Biosynthesis of plant-specific stilbene polyketides in metabolically engineered Escherichia coli

BACKGROUND: Phenylpropanoids are the precursors to a range of important plant metabolites such as the cell wall constituent lignin and the secondary metabolites belonging to the flavonoid/stilbene class of compounds. The latter class of plant natural products has been shown to function in a wide range of biological activities. During the last few years an increasing number of health benefits have been associated with these compounds. In particular, they demonstrate potent antioxidant activity and the ability to selectively inhibit certain tyrosine kinases. Biosynthesis of many medicinally important plant secondary metabolites, including stilbenes, is frequently not very well understood and under tight spatial and temporal control, limiting their availability from plant sources. As an alternative, we sought to develop an approach for the biosynthesis of diverse stilbenes by engineered recombinant microbial cells. RESULTS: A pathway for stilbene biosynthesis was constructed in Escherichia coli with 4-coumaroyl CoA ligase 1 4CL1) from Arabidopsis thaliana and stilbene synthase (STS) cloned from Arachis hypogaea. E. coli cultures expressing these enzymes together converted the phenylpropionic acid precursor 4-coumaric acid, added to the growth medium, to the stilbene resveratrol (>100 mg/L). Caffeic acid, added in the same way, resulted in the production of the expected dihydroxylated stilbene, piceatannol (>10 mg/L). Ferulic acid, however, was not converted to the expected stilbene product, isorhapontigenin. Substitution of 4CL1 with a homologous enzyme, 4CL4, with a preference for ferulic acid over 4-coumaric acid, had no effect on the conversion of ferulic acid. Accumulation of tri- and tetraketide lactones from ferulic acid, regardless of the CoA-ligase expressed in E. coli, suggests that STS cannot properly accommodate and fold the tetraketide intermediate to the corresponding stilbene structure. CONCLUSION: Phenylpropionic acids, such as 4-coumaric acid and caffeic acid, can be efficiently converted to stilbene compounds by recombinant E. coli cells expressing plant biosynthetic genes. Optimization of precursor conversion and cyclization of the bulky ferulic acid precursor by host metabolic engineering and protein engineering may afford the synthesis of even more structurally diverse stilbene compounds

Discovery of a Substrate Selectivity Switch in Tyrosine Ammonia-Lyase, a Member of the Aromatic Amino Acid Lyase Family

SummaryTyrosine ammonia-lyase (TAL) is a recently described member of the aromatic amino acid lyase family, which also includes phenylalanine (PAL) and histidine ammonia-lyases (HAL). TAL is highly selective for L-tyrosine, and synthesizes 4-coumaric acid as a protein cofactor or antibiotic precursor in microorganisms. In this report, we identify a single active site residue important for substrate selection in this enzyme family. Replacing the active site residue His89 with Phe in TAL completely switched its substrate selectivity from tyrosine to phenylalanine, thereby converting it into a highly active PAL. When a corresponding mutation was made in PAL, the enzyme lost PAL activity and gained TAL activity. The discovered substrate selectivity switch is a rare example of a complete alteration of substrate specificity by a single point mutation. We also show that the identity of the amino acid at the switch position can serve as a guide to predict substrate specificities of annotated aromatic amino acid lyases in genome sequences

Clarifications to the limitations of the s-α equilibrium model for gyrokinetic computations of turbulence

In the context of gyrokinetic flux-tube simulations of microturbulence in magnetized toroidal plasmas, different treatments of the magnetic equilibrium are examined. Considering the Cyclone DIII-D base case parameter set [Dimits et al., Phys. Plasmas 7, 969 (2000)], significant differences in the linear growth rates, the linear and nonlinear critical temperature gradients, and the nonlinear ion heat diffusivities are observed between results obtained using either an $s-\alpha$ or an MHD equilibrium. Similar disagreements have been reported previously [Redd et al., Phys. Plasmas 6, 1162 (1999)]. In this paper it is shown that these differences result primarily from the approximation made in the standard implementation of the $s-\alpha$ model, in which the straight field line angle is identified to the poloidal angle, leading to inconsistencies of order $\varepsilon$ ($\varepsilon=a/R$ is the inverse aspect ratio, $a$ the minor radius and $R$ the major radius). An equilibrium model with concentric, circular flux surfaces and a correct treatment of the straight field line angle gives results very close to those using a finite $\varepsilon$, low $\beta$ MHD equilibrium. Such detailed investigation of the equilibrium implementation is of particular interest when comparing flux tube and global codes. It is indeed shown here that previously reported agreements between local and global simulations in fact result from the order $\varepsilon$ inconsistencies in the $s-\alpha$ model, coincidentally compensating finite $\rho^*$ effects in the global calculations, where $\rho^*=\rho _s / a$ with $\rho_s$ the ion sound Larmor radius. True convergence between local and global simulations is finally obtained by correct treatment of the geometry in both cases, and considering the appropriate $\rho^* \rightarrow 0$ limit in the latter case

Flux- and gradient-driven global gyrokinetic simulation of tokamak turbulence

The Eulerian gyrokinetic turbulence code GENE has recently been extended to a full torus code. Moreover, it now provides Krook-type sources for gradient-driven simulations where the profiles are maintained on average as well as localized heat sources for a flux-driven type of operation. Careful verification studies and benchmarks are performed successfully. This setup is applied to address three related transport issues concerning nonlocal effects. First, it is confirmed that in gradient-driven simulations, the local limit can be reproduced-provided that finite aspect ratio effects in the geometry are treated carefully. In this context, it also becomes clear that the profile widths (not the device width) may constitute a more appropriate measure for finite-size effects. Second, the nature and role of heat flux avalanches are discussed in the framework of both local and global, flux-and gradient-driven simulations. Third, simulations dedicated to discharges with electron internal barriers are addressed. (C) 2011 American Institute of Physics. [doi: 10.1063/1.3567484

Particle transport in density gradient driven TE mode turbulence

The turbulent transport of main ion and trace impurities in a tokamak device in the presence of steep electron density gradients has been studied. The parameters are chosen for trapped electron (TE) mode turbulence, driven primarily by steep electron density gradients relevant to H-mode physics, but with a transition to temperature gradient driven turbulence as the density gradient flattens. Results obtained through non-linear (NL) and quasilinear (QL) gyrokinetic simulations using the GENE code are compared with results obtained from a fluid model. Main ion and impurity transport is studied by examining the balance of convective and diffusive transport, as quantified by the density gradient corresponding to zero particle flux (peaking factor). Scalings are obtained for the impurity peaking with the background electron density gradient and the impurity charge number. It is shown that the impurity peaking factor is weakly dependent on impurity charge and significantly smaller than the driving electron density gradient.Comment: 11 pages, 6 figures. Submitted to Nuclear Fusion SP