학술논문
Molecular characterization and structure-based engineering of arachidonate 15-lipoxygenases and their applications in the production of hydroxy fatty acids / 아라키도네이트 15-리폭시게네이즈의 분자특성화 및 구조기반 엔지니어링에 따른 하이드록시 지방산 생산에 대한 응용
Document Type
Dissertation/ Thesis
Source
Subject
Language
Korean
Abstract
구조적 및 생물학적으로 기능성 물질으로서 산소화된 분자의 그룹인 옥시리핀은 식물, 동물, 곰팡이 및 미생물을 포함한 다양한 생물체에서 흔히 발견됩니다. 이 화합물들은 특화된 염증해소 촉진전달 인자 (Specialized pro-resolving mediators, SPMs)를 포함하여 항상성, 감염 및 염증에 대한 치유 과정과 관련된 생리적 반응에 관여하며 생체 내 세포 소통, 화학적 합성 또는 생체 전환에 의해 C18, C20 및 C22 다가불포화 지방산에서 유래된 것입니다. 그러나 생체 내 생합성은 미량 생성하고, 생성에 관여하는 효소 활성이 낮고, 화학적 합성은 독성 물질, 유기 용매 및 고온의 사용 및 중금속 촉매 작용과 함께 많은 (20-30) 단계를 포함하며, 수율이 낮은 한계를 가집니다. 또한 박테리아 효소를 이용한 생체 전환은 낮은 농도, 기질의 낮은 용해도 및 플라스크 내 산소 공급 부족을 포함합니다. 문제 및 한계를 극복하기 위해 활성이 높은 효소의 발견, 효소 공학의 적용, 생산 공정의 최적화 및 공정 개발기술을 적용했습니다.2장에서, 이 장은 염증해소 촉진전달 인자 생산 미생물인 아라키도네이트 15S-리폭시게네이즈를 발견했습니다. 이는 포유류 리폭시게네이즈보다 13,000배 높은 이중 산소 활성을 가지고 있습니다. C20 및 C22 다가불포화 지방산은 S,S-디하이드록시 지방산으로 20% (w/w) 전환되었습니다. 선호되는 기질-잔기 상호 작용 분석 및 돌연변이 연구에서 Leu429 및 Leu430잔기가 위치 특이성의 결정적인 잔류물임을 밝혀냈습니다.3장에서, 이 장은 보고된 리폭시게네이즈 중 가장 높은 단일 및 이중 산소 활성을 갖는 새로운 입체 선택적 아라키도네이트 15R-리폭시게네이즈를 발견했습니다. C20 및 C22 다가불포화 지방산은 염증해소 촉진전달 인자의 이성질체로서 3가지 유형의 R,R-디하이드록시 지방산으로 전환되었으며, 농도는 1.7 g/L 이상, 전환율은 83% (w/w)이상으로 전환되었습니다. 레졸빈 D5 및 17R-하이드록시도코사헥사엔산의 이성질체로서 오일 가수분해물을 값비싼 지질 조절제로 효율적으로 생체 변환하기 위해 선호되는 기질-잔기 상호 작용 분석 및 돌연변이 연구를 진행하였습니다. 이를 통해 Leu606 잔기가 두 번째 산소화반응의 결정적 잔류물임을 밝혀냈습니다.4장에서, 수지와 용매 추출을 통한 리파아제 처리로 어유를 도코사헥사엔산 농축유로 가수분해하였다. 농축된 어유 가수분해물이 염증해소 촉진전달 인자인 고가의 레졸빈 D5로 효율적인 생물 전환을 위하여 기질의 용해도를 높이고자 하였고, 추가적으로 고분자와 용매를 첨가와 생물반응기를 적용하여 생물 전환 시 충분한 산소를 공급하였다. RvD5는 1.0 g/L 이상의 농도로 생산되었으며, 수율은 87% (w/w)이상이었습니다.5장에서, 플라스크에서 전환 수율 (60% 이상)이 높은 대장균 세포를 이용하여 다가불포화 지방산으로부터 에폭시 하이드록시 지방산을 통한 식물 옥시리핀인 트리하이드록시 지방산으로 전환했습니다. 홍화유는 수지로 리파아제 처리를 통해 리놀레산을 포함하는 홍화유 가수분해물로 가수분해되었습니다. 다가불포화 지방산를 트리하이드록시 지방산으로 효율적인 생체 전환을 위해 흡착제 수지를 첨가하여 리놀레산의 함량을 높였습니다. 생물반응기를 사용하여 홍화유에서 얻은 수지 처리 홍화유 가수분해물의 250 mM 리놀레산을 플라스크에서 보다 2배 높은 230 mM의 11R,12R,13S-트리하이드록시-9Z-옥타데센산으로 전환했습니다.6장에서, 고분자와 용매를 사용한 반응 최적화에 의해 에이코사펜타엔산을 레졸빈 E4와 레졸빈 E4의 거울상 이성질체로 고농도의 효율적인 생체 변환을 수행했습니다. 돌연변이는 구조 유도 엔지니어링을 사용하여 각각 L429W/L430M/L575M과 L423W/L424M/L568M 돌연변이에 의해 15R- 및 15S-리폭시게네이즈를 18R- 및 18S-리폭시게네이즈로 각각 변경했습니다. 엔지니어링된 18S- 및 18R-리폭시게네이즈를 발현하는 대장균 세포는 에이코사펜타엔산를 18S- 및 18R-하이드록시에이코사펜타엔산으로 전환했으며, 이는 제브라 다니오 5S-리폭시게네이즈를 발현하는 대장균 세포에 의해 각각 18S-레졸빈 E2 및 레졸빈 E2로 전환되었습니다.본 장에서는 지질 조절제 및 염증해소 촉진전달 인자를 포함한 옥시리핀의 효율적인 생산을 위한 고활성 효소 발견, 효소공학의 적용 및 공정 최적화를 진행하였으며, 이는 다가불포화 지방산 또는 재생성 오일로부터 구조적으로 다양한 옥시리핀의 생체 전환을 적용할 수 있습니다. 이러한 결과는 화학적 합성을 위한 환경 오염을 극복하기 위한 그린 경로로서 다가불포화 지방산 또는 오일로부터 지질 조절제 및 염증해소 촉진전달 인자를 포함한 생체 활성 옥시리핀의 효율적, 비용 효율적 및 친환경적인 생체 촉매 합성을 나타냅니다.
Molecular characterization and structure-based engineering of arachidonate 15-lipoxygenases and its application in production of hydroxy fatty acids Lee, Jin Department of Bioscience and Biotechnology Graduate School of Konkuk University Oxylipins, a group of structurally and biologically active oxygenated molecules, are commonly found in a variety of organisms, including plants, animals, fungus, and microorganism. These compounds, which are essential signaling compounds such as lipid mediators (LMs), including specialized pro- resolving mediators (SPMs), are involved in physiological responses related to homeostasis and healing processes against infection and inflammation and are produced from which originate from C18, C20, and C22 polyunsaturated fatty acids (PUFAs) by cell communication in vivo, chemical synthesis or bioconversion. However, biosynthesis in vivo produces trace amounts, the enzyme activity involved in generation is low, and chemical synthesis involves many multi (20−30) steps with toxic agents, organic solvents, high temperature, and heavy-metal catalysis, and has low yields. Alternatively, the bioconversion using bacterial enzymes involves low concentrations, with low solubility of the substrate, and insufficient supply of oxygen in a flask. To overcome problems and limitations, the discovery of highly active enzymes, enzyme engineering, production process optimization, and process development was applied. In Chapter 2, this chapter discovered a SPM-producing microorganism, arachidonate 15S-lipoxygenase (LOX). It has a high double-oxygenating activity of ARA 15S-LOX, which is 13,000-fold higher than that of the mammalian LOX. C20- and C22-PUFAs were converted into S,S-dihydroxy fatty acids (DiHFAs), with conversions of >20% (w/w). Favorable substrate- residue interaction analysis and mutation studies revealed that Leu429 and Leu430 were a determinant residue for the regiospecificity. In Chapter 3, this chapter discovered a novel stereoselective arachidonate 15R-LOX the highest single- and double-oxygenating activity among the reported LOXs. C20- and C22-PUFAs were converted into R,R-DiHFAs of 3- types as SPM isomers, with concentrations of >1.7 g/L and conversions of >83% (w/w). For the efficient bioconversion of the oil hydrolysate into expensive LMs, as isomer of RvD5 and 17R-hydroxydocosahexaenoic acid. Favorable substrate-residue interaction analysis and mutation studies revealed that Leu606 was a determinant residue for the second oxygenation. In Chapter 4, fish oil was hydrolyzed to docosahexaenoic acid (DHA)- enriched oil hydrolysate by lipase treatment with resin and solvent extraction. For the efficient bioconversion of the oil hydrolysate into expensive resolvin D5 (RvD5) as SPMs, the polymer and solvent were added to increase the solubility of substrate, and a bioreactor was applied to supply sufficient oxygen during bioconversion. RvD5 was produced at the >1.0 g/L level with a yield of >87% (w/w). In Chapter 5, Escherichia coli cells converted PUFAs into plant oxylipin trihydroxy fatty acids (THFAs) via epoxy hydroxy fatty acids (EHFAs) with high conversion yields (>60%) in a flask. Safflower oil was hydrolyzed to safflower oil hydrolysate, containing linoleic acid (LA), by lipase treatment with resin. For the efficient bioconversion of PUFAs into trihydroxy fatty acids (THFAs), the adsorbent resin was added to increase the content of LA. Using a bioreactor, 250 mM linoleic acid (LA) in resin-treated safflower oil hydrolysate obtained from safflower oil was converted into 230 mM 11R,12R,13S- trihydroxy-9Z-octadecenoic acid, which is 2.0-fold higher than that in a flask. In Chapter 6, this chapter performed high-concentration and efficient bioconversion of eicosapentaenoic acid (EPA) into RvE4 and enantiomer of RvE4 by reaction optimization using polymer and solvent. The mutations changed the 15R- and 15S-LOXs into a 18R- and 18S-LOXs by L429W/L430M/L575M and L423W/L424M/L568M mutations, respectively, using structure-guided engineering. E. coli cells expressing engineered 18S- and 18R- LOXs converted EPA into 18S- and 18R-hydroxyeicosapentaenoic acids (18R- HEPAs), which were converted into 18S-RvE2 and RvE2 by E. coli cells expressing 5S-LOX from Danio rerio, respectively. In present chapters, discovery of highly active enzyme, enzyme engineering, and process optimization for the efficient production of oxylipins, including LMs and SPMs, can apply the bioconversion of structurally diverse oxylipins from PUFAs or renewable oils, and produced industrial concentrations. These results represent an efficient, cost-effective, and eco-friendly biocatalytic synthesis of bioactive oxylipins, including LMs and SPMs, from PUFAs or oils as a green route to overcome the environmental pollution for chemical synthesis. Keywords: Lipoxygenase, Lipid mediators, Specialized pro-resolving mediators, Molecular determination, Enzyme engineering, Enhanced bioconversion, Sustainable oils
Molecular characterization and structure-based engineering of arachidonate 15-lipoxygenases and its application in production of hydroxy fatty acids Lee, Jin Department of Bioscience and Biotechnology Graduate School of Konkuk University Oxylipins, a group of structurally and biologically active oxygenated molecules, are commonly found in a variety of organisms, including plants, animals, fungus, and microorganism. These compounds, which are essential signaling compounds such as lipid mediators (LMs), including specialized pro- resolving mediators (SPMs), are involved in physiological responses related to homeostasis and healing processes against infection and inflammation and are produced from which originate from C18, C20, and C22 polyunsaturated fatty acids (PUFAs) by cell communication in vivo, chemical synthesis or bioconversion. However, biosynthesis in vivo produces trace amounts, the enzyme activity involved in generation is low, and chemical synthesis involves many multi (20−30) steps with toxic agents, organic solvents, high temperature, and heavy-metal catalysis, and has low yields. Alternatively, the bioconversion using bacterial enzymes involves low concentrations, with low solubility of the substrate, and insufficient supply of oxygen in a flask. To overcome problems and limitations, the discovery of highly active enzymes, enzyme engineering, production process optimization, and process development was applied. In Chapter 2, this chapter discovered a SPM-producing microorganism, arachidonate 15S-lipoxygenase (LOX). It has a high double-oxygenating activity of ARA 15S-LOX, which is 13,000-fold higher than that of the mammalian LOX. C20- and C22-PUFAs were converted into S,S-dihydroxy fatty acids (DiHFAs), with conversions of >20% (w/w). Favorable substrate- residue interaction analysis and mutation studies revealed that Leu429 and Leu430 were a determinant residue for the regiospecificity. In Chapter 3, this chapter discovered a novel stereoselective arachidonate 15R-LOX the highest single- and double-oxygenating activity among the reported LOXs. C20- and C22-PUFAs were converted into R,R-DiHFAs of 3- types as SPM isomers, with concentrations of >1.7 g/L and conversions of >83% (w/w). For the efficient bioconversion of the oil hydrolysate into expensive LMs, as isomer of RvD5 and 17R-hydroxydocosahexaenoic acid. Favorable substrate-residue interaction analysis and mutation studies revealed that Leu606 was a determinant residue for the second oxygenation. In Chapter 4, fish oil was hydrolyzed to docosahexaenoic acid (DHA)- enriched oil hydrolysate by lipase treatment with resin and solvent extraction. For the efficient bioconversion of the oil hydrolysate into expensive resolvin D5 (RvD5) as SPMs, the polymer and solvent were added to increase the solubility of substrate, and a bioreactor was applied to supply sufficient oxygen during bioconversion. RvD5 was produced at the >1.0 g/L level with a yield of >87% (w/w). In Chapter 5, Escherichia coli cells converted PUFAs into plant oxylipin trihydroxy fatty acids (THFAs) via epoxy hydroxy fatty acids (EHFAs) with high conversion yields (>60%) in a flask. Safflower oil was hydrolyzed to safflower oil hydrolysate, containing linoleic acid (LA), by lipase treatment with resin. For the efficient bioconversion of PUFAs into trihydroxy fatty acids (THFAs), the adsorbent resin was added to increase the content of LA. Using a bioreactor, 250 mM linoleic acid (LA) in resin-treated safflower oil hydrolysate obtained from safflower oil was converted into 230 mM 11R,12R,13S- trihydroxy-9Z-octadecenoic acid, which is 2.0-fold higher than that in a flask. In Chapter 6, this chapter performed high-concentration and efficient bioconversion of eicosapentaenoic acid (EPA) into RvE4 and enantiomer of RvE4 by reaction optimization using polymer and solvent. The mutations changed the 15R- and 15S-LOXs into a 18R- and 18S-LOXs by L429W/L430M/L575M and L423W/L424M/L568M mutations, respectively, using structure-guided engineering. E. coli cells expressing engineered 18S- and 18R- LOXs converted EPA into 18S- and 18R-hydroxyeicosapentaenoic acids (18R- HEPAs), which were converted into 18S-RvE2 and RvE2 by E. coli cells expressing 5S-LOX from Danio rerio, respectively. In present chapters, discovery of highly active enzyme, enzyme engineering, and process optimization for the efficient production of oxylipins, including LMs and SPMs, can apply the bioconversion of structurally diverse oxylipins from PUFAs or renewable oils, and produced industrial concentrations. These results represent an efficient, cost-effective, and eco-friendly biocatalytic synthesis of bioactive oxylipins, including LMs and SPMs, from PUFAs or oils as a green route to overcome the environmental pollution for chemical synthesis. Keywords: Lipoxygenase, Lipid mediators, Specialized pro-resolving mediators, Molecular determination, Enzyme engineering, Enhanced bioconversion, Sustainable oils