Increasing diterpene yield with a modular metabolic engineering system in E. coli: comparison of MEV and MEP isoprenoid precursor pathway engineering

Engineering biosynthetic pathways in heterologous microbial host organisms offers an elegant approach to pathway elucidation via the incorporation of putative biosynthetic enzymes and characterization of resulting novel metabolites. Our previous work in Escherichia coli demonstrated the feasibility of a facile modular approach to engineering the production of labdane-related diterpene (20 carbon) natural products. However, yield was limited (<0.1 mg/L), presumably due to reliance on endogenous production of the isoprenoid precursors dimethylallyl diphosphate and isopentenyl diphosphate. Here, we report incorporation of either a heterologous mevalonate pathway (MEV) or enhancement of the endogenous methyl erythritol phosphate pathway (MEP) with our modular metabolic engineering system. With MEP pathway enhancement, it was found that pyruvate supplementation of rich media and simultaneous overexpression of three genes (idi, dxs, and dxr) resulted in the greatest increase in diterpene yield, indicating distributed metabolic control within this pathway. Incorporation of a heterologous MEV pathway in bioreactor grown cultures resulted in significantly higher yields than MEP pathway enhancement. We have established suitable growth conditions for diterpene production levels ranging from 10 to >100 mg/L of E. coli culture. These amounts are sufficient for nuclear magnetic resonance analyses, enabling characterization of enzymatic products and hence, pathway elucidation. Furthermore, these results represent an up to >1,000-fold improvement in diterpene production from our facile, modular platform, with MEP pathway enhancement offering a cost effective alternative with reasonable yield. Finally, we reiterate here that this modular approach is expandable and should be easily adaptable to the production of any terpenoid natural product

Rational Design of Pathogen-Mimicking Amphiphilic Materials as Nanoadjuvants

An opportunity exists today for cross-cutting research utilizing advances in materials science, immunology, microbial pathogenesis, and computational analysis to effectively design the next generation of adjuvants and vaccines. This study integrates these advances into a bottom-up approach for the molecular design of nanoadjuvants capable of mimicking the immune response induced by a natural infection but without the toxic side effects. Biodegradable amphiphilic polyanhydrides possess the unique ability to mimic pathogens and pathogen associated molecular patterns with respect to persisting within and activating immune cells, respectively. The molecular properties responsible for the pathogen-mimicking abilities of these materials have been identified. The value of using polyanhydride nanovaccines was demonstrated by the induction of long-lived protection against a lethal challenge of Yersinia pestis following a single administration ten months earlier. This approach has the tantalizing potential to catalyze the development of next generation vaccines against diseases caused by emerging and re-emerging pathogens

INEEL ENVIRONMENTAL MANAGEMENT NONDESTRUCTIVE ASSAY ACTIVITIES

ABSTRACT Successful management of the U. S. Department of Energy (DOE) environmental management wastes at the Idaho National Engineering and Environmental Laboratory (INEEL) has required the development of advanced nuclear based nondestructive assay (NDA) technologies and methodologies. These activities have been directed towards addressing current and future NDA needs associated with (a) spent nuclear fuel, (b) contact-handled transuranic (TRU) waste, (c) remote-handled transuranic waste, and (d) environmental monitoring and remediation programs. The types of activities undertaken include: 1. Fission physics research to develop NDA technology based on correlated gamma and neutron data for assay of high activity wastes. 2. Radiation transport analysis to support development of quantitative prompt gamma neutron activation analysis and gamma ray transport analysis for simple gamma assay systems. 3. Development of gamma and neutron based down-hole probes for subsurface characterization. 4. Development of accelerator based, high-energy x -ray fluorescence to support subsurface contaminant modeling. 5. NDA applied to radionuclide characterization of TRU waste. 6. Validation of NDA characterization data for waste containers. This paper presents an overview and discusses the application and benefits of these activities

The effect of geometry on ice shelf ocean cavity ventilation: a laboratory experiment

A laboratory experiment is constructed to simulate the density-driven circulation under an idealized Antarctic ice shelf and to investigate the flux of dense and freshwater in and out of the ice shelf cavity. Our results confirm that the ice front can act as a dynamic barrier that partially inhibits fluid from entering or exiting the ice shelf cavity, away from two wall-trapped boundary currents. This barrier results in a density jump across the ice front and in the creation of a zonal current which runs parallel to the ice front. However despite the barrier imposed by the ice front, there is still a significant amount of exchange of water in and out of the cavity. This exchange takes place through two dense and fresh gravity plumes which are constrained to flow along the sides of the domain by the Coriolis force. The flux through the gravity plumes and strength of the dynamic barrier are shown to be sensitive to changes in the ice shelf geometry and changes in the buoyancy fluxes which drive the flow