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Global interest in microalgae cultivation continues to escalate in response to the world’s quest for sustainable biofuel feedstocks, protein feeds and nutraceuticals. However, significant challenges remain in the large scale photoautrophic production of microalgal biomass at reasonable cost, and multifarious threats exist to achieving consistent, robust cultivation systems in an integrated technology line-up. Emphasis must be placed on the generation of intracellular lipid biofuel precursors, protein, and high value products, such as omega-3 fatty acids, if the techno-economics are to be viable for commercialization of the technology. Adopting a biorefinery approach to microalgae cultivation must be emphasized, as well as application of advanced biotechnologies to boost productivity. Techniques such as the use of fixed-carbon sources in a mixotrophic system, and the use of cell selection techniques such as flow cytometry for improved strain phenotypes will be highlighted.

In the first part of talk, I will present biophotonic nanosatellites that have multiple functions: targeting, imaging, gene delivery & regulationsin living systems. For the remote optical control of gene regulations and therapeutic applications, we have developed Oligonucleotides on a Nanoplasmonic Carrier Optical Switch (ONCOS). We also accomplished photonic gene circuits thatutilized RNA-medicated gene silencing to multistep bidirectional control of specific gene expression by taking advantage of the tunability of the longitudinal plasmon resonance wavelength in gold optical nanoantennas.In the second part of talk, I will discuss (1) Cellular BASICs (Biologic Application Specific Integrated Circuits) for single stem cell analysis, dynamic cell culture array, and self-powered integrated microfluidic blood analysis system;(2) Biologically inspired 3D tissues on chip: disease-specific integrated microphysiological human tissue models via normal and patient-specific human induced pluripotent stem cells for drug development and safety; (3) Integrated Molecular Diagnostic Systems (iMDx) for global healthcare. In summary, I will share my vision for the convergence of science, engineering, and medicine to transform life sciences, and finding the solutions for preventive personalized medicine and low-cost healthcare systems.

Biodiesel production from microalgae has been recently expected to replace the petroleum-based energy sources owing to its high areal productivity and lipid contents. However, large-scaled algae cultivation for biodiesel production should be significantly improved to compete with the cost of present fossil-based energy. In order to reduce the input cost for microalgal cultivation, we investigated the utilization of blended wastewaters taken from domestic and livestock wastewater treatment plants (WWTP). A Chlorella species isolated from Nacdong River effectively utilized the nutrients dissolved in the blended wastewaters. After 10 days cultivation under 200 μmol/m2/s cool-white fluorescent light and 25±1 ℃, significantly high maximum biomass production of c.a. 5.0 g-dry cell weight/L was obtained from the combined wastewater of a domestic WWTP influent and the livestock WWTP effluent without any pretreatment of those wastewaters. The optimal concentration range of the total nitrogen was 100-200 mg/L. These results demonstrated that blended wastewaters would be utilized for mass cultivation of microalgae as both water and nutrient sources.

Since Korea is highly relying on imported fossil fuels for transportation, sourcing alternative renewable and eco-friendly transportation fuel remains highly pertinent. Long been microalgae are looked at as a suitable source of alternatives for fossil fuel and efficient CO2 mitigators. In spite of the remarkable advantages, microalgal biodiesel production still remains an expensive technology, and commercially viable large -scale algal production has yet to be achieved.This study focused on mass-cultivation of oleaginous microalgae under autotrophic and mixotrophic conditions with low-cost photobioreactors. Several species of microalgae from natural environments and microalgal culture bank were screened for their ability to grow autotrophically and mixotrophically (glucose/glycerol) with high CO2 concentrations (often 10% and coal-fired flue gas). As a result, a few microalgae spp. were identified with high growth rates and lipid content (~40 % to their dry cell mass). When the selected isolates were grown in 1 L bubble column under different nutritional modes, mixotrophic mode yielded the maximum growth when compared to the autotrophic or heterotrophic mode of cultivation. Several analyses are being instigated for detailed characterization of the cultivation of microalgal isolates, which includes; 18S rRNA gene sequencing, changes in pH and optical density of the culture, microscopic analyses (light microscopy, SEM and TEM analysis), estimation of lipid content, characterization of Fatty Acid Methyl Ester (FAME) profiles, estimation of carbohydrates, protein and chlorophyll, measurement of cell size/numbers, rate of glucose/glycerol consumption, rate of nitrate, phosphate and CO2 consumption, production of organic acid metabolites, and Denaturing Gradient Gel Electrophoresis (DGGE) for community analysis of epiphytic bacteria associated with microalgae in culture. For mass cultivation studies, photobioreactors were designed with transparent films and various prototypes of working volume 3 to 30 L are being evaluated for their efficiency to utilize CO2 and flue gas from a 2 MW coal power plant under indoor and outdoor conditions. This talk would discuss the recent experimental data and future directions of the study.

The demand for the biomass-based drop-in fuels and chemicals that can replace the existing petrochemical products has encouraged policy makers throughout the world to introduce new legal mandates and researchers to identify ground-breaking solution to the challenging problems. Korea is not an exception to this new challenge and has been actively establishing national agenda such as Green Technology Policy and Biomass-based Energy Technical Roadmap, and investing heavily in R&D efforts to produce biofuels and chemicals using cellulosic and microalgal biomass. The Advanced Biomass R&D Center (ABC) is the largest research consortium (US$110 Million) in Korea that focuses its efforts to develop break-through basic and applied research and engineering solutions to produce the biomass-based biofuels economically and sustainably. Over 300 active participants work together to devise scientific and engineering solutions to solve the challenges that are associated with the production of biomass-based fuels and chemicals. In the present talk, the current policy and funding strategies in Korea in biomass-based alternative energy, and the recent technical progress that has been made in ABC will be discussed. (This work was supported by the Advanced Biomass R&D Center (ABC) of Korea Grant funded by the Ministry of Education, Science and Technology (ABC-2011-K000908).

We are in the process of selecting elite lines of Korean domestic microalgae for mass cultivation and biodiesel production in Korea. Twenty microalgal strains from various domestic streams and lakes were collected, axenically purified, and tested for physiological characteristics and lipid production. Many microalgae were able to autotrophically synthesize hydrocarbon chains such as pentadecane (C15H34) and heptadecane (C17H36), which can be directly used as fuel without requiring a transesterification step. Consequently, the genes involved in alkane biosynthesis such as an aldehyde decarbonylase and an acyl-acyl carrier protein (ACP) reductase were present in this microalgae. Genetic clonings and characterization of those domestic genes are currently underway. In addition, a high value fatty alcohol such as hexadecenol (C20H40O) was present in this photosynthetic microorganism. Other common algal biodiesel constituents, palmitic acid (C16:0), palmitoleic acid (C16:1), linoleic acid (C18:2) and linolenic acid (C18:3) were also produced by many strains as major fatty acids. Therefore, numerous domestic microalgae appear to show promise for their application in cost-effective production of microalgae-based biofuel and high-value products. Also we are investigating Korean type microalgae mass cultivation system to grow and induce lipids in various conditions. Over 100-ton raceways have been operated by our laboratory for field cultivation and currently approximately 1000-ton raceways are under construction. A few microalgal strains that are suitable for field mass-cultivation were selected.

In the present study, spent yeast from a brewery was used as the growth substrate for the docosahexaenoic acid (DHA)-rich microalga, Aurantiochytrium sp. KRS101. A significant biomass yield (6.69 g/l/d) was obtained using only spent yeast as the growth substrate, with simple stirring as pretreatment. Maximization of nutrient utilization through the use of stepwise cultivation increased the yield to 31.8 g/l of biomass. DHA constituted 38.2% (w/w) of the total fatty acids, and the highest DHA productivity was observed when the C/N ratio was 20:1 (w/w). Spent yeast thus served as a good growth substrate for the production of DHA. Economic assessment revealed that stepwise cultivation using spent yeast as either the sole growth substrate or as a nutrient source could substantially reduce the production cost of microalgal DHA. (This work was supported by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Ministry of Education, Science and Technology. ABC-2010-0029728)

Biofuel has received a lot of attention as a main viable technology that can reduce the greenhouse gas (GHG) emissions and replace petroleumbased transport fuel. It has been commonly known to be sustainable and carbon-neutral because it can be produced from biomass, which is plentiful and absorbs CO2 to grow through photosynthesis. However, some biomass sources are evaluated to show limitations in aspects of sustainability. Especially, the high demand of food-derived biofuels may have been associated with the recent hikes in food prices, some adverse impacts on environment and issues of land use change. Under these conditions, Global Bioenergy Partnership makes efforts to promote environmentally-friendly biofuel production and use through the development of “Sustainability standards”, which contains some criteria such as GHG emissions, net energy balances, biodiversity, food security, and socioeconomic impacts. Korean government introduced Renewable Fuel Standard (RFS) in 2012. It is required to develop more specified RFS that considers the sustainability issues of the biofuel and reflects the levels of life-cycle GHG reductions of each biomass.

Over years, cyanobacteria have been regarded as ideal model system for studying fundamental biochemical processes like oxygenic photosynthesis, carbon and nitrogen assimilation. They have been also used as human foods, sources for vitamins, proteins, fine chemicals, and bioactive compounds. Additionally, aiming to increase plant productivity as well as nutritional values, cyanobacterial genes involved in carbon metabolism, fatty acid biosynthesis, and pigment biosynthesis have been intensively exploited as alternatives to homologous gene sources (Park et al.. 2009). Cyanobacterial photosynthesis is currently drawing strong interests among bioenergy society as model organisms for developing ideal oil algae with various characteristics including high light and antibiotic resistance, and inducible oil secretion and flocking, etc (Machado and Atsumi, 2012). In this talk, I’ll introduce on-going researches related to functional genomics approach to the regulation of photosynthesis by means of signal transduction networks to various environmental cues and metabolic engineering to manipulate assimilated carbon partitioning between carbohydrate and lipid in the oxygenic cyanobacterium Synechocystis sp. PCC 6803.

Conversion of microalgae to biodiesel typically includes the following four steps: microalgae cultivation, cell harvesting, lipid extraction, and biodiesel conversion. A number of methods can be used to achieve lipid extraction from microalgae, including solvent extraction, cracking, supercritical extraction, pyrolysis, enzymatic hydrolysis, and osmotic shock. Many researchers have recovered microalgal lipids from dried microalgae. However, the lipid extraction from wet microalgae has to be developed because the dewatering process to obtain dried microalgae is costly. The studies on wet lipid extraction have been very limited. In this study, the feasibility of lipid extraction and biodiesel production from wet microalgae was investigated. The microalgal lipid was extracted from wet microalgae by hot-water pretreatment. To enhance the lipid extraction yield, chemicals were added to microalgal solution. The lipid extraction yields under various experimental conditions were examined. After that, the microalgal biodiesel was produced by transesterification using sulfuric acid-methanol solution. After distillation, light-yellow biodiesel was obtained from dark-green biodiesel.

Antifreeze proteins (AFP) have an ability to bind ice crystals and cause thermal hysteresis (TH) which lowers the freezing temperature without affecting the melting point. Thereby, AFPs have already been applied in various areas (food, medical, cryopreservation and cold hardness of crop plant). Immunolocalization, western blot and antifreeze activity assay clearly showed that antartic diatom AFPs were located in the intracellular area, apoplastic region near to the membrane and flagella. When, the AFPs activities were measured, the maximum TH values of recombinant diatom AFP was measured as 1.28℃ at the protein concentration of 10 mg/ml protein. To predict the ice binding site, 3-dimensional structure of diatom AFP was simulated in silico. Amino acid substitution by point mutations was performed to verify the putative ice-binding surface. Mutant diatom AFPs which had substituted amino acids of the predicted ice binding site revealed 10% TH activity of that of the wild type AFP and exhibited changed ice crystal form at various protein concentrations compared to that of the wild type AFP. The possible explanation of the ice binding mechanism and its biotechnological application will be discussed.

An indigenous crude extract was used to produce calcite from Canavalia ensiformis and its ability to precipitate calcite with urea and calcium chloride was investigated. X-ray diffraction and scanning electron microscopy were employed to elucidate the mechanism of calcite formation from the plant crude extracts. The results revealed that urease in the plant crude extracts catalyzed the hydrolysis of urea in liquid state cultures. The procedure described herein is a simple method of calcite biomineralization without cultivation of microorganisms or further purification of crude extracts. The unconfined compressive strength of a specimen injected once with Canavalia ensiformis extract showed a 10.6 fold increase when compared to an untreated specimen. In addition, the manufactured plant crude extracts showed significant heavy metal removal efficiency and acidified soil remediation ability in the mine impacted soil. This study showing successful biomineralization of calcite and soil remediation using indigenous Canavalia ensiformis crude extracts without further enzyme purification.

Lysosomes are heterogeneous cell organelles consisting of vacuoles that vary in size, form, and density, and lysosomes release lysosomal enzymes to digest intracellular and extracellular waste material. Lysosomal enzymes degrade bacteria cell walls, demonstrating the antimicrobial activity of lysosomes and they have anticancerous effect when mammalian cells progress apoptosis. Lysosomes are not silent organelles, but dynamic organelles in which lysosomal enzymes are easily integrated or released when exposed to stressful conditions, especially oxidative stress. Understanding in vitro lysosomal function in cells following hydrogen peroxide (H2O2), 6-hydroxydopamine (6-OHDA), or UVB irradiation may elucidate lysosomal mechanisms associated with oxidative stress and lysosomal activation. We exposed HeLa cell lines to three oxidative causing agents and isolated lysosomes from HeLa cells exposed to these stressors. It was found that the lysosomes have antimicrobial activity against seven different microorganisms, including E. coli, and oxidative stress-induced lysosomes showed enhanced antimicrobial activity compared to those from normal lysosomes. These results suggest the possibility that lysosomal alterations during starvation may induce conditions that activate lysosomes for future development of efficient antimicrobial agents. This study suggests a new methodology for enhancing lysosomal activity in a lysosome-source strain through special treatment, such as starvation.

This study will present efficient and environmental-friendly methods for the pretreatment of rice straw by using planetary or attrition mills. Both mill modes are effective to reduce size and cellulose crystallinity in rice straw. The relative crystallinity indexs were 0.20 and 0.69 for planetary milled and attrition milled samples. Enzymatic treatment of the milled rice straw produced both glucose and xylose. Total monosaccharides produced in enzymatic hydrolysis were 0.39 and 0.37 g/g rice straw for planetary and attrition mill treated, respectively. In comparison experiment to chemical pretreatments, none of the rice straw biomass was lost during the milling processes but it showed a 32.3 % and 10.3 % reduction in the SAA and NaOH pretreatments, respectively. Soaking the rice straw in both the aqueous ammonia and sodium hydroxide solutions led to the production of 0.95 and 2.34 g/l of freely soluble phenolic compounds, respectively. In contrast, the planetary and attrition mill pretreatments produced 3- to 9-fold less phenolics. To further investigate, the toxicity of each hydrolysate samples, a recombinant bacterium E. coli strain DH5α/pDMA3 was employed. The hydrolysates from the NaOH and SAA treatment elicited a 31-fold and 6-fold induction in bioluminescence while no induction was seen for both the planetary and attrition mills.

Metabolic processes in biological systems can largely be classified into primary and secondary metabolisms. Notably, via secondary metabolism, a wide variety of metabolites with low-molecular weight can be synthesized, some with potent physiological activities. Fungi or microalgae are of particular interest due to their capacity to produce an extensive array of secondary metabolites, which have tremendous importance for humans with beneficial (e.g., antibiotics) or detrimental (e.g., mycotoxins) properties (Demain & Fang, 2000). In this study, we utilized functional genomics, forward and reverse genetics, proteomics, and high efficiency homologous recombination, to better understand the complex secondary metabolism regulations in fungi. By doing so, several fungal mutant strains with significantly altered secondary metabolites could be identified and these strains will have a high potential for subsequent biotechnological applications. In microalgae, our results reveal that MAP kinase signaling pathways are highly conserved, since MAP kinase pathways tested in several microalgal species are all negatively implicated in carotenoid biosynthesis. Currently, we are striving to develop the promising microalgal strains and these strains will be utilized in downstream process based on photobioreactors. Some current research efforts in our research group are also focused on developing the appropriate microalgal cultivation strategy using LED light.

A microchip that integrates the sol-gel affinity column of a SELEX (Systematic Evolution of Ligands by Exponential Enrichment) system is developed. SELEX has been widely used for identification and characterization of interactions between aptamers and protein, peptides or chemical compounds. Aptamers, nucleic acid species that have been selected through repeated rounds of SELEX offer utility for biotechnological and therapeutic applications as they offer molecular recognition properties that rival that of the commonly used antibodies. To obtain a high-quality aptamer requires typically 8-12 rounds of SELEX, where each round includes both positive and negative selection. This can require a few to several weeks of effort. Therefore, multiplexing and more efficient SELEX technologies could significantly reduce the cost of generating these reagents. Our microchip includes electric heaters for individual heat elution from sol-gels which contain target molecules. To test the microfluidic system for SELEX compatibility, agrochemicals, azoxistrobin, was delivered, then binding aptamers were eluted from sol-gels and amplified by PCR. We also demonstrated the multiplex SELEX in this microfluidic system by adding pneumatic air valves between incubation chambers.We could specifically bind and elute the corresponding aptamers from each target embedded sol-gels. Comparing to the conventional SELEX, our microfluidic SELEX system improved cycle efficiency.

Bioelectrochemical systems (BES) use electrochemically active biofilm on the electrode as biocatalyst for electrical energy recovery and useful products. BES has been extensively investigated for application to recover bioenergy in the from of electricity and biohydrogen from biodegradable organic matter, biosensor, bioremediation and microbial desalination. The biofilm of BES can exchange electron with the electrode which makes different biological application and metabolic capability of attached microorganisms from the conventional biofilm process. The microbial oxidation and reduction can be combined with ion exchange capability by membrane and separator in the processes, therefore it might be able to facilitate production and separation in the same process. Recently, BES has also been designed to Microbial electrosynthesis (MES) which can reduce proton in cathode chamber into hydrogen and higher organic compounds such as volatile fatty acids and alcohol with power boost-up using external power supply. It was reported that the dual energy source in MEC using both electrical energy from bio- oxidation of organics and power supply can increase energy and coulombic efficiency to 80% and 92%, respectively. Furthermore, the BES concept recently has been introduced into biorefinery which synthesize chemicals by using microbial electron transport between electrode and live bacterial cell. In this process, the cathodic reduction is more focused than the anodic oxidation of organic matters. Alkaline and commodity chemicals such as methane, acetate, ethanol and buthanol can be produced by biotic cathodic reduction reaction using controlled poised potential. This report will investigate the recent findings of BES and its future application for useful product and biorefinery.

Due to the structural variability of hemicelluloses, a variety of enzymes is involved in their efficient degradation. The complete hydrolysis of hemicellulosic biomass can be achieved via the cooperative reactions of various enzymes including the main chain-cleaving endo- and/or exo-hydrolases and the side chain-removing accessory enzymes. For example, Thermotoga spp. are well-known hyperthermophiles which possess a number of carbohydrate-active enzyme genes, such as endo-xylanase, exo- xylosidase, α-L-arabinofuranosidase, and acetyl xylan esterase, in their genomes. Endo-xylanase can randomly cleave the main chain of xylans to produce a mixture of xylooligosaccharides, while the other accessory enzymes can remove each modified linkages in xylans to release xyloses, arabinoses or acetates. Meanwhile, some Bacillus spp. are known to share different sets of exo- and endo-hydrolase genes for the efficient degradation of arabinan polymers. Due to their unique action patterns, these series of enzymes act in concert to catalyze specific hydrolysis of various hemicellulosic polymers, including xylans and arabinans. In the present study, it is proposed that the simultaneous enzymatic treatments work synergistically and produce much higher amount of pentose sugars from various polymers. In addition, some versatile step-wise enzymatic processes are suitable for the cost-effective production of various oligosaccharide derivatives.

Sialylated sugar chains are present at the cell surface of various animal species. Due to their position, they are thought to serve important roles in a large variety of biological functions such as cell–cell and cell– substrate interactions, bacterial and virus adhesion, and protein targeting. We present a bio-conversion process for the conversion of N- acetylglucosamine and CMP into CMP-neuraminic acid with five enzymes. Key enzyme is N-acyl-D-glucosamine 2-epimerase from Baceroides fragilis. 2,3-Sialyllactose and 2,6-sialyllactose are synthesized by one-pot reaction from lactose, N-acetyl-glucosamine and CMP with CMP recycling. CMP-neuraminic acid was used to synthesize sialyllactose derivatives (vancomycin sialoside, biotin-sialoside, flavonoid sialoside and multi-ligand sialosides).

Recent advances in directed evolution techniques have enabled us to engineer biocatalysts for broadening their industrial uses. Nevertheless, most engineering strategies still require labor-intensive and complicated procedures to obtain tailor-made enzymes to meet industrial demands. Instead of conventional rational design and directed-evolution approaches, here we used a semi-rational approach based on amino acid sequence comparisons of homologous enzymes for engineering their intrinsic properties (i.e., pH optimum, thermostability, metal requirement etc.). Semi-rational ‘consensus concept’ led us to engineer the physicochemical properties of L-arabinose isomerases (AIs) for the production of a novel and natural sweetener, D-tagatose at elevated temperatures. Our case studies with a variety of AIs suggest that such a simple semi-rational strategy could be very effective and powerful for engineering AIs.

Human milk contains a large variety of oligosaccharides (HMOs) that have the potential to modulate the gut flora, affect different gastrointestinal functions, and influence inflammatory processes. This review introduces the recent advances in the microbial and coupled enzymatic methods to produce HMOs with grouping them into trisaccharides (sialyllactose and fucosyllactose) and complex oligosaccharides (lacto-Nbiosederivatives). Particularly, a whole cell biosynthesis of fucosyllactose by using recombinant Escherichia coli isr egarded as a promising method.

We are developing a new technology to research the micro-biomechanics of breast cancer more fully. New insights from this field will have clinical applications. The device will apply mechanical forces to cultured breast tissues that will mimic the pattern observed in breast cancer tumorigenesis. The device thus reproduce key structural, functional, and biomechanical properties of breast tissues with greater fidelity to in vivo tissues than current laboratory methods. Information gathered provide a bridge to clinical diagnosis and treatment. This low-cost, high-throughput technology provide alternatives to animal and clinical studies in medical research and new tools for the individualized treatment of breast cancer. This also provide valuable multidisciplinary education and research opportunities in newly developing fields.

As the concerns about environmental problems, climate change and limited fossil resources increase, bio-based production of chemicals and polymers from renewable resources gains much attention as one of the promising solutions to deal with these problems. Polyhydroxyalkanoates (PHAs) are polyesters produced and accumulated in various microorganisms. PHAs are synthesized using monomers provided from metabolite precursors of diverse metabolic pathways and are accumulated as distinct granules inside the cells. On the other hand, most so called bio-based polymers including polybutylene succinate (PBS), polytrimethylene terephthalate (PTT), and polylactic acid (PLA) are synthesized by a chemical process using fermentation-derived monomers. PLA, an attractive biomass-derived thermoplastic, is currently synthesized by ring opening polymerization (ROP) of (L)-lactide that is made from fermentation-derived (L)-lactic acid. Recently, we have developed one-step fermentative synthesis process for the production of PLA and PLA copolymers from renewable resources employing recombinant bacteria equipped with PHA biosynthesis pathways coupled with a novel metabolic pathway. This could be accomplished by establishing a pathway for generating lactyl- CoA and engineering PHA synthase to accept lactyl-CoA as a monomer combined with systems metabolic engineering. In this presentation, we report recent advances in the production of lactate-containing homo- and co- polyesters, and furthermore 2-hydroxyacid containing polyesters in recombinant microorganisms. We will discuss challenges remaining to efficiently produce 2-hydroxyacid containing polyesters such as PLA, PLA copolymers and 2-hydroxybutyrate containing PHA and development of the metabolic engineering strategies to overcome these challenges.

The use of plant biomass for biofuel production will require efficient utilization of all sugars in cellulosic biomass, primarily glucose and xylose. While Saccharomyces cerevisiae can be engineered to ferment xylose, the engineered S. cerevisiae cannot utilize xylose until glucose is completely consumed. This sequential utilization of glucose and xylose results in lower yields and productivities of ethanol. To overcome this problem, we engineered S. cerevisiae to co-ferment cellobiose and xylose by introducing cellodextrin transporter (cdt-1) and intracellular β-glucosidase (gh1-1) from Neurospora crassa (1). The cellobiose fermenting S. cerevisiae was also able to co-ferment cellobiose and galactose with a higher ethanol yield and productivity, suggesting that the strain can be applied for utilizing marine biomass (2). Energetic benefits were improved through phosphorolysis pathway instead of hydrolysis pathway for cellobiose utilization (3). HXT2.4, the poor cellodextrin transporter from Scheffersomyces stipitis was drastically improved by substitutions of alanine (A291) to charged amino acids in HXT2.4 (4). These results suggest that efficient and rapid co-fermentation of cellobiose and other sugars can be achieved using the cellobiose-fermenting S. cerevisiae strains (5).

The genomes of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) consist of single-stranded RNA encoding polyproteins, which are processed to individual functional proteins by virus-encoded specific proteases. These proteases have been used as targets for drug development. Here, instead of targeting these proteases to inhibit viral infection, we utilized the protease activity to activate a toxic protein to prevent viral infection. We engineered the MazE-MazF antitoxin-toxin system of Escherichia coli to fuse a C-terminal 41-residue fragment of antitoxin MazE to the N-terminal end of toxin MazF with a linker having a specific protease cleavage site for either HIV PR (HIV-1 protease), NS3 protease (HCV protease), or factor Xa. These fusion proteins formed a stable dimer (instead of the MazF2-MazE2-MazF2 heterohexamer in nature) to inactivate the ACA (sequence)-specific mRNA interferase activity of MazF. When the fusion proteins were incubated with the corresponding proteases, the MazE fragment was cleaved from the fusion proteins, releasing active MazF, which then acted as an ACA-specific mRNA interferase cleaving single-stranded MS2 phage RNA. The intramolecular regulation of MazF toxicity by proteases as demonstrated may provide a novel approach for preventive and therapeutic treatments of infection by HIV-1, HCV, and other single-stranded RNA viruses.

Antibody IgG has been evolved in natural immune system against a variety of pathogens and widely used for therapeutics, diagnostics, and research reagents due to its high evolvability which adopts new functions at ease with tolerating destabilizing mutations. In IgG molecules, removal of the invariant glycan at Asn297 abolishes binding to FcγRs (Fc gamma receptors) expressed on the surface of various immune cells. Therefore, aglycosylated antibodies produced in bacteria have not been used for therapeutic antibodies requiring effector functions such as clearance of tumor cells. In this lecture, I will present a set of Fc (fragment crystallizable) engineered versions of aglycosylated antibody with various FcγRs selectivities and unique therapeutic effector functions that clinical grade glycosylated antibodies do not display. Additionally, cytochrome P450 enzyme, a biocatalyst transforming a broad scope of substrates, has been evolved for a novel diagnostic tool. Strategies to increase sensitivity of the biomedical agent have been developed and will be discussed.

By controlling their physical and chemical properties, diverse materials can exhibit unique characteristics which have high potentials to overcome various obstacles in current clinical approaches. Design and corresponding engineering of nanobiomaterials including diverse nanoparticles and three dimensional polymer scaffolds allow us to modulate cells and tissues on demand. These materials are used in diagnostics, drug delivery, cancer therapy, and tissue engineering. This presentation will introduce some of the examples how we are able to control the living systems through designed materials for therapeutic applications.

Recently, boron-doped diamond (BDD) thin films have been used as active electrodes for various electrochemical sensors, because of their unique chemical, physical, optical and mechanical properties [1]. Electrochemical sensors based on the BDD electrodes show an enhanced signal-to-noise ratio (S/N), a low detection limit and a wide linear range due to their unique advantages such as a low background current and a wide potential window [2-3]. In addition, the low adsorption of organic contaminants on the BDD surface results in low fouling of the functional proteins on the electrode surface [4]. However, it is difficult to synthesize the BDD materials with 3-D nanostructures and to modify their surface for immobilization of biomaterials. So, various nanomaterials on the BDD electrode are introduced for the enhanced electrical performance and effective functionalization. Electrochemical sensor based on the BDD electrodes with various nanomaterials exhibited the improved performances, i.e. higher sensitivity, a lower detection limit, and a wide linear range.

Surface enhanced Raman scattering (SERS) sensing has attracted considerable attention for ultrasensitive, highly specific and multiplex biomolecular assays. Recently, new optofluidic approaches have been developed for the reliable and reproducible on-chip SERS detection. These optofluidic systems have opened up possibilities of small sample requirements, fast mixing, and enhanced sensing signals. In accordance with these requirements, we here describe the development of an electrokinetic optofluidic SERS chip using the electro-active microwell for sensitive and multiplex SERS detection. This device allows for both efficient mixing to enhance the rate of binding between the SERS enhancers and the biomolecular targets and sample enrichment to improve detection sensitivity through the use of electroactive microwells. As another approach, we here present an optofluidic surface enhanced Raman spectroscopy (SERS) device for on-chip detection of vasopressin using an aptamer based binding assay. To create the SERS-active substrate, densely packed, 200 nm diameter, metal nanotube arrays were fabricated using an anodized alumina nanoporous membrane as a template for shadow evaporation. We explore the use of both single layer Au structures and multilayer Au/Ag/Au structures and also demonstrate a facile technique for integrating the membranes with all PDMS microfluidic devices. Using the integrated device, we demonstrate a linear response in the main detection peak intensity to solution phase concentration and a limit of detection on the order of 50 ng/mL. This low limit of detection is obtained with device containing the multilayer SERS substrate which we show exhibits a stronger Raman enhancement while maintaining biocompatibility and ease or surface reactivity with the capture probe.

Influenza is a vaccine-preventable disease, but remains a major health problem world- wide. Morbidity and mortality due to influenza could be reduced by development of simple and effective vaccination methods. Immunization via the skin is attractive, because, in large part, the skin is replete with antigen-presenting cells such as Langerhans and dermal dendritic cells. Arrays of metal micron-scale needles were coated with influenza inactivated virus vaccines suitable for simple, manual application. A single dose of influenza vaccine from microneedles (MNs) generated strong antibody and cellular immune responses in mice and provided superior protection against lethal viral challenge at the main site of viral replication in the lung, as evidenced by virus clearance below the detection limit. Additionally, microneedle vaccination resulted in enhanced cellular recall responses after challenge. In contrast to conventional egg-based vaccine production, cell-based vaccines are being developed to expedite vaccine manufacturing and thereby reduce the threat of insufficient supply. Virus like particles (VLPs) and DNA vaccines are attractive cell-based vaccines and the vaccinations using MN patch coated with VLP or DNA demonstrated dose-sparing effects of influenza vaccine in comparison with intramuscular (IM) injection. Apart from immunologic advantages, microneedles also offer potential logistic opportunities. The small size of microneedles should facilitate storage, stockpiling and transportation of influenza vaccines. Vaccination should be faster and simpler because microneedles are painless and suitable for self administration. Mass-produced microneedles would be cost-competitive with hypodermic needle and syringe. In summary, our results suggest that influenza vaccine delivery to the skin using microneedle patches may provide a new modality to increase patient coverage and improve immunogenicity of influenza and other vaccines.