Cultivation and functional characterization of 79 planctomycetes uncovers their unique biology
Wiegand, Sandra ; Jogler, Mareike ; Boedeker, Christian ; Pinto, Daniela ; Vollmers, John ; Rivas-Marín, Elena ; Kohn, Timo ; Peeters, Stijn H. ; Heuer, Anja ; Rast, Patrick ; Oberbeckmann, Sonja ; Bunk, Boyke ; Jeske, Olga ; Meyerdierks, Anke ; Storesund, Julia E. ; Kallscheuer, Nicolai ; Lücker, Sebastian ; Lage, Olga M. ; Pohl, Thomas ; Merkel, Broder J. ; Hornburger, Peter ; Müller, Ralph Walter ; Brümmer, Franz ; Labrenz, Matthias ; Spormann, Alfred M. ; Camp, Huub J.M. Op den; Overmann, Jörg ; Amann, Rudolf ; Jetten, Mike S.M. ; Mascher, Thorsten ; Medema, Marnix H. ; Devos, Damien P. ; Kaster, Anne Kristin ; Øvreås, Lise ; Rohde, Manfred ; Galperin, Michael Y. ; Jogler, Christian - \ 2019
Nature Microbiology 5 (2019). - ISSN 2058-5276 - p. 126 - 140.
When it comes to the discovery and analysis of yet uncharted bacterial traits, pure cultures are essential as only these allow detailed morphological and physiological characterization as well as genetic manipulation. However, microbiologists are struggling to isolate and maintain the majority of bacterial strains, as mimicking their native environmental niches adequately can be a challenging task. Here, we report the diversity-driven cultivation, characterization and genome sequencing of 79 bacterial strains from all major taxonomic clades of the conspicuous bacterial phylum Planctomycetes. The samples were derived from different aquatic environments but close relatives could be isolated from geographically distinct regions and structurally diverse habitats, implying that ‘everything is everywhere’. With the discovery of lateral budding in ‘Kolteria novifilia’ and the capability of the members of the Saltatorellus clade to divide by binary fission as well as budding, we identified previously unknown modes of bacterial cell division. Alongside unobserved aspects of cell signalling and small-molecule production, our findings demonstrate that exploration beyond the well-established model organisms has the potential to increase our knowledge of bacterial diversity. We illustrate how ‘microbial dark matter’ can be accessed by cultivation techniques, expanding the organismic background for small-molecule research and drug-target detection.
Capturing of the monoterpene olefin limonene produced in Saccharomyces cerevisiae
Jongedijk, E.J. ; Cankar, K. ; Ranzijn, J. ; Krol, A.R. van der; Bouwmeester, H.J. ; Beekwilder, M.J. - \ 2015
Yeast 32 (2015)1. - ISSN 0749-503X - p. 159 - 171.
monoterpene biosynthesis - escherichia-coli - synthase - precursor
Monoterpene olefins such as limonene are plant compounds with applications as flavouring and fragrance agents, as solvents and potentially also in polymer and fuel chemistry. We engineered baker's yeast Saccharomyces cerevisiae to express a (-)-limonene synthase from Perilla frutescens and a (+)-limonene synthase from Citrus limon. Both proteins were expressed either with their native plastid targeting signal or in a truncated form in which the plastidial sorting signal was removed. The yeast host strain for expression was AE9 K197G, which expresses a mutant Erg20 enzyme. This enzyme catalyses the formation of geranyl diphosphate, which is the precursor for monoterpenes. Several methods were tested to capture limonene produced by the yeast. Extraction from the culture medium by pentane, or by the addition of CaCl2 followed by solid-phase micro-extraction, did not lead to detectable limonene, indicating that limonene is rapidly lost from the culture medium. Volatile terpenes such as limonene may also be trapped in a dodecane phase added to the medium during fermentation. This method resulted in recovery of 0.028¿mg/l (+)-limonene and 0.060¿mg/l (-)-limonene in strains using the truncated Citrus and Perilla synthases, respectively. Trapping the headspace during culture of the limonene synthase-expressing strains resulted in higher titres, at 0.12¿mg/l (+)-limonene and 0.49¿mg/l (-)-limonene. These results show that the volatile properties of the olefins produced require specific methods for efficient recovery of these molecules from biotechnological production systems. Gene Bank Nos were: KM015220 (Perilla limonene synthase; this study); AF317695 (Perilla limonene synthase; Yuba et al., 1996); AF514287.1 (Citrus limonene synthase; Lucker et al., 2002).
|Metabolic engineering of terpenoid biosynthesis in plants
Lücker, J. ; Bouwmeester, H.J. ; Aharoni, A. - \ 2007
In: Applications of plant metabolic engineering / Verpoorte, R., Alferman, W., Johnson, T.S., Berlin : Springer Verlag - ISBN 9781402060304 - p. 211 - 218.
|Molecular engineering of floral scent
Lücker, J. ; Verhoeven, H.A. ; Plas, L.H.W. van der; Bouwmeester, H.J. - \ 2006
In: Biology of floral scent / Dudareva, N., Pichersky, E., Atlanta, USA : CRC Press - ISBN 9780849322839 - p. 321 - 337.
Increased and altered fragrance of tobacco plants after metabolic engineering using three monoterpene synthases from lemon
Lücker, J. ; Schwab, W. ; Hautum, B. van; Blaas, J. ; Plas, L.H.W. van der; Bouwmeester, H.J. ; Verhoeven, H.A. - \ 2004
Plant Physiology 134 (2004)1. - ISSN 0032-0889 - p. 510 - 519.
s-linalool synthase - biosynthetic-pathway - volatile compounds - nicotiana-tabacum - gene-expression - floral scents - flowers - transformation - emission - clarkia
Wild-type tobacco (Nicotiana tabacum) plants emit low levels of terpenoids, particularly from the flowers. By genetic modification of tobacco cv Petit Havana SR1 using three different monoterpene synthases from lemon (Citrus limon L. Burm. f.) and the subsequent combination of these three into one plant by crossings, we show that it is possible to increase the amount and alter the composition of the blend of monoterpenoids produced in tobacco plants. The transgenic tobacco plant line with the three introduced monoterpene synthases is emitting -pinene, limonene, and -terpinene and a number of side products of the introduced monoterpene synthases, from its leaves and flowers, in addition to the terpenoids emitted by wild-type plants. The results show that there is a sufficiently high level of substrate accessible for the introduced enzymes
Metabolic engineering of monoterpende biosysnthesis: two step production of (+)-trans-Isopiperitenol by tobacco
Lücker, J. ; Schwab, W. ; Franssen, M.C.R. ; Plas, L.H.W. van der; Bouwmeester, H.J. ; Verhoeven, H.A. - \ 2004
The Plant Journal 39 (2004)1. - ISSN 0960-7412 - p. 135 - 145.
peppermint mentha-piperita - functional expression - limonene enantiomers - linalool synthase - cdna isolation - s-linalool - plants - mint - (-)-limonene - cloning
Monoterpenoid biosynthesis in tobacco was modified by introducing two subsequent enzymatic activities targeted to different cell compartments. A limonene-3-hydroxylase (lim3h) cDNA was isolated from Mentha spicata L. 'Crispa'. This cDNA was used to re-transform a transgenic Nicotiana tabacum'Petit Havana' SR1 (tobacco) line expressing three Citrus limon L. Burm. f. (lemon) monoterpene synthases producing (+)-limonene, gamma-terpinene and (-)-beta-pinene as their main products. The targeting sequences of these synthases indicate that they are probably localized in the plastids, whereas the sequence information of the P450 hydroxylase indicates targeting to the endoplasmatic reticulum. Despite the different location of the enzymes, the introduced P450 hydroxylase proved to be functional in the transgenic plants as it hydroxylated (+)-limonene, resulting in the emission of (+)-trans-isopiperitenol. Some further modifications of the (+)-trans-isopiperitenol were also detected, resulting in the additional emission of 1,3,8-p-menthatriene, 1,5,8-p-menthatriene, p-cymene and isopiperitenone.
The influence of monoterpene synthase transformation on the odour of tabacco.
Tamer, M.K. el; Smeets, M.A.M. ; Holthuysen, N.T.E. ; Lucker, J. ; Tang, A. ; Roozen, J.P. ; Bouwmeester, H.J. ; Voragen, A.G.J. - \ 2003
Journal of Biotechnology 106 (2003)1. - ISSN 0168-1656 - p. 15 - 21.
linalool synthase - gene-expression - s-linalool - biosynthesis - plants - cancer
Monoterpenes are an important class of terpenoids that are commonly present in plant essential oils. These can be extracted from plants and are used in the flavouring and perfumery industry. Monoterpene synthases are the key enzymes in monoterpene biosynthesis, as they catalyse the cyclisation of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. Tobacco is one of the most studied model plants, it can easily and efficiently be transformed, and is a suitable model to study the release of plant volatiles. Thus, we have isolated monoterpene synthases from lemon, transformed tobacco with these cDNAs and have used human panelists to study the change in fragrance of the transgenic in comparison to the wild type plants. In a triangle test, we found that subjects were capable of smelling significant differences between leaf samples. However, as a result of variability in panel ratings, no significant difference between two sets of transgenic flowers and the wild type tobacco flowers was found for the generated attributes in a descriptive test.
Domain swapping of Citrus limon monoterpene synthases: impact on enzymatic activity and product specifity.
Tamer, M.K. el; Lucker, J. ; Bosch, D. ; Verhoeven, H.A. ; Verstappen, F.W.A. ; Schwab, W. ; Tunen, A.J. van; Voragen, A.G.J. ; Maagd, R.A. de; Bouwmeester, H.J. - \ 2003
Archives of Biochemistry and Biophysics 411 (2003). - ISSN 0003-9861 - p. 196 - 203.
site-directed mutagenesis - 5-epi-aristolochene synthase - trichodiene synthase - linalool synthase - germacrene-a - biosynthesis - expression - biology
Monoterpene cyclases are the key enzymes in the monoterpene biosynthetic pathway, as they catalyze the cyclization of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. From Citrus limon, four monoterpene synthase-encoding cDNAs for a P-pinene synthase named Cl(-)betaPINS, a gamma-terpinene synthase named ClgammaTS, and two limonene synthases named Cl(+)LIMS1 and Cl(+)LIMS2 were recently isolated [J. Lucker et al., Eur. J. Biochem. 269 (2002) 3160]. The aim of our work in this study was to identify domains within these monoterpene synthase enzymes determining the product specificity. Domain swapping experiments between Cl(-)betaPINS and ClgammaTS and between Cl(+)LIMS2 and ClyTS were conducted. We found that within the C-terminal domain of these monoterpene synthases, a region comprising 200 amino acids, of which 41 are different between Cl(-)betaPINS and ClgammaTS, determines the specificity for the formation of P-pinene or gamma-terpinene, respectively, while another region localized further downstream is required for a chimeric enzyme to yield products in the same ratio as in the wild-type ClgammaTS. For Cl(+)LIMS2, the two domains together appear to be sufficient for its enzyme specificity, but many chimeras were inactive probably due to the low homology with ClyTS. Molecular modeling was used to further pinpoint the amino acids responsible for the differences in product specificity of ClyTS and Cl(-)betaPINS. (C) 2003 Elsevier Science (USA). All rights reserved.
Exploring multi-trophic plant-herbivore interactions for new crop protection methods
Bouwmeester, H.J. ; Verstappen, F.W.A. ; Aharoni, A. ; Lücker, J. ; Jongsma, M.A. ; Kappers, I.F. ; Luckerhoff, L.L.P. ; Dicke, M. - \ 2003
In: Proceedings of the International Congress Crop Science and Technology, 10-12 November 2003 Glasgow, British Crop Protection Council, Alton, UK, 2003 - p. 1123 - 1134.
Monoterpene biosynthesis in lemon (Citrus limon) cDNA isolation and functional analysis of four monoterpene synthases
Lücker, J. ; Tamer, M.K. El; Schwab, W. ; Verstappen, F.W.A. ; Plas, L.H.W. van der; Bouwmeester, H.J. ; Verhoeven, H.A. - \ 2002
European Journal of Biochemistry 269 (2002). - ISSN 0014-2956 - p. 3160 - 3161.
Citrus limon possesses a high content and large variety of monoterpenoids, especially in the glands of the fruit flavedo. The genes responsible for the production of these monoterpenes have never been isolated. By applying a random sequencing approach to a cDNA library from mRNA isolated from the peel of young developing fruit, four monoterpene synthase cDNAs were isolated that appear to be new members of the previously reported tpsb family. Based on sequence homology and phylogenetic analysis, these sequences cluster in two separate groups. All four cDNAs could be functionally expressed in Escherichia coli after removal of their plastid targeting signals. The main products of the enzymes in assays with geranyl diphosphate as substrate were ( )-limonene (two cDNAs) (-)--pinene and -terpinene. All enzymes exhibited a pH optimum around 7; addition of Mn2 as bivalent metal ion cofactor resulted in higher activity than Mg2 , with an optimum concentration of 0.6 mm. Km values ranged from 0.7 to 3.1 ?m. The four enzymes account for the production of 10 out of the 17 monoterpene skeletons commonly observed in lemon peel oil, corresponding to more than 90␘f the main components present
Metabolic engineering of monoterpene biosynthesis in plants
Lücker, J. - \ 2002
Wageningen University. Promotor(en): L.H.W. van der Plas; H.A. Verhoeven; H.J. Bouwmeester. - S.l. : S.n. - ISBN 9789058087171 - 158
nicotiana - petunia - citrus limon - monoterpenen - biosynthese - genetische modificatie - metabolisme - transgene planten - plantenfysiologie - nicotiana - petunia - citrus limon - monoterpenes - biosynthesis - genetic engineering - metabolism - transgenic plants - plant physiology
Monoterpenes are a large group of compounds that belong to the terpenoid family of natural compounds in plants. They are small, volatile, lipophilic substances of which around one thousand different structures have been identified. Monoterpenes are involved in plant-insect, plant-microorganism and plant-plant interactions. Many monoterpenes, such as menthol, carvone, limonene and linalool, are of commercial interest as they are commonly used in foods, beverages, perfumes and cosmetics and in many cleaning products. In flowers they also contribute to the characteristic scent. Monoterpene synthases and subsequent modifying enzymes such as cytochrome P450 hydroxylases, dehydrogenases, reductases and isomerases are responsible for the production of the variety of different carbon skeletons of monoterpenes that are found in nature. In this thesis the use of genetic engineering to introduce or alter the production of monoterpenes by plants was explored.
Initially, as described in Chapter 2, S -linalool synthase from Clarkia breweri was introduced in Petunia plants regulated by a constitutive promoter. Expression was obtained in all tissues analysed, but formation of linalool was restricted to leaves, sepals, corollas, stems and ovaries, and could not be detected in nectaries, roots, pollen and style. Although it was expected that the formation of linalool would result in an alteration of the scent of the plants, no linalool was detected in the headspace. Instead, all the S -linalool produced was efficiently converted by an endogenous glucosyltransferase present in the petunia tissues to the non-volatile S -linalyl-b-D-glucopyranoside. These results showed that genetic engineering of plants for monoterpene biosynthesis is possible, but that it can lead to unexpected conversions of the produced metabolites by endogenous enzyme activities.
In order to obtain new monoterpene synthases for the genetic engineering of plants, a cDNA library was made of the fruit peel of lemon, a plant species producing many different monoterpenes. From this library four different monoterpene synthases were obtained as described in Chapter 3, which together showed to be responsible for more than 90% of the total number of components present in lemon oil. The product specificity of the enzymes could be analysed after heterologous expression in Escherichia coli . Two of the four cDNA-encoded enzymes were producing (+)-limonene, the main component present in lemon. One cDNA-encoded enzyme was mainly producing (-)-b-pinene and the fourth cDNA-encoded enzyme was mainly producingg-terpinene. The latter two enzymes were both producing traces of multiple side products as well. Also other properties of the heterologously expressed enzymes were determined, which are described in Chapter 3.
Three monoterpene synthases responsible for the production of different main products were chosen for the genetic engineering of Nicotiana tabacum 'Petit Havana' SR1, described in Chapter 4. The wild type of this tobacco variety produces one monoterpene, linalool that is only emitted from the flowers. After the transformation with the three monoterpene synthases and subsequent crossings, a plant was obtained that emitted all the three main products of the three introduced monoterpene synthases in addition to the endogenous linalool in the flowers. The levels of limonene,b-pinene andg-terpinene emitted from the leaves and flowers of the plant were higher than the level of the endogenous monoterpene. Also the side products of the monoterpene synthases were detected. The extensive modification of the volatile profile of the tobacco plants that we obtained indicates that there is a sufficient amount of substrate available to the introduced enzymes.
In Chapter 5 the transgenic tobacco plant emitting the products of three monoterpene synthases, was used in a subsequent transformation experiment in order to modify the already introduced pathway. A second step in the pathway was introduced by transformation of the plant material with a limonene-3-hydroxylase isolated from spearmint, which is supposed to be localised in the endoplasmatic reticulum (ER) in the cytosol of the plant cells, while the primarily introduced monoterpene synthases were most likely localised in the plastids in the transgenic plants. The introduction of the cytochrome P450 monoterpene hydroxylase and the resulting formation of the hydroxylated product of (+)-limonene, (+)- trans -isopiperitenol demonstrates that there is intracellular trafficking of limonene from the plastids to the ER in the cytosol. That this trafficking mechanism would be present in plants normally producing these hydroxylated monoterpenes could be expected, but that it is apparently also present in plants not specialised for the production of these compounds is an exciting discovery. Apart from the production and subsequent emission of high further oxidised conversion product isopiperitenone was detected. In addition, an increase in the p -cymene level and the formation of the new products 1,3,8- p -menthatriene and 1,5,8- p -menthatriene were detected. The occurrence of these latter two products and the increase of the p -cymene level could be a consequence of the metabolic engineering of the biosynthetic route into a cell compartment not adapted to the production of these compounds. Endogenous enzymes and pH differences were suggested to be the main cause the formation of these products.
Chapter 6 discusses the various strategies followed for the metabolic engineering of monoterpene biosynthesis in this thesis and by other groups. Functional implications are discussed such as ecological and physiological consequences of the new metabolites for the transgenic plants. The commercial aspects and interesting opportunities for further research are also discussed.
Expression of Clarkia S-linalool synthase in transgenic petunia plant results in the accumulation of S-linalyl-b-D-glucopyranoside
Lücker, J. ; Bouwmeester, H.J. ; Schwab, W. ; Blaas, J. ; Plas, L.H.W. van der; Verhoeven, H.A. - \ 2001
The Plant Journal 27 (2001). - ISSN 0960-7412 - p. 315 - 324.
Petunia hybrida W115 was transformed with a Clarkia breweri S-linalool synthase cDNA (lis). Lis was expressed in all tissues analysed, and linalool was detected in leaves, sepals, corolla, stem and ovary, but not in nectaries, roots, pollen and style. However, the S-linalool produced by the plant in the various tissues is not present as free linalool, but was efficiently converted to non-volatile S-linalyl--d-glucopyranoside by the action of endogenous glucosyltransferase. The results presented demonstrate that monoterpene production can be altered by genetic modification, and that the compounds produced can be converted by endogenous enzymatic activity.
Functional genomic analysis of potato tuber life-cycle
Bachem, C.W.B. ; Hoeven, R. van der; Lucker, J. ; Oomen, R. ; Casarini, E. ; Jacobsen, E. ; Visser, R. - \ 2000
Potato Research 43 (2000). - ISSN 0014-3065 - p. 297 - 312.
|Milieuaansprakelijkheid bij bodemverontreiniging. Van financiering naar gedragsbeïnvloeding.
Lücker, F. - \ 1991
Wageningen : Unknown Publisher - 66 p.
Food Choice and Caries Experience in Dutch Teenagers as a Function of the Level of Education of Their Parents
Vries, H.C.B. de; Lucker, T.P.C. ; Cremers, S.B.L. ; Katan, M.B. - \ 1990
European Journal of Clinical Nutrition 44 (1990)11. - ISSN 0954-3007 - p. 839 - 846.
The relation of the level of education attained by the father and mother with caries prevalence and food intake in the child was investigated in 284 fourteen-year-old Dutch schoolchildren.Foods were classified into three categories: 'health food', 's
Koffie en gezondheid III: Effecten op groei en ontwikkeling van de foetus, en op borstgezwellen.
Cremers, S.B.L. ; Lucker, T.P.C. ; Katan, M.B. - \ 1988
Voeding 49 (1988). - ISSN 0042-7926 - p. 106 - 110.