研究業績集

タイトル

Building a model system for development of enhanced biohydrogen production

発表者

Patrik R. Jones

学会名

Asian Biohydrogen Symposium

内容

Microbial fermentation is a potentially promising method for renewable production of hydrogen. A major factor currently limiting commercial viability of hydrogen production is poor stoichiometric yield per mole of sugar. Although several approaches have been tried to enhance microbial hydrogen productivity, genetic engineering technologies for most promising microorganisms are either unavailable or severely limited. Engineering of native H2-producing microorganisms is further limited by narrow or undefined substrate specificities and directionalities of most endogenous H2-pathways, setting an upper absolute limit to achievable H2-yield well below theoretical potential. An alternative approach instead is to choose an engineerable host, without regard for native H2-synthesis capabilities. Hydrogenases and pathways channelling electrons between carbon catabolism and hydrogenases therefore need to be introduced. However, mere introduction of new pathways is unlikely to be sufficient, as native host metabolism also will have to be engineered to favour the new pathways. Ultimately, we envision the following five-step process in order to generate H2-producing strains with a production capability that is superior to existing genetically unmodified strains;
(i) Introduction of recombinant hydrogenases, (ii) Introduction of recombinant oxidoreductases and electron acceptor/donors, (iii) Modification of native metabolism to redirect flux through introduced pathways, (iv) Combination of multiple pathways and engineering strategies for redirecting native metabolism, and (v) Transfer to alternative hosts or additional modifications to convert laboratory model strains to industrially applicable strains.
I present a review of current work in my group aimed at developing a model system for improving microbial H2-production by genetic engineering based on the above principles. This includes the description of an experimental verification of thermodynamic limits imposed upon synthetic NAD(P)H:H2-pathways and a novel approach for enhancing hydrogenase activity and hydrogen production in the presence of oxygen.

タイトル

Modular architecture of protein structures and allosteric communications: potential implications to signaling proteins and regulatory linkages

発表者

Antonio del Sol1

学会名

2007.01.22
5th European Conference on Computational Biology

内容

In recent years, the number of proteins displaying modular allosteric mechanisms has grown. The modular configuration allows signaling proteins to generate new regulatory linkages. We show that protein domains consist of modules interconnected by residues, which mediate signaling through the shortest pathways. The mediating residues tend to be located at the inter-modular boundaries. The boundaries are more rigid and display a larger number of long-range interactions compared to intra-modular regions. Our approach relies on a representation of protein structures as residue interacting networks, and removal of the most central residue contacts, which are assumed to be crucial for the allosteric communications. The analysis of 13 allosteric proteins unveiled that modules characterize experimentally identified functional regions. Based on the study of an additional functionally-annotated dataset of 115 proteins, we propose that high modularity modules include functional sites and are the basic functional units. We provide examples (the G subunit and Cytochrome P450eryF) illustrating that the modular architecture of active sites is linked to their functional specialization. A modular configuration is advantageous: it would allow signaling proteins to expand their regulatory linkages and may elicit a broader range of control mechanisms either via modular combinations or through modulations of inter-modular linkages.

タイトル

Multifunctional Bio-Nano Patterns Derived from Colloidal Self-Assembly as Model Surfaces to Study Antigen-Antibody Interactions.

発表者

S. Krishnamoorthy

学会名

NSTI Nanotech 2007 (Oral Presentation)

内容

Nanosphere lithography (NSL) has become a popular tool as a patterning technique recently because the method is cheap and involves extremely simple procedures compared with other nanopatterning techniques. NSL can also be applied to a wide range of organic and inorganic materials. If combined with selective self-assembly of organic monolayers, NSL provides a feasible tool for the bio-functionalization of substrates as future biosensors or other bio-mimetic devices. This paper presents a simple and effective means of preparing nanopatterns made of organic self-assembled monolayers on large areas by means of NSL and exploring the capability of these surfaces for oriented immobilization of antibodies. The activity of antibodies on the chemically patterned surface is compared with their un-structured counterparts, and the influence of surface topography and chemistry on the activity of the surfaces towards ELISA assays is presented. The antibody immobilization, orientation and the surface activity were followed using UV/visible, FT-IR, and surface plasmon resonance spectroscopies.

タイトル

Metabolic engineering of NAD(P)H-dependent H2 synthesis in Escherichia coli

発表者

Andrea Veit

学会名

Gordon Research Conference for Applied and Environmental Microbiology

内容

Hydrogen is a promising energy carrier for both mobile and stationary applications in the future. H2 can be produced by micro organisms using biomass as a substrate. Fermentative H2 production is characterized by relatively poor yields per substrate and sensitivity to end-product accumulation. Although complete oxidation of glucose in theory yields 12 moles H2 per mole of glucose, only a maximum threshold of 4 moles of H2 is currently obtained by known pathways. Microbial H2 production with a yield above 4 H2 per mole of glucose has not yet been characterized to the stage that the process is understood. In our study we investigated the thermodynamic limitations of NAD(P)H dependent H2 synthesis and evaluated its potential for further engineering towards eventual commercial application. As micro-organisms with native NAD(P)H:H2 pathways are either not genetically tractable and/or contain multiple H2 synthesis/consumption pathways, we chose a novel approach: Instead of investigating H2 metabolism in a native H2 producing micro -organism, we engineered a synthetic H2 synthesis pathway consisting of a ferredoxin-dependent hydrogenase, three hydrogenase maturation factors, a [4Fe4S]-ferredoxin as intermediate electron acceptor/donor and ferredoxin-dependent oxidoreductases with defined specificity for either NADPH or NADH and NADPH. Co-expression of the pathway components in E. coli resulted in functional implementation of NADPH-dependent H2-synthesis. When the substrate specificity was extended to NADH under closed batch conditions only the reverse pathway, H2-consumption, was observed. By testing the thermodynamic limitations, we could show that nucleotide pyridine dependent H2 formation is highly sensitive to end-product accumulation.

タイトル

Modulating expression of the E. coli Isc-operon enhances Fe-Fe hydrogenase activity in the presence of oxygen

発表者

Mohammed Kalim Akhtar

学会名

The 8th International Hydrogenase Conference

内容

For the majority of known Fe-only hydrogenases, assembly and insertion of as many as five Fe-S clusters are required to ensure functional activity. Considering the unusually high demand of Fe-S clusters for the synthesis of C. acetobutylicum HydA, we speculated whether Fe-S cluster synthesis and assembly limited the ability of E. coli to synthesize active HydA. To test this hypothesis, the gene products of the isc operon, (IscS, IscU, IscA, HscA, HscB and Fdx) were overexpressed by plasmid-based induction or chromosomal in-frame deletion of the Isc transcriptional regulator, IscR. A 2- to 9- fold reduction in specific HydA activity was observed under strictly anoxic conditions with Isc overexpression. In contrast, under partially oxic conditions, the specific and total activity of Fe-only hydrogenase was increased by 5- and 65-fold, respectively, with a concomitant 3- to 18-fold increase in HydA protein yields. Given the vulnerability of Fe-S clusters toward oxidative damage, the enhanced effect, observed under oxic conditions, with Isc coexpression strongly suggests the possibility of a repair-mediated effect of the ISC machinery. Such a proposed role of the ISC machinery is currently under investigation. Furthermore, these findings also demonstrate an alternative strategy in sustaining hydrogenase activity in an oxic environment.