Earticle

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important energy-rich resources that can be utilized for the production of bioethanol by fermentation. The yeast Saccharomyces cerevisiae is widely used for commercial production of ethanol from sucrose or starch-derived glucose. While glucose can be fermented to ethanol efficiently, the major pentose sugar xylose remains unutilized. In most natural xylose-ssimilating yeasts and fungi, xylose is metabolized via two consecutive redox reactions catalyzed by the predominantly NADPH-dependent xylose reductase (XR) followed by the NAD+-dependent xylitol dehydrogenase (XDH), with xylitol as the pathway intermediate. Such yeasts and recombinant S. cerevisiae strains harboring these genes can ferment xylose to ethanol anaerobically, but also produce considerable amounts of xylitol as byproduct due to an impairment of the cofactor balance resulting from the different coenzyme specificities of XR and XDH. In contrast, bacteria convert xylose directly to xylulose using the enzyme xylose isomerase which requires divalent metal ions as cofactors. However, bacterial xylose isomerases (XylA) are mostly thermophilic in nature and inactive or misfolded when expressed in yeast. In our studies, we have constructed a recombinant xylose-utilizing S. cerevisiae strain by co-expressing the genes for xylose isomerase (xylA) from the anaerobic fungus Orpinomyces, the endogenous S. cerevisiae xylulokinase (XKS) and the SUT1 sugar transporter from Pichia stipitis. The recombinant strain, designated as INVSc1/ pRS406XKS/pILSUT1/pWOXYLA, consumed 15.55 g l-1 xylose to produce 6.05 g l-1 ethanol, during 140 h fermentation, with an ethanol yield of 0.39 g (g xylose consumed)-1. The accumulation of byproduct xylitol (1.28 g l-1) was significantly low compared to other recombinant strains of S. cerevisiae expressing the XR-XDH pathways1. Additionally, this strain was adapted for improved xylose utilization by serial transfer in aerobic shake flask cultures in xylose containing minimal medium. The xylose-adapted strain, designated as ADAP8, was used for subsequent fermentation studies. In order to improve the ethanol productivity from xylose using the strain ADAP8, we studied the effect of temperature and addition of sugar-complexing agent sodium tetraborate on the fermentation performance of the recombinant yeast. Consistent with previous studies2, fermentation at 35 ºC led to a marked improvement in the ethanol yield and productivity. Xylose was also fermented efficiently in non-selective complex yeast extract-peptone medium. The addition of borate resulted in further enhancement of ethanol yield and productivity. During fermentation in complex medium at 35 ºC and in the presence of an optimal concentration of borate, 15.99 g l-1 xylose was consumed to produce 7.63 g l-1 ethanol after 40 h.

Polyhydroxyalkanoates (PHAs) are biological polyesters that are produced by a wide variety of bacteria as an intracellular storage material of carbon and energy. PHAs have attracted industrial attention because of their potential use as practical biodegradable thermoplastics. Xylose is the most abundant sugar in the hemicelluloses of hardwoods and crop residues, and the efficient production of PHA from xylose reduces production cost. Certain bacteria can produce PHA from xylose; however, high production rate and yield of PHA from xylose have not been achieved. Some lactic acid bacteria are capable of efficiently fermenting xylose into lactic acid and acetic acid at high production rate. These organic acids produced from xylose can be used as carbon sources for R.eutropha, the most famous PHA producer, and able to convert into PHA. We investigated the feeding strategy of the mixture of lactic acid and acetic acid for fed-batch culture of R. eutropha to efficiently produce PHA from xylose by way of such organic acids. Vegetable oils are also desirable feedstock for PHA production. Since vegetable oils compose a much higher number of carbon atoms per weight than glucose and sucrose, the yield coefficients of PHA production from vegetable oils are as high as over 0.7 g-PHA per g-vegetable oils (sugars are in the range of 0.2-0.4 g-PHA per g-substrate). To produce PHA from soybean oil as a model substrate, recombinant R. eutropha was generated and cultured in 10-l laboratory scale fermentor. As a result, high dry cell weight (138 g/l) and high PHA content (74% w/w) were achieved after 96 h of cultivation, with high production yield ranging from 0.72 to 0.74 g-PHA per g-soybean oil. Furthermore, we evaluated whether PHA production form vegetable oils is environmentally friendly bioprocess by life cycle assessment-based approach.

The application of an alkaline catalyst in the transesterification of low-quality feedstock oil is somewhat limited, because the FFA in vegetable oil reacts with the alkaline catalyst to forms soap. In order to overcome the disadvantages of alkaline catalyzed biodiesel production, comprehensive research on biocatalyst (lipase) bases has been performed. Lipase-catalyzed reactions have advantages over traditional chemical-catalyzed reactions: insensitive to FFA and water content in waste cooking oil. Lipase is capable of converting all the FFA contained in WCO to their respective fatty acids methyl esters (FAME). The objective of this study was to evaluate the immobilization of lipase in the membrane support, aiming at its application in biodiesel synthesis. Lipase was immobilized by adsorption-filtration in asymmetric membrane made of polyethersulfone. Effect of enzyme concentration and incubation time on enzyme loading was studied. The catalytic properties of immobilized lipase in membrane reactor for the conversion of palm oil into biodiesel were analyzed experimentally. The profile scanning electron microscopy (SEM) technique was used to visualize of lipase immobilized membrane (LIM).

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. Futhermore, recent development in various algal biotechnology found algal mass culture could be a useful solution in treatment of wastewater, fixation of carbon dioxide and production of biofuel. 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. However, the limitation of large scale cultivation and posttreatment of algal biomass hampers wide-spread use of microalgae for CO2 reduction. Microalgae commonly double their biomass within 24 h and some even can double as short as 3.5 h at optimal condition. Oil content in microalgae can exceed 70% by weight of dry biomass biomass productivity in photobioreactors. We have tested the possibility of applying microalgae for biological CO2 sequestration. Some microalgal species such as Botryococcus braunii and Chlorella emersonii appear to be attractive solution for CO2 sequestration. Statistical optimization of culture conditions and engineering optimization of photobioreactors will be reported.

대표적 생명공학 벤쳐 회사인 암젠은 1980년 설립된 이후, 매년 10% 이상 성장하고 있으며, 최근에도 골다공증 신약 ‘조메타’, 혈소판 촉진제 신약 ‘엔플레이트’등을 시장에 선보이고 있다. 생명공학 분야, 특히 신약 개발 분야의 경우 유전자 서열 확보, 기능 확인, 타겟 수용체 및 후보 물질 확보등 일련의 과정이 별도로 존재하는 것이 아니라 서로 깊이 연관됨에 따라 유전자 서열 확보시 향후 개발될 발명(DOWNSTREAM INVENTION)에 대한 다양한 카테고리의 특허청구범위를 확보하는 것이 필요하다. EPO는 빈혈치료제로 사용되는 단백질로 이미 뇨로부터 추출한 EPO가 존재하고 있었으므로 EPO 자체에 대해서는 특허가 거절되었으나, EPO를 코드하는 유전자와 이 유전자를 함유하는 재조합벡터 및 형질전환 미생물 등에 대해서는 특허가 인정되었다. 미국의 유명한 바이오회사인 암젠 (Amgen)은 이 유전자를 이용하여 유전공학적으로 EPO를 생산하고 있다. EPO의 세계 시장규모는 약30억$이며, 그 가격이 무려 67만$/g에 이른다. 이는 금값의 수 만배로 그 부가가치는 상상을 초월한다. tPA도 단백질 자체에 대해서는 특허가 거절되었지만, tPA를 코드하는 유전자에 대해서는 특허가 인정되었다. 제넨테크 (Genentech)는 이 유전자 특허권을 가지고 세계 tPA 시장을 주도하고 있다. 현재 산업에 적용되고 있는 암젠의 EPO. 제넨테크의 tPA 특허청구범위를 통해 생명공학 분야 특허의 청구범위 작성법에 대해 살펴보고자 한다.

Bio-butanol has become one of the possible alternatives to gasoline fuel. Its better haracteristics such as higher energy content, less hydroscopic and volatile which make it possible to use pre-existing transport infrastructure, than ethanol is regaining the reputation of conventional ABE (Acetone-Butanol-Ethanol) fermentation. However, the potent toxicity of butanol on the main ABE producers derived from Clostridium acetobutylicum ATCC 824, Clostridium beijerinckii NCIMB 8052, Clostridium saccharoperbutylacetonicum N1-4 etc., is still a main drawback of commercialization as a fuel. Extensive studies have been done only to reach the best titer around 19.2 g/L. In this study, we developed new HTS (High-Throughput-Screening) systems specific to butanol based on designed enzymatic reaction. Along with HTS system, we designed a programmed chemical mutagenesis system suitable for screening strains resistant to membrane toxicity. Using our original HTS system coupled with programmed random mutagenesis, we successfully developed a mutant strain, Clostridium beijerinckii CBP-381, which produced at least 30% more butanol under the optimized fermentation condition.

신정부 출범에 따라 투자대비 성과 미흡한 분야로 항공, 바이오, 에너지가 선정된 바 있다. 구 과학기술혁신본부의 기능이 국가과 학기술위원회에 이관되면서 BT관련 부처에서 수행하는 사업에 대한 예산조정이 이루어졌다. 초기에는 바이오괴담 설이 나올 정도로 대폭적인 예산감소 징후가 있었다. 하지만 관련 부처의 담당공무원 등의 노력으로 우려할 만큼의 예산조정이 이루어지지 않았다. 이러한 일면 속에는 신정부가 바이오에 대한 중요성을 인식하고 있음을 내포하고 있으며, 보다 효율적인 성과창출에 역점을 두고 있는 것 같다. 이명박 정부의 바이오관련 정책방향과 추진전략, 그간의 바이오현황과 문제점에 대한 소개로 공감대를 형성하고자 한다. 또한, 정부의 육성방향과 바이오 기술과 산업 발전을 위해 연구자를 비롯한 바이오종사자들이 어떻게 해야 하며, 그 대응방안에 대해서 고민하고자 한다. BT는 우리나라가 세계 속에서 잘 사는 기술 강국으로 가기위한 성장동력이며, 국민의 복지와 건강에 필수적인 기술 분야이다. 앞으로 정부가 지속적으로 지원할 수 있도록 실질적인 성과 창출을 이룩하고, 이를 통해 바이오를 국가기간산업으로 발전시켜 우리나라 경제성장의 핵심분야로 자리매김하여야 한다.

Assurance of product quality is derived from careful attention to a number of factors including selection of quality parts and materials, adequate product and process design, control of the process ,and in-process and end-product testing. Due to the complexity of today’s medical products, routine end product testing alone often is not sufficient to assure product quality for several reasons, limited sensitivity, destructive testing. Process validation should be performed to assure quality of product through life cycle approach.

The impurities of a drug substance and drug product should be controlled. This presentation is intended to provide guidance for registration applications on content and qualification of impurities in new drug substances. Impurities in new drug product and drug product are addressed from two perspectives: Chemistry aspects include classification and identification of impurities, reporting generation, listing of impurities in specifications, and brief discussion of analytical procedures; and Safety aspects include specific guidance for qualifying those impurities.

The objective of this presentation is to provide basic information necessary to produce sterile products by aseptic processing. Aseptic processing isprocess by which a sterile (aseptic) product is packaged in a sterile container in a way which maintains sterility. It is not only any technique but an integrated art through validations & risk assessment. It is included from establishing building and facility, process design, EM control, personnel training, disinfectant method, validation & qualification to CCM (closure and container monitoring method). It is important to make application of aseptic process practically after setting up each process through validation, for example AVP and autoclave PQ and so on. So this presentation will put emphasis on the nexus between the system & validation (risk assessment).

Single-use disposable systems offer increased flexibility and versatility while mitigating the risk of cross-contamination during biopharmaceuticals manufacturing in a multi-product facility. In this presentation, the rationales for use of disposables in API manufacturing, suitable applications for single-use manufacturing, and its practice examples in KBCC will be introduced.

의약바이오산업은 비약적으로 발전하고 있으며, 세계시장도 급속도로 증가하고 있어 국내 바이오의약품을 생산하는 업체의 지원이 절실히 필요한 실정입니다. 산업체를 지원하는 방법에는 여러 수단 들이 있을 수 있지만, 그중에서도 세계시장 점유를 위해서는 표준에 대한 지원도 절실히 필요한 실정입니다. 따라서, 본 발표에서는 표준의 필요성 및 중요성, cGMP 기술의 표준화를 국내 도입시의 문제점과 이를 해결하기 위한 방안 및 표준화 정책 등을 제시하고자 합니다.

Aromatic nitro groups are found in diverse types of important antibiotics, but the biosynthesis is poorly understood. Spectinabilin with antiviral and antimalarial activities is one of nitrophenyl-containing compounds. Here, we cloned and characterized a spectinabilin biosynthesis gene cluster of 45.4 kb from Streptomyces spectabilis and heterologously produced spectinabilin in Streptomyces lividans. Fourteen genes including type I modular polyketide synthases are expected to be related to spectinabilin biosynthesis. Remarkably, an iterative modular type I polyketide synthase is involved in the biosynthesis of spectinabilin. An ADC lyase with p-aminobenzoate N-oxygenase and pABA synthase catalyzes the generation of a rare p-nitrobenzoate start unit. An additional bimodular polyketide synthase catalyzes two additional successive chain extensions, compared to aureothin cluster. The results provide insights of biosynthetic pathways of other spectinabilin derivatives.

Oxanine (Oxa), generated as one of the major products from guanine by nitrosative oxidation, has been studied as a mutagenic lesion [1]. Here, Oxa was focused in terms of its unique property to react with NH2 group. Especially, Oxa having an O-acylisourea structure was explored to see if its reactivity with amino group is useful in DNA microarray fabrication. The nucleotide unit of oxanine (Oxa-N) was incorporated into the 5'-end of probe DNA by the chemical synthesis method [2] and the synthetic probe DNA was spotted onto the NH2-functionalized glass slide. It was found that the Oxa-DNA probe was immobilized on the glass surface by one-pot reaction, producing the high-efficiency of the target hybridization. Thanks to activation-free reactivity of Oxa, the post-spotting treatment can be performed under the mild conditions (at 25 or 42C), which could be favorable for maintaining effective hybridization performance of the immobilized DNA probe. Further, it has been determined under the mild conditions that the humidity and time of the post-spotting treatment, pH of the spotting solution and the synergistic effects with UV-irradiation largely contribute to the desired immobilization and resulting target recognition [3]. Immobilization of DNA oligomer by use of Oxa-N on the NH2-functionalized surface without any activation step can be used as one of the advanced methods for generating DNA-conjugated solid surface.

Biological hydrogen production, using microorganisms, is an exciting field of technology development that offers potential production of usable hydrogen from a variety of renewable resources. Microbial hydrogen production is mainly based on H2 evolving enzyme, hydrogenase, specifically [Fe] hyrogenase. However, [Fe] hydrogenase has substantial limitation because of its oxygen-sensitive property. Thus in this work, we focused on [NiFe] hydrogenases as another target enzymes for microbial H2 production. Even they have relatively lower activity to [Fe] hydrogenase, they have high oxygen tolerance. This property is important to build more robust process for biohydrogen production. As a model system, recombinant E. coli which expressed [NiFe] hydrogenase was investigated for biohydrogen production. As target enzymes, [NiFe] hydrogenases from E. coli and H. marinus was cloned and expressed in E. coli BL21 because it is a non H2-producing strain. Up to now, it is generally known as [NiFe] hydrogenase is involved in H2-uptake process. However, in this work, E. coli BL21 expressing [NiFe] hydrogenase produced hydrogen actively compared to wild type BL21. Recombinant [NiFe] hydrogenase was also isolated as extract form or purified form and in vitro activity was investigated. Even [NiFe] hydrogenase origated from E. coli has oxygen tolerance, that from H. marinus has more highly tolerant property to oxygen. It showed 2-fold higher activity than E. coli [NiFe] hydrognease under normal atmospheric condition.

Basic characteristics of astaxanthin and H aematococcus pluvialis cracked cell, including solubility and stability were investigated, and the extraction conditions were optimized for the astaxanthin production from H . pluvialis cracked cells. Acetone was selected as the best solvent for the extraction of astaxanthin from H . pluvialis. Response surface methodology (RSM), based on a two-factor interaction model, was applied, and optimization of the extraction parameters such as β-glucanase concentration and sonication time was performed. Astaxanthin yields was the highest when it was extracted after H . pluvialis cell was treated by β-g lucanase(2%) and the sonication w as carried out spontaneously with the extraction for 75 min. Additionally, inclusion complexes of astaxanthin with various cyclodextrins were prepared and characterized with respect to water-solubility and stability. The best host material for the inclusion of astaxanthin was selected as β-cyclodextrin. The optimum ratio of astaxanthin and cyclodextrin for the inclusion complex formation was found to be 1 : 200. As a results, the solubility of the inclusion complex (As-β-CDIC) was about 110 times higher than free astaxanthin at pH 6.5 and 25℃, respectively.

The main focus of the present study was to develop an efficient heterologous host for the production of valuable macrolide antibiotics using Streptomyces venezuelae. The S. venezuelae strain is amenable to genetic manipulation and has fast growth rate characteristics, thus it required a short culture period for metabolite production compared to other Streptomyces species. In the pikromycin PKS deletion mutant of S. venezuelae, tylosin polyketide synthase (PKS) was heterologously expressed using two compatible low-copy plasmids. The mutant strain produced 0.5 mg/l of the 16-membered ring macrolactone, tylactone and a small amount of its derivative, desosamine-glycosylated tylactone. To improve the production level of tylactone and desosaminyl tylactone, additional copy of the positive regulatory gene, pikD, was introduced into the pikromycin PKS deletion mutant of S. venezuelae expressing tylosin PKS genes, leading to 2.7-fold and 17.1-fold enhanced production, respectively. Another approach to improve the productivity of tylactone was to enhance ethylmalonyl-CoA pool, which is not used to generate the native polyketide, pikromycin but required the biosynthesis of tylactone. Genome analysis of S. venezuelae revealed that the crotonyl-CoA pathway genes involved in the biosynthesis of ethylmalonyl-CoA are located in the genome of S. venezuelae. RT-PCR and CoA analysis showed that L-lysine triggered the expression of crotonyl-CoA pathway genes and led to an increase of the ethylmalonyl-CoA pool and consequently resulted in the enhanced production of tylactone. Disruption of putative ethylmalonyl-CoA mutase gene (meaA) also contributed to overproduction of tylactone. These results introduce S. venezuelae as a rapid heterologous expression system for the production of valuable secondary metabolites.

A bacterial strain, MBBL 2-9, displaying potent antimicrobial activity against Grampositive bacteria, including Staphylococcus aureus, was isolated from vaginal microbiota of healthy woman. Based on its morphological properties and 16S rRNA sequence analysis, the bacterium was identified as Staphylococcus hominis. A new bacteriocin named as hominicin was purified by chloroform extraction, cation-exchange column chromatography and reverse-phase HPLC. Hominicin was heat stable up to 121°C, 15 min and active under conditions of acid/alkali (pH 2.0 to 9.0). Antimicrobial activity was lost after treatment with proteinase K, but trypsin, chymotrypsin, pepsin, and lipase had no effect on hominicin activity. Also, hominicin not showed cytotoxicity on blood agar plate after incubation for 24 h. The molecular mass of purified hominicin as determined by mass spectrometry was 2,038 Da. MS/MS results suggested that hominicin consist of peptidic and non-peptidic moiety. De novo sequencing of peptidic moiety revealed that Ala-Ala-Val-Leu/Ile-Ala-Gly and no identity with previously reported bacteriocins. Plasmid curing result indicated that a plasmid (about 5 kb in size), designated as pSH29, seemed to be responsible for hominicin production. This present study is the first report of a novel bacteriocin, hominicin, produced by S. hominis that has potential for use as an alternative antimicrobial agent for the treatment of infection with Gram-positive bacteria. This work was supported by a grant (20080401034009) from BioGreen 21 Program, Rural Development Administration, Republic of Korea.

The signal sequence of organophosphorus hydrolase (OPH SS) from Flavobacterium sp. is composed of 29 amino acids and has a twin arginine (RR) amino acids sequence in its hydrophobic region near the N-terminal that is analogous to the twin arginine consensus motif of twin arginine translocastion (Tat) pathway. To investigate that OPH SS is dependent on which pathway between general secretion (Sec) and Tat pathways, green fluorescent protein as a Tat substrate and alkaline phosphatase as a Sec substrate were fused with OPH signal sequence, and we compared their expressions and localizations with the cases of TorA signal sequence of Tat pathway and PelB signal sequence of Sec pathway. From the results, we found interesting results because both Tat and Sec substrates were successfully translocated into the periplasmic space with comparable secretion efficiencies compared to the control sequences. We surmised that OPH SS switched its translocation pathway according to type of target protein in Escherichia coli.

Immune system is composed of multiple cells with distinct functions, and immune responses are orchestrated by complex and dynamic cell-cell interactions. Therefore, each cell behavior and function should be understood under right spatio-temporal context. Studying such complexity and dynamics has been challenging with conventional biological tools. Recent development of new technologies such as state of art imaging instruments and microfabrication techniques compatible with biological systems have provided many exciting opportunities to dissect complex and dynamic immune cell interactions; new microscopy techniques enable us to observe stunning dynamics of immune system in real time. Microfabrication permits us to manipulate microenvironments governing molecular/cellular dynamics of immune cells to study detailed mechanisms of phenomena observed by microscopy. Also, microfabrication can be used to engineer microenvironments optimal for specific imaging techniques. In this presentation, I am going to present two examples of how these two techniques can be combined to tackle challenging problems in immunology. Obviously, this strategy can readily be applied to many different fields of biology other than immunology.

The relationship between genotype and phenotype is a central issue in genetics, and approaches are needed that allow us to interpret the increasing collection of data on genotypic variation in terms of the affect on organismal phenotypes. Our understanding of these relationships came historically from forward-genetics and reverse-genetics approaches, which have proved remarkably powerful, but which are still difficult in complex animals. Here, we present a method, network-guided genetics, for predicting gene function and loss-of-function phenotypes that can be applied to extend genetic screens and prioritize candidate genes for focused testing. We demonstrate that genes with similar function or loss-of-function phenotypes are often clustered in functional gene network of yeast, thus allowing the prioritization of new candidate genes by their network connectivity. We tested a large set of available loss-of-function phenotypes from genome-wide assays of yeast, showing predictability for broad range of phenotypes spanning cellular morphology, growth, metabolism, cell-type specific processes, and even quantitative features of cell shape.

Brassinosteroid (BR) and light play key roles antagonistically in photo- and skotophotomorphogenesis of Arabidopsis, but a molecular mechanism underlying their signaling interaction is not yet fully understood. We selected differentially expressed genes (geneset-1) previously well characterized in light or BR signaling. These genes are light-induced and BR-suppressed, or vice versa. To identify crosstalk components between light and BR signaling pathways, we then selected 11 genes (geneset-2) with strong co-expression pattern with geneset-1 during application of various phytohormones or wavelengths of light from Arabidopsis macroarray data by combined P-value analysis. Genetic ablation of a putative phosphatase 2C (PP2C), one of geneset-2, leads to insensitivity to brassinazole (BRZ), BR biosynthesis inhibitor, during etiolation and abscisic acid (ABA) during seed germination. This observation suggests that PP2C is an important modulator for crosstalking among BR-, light-, and ABA signaling pathways. Integrative analysis of microarray data of different conditions enables us to unravel a complex regulatory network of plant signal transduction.

The first thing that comes to mind when trying to bring genes from a list into some biologically meaningfully context is gene ontology(GO), an expert-curated database assigning genes to various functional categories. And there are two ways to carry the analysis beyond GO classification deeper into biology: going to the molecular level, which is promoter and regulatory network analysis, or employing the vast-accumulated knowledge from the literature to carry out pathway analysis. This knowledge is scattered throughout numerous scientific publications. Scientific literature is a source of the most reliable and comprehensive knowledge about molecular interaction networks. Pathway and network analyses are rapidly becoming the mainstream tools for functional interpretation of high-throughput data. Pathway analysis tools are also popular for the analysis of drug action and validation of drug efficacy and toxicity. In this talk we will discuss pathway-centric methods we apply to high throughput, genomic scale experiments. Using several case studies, we will illustrate how pathway-centric methods allow us to extract insights from multiple experiments that may otherwise be overlooked.

The discovery and analysis of interacting proteins are essential for delineating biological networks, the understanding of cellular function and for drug discovery. We have developed a bacterial system for the discovery of interacting proteins that, unlike other 2-hybrid systems, allows for the selection of protein pairs on the basis of affinity or expression. This new technology, called APEx 2-hybrid, relies on the Anchored Periplasmic Expression (APEx)1) of one protein on the periplasmic side of the inner membrane of Escherichia coli and its interacting partner as a soluble, epitope-tagged, periplasmic protein2). After removal of outer membrane and labelling with fluorescent probe, cells expressing protein pairs exhibiting different affinities can be readily distinguished by flow cytometry. APEx 2-hybrid system coupled with multi-color fluorescence activated cell sorting (FACS) enables protein-protein interaction study in periplasm of E. coli as well as high thrroughput screening of highly interacting proteins from combinatorial libraries. Various applications of APEx 2-hybrid system toward antibody engineering (affinity/expression maturation of antibody and antibody fragments, disulfide bond-free intrabody etc.) will be presented.

Through X-omics researches, a lot of genes with differential expression between control and test could be selected. Lots of genes among them are not functionally annotated and identified. From the systems biology viewpoint, there are highly connected biological networks with scale-free topology among biological identities such as genes, mRNAs, proteins, and metabolites. Hence unknown genes could be functionally interacted with known genes through interaction networks. Some kind of interaction networks like transcriptional regulatory networks between transcription factor and its regulons, protein-protein interaction networks, genetic networks among genes, integrative gene-metabolite networks, functional interaction networks based on genome context methods like phylogenetic profiling method, Rosetta stone method, gene cluster method and gene neighbour method, etc could be possibly constructed. In this presentation, some methods to construct biological interaction networks will be explained and some kinds of interaction network maps using Cytoscape will be shown. And also some gene mining based on various kinds of biological interaction networks for the development of industrial microorganisms will be demonstrated.

Glycerol is an abundant, inexpensive, and highly reduced molecule generated as inevitable by-product of biofuels production. Its use as carbon source in fermentation processes offers the opportunity to produce reduced chemicals at yields higher than those obtained with the use of common sugars. Fully realizing this potential, however, would require the metabolism of glycerol in the absence of electron acceptors. Unfortunately, fermentative utilization of glycerol is restricted to a small group of microorganisms, most of them not amenable to industrial applications. The synthesis of 1,3-propanediol (1,3-PDO) by these organisms has long been considered the metabolic property that determines their ability to ferment glycerol. We have recently discovered that E. coli can ferment glycerol in a 1,3-PDO-independent manner. We identified 1,2-propanediol (1,2-PDO) as a fermentation product and established the pathway that mediates its synthesis as well as its role in the metabolism of glycerol. Based on our findings, we proposed a new model for the fermentative utilization of glycerol in which: (1) the production of 1,2-PDO provides a means to consume reducing equivalents generated in the synthesis of cell mass, thus facilitating redox balance, and (2) the conversion of glycerol to ethanol, through a redox-balanced pathway, fulfills energy requirements by generating ATP via substrate-level phosphorylation. Other auxiliary or enabling pathways facilitating this metabolic process were identified along with the culture conditions triggering them, and therefore facilitating glycerol fermentation. We have also shown that this model for the 1,2-PDO-dependent fermentation of glycerol is valid for other microorganisms. The knowledge base created by the aforementioned studies has been instrumental in the implementation of metabolic engineering strategies to convert glycerol to a variety of fuels and reduced chemicals, including ethanol, hydrogen, formate, succinate, and 1,2-PDO. We will present at the meeting our latest results in the understanding of this new metabolic competency and the metabolic engineering of these microorganisms to produce different chemicals from glycerol.

Concerns about the pollution and global warming, as well as the depletion of fossil fuel reservoir necessitate search for renewable energy sources. Mono-alkyl esters of long chain fatty acids, termed as biodiesel, are promising alternative to petroleum-based fuels. They are produced from triacylglycerides contained in plant oils, animal fats, and waste oils through transesterification with alcohols. Currently, biodiesel is commercially produced by chemical processes using hydroxides or alcoholates catalysts. However, they have several problems. First of all, in the chemical process, water and free fatty acids in raw materials and catalyst preparations should be completely removed to prevent the formation of soaps. Furthermore, the alcoholate catalysts are highly flammable and toxic. There have been constant efforts to replace the catalysts with “greener” ones like lipases. Lipases can convert both triglycerides and free fatty acids to alkyl esters even in the presence of water, providing more flexibility in feedstock selection. In addition, using lipase can minimize the handling of hazardous chemicals and simplify the downstream processes. In this presentation, recent progresses in the research project aiming to make the enzymatic biodiesel production more economic and competitive will be summarized. Focus will be placed on the prevention of enzyme inhibition, supercritical processes for oil extraction and enzymatic transesterification, and design of lipases for high stability.

3-Hydroxypropionic acid (3-HP) is an important platform chemical having diverse industrial applications, but its biological production has not been well studied. We have developed various recombinant Escherichia coli biocatalysts for the production of 3-HP from glycerol, and demonstrated that the strain E. coli SH254, expressing glycerol dehydratase (dhaB1, dhaB2, and dhaB3) and aldehyde dehydrogenase (aldH), could effectively produce 3-HP at 6.5 mmol 1  . The recombinant E. coli SH254 strain was further improved by changing the expression vectors for glycerol dehydratase and aldehyde dehydrogenase and cloning the additional genes (gdrAB) which encode dehydratase reactivase. The resultant recombinant SH-BGA1 exhibited improved activities of the two relevant enzymes in vivo and a higher 3-HP titer of 51 mmol 3-HP 1  in shake-flask conditions. When a pH-stat, fed-batch culture was conducted in 5-L bioreactor, the recombinant E. coli SH-BGA1 produced 3-HP at 346±20 mmol or 31±1.8g 3-HP per liter in 72h. The final yield of 3-HP was 0.35 mol mol  glycerol. The maximum specific rate of 3-HP production was estimated at 1.43 mmol g  cdw h   between 8 and 72 h. Here, for the first time, we report the high titer of 3-HP production using the recombinant E.coli SH-BGA1.

We have developed a novel strategy for rapid, combinatorial optimization of hot spots using the lipase B from Candida antartica (CalB) as a model enzyme. After combinatorial randomization of target locations that are known to affect the enzymatic activity of CalB, individual variant genes isolated in the E.coli cells were PCRamplified and expressed in a cell-free protein synthesis system to analyze the enzymatic activity of the CalB variants. Through an extensive expression screening, we were able to find a series of variant CalB enzymes whose activity was improved as much as 20 folds compared to that of wild-type enzyme. Furthermore, similar extent of increase in enzymatic activity was observed when the selected clones were expressed in Pichia pastoris indicating that amino acid substitutions give similar effects to the variant enzymes either in vivo or in vitro. Based on these results, we expect that the proposed strategy can be used as a powerful platform for engineering the enzymes of industrial importance.

Glycerol carbonate is a key bifunctional compound employed as solvent, additive, monomer, and chemical intermediate. Lipase originated from Candida antarctica (CALB) was used to produce the benign monomer glycerol carbonate from renewable starting materials: glycerol, triglyceride and dimethyl carbonate. Various organic solvents including t-butanol and tetrahydrofuran were used as the reaction medium to enhance the formation rate of glycerol carbonate and conversion approached quantitative yield. Removal of methanol produced as byproduct during reaction increased the yield of glycerol carbonate. The total amount of by-products was minute compared with that of glycerol carbonate, which means that lipase-catalyzed reaction is very regioselective for the synthesis of glycerol carbonate.