Earticle

바이오벤처기업인 크레아젠㈜가 개발한 수지상세포를 이용한 항암 세포치료제인크레아박스-알씨씨(CreaVax-RCC)가 세포치료제로는 세계 최초로 품목허가를획득하고 시판되었다. 이에 따라 크레아젠은 전세계에서 첫 번째로 수지상세포를이용한 항암 세포치료제를 상품화한 기업이 되었다. 크레아박스-알씨씨는 환자의 면역세포로 항암면역을 유도하여 암을 치료하는 신개념의 생물의약품이다. 즉 환자자신의 말초혈액 단핵세포(PBMC)로부터 수지상세포를 분화시킨 후 환자에서 적출한 암세포 분쇄물을 수지상세포에 감작하여 활성화된 수지상세포를 주사제 형태로 환자에게 다시 투여하는 환자유래 세포치 료제이다. 수지상세포 항암치료제인 크레아박스-알씨씨의 특징은 다음과 같다. 1. 뛰어난 생명연장 효능 (평균생존기간 28.9 개월) 2. 부작용 없는 항암치료제 (Grade 3-4 독성 없음) 3. 삶의 질(Quality of Life)을 유지하는 항암치료제 (통원 치료) 4. 기억면역 유도로 암의 전이 및 재발을 억제하는 근원적 치료제 크레아젠은 신장암(신세포암)을 첫 번째 적응증으로 치료제를 개발하였다. 신장암 세포치료제로 수지상세포 기반기술의 효능이 증명될 경우 타 암종에 대해서도 비교적 용이하게 적응증을 확대할 수 있다. 즉, 환자의 혈액에서 단핵세포를 추출하여 수지상세포로 분화시키고 암항원을 감작시켜 항암 세포치료제를 만드는 기본 과정은 동일하다. 다만 어떠한 암항원을 감작시키느냐에 따라 치료제의 적응증이 달라진다. 크레아젠은 전립선암치료제인 크레아박스-피씨(CreaVax-PC)를 개발하여 2006년 7월 식약청의 임상승인 (IND)을 받았으며 현재 국립암센터와 삼성 서울병원에서 임상을 준비 중에 있다. 또한 간암치료제인 크레아박스-에취씨씨(CreaVax-HCC)및 유방암치료제인 크레아박스-비씨(CreaVax-BC)가 전임상 시험을 진행 중에 있으며 늦어도 내년 초에는 임상 진입을 목표로 하고 있다. 한편 면역 조절용 수지상세포를 이용한 류마티스관절염 치료제도 동물실험에서 좋은 성적을 보여 전 임상시험을 준비 중에 있다. 수지상세포를 이용할 경우 현재까지 마땅한 치료제가 없는 류마티스관절염에 대해서도 조절면역을 유도하여 병을 근원적으로 치료할 수 있는 생물의약품을 개발할 수 있을 것으로 기대하고 있다.

기후 변화와 자원 고갈이 인류의 현안으로 대두되면서 극지는 지구상의 유일한 미개발 , 비오염 지역으로서 지구환경 변화를 연구할 수 있는 최고의 실험장이자 마지막 남은 인류자원의 보고이다. 이러한 극지의 중요성을 인식한 세계 각국에서는 막대한 재정과 첨단기술를 투입해 개발에 경쟁을 다하고 있다. 우리나라도 남극의 세종기지와 북극의다산기지를 거점으로 극지의 과학탐사 및 연구 활동을 활발히 수행 중이다. 극지에는 유전 및 가스와 같은 지하자원외의 극지해양생태계가 포함하는 막대한 양의 생물자원을 가지고 있다. 미생물학자로서 극지의 유용생물자원을 개발하기 위해서 새로운 해양미생물과 극지미생물을 탐색하고 산업적으로 활용 가능하도록 연구해온 지난 20 여년 동안의 활동을 소개하고자 한다.

The conventional live or killed vaccines, though quite successful, have a few shortcomings which make a chance to develope new kinds of vaccines1). For some reasons, new approaches are being considered for vaccine development, which are not based on the entire organism. These include recombinant vaccines, DNA vaccines, peptide vaccines and therapeutic vaccines. I will mainly focused on recombinant vaccines are created by utilizing bacteria or yeast to produce large quantities of a single viral protein. Up to now, recombinant vaccines licensed in our country are hepatitis B vaccine and HPV vaccine. The success of the first VLP vaccine in humans, the yeast-grown HBV vaccine, was encouraging for the development of additional VLP vaccines. The subsequent successful immunization programs for HPV suggest that VLP made by expression of recombinant capsid proteins may be a platform technology for all non-enveloped viruses that emerge as candidates for vaccine protection2). I will also mention a recent direction, namely, the development of recombinant virus vaccines as well as in manufacturing and quality control for recombinant vaccines according to related guideline published by KFDA.

성공하는 여성 리더가 되기 위한 개인 내적 요인 및 사회적 요인을 알아본다 실제 . 현장에서 학습하고 적용하고 있는 리더십 이론의 상당 부분이 남성 리더십 이론에서 남성 부분을 여성으로 치환한 것인 경우가 많다. 효과적이고 효율적인 여성 리더의 필요성이 요구되는 현시점에서 남성 리더십의 대척점으로서의 여성 리더십이 아니라 여성의 생물학적, 심리적, 사회적 특성을 부가적으로 고려한 리더십이 필요하다. 또한 여성리더간의 조화로운 균형과 통합을 위한 마음 갖춤새에 관하여 알아본다.

Gene therapy products are products in which nucleic acids are used to modify the genetic material of cells. Gene therapy products can be broadly classified on the basis of their delivery system. Means for delivering gene therapy products include viral vectors, nucleic acids(naked DNA), or nucleic acids formulated with agents, such as liposomes, that enhance their ability to penetrate the cell. Current gene therapy is experimental and has not proven very successful in clinical trials. Little progress has been made since the first gene therapy clinical trial began in 1990. Since gene therapy products contain genetic and other biological materials, many of the safety considerations, issues and principles. In all cases the product should be sufficiently characterized to provide reassurance that the preclinical studies have been conducted with material that is representative of that to be administered to humans in the clinical studies. The potential impact of any modifications to the manufacturing process and the test material during the development programme, for the extrapolation of the animal findings to humans should be considered.

A bacterial strain isolated from soil for its potential to control fungal plant pathogens was identified as a Bacillus subtilis. Bacillus subtilis CMB32 produced antifungal agents on M9 broth at 30°C. Biosurfactant lipopeptides produced by Bacillus subtilis CMB32 were precipitated by adjusting to pH 2 and extracting using chloroform/methanol, and then were purified using column chromatography and reversed-phase HPLC. Molecular masses of the lipopeptides were estimated by MALDI-TOF mass spectrometry as (a) 1080, (b) 1486, and (c) 1044 Da, respectively. They had cyclic structures and amino acid compositions of (a) Pro, Asx, Ser, Tyr, Glx, (b) Glx, Tyr, Thr, Ala, Pro, Ile, and (c) Glx, Leu, Val, Asx, respectively. Further analysis revealed that Bacillus subtilis CMB32 produced three antifungal lipopeptides: (a) iturin A, (b) fengycin, and (c) surfactin A.

Biopharmaceutical industry was designated as one of the Korea's ten future growth industries and developing biological new drugs itself is high technology. The biological products arising from their natures request the compliance with the strict manufacturing and quality control standards for commercialization. Considering this, many countries including Korea have developed and complied with separate GMP(Good manufacturing Practice) for biological products in addition to GMP for conventional pharmaceutical products. Today, I am going to briefly explain about Korea's GMP for biological products and related policy agendas in the future. First, I will review the definition, history of GMP and the legal framework of GMP system in Korean pharmaceutical laws and regulations. Next, I will discuss the point when the GMP Should be applied to during the process of the research & development, pre-clinical trials, clinical trials, registration of products and licence approval of biological products and the reason for these applications. The difference of biological and conventional pharmaceutical products in applying the relevant regulations, scope of GMP for biologicals, additional points to consider in GMP for biologicals compared with conventional products and the major inspection check points & the most common inspection findings will be dealt with. Finally, the initiative policies and their progress for GMP for biological products by us, Biologics Management Team will be introduced.

GMP란? 품질 즉, 안전성, 안정성, 유효성이 보증된 의약품을 재현성있게 제조하기 위하여 제조시설 및 설비, 원자재구입, 제조, 포장, 출하까지 생산공정 전반에 걸쳐 충분한 조직적 관리를 하기 위한 필요 요건을 명기해놓은 규정로 정의 되어진다. 이러한 GMP는 미국에서 1937년 “Elixir Sulfanilamide”의 독성으로 인하여 107명이 사망한 뒤로 안전성자료의무화 법을 시행하면서 1953년 실사 의무화 FDA 483 form 법적 효력을 가지고 1962년에 의약품의 유효성을 입증하는 것이 의무화되면서 GMP가 점점 강하게 적용 되었다. 현재는 프로세스 분석 기술 (PAT)를 거쳐 의약품 개발에서의 GMP 적용 및 관리가 Quality Risk Management의 개념을 도입하여 적용 하고 있다. 의약품 개발의 전체를 시스템적으로 관리 감독하는 것 뿐만 아니라 위해요소평가를 통하여 위험을 예방하는 측면이 강하게 대두되고 있다. GMP는 단순히 의약품을 제조하는 생산 사이트에만 적용되는 것이 아니라 의약품의 연구 개발 단계부터 필요 부분 적용해야 하며 허가 단계별 GMP적 관리가 강화 되어야 한다. 연구개발 단계에서는 기초데이터의 철저한 기록, 분석기기의 정기적 검교정 실시, 분석법 밸리데이션, 시험약 관리, 안정성 시험 수행, 기술이전 수행 등에 대하여 GMP개념을 적용할 수있다. 또한 허가의 임상시험 단계별 시험약제조 및 관리에 대해 GMP적용이 강화되어 임상3상 시험약은 full GMP 시스템 하에서 생산해야 한다.

테고사이언스(주)는 세계수준의 세포배양 기술을 기반으로 화상, 피부궤양, 백반증, 선천성 반점 및 각막손상 등에 효능을 가진 피부세포치료제를 연구, 생산, 제공하는 전문 생명공학기업이다. 2001년 3월에 처음 설립되었으며, 국내에서 최초로 전문의약품인 자기유래배양피부와 동종유래배양피부 제품을 개발하여 상용화하는데 성공하였다. 이와 같은 회사의 설립배경과 회사에 대한 소개를 통하여 실험실에서의 연구내용이 환자에게 직접적인 도움이 되고 있는 그동안의 경험을 나누고자 한다.

Nowadays, the consumption of botulinum toxin products has been steeply increased in both clinical objective and cosmetic objective. Also the indication of botulinum toxin has been expanded over cervical dystonia to prostatism , migrein and other nuerological disease such as Parkinson's disease. In Korea, four products of botulinum toxin are launched in market and several companies are trying to acquire the approval of their new botulinum products. To develop and provide safe and effective products, it is very important to control the quality of products at several stages such as initial researches, development, production, and lot release testing. In this presentation, first the status and prospece of botulinum product will be introduced. Secondly, how to control the quality of botulinum products will be showed in the regulatory perspective of KFDA.

Versatile technology for manipulation of chromosome is playing an increasingly important role in functional analysis of genomes. Here we describe a simple but innovative method to split chromosomes in Saccharomyces cerevisiae, which we call PCR-mediated chromosome splitting (PCS). The PCS method combines a streamlined procedure (two-step PCR and one transformation per splitting event). Moreover, this method was applied to analyze yeast rDNA-concerned cellular physiology. The rDNA cluster in S. cerevisiae is located in chromosome XII and consists of ca.150 tandemly repeated copies of a 9.1-kb rDNA unit. To explore the biological significance of this specific chromosomal context, chromosome XII was split at both sides of the rDNA cluster by using PCS method and strain harboring chromosome XII consisting of rDNA chromosome (1,500-kb rDNA cluster only) was created. In the strain harboring the rDNA chromosome, the copy number and size of rDNA cluster were decreased and life span also was shorted. These observations suggest that the context of chromosome XII is important for rDNA function and the splitting method is useful tool to study the functional analysis of eukaryotic genome.

Bacterial community shift was examined in relation with changes in process performance throughout a lab-scale anaerobic reactor treating whey permeate. The operation was successfully performed with the complete mineralization of initial substrate after 42 days of incubation. Bacterial and archaeal population dynamics were quantitatively monitored using real-time quantitative PCR (qPCR). Bacterial community structure was investigated using denaturing gradient gel electrophoresis (DGGE) based on 16S rRNA gene. An Aeromonas-like microorganism was supposed to be responsible for the rapid fermentation of carbohydrate. Several Clostridium-related bacteria with putative roles in carbohydrate fermentation, lactate oxidation, ethanol oxidation, or homoacetogenesis were found. The statistical analysis of the DGGE profiles provided a comprehensive insight into changes in bacterial community structure. The bacterial community in the reactor shifted anticlockwise and returned close to the initial location on the non-metric multidimensional scaling (NMDS) plot. In conclusion, these results suggest the possibility of process diagnosis and performance prediction by monitoring bacterial community1).

Previously, we designed and constructed a hybrid of the mussel adhesive protein (MAP) fp-151, which is a fusion protein with six type 1 (fp-1) decapeptide repeats at each type 5 (fp-5) terminus. Through various cell-adhesion analyses, we previously demonstrated that fp-151 has the potential to be used as a cell or tissue bioadhesive. In the present study, to improve the cell-adhesion properties of fp-151, we designed a new cell-adhesive protein, fp-151-RGD, which is a fusion with the GRGDSP residues, a RGD peptide sequence that has previously been identified at the cell-attachment site of fibronectin, at the C-terminus of fp-151. Although recombinant fp-151-RGD maintained the advantages associated with fp-151, such as a high production yield in Escherichia coli and simple purification, it showed superior spreading ability, which is important for cell proliferation under serum-free conditions, as well as better cell-adhesion ability compared with other commercially produced cell-adhesion materials such as poly-L-lysine (PLL) and the naturally extracted MAP mixture Cell-Tak.

The biotechnology industry demands new and high-performance online measurements to identify important process variables. The ultrasonic measurement in combination with mathematical models provides online control of bioprocesses. Characteristically, yeast cultivations show different process phases such as glucose, ethanol and acetate consumption which can be identified via ultrasonic velocity measurements. In addition, a prediction was carried out with measured and simulated data and thus, the process variables were calculated. Because the ultrasonic signals depends on several unspecific variables, this research uses theoretic and chemometric models to correlate the online measurement of the ultrasonic velocity with the offline measurements of the process variables. The prediction of different process variables were performed by so-called software sensors, which are based on multilinear regression, principal component analysis, extended Kalman filter and neural networks. The simulated values and the measurements of ultrasonic velocity were used for the evaluation as well as the prediction of ethanol concentration.

White spot syndrome virus (WSSV) is a major disease causing virus to shrimp. WSSV infection causes high mortality and large economic loss in shrimp culture industry.1) The objective of this study was to clone WSSV envelope protein gene and to express the protein for the development of vaccines against WSSV. The virus was consist of at least five major proteins VP19, VP26, VP28, VP281 and VP466.2) The envelope protein VP466 was cloned into a constitutive expression vector, pHCE II B and cold-shock expression vector, pCold I. The recombinant plasmids were transformed into E.coli BL21 and RIPL. Product of pCold-VP466 was purified by affinity chromatography using His-tag column (Amersham Biosciences). Recombinant proteins VP466 were used to vaccinate shrimps against WSSV. Recombinant proteins VP466 were injected into Penaeus chinensis shrimps intramuscularly. At 1 week after injection, the shrimps were challenged. Positive control, crude rVP466 (product of pHCE-VP466) and purified rVP466 (product of pCold-VP466) showed a final cumulative mortality of 100%, 90% and 61%, respectively. Purified rVP466 has high survival ratio than crude rVP466. This result showed that protection can be obtained in shrimp against WSSV using rVP466 protein as a recombinant protein vaccine.

Laboratory evolution has become a commonly used approach to address many of the fundamental long-standing questions about evolution as well as to provide an experimental basis for delineating the mechanistic basis of adaptive evolution, such as antibiotic resistance. We have got 7 independent glycerol evolved strains from wild type E. coli K-12 MG1655 by laboratory evolution. We have fully resequenced 5 independent glycerol evolved strains using the microarray-based Comparative Genome Sequencing service provided by Nimblegen Systems Inc. Surprisingly, one of them has only three mutations of whole genome in the endpoint. We added two mutations out of them into the wild type strain by site-directed mutagenesis. The double knock-in mutant, which had mutations in glpK and rpoC, had similar specific growth rate of the evolved strain. The two mutations thus seem to fully be responsible for the growth rate change during adaptive evolution. For intracellular metabolome analysis, we used capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). The double knock-in mutant and the evolved endpoint strain showed very similar intracellular metabolite profile at mid exponential phase. Especially, concentrations of N-Carbamoyl-L-aspartate and N-Acetyl-L-aspartate, which participate in alanine and aspartate metabolism, were significantly decreased.

Soy sauce is mainly used as a salty condiment in Eastern Asia. It is containing lipids which have an effect on its storage because the lipid in food is easily deteriorated by oxidation with atmospheric oxygen.1) Consumer's various demands about functionality and public fancy of soy sauce isn't satisfied. Therefore the development of technology on long-term storage for soy sauce is needed and very important. It is also necessary to produce a soy sauce containing functional materials. 2) Supercritical fluid extraction(SFE) has distinct advantages for removal of liquids because of the variable selectivity of the process, use of non-toxic solvent. A supercritical fluids as solvents, especially carbon dioxide(CO2), offers new opportunities for the solution of separation problems. By using a countercurrent extraction column, the sample is exposed to a greater contact area between the liquid and the CO2.3) The objective of the present work is to research the change of chemical properties of the soy sauce treated countercurrent SCO2(C-SCO2) and is to investigate antioxidation and haracteristics adding natural functional compounds extracted from natural resources such as crab shell, brown seaweed and wasabi by SCO2.

Cell-free protein synthesis is a versatile tool for the rapid and parallel translation of genetic information into protein molecules1. However, the potential of this technology can only be fully exploited when protein molecules can be produced at high productivity and low cost. This study focuses on the development of a cost-effective and highly productive cell-free protein synthesis system derived from Escherichia coli. We developed a new scheme for supplying energy using dual energy sources to enhance the regeneration of ATP and lower the rate of phosphate accumulation2. In the proposed scheme, where creatine phosphate and glucose were simultaneously used as the energy sources, the phosphate released from the creatine phosphate was subsequently used in the glycolytic pathway for the utilization of the glucose, which enhanced the ATP supply and reduced the rate of inorganic phosphate accumulation. When tested against different proteins, the developed method produced 2-3 times more protein than the conventional ATP regeneration methods using single energy sources.

To investigate the effect of low culture temperature on cell growth and recombinant protein production, Chinese hamster ovary (CHO) cells producing erythropoietin (EPO) were cultivated between 25ºC to 37ºC with perfusion mode. CHO cells were cultivated at 37ºC until viable cell concentration reached 1 x 107 cells/mL. After that, temperature was shifted to 25ºC, 28ºC, 30ºC, 32ºC, 37ºC (control), respectively. Among five perfusion cultures, perfusion at 32ºC seems to be optimal since it showed the highest specific EPO productivity (qEPO) and cumulative EPO production. In addition, the heterogeneity, antennary structure and charge profile of EPO were investigated after the secreted EPO during perfusion culture at various culture temperatures was purified with ion exchange chromatography. A detailed result will be discussed.

This paper addresses biosynthetic responses of gonadotropes, which utilizes several signal transduction pathways to mediate gonadotropin-releasing hormone (GnRH)-mediated signaling from brain1). Experimental studies for the biosynthetic response of the gonadotrope to varying GnRH concentrations showed that the overall average level of ERK activation in populations of cells increased non-cooperatively with increasing GnRH. However, it is observed from the single-cell responses that individual gonadotropes exhibited two response states, inactive and active, while both the probability of activation and the average response in activated cells increased with increasing GnRH concentration. These data indicate a hybrid single-cell response having both digital (switch-like) and analog (graded) features2). In order to provide a plausible mechanism for the hybrid response, we have employed mathematical modeling approach based on the detailed kinetic mechanism, and it was found that the hybrid response can be explained by cell-to-cell variations for total concentrations of proteins as well as indirect thresholding of ERK activation resulting from the distributed structure of the signaling network. In addition, on the basis of the developed model, we investigated the propagation of cell to cell response variation through this pathway and found that the noise scales in proportion to the average response as observed in the experimental data.

Biokinetics of Aeromonas hydrophila utilizing glucose in an anaerobic biosystem were evaluated. A sequential approach with a fitting specific growth rates to the initial glucose concentrations followed by a solving differential equations numerically using the 4th order Runge-Kutta method was used to estimate the parameter values.1 During the batch culure of Aeromonas hydrophila a complete mineralization of glucose, 5000 mg L-1, occurred in approximately 34 hours of incubation. Ethanol, acetate, succinate, and formate were the metabolic products. The hypothetical maximum growth rate, μm, half saturation coefficients, Ks, microbial yield, Y, cell mass decay rate, kd, and substrate inhibition coefficient, Ksi, were evaluated as being 0.25 ± 0.03 hr-1, 118 ± 31 mg glucose L-1, 0.12 μg DNA mg glucose-1, 0.01 hr-1, and 3108 ± 1152 mg glucose L-1, respectively. Using the estimated parameters, the theoretical performance of the continuous operation with an influent glucose concentration of 5000 mg L-1 was also illustrated. The expected glucose utilizing efficiencies were greater than 95% at 7 hours or longer HRTs. Aeromonas hydrophila was expected to be washed out at 6.7 hours of HRT.

It has been reported that the acidogenesis was very important due to the fact that volatile fatty acids generated through this process, especially acetate and butyrate, were preferable substrates for methanogens. For this process of swine wastewaster with ca. 40,000 mg/L of COD, a series of experiments were carried out with laboratory-scale continuously stirred tank reactors to obtain short chain volatile fatty acids. The profile of total volatile fatty acids production was investigated with simultaneous variations in hydraulic retention time (HRT) (0.5 to 2.5 days) and temperature (40 to 60?). Response surface methodolgy made an application successfully to approximate the responses of the production of total volatile fatty acids. The optimum physiological conditions the production were 2.0 days HRT and 50? using the quadratic model. The calculated model output value of volatile fatty acids production at the optimum condition was 1,830 ± 230 mg/L. These results exhibited that the optimal condition induced by thermophilic microorganisms was different from that by mesophilic microorganisms.

To test impact of commercial nanomaterials on environment and human health, the potential eco-toxicity of nanosized silver powder was investigated with bacteria, which contains Gram-negative Escherichia coli and Gram-positive Bacillus subtilis, and yeast strain. Commercial product of silver nanopowder prepared in water contains both silver nanoparticles (AgNPs) and silver ions. AgNPs and Ag ion contents were measured by ICP (Inductively Coupled Plasma) and ISE (Ion Selective Electrodes). Toxic effects to cell after direct exposal with silver powder solution were confirmed by counting colony forming units (CFU) on agar plates and by images of transmission electron microscope (TEM).

Escherichia coli heat-labile enterotoxin B subunit (LTB) is known not only to induce strong immune responses but also to be an efficient mucosal adjuvant towards either co-administrated unrelated antigens or tolerogenic T-cell epitopes. Although it has already been expressed in several different systems including prokaryotic as well as eukaryotic organisms, studies on the synthesis of the LTB into oligomeric structure of pentameric size in budding yeast Saccharomyces cerevisiae has been limited. In these studies, with a functional signal peptide of amylase 1A protein from rice, the yeast-expressed LTB was targeted to endoplasmic reticulum to oligomerize with the expected size of pentamer. The xpression and assembly of recombinant TB was observed in both cell-free extract as well as culture filtrate of recombinant strain by Western blot analysis. The binding of LTB pentamers to intestinal epithelial cell membrane glycolipid receptors was further confirmed by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). Based on the results of GM1-ELISA, pentameric LTB proteins comprise approximately 0.1 - 0.2% of total soluble protein and the maximum amount of secreted LTB was estimated to be 100mg/L after the 3-day cultivation.

Besides a role as a causal agent of leaf spot disease of timothy (Phleum pratense), the hypomycetous fungus Cladosporium phlei (C. T. Gregory) de Vries is now considered as a potential provider of a key intermediate, phleichrome, which is a fungal pigment belonging to a group of perylenequinone and known to be further converted to a photodynamic therapeutic (PDT) agent.1) In this study, protoplasts of C. phlei were prepared and transformed to hygromycin B resistance using a plasmid, which contains a 2.4 kb SalI fragment of pDH25. The transformation efficiency ranged from approximately 20-100 transformants per experiment. Southern blot analysis of stable transformants revealed integrative transformation of the vector at different sites and more than two copies of the inserted marker gene appeared to be common. The establishment of easy protoplast-mediated transformation method of C. phlei should greatly facilitate the various molecular breeding of this strain for better production of bioactive materials such as fungal pigment.

Cryparin is an abundant cell wall-associated hydrophobin of the chestnut blight fungus, Cryphonectria parasitica. Although cryparin is encoded as a single copy gene (Crp), it isthe most abundant protein produced by this fungus when grown in liquid culture1), 2). As the results of previous studies to characterize the transcriptional regulatory element(s) for strong expression and hypoviral regulation, it was suggested that the fragment between nt -188 and the translation start codon appeared to be a minimal but sufficient promoter element for the efficient expression of the cryparin gene. To examine whether this small fragment is necessary and sufficient for a high level expression of the fungal gene, three different chimeric reporter genes using ORF’s of a hygromycin B resistance gene (hph), an inducible laccase (lac3) of C. parasitica and glucose oxidase (Go) of Aspergillus niger were tested3),4). When using the hphgene as a reporter gene, more than 1,000 stable transformants were obtained indicating that the 188 bp-fragment is comparable to that of trpC promoter of A. nidulans. When using the endogenous lac3 gene as a reporter gene, which requires the presence of tannic acid forthe transcriptional induction, the lac3 transcript was observed without any induction by tannic acid. In addition, glucose oxidase as a secretory protein was also tested for the expression. The enzymatic level of recombinant C. parasitica was within a range of two to threefold more than that of A. niger. These high expression levels were further confirmed by real-time PCR as well as comparison to other promoter. Moreover, the 188 bp-fragment was also examined for the promoter activity in other fungus Cochliobolus heterostrophus. Transformation of C. heterostrophus to hygromycin B resistance was successful indicating that this fragment can be applied as a strong promoter for various fungal biotechnology.

Quantum Dots-oligonucleotide conjugates, which are complementary to small region of target DNA, can probe interest microorganism in water environment. Luminescent Quantum Dots (QD) has narrower emission spectra and a continuous broad-band excitation compared with conventional fluorescent dyes (such as FITC and Cy3) so it has been used for many years for biological staining and diagnostics.1) It is easy to make QD-oligonucleotide conjugates by incubating QD coated with streptavidin and oligonucleotides modified with biotin. After incubation reaction free oligonucleotides, the unbound with QD, have to be removed from the mixture because hybridization efficiency decreases if unbound (unconjugated) oligonucleotides coexist with QD-oligonucleotide conjugates in the hybridization reaction. This study represents the application of field flow fractionation (FFF) for enabling easy separation of QD-oligonucleotide conjugates, which are required for effective-hybridizing to complementary target sequences. The FFF is one of the most versatile families of separation technique known. It has been successfully applied to studies of the environmental colloids, bacteria, proteins, and etc in a wide size range.2)3) The oligonucleotide used here is specific for Microcystis sp. to be able to produce microcystin and purified QD-oligonucleotide conjugates was applied to detect Microcystis sp. in the environmental sample.

Organophosphorus hydrolase (OPH) from Flavobacterium sp. is a membrane associated homodimeric metalloenzyme and has a signal sequence in its N-terminus. We found that the signal sequence of OPH is composed of 29 amino acids and has a twin arginine (RR) amino acid sequence in its hydrophobic region near the N-terminus that is analogous to the consensus motif (S-R-R-x-F-L-K) of twin arginine translocase (Tat) pathway. To investigate whether this sequence is dependent on Tat pathway, we used green fluorescent protein (GFP) as a cytoplasmic folding reporter. Unlike Sec pathway, Tat pathway can export correctly folded GFP into the periplasm of Escherichia coli. By using the novel Tat signal sequence of OPH, we demonstrated that foreign GFP was translocated with fully active form into the periplasmic space of E. coli.

The polyene antifungal antibiotics, mostly produced by Gram-positive soil actinomycetes, are a family of type I polyketide macrolide ring compounds with 20-40 carbon backbone contain 3-8 conjugated double bonds. Using polyene-specific genomic screening strategy, we previously isolated three novel polyene-producing actinomycetes strains from soil.1,2 Among three isolated strains, we have cloned and completely sequenced approximately 125.7 kb of nystatin-like CPP (Cryptic Pseudonocardia Polyene) biosynthetic gene cluster consisting of six genes for modular PKS and 17 additional genes from P. autotrophica KCTC9441.3 In addition, a 44.8kb FR008-like polyene biosynthetic gene cluster in Streptomyces sp. MMBL003 were isolated and genetically characterized.4 We also confirmed the production of these polyene compounds via culture media optimization along with antifungal bioassay as well as DAD-HPLC analysis, revealing that CPP is a novel antifungal compound different from nystatin.3 Disruption of a putative CPP PKS gene completely abolished CPP biosynthesis as well as antifungal activity. These results suggest that the polyene-specific genome screening as well as characterization of the cryptic polyene compound may constitute an efficient method for isolation of potentially valuable cryptic polyene biosynthetic genes and compounds from various rare actinomycetes.

Bioconverted omega-3 hydroxy fatty acid, eicosapentaenoic acid (bEPA), obtained from the microbial conversion by Pseudomonsa aeruginosa PR3 was evaluated for its antifungal potential. bEPA at 5 ㎕/ml exhibited 34-60% fungal mycelium growth inhibition against R. solani, B. cinerea, F. oxysporum, F. solani, P. capsici and C. capsici. bEPA showed antifungal activity with minimum inhibitory concentration, ranging from 250 to 500 ㎍/ml. Also, the screening was carried out using varied concentrations of bEPA for determining the inhibitory effect on the spore germination. Concerning the in vitro antifungal effect of bEPA, three plant pathogens,such as C. capsici, P. capsici and F. oxysporum were subjected to in vivo antifungal screening. Further, elaborative column chromatographic study conducted on bEPA expanded to the isolation of one bioconverted compound, designated as MS1. The structure of the compound was elucidated as eicosahexaenoic acid on the basis of instrumental analysis (MS, H-1NMR and 13NMR). MS1 displayed promising antifungal effect against C. capsici and F. solani with their respective MIC values 30 ㎍/ml for each fungal strain.