Engineering synthetic pathways for adipic acid biosynthesis

Utilization of petroleum in consumer product manufacturing is causing irreversible environmental damage. Its impact on land, sea, and air calls for the development of more sustainable technologies based on the use of renewable materials such as lignocellulosic biomass and its conversion into platform chemicals. Engineering microorganisms to produce chemicals is an important undertaking to address such issues and bio-based production of adipic acid especially has gained recent attention. In the present thesis I assess the in vivo and in silico action of enzymes involved in microbial production of adipic acid from simple sugar molecules. The aim of this work was to comprehensively map out the metabolic pathways leading to adipic acid biosynthesis and to investigate the enzymatic components of the L-lysine pathway, the reverse β-oxidation pathway, and cis,cis-muconic acid reduction.Investigation of theoretical and in silico aspects in the deamination step in the L-lysine pathway revealed deamination of L-lysine was determined to be chemically difficult to occur. Removal of the β-amino group from β-D-lysine was deemed more feasible than the α-amino group from L-lysine, and an alternative route via β-D-lysine deamination was suggested. Homology modeling and molecular docking studies shed light on the substrate binding mechanisms of enzymes responsible for the reduction of the intermediates in the L-lysine pathway. Potential mechanism and feasibility of α,β-reduction were explained in terms of substrate interaction in the enzyme-binding pockets. Corynebacterium glutamicum was chosen as the host chassis for achieving adipic acid synthesis via reverse β-oxidation. Stepwise construction of a five-step synthetic pathway demonstrated functionality of each step in C. glutamicum. Biosynthesized and secreted 3-hydroxyadipate was detected in the cultivation broth using GC/MS. Weak trans-2-hexenedioic acid and adipic acid signals was observed using LC/MS after concentrating the cultivation broth. Dehydration of 3-hydroxyadipyl-CoA was identified as a potential bottleneck hindering this pathway. While implementing the reverse β-oxidation pathway, a new pathway involving cis,cis-muconic acid and 3-oxoadipic acid was observed and experimented on. The modified strategy for bio-conversion of benzoic acid to cis,cis-muconic acid was successful and molecular docking studies were carried out to better understand how oxidoreductases might reduce cis,cis-muconic acid.Taking multiple approaches to generate adipic acid revealed different challenges in each pathway. One approach led to biosynthesis of adipic acid. Further investigation will allow multiple options for bio-based adipic acid production for better sustainability

Low-cost, open-source contact angle analyzer using a mobile phone, commercial tripods and 3D printed parts

Measurement of contact angle is important in many areas of science and engineering research. Contact angle analyzers are however not easily accessible due to their expensive cost, which hinders their use in research and also in education. In this study we propose a low-cost contact angle analyzer that can be assembled with 3D printed parts. Mobile phone is used for imaging, and the image is analyzed using an open-source ImageJ plugin. Commercial camera tripods are used as platform that provides movement in many degrees of freedom, which are important in leveling of the substrate and proper imaging of droplets. We utilize the tripods to build imaging modules, sample plate module and volume metering module, each of which perform distinct tasks. Especially, we characterize the usefulness of the volume metering module, which helps users dispense same volume of liquid to reduce human error during measurement. The cost of an analyzer is $255.10, which is an order of magnitude lower compared to commercial products. With the advancement in open source software and upgrades in the hardware modules, we expect that the proposed contact angle analyzer to have a positive impact in resource limited research labs and educational environments

DNA unwinding assay using streptavidin-bound oligonucleotides

BACKGROUND: Helicases play essential roles in many cellular processes including replication, transcription and translation. Most helicases translocate along one strand of the duplex while displacing the complementary strand (of either DNA or RNA). Thus, helicases have directionality. They move along nucleic acids in either the 3'→ 5' or 5'→ 3' direction. The directionality of helicases with low activity or of those that cannot initiate duplex unwinding from a substrate that contains only one single-stranded overhang region is difficult to determine. RESULTS: An improved assay to determine helicase directionality was developed that uses a substrate containing biotinylated oligonucleotides. As a proof of concept, it was shown that the substrates substantially improve helicase activity and directionality determination for several DNA helicases in comparison to more traditional substrates. In addition, a universal substrate that can be used to determine the directionality of both 3'→ 5' and 5'→ 3' helicases was developed. CONCLUSION: It is shown here that the use of a biotin-streptavidin complex as a helicase substrate improves helicase activity and the determination of helicase directionality. The method described is simpler that the currently available techniques

PDB-Ligand: a ligand database based on PDB for the automated and customized classification of ligand-binding structures

PDB-Ligand (http://www.idrtech.com/PDB-Ligand/) is a three-dimensional structure database of small molecular ligands that are bound to larger biomolecules deposited in the Protein Data Bank (PDB). It is also a database tool that allows one to browse, classify, superimpose and visualize these structures. As of May 2004, there are about 4870 types of small molecular ligands, experimentally determined as a complex with protein or DNA in the PDB. The proteins that a given ligand binds are often homologous and present the same binding structure to the ligand. However, there are also many instances wherein a given ligand binds to two or more unrelated proteins, or to the same or homologous protein in different binding environments. PDB-Ligand serves as an interactive structural analysis and clustering tool for all the ligand-binding structures in the PDB. PDB-Ligand also provides an easier way to obtain a number of different structure alignments of many related ligand-binding structures based on a simple and flexible ligand clustering method. PDB-Ligand will be a good resource for both a better interpretation of ligand-binding structures and the development of better scoring functions to be used in many drug discovery applications

Synthesis and characterization of nitric oxide-releasing silica materials for sensing applications

Nitric oxide (NO), a free radical endogenously synthesized in the human body, regulates a range of biological processes in the cardiovascular, genitourinary, respiratory, and nervous systems. With the discovery of NO as a potent inhibitor of platelet activation and its identification as an antibacterial agent, the utility of NO has been expanded to developing more biocompatible materials that resist to biofouling. My research has focused on the development of NO-releasing glucose biosensors via xerogel/polyurethane hybrid membranes that release NO in a controlled fashion. This new class of glucose biosensors was characterized by both adequate analytical response to glucose and improved bacterial adhesion resistance at NO fluxes greater than or equal to 5 pmol·cm-2·s-1 for 20 h. To further elucidate the complex and wide ranging roles of NO in physiology, an amperometric xerogel-based NO microsensor was fabricated. Several silicon-based NO sensor membranes were synthesized by doping alkyl/amino-alkoxysilane-based xerogels with Nafion. The performance of xerogel-based NO sensors was then evaluated to identify the optimum xerogel composition that maximized NO permeability and provided sufficient selectivity for NO. Xerogel permeability and selectivity were further manipulated as a function of specific reaction/processing conditions. The analytical performance of the xerogel-based sensor far exceeded that of current commercial sensors. Silica nanoparticles capable of controlled NO release were also synthesized via sol-gel chemistry. Control over both the structure and concentration of the silane precursors (i.e., tetraalkoxy- and aminoalkoxysilanes) and the synthetic conditions used to prepare the silica allowed for the preparation of NO donor-modified silica nanoparticles of widely varying size (d = 20 - 500 nm), NO payloads (50 - 74000 nmol·mg-1), maximum NO release amounts (10 - 103000 ppb·mg-1), half-lives (0.1 - 12 h), and NO release durations (up to 30 h). Nitric oxide-releasing silica nanoparticles may prove useful in the development of glucose sensors with extended NO release durations and as new NO-derived anti-tumor chemotherapeutics. Preliminary studies evaluating the anti-cancer efficacy of NO-releasing silica nanocom-posites against human ovarian surface epithelial (HOSE) cancer cells were conducted. The viability of HOSE cancer cells was significantly reduced upon exposure to the NO-releasing silica nanoparticles. Such scaffolds may allow for future tissue/cell targeting via particle size (enhanced permeability/retention effect) and/or ligand-receptor binding chemistry

Use of a restriction enzyme-digested PCR product as substrate for helicase assays

DNA helicases play essential roles in many cellular processes. The currently available techniques to generate substrates for helicase assays are fairly complicated and need some expertise not available in all laboratories. Here, a PCR-based method to generate a substrate for a helicase assay is described, and its application for several archaeal, bacterial and viral enzymes is demonstrated

Delayed presentation of cerebellar and spinal cord infarction as a complication of computed tomography-guided transthoracic lung biopsy: a case report

INTRODUCTION: Computed tomography-guided transthoracic needle biopsy is a common diagnostic procedure that is associated with various complications including pneumothorax, parenchymal hemorrhage, and hemoptysis. A systemic air embolism is a very rare (0.06 to 0.21%) but potentially fatal complication. CASE PRESENTATION: A 70-year-old Korean male was admitted to our hospital for evaluation of a solitary pulmonary nodule located adjacent to the right inferior pulmonary vein in the medial basal segment of the right lower lobe. A computed tomography-guided needle biopsy was performed by a radiologist using a coaxial needle. A computed tomography image obtained immediately after the biopsy showed intraluminal free air in the proximal ascending aorta. He complained of a mild electrical current sensation in both lower extremities. After three hours he complained of neurological deficit in both lower extremities as well as voiding difficulty. The brain and spine magnetic resonance images showed a right cerebellar and spinal cord infarction at the T8-10 levels. CONCLUSIONS: We report a case of air embolism to the cerebellum and spinal cord causing infarction presenting with an initial symptom of mild electrical current sensation in both lower extremities during the transthoracic needle biopsy. For this potentially fatal complication, early recognition, followed by prompt therapy is critical to reducing morbidity and mortality

Protein kinase CK2 phosphorylates and activates p21-activated kinase 1

Activation of the p21-activated kinase 1 (PAK1) is achieved through a conformational change that converts an inactive PAK1 dimer to an active monomer. In this paper, we show that this change is necessary but not sufficient to activate PAK1 and that it is, rather, required for CK2-dependent PAK1S223 phosphorylation that converts a monomeric PAK1 into a catalytically active form. This phosphorylation appears to be essential for autophosphorylation at specific residues and overall activity of PAK1. A phosphomimetic mutation (S223E) bypasses the requirement for GTPases in PAK1 activation, whereas the constitutive activity of the PAK1 mutant (PAK1H83,86L), postulated to mimic GTPase-induced structural changes, is abolished by inhibition of S223 phosphorylation. Thus, S223 is likely accessible to CK2 upon conformational changes of PAK1 induced by GTPase-dependent and GTPase-independent stimuli, suggesting that S223 phosphorylation may play a key role in the final step of the PAK1 activation process. The physiological significance of this phosphorylation is reinforced by the observations that CK2 is responsible for epidermal growth factor–induced PAK1 activation and that inhibition of S223 phosphorylation abrogates PAK1-mediated malignant transformation of prostate epithelial cells. Taken together, these findings identify CK2 as an upstream activating kinase of PAK1, providing a novel mechanism for PAK1 activation

Low energy proton-proton scattering in effective field theory

Low energy proton-proton scattering is studied in pionless effective field theory. Employing the dimensional regularization and MS-bar and power divergence subtraction schemes for loop calculation, we calculate the scattering amplitude in 1S0 channel up to next-to-next-to leading order and fix low-energy constants that appear in the amplitude by effective range parameters. We study regularization scheme and scale dependence in separation of Coulomb interaction from the scattering length and effective range for the S-wave proton-proton scattering.Comment: 23 pages, 6 eps figures, revised considerably, accepted for publication in Phys. Rev.