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Our lab is broadly interested in the discovery and preclinical/clinical development of enzymes andantibodies for therapeutic purposes, especially cancer. We employ integrated approach to thedevelopment of protein therapeutics that begins with the invention of platform technologies tofacilitate discovery, continues with tissue and animal testing of efficacy and then with bioprocessdevelopment, GMP production and toxicology, as required for clinical studies. Examples of our recentstudies in protein therapeutics will be discussed: 1) Enzyme therapeutics: The use of enzymes to systemically deplete metabolites required for thegrowth of tumor cells but not of normal tissues, has been pursued for many years. However thistherapeutic approach had been stymied by poor therapeutic properties and immunogenicity of theenzymes investigated. We have engineered and validated in xenograft models: (i) human arginasefor L-Arg depletion therapy for the treatment of hepatocellular carcinomas, metastatic melanomasand other L-Arg auxotrophic tumors. A phase I clinical trial of this drug is scheduled to begin in July2012. (ii) A human methionine g-lyase for L-Met restriction in tumors of neurological origin. 2) Engineered aglycosylated antibodies displaying novel effector functions and enhanced ADCC:Antibody- Dependent Cell Cytotoxicity (ADCC), i.e. the ability of the antibodies to activate innateimmune cells to destroy pathogens or diseased cells is critical for the therapeutic action of severalantibodies. The potency of ADCC depends on very subtle molecular effects that dictate the bindingof the antibody to inhibitory or activating receptors on immune cells. We have succeeded inengineering antibodies that engage exclusively (or preferentially) the activating receptors only andthus, display unique abilities to induce the efficient killing of cancer cells. Remarkably, theseengineered antibodies are aglycosylated, i.e. they lack the normally invariant carbohydrate chainwithin the Fc domain, a property that greatly facilitates bioprocessing. 3) Deconvolution of the monoclonal antibodies that comprise the polyclonal serum response:Remarkably, after 100 years of intense research in immunology, there is nothing known about themonoclonal antibodies that comprise the antigen-specific immunoglobulin pool in serum. We havenow developed a technology that enables the identification and relative quantitation of specificantibodies in serum samples from patients. This technology can guide the discovery of noveltherapeutic antibodies to infectious agents and other diseases, via the mining of the serum responsesthat have overcome the disease.

Micro and extended-nano (EN) fluidic device technology has brought evolutional progress in chemistry and medical biology although it is still on the way of development for practical use. In terms of the sample volume, analytical instrumentation technology is getting new tools to reduce the volume evolutionally, that is the micro and EN fluidic device providing nano, pico, femt and atto litter orders. Micro fluidic devices are almost in practical use. We proposed the micro unit operation (MUO) and continuous flow chemical processing (CFCP) concept for realizing chemical circuit on a microchip, and it became a chemical CPU for an analytical instrument in which the sample volume is nano to pico litter which is almost the same as single cell volume. The size region of 101-102 nm, that is EN space, is still unexplored area. In our group, we fabricated extended-nano channels into glass microchips, and succeeded in fluidic control and chemical processing such as chromatographic separation. In those EN fluidics, the MUO and CFCP concepts are kept. Actually, we applied the extended-nano channels and spaces to pico-litter immunoassay and atto-litter chromatography. Pico-litter is smaller than the volume of a living cell, and therefore, single cell single molecule analysis will be available. The atto-litter chromatography is really challenging, and some examples will be introduced in this talk. One of the critical issues of micro/EN fluidic technology is detection and determination. Especially, optical detection of non-fluorescent analytes is quite difficult, because optical pass length of channels is extremely short. Therefore, we developed the thermal lens micro-detection (TLM) method, and it was proved to be a very powerful detector even at zmol regions. However, optical pass length is a couple of orders shorter in EN, and it was still very difficult. Therefore, we got an idea of introducing differential interference contrast (DIC) optical configuration into TLM. DIC-TLM enabled us to detect optical absorption in EN optical pass length, and we could obtain chromatogram of non-fluorescent molecules. Principle of DIC is based on wave optics, and EN optical pass length was still sufficiently long comparing to wavelength for detection of phase shift induced by TL effect in EN channels. The combination of EN fluidics and DIC-TLM will be a powerful tool for single cell analysis and other various kinds of nano technologies.

The induced pluripotent stem cell (iPSC) technology pioneered by Yamanaka and his colleagues in 2006 has ignited an explosion of scientific and public interest because these cells can potentially offer an ideal cell source to study and treat numerous human diseases by providing patient- and disease-specific cells. However, widely established methods to generate iPSCs require the use of viral and/or genetic materials that likely integrate into the chromosomal DNAs with unknown genetic changes. Indeed, recent evidence demonstrated that viral-based iPS cells compromise genomic integrity and exhibit abnormal phenotypes. Thus, to realize the therapeutic and biomedical potentials of iPSCs, it is critical to develop reprogramming methods that can avoid or minimize these potential abnormalities. As a potential approach, we attempted to generate iPSCs without the use of viruses or DNA transfection by directly delivering four reprogramming proteins (Oct4, Sox2, Klf4, and c-Myc) fused with a cell penetrating peptide. We also characterized these protein-based iPSCs along with virus-based iPSCs for their molecular and differentiation properties. This presentation will discuss current limitations as well as the potential of protein-based iPSC reprogramming with the long-term goal to use them for eventual personalized medicine.

Apoptosis is a biological process of cell suicide that plays critical roles in development and homeostasis of metazoan organisms. Apoptosis is activated via the cell-intrinsic and cell-extrinsic signaling pathways. Cancer cells often acquire mutations that enable them to escape proapoptotic signals induced by conventional therapies. Hence, intense efforts have been underway to develop treatment strategies that can overcome hurdles to apoptosis in cancer cells. The cell-extrinsic pathway is triggered at the cell surface via pro-apoptotic death receptors such as DR4 and DR5. Human monoclonal antibodies (MAbs) that activate these receptors display potent activity in various preclinical tumor models, and are studied in cancer clinical trials. We have discovered that Fγ receptors (FγRs), expressed on tumor-infiltrating leukocytes, provide a dynamic cross-linking platform to mediate the apoptosisinducing activity of DR5 MAbs. Our findings provide novel insights into the mechanism of action of death receptor-agonistic MAbs against tumors and suggest that endowing such molecules with stronger FγR binding may further enhance their clinical utility.

I will discuss about our preclinical data of anti-KDR neutralizing fully human IgG1, Tanibirumab which has cross-species cross reactivity, and about our recent progress from dual target neutralizing multifunctional Ig like next generation protein therapeutics, DIG-KT (KDR &Tie2) from DIG-bodies platform, and PIG-KM (KDR &cMET) from PIG-bodies platform.

Antibodies and related products are the fastest growing class of therapeutic agents. The currently marketed antibody-based drugs have been approved not only to treat diseases affecting large numbers of patients (such as cancer and autoimmune diseases) but also for more specialized indications (such as orphan diseases). Antibodies have been the highest earning category of all biological drugs; several monoclonal antibodies generated multi billion dollar revenues including Rituxan, Herceptin and Avastin from Genentech. In this presentation, I will presentation our strategies of selecting and optimizing potential therapeutic antibodies at Genentech. Specific case studies will be provided for engineering specific and potent antibodies to dissect complex biological systems, and development of therapeutic antibodies with good CMC properties for clinical trials.

Despite recent advances in imaging techniques and molecular imaging tools, biomarkers considered appropriate for detection of cancer are severely limited to a few well known surface molecules, mainly belonging to growth factor or receptor tyrosine kinase receptors. Particularly, ever-increasing knowledge of the linkage between tumor and inflammation has yet to be translated into novel approaches to better diagnose and treat cancers. In this talk, I"ll present our recent work on developing molecules against cancer biomarkers prevalent not only in tumors but also in tumor inflamed stroma, i.e., tumor endothelium and tumor-associated macrophages. These tumor-selective moieties are then formulated to provide highest sensitivity and specificity in consideration of biological and physiological barriers.

Hepatitis B virus (HBV) is one of the main pathogens responsible for hepatitis and hepatocellular carcinoma. Human plasma-derived hepatitis B immune globulin (HBIG) is being used for prophylactic and liver transplantation currently. However, it may be necessary to replace a HBIG with a recombinant one because of limited availability of human plasma with high anti-HBs antibody titer. In order to meet these requirements, we developed a recombinant HBIG (rHBIG) using antibody engineering technologies. The activity of rHBIG is much higher than that of HBIG from human plasma; 3,500 vs 10 units/mg. The rHBIG does not bind to normal human tissues examined, although it binds to HBV-infected human liver tissue. Neutralization of the HBV by rHBIG was proved in a chimpanzee model where the rHBIG treated chimpanzees did not show any signs of HBV infection during one year of follow up. We also demonstrated that this rHBIG is able to neutralize G145R mutant of the HBV in a hydrodynamic mouse model; the G145R mutant is a typical mutant known to escape from HBIG treatment. We identified that the rHBIG recognizes strictly conserved amino acid residues of HBsAg, which plays a key role in neutralizing the diverse HBV mutants. Safety of the rHBIG was proved in monkeys. In healthy human volunteers, the rHBIG was well tolerated without any serious adverse events up to 8 folds of clinical dose and current stage is in clinical phase II/III for liver transplantation in Korea. Further we plan to apply this rHBIG to chronic hepatitis treatment caused by HBV with combination of a current polymerase inhibitor. This rHBIG has several advantages compared to plasma-derived HBIG in activity, safety and availability.

We have generated neutralizing monoclonal antibodies directly from human B-cells of convalescent patients who had been infected with a new H1N1 virus. The generated antibodies could neutralize various A type influenza viruses and were efficacious in animal survival tests. One antibody, CT120, from the first screening using hemagglutinin (HA) of H1N1 showed microneutralization (MN) activity against H1, H2, H5, and H9 of group 1. Another antibody, CT149, from the second screening using HA of H3N2 from the same antibody pool showed MN activity against H1, H5, and H9 of group 1 and H3 and H7 of group. Interestingly this antibody could neutralize H3N2 isolates over the past 40 years. These antibodies cannot inhibit hem-agglutination induced by HA, but can inhibit HA associated cell fusion which indicate that these antibodies are targeting the stem region of HA. Animal passive protection test indicated that CT120 was effective prophylactically and therapeutically against H1N1 and H5N1. CT149 was also effective in prophylactically and therapeutically against H3N2. Taken together, the aforementioned two neutralizing monoclonal antibodies can be useful prophylactic and therapeutic agents against epidemic and pandemic influenza virus infection. This work was performed mainly under the collaboration of Dr. Ruben Donis (Chief, Molecular Virology and Vaccines Branch, Influenza Division, CDC, Atlanta, GA USA).

The past 15 years have seen considerable progress in the development of microfabricated systems for use in the chemical and biological sciences. Interest in microfluidic technology has in large part been driven by concomitant advances in the areas of genomics, proteomics, drug discovery, high-throughput screening and diagnostics, with a clearly defined need to perform rapid measurements on small sample volumes. At a basic level, microfluidic activities have been stimulated by the fact that physical processes can be more easily controlled when instrumental dimensions are reduced to the micron scale. The relevance of such technology is significant and characterized by a range of features that accompany system miniaturization. Such features include the ability to process small volumes of fluid, enhanced analytical performance, reduced instrumental footprints, low unit costs, facile integration of functional components within monolithic substrates and the capacity to exploit atypical fluid behaviour to control chemical and biological entities in both time and space. Based on these advantageous characteristics, microfluidic systems have been used to good effect in a wide variety of applications including nucleic acid separations, protein analysis, process control, small-molecule synthesis, DNA amplification, immunoassays, DNA sequencing, cell manipulations, nanomaterial synthesis and medical diagnostics. The exploitation of microdroplets produced within microfluidic environments has recently emerged as a new and exciting technological platform for many. Microfluidic systems that generate and utilize a stream of sub-nanolitre droplets dispersed within an immiscible continuous phase have the advantage of allowing ultra high-throughput experimentation and being able to mimic conditions similar to that of a single cell thereby compartmentalizing biological and chemical reactions. Moreover, since they are isolated from channel surfaces and other droplets, each one acts as an individual reaction vessel. Variation of the cross-sectional dimensions of microchannels can be used to regulate droplet volumes, and flow rate variation allows control of reagent concentrations. Importantly, droplets can be generated at kHz frequencies, meaning that millions of individual reactions can be processed in very short times. We have developed a range of functional components and techniques for use in such systems. These include tools for droplet generation, droplet merging, droplet dilution, droplet splitting and phase separation. These tools can then be integrated to address key problems in the fields of genetics, proteomics, high-throughput screening and cellular analyses. My lecture will describe recent studies in all the above areas.

A series of microfluidic based point of care technologies were developed in Dalian Lab, some of them are shown as following. 1. An array microfluidic immunoassay chip integrated with micro-valves (pumps) was developed for automatic analysis of clenbuterol and testosterone. 2. A microfluidic device integrated with microvalves and micropumps and a microfluidic compact disk (CD) chip based on one-step reciprocating flow were fabricated and applied for rapid DNA hybridization analysis. These devices were used for discrimination of single nucleotide polymorphisms and detection of 48 Dengue Virus samples, the hybridization time was shortened to 90s. 3. A droplet based processing was designed and developed for on-line SPE of DNA. Multiple reagent addition-droplet splitting units were integrated on a single chip to perform the reagent addition and waste removal processes in SPE. The hepatitis B virus (HBV)-DNA extraction from serum samples was performed and followed by chip PCR. 4. The fabrication of paper based microfluidic devices in nitrocellulose membrane by wax printing for immobilization related applications was demonstrated, clenbuterol was tested as an example. It looks close to see the possibility of commercialization.

Microsystems have the potential to impact biology by providing new ways to manipulate cells and the microenvironment around them. Simply physically manipulating cells or their environment—using microfluidics, electric fields, or optical forces—provides new ways to separate cells and organize cell-cell interactions. One example illustrating the power of microscale manipulation of cells is to sort cells based on their intrinsic electrical properties. Electrical properties have previously been correlated with important biological phenotypes (apoptosis, cancer, etc.), but a sensitive and specific method approach has been lacking. We have developed a method called iso-dielectric separation that uses electric fields to drive cells to the point in a conductivity gradient where they become electrically transparent, resulting in a continuous separation method specific to electrical properties. With this method, we have screened the entire genome of an organism to understand the biological basis of electrical properties, finding that the relationship between genetics and intrinsic properties has both intuitive and non-intuitive features. Microfluidics can also be used to manipulate the environment around cells. For example, we have developed arrays of microfluidic perfusion culture chambers that use fluid flow to create a convection-dominated transport environment, allowing control over local cell-cell diffusible signaling. This in turn provides a more controlled soluble microenvironment in which to study diffusible signaling in cell systems. In particular, we have examined the impact of diffusible signaling on self-renewal and neural specification of embryonic stem cells. Using these microsystems, we have identified the existence of previously unknown autocrine loops involved in fate specification, and have delineated the effects of shear itself on self-renewal. Together, these new microscale tools provide ways to exploit cells’ potential for both basic science and applied biotechnology.

In this talk, I will present the use of physically modified microfluidic channels towards various biomedical applications including single cell analysis and organ chips. The physical modification can be realized either by embedding physical micro/nanostructures or including a topographically patterned substrate at micro- or nanoscale inside a channel. To enable micro/nanofabrication with microfluidics, a simple capillarity-driven lithographic method termed “capillary force lithography” is introduced for fabricating micro/nanostructures onto a surface or inside a microfluidic channel. In the first part, hollow micro-wells are integrated inside a microfluidic channel to capture cells at single cell level. Using this microfluidic platform, we carried out programmable time-course live-cell imaging aided by fluorescent image processing. In the second part, the method is applied for mimicking various aspects of in-vivo environments for cells or tissues. Two organ chips are presented for potential drug screening and advanced tissue engineering applications: heart and kidney chips.

For the tissue engineering, the regulation of 3D microenvironment is important for the cell culture under in vivo like microenvironment and the study of cell pathophysiology, and it has been a great challenge by using conventional cell culture platform. Recent progress of microtechnology enabled the construction of microenvironment for the regulation of cellular behavior and differentiation. However, it is still difficult to build 3D in vivo mimicking microenvironments. Here, we propose the microscale arrayed concave PDMS wells for the formation and culturing of artificial cell spheroid in a easy and cost effective way and its use of 3D small tissue and further larger organ model. By the proposed technology, we created 3D micro tissue having uniform size and shape in a large scale using several kinds of cells including cancer cell, stem cell, neuron cell, hepatocyte and beta cells. In this presentation, we introduce the mass production of diverse cell spheroid: 1) embryoid body of mouse ES cell and their differentiation to neuron and cardiac cells, 2) hepatocyte spheroid, and hetero-spheroid including hepatocyte and hepatic stellate cell for the assisting liver function, and 3) islet spheroid production and in situ encapsulation with and collagen-alginate composite materials and in vivo evaluation of glucose control for 1 months. Finally, the new fibers which were coded with different chemicals or morphologies were fabricated using the microfluidic chip combined with computer controlled arrayed valve system. The applicability of these fibers to tissue engineering was evaluated through diverse cell culture in and on the fiber.

Isomagnetophoresis can be used to discriminate subtle differences in magnetic susceptibility by using a magnetic susceptibility gradient in a microfluidic channel [1]. In isomagnetophoretic immunoassays, the magnetic nanoparticles are used as labels on microbeads in sandwich-type immunoassay, detecting the amount of bound analytes by isomagnetophoretic focusing the solid-support microbeads under the magnetic susceptibility gradient and magnetic field in a microchannel. One advantage of the method is that the dynamic range of the isomagnetophoretic immunoassay system can be adjusted by altering the magnetic susceptibility gradient. In addition, this isomagnetophoretic immunoassay system can be used to analyze the selected concentration of target analytes in detail by tuning the dynamic ranges. The proposed immunoassay can be useful to accurately quantify the concentrations of biomarkers over the whole range of analyte concentrations, based on the current status and needs of the patient [2]. In this presentation, multiplexed immunoassay results based on isomagnetophoresis as well as magnetophoresis will be addressed.

The goal of “Synthetic Biology” is to synthesize whole biological system or its subsystem intentionally and designing elements such as the regulatory elements and functional gene should be necessarily predictable. To achieve the successful design or redesign of the biological systems, robustness of naturally occurring biological systems has to be relieved so that cells can be easily redesigned. Bacterial cells are generally evolved at the various levels from DNA to protein for maintaining their robustness against the changing circumstances. Therefore, general strategy to modify cellular physiology depending the robustness or flexibility of the biological systems should be required. In this study, we developed the general tools to modify the biological robustness at the various levels including translation and protein levels – “UTR Designer”, “Riboselector”, and “Allosteric Deregulator”. The potentials of the platform technology developed in this study for the application to the production of biofuels and commodity chemicals.

Programming living organisms with new biological functionalities requires coordinated recombination of heterologous genetic elements into a host cell. Several approaches have been investigated to provide logical connectivity between well-defined binding events among biomolecules and the way how the biomolecular interactions lead to programmable circuit behaviors. In this study, E. coli and S. serevisiae cells were engineered to achieve small chemical-induced gene regulation. The engineered cell undergoes programmable biological behaviors through translational control of gene expression. This study may open a new avenue to construction of synthetic cells amenable to gene regulation via riboregulatory elements.

The recent advances in metabolic engineering to reprogram industrially competitive microorganisms is being accelerated by a new paradigm of integrating systems-level high throughput omics technologies with emerging synthetic biology-based strain design tools. Accurate prediction and control of physiological and metabolic characteristics of microorganisms is expected to realize the next generation of metabolic engineering. Here we present a good example of systematically engineering microorganisms efficiently producing biochemicals, taking an amino acid producing Escherichia coli as an example. We are particularly interested in integration of genome-scale metabolic model with high throughput omics data that can purposefully engineer the strain for the production of biochemicals at near industrially competitive level.

Metagenomics promises to provide new enzymes with diverse functions, however, current methods for the screening and cloning of novel metagenome-derived genes are limited to the examination of halo on solid media or well-plate assay. In this study, we developed a new platform for the screening of diverse enzyme genes from metagenome using a synthetic genetic circuit, based on the recognition of phenol derivatives, which are produced from aromatic substrates by many enzyme reactions. The phenol derivatives activates a transcriptional factor, DmpR, to express a fluorescent reporter gene, resulting in fluorescence emission. In cells harboring this genetic circuits, enzyme activities, such as those of tyrosine phenol-lyase, lipase, cellulase, and methyl parathion hydrolase, were detected by the fluorescence emitted. Finally, by applying the screening method to metagenome libraries, a novel phosphatase was successfully isolated using flow cytometry. These results show that phenol-mediated genetic enzyme screening system (GESS) is a widely applicable tool for high throughput and quantitative screening of diverse enzymes from the genetic pools.

Precise and temporal control of the expression of bacterial genes in response to an external stimulus is an essential tool for understanding and manipulating complex biological systems. Efficient control of biological systems would, in turn, help achieve maximal production of the desired products. Light-inducible gene expression systems are indispensable tools in systems biology, metabolic engineering, and synthetic biology, as they provide an easily switchable control over gene expression. They have several advantages over previously developed physically or chemically inducible gene expression systems in that they can help avoid toxic, unintended, or pleiotropic effects on bacterial physiology. However, previous light-inducible gene expression system in Escherichia coli could only repress target gene expression in the presence of light. Here, we have developed a light-switchable gene expression system in E. coli that could favor both inducible and oscillatory control of gene expression in the presence of light. Theλ cI repressor gene was expressed under the control of a previously constructed light-repressible ompC promoter. The green fluorescent protein (GFP) gene fused to a λ repressor-repressible promoter was used as a reporter. This light-switchable system allows rapid turn-on or turn-off of the target gene expression, at any desired time. It could also favor efficient reversible induction and repression of gene expression (switchable gene expression). This system would be a good tool for flexible control and fine-tuning of gene expression. It could be also used to study gene function, optimize metabolic pathways, and control biological systems both spatially and temporally.

Although metabolic networks have been reconstructed on a genome scale, the corresponding reconstruction and integration of governing transcriptional regulatory networks has not been fully achieved. Here we reconstruct such an integrated network for amino acid metabolism in Escherichia coli. Analysis of ChIP-chip and gene expression data for the transcription factors ArgR, Lrp and TrpR showed that 19 out of 20 amino acid biosynthetic pathways are either directly or indirectly controlled by these regulators. Classifying the regulated genes into three functional categories of transport, biosynthesis and metabolism leads to the elucidation of regulatory motifs that constitute the integrated network’s basic building blocks. The regulatory logic of these motifs was determined on the basis of relationships between transcription factor binding and changes in the amount of transcript in response to exogenous amino acids. Remarkably, the resulting logic shows how amino acids are differentiated as signaling and nutrient molecules, revealing the overarching regulatory principles of the amino acid stimulon.

Multiplex Automated Genome Engineering (MAGE) employs short oligonucleotides to scarlessly modify genomes. However, insertions of >10 bases are still inefficient, but can be improved significantly by selection of highly modified chromosomes. Here, we describe Co-Selection MAGE (CoS-MAGE) to optimize biosynthesis of aromatic amino acid derivatives by combinatorially inserting multiple T7 promoters simultaneously into 12 genomic operons. Libraries of promoter variants can be easily generated in several days to study gain-of-function epistatic interactions in gene networks.

Cellulosic biomass is the most attractive renewable source for biorefinery processes producing a wide range of products, such as fuels, commodity chemicals or bioplastics. However, traditional biomass bioconversions are economically inefficient multistep processes due to the recalcitrance of cellulose (1). Thus far, no microorganisms able to perform single-step fermentation into products (consolidated bioprocessing; CBP) have been isolated. Metabolic engineering is currently the most attractive way to develop recombinant microorganisms suitable for CBP. In order to develop a system to be used for consolidated bioprocessing of cellulose, a cellodextrin (including cellobiose) utilization system must be established in recombinant microorganism. In this presentation, we describe efforts in our laboratory toward the cellulosic biorefinery products, 2,3-butanediol(2,3-BDO), lactic acid and ethanol. We have successfully engineered an E. coli to utilize cellodextrins, the hydrolysis intermediates from cellulose depolymerization by periplasmic expression of a cellodextrinase. The resulting strain grew well on cellodextrins with varying degrees of polymerization. A synthetic operon for BDO biosynthesis was engineered into the cellodextrinase-expressing cells, resulting in a biocatalyst capable of converting cellodextrin to 2,3-BDO with 84% conversion yield without exogenous β-glucosidase(2). This periplasmic cellodextrinase also conferred the E. coli cells the ability to direct ferment cellodextrin to lactic acid at about 80% of theoretical yield (3). Furthermore, we successfully engineered E. coli biocatalysts to assimilate cellobiose through a phosphorolytic mechanism. Cytoplasmic expression of the cellobiose phosphorylase allowed E. coli to use cellobiose. Subsequent knockout and complementation studies indicated that the endogenous LacY was responsible for the transport of cellobiose (4). This is an important step toward consolidated bioprocessing for production of biofuel and biorefinery products from lignocelluloses. The ability for the biocatalyst to directly use cellodextrins eliminates the need for exogenous β- glucosidase and removes from hydrolysate cellobiose and cellodextrins, potential inhibitors for the cellulases.

In spite of advantages in bio-based chemical and energy production, lignocellulosic biomass is still not a top choice of carbon source for microorganisms. Sugar heterogeneity derived from lignocelluloses hamper the flexibility of process design and decrease the productivity and yield. Prioritization of sugar consumption is a common theme in bacterial growth and a problem for complete utilization of five and six carbon sugars. Growth studies show that Lactobacillus brevis, Lactobacillus buchneri, and Lactobacillus pentosus JH5XP5 simultaneously consumes numerous carbon sources and appears to lack normal hierarchical control of carbohydrate utilization. Analysis of the proteome of L. brevis cells grown on glucose, xylose or a glucose/xylose mixture revealed the constitutive expression of the enzymes of the heterofermentative pathway. In addition, fermentative mass balances between mixed sugar inputs and end-products indicated that both glucose and xylose are simultaneously metabolized through the heterofermentative pathway. Proteomic and mRNA analyses revealed that genes in the xyl operon were expressed in the cells grown on xylose or on glucose/xylose mixtures but not in those grown on glucose alone. However, the expression level of XylA and XylB proteins in cells grown on a glucose/xylose mixture was reduced 2.7-fold from that observed in cells grown solely on xylose. Genomics and proteomics analysis of L. buchneri and L. pentosus JH5XP5 exhibit the same trait showing the constitutive expression of xyl operons. It suggests regulation of xylose utilization in is not stringently controlled as seen in other lactic acid bacteria, where carbon catabolite repression operates to prioritize carbohydrate utilization more rigorously. Also, the important of transport system to overcome the hierarchical regulation of sugar utilization.

Lignocellulosic biomass is an abundant renewable natural resource that can serve as an important raw material for the production of biofuels and other bioproducts. However, the costs associated with enzymatic hydrolysis of lignocellulose limits its use as a source of fermentable sugars. Protein engineering1 and bioprospecting represent promising approaches to obtain cellulases with improved properties under conditions of practical interest. A major bottleneck in screening cellulases is the development of efficient protein-expression methods and techniques for rapid assay of protein libraries2. Conventional cell-based cellulase expression methods are time-consuming and laborious. On the other hand, cell-free protein expression is an attractive alternative because of its flexibility and suitability for high-throughput automation. We have developed a high-throughput expression and screening platform by coupling enzyme expression to biocatalytic activity. This approach was applied to identify novel carbohydrate-active enzymes in metagenomic libraries and improved cellulases in directed evolution experiments. The new method represents a powerful platform for high-throughput screening of cellulases and related enzymes.

Cellulosic biomass, the most abundant organic matter on earth, is comprised of cellulose, hemicellulose, and lignin. Its structural complexity, along with the existence of lignin, makes it recalcitrant to enzymatic and microbial attacks and thus serves a serious obstacle to the commercialization of biomass-based fuels [1, 2]. To make cellulose content more susceptible to subsequent enzymatic hydrolysis, therefore, raw biomass feedstocks must go through a pretreatment process. Thus far, a substantial number of such pretreatment techniques have been extensively examined to process various biomass feedstocks on scale of both laboratory and pilot plants [3, 4, 5]. Nevertheless, none of those options has demonstrated to be fully satisfactory, because each method has its intrinsic benefits and drawbacks [6]. Our research was aimed at the development of economical pretreatment of cellulosic biomass for bioethanol production. Three innovative approaches were investigated to pretreat rice straw such as FeCl3, alkali, and sono-assisted dilute sulfuric acid (SADA) pretreatment process. Ferric iron (Fe3+), a strong catalyst on hydrolysis of hemicellulose, was found to be reduced to ferrous iron (Fe2+) by oxidizing xylose and lignin. Furthermore, this ferrous iron was completely oxidized at anode in a fuel cell generating a power of 1110 mW/m2 at optimal conditions; pH of 7.0 and ferrous iron concentration above 0.008 M. Thus, FeCl3 pretreatment combining with fuel cell system proved a green process that could be successfully employed for integrated energy production system using waste species (agriculture residue, Fe2+). Pretreatment via alkali and sono- assisted dilute acid process was studied using response surface methodology (RSM). Pretreatment time, concentration and temperature were selected as process factors. For alkali pretreatment, NaOH concentration (1.0−4.0%), temperature (60−100°C) and pretreatment time (30−90 min) were employed. The maximum glucose yield of 254.5 ± 1.2 g/kg was obtained at the optimal pretreatment factor levels of 2.96%, 81.79°C and 56.66 min respectively. In case of SADA pretreatment, sulfuric acid concentration (5−10%), temperature (70−90°C) and sonication time (30−50 min) were employed. The process was optimized at factor levels (10%, 80°C and 50 min) respectively against a sugar yield of 31.78 g/100g. The results suggested that SADA process was probably feasible for simultaneous pretreatment and saccharification of biomass. Further research was needed to investigate the recovery of spent acid and application of hydrodynamic cavitation than sonication in order to improve the process economics. Thus, the study of three pretreatment processes presented innovative and economical pretreatment technologies for cellulosic biomass.

Production of bio-based fuels and chemicals from renewable resources is becoming more important as fossil resources diminish and global climate changes threaten environmental safety and human life. One such chemical, 2,3-butanediol (2,3-BD) possesses a wide range of industrial applications, and its production by microbial fermentation has been extensively studied for more than 100 years. However, the secretion of mucoid substances by Klebsiella pneumoniae, a natural 2,3-BD hyper-producer, hinders its application in large-scale fermentation because of pathogenicity, fermentation instability, and downstream difficulty. In this study, a number of mucoid-free K. pneumoniae strains were isolated from a waste water treatment plant, and their lactate dehydrogenase (ldhA-) mutants were made using pKOV plasmid which was originally developed for Escherichia coli. Fed-batch fermentation of the mutants resulted in the production of 2,3-BD at more than 70 g l-1 with an 2,3-BD yield of 0.40 g g-1 glucose. These results strongly suggest that the newly isolated mucoid-free K. pneumoniae strains have a great potential for industrial 2,3-BD production.

Room temperature ionic liquids (ILs) have recently been very popular as green solvents due to their unique physicochemical properties of negligible vapor pressure, non-flammability, excellent thermal stability and a strong ability to dissolve a wide range of organic and inorganic compounds. They also have great potential as reaction media or co-solvent for enzymatic bioconversion including biodiesel production. Cheap glucose from cellulose opens a wide window of opportunities for the production of bioenergy and plartform chemicals. Nevertheless, the recalcitrance of cellulose poses a key problem for chemcial and biological processes in biorefinery schemes. Dissolving cellulose in ionic liquids, however, helps to overcome the hurdles of the low reactivity of the cellulose fibers resulting in the enhancement of enzymatic saccharification and fermentation. In this presentation, the application of ILs in biomass pretreatment will be mainly addressed. ILs have been used as alternative solvent for cellulose pretreatment. Pretreatment of biomass is key factors for enhance enzymatic saccharification and fermentation of biomass for biofuel and bio-based chemical industry. The ILs-pretreated celluloses become less crystalline and in somewhat condition have lower degree of polymerization (DP) than that of the nature. Microwave heating could cause a significant decrease in DP of cellulose dissolved in ILs which led to a great improvement on cellulase-catalyzed cellulose hydrolysis.

Plant expansins are capable of inducing plant cell wall extension and disrupting cellulose. BsEXLX1 found in Bacillus subtilis is a bacterial expansin, a structural homolog to a plant expansin ZmEXPB1. In our previous study, BsEXLX1was found to possess synergism with cellulase in the enzymatic hydrolysis of cellulose (Kim, E. S. et al., 2009, Biotechnol. Bioeng. 102(5):1342-1353). The thermodynamic analysis of binding of BsEXLX1 to micrcrystalline cellulose (e.g., Avicel) revealed that the binding mode of BsEXLX1 to Avicel was similar to those of other Type A surface-binding carbohydrate binding modules (CBMs). BsEXLX1 bound to Avicel in an entropy-driven mode possibly due to the increased mobility of water molecules released from the interface between the protein and the substrate. BsEXLX1 did not bind to soluble cellooligosaccharides, which was also similar to the behavior of other Type A CBMs. In addition, cellulose and xylan had much lower binding for BsEXLX1 than for CtCBD3, a Type A CBM. When the binding studies of BsEXLX1 were performed against pretreated or unpretreated Miscanthus x giganteus using CtCBD3, the amounts of BsEXLX1 bound to lignin-rich substrates were much higher compared to those of CtCBD3. Also, a binding competition assay demonstrated BsEXLX1 binding decreased in the presence of CtCBD3 against Avicel but not against alkali lignin. The mutagenesis of each of the three hydrophobic amino acid residues on the binding domain on the C-terminus of BsEXLX1 that are presumed to be responsible for cellulose binding did not affect the lignin binding activity but substantially decreased the cellulose binding activity. The findings in this work suggest that BsEXLX1 could be industrially applicable as a lignin blocker during the enzymatic hydrolysis of lignocellulose.

Various RNAi delivery systems have been developed using virus, liposomes and polymer based nanoparticles, but issues like difficulties in production scale up, drug leakage and unwanted toxicity propels the consideration for development of robust vector system of biological origin. In this study, we therefore focused on to development of bacterially derived nano sized outer membrane vesicles (OMVs) for siRNA encapsulation and specifically targeting of cancer cell surface via receptor specific Affibody molecules. For this purpose, HER2 receptor specific Affibody was co-localized on outer membrane of secreted OMVs (HER2-AffiOMVs) with the help of ClyA, a pore forming toxin which shows no cytotoxicity upon fusion and acts as transporter for fusion partners. HER2-AffiOMVs si-KSP treated SKOV3 cells showed a significant KSP gene silencing along with substantial inhibition of KSP protein expression and massive cell death, confirmed by MTT assay. In summary, we developed a novel siRNA carrier of bacterial origin which is inert, rigid and stable therefore can overcome undesirable side effects seen due to cargo leakage. The possibility to re-engineer OMVs and express targeting ligands on their surface makes it suitable carrier system for targeted delivery.