Development of metabolic pathways for the biosynthesis of hydroxyacids and lactones

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, February 2010.Cataloged from PDF version of thesis.Includes bibliographical references (p. 178-210).In this thesis, metabolic routes were developed for the production of hydroxyacids and their lactones in multiple microbial systems. These compounds see widespread use in the production of pharmaceuticals, polymers, and fine chiral intermediates. First in this thesis, strategies and tools for metabolic pathway design are discussed. This is followed by the descriptions of and data for each microbial production system. The compounds produced in this thesis and their highest titers obtained are shown below: 3-Hydroxybutyrate (3HB) 2.9 g/L 3-Hydroxyvalerate (3HV) 5.3 g/L 4-Hydroxyvalerate (4HV) 27.1 g/L 4-Valerolactone (4VL) 8.2 g/L 3,4-Dihydroxybutyrate (DHBA) 3.2 g/L 3-Hydroxybutyrolactone (3-HBL) 2.2 g/L The production of the two hydroxyvalerates was accomplished through the reduction of the renewable substrate levulinate in Pseudomonas putida by endogenous host enzymes followed by the liberation of the hydroxyvalerate product through the recombinant expression of thioesterase B (tesB). The production of 4VL was accomplished from levulinate by adding the lactonase paraoxonase I (PON1) to the P. putida hydroxyvalerate production system. Because 4VL was found to exist in a pH-dependent equilibrium with 4HV, the lactonase was expressed extracytosolically in acidic media to achieve significant titers of 4VL. The addition of a second resin phase to 4VL-producing cultures with a high affinity for 4VL substantially enhanced lactone production. 3HB, 3HV, DHBA, and 3-HBL were all produced in Escherichia coli through the expression of an acetoacetyl-CoA thiolase (thil, bktB, or phaA), a 3-hydroxybutyryl-CoA reductase (phaB or hbd), and tesB.(cont.) Supplying glucose to E. coli expressing these enzymes resulted in 3HB production, while supplying glucose and propionate results in 3HV production. Supplying glucose and glycolate resulted in DHBA production with some 3-HBL, but only with the help of a fourth gene - propionyl-CoA transferase (pct). Removing the tesB gene from this four-gene system substantially increases 3-HBL titers at the expense of DHBA. This work represents the first successful production of DHBA and 3-HBL in a biological system from carbohydrate-based substrates. In each of these systems, several broadly-applicable tools and strategies were developed to enhance product titer or discover new metabolic activities. In the P. putida system, cytosolic and extracytosolic biocatalysis were combined in a single metabolic pathway to realize lactone production. This catalytic strategy, termed integrated bioprocessing, is applicable to other metabolic pathways whose production suffers due to a suboptimal cytosolic enzyme. Also in the P. putida system, two-phase cultures were used to sequester the lactone product away from the lactonase, helping to drive lactonehydroxyacid equilibrium towards the lactone. This methodology allows one to overcome equilibrium-based limitations of product titer. Finally in the E. coli work, a promiscuous pathway normally used for polyhydroxyalkanoate synthesis was exploited to give a wide range of hydroxyacid products. This substrate promiscuity was critical in achieving the production of new compounds biologically and thus substrate promiscuity was identified as a key component for metabolic pathway design and construction.by Collin H. Martin.Ph.D

Biosynthesis of chiral 3-hydroxyvalerate from single propionate-unrelated carbon sources in metabolically engineered E. coli

Background The ability to synthesize chiral building block molecules with high optical purity is of considerable importance to the fine chemical and pharmaceutical industries. Production of one such compound, 3-hydroxyvalerate (3HV), has previously been studied with respect to the in vivo or in vitro enzymatic depolymerization of biologically-derived co-polymers of poly(3-hydroxybutyrate-co-3-hydroxyvalerate). However, production of this biopolymeric precursor typically necessitates the supplementation of a secondary carbon source (e.g., propionate) into the culture medium. In addition, previous approaches for producing 3HV have not focused on its enantiopure synthesis, and thus suffer from increased costs for product purification. Results Here, we report the selective biosynthesis of each 3HV stereoisomer from a single, renewable carbon source using synthetic metabolic pathways in recombinant strains of Escherichia coli. The product chirality was controlled by utilizing two reductases of opposing stereoselectivity. Improvement of the biosynthetic pathway activity and host background was carried out to elevate both the 3HV titers and 3HV/3HB ratios. Overall, shake-flask titers as high as 0.31 g/L and 0.50 g/L of (S)-3HV and (R)-3HV, respectively, were achieved in glucose-fed cultures, whereas glycerol-fed cultures yielded up to 0.19 g/L and 0.96 g/L of (S)-3HV and (R)-3HV, respectively. Conclusions Our work represents the first report of direct microbial production of enantiomerically pure 3HV from a single carbon source. Continued engineering of host strains and pathway enzymes will ultimately lead to more economical production of chiral 3HV.Synthetic Biology Engineering Research CenterNational Science Foundation (Grant EEC-0540879)Massachusetts Institute of Technology. Energy InitiativeShell Oil Compan

The X-ray spectrum of a disk illuminated by ions

The X-ray spectrum from a cool disk embedded in an ion supported torus is computed. The interaction of the hot ions with the disk increases the hard X-ray luminosity of the system}. A surface layer of the disk is heated by the protons from the torus. The Comptonized spectrum produced by this layer has a shape that depends only weakly on the incident energy flux and the distance from the accreting compact object. It consists of a `blue bump' of unComptonized soft photons and a flat high energy tail, reminiscent of the observed spectra. The hard tail becomes flatter as the thermalization depth in the cool disk is increased. Further evidence for ion illumination are the Li abundance in the secondaries of low mass X-ray binaries and the 450 keV lines sometimes seen in black-hole transient spectra.Comment: 7p, to appear in Monthly Notice

BioMAJ: a flexible framework for databanks synchronization and processing

Large- and medium-scale computational molecular biology projects require accurate bioinformatics software and numerous heterogeneous biological databanks, which are distributed around the world. BioMAJ provides a flexible, robust, fully automated environment for managing such massive amounts of data. The JAVA application enables automation of the data update cycle process and supervision of the locally mirrored data repository. We have developed workflows that handle some of the most commonly used bioinformatics databases. A set of scripts is also available for post-synchronization data treatment consisting of indexation or format conversion (for NCBI blast, SRS, EMBOSS, GCG, etc.). BioMAJ can be easily extended by personal homemade processing scripts. Source history can be kept via html reports containing statements of locally managed databanks

Protecting nickel with graphene spin-filtering membranes: A single layer is enough

We report on the demonstration of ferromagnetic spin injectors for spintronics which are protected against oxidation through passivation by a single layer of graphene. The graphene monolayer is directly grown by catalytic chemical vapor deposition on pre-patterned nickel electrodes. X-ray photoelectron spectroscopy reveals that even with its monoatomic thickness, monolayer graphene still efficiently protects spin sources against oxidation in ambient air. The resulting single layer passivated electrodes are integrated into spin valves and demonstrated to act as spin polarizers. Strikingly, the atom-thick graphene layer is shown to be sufficient to induce a characteristic spin filtering effect evidenced through the sign reversal of the measured magnetoresistance.We acknowledge the Helmholtz-Zentrum-Berlin Electron storage ring BESSY II for provision of synchrotron radiation at the ISISS beamline and we thank the BESSY staff for continuous support of our experiments. R.S.W. acknowledges a Research Fellowship from St. John’s College, Cambridge. S.H. acknowledges funding from ERC grant InsituNANO (No. 279342) and EPSRC grant GRAPHTED (EP/K016636/1). P.S. acknowledges the Institut Universitaire de France for a junior fellowship. This research was partially supported by the EU FP7 Work Programme under Grant GRAFOL (No. 285275) and Graphene Flagship (No. 604391).This is the final published version. It first appeared at http://scitation.aip.org/content/aip/journal/apl/107/1/10.1063/1.4923401

A Structure for Quasars

This paper proposes a simple, empirically derived, unifying structure for the inner regions of quasars. This structure is constructed to explain the broad absorption line (BAL) regions, the narrow `associated' ultraviolet and X-ray warm absorbers (NALs); and is also found to explain the broad emission line regions (BELR), and several scattering features, including a substantial fraction of the broad X-ray Iron-K emission line, and the bi-conical extended narrow emission line region (ENLR) structures seen on large kiloparsec scales in Seyfert images. Small extensions of the model to allow luminosity dependent changes in the structure may explain the UV and X-ray Baldwin effects and the greater prevalence of obscuration in low luminosity AGN.Comment: 35 pages, including 8 color figures (figures 4abc are big). Astrophysical Journal, in press. Expanded version of conference paper astro-ph/000516

Slicing the Torus: Obscuring Structures in Quasars

Quasars and Active Galactic Nuclei (AGNs) are often obscured by dust and gas. It is normally assumed that the obscuration occurs in an oblate "obscuring torus", that begins at the radius at which the most refractive dust can remain solid. The most famous form of this torus is a donut-shaped region of molecular gas with a large scale-height. While this model is elegant and accounts for many phenomena at once, it does not hold up to detailed tests. Instead the obscuration in AGNs must occur on a wide range of scales and be due to a minimum of three physically distinct absorbers. Slicing the "torus" into these three regions will allow interesting physics of the AGN to be extracted.Comment: 8 pages, 1 figure. To appear in the proceedings of the conference "The central kiloparsec in Galactic Nuclei:Astronomy at High Angular Resolution 2011", open access Journal of Physics: Conference Series (JPCS), published by IOP Publishin

A human MAP kinase interactome.

Mitogen-activated protein kinase (MAPK) pathways form the backbone of signal transduction in the mammalian cell. Here we applied a systematic experimental and computational approach to map 2,269 interactions between human MAPK-related proteins and other cellular machinery and to assemble these data into functional modules. Multiple lines of evidence including conservation with yeast supported a core network of 641 interactions. Using small interfering RNA knockdowns, we observed that approximately one-third of MAPK-interacting proteins modulated MAPK-mediated signaling. We uncovered the Na-H exchanger NHE1 as a potential MAPK scaffold, found links between HSP90 chaperones and MAPK pathways and identified MUC12 as the human analog to the yeast signaling mucin Msb2. This study makes available a large resource of MAPK interactions and clone libraries, and it illustrates a methodology for probing signaling networks based on functional refinement of experimentally derived protein-interaction maps

Sensitivity of the IceCube Detector to Astrophysical Sources of High Energy Muon Neutrinos

We present the results of a Monte-Carlo study of the sensitivity of the planned IceCube detector to predicted fluxes of muon neutrinos at TeV to PeV energies. A complete simulation of the detector and data analysis is used to study the detector's capability to search for muon neutrinos from sources such as active galaxies and gamma-ray bursts. We study the effective area and the angular resolution of the detector as a function of muon energy and angle of incidence. We present detailed calculations of the sensitivity of the detector to both diffuse and pointlike neutrino emissions, including an assessment of the sensitivity to neutrinos detected in coincidence with gamma-ray burst observations. After three years of datataking, IceCube will have been able to detect a point source flux of E^2*dN/dE = 7*10^-9 cm^-2s^-1GeV at a 5-sigma significance, or, in the absence of a signal, place a 90% c.l. limit at a level E^2*dN/dE = 2*10^-9 cm^-2s^-1GeV. A diffuse E-2 flux would be detectable at a minimum strength of E^2*dN/dE = 1*10^-8 cm^-2s^-1sr^-1GeV. A gamma-ray burst model following the formulation of Waxman and Bahcall would result in a 5-sigma effect after the observation of 200 bursts in coincidence with satellite observations of the gamma-rays.Comment: 33 pages, 13 figures, 6 table