the Ginzburg-Landau equations. It is shown that a small enhancement of the superheating field is possible, on the order of a few percent, for the SIS' structure relative to a bulk superconductor of the film material, if the materials and thicknesses are chosen appropriately.Comment: 7 pages, 5 figure

Sensitive protein detection using an optical fibre long period grating sensor anchored with silica core gold shell nanoparticles

Copyright ©2014 Society of Photo-Optical Instrumentation Engineers. This paper was published in the Proceedings of SPIE and is made available with permission of SPIE. One print or electronic copy may be made for personal use only. Systematic electronic or print reproduction and distribution, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper are prohibited.An optical fibre long period grating (LPG), modified with a coating of silica gold (SiO2:Au) core/shell nanoparticles (NPs) deposited using the layer-by-layer (LbL) method, was employed for the development of a bio-sensor. The SiO2:Au NPs were electrostatically assembled onto the LPG with the aid of a poly(hydrochloride ammonium) (PAH) polycation layer. The LPG sensor operates at the phase matching turning point to provide the highest sensitivity. The SiO2:Au NPs were modified with biotin, which was used as a ligand for streptavidin (SV) detection. The sensing mechanism is based on the measurement of the refractive index change induced by the binding of the SV to the biotin. The lowest detected concentration of SV was 19 nM using an LPG modified with a 3 layer (PAH/SiO2:Au) thin film