Earticle

Proteins are always at the center of any biological systems. As the number of protein sequences with unknown function increases, it has become an important issue to assign accurate functions to those proteins. To understand their functions in a more complete sense, it is important to understand how they look, what they do, what they interact with, and how they do all those things. It is also important to understand what the consequences of specific structures, functions and interactions of proteins are. In this presentation, we will discuss recent research developments on protein’s structures, functions, and interactions. We will also discuss its implications on drug discovery and protein design.

The Designer’s Synthetic Biology Cluster is dedicated to provide total synthetic biology solutions - from Gene Design to Directed Protein Evolution & Expression, which is sponsored and supported by DDI (Daedeok Innopolis) and MKE (Ministry of Knowledge Economy) of Korea. This program is aimed at establishing a foundation for artificial synthetic technologies from genes to proteins by integrating all the steps, from the design of a functional new gene to the high speed synthesis of the gene to the directed evolution of the protein and gene and its expression. It also aims to synthesize the total genome and develop the functionally improved proteins that are needed in the White, Red, and Green Bio fields. The Designer’s Synthetic Biology Cluster, which will lead the era of Bio 2.0, the nextgeneration bio-industry era, will be created and be expanded into enterprises and research institutes that need the synthetic biology-based technology developed by this project. In this presentation, we will report our works – Gene Design Software, HT Gene Synthesizer and Kit, Mega-base Oligo Synthesizer- ranging from the design of a functional new gene to the high speed synthesis of the gene. One of the most important platform technologies in implementing Synthetic Biology is the software used to design the synthetic gene sequence necessary for the gene or set of genes that the customer wants. We firstly developed, in collaboration with KAIST, this software, which is composed of codon optimization, mRNA secondary structure estimation and oligonucleotide design. This software enables the design of genes by optimizing the codon specific for the host and different factors related to protein expression so that the expression of the protein, which is the final target of gene synthesis, can be implemented. The designed genes are automatically and high-Throughput synthesized on the HT Gene Synthesizer, on the basis of Accuracy Position Control Technology and Micro Sealing Technology, and the Peltier method. The gene synthesis kit, a ready-to-use format including all components for gene synthesis was also developed. Sub-nanomole Scale Oligo Synthesizer is under development, having the capability to synthesize with sub nano mole at an initial synthesis scale in a rapid and economical cycle time. It ensures the quality and purity of the synthesized oligonucleotides and is optimized to synthesize oligos for gene synthesis, gene libraries and codon bias adjustments. On this platform, we will discuss in detail during the presentation.

Nearly two decades ago, I started some genetic as well as genomic study using a eubacterium, Bacillus subtilis 168. Her ability drew my great attention, to take up DNA given outside and integrate it into her genome via homologous recombination. This research style later lead to development of a novel cloning vector. Most DNA cloning vectors can not handle a large number of genes, equivalent to giant DNA, at one time. Several works merged to provide the novel cloning system based on the B. subtilis genome as a cloning vehicle. The Bacillus GenoMe (BGM) vector derived from the 4.2-Mb genome of B. subtilsi168 was demonstrated to accommodate fairly large DNAs and is highlighted by the successful stable cloning of a whole 3.5-Mb genome of the nonpathogenic, unicellular photosynthetic bacterium Synechocystis (1) and any sequence-known DNA (2-3). Attempts to build a man-made synthetic genome will also be introduced. (1) Itaya, M., Tsuge, K. Koizumi, M., and Fujita, K. Combining two genomes in one Cell: Stable cloning of the Synechosystis PCC6803 genome in the Bacillus subtilis 168 genome. Proc. Natl. Acad. Sci., USA., 102, 15971-15976 (2005) (2) Tsuge, K.、Matsui, K., and Itaya, M. One step assembly of multiple DNA fragments with designed order and orientation in Bacillus subtilis plasmid. Nucleic Acids Res. 31: No. 21, e-133 (2003). (3) Itaya, M., Fujita, K., Kuroki, A., and Tsuge, K. Bottom-up genome assembly using the Bacillus subtilis genome vector. Nature Methods 5, 41-43 (2008).

The fission yeast, Schizosaccharomyces pombe, has served as an excellent model organism for mechanism studies of cell cycle control, mitosis and meiosis, DNA repair and recombination, the checkpoint controls and genome stability. The systematic generation of deletion mutants by targeted mutagenesis accelerates the use of S. pombe for functional and comparative studies of eukaryotic cell processes. We report here that more than 90% of total genes of S. pombe have been deleted by using PCR-generated deletion cassettes, which were designed to facilitate the later HCS procedures. Lowering the dosage of a single gene from two copies to one copy in diploid S. pombe results in HAPLOINSUCFFICIENCY, that is sensitized to any drug that acts on the product of this gene. With the genome-wide gene deletion heterozygotic mutants, we have setup a chip assay system, which is useful when only small amount of chemical is available. The HCS method using the systematic S. pombe deletion mutants can be exploited for the identification of drug targets. *This work has been supported by Bioneer (Daejeon, Korea), KRIBB, and MOST. The mutants are property of both Bioneer and KRIBB.

In the past decade methods of directed evolution has become a widely accepted and broadly applied method for biocatalyst engineering. A directed evolution experiment comprises two main steps: generating diverse mutant libraries and screening for improved protein variants. Although the vast majority of reported directed evolution experiments use a combination of error-prone PCR and DNA shuffling, methods for constructing diverse molecular libraries continue to accumulate. More important than the motivation to circumvent patent laws, these new approaches aim to generate more comprehensive, less biased libraries, because the quality of a mutant library is decisive for the success of a directed evolution experiment. This emerging focus on library quality is also reflected in new computational methods. In parallel with experimental techniques, computational predictive frameworks have been dramatic advances in design and analysis of directed evolution experiments. Recently, chemical DNA synthesis has been adopted to make various controlled mutant libraries with low bias and high fidelity, because of high efficiency, fidelity, cost reduction and process automation. Advanced mathematical and data-mining tools in computer modeling have been applied to analyze sequence-activity or sequence-stability relationships and to assist in the design of proteins with specified properties. It is unclear now, however, whether it is more efficient to mutate an enzyme randomly or to mutate active sites or key sites specifically designed by computer modeling. We expect that the powerful combination of in silico and in vitro methods based on DNA synthesis and computational analysis for directed evolution will further accelerate the successful development of desired biocatalysts.

Ability to write and synthesize DNA is fundamentally changing the way of research and approach in biotechnology research. However purposeful modification of metabolic pathways, metabolic engineering, is still limited by tuned expression of genes. In this presentation, we will review basic needs of expression systems in the era of synthetic biology and report our work on autoinduction expression systems. Firstly, we developed autoinduction system by using acs promoter. Acetyl-CoA synthetase(Acs) is responsible for the consumption of acetate accumulated during growth phase. Repressed in the presence of glucose and highly expressed at the stationary phase or at the limited growth phase, it suits many aspects of the ideal expression system in synthetic biology applications. pACE plasmid vectors derived from a pUC containing acs promoter was constructed and tested for the expression of LacZ and GFPuv as reporter proteins. Expression of the reporter proteins was tightly controlled with the carbon source: When glucose was used as a sole carbon source, the expression of LacZ was completely repressed during exponential growth phase and induced only at the stationary phase. When glycerol or organic acids were used, the LacZ was constitutively expressed. The uniform expression of GFPuv in each cell at the stationary phase was confirmed by flow cytometry analysis. This promoter was further evolved to display diverse expression profiles. This acs-based expression would provide a novel autoinduction system, enriching various expression systems available currently in Escherichia coli. A similar expression system is under development in Bacillus subtilis, which will be discussed in detail during the presentation. (Supported by SynBio Cluster Program, MOKE, Korea)

Synthetic biology is an emerging field that aims to design and build synthetic networks of biological reactions using well-characterized biomolecular components and genetic modules. While the current efforts in synthetic biology rely mostly on the engineering of living host cells, progress in the in vitro synthesis of DNA, RNA or proteins make it possible to construct truly 'synthetic' networks of biological functions. In particular, recent advances in the techniques of cell-free protein synthesis are expected to provide a versatile platform for the in situ construction of in vitro networks of important proteins. Compared to in vivo synthetic biology, in vitro synthetic biology offers substantial advantages including increased flexibility of engineering and less background interference with the synthetic enzyme network. Herewith, we present and discuss our recent achievements in the development of cell-free protein synthesis systems, which have been modified in various configurations to allow the flexible manipulation of gene expression and reaction conditions. We expect that the developed systems will be used for studying focused and intentional regulation of protein synthesis and other biological functions in the context of synthetic biology.

The number of cloned and sequenced genes involved in natural product biosynthesis has increased rapidly during the past decade, and increasing knowledge on genetic information has enabled the manipulation of their biosynthetic pathways to yield novel compounds. However, many of the original producers are difficult to manipulate genetically, have poor growth characteristics, or yield poor titers. The transfer of biosynthetic genes from the original producers to a more amenable and robust heterologous host therefore becomes an attractive alternative to producing high levels of desired compounds or to providing a basis for subsequent combinatorial biosynthetic approaches. Streptomyces venezuelae has been recently developed as a heterologous host that requires a short culture period for metabolite production compared to other Streptomyces species. It is also amenable to genetic manipulation and has high transformation efficiency. These characteristics make S. venezuelae an alternative system for a rapid heterologous production of desired compounds from benchtop genetic manipulation to product fermentation. For diversifying the structures of microbial natural products or increasing titers, the combinatorial biosynthetic approaches combined with metabolic engineering generated a wide range of unnatural polyketides with increased yield. This approach also enabled us to dissect the biosynthetic pathway of natural products produced by genetically non-amenable bacterial strains such as aminoglycoside-producing actinomycetes. We show here that heterologous expression of different combinations of genes from the 4,6-disubstituted aminoglycoside biosynthetic gene cluster in a non-aminoglycoside producing strain of S. venezuelae caused production of intermediates from the biosynthetic pathway. This provided experimental evidence for assigning the functions of individual gene products and also allowed us to recognize a complete biosynthetic pathway for generation of 4,6-disubstituted aminoglycosides. References 1. Je Won Park, Jay Sung Joong Hong, Niranjan Parajuli, Won Seok Jung, Sung Ryeol Park, Si-Kyu Lim, Jae Kyung Sohng and Yeo Joon Yoon "Genetic dissection of the biosynthetic route to gentamicin A 2 by heterologous expression of its minimal gene set" (2008) Proceedings of the National Academy of Science of the United States of America 105(24) 8399-8404.

Redesign or modification of the cellular physiology requires a quantitatively well-controlled expression system known as the “tunable expression.” Although the modification of promoters demonstrates the great impact on the translation efficiency, it is difficult to detect the proper variants required for tunable expression. The 5′-untranslated region (UTR), however, can be an important target for tunable expressions because the ribosome binding affinity is directly modulated by the sequence variants of the Shine-Dalgarno (SD) sequence and the AU-rich sequence, which are the ribosome binding sites and a SD-sequence-independent translation enhancer, respectively. Additionally, downstream region (DR) of translation initiation codon also affect on the expression level depending on its secondary structure. This study developed a simple method to obtain numerous 5′UTR variants and analyze their translation efficiency based on the PCR-based site-directed mutagenesis and the expressional PCR using coupled in vitro transcription/ translation system derived from E. coli and eGFP gene as a template. SD sequence variants and AU-rich sequence variants, which have a wide range of relative expression levels ranging from 0.1 to 2.0, were obtained. Furthermore, silent mutations around DR showed a significant difference in the expression level more than 10 times. We found that the translation efficiency was affected by the ribosome binding affinity and its accessibility that is dependent on the secondary structure around the 5′UTR and DR. The results in this study illustrates the potentials fine control of expression level based on the modifications in 5’ UTR and DR structures.

Algal biotechnology is drawing increasing interest due to its potential as a source of valuable pharmaceuticals, pigments, carbohydrates, and other fine chemicals. Its application has been extended to the areas of wastewater treatment and agriculture. Recent development in various algal biotechnology found microalgal mass culture can be a useful solution in treatment of wastewater, fixation of carbon dioxide and production of biofuel. Furthermore, the fact that microalgae can conquer the top three environmental problems in a simultaneous manner is even more exciting. Green house gases such as CO2 in the atmosphere became a serious problem for the whole mankind. The signs of global warming have boosted many researches on sequestration and removal of CO2. One of the possible solutions is a biological CO2 fixation using the photosynthesis of microalgae. Microalgae have been utilized for several decades for the treatment of municipal and other wastewaters. The removal of heavy metals and/or residual nutrients from wastewaters, especifically N and P, has also been studied with a variety of processes. Renewable, carbon neutral, transport fuels are necessary for environmental and economic sustainability. Oil productivity of many microalgae greatly exceeds the oil producti vity of the best producing oil crops. Approaches for making microalgal biodiesel economically competitive with petrodiesel would be possible in the near future.

Supercritical water gasification of biomass is expected to be an effective measure to convert wet biomass into combustible gas. Since supercritical water is a high-temperature, high-pressure water, the reaction engineering for this technology is difficult to develop. In Japan, a 1 t-wet/d pilot plant has been built, and is operated as a part of NEDO project, and successfully operated for chicken manure gasification. The concept and result obtained from this project is introduced, which led to the reaction engineering of supercritical water gasification.

바이오에너지는 바이오매스 자원을 에너지화한 것을 말하는데 바이오매스 자원은 식물과 미생물의 광합성에 의하여 생성되는 식물체, 균체와 이를 먹고 살아가는 동물체를 포함하는 생물유기체를 말한다. 바이오에너지는 크게 세가지로 분류되는데 바이오 디젤, 바이오 가스, 바이오 에탄올로 나뉜다. 식물로 대표되는 바이오매스는 지구상에서 1 년간 생산되는 양이 전체 석유매장량과 비슷해 고갈될 염려가 없는 에너지로 평가 받고 있다. 바이오에너지 생산 원료로 사용되는 바이오매스는 유체, 콩, 팜, 자트로파, 옥수수, 사탕수수, 카사바 등 농작물과 간벌목 볏짚 왕겨 등 농임산 부산물, 그리고 음식물쓰레기 축산분뇨 등 유기성 폐기물로서 농임업과 밀접한 관계에 있다. 우리나라의 신재생에너지 보급율은 2006 년 말 기준 총 1 차에너지공급량 대비 2.24%를 차지하고 있으며, 2030년에는 이를 11%로 확대할 계획이다. 국내의 바이오에너지 보급현황을 수송용 바이오에너지인 바이오디젤과 바이오에탄올을 중심으로 살펴보면, 바이오디젤은 2002 년 시범사업을 거쳐 2009 년 현재 1.5%를 혼합하고 있으며 2012 년까지 3%까지 혼합할 예정이다. 바이오에탄올의 경우는 바이오에탄올 혼합연료의 보급에 적합한 유통시스템 구축을 위한 실증연구를 수행하는 등 국내 바이오에탄올 보급을 시행하기 위한 검토를 활발히 진행 중에 있다. 한편, 이러한 바이오에너지공급에 있어서 가장 핵심적인 현안은 원료(Feedstock)의 안정적 확보인데, 우리나라의 경우 대규모의 재배면적확보를 비롯하여 에너지작물의 생장조건에 적합한 기후 및 토양 등의 조건 충족에 사실상 한계가 있어, 불가피하게 이에 적합한 요건을 갖추고 있는 동남아를 비롯한 해외지역에 대규모 플랜테이션 확보를 추진할 수 밖에 없다. 정부 역시 이러한 상황을 인지하고 해외자원개발법에 따른 해외자원개발사업분야에 “바이오연료의 원료작물”을 포함시킴으로써 바이오원료작물 개발사업을 지원할 수 있는 근거를 마련하고 있다. 최근 우리나라의 여러 기업들은 바이오에너지작물의 해외플랜테이션 확보를 위해 많은 노력을 해오고 있으며, 이제 서서히 그 가시적 성과를 올리고 있다. 주로 말레이시아, 인도네시아, 필리핀, 캄보디아, 라오스, 파푸아뉴기니 등 동남아에 집중적으로 진출하고 있으며, 바이오디젤 원료인 팜 및 자트로파농장을 비롯하여 바이오에탄올 원료인 카사바농장확보에 주력하고 있다. 본 발표에서는 바이오에너지 작물 중에서 우리나라기업들이 동남아에서 주로 추진하고 있는 자트로파 및 카사바농장에 대하여 각 작물의 특성, 식재 및 재배방법, 농장운영방법 및 고려요소, 현안문제점 등을 살펴보도록 함으로써, 국내외 인식제고 및 향후 긴밀한 산학협력을 통한 해외플랜테이션의 성공적 확보를 달성하여, 우리나라 바이오에너지보급사업에 도움이 되고자 한다.

최근에 전 세계적으로 화석연료의 과다 사용에 따른 자원고갈 및 환경오염에 대한 우려가 증가하면서 자연과 공존하며 안정적으로 발전하자는 “지속 성장(sustainable development)"의 개념이 화두가 되고 있으며, 불안정한 유가에 대응하면서 지구온난화의 주범이 되고 있는 온실효과가스(GHG: Greenhouse effect Gas) 배출량 감소를 위하여 친환경 대체에너지 개발에 박차를 가하고 있다. 특히 석유 대체연료로서 생물자원을 원료로 하는 바이오연료의 생산량이 급증하고 있다. 선진국의 바이오 에너지 정책, 개도국의 높은 경제성장 등에 의해 곡물 수요가 항구적으로 늘어난 반면, 지구 온난화, 농업 생산성 증대의 한계 등으로 인하여 곡물 공급을 안정화시키기가 어려운 실정이다. 따라서 바이오연료(바이오에탄올 및 바이오디젤 등) 확대 보급에 따라 곡물가격 폭등으로 가축사료 가격 상승 등 시장 왜곡 현상이 야기될 수 있으므로 바이오에너지 보급확대의 순기능적 측면 이외에 역기능적 측면을 동시에 고려하면서 합리적 보급 방안 마련이 필요하다. 주요 선진국들은 곡물이 아닌 비식용의 자원을 비롯한 차세대 친환경 원료의 활용에 집중하고 있으며, 이에 대해 원료 수송부터 최종제품 유통까지 전체 과정을 고려한 정책을 펴고 있다. 우리나라에서도 저탄소 녹색성장의 정책목표를 두고 바이오에너지 국산화율을 높이고자 이미 바이오디젤의 사용기준을 법제화 하였고, 바이오에탄올의 사용기준도 법제화를 마친 상태이며, 바이오에너지를 생산하는데 필요한 원료의 자급화를 위해 비식량 바이오매스 자원 개발을 강력하게 추진하고 있다. 또한 기술발전과 효율성 증진(바이오매스 단수 증가와 바이오매스에서 생산할 수 있는 바이오연료의 양 증가)이 이루어지면 경제적 비용과 환경에 미치는 영향이 점차 줄어들 것이다. 바이오연료의 미래를 결정하는 수익성은 몇 가지 상호 연관된 요인들의 영향을 받는데 가장 중요한 변수는 국제원유 가격이다. 더욱이 경지면적이라는 제한 때문에 세계 에너지 공급부문에서 바이오연료가 차지하는 비중은 일정 수준을 넘기 어려울 것이다. 이러한 한계를 극복하기 위해서는 새로운 기술의 개발이 필수적이다. 광범위하게 사용되는 셀룰로오스계 바이오매스를 이용한 경제적인 바이오연료 개발이 상용화된다면 바이오연료의 비중은 현재보다 높아질 것이다. 우리나라의 경우 에너지 소비 양상을 볼 때 바이오연료의 파급효과가 적지않으며, 차세대 원료를 바탕으로 한 바이오연료 시장의 잠재력이 매우 크다는 점을 감안한다면 보다 적극적인 바이오연료 정책이 필요하다. 우리나라에서도 바이오디젤 보급 단기목표로 2012년에 54만㎘를 보급할 계획이며, 여기에 필요한 원료를 확보하고자 2011년에 45천ha 규모의 동절기 유채 재배를 계획하고 있다.

우리 인류가 이룩한 산업의 발전은 화석원료에 기반을 두고 있다. 현재 인류가 사용하고 있는 대부분의 정밀 화학제품, 의약품, 각종 화학 소재, 플라스틱, 연료 등은 화석원료 특히 석유를 원료로 생산되고 있다. 그러나 석유는 수요의 지속적 증가와 매장량의 한계 때문에 최근 그 가격이 급격히 상승하고 있으며 이로 인해 세계 경제의 발전이 크게 위협받고 있다. 또한 화석연료나 화석원료 이용하는 화학제품의 제조공정은 지구온난화 가스 및 폐기물을 대량생산하여 인류에게 심각한 환경문제를 야기하고 있다. 이에 바이오매스를 원료로 사용하는 새로운 생물화학공정, 즉 산업 BT 의 필요성이 대두되었다. 미국 에너지성(US DOE)은 2004 년 8 월 미래의 대체 화학물질의 목록을 발표하였다. 먼저 300 개 이상의 후보군을 선정하고, 이를 30 개의 잠재적인 후보군으로 압축하고 최종적으로 시장성과 합성방법의 기술적인 복잡성을 검토하여 당으로부터 생산될 수 있는 12 개의 기본요소(Building block)를 선정하였다. 이들은 보통 12 개의 Top valueadded chemicals 로 불리며 대체화학원료를 논할 때 가장 보편적으로 인용되고 있다. 아직까지 Biotech 을 이용하여 생산되는 대체 화학원료의 수와 양은 제한적이다. Ethanol(12.6 million ton/year)과 구연산(750,000 ton/year)을 제외하면 몇 종류의 아미노산(L-lysine, L-glutamine)이 연간 10 만 톤 규모로 생산되고 있으며 유기산(acetic, itaconic, lactic acid)이 연간 1 만 톤 규모, 그리고 비타민(B2, B12)과 항생제들이 연간 약 1000 톤 규모로 생산되고 있을 뿐이다. 그러나 장기적으로 그 수와 양은 급격히 증가할 것으로 예상된다. 본 발표에서는 12 개 기본요소를 중심으로 현황과 전망을 소개한다.

Biodiesel, one of the alternative fuels, is produced from the transesterification of vegetable or animal oil with alcohol and has become more attractive recently because of its benefits, i.e., it is non-toxic, biodegradable, and renewable. Among various biodiesel production processes, biological process overcomes defect of chemical process such as energy intensive, difficulty of the glycerol and catalyst separating. However, it has not been commercialized because of the high price, the instability of biological catalysts and slow reaction rate. To overcome these disadvantages of biological process, various processes have been investigated. Therefore, in this talk, current state and development of various biological processes were reviewed for biodiesel production.

energy sources is a major issue that bridges the academic endeavors of the scientific community to the real problems of the global community. In this light, bio-hydrogen stands as a remarkably useful energy source for alleviating the pressures of limited resources as well as promoting the use of environmentally friendly technology. Produced from renewable resources such as water and organic wastes by biological means, it represents a hopeful effort to address some of the everyday problems that people meet in terms of basic energy needs across the world. Various biological hydrogen production processes are in the R&D phase and substantial research is required to enable commercialization of technology. Several processes are currently under development ranging from anaerobic fermentations to photobiological processes with a promising aspect for the economical feasibility. In this symposium, an overview of biological hydrogen production processes, such as dark anaerobic fermentation from organic wastes, photo-biolysis of water, photo-fermentation, and in vitro systems will be introduced briefly for their fundamental and applied research. In addition, the combination of two consecutive fermentation processes, dark anaerobic fermentation of organic wastes and photo-fermentation, which is known to be the most economical and efficient technology for complete use of chemical energy in the substrate, will be presented in detail.

기존의 화석에너지를 대신할 새로운 청정/대체에너지의 개발에 대한 요구가 지속적으로 증가하면서 새로운 대체에너지 자원으로서 목질계/섬유소계 바이오매스가 다시 주목받고 있다. 특히 수송용 에너지를 대체할 수 있는 바이오 연료 생산 기술은 저장이 가능한 에너지원의 확보 및 화학 원료 생산을 위한 재료로서 그 연구 가치가 증가하고 있다. 하지만 현재의 수송용 연료로 적용되고 있는 바이오 연료는 대부분 전분계인 옥수수와 당질계인 사탕수수로부터 얻어지는 것으로 이들은 식량자원과의 가격 경쟁의 문제점을 갖고 있으며, 상당 수준의 기술 개발이 완료되어 기술 향상의 가능성이 적다. 또한 에너지 생산을 위한 투입 에너지 비율이 높아 환경 개선 효과가 미비하며, 생산 비용 측면에서도 기존의 석유 연료보다 높다는 여러 가지 단점을 갖고 있다. 이러한 생산비용 및 환경 개선 그리고 재료의 확보라는 여러 가지 문제점들을 극복하기 위한 대안으로 목질계/섬유소계 바이오매스를 이용한 바이오 연료에 많은 관심이 집중되고 있으며 그 가능성이 확인되고 있다. 목질계 바이오 연료의 생산을 위한 공정은 1. 당화 저해 물질을 제거하고 셀룰로오스의 접근 가능성을 증가시키기 위한 전처리 공정(Pretreatment), 2. 셀룰로오스의 가수분해를 통해 발효 가능한 당류로 변환하는 당화 공정(Saccharification), 3. 생성된 당을 효모,박테리아를 이용하여 에탄올 및 부탄올로 전환 시키는 발효 공정(Fermentation)으로 구분할 수 있다. 이러한 바이오 연료의 성공적인 생산을 위해서는 전처리 공정을 통하여 바이오매스의 분별 및 당화 공정과 효소 당화 공정이 필수적이다.

식용 원료로부터 바이오에탄올 생산은 곡물 가격 상승을 비롯하여 다양한 문제점을 야기하고 있는 시점에서, 비식용 원료를 사용하고자 하는 시도가 이루어지고 있다. 목질계 바이오매스로부터 전처리, 당화, 발효 과정을 거쳐 생물학적으로 바이오에탄올을 생산하는 연구가 한창 진행되고 있지만, 목질계 원료의 전처리 과정에 많은 어려움이 있는 것이 사실이다. 또한 목질계 원료의 리그닌 성분은 전통적인 생물학적 발효 공정에서는 에탄올로의 전환이 어려워 보통 열원으로써 사용되고 있다. 소개하고자 하는 합성가스 발효 공정은 목질계 바이오매스의 가스화를 통해 셀룰로스, 헤미셀룰로스 뿐만 아니라 리그닌까지도 합성가스로 전환시키는 공정을 포함하고 있다. 합성가스의 정제 후 미생물을 사용하여 발효 공정에 의해 바이오에탄올을 생산한다. 에탄올 생산 공정의 반응식은 다음과 같다. 6CO + 3H2O → C2H5OH + 4CO2 2CO2 + 6H2 → C2H5OH + 3H2O 합성가스가 생물 반응기로 유입되기 전 가스 정제를 통하여 미생물에 대한 독성을 제거해야 하며, 발효 과정에서 에탄올 외에 다른 부산물이 생성되기도 하므로 부산물보다 에탄올을 높은 농도로 생산할 수 있는 미생물의 선정 및 반응 조건의 도출이 요구된다. 또한 기-액 사이에서 일어나는 반응이므로 기-액 물질전달을 효율적으로 수행할 수 있는 생물 반응기의 설계가 중요한 요소라 할 수 있다. 현재 Coskata, Conocophillips 등의 회사가 합성가스 발효에 관심을 가지고, 실증 플랜트 건설 및 상용화를 목적으로 연구를 진행하고 있다.

For several decades, Escherichia coli has been considered as the most important host for recombinant protein production because of rapid growth, easy gene manipulation, easy scale-up etc. In addition, recent developments of various protein display system have allowed E. colI to be an useful tool in protein engineering. Previously we have developed new protein display system, Anchored Periplasmic Expression (APEx) system, in which protein is anchored on the inner membrane of E. coli via lipoprotein fusion. Combination of flow cytometry (FC) and APEx system make it possible to do high throughput screening of protein variant with odified/improved properties such as different specificity, maturated affinity, enhanced stability etc. Recently, based on APEx system, we have developed new 2-hybrid system (APEx 2-hybrid) in which two interacting proteins are coexpressed and is suitable for proteomic study as well as high throughput screening for protein engineering. Various applications of APEx and APEx 2-hybrid systems toward antibody engineering (affinity maturation of antibody fragments and full-length IgG, development of intrabody etc.) will be presented.

CD24 is a glycosylphosphatidylinositol(GPI)-anchored membrane protein which consists of a small protein core comprising 27 amino acids and is extensively glycosylated. Almost half of the amino acid in CD24 are composed of Ser and Thr residues which can be potential sites for Olinked glycans. CD24, originally described as a B cell marker, started drawing considerable attention as anti-cancer target showing that CD24 overexpressions in many carcinomas are significantly associated with a more aggressive course of the disease. In our previous studies, we found that expression of CD24 in ovarian cancer had association with tumor grade and stage. In this study, we tried to validate CD24 as anti-cancer target in ovarian cancer. From microarray result, ovarian cancer had the most relevant CD24 overexpression level difference from normal. To study CD24 targeting effect in ovarian cancer, we screened CD24 overexpression cell with various method like RT-PCR, qPCR, WB, FACS. And then ADCC and CDC effects, which were considered as major mechanism for mAb therapeutic effect, were measured with CD24 overexpression cells, OVCAR3 and SK-OV3 using commercial anti-CD24 mAb, SN3. Targeting of CD24 in those cell lines showed dramatic cytotoxic effects more than 50% through natural immune system, ADCC and CDC. Another mechanism for anti-cancer effect, which we have taken notice, is P-selectin binding to cancer cell via CD24 which could facilitate cancer metastasis. From this point of view, Anti-CD24 mAb could also play a metastasis-inhibiting role by preventing CD24 from binding to P-selectin. Practically, we confirmed that CD24 could interact to P-selectin from sandwich ELISA and FACS. Based on these results, we will have further study to confirm anti-metastatic effect of CD24 mAb in ovarian cancer metatastasis mouse model. Finally we conducted in vivo experiment with human ovarian cancer xenograft nude mouse. We observed that mouse with treatment of anti-CD24 antibody had a propensity to decrease the tumor growth rate compared with control group treated with PBS. In conclusion, CD24 may have possibility as a validated target for monoclonal antibody therapy in ovarian carcinoma.

By immunizing rabbits with HGF/cMet complex, we successfully generated a monoclonal antibody that inhibits HGF/cMet interaction, and blocks the biological function mediated by HGF in vitro and in vivo. To define epitope, we screened out an epitope-mimicking peptide, KSLSRHDHIHHH, from a phage display of combinatorial peptide library. In molecular mimicry this peptide bound to cMet and inhibited HGF/cMet interaction. No humoral response was induced to this epitope-mimicking peptide when immunization was done with HGF alone. This rabbit monoclonal antibody was successfully humanized, optimized for process development and superior in vivo characteristics and now under active characterization.

Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. Models of murine tumor angiogenesis and receptor-specific antibodies are required to evaluate roles of VEGF receptors in mouse xenograft models of human cancer. Human VEGFR-2 (also known as kinase insert domaincontaining receptor, KDR) and murine VEGFR-2 (or Fetal liver kinase-1, Flk-1) share 85% amino acid sequence identity in their extracellular domain. However, sequence homology of VEGF binding domain of KDR and Flk1 is less than 75%. Until now, none of available KDR neutralizing antibodies has species crossreactivity. We first describe here the development of fully human antibodies that cross-react with mouse, rat and human VEGFR-2. High-affinity, species cross-reactive, ScFv antibodies specific for KDR/Flk-1 were selected from fully human naïve antibody phage display library we constructed. The selected and converted fully human IgG antibodies were found to bind to purified KDR with sub-nanomolar affinity. Their binding epitope locate in the IgG like domain 3 of extracellular domain of VEGFR-2, which is responsible for neutralizing effect of KDR function. I will discuss about recent in vivo data and part of preclinical data of anti-KDR antibody.

Development of protein binders that interact specifically with a given target molecule with high affinity has been of interest for their pharmaceutical and industrial applications. For this purpose, antibodies (Abs) are the primary choice for proteins capable of specific high affinity binding to target molecules, which can be isolated by immunization or from synthetic antibody library in vitro. As an alternative to Abs, nonimmunoglobulin proteins have been extensively exploited as structural scaffold to isolate in vitro target specific high affinity binders from the synthetic library randomized particular regions on the constant scaffold. Here we report a novel target-specific protein binder developed based on the scaffold of kringle domain (KD), which is present as a structual modular unit in 31 human proteins. By exploiting extensive divergence of the primary structure in the 7 loop regions, but the highly conserved backbone folding by core residues and disulfide bond linkages of the naturally occurring human KDs, we generated synthetic KD library on the yeast cell surface by randomizing the residues in the highly variable and surface exposed loops. From the KD scaffold library, we screened to isolate agonistic KD variants specfically binding to anticancer target proteins of human death receptor 4 (DR4) and/or DR5 and inducing apoptotic cell death as a single agent for several cancer cells in vitro and their in vivo tumor xenograft models. In addition to the agonists, we can also isolate from the libary an antagonistic KD variant against human tumor necrosis factor-α (TNFα), which efficiently neutralized the TNFα-induced cytotoxicity in vitro and in vivo. The selected KD variants retained the secondary strucutre and high thermal stability, comparable to those of wild type KD, suggesting that KD scaffold are strongly seqeunce-tolerant. Our results suggest that KD scaffold can be developed as an attractive target-specific protein binder functioning as agonists or antagonists to given target molecules to modulate their biological activity.

Glycosylation is a major post-translational protein modification, which alters physicochemical properties of the protein, affecting the folding, distribution, stability and thus biological function and efficiency of protein. Nglycosylation begins in the endoplasmic reticulum (ER) with the synthesis and the co-translational transfer of a lipidlinked oligosaccharide precursor to specific asparagine (N) residues of the nascent polypeptide chain. During the maturation in the ER and the Golgi complex, N-glycans can be further modified by glycosidases and glycosyltransferases until the glycoprotein arrives in its final destination. After their maturation, some plant type complex N-glycans are distinctive from those found in mammalian because they contain β1,2-xylose and core α1,3-fucose residues attached to the pentasaccharide (Man3GlcNAc2) core structure but no sialic acid residues. The presence of β1,2-xylose and core α1,3-fucose residues on plant type complex N-glycans has long been an irritating limitation in the use of plant-made pharmaceuticals (PMPs) in human therapy, as these N-glycan epitopes are potentially immunogenic in mammals. However, considerable improvement has been made towards humanization of N-glycosylation in plants to remove the potential limits of plant cells as a factory for the production of pharmaceutical glycoproteins. The addition of the potentially immunogenic β1,2-xylose and core α1,3-fucose residues on plant-made pharmaceuticals may be avoided when the expressions of responsible glycosyltransferases have been interfered or removed by RNA interference (RNAi) or gene knockout techniques, respectively. Alternatively, cgl mutants lacking N-acetylglucosaminyltransferase I (GnTI) activity synthesize only oligomannosidic N-glycans and ER retention of the recombinant protein may be used to restrict glycosylation of target proteins to only high-mannose type N-glycans. In addition, N-glycosylation also plays important roles in the quality control (QC) of glycoprotein folding in the ER lumen and in ER-associated degradation (ERAD) of proteins by cytosolic proteasomes. Protein QC in the ER lumen is functionally linked to unfolded protein response (UPR), namely, an increase of misfolded protein in the ER is sensed as an ‘ER stress’ and induces increased synthesis of UPR- and ERAD-associated genes. Plants are emerging as a substitute system for the pharmaceutical protein production because of their practical, economic and safety advantages. However, the limitations of plants as a pharmaceutical protein production system should be carefully considered and ameliorated. (Supported by BK21 program)

To develop potential plant-derived vaccine, Canine Parvovirus (CPV) virion protein 2 (VP2) and Avian influenza virus A/H5N1 nucleoprotein (NP), were expressed in Esherichia coli and Nicotiana benthamiana, respectively. A full-length of VP2 gene was engineered to be expressed by a bacterial expression vector pET-28a, while a partial length of NP containing antigenic domain was constructed in pET28a vector. Both recombinant proteins fused with His-tag were purified from E. coli using Ni-NTA chromatography under denaturing conditions. The purified VP2 was injected to produce rabbit polyclonal antisera, and the purified NP was injected to produce polyclonal IgG from rat and IgY from chicken egg yolk. The full-length of VP2 gene and the antigenic domain of NP were also engineered to be expressed by a plant expression binary vector pPZP212 under the control of Cauliflower mosaic virus (CaMV) 35S promoter, and transformed with Agrobacterium. Therefore, both gene constructs were introduced into binary vector containing an expression cassette, CaMV 35S promoter, a TEV leader sequence, the PCR-amplified target gene fragments and a 35S terminator. Each gene construct was tested for its ability to express target protein after transient expression by agroinfiltration into N. benthamiana leaves, and infected onto leaf discs for the production of transgenic plants. Genomic DNA and mRNA analyses of the transformed plantlets confirmed the integration of the target cDNA into the N. benthamiana genome, as well as its transcription. The expression of each recombinant protein was then observed in transgenic plants qualitatively by Western blot and quantitatively by ELISA. The morphological characteristics, including shoot and root growth were investigated, were shown no significant differences between non-transgenic and transgenic plants. Plant-expressed VP2 was strongly cross-reacted with rabbit polyclonal antisera against E. coli-produced recombinant VP2, and expressed as a 65 kDa band which is corresponding to the predicted molecular weight of recombinant protein from the cDNA sequence. The recombinant NP expressed in N. benthamiana was well cross-reacted with rat polyclonal antisera and chicken IgY against E. coli-produced NP. However, its size was 45 kDa which is much higher than the predicted molecular weight of recombinant protein from the cDNA sequence. It could be experienced the protein modification in plant cells, possibly by glycosylation. Both VP2 and NP genes were constructed in the same vector system under the same conditions, and probably both gene sequences of animal viruses contain the specific sequences which may not be favorable to be expressed in plants. Nevertheless, the expression of NP gene construct only undergoes the modification in plant. It suggests that a foreign gene expression in plant can be varied depending on the inserted gene source.

The Nexgen is focusing on development of plant molecular farming based diagnostic kits which could be fast marketable. These kinds of products may have advantages over injectible pharmaceuticals as follow: 1) part of pharmaceutical industries, 2) less regulatory affairs, 3) fast market penetration, 4) shortens investment pregnancy period, 5) requires tiny amount of proteins, 6) requires less facilities, 7) removes false positive reaction caused by E.coli produced antigenic proteins, 8) applicable to diagnostic system for epidemic and autoimmune diseases, 9) over $30B world market Plantized(amino acid sequences as original but optimized DNA sequences with plant codon usage and removing of intron like sequences in plant) human Thyroid Stimulating Hormonal Receptor(TSHR) gene and nucleocapsid genes of Epidemic Hemorrhagic Fever viruses such as Hantaan, Puumala and SinNombre were designed and synthesized then cloned into marker free constructs. Proteins of interest were successfully expressed in plant by Agrobacterium -mediated transient expression system which was suitable for small scale production in the lab to avoid neither environmental nor regulatory matters. Plant-made antigenic proteins were used to prepare ELISA and Rapid Strip kits that are currently being reviewed by KFDA and USFDA. Transgenic plants using oriental melon and cucumber are being produced in case of further mass production of antigenic proteins in the lab then some parts of transgenic tissues will be relocated into the enclosed vessel system for organ culture in the surely secured in-house facility. There would not be open field trials for transgenic plants to avoid public concerns.

The commercial marketing of a series of GM (genetically modified) crops has increased continuously every year due to the transformation technology. According to the annual global review of commercialized GM crops conducted by ISAAA, the estimated global area occupied by transgenic or GM crops in 2008 was 125 million hectares, almost 5.7 times the land area of the Korean peninsula, which is about 65 fold during the ten-year period from 1996 to 2008. ISAAA found that 13.3 million farmers in a record 25 countries planted 125 million hectares of biotech crops last year, the sixth largest growth spurt in 13 years of reporting. The global market value of biotech crops was $6 billion in 2008 and will be about $15 billion in 2015. Presently, soybean, corn, cotton and canola are 4 major GM crops and 8 more plants such as carnation, sugar beet, tomato, popular, petunia, papaya, sweet pepper, squash and alfalfa are cultivating as minor. The biotech traits are herbicide tolerant gene, Bt gene, stacking of both herbicide tolerance and Bt, virus resistance and delayed ripening. Here in Korea, researchers in universities and national institutes have poured money and labor during the last 15 years or so to develop valuable GM crops. However, none of the Korean GM crops has ever been commercially available and even not a single crop has been approved by the safety risk assessment yet. Therefore, it will take several years until any GM crop is on the Korean market. There are several major reasons why the development of GM crop is far behind worldwide levels. Those would be the lack of recognition about GMO’s power, the lack of an entire process from GM development to marketing, the lack of the private sector involvement and the huge negative concern from public. Very recently, there are signs of progress that the private sectors have also been actively working in this business with vegetables in Korea. In order to catch up in the race for the new agricultural biotechnology industry, each one of us in private sectors alongside academia and national research institutes needs to focus altogether on what can be done best in terms of developing a promising GM crop, that represents a Korean product. For this purpose, here, I address a road map for GM crop commercialization. It includes: selection of an ideal target gene for crop transformation, development of GM crop, transgenic breeding, biosafety assay and the time table from the beginning of GMO development to commercialization. Especially the subject called transgenic breeding that is not well known in most of the plant science arena will be mainly discussed.

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig), a fusion recombinant protein, was expressed in transgenic rice cell suspension cultures. The expression vector pMYN409 was constructed to express hCTLA4Ig under the control of RAmy3D promoter. Under the induction condition by sugar starvation, suspension-cultured rice cells secreted hCTLA4Ig into the media. The rice-derived hCTLA4Ig could be purified with affinity chromatography using protein A resin. Recombinant hCTLA4Ig has molecular weight of ~50 kDa on SDS-PAGE under reducing condition. hCTLA4Ig was produced inducibly by sugar starvation in stirred tank bioreactors with various process strategies. Marine, turbine, and hollowed paddle impeller were compared for the cultivation of transgenic rice cells and optimization of culture medium was performed. Two-stage cultivation method was applied to achieve high cell density during cell growth phase and to enhance the production of hCTLA4Ig. Two kinds of media exchange methods (perfusion and direct exchange) were compared in a 5-L stirred tank bioreactor. In the production phase, inducible production of hCTLA4Ig caused cell death because of sugar depletion, and media exchange induced cell lysis by additional shear stress. It was essential to reduce shear stress for increasing productivity because protease activity was increased by cell lysis. High density cultivation was also investigated in the bioreactor. With fed-batch cultivation, maximum dry cell weight reached 21.2 g/L and maximum hCTLA4Ig production reached 13.4 mg/L which were 2.18-fold and 2.14-fold higher than those of the direct media exchange, respectively. Therefore, we can conclude that the minimization of cell lysis and achievement of high cell density could be necessary to enhance the production of hCTLA4Ig with RAmy3D promoter system. Control of oxygen level was found to be important. In addition, effect of shear was significant. S225

In this study, a high performance biosorbent, PEI-modified biosorbent, was developed by cross-linking with polyethylenimine (PEI) on the surface of inactive waste biomass of Escherichia coli, which is generated from a Lphenylalanine fermentation plant. Platinum-bearing wastewater was collected from industrial analytical laboratory and was used as a model platinum waste solution. Sorption performance of the PEI-modified biosorbent was evaluated using kinetic and isotherm experiments and compared with that of the raw biomass. The maximum platinum uptake of PEI-modified biomass was as high as 108.8 mg/g compared to 21.4 mg/g of raw biomass. The kinetic experiments revealed that sorption equilibrium could be attained within 60 min. The results of FTIR and XPS analyses of Pt-unloaded and Pt-loaded PEImodified biosorbents showed that the electrostatic interaction would be the main binding mechanism between the platinum ions and binding sites on the surface of biosorbents. Metallic form of platinum in ash could be recovered by incineration, with over 98.7% of recovery efficiency. Furthermore, TEM, XPS and XRD results confirmed that the forms of platinum were existed as Pt0 and Pt2+ (as platinum oxide) in the incineration ash. Therefore, it can be concluded that the developed PEI-modified biosorbent can be used for platinum recovery through a combined process of biosorption and incineration.

Stem cells have significant promise for regenerative medicine, but they first require an understanding of molecular or environmental cues that can regulate their proliferation and differentiation, as well as means to manipulate these signals to control cell behavior. Delivery of genes encoding molecules capable of regulating stem cell function can serve as an effective means to both investigate stem cell biology and to control cell fate for therapeutic applications. Additionally, gene delivery coupled with gene targeting has the potential to introduce mutations, and thereby generate disease models, as well as to correct deleterious genetic mutations in stem cell populations. However, a major obstacle to such applications continues to be the development of efficient and safe gene delivery vectors. Adenoassociated viral (AAV) vectors, which are being broadly explored in clinical trials, have significant promise as therapeutic vectors due to their safety and delivery efficiency, as well as their potential for gene targeting. Unfortunately, no natural AAV variants have been found with optimal properties for infecting stem cells. Due to the significant advantages of the vector, however, engineering AAV vectors to overcome rate limiting steps (i.e., cellular binding, intracellular trafficking, viral unpackaging, etc.) in stem cell transduction may have a high impact for stem cell investigations. Current approaches to design custom AAV vectors are limited to rational peptide insertion into or chemical modifications of the viral capsid structure. However, since the structure-function relationships of the complex AAV capsids are not fully understood, rationally designing and modifying the AAV capsid to meet specific needs are still challenges. In this presentation, a powerful tool (i.e. directed evolution) to create de novo bio-inspired nanoparticles (i.e., AAV vectors) that can significantly enhance the capabilities of gene delivery as well as gene targeting within stem cells will be introduced, and a variety of potential applications using gene-targeted stem cells will be discussed.