Study on antibacterial activity of lactic acid bacteria against different plant pathogenic bacteria

Anupama Shrestha 강원대학교 2008 국내석사

Lactic acid bacteria (LAB) are a phylogenetically diverse group of bacteria that are gram-positive usually non-motile, non-spore-forming rods and cocci with less than 50 mol% G+C in their DNA. They lack the ability to synthesize cytochromes and therefore cannot generate ATP by creation of a proton gradient. LAB includes the genera Lactobacillus, Lactococcus, Pediococcus, Leuconostoc, Carnobacterium, Propionibacterium and Bifdobacterium. Many LAB have important roles in the production of fermented foods, and some of these bacteria have been shown to be capable of inhibiting the growth of a wide variety of food spoilage organisms. In this study, strains were isolated from the soil around healthy tomato plants in a polyhouse, where most of the other plants showed bacterial wilt symptoms. In vitro antagonistic effect of culture fractionated in bacteria free supernatant and bacterial suspension of the isolated strain KLF01, Lactococcus strain KLC02 and Pediococcus strain KPD03 were tested against different plant pathogenic bacteria. The antibacterial substances from all three bacteria were subjected to different physical conditions as different pH and temperature. Furthermore, greenhouse experiments were conducted to determine the effectiveness of the Lactococcus strain KLC02, Pediococcus strain KPD03 and Lactobacillus strain KLF01 to control the bacterial wilt caused by Ralstonia solanacearum. Biochemical and 16S rRNA gene analysis of bacterial isolate KLF01 from garden soil showed relatedness to Lactobacillus sp. The supernatant was found to show no antibacterial effect, indicating that live bacteria are responsible for the activity in case of Pediococcus strain KPD03 and Lactobacillus strain KLF01 while the supernatant of Lactococcus strain KLC02 showed antibacterial activity against Xanthomonas axonopodis pv. citri. Among different plant pathogenic bacteria investigated, the zone of inhibition against Xanthomonas axonopodis pv. citri, Xanthomonas campestris pv. vesicatoria and R. solanacearum were significant therefore, selected for further study. The results showed that the antibacterial substance from Lactococcus strain KLC02 was found to consistently retain its biological activity after exposure to wide range of pH (2.0-9.0) and heat. Drench application of these strains (4×108 C.F.U/ml) into the experimental plot containing tomato (Solanum lycopersicum L.) cultivar ‘Lokkusanmaru’ and pepper (Capsicum annuum L.) cultivar ‘Buja’ plants, post-inoculated with the pathogen, R. solanacearum, drastically reduced the disease severity, compared to the non-treated plants. Since the above mentioned bacterial pathogens are critical in the initiation and progression of wide variety of plant diseases, the present findings suggest that the implementation of LAB may offer a novel approach in the biological control of these diseases. 계통 발생적으로 Lactic acid bacteria (LAB)는 다양성을 가지는 세균의 한 그룹으로써 gram-positive 이고 이동성이 없으며 포자를 형성하지 않는 rods 와 cocci 모양을 띄고 DNA 에는 50 mol% 보다 적은 G+C를 가지고 있다.이와 같은 세균은 cytochromes 을 생성 할 수 없다. 그러므로 proton gradient를 이용하여 ATP를 만들지 못한다. LAB의 속에 포함된 세균들은 다음과 같다: Lactobacillus, Lactococcus, Pediococcus, Leuconostoc, Carnobacterium, Propionibacterium and Bifdobacterium. 많은 LAB 은 발효식품 가공 시 중요한 역할을 한다 또한 이균들 중 음식을 상하게하는 미생물을 억제할수 있는 능력을 지녔다. 이번 연구를 위해 ployhouse 의 시들음병 에걸린 토마토 식물체중 건강한 토마토 식물의 토양에서 strain 을 분리 (채취) 하였다. 세균이 없는 supernatant 와 분리된 KLF01, Lactococcus strain KLC02 and Pediococcus strain KPD03 계통의 bacterial suspension이 병원성 세균에 대한 대립성을 알아 보기 위해 in vitro 시험되었다. 이 세종류의 항생성 은 pH와 온도 와 같은 물리적인 조건에 따라 다르게 나타난다. 또한, Lactococcus strain KLC02, Pediococcus strain KPD03 and Lactobacillus strain KLF01 의 R. solanacearum 에 의해 시들음 병을 억제할 수 있는 능력의 효과를 알아보기 위해 온실에서 실험되었다. KLF01 계통의 16S rRNA gene 와 생화학적 분석을 통해 Lactobacillus sp. 와의 연관성을 알수있었다. Supernatant에서는 항생 효과가 없었다. 이는, 살아 있는 세균만이 KPD03 계통의 Pediococcus 와 KLF01 계통의 Lactobacillus에서 제 역할을 하는 것으로 나타났다. 그런 반면, KLC02 계통의 Lactococcus supernatant는 Xanthomonas axonopodis pv. citri. 에데해 항생성을 보였다. 다른 식물 병원성 세균들도 조사한결과 Xanthomonas axonopodis pv. citri, Xanthomonas campestris pv. vesicatoria와 R. solanacearum 의 저항성은 눈에띄게 나타났다. 그러므로 더 연구 해볼 필요가 있다. KLC02 계통의 Lactococcus 의 항생성분 은 pH (2.0-9.0) 와 열에 노출되었음에도 불구하고 모든 생물적인 활동은 잃지 않았다. 위의 strains (4×108 C.F.U/ml)에 Drench application을 tomato (Solanum lycopersicum L.)의 재배종 ‘Lokkusanmaru’ 와 pepper (Capsicumannuum L.) 의 재배종 ‘Buja’ 식물들이 병원성 R. solanacearum에 접종되었을 때가 접종되지 않았을 때 보다 병징을 확연히 줄였음을 확인할수 있었다. 그러므로 위와 같은 세균으로부터 시작, 발전되는 다양한 식물병을 control 할수있는 방법 등에 LAB은 많은 도움과 성과를 불러올 것으로 판단된다.

Si BASED INTERFACES TO MATCH WITH PRINTED ELECTRONIC DEVICES

Kiran Shrestha 순천대학교 대학원 2019 국내석사

Printed electronic devices has very large potential in IT markets. In the near future, the printed electronics will replace silicon devices in most of the low-cost and large area applications such as smart packaging and large digital signage. However, the current status of the printed devices is not as matured as the silicon-based electronics devices in stability and power consumption. Thus, this thesis presents the methods of interfacing the printed devices with Si based devices for providing the device stability and low power operation. As typical examples, the printed sensors and antennas were bridged with the silicon devices for the wireless communication with low operation voltage. In addition, all R2R gravure printed TFT active matrix was bridged with Si based driving IC to display flexible E-paper. Especially, driving the E-papers with printed TFT active matrix will revolutionize the display industry with high throughput production, limitless size and all additive process (no etching).

Establishment of hybrid breeding system in Sweet Pepper (Capsicum annum L.)

Surendra Lal Shrestha 강원대학교 대학원 2009 국내박사

파프리카는 가지과에 속하는 온대성 작물로써 주변에서 손쉽게 볼 수 있다. 이국적인 특징을 가지고 있으며 최근 고소득작물로 대부분 종자가 비싼 값으로 수입되고 있다. 현재 약 70%정도가 강원도에서 재배되고 있다. 본 연구의 목적은 새로운 유전형질을 가진 개채의 선발과 현존하는 파프리카 품종을 육성하는데 있다. 파프리카의 약배양 실험은 강원대학교 원예학과에서 하였으며 옥외 실험은 화천에서 실시하였다. 현재 널리 재배되고 있는 품종인Derby, Debora, Fiesta, Special, Bossanova, Minipaprika, Clarity, Kufrah, Boogie, Phenlene를 약배양을 하였는데 Kufrah가 69.8%로 가장 높은 캘러스 형성율을 보였으며 재분화율은 Phenlene(2.67%)가 가장 높았으며 다음으로 Bossanova(2.41%)품종이었다. 이것은 모식물(donor plant)의 유전형이 약배양에 있어 중요하다는 것을 보여준다. 약배양을 통해 얻어진 314개체는 형태학적과 세포학적인 특성 조사를 한 결과 25% 묘가 반수체로서 2배체에 비해 낮은 초장과 절간 특징을 보였으며 작은 잎 구조와 화기, 기공크기를 가졌다. 또한 대체적으로 활력이 없었다. 과실도 크기가 작았으며 종자가 거의 없었다. 그리고 많은 용해성 고형물을 가지고 있었다. 이중 3개체가 웅성불임성이 관찰되었다. 배수성을 검정하기 위해 flow-cytometry로 뿌리끝 조직을 관찰하여 염색체 수를 확인하였다. 기공의 크기는 반수체와 2배체 사이에 뚜렷한 차이를 보였다.각 나라에서 이미 생육 평가(초장, 마디수, 마디색, 성숙도)와 화기와 과실의 특성(개화일수, 착과일수, 과실형태, 과실색깔, 경와, 배꼽굴곡) 그리고 수량에 관련된 요소(과실의 평균 무게, 식물 개체당 과실 수확량, 과실당 종자수)가 이미 밝혀진 123가지의 내혼 계통이 국외로부터 수집되었는데 그 중에 계통에 따라서 다양한 식물체와 과실, 그리고 수량 특성이 관찰되었다. 17종류의 계통은 높은 TSS함량(10.0%이상)을 가지고 있었으며 그 중 #133과 #91은 각각 11.8%, 11.7%의 함량을 보였다. 4계통(SP48, C01541, KNU3002, and SP142)은 두꺼운 과피(6.0mm이상)를 가지고 있었다. 따라서 재배에 적합한 계통으로 SP8, SP20, SP21, SP41, SP91, SP115, SP124, SP142, SP143, KNU1009가 선발되었다. 이러한 특성을 이용한 계통선발은 다양한 계통의 동정과 순계 선발에 있어서 유용할 뿐만 아니라 육종가에게 우수한 육종 소재를 제공해줄 것으로 추측된다. 38가지의 모본계통과 29가지의 부본계통을 교잡하여 133가지 조합의 잡종 종자를 얻었다. 그 중 4가지 조합은 종자가 없는 과실이었다. 이 잡종들은 비닐하우스에서 2007년과 2008년, 4월 마지막 주부터 10월까지 재배되었다. 각 식물마다 3개의 방아다리로 관리하였고 관비와 관수는 점적관수 방법을 이용하였다. 위의 양친으로부터 교잡해서 나온 F1은 다음과 같은 특징을 보였다 ; 작은 것과 큰 과실을 가진 개체를 교배할 경우 중간크기의 과실을 가진 F1이 나온다. 빨간색과 노란색 과실의 경우 전부 빨간색계통이며 오렌지색과 노란색의 경우는 전부 노란색을 가진 형질을 보였다. 둥근것과 볼록한 형질 사이에서는 중간 형태의 과실이 나왔다. 이런 모든 잡종 계통들은 각각 2007,08년도에 평가 되어 졌는데 그 중에 30 계통이 우수한 형질을 보였다. 2007년에 Special과 President의 교잡종이 대조군과 같은 조건으로 재배되었다. 그에 반에 2008년에는 11개의 상업적 품종인 Derby, Debla, Fiero, Special, Cupra, Fiesta, Jirisan, Maserati, Boogie, Plenty, President 를 위의 실험군 품종과 비교하기 위해 재배하였다. 우수한 과실형태와 맛, 두꺼운 과피, 높은 TSS함량과, 수확량 즉 잡종강세를 가진 특성을 기초로 하여 가장 우수한 교잡계통인 KNU1017(SP27), 5AVS5(SP27), 5AVS8(SP48), 5AVS7(SP45), 5AVS8(SP45), SP27(SP25), SP12(SP38)을 선발하였다. 이러한 잡종강세를 가진 계통들의 수확량은 각각 86%, 42%, 85%, 130%, 21%, 67% 그리고 126% 였다. 이중에서 5AVS5(SP27)은 노란색 과실을 가지고 있었다. 게다가 5AVS1(SP43)은 미니파프리카 타입으로 높은 TSS함량, 풍미, 원뿔과실모양을 보였다. 다른 계통인 KNU2006(SP14), KNU1006(SP14), KNU1009(SP27), KNU1015(SP32), 5AVS7(SP10), 5AVS8(SP32), 5AVS7(SP34), 5AVS7(SP27), 5AVS7(SP32), 5AVS8(SP10), 5AVS8(SP51), SP10(SP11), SP11(SP27), KNU1015(SP27)은 TSS함량과 과실내 종자수 간에 상관관계가 있었다. 즉 종자수가 감소할수록 높은 TSS함량과 풍미를 보였다. Sweet pepper (Capsicum annum L.) is a warm season crop that belongs to the solanaceae family. It is a fast emerging vegetable and cash crop in Korea where seeds are quite expensive and most of the growing cultivars are exotic. Seventy percent of growing area is under Kangwon Do in summer season crop. The main objective of this study was to create and select new genotypes of sweet pepper equal or better than the existing hybrid cultivars. Anther culture was performed in Kangwon National University, Horticulture Laboratory and rests of the field experiment were performed in Hwacheon area. The anthers of widely grown hybrid cultivars (Derby, Debora, Fiesta, Special, Bossanova, Minipaprika, Clarity, Kufrah, Boogie, and Phenlene) were used in anther culture, where cultivar Kufrah had the highest percentage of callus formation (69.8%) but the percentage of plant regeneration were higher in Phenlene (2.67%) followed by Bossanova (2.41%). Even though there is the effect of genotype of donor plants on the success of anther culture, plantlets could be developed from all of these genotypes. These anther culture derived three hundred fourteen plantlets were morphologically and cytologically characterized. Twenty five percent plantlets were haploid which had shorter height and internodes, smaller leaves, plant structure, flower buds, fruit and stomata size, weak and poor in vigor and mostly seedless fruits with high total soluble solid contents as compared to diploid plants. Five plants were found male sterile. To determine the ploidy level of the plants obtained from in vitro anther culture, chromosome counts in root tip squashes and flow-cytometery were used. Differences in stomata length was also noticed between haploid and diploid plant. One hundred twenty three inbred lines were characterized on vegetative parameter; plant height, number of nodes to first branch, nodal color, maturity, flower and fruit characteristics; days to flowering, fruit set, fruit shape, fruit color, stalk cavity, apex grove, yield parameter; average weight of fruit, yield per plant, and number of seeds per fruit. These inbred lines were collected from abroad, inside the country and developed in the university in the past years. Variation on vegetative, fruit, and yield characteristics was observed. Among them, seventeen lines were recorded with higher TSS content (>10.0%) where SP133, and SP91 had TSS 11.8% and 11.7% respectively. Four lines (SP48, C01541, KNU3002, and SP142) had thicker pericarp (>6.0 mm).With respect to their over all performance, SP8, SP20, SP21, SP41, SP91, SP115, SP124, SP142, SP143, and KNU1009 were selected as superior inbred lines for cultivation. This characterization will help to identify the variety and to maintain purity in future. Beside this, it will provide the information to the breeders on the Sweet pepper varietal improvement work. Thirty eight female lines and twenty nine male lines were crossed and hybrid seeds of one hundred twenty nine combinations were collected. These hybrid lines were grown in plastic house condition on field soil from last week of April to October in 2007 and 2008. Training and pruning were done by keeping three stems per plant. Nutrient and water were supplied through drip irrigation. The effect of crossing between male and female parents to the expression of characteristics in F1 showed that; when small and big sized fruiting plants were crossed, F1 plants beard medium sized fruits, between red and yellow color, all red color, between orange and yellow all yellow color fruits, between round and lamuyo fruits it became glamour shaped fruits. Most of the hybrid lines were evaluated in 2007 and 2008. Among the hybrid lines, thirty lines showed superior performance. In the first year, only hybrid cultivars; special and President, were grown as a control whereas in 2008, eleven commercial hybrid cultivars (Derby, Debla, Fiero, Special, Cupra, Fiesta, Jirisan, Maserati, Boogie, Plenty, and President) were grown for comparison. On the basis of good shape, taste, thick pericarp, high total soluble solid content, yield, and heterosis, seven hybrid lines; KNU1017(SP27), 5AVS5(SP27), 5AVS8(SP48), 5AVS7(SP45), 5AVS8(SP45), SP27(SP25) and SP12(SP38) were found superior, and selected for cultivation. These hybrid lines had heterosis on yield were 86%, 42%, 85%, 130%, 21%, 67%, and 126% respectively. Among them, 5AVS5(SP27) had yellow fruit and the rest were red. In addition to this, 5AVS1(SP43) had shown excellent performance as a minipaprika type containing high TSS, good taste, and conical shape. The other promising lines were KNU2006(SP14), KNU1006(SP14), KNU1009(SP27), KNU1015(SP32), 5AVS7(SP10), 5AVS8(SP32), 5AVS7(SP34), 5AVS7(SP27), 5AVS7(SP32), 5AVS8(SP10), 5AVS8(SP51), SP10(SP11), SP11(SP27), and KNU1015(SP27).

Anti-amyloidogenic effect of ethanol extract from the mixture of leaves and stem of Mallotus japonicus (L. F.) Müll. Arg. on β-amyloid induced memory impairment in Alzheimer’s disease mouse model

Abinash Chandra, Shrestha 우석대학교 대학원 2019 국내석사

Development of Versatile Drug Delivery Systems for Cell Therapy

Shrestha Prakash 영남대학교 대학원 2022 국내박사

A study on sustainability of Nepalese fisheries and aquaculture

SHRESTHA, SAMEK 부경대학교 대학원 2016 국내석사

The overview of this research has been based on sustainability of Nepalese fisheries and aquaculture sector in the context of Nepal using three fundamental approaches as per Nepal government rules and regulation under Ministry of Agriculture internal plans, policies and agreements. Firstly, it is about overall basic information of the nation with status of fishing industry of Nepal, which basically is about aquatic resources and contains about its origin, history, species, structure, systems, practices, nature and resources of fisheries sector. Secondly, an overview of fisheries production, markets, trade and economic role. Similarly, focuses on the role for sustainable development, policies and plans for the general development of the sector with opportunities and challenges in the management process of fisheries and aquaculture. Although, Nepal fishery does seems to be started (1940s) from long back history but the modern technique of producing fish and systematic process of doing aquaculture has just being developing in recent years. Aquaculture is completely a new and modern way of fishery activity in Nepal. The potentials for aquaculture development in the country are considerable high because of abundant availability of natural resources such as; fisheries species, fresh water resources etc. Nepal is a habitat of 186 indigenous among which 56 are cold water species and 11 exotic fish species and has more than 5000 natural water bodies with very suitable climate condition for aquaculture. It has got potential of development because of excellent climate condition for fish breeding and nursery grounds along with fish feeds and as well as is favorable to aquatic ecosystems. Similarly, as Nepal being a Himalayan nation, the glaciers ice when melted down, it forms different natural water bodies like: rivers, lakes and other various natural fresh water resources. These fresh natural water bodies are the suitable for 59 cold water fisheries species found in Nepal and these species are even useful for aquaculture. Poor technological adaptation, poor budget funding’s, lack of proper management strategies, lack of quality fish feed and fingerlings and low market availability are some of the major problems and challenges that currently Nepalese freshwater fisheries is facing. Long-term sustainable plans, modern or scientific and technological study on various indigenous and exotic aquatic species proper hygiene management and effective disease control might improve the current and future aquaculture status of management of Nepalese fisheries sector. In its initial stage, aquaculture was done with pond culture by introducing some Indian carps because of similar geographical background and climate. Specially, the climatic condition of terai or plain region where about 94 percent of the fish ponds are located has similar weather nature with Indian climate because of common geographical nature so that, it is easy to acquire fish seeds and fish feeds. Beside, Pond culture cage fish culture and enclosure fish culture in lakes and reservoirs as well as rice-fish culture are popular production systems. Having enclosure aquaculture system means minimum management of the resources. Now a day, aquaculture has become much popular activity for being self-employed and has high returns on investment. A preliminary analysis of employment and income generation has shown that people employed in aquaculture sector have higher income potential compared to people employed in other agriculture sectors. Nepal employed about 504000 people in the country during 2003/04 fisheries and aquaculture development activities. However, there is always a question about the sustainability of these resources because of over-exploitation, managerial system, techniques and knowledge regarding fisheries management. An overview of this thesis show, that like various nation in the world, the fishing sector in Nepal mostly considered as a crucial and inseparable segment of the Nepalese community which has being passing through-out the history of Nepalese agriculture system and is evolving rapidly and contributing in economic growth, source of an employment, protein food and so on. Therefore, the potential of Nepalese fisheries and aquaculture has greater due to following factors like; the fresh water, suitable environment, aquatic resources, low labor cost, manpower and as well as on the basis of possible low investments with good return. Finally, for sustainable development of the fisheries resources the important aspects such as; climate change, conservation and protection, human impacts, managerial system, skill manpower and clear plans and policies should be taken in to consideration.

Identification of the hrp gene cluster in Erwinia pyrifoliae and molecular characterization of HrpNEp protein

Shrestha, Rosemary Kangwon National University 2006 국내박사

Dealing with a Deadly Issue : Government’s Efforts on Combating Human Trafficking in Nepal

SHRESTHA, Scheme 이화여자대학교 국제대학원 2019 국내석사

Human trafficking is a shame on humanity. It is a fast-growing illegal trade with a high profit at minimal risk. Nepal is a source, transit, and destination country in terms of human trafficking. Nepal is, thus, recognized as a Tier-2 country as categorized by UNODC. Hundreds of Nepalese, especially women and girls, are being trafficked internally and externally. The internal human trafficking in the entertainment sector has become a serious concern for the Government of Nepal. The government has increased its efforts to combat human trafficking and transportation. Despite this, it has been increasing annually. Since 2012, the NPA has been implemented by the government focusing on five thematic areas: Prevention, Protection, Prosecution, Capacity development, and Cooperation, Coordination, and Collaboration. This paper intends to analyze the challenges faced by the Government of Nepal to combat human trafficking domestically as well as internationally. This paper argues that the NPA is well formulated, but it lacks effective implementation because of the inadequate programs and budgetary allocation. Lack of an integrated information management system, education and awareness, competent and sensitized government officials and so on are more responsible for women trafficking. The limited employment opportunities, the unbalanced power structure of the society, armed conflict, natural disaster, corruption, rampant poverty, and violence are against women are some of the challenges for the Government of Nepal to complete eradication of human trafficking. Therefore, to combat human trafficking, Government of Nepal has to focus on coordination, cooperation, and collaboration in anti-human trafficking activities to strengthen the networking among stakeholders for transforming collective energy into synergy. Keywords: Human trafficking, Sexual exploitation, Labor exploitation, Prevention, Protection, Prosecution 인신매매는 인류의 큰 부끄러움이지만 낮은 위험성과 높은 수익으로 인해 불법 거래가 급성장하고 있습니다. 네팔은 이러한 인신매매의 시작과 중간, 그리고 끝 모두가 이루어지고 있습니다. 따라서 네팔은 UNODC의 2단계 지역으로 지정되어 있습니다. 주로 여성과 어린 소녀들로 이루어진 수 백명 이상의 네팔 사람들이 네팔 안쪽으로, 또는 바깥쪽으로 인신매매를 당합니다. 네팔 정부는 여성들이 유흥지역에 인신매매되는 것을 매우 걱정하고 있으므로 인신매매와 수송을 막기 위해 적극적으로 대처하고 있습니다. 그럼에도 불구하고 인신매매는 매년 증가하는 추세입니다. 2012년 이후로 정부는 예방, 보호, 고발, 역량 개발, 그리고 협력과 협동, 조정이라는 다섯 포인트에 초점을 맞춘 NPA를 시행하고 있습니다. 이 보고서는 네팔 정부가 국제 인신매매 뿐만 아니라 자국내의 인신매매에 직면한 상황을 분석하기 위해 쓰였습니다. 그리고, NPA가 얼마나 잘 구성되어 있는지와 그럼에도 적절하지 못한 프로그램 및 예산배정 등으로 효율적이지 못한 부분에 대해서도 다루고 있습니다. 여성 인신매매에 대해서는, 통합된 정보관리 시스템, 교육과 인식수준, 유능한 공무원 등의 부재가 큰 원인입니다. 제한된 취업의 기회와 사회의 무너진 권력구조, 무장단체와의 갈등, 자연재해, 부패, 만연하는 가난과 여성에 대한 폭력은 네팔 정부가 인신매매를 뿌리뽑기 위한 필수사항입니다. 그러므로 네팔 정부는 인신매매에 맞서기 위해 반인신매매 활동의 협력과 협동, 조정에 초점을 맞추고 당사자들간의 유대를 통해 시너지를 낼 수 있어야 합니다.

METABOLIC ENGINEERING OF Escherichia coli FOR THE MODIFICATIONS OF POLYPHENOL BY C-GLYCOSYLATION AND HYDROXYLATION

Shrestha, Anil 선문대학교 일반대학원 2018 국내박사

Two plant-originated C-glucosyltransferases (CGTs) UGT708D1 from Glycine max and GtUF6CGT1 from Gentiana triflora for glucosylation of selected flavones chrysin and luteolin. UDP-glucose pool was enhanced in E. coli cell cytosol by introducing heterologous UDP-glucose biosynthestic genes i.e., glucokinase (glk), phosphoglucomutase (pgm2), and glucose 1-phosphate uridylyltransferase (galU), along with glucose facilator diffusion protein from (glf) from different organisms, in a multi-monocistronic vector. The C-glucosylated products were analyzed by high performance liquid chromatography-photo diode array, high-resolution quadruple time-of-flight electrospray ionization mass spectrometry, and one-dimensional nuclear magnetic resonance analyses. Fed-batch shake flask culture showed 8% (7 mg/L; 16 µM) and 11% (9 mg/L; 22 µM) conversion of chrysin to chrysin 6-C-β-D-glucoside with UGT708D1 and GtUF6CGT1, respectively. Moreover, the bioengineered E. coli strains with exogenous UDP-glucose biosynthetic genes and glucose facilator diffusion protein enhanced the production of chrysin 6-C-β-D-glucoside by approximately 1.4–fold, thus producing 10 mg/L (12%, 24 µM) and 14 mg/L (17%, 34 µM) by UGT708D1 and GtUF6CGT1, respectively, without supplementation of additional UDP-glucose in the medium. The biotransformation was further elevated when the bioengineered strain was scaled up in lab scale fermentor at 3-L volume. HPLC analysis of fermentation broth extract revealed 50% (42 mg/L, 100 µM) conversion of chrysin to chrysin 6-C-β-D-glucoside at 48h upon supplementation of 200 µM of chrysin. The maximum conversion of luteolin was 38% (34 mg/L, 76 µM) in 50 mL shake flask fermentation at 48 h. C-glucosylated derivative of chrysin was found to be more soluble, and more stable to high temperature, different pH range, and β-glucosidase enzyme, than O-glucosylated derivative of chrysin. Resveratrol, a major stilbene phytoalexin is valuable polyphenol having beneficial effects to human health. It has potent antioxidant and antitumor effects and promotes longevity. In this study, resveratrol and its ortho-hydroxylated derivative piceatannol were biosynthesized by modular pathway engineering in E. coli. The biosynthetic pathway was divided into three different modules. Module I includes polyketide biosynthetic genes; module II genes include acetyl-CoA and malonyl-CoA pool-enhancing genes from three different organisms, and module III genes are regiospecific 3′- hydroxylating enzymes. Biosynthetic genes include 4-coumarate:CoA ligase from Petroselinum crispum (Pc4CL2) and a codon optimized stilbene synthase from Vitis vinifera (VvSTS) (module I) accompanied by acetyl-CoA and malonyl-CoA building blocks supplying enzyme complexes (module II) from Nocardia farcinica (nfa9890, nfa9940, nfa9950, nfa3550; module IIa), E. coli (accCADB; module IIb), and Streptomyces venezuelae (matBC; module IIc). HpaBC complex (module III) from E. coli was overexpressed for the hydroxylation. E. coli BL21(DE3) with module I produced 8.6 mg/L of resveratrol from exogenously fed 1 mM p-coumaric acid. Combination of module I and acetyl-CoA supplementing module IIa genes from N. farcinica produced 2.5-fold higher (60 mg/L) titer of resveratrol than the module IIb genes from E. coli. The exogenous supplementation of sodium acetate further enhanced production to 64 mg/L. Furthermore, module I with module IIc harboring matBC from S. venezulae produced 73 mg/L of resveratrol, which was elevated to 151 mg/L upon supplementing disodium malonate exogenously. This increment is 17.5-fold higher than module I harboring E. coli BL21(DE3). The combination of module I and two different module II genes yielded 137 mg/L resveratrol when supplemented with both sodium acetate and disodium malonate. The high resveratrol-producing combination module was further modified with incorporation of hpaBC for the ortho-hydroxylation of resveratrol to produce piceatannol. The engineered strain harboring modules I, IIc and III produced 124 mg/L of piceatannol, the highest titer after 72 h in disodium malonate-supplemented strain, which is 2-fold higher than in non-supplemented strain. The remaining resveratrol was about 30 mg/L. Furthermore, caffeic acid (85.5 mg/L) was also produced in the same strain. Resveratrol and piceatannol were biosynthesized along with caffeic acid by three different modules overexpressing acetate and malonate assimilation pathway genes from three different sources. The production titer of both resveratrol and piceatannol may be enhanced by blocking acetyl-CoA and malonyl-CoA utilizing genes in host strain. Moreover, we attempted to produce diverse derivatives of diadzein by biotransformation. O-methyltransferase from S. avermetilis was used for methylation. YjiC, a substrate promiscious and regio-flexible glycosyltransferase, from Bacillus was used for glycosylation. In order to tune the glycosylation, the glycosyltransferase was expressed under theophylline inducible system, where the methylation and glycosylation steps were controlled for production of methylated, glycosylated and both methylated/glycosylated derivatives. Hence, with this approach, we could generate formonentin and ononin. Particularly, the tuning by controlled expression provided a platform for generating the onionin in higher titer. The cytotoxic effect and wound healing assay were tested for all the diadzein derivatives. The deriavative compounds inhibited proliferation of each cancer cell line with different sensitivity to growth inhibition. Notably, methylated product and double modified glycosylated/methylated showed comparatively better growth inhibition than control product. Methylated derivative also reduced the migration capacity of AGS gastric cancer cells.

Identification of cytochrome P450 and redox partners in Streptomyces peucetius ATCC 27952 : understanding electron transfer system and application

Shrestha, Pramod Sun Moon Univ. 2008 국내박사

Nineteen cytochrome P450s (CYP) along with two ferredoxins (FDX) and four ferredoxin reductases (FDR) have been reported in Streptomyces peucetius ATCC 27952. However, genome annotations need to be regularly updated if the information they contain is to remain accurate and relevant. Following the homology searches based on conserved signature sequences necessary for CYP, revised analyses of S. peucetius ATCC 27952 genome database available in our laboratory revealed four more CYPs and correction in sequences of few previously known CYPs making twenty three CYPs in total. We also found complete sequences of three pseudo CYP whose heme-binding motifs were either missing or unidentified. Moreover, one FDR and two previously reported FDXs were found to be mistaken. Fdx808 reported as ferredoxin has been found as NADH oxidoreductase, experimentally. We also identified four more FDRs and six new FDXs. So, seven FDRs and six FDXs were found to be present totally in S. peucetius ATCC 27952 to support twenty three cytochrome P450s. Even though numbers of FDXs and FDRs have been reported, sufficient details about the relevant homologous electron transfer system in Streptomyces P450s is unclear. Since CYPP166B1 was clustered with putative ferredoxin FDX6, we expressed and purified six ferredoxin reductase along with them and envisaged the primary pathway NADH→FDR4→FDX6→CYP166B1 for dealkylation of 7- ethoxycoumarin, however, we are unsuccessful to express FDR1. Consequently, further mechanistic studies with the identification of endogenous substrate for CYP166B1 would give more clear explanation. As we know that cytochrome P450s are responsible for the oxidative metabolism of a wide variety of endogenous and exogenous compounds. We expressed several CYPs from S. peucetius and CYP105F2 overexpress in E. coli BL21 (DE3) pLysS, indicated by alkali treated assay, could have activity with oleandomycin. A 3D model was constructed based on the known crystallographic structures of cytochrome P450, and comparison with PikC and MoxA further signified its broad substrate specificity towards structurally diverse compounds. Invitro hydroxylation of oleandomycin by purified CYP105F2 observed in liquid chromatography/mass spectrometry and mass/mass spectrometry indicated its flexibility towards alternative polyketides for the structural diversification of the macrolide by post-polyketide synthase hydroxylation. Further, a biotransformation system was designed to co-express CYP107P3 (CSP4), cytochrome P450, from S. peucetius, along with CamA (putidaredoxin reductase) and CamB (putidaredoxin) from Pseudomonas putida, the necessary reducing equivalents; in a class I type electron-transfer system in E. coli BL21 (DE3). This was carried out using two plasmids with different selection markers and compatible origins of replication. The study results showed that this biotransformation system was able to mediate the O-dealkylation of 7- ethoxycumarin. Key words: Streptomyces peucetius ATCC 27952, Cytochrome P450, Sequence analysis, Ferredoxin/Ferredoxin reductase, electron transport system, whole-cell biotransformation