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On the way of making plants smell like moths - a synthetic biology approach

Ding, Bao-Jian LU (2014)
Abstract (Swedish)
Popular Abstract in Chinese

在世界許多地方蛾類幼蟲是主要的農業害蟲,但也有蛾類養殖業為獲取其絲綢蠶繭,例如家蠶。其實它也是第一個也是最好的研究揮發性有機分子用於傳遞信息的昆蟲研究模型。一般來說是雌蛾釋放信息素通過很長的距離來吸引雄性同類,而且具有出色的靈敏度和特異性。至今有記載的蛾類信息素都屬於同一類化合物(脂肪酸衍生物), 在信息素腺體內合成。信息素的生物合成涉及到脂肪酸的脫飽合,碳鏈縮短,並最終還原,乙酰化或氧化成醛等。這些反應過程中的酶已經進化到具有專一性,專門用於生產複雜的化學信號,使得蛾類的信息素存在豐富的化學多樣性。在本論文中,首先對幾個脫飽和酶進行研究。第一篇文章對冬尺蠖的脫飽合酶進行克隆並在酵母中通過異源表達證明了它具有把順十一,順十四,順十七-二十碳三烯酸轉化成順十一,順十四,順十七,十九-二十碳四烯酸的能力。這是有史以來第一個對甲基端脫飽合酶的報導。第二篇文章報導了來自蘋果蠹蛾和梨小食心蟲的脫飽合酶。它們都使用不飽和的十二碳烯醇和乙酸酯作為性信息素成分。在蘋果蠹蛾中發現兩個脫飽和酶連續工作產生共軛雙鍵。但在梨小食心蟲中,發現是由同一個脫飽和酶工作兩次產生的共軛雙鍵。這項研究提供了在蛾信息系素腺體內可以生產共軛二烯的又一個例子。第三篇文章對十一位脫飽和酶的立體特異性進行了調查。薔薇斜條捲葉蛾的十一位脫飽和酶作用於飽和十四酸,產生的順十一十四酸比反十一十四酸等於一百比四十七的混合物;... (More)
Popular Abstract in Chinese

在世界許多地方蛾類幼蟲是主要的農業害蟲,但也有蛾類養殖業為獲取其絲綢蠶繭,例如家蠶。其實它也是第一個也是最好的研究揮發性有機分子用於傳遞信息的昆蟲研究模型。一般來說是雌蛾釋放信息素通過很長的距離來吸引雄性同類,而且具有出色的靈敏度和特異性。至今有記載的蛾類信息素都屬於同一類化合物(脂肪酸衍生物), 在信息素腺體內合成。信息素的生物合成涉及到脂肪酸的脫飽合,碳鏈縮短,並最終還原,乙酰化或氧化成醛等。這些反應過程中的酶已經進化到具有專一性,專門用於生產複雜的化學信號,使得蛾類的信息素存在豐富的化學多樣性。在本論文中,首先對幾個脫飽和酶進行研究。第一篇文章對冬尺蠖的脫飽合酶進行克隆並在酵母中通過異源表達證明了它具有把順十一,順十四,順十七-二十碳三烯酸轉化成順十一,順十四,順十七,十九-二十碳四烯酸的能力。這是有史以來第一個對甲基端脫飽合酶的報導。第二篇文章報導了來自蘋果蠹蛾和梨小食心蟲的脫飽合酶。它們都使用不飽和的十二碳烯醇和乙酸酯作為性信息素成分。在蘋果蠹蛾中發現兩個脫飽和酶連續工作產生共軛雙鍵。但在梨小食心蟲中,發現是由同一個脫飽和酶工作兩次產生的共軛雙鍵。這項研究提供了在蛾信息系素腺體內可以生產共軛二烯的又一個例子。第三篇文章對十一位脫飽和酶的立體特異性進行了調查。薔薇斜條捲葉蛾的十一位脫飽和酶作用於飽和十四酸,產生的順十一十四酸比反十一十四酸等於一百比四十七的混合物; 而來自於斑點捲葉蛾的脫飽和酶也作用於十四個碳飽和酸,但只生產純的反十一十四酸。通過對這兩個脫飽和酶的定點突變研究證明,一個在蛋白的羧基末端的氨基酸對脫飽和酶的順式活性至關重要。第四篇文章的研究集中在脂肪醇乙酰轉移酶,其在許多蛾類性信息素的生物合成途徑中承擔很大的作用。它催化的反應是轉移乙酰基給脂肪醇形成乙酸酯。由於此酶沒有在昆蟲中被克隆到,本研究異源表達了植物來源的乙酰基轉移酶,在酵母系統中測試其轉化蛾類性信息素脂肪醇的功能。結果表明其可以轉化碳鍊長度範圍從十個碳到十八個碳,不同雙鍵位置的各種脂肪醇,成為其相應的乙酸酯。第五篇文章利用下一代測序技術對黃地老虎信息素腺體進行分析,鑑定出許多與信息素合成相關的基因及過程,例如脂肪酸合成酶、脫飽合酶、貝塔氧化、還原酶、乙酰基轉移酶等等。在第六篇文章中利用煙草作為生物反應器來生產蛾類信息素來證明這個理論是可行的。通過瞬時表達四個蛾類信息素合成基因本研究成功生產出典型的蛾類信息素並證明了其具有能吸引雄蛾的生物活性。雖然利用植物生產的信息素的成份和比例沒有經過優化,但還是誘捕到大量的稠李巢蛾和蘋果巢蛾,其效果與有機合成的信息素相匹配。我們的長期目標是利用轉基因植物來生產任何類型的蛾類信息素。這種新穎的利用合成生物學的技術來製備信息素的方法將來也許會成為害蟲綜合防治的首選。



Popular Abstract in Swedish

Nattfjärilslarver är viktiga jordbruksskadedjur i många delar av världen. Men det finns nattfjärilarar som odlas för deras silkeskokong, till exempel den anmärkningsvärda silkesfjärilen, Bombyx mori. Denna art är den första och bäst studerade insektsarten som använder flyktiga organiska molekyler att kommunicera med, och med oerhört hög sensitivitet och specificitet. I allmänhet är det nattfjärilshonorna som lockar hannarna med sitt könsferomon över långa avstånd. De flesta beskrivna nattfjärilskönsferomonerna hör till samma klass av kemiska föreningar, nämligen fettsyrederivat som produceras de novo i feromonkörteln. Deras feromonbiosyntes innebär desaturation, kedjeförkortning med β-oxidation och slutligen reduktion, acetylering eller oxidation. Dessa enzymer har utvecklats för att fungera i produktionen av de komplexa kemiska signaler som används för sexuell attraktion, vilket bidrar till den kemiska mångfalden av nattfjärilsferomoner.

I denna avhandling har flera desaturaser undersökts. Först är det en terminalfett-acyl-CoA-desaturas från frostfjärilen Operophtera brumata som klonats och uttryckts heterologt i jäst och visat sin förmåga att omvandla Z11,Z14,Z17-eikosatriensyra till Z11,Z14,Z17,19-eikosatetraensyra. Detta är den första rapporten om metyl-terminal-desaturas någonsin.

I det andra manuskriptet har jag visat de desaturationssteg i Cydia pomonella och Grapholita molesta som använder omättad dodekenylalkohol och/eller -acetat som sexualferomonkomponent(er). Jag fann desaturaser i C. pomonella som arbetar i följd och bidrar till att producera en konjugerad dubbelbindning i fettsyrekedjan (E8,E10-12:CoA). Denna studie är ett exempel på hur en konjugerad dien kan produceras i en nattfjärilferomonkörtel. Men för G. molesta upptäckte jag att ∆10 desaturaser använder mättat 14-kol. Den E/Z10-14:Acyl genomgå kedjeförkortning, slutligen reduktion och acetylering kan producera G. molesta feromon som består av Z8-12:OAc, E8-12:OAc, och Z8-12:OH.

Det tredje manuskriptet handlar om stereospecificiteten hos Δ11-desaturaser. En Δ11-desaturas från Choristoneura rosaceana använder mättat 14-kol och producerar en blandning av Z/E11(65:35) C14-syra. En desaturas från C. parallela tar också mättad 14-kol, men producerar enbart ren E11-14C. Ömsesidiga riktade mutationer på dessa desaturaser avslöjade att en aminosyra vid C-terminalen av proteinet är kritisk för Z-aktiviteten av desaturaset (vinst eller förlust av funktion). Denna studie är fundamental för förståelsen av stereospecificiteten av desaturas.

Den fjärde studien fokuserade på fettsyrealkohol-acetyltransferas som har stor påverkan på många nattfjärilsferomoners biosyntesvägar. Den katalyserar bildningen av acetat-ester genom att överföra acetatgruppen från acetyl-CoA till fettsyrealkohol. Eftersom inget feromonbiosyntetiskt acetyltransferas som härstammar från insekter klonats, uttryckte jag en heterologiskt acetyltransferas, EaDAcT, utvecklat från växter istället, i ett jästsystem för att testa funktionaliteten och giltigheten i att omvandla fjärilsferomonintermediärer, fettsyrealkoholer, till den slutliga feromonprodukten, nämligen fettsyrealkohol-acetatestrar. Resultaten visade att EaDAcT kunde konvertera olika fettsyrealkoholer med kedjelängd från 10 till 18 kolatomer, med dubbelbindningar vid olika positioner i motsvarande acetatestrar. EaDAcT föredrar kortare kedjelängd över de långa, dvs. omättade över mättade.

I den femte studien använde jag ”next-generation sequenzing” av feromonproducerande vävnad för att kunna identifiera gener som kan vara involverade i den feromonbiosyntetiska processen för sädesbroddflyet, Agrotis segetum, till exempel fettsyresyntas, β-oxidationsenzymer, desaturaser, fettacylreduktaser , acetyltransferaser, etc.

Den sista studien var ”montering” av delar som tidigare identifierats i ett ”chassi” för att göra nattfjärilsferomoner. Genom att använda tobaksplantan, Nicotiana benthamiana, som en ”växtfabrik” producerade vi typiska nattfjärilsferomon-komponenter genom ”transient expression” av upp till fyra gener som kodar för konsekutiva biosyntetiska steg. Vi producerade specifikt biologiskt aktiva flerkomponentsferomoner för två arter spinnmalar, Yponomeuta. Fettsyrealkohol-fraktioner från de genetiskt modifierade växterna acetylerades och blandades för att efterlikna respektive könsferomon från Y. evonymella och Y. padella. Även om sammansättningen av de från växter härstammande blandningarna inte var optimerad, var dessa blandningar mycket effektiva och specifika för fångst av nattfjärilshanar, och matchar mycket väl aktiviteten av konventionellt framställda syntetiska feromoner. Vår långsiktiga vision är att utforma skräddarsydd produktion av valfria nattfjärilsferomonkomponenter i genetiskt modifierade växter. Sådan semisyntetisk beredning av könsferomoner kan vara ett nytt och kostnadseffektivt sätt att producera måttliga till stora mängder feromoner för integrerat växtskydd, med hög renhet och ett minimum av farligt avfall. (Less)
Abstract
Moth caterpillars are major agricultural pests in many parts of the world. In general female moths attract male mates with their pheromone over long distance. Most of the described moth pheromones belong to the same class of chemical compounds, fatty acid derivatives that are produced de novo in the pheromone gland. The pheromone biosynthesis involves desaturation, chain-shortening by β-oxidation and functional group modification such as reduction, acetylation or oxidation, etc. These enzymes have evolved to function in the production of the complex chemical signals used for sex attraction, thus contributing to the chemical diversity of moth pheromones. In the current thesis, several desaturases were characterized. Firstly, a terminal... (More)
Moth caterpillars are major agricultural pests in many parts of the world. In general female moths attract male mates with their pheromone over long distance. Most of the described moth pheromones belong to the same class of chemical compounds, fatty acid derivatives that are produced de novo in the pheromone gland. The pheromone biosynthesis involves desaturation, chain-shortening by β-oxidation and functional group modification such as reduction, acetylation or oxidation, etc. These enzymes have evolved to function in the production of the complex chemical signals used for sex attraction, thus contributing to the chemical diversity of moth pheromones. In the current thesis, several desaturases were characterized. Firstly, a terminal fatty-acyl-CoA desaturase from winter moth (Operophtera brumata) was cloned and expressed heterologously in yeast and proved its ability to convert Z11,Z14,Z17-eicosatrienoic acid to Z11,Z14,Z17,19-eicosatetraenoic acid. This is the first report on methyl terminal desaturase ever. Secondly, desaturation steps in Cydia pomonella and Grapholita molesta, which use unsaturated dodecenyl alcohol and/or acetate as sex pheromone component(s), were characterized. We found the desaturases in C. pomonella work consecutively, account for the production of conjugated double-bond in the fatty acyl chain (E8,E10-12:CoA). But in the case of G. molesta, we found ∆10 desaturase on myristic acid. The E/Z10-14:Acyl, which after chain-shortening, reduction and acetylation may produce the G. molesta pheromone consisting of Z8-12:OAc, E8-12:OAc and Z8-12:OH. Thirdly, the stereospecificity of two ∆11 desaturases was investigated. A ∆11 desaturase from Choristoneura rosaceana takes saturated 14 carbon and produce a mixture of (E)-11-tetradecenoate and (Z)-11-tetradecenoate with an excess of the Z isomer. A desaturase from C. parallela also takes saturated 14C but produce almost pure E11-14:Acyl. Reciprocal site-directed mutations on this two desaturases revealed that one amino acid at the C-terminal of the protein is critical for the Z activity of the desaturase (gain or lose of function). This study shed light on cracking the stereospecificity of desaturase. The next study focused on fatty alcohol acetyltransferase that bears great implications in many moth pheromone biosynthesis pathways. It catalyzes the formation of acetate ester by transferring acetate group from the acetyl-CoA to the fatty alcohol. Since no insect-derived pheromone biosynthetic acetyltransferase has been cloned, we heterologously expressed a plant derived acetyltransferase, EaDAcT, in a yeast system, to test the functionality and validity in converting moth pheromone intermediates, fatty alcohols, into final pheromone product, fatty alcohol acetate esters. The results showed EaDAcT could convert various fatty alcohols with chain length range from 10 to 18 carbons, with double bound at varying positions, into their corresponding acetate esters. EaDAcT prefers shorter chain length to the long ones, unsaturated to the saturated ones. The microsome preparations showed an activity pattern similar to the activity observed in the in vivo experiments. Next, through massive sequencing of pheromone producing tissue, we identified genes that might be involved in the pheromone biosynthetic process of the turnip moth (Agrotis segetum), such as: fatty acid synthase, β-oxidation enzymes, desaturase, fatty acyl reductase, acetyltransferase, etc. The final study was assembling the parts identified previously in a chassis to make moth pheromones. Using Nicotiana benthamiana, as a plant factory, we produced typical moth sex pheromone components by transient expression of up to four genes coding for consecutive biosynthetic steps. We specifically produced biologically active multi-component sex pheromones for two species of small ermine moths. The fatty alcohol fractions from the genetically modified plants were acetylated and mixed to mimic the respective sex pheromones of Yponomeuta evonymella and Y. padella. Although the composition of the plant-derived mixtures was not optimized, these mixtures were very efficient and specific for trapping of male moths, matching the activity of conventionally produced synthetic pheromones. Our long-term vision is to design tailor-made production of any moth pheromone component in genetically modified plants. Such semi-synthetic preparation of sex pheromones may be a novel and cost-effective way of producing moderate to large quantities of pheromones for integrated pest management, with high purity and a minimum of hazardous waste. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Newcomb, Richard, The New Zealand Institute for Plant & Food Research
organization
publishing date
type
Thesis
publication status
published
subject
keywords
moth pheromone, transcriptome, fatty acyl desaturase, fatty acyl reductase, acetyltransferase, chain shortening, chain elongation, heterologous expression, synthetic biology, plant expression
pages
180 pages
publisher
Lund University, Faculty of Science, Department of Biology
defense location
Blue Hall, Ecology Building, Sölvegatan 37, Lund
defense date
2014-02-14 09:30
ISBN
978-91-7473-836-0
project
The insect pheromone brewery
language
English
LU publication?
yes
id
349de72f-99b2-4dc9-a0ea-daa35cbd3b5d (old id 4276815)
date added to LUP
2014-01-30 16:25:53
date last changed
2016-09-26 16:24:34
@phdthesis{349de72f-99b2-4dc9-a0ea-daa35cbd3b5d,
  abstract     = {Moth caterpillars are major agricultural pests in many parts of the world. In general female moths attract male mates with their pheromone over long distance. Most of the described moth pheromones belong to the same class of chemical compounds, fatty acid derivatives that are produced de novo in the pheromone gland. The pheromone biosynthesis involves desaturation, chain-shortening by β-oxidation and functional group modification such as reduction, acetylation or oxidation, etc. These enzymes have evolved to function in the production of the complex chemical signals used for sex attraction, thus contributing to the chemical diversity of moth pheromones. In the current thesis, several desaturases were characterized. Firstly, a terminal fatty-acyl-CoA desaturase from winter moth (Operophtera brumata) was cloned and expressed heterologously in yeast and proved its ability to convert Z11,Z14,Z17-eicosatrienoic acid to Z11,Z14,Z17,19-eicosatetraenoic acid. This is the first report on methyl terminal desaturase ever. Secondly, desaturation steps in Cydia pomonella and Grapholita molesta, which use unsaturated dodecenyl alcohol and/or acetate as sex pheromone component(s), were characterized. We found the desaturases in C. pomonella work consecutively, account for the production of conjugated double-bond in the fatty acyl chain (E8,E10-12:CoA). But in the case of G. molesta, we found ∆10 desaturase on myristic acid. The E/Z10-14:Acyl, which after chain-shortening, reduction and acetylation may produce the G. molesta pheromone consisting of Z8-12:OAc, E8-12:OAc and Z8-12:OH. Thirdly, the stereospecificity of two ∆11 desaturases was investigated. A ∆11 desaturase from Choristoneura rosaceana takes saturated 14 carbon and produce a mixture of (E)-11-tetradecenoate and (Z)-11-tetradecenoate with an excess of the Z isomer. A desaturase from C. parallela also takes saturated 14C but produce almost pure E11-14:Acyl. Reciprocal site-directed mutations on this two desaturases revealed that one amino acid at the C-terminal of the protein is critical for the Z activity of the desaturase (gain or lose of function). This study shed light on cracking the stereospecificity of desaturase. The next study focused on fatty alcohol acetyltransferase that bears great implications in many moth pheromone biosynthesis pathways. It catalyzes the formation of acetate ester by transferring acetate group from the acetyl-CoA to the fatty alcohol. Since no insect-derived pheromone biosynthetic acetyltransferase has been cloned, we heterologously expressed a plant derived acetyltransferase, EaDAcT, in a yeast system, to test the functionality and validity in converting moth pheromone intermediates, fatty alcohols, into final pheromone product, fatty alcohol acetate esters. The results showed EaDAcT could convert various fatty alcohols with chain length range from 10 to 18 carbons, with double bound at varying positions, into their corresponding acetate esters. EaDAcT prefers shorter chain length to the long ones, unsaturated to the saturated ones. The microsome preparations showed an activity pattern similar to the activity observed in the in vivo experiments. Next, through massive sequencing of pheromone producing tissue, we identified genes that might be involved in the pheromone biosynthetic process of the turnip moth (Agrotis segetum), such as: fatty acid synthase, β-oxidation enzymes, desaturase, fatty acyl reductase, acetyltransferase, etc. The final study was assembling the parts identified previously in a chassis to make moth pheromones. Using Nicotiana benthamiana, as a plant factory, we produced typical moth sex pheromone components by transient expression of up to four genes coding for consecutive biosynthetic steps. We specifically produced biologically active multi-component sex pheromones for two species of small ermine moths. The fatty alcohol fractions from the genetically modified plants were acetylated and mixed to mimic the respective sex pheromones of Yponomeuta evonymella and Y. padella. Although the composition of the plant-derived mixtures was not optimized, these mixtures were very efficient and specific for trapping of male moths, matching the activity of conventionally produced synthetic pheromones. Our long-term vision is to design tailor-made production of any moth pheromone component in genetically modified plants. Such semi-synthetic preparation of sex pheromones may be a novel and cost-effective way of producing moderate to large quantities of pheromones for integrated pest management, with high purity and a minimum of hazardous waste.},
  author       = {Ding, Bao-Jian},
  isbn         = {978-91-7473-836-0},
  keyword      = {moth pheromone,transcriptome,fatty acyl desaturase,fatty acyl reductase,acetyltransferase,chain shortening,chain elongation,heterologous expression,synthetic biology,plant expression},
  language     = {eng},
  pages        = {180},
  publisher    = {Lund University, Faculty of Science, Department of Biology},
  school       = {Lund University},
  title        = {On the way of making plants smell like moths - a synthetic biology approach},
  year         = {2014},
}