Pathway transfer in fungi: Transporters are the key to success
Straat, L. van der; Graaff, L.H. de - \ 2014
Bioengineered 5 (2014)5. - ISSN 2165-5979 - p. 335 - 339.
itaconic acid production - saccharomyces-cerevisiae - lactococcus-lactis - aspergillus-niger - cephalosporin production - penicillium-chrysogenum - functional expression - membrane-proteins - ceft gene - overproduction
Itaconic acid is an important building block for the chemical industry. Currently, Aspergillus terreus is the main organism used for itaconic acid production. Due to the enormous citric acid production capacity of Aspergillus niger, this host is investigated as a potential itaconic acid production host. Several strategies have been tried so far: fermentation optimization, expression of cis-aconitate decarboxylase (cadA) alone and in combination with aconitase targeted to the same compartment, chassis optimization, and the heterologous expression of two transporters flanking the cadA gene. We showed that the heterologous expression of these two transporters were key to improving itaconic acid production in an A. niger strain that was unable to produce oxalic acid and gluconic acid. The expression of transporters has increased the production levels of other industrially relevant processes as well, such as ß-lactam antibiotics and bioethanol. Thus far, the role of transporters in production process optimization is a bit overlooked.
Gene Coexpression Analysis Reveals Complex Metabolism of the Monoterpene Alcohol Linalool in Arabidopsis FlowersW
Ginglinger, J.F. ; Boachon, B. ; Hofer, R. ; Paetz, C. ; Kollner, T.G. ; Miesch, L. ; Lugan, R. ; Baltenweck, R. ; Mutterer, J. ; Ullman, P. ; Verstappen, F.W.A. ; Bouwmeester, H.J. - \ 2013
The Plant Cell 25 (2013)11. - ISSN 1040-4651 - p. 4640 - 4657.
cytochrome-p450 limonene hydroxylases - avocado persea-americana - functional expression - endoplasmic-reticulum - terpene synthases - mint mentha - saccharomyces-cerevisiae - beta-glucuronidase - essential oil - floral scent
The cytochrome P450 family encompasses the largest family of enzymes in plant metabolism, and the functions of many of its members in Arabidopsis thaliana are still unknown. Gene coexpression analysis pointed to two P450s that were coexpressed with two monoterpene synthases in flowers and were thus predicted to be involved in monoterpenoid metabolism. We show that all four selected genes, the two terpene synthases (TPS10 and TPS14) and the two cytochrome P450s (CYP71B31 and CYP76C3), are simultaneously expressed at anthesis, mainly in upper anther filaments and in petals. Upon transient expression in Nicotiana benthamiana, the TPS enzymes colocalize in vesicular structures associated with the plastid surface, whereas the P450 proteins were detected in the endoplasmic reticulum. Whether they were expressed in Saccharomyces cerevisiae or in N. benthamiana, the TPS enzymes formed two different enantiomers of linalool: (-)-(R)-linalool for TPS10 and (+)-(S)-linalool for TPS14. Both P450 enzymes metabolize the two linalool enantiomers to form different but overlapping sets of hydroxylated or epoxidized products. These oxygenated products are not emitted into the floral headspace, but accumulate in floral tissues as further converted or conjugated metabolites. This work reveals complex linalool metabolism in Arabidopsis flowers, the ecological role of which remains to be determined.
Potential of proton-pumping rhodopsins: engineering photosystems into microorganisms
Claassens, N.J.H.P. ; Volpers, M. ; Martins Dos Santos, V.A.P. ; Oost, J. van der; Vos, W.M. de - \ 2013
Trends in Biotechnology 31 (2013)11. - ISSN 0167-7799 - p. 633 - 642.
escherichia-coli - functional expression - microbial rhodopsins - carbon-dioxide - schizosaccharomyces-pombe - gloeobacter rhodopsin - purple membrane - proteorhodopsin - bacteriorhodopsin - gene
A wide range of proton-pumping rhodopsins (PPRs) have been discovered in recent years. Using a synthetic biology approach, PPR photosystems with different features can be easily introduced in nonphotosynthetic microbial hosts. PPRs can provide hosts with the ability to harvest light and drive the sustainable production of biochemicals or biofuels. PPRs use light energy to generate an outward proton flux, and the resulting proton motive force can subsequently power cellular processes. Recently, the introduction of PPRs in microbial production hosts has successfully led to light-driven biotechnological conversions. In this review, we discuss relevant features of natural PPRs, evaluate reported biotechnological applications of microbial production hosts equipped with PPRs, and provide an outlook on future developments
Aspergillus nidulans-galactosidase of glycoside hydrolase family 36 catalyses the formation of -galacto-oligosaccharides by transglycosylation
Nakai, H. ; Baumann, M.J. ; Petersen, B.O. ; Westphal, Y. ; Hachem, M.A. ; Dilokpimol, A. ; Duus, J.O. ; Schols, H.A. ; Svensson, B. - \ 2010
FEBS Journal 277 (2010)17. - ISSN 1742-464X - p. 3538 - 3551.
molecular-cloning - bifidobacterium-adolescentis - n-acetylgalactosaminidase - escherichia-coli - pichia-pastoris - phanerochaete-chrysosporium - clostridium-perfringens - functional expression - lactobacillus-reuteri - thermotoga-maritima
The -galactosidase from Aspergillus nidulans (AglC) belongs to a phylogenetic cluster containing eukaryotic -galactosidases and -galacto-oligosaccharide synthases of glycoside hydrolase family 36 (GH36). The recombinant AglC, produced in high yield (0.65 g·L-1 culture) as His-tag fusion in Escherichia coli, catalysed efficient transglycosylation with -(1¿6) regioselectivity from 40 mm 4-nitrophenol -d-galactopyranoside, melibiose or raffinose, resulting in a 37–74% yield of 4-nitrophenol -d-Galp-(1¿6)-d-Galp, -d-Galp-(1¿6)--d-Galp-(1¿6)-d-Glcp and -d-Galp-(1¿6)--d-Galp-(1¿6)-d-Glcp-(1¿ß2)-d-Fruf (stachyose), respectively. Furthermore, among 10 monosaccharide acceptor candidates (400 mm) and the donor 4-nitrophenol -d-galactopyranoside (40 mm), -(1¿6) linked galactodisaccharides were also obtained with galactose, glucose and mannose in high yields of 39–58%. AglC did not transglycosylate monosaccharides without the 6-hydroxymethyl group, i.e. xylose, l-arabinose, l-fucose and l-rhamnose, or with axial 3-OH, i.e. gulose, allose, altrose and l-rhamnose. Structural modelling using Thermotoga maritima GH36 -galactosidase as the template and superimposition of melibiose from the complex with human GH27 -galactosidase supported that recognition at subsite +1 in AglC presumably requires a hydrogen bond between 3-OH and Trp358 and a hydrophobic environment around the C-6 hydroxymethyl group. In addition, successful transglycosylation of eight of 10 disaccharides (400 mm), except xylobiose and arabinobiose, indicated broad specificity for interaction with the +2 subsite. AglC thus transferred -galactosyl to 6-OH of the terminal residue in the -linked melibiose, maltose, trehalose, sucrose and turanose in 6–46% yield and the ß-linked lactose, lactulose and cellobiose in 28–38% yield. The product structures were identified using NMR and ESI-MS and five of the 13 identified products were novel, i.e. -d-Galp-(1¿6)-d-Manp; -d-Galp-(1¿6)-ß-d-Glcp-(1¿4)-d-Glcp; -d-Galp-(1¿6)-ß-d-Galp-(1¿4)-d-Fruf; -d-Galp-(1¿6)-d-Glcp-(1¿1)-d-Glcp; and -d-Galp-(1¿6)--d-Glcp-(1¿3)-d-Fruf.
Metabolomic and genetic analyses of flavonol synthesis in Arabidopsis thaliana support the in vivo involvement of leucoanthocyanidin dioxygenase
Stracke, R. ; Vos, R.C.H. de; Bartelniewoehner, L. ; Ishihara, H. ; Sagasser, M. ; Martens, S. ; Weisshaar, B. - \ 2009
Planta 229 (2009)2. - ISSN 0032-0935 - p. 427 - 445.
phenylpropanoid biosynthesis - anthocyanidin synthase - functional expression - transcription factor - petunia-hybrida - flavanone 3-beta-hydroxylase - differential regulation - conserved histidine - mass-spectrometry - escherichia-coli
Flavonol synthase (FLS) (EC-number 220.127.116.11), the enzyme that catalyses the conversion of flavonols into dihydroflavonols, is part of the flavonoid biosynthesis pathway. In Arabidopsis thaliana, this activity is thought to be encoded by several loci. In addition to the FLAVONOL SYNTHASE1 (FLS1) locus that has been confirmed by enzyme activity assays, loci displaying similarity of the deduced amino acid sequences to FLS1 have been identified. We studied the putative A. thaliana FLS gene family using a combination of genetic and metabolite analysis approaches. Although several of the FLS gene family members are expressed, only FLS1 appeared to influence flavonoid biosynthesis. Seedlings of an A. thaliana fls1 null mutant (fls1-2) show enhanced anthocyanin levels, drastic reduction in flavonol glycoside content and concomitant accumulation of glycosylated forms of dihydroflavonols, the substrate of the FLS reaction. By using a leucoanthocyanidin dioxygenase (ldox) fls1-2 double mutant, we present evidence that the remaining flavonol glycosides found in the fls1-2 mutant are synthesized in planta by the FLS-like side activity of the LDOX enzyme
Induction of a leaf specific geranylgeranyl pyrophosphate synthase and emission of (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene in tomato are dependent on both jasmonic acid and salicylic acid signaling pathways
Ament, K. ; Schie, C.C. ; Bouwmeester, H.J. ; Haring, M.A. ; Schuurink, R.C. - \ 2006
Planta 224 (2006)5. - ISSN 0032-0935 - p. 1197 - 1208.
farnesyl diphosphate synthase - coenzyme-a reductase - arabidopsis-thaliana - carotenoid biosynthesis - differential induction - functional expression - molecular-cloning - methyl salicylate - chain elongation - wound response
Two cDNAs encoding geranylgeranyl pyrophosphate (GGPP) synthases from tomato (Lycopersicon esculentum) have been cloned and functionally expressed in Escherichia coli. LeGGPS1 was predominantly expressed in leaf tissue and LeGGPS2 in ripening fruit and flower tissue. LeGGPS1 expression was induced in leaves by spider mite (Tetranychus urticae)-feeding and mechanical wounding in wild type tomato but not in the jasmonic acid (JA)-response mutant def-1 and the salicylic acid (SA)-deficient transgenic NahG line. Furthermore, LeGGPS1 expression could be induced in leaves of wild type tomato plants by JA- or methyl salicylate (MeSA)-treatment. In contrast, expression of LeGGPS2 was not induced in leaves by spider mite-feeding, wounding, JA- or MeSA-treatment. We show that emission of the GGPP-derived volatile terpenoid (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) correlates with expression of LeGGPS1. An exception was MeSA-treatment, which resulted in induction of LeGGPS1 but not in emission of TMTT. We show that there is an additional layer of regulation, because geranyllinalool synthase, catalyzing the first dedicated step in TMTT biosynthesis, was induced by JA but not by MeSA.
Identification of a novel alpha-galatosidase from the hyperthermophilic archaeon Sulfolobus solfataricus
Brouns, S.J.J. ; Smits, N. ; Wu, H. ; Wright, P.C. ; Snijders, A.P.L. ; Vos, W.M. de; Oost, J. van der - \ 2006
Journal of Bacteriology 188 (2006)7. - ISSN 0021-9193 - p. 2392 - 2399.
catalytic nucleophile - phanerochaete-chrysosporium - thermoacidophilic archaeon - functional expression - glycosyl hydrolases - crystal-structure - escherichia-coli - genus sulfolobus - complete genome - melon fruit
Sulfolobus solfataricus is an aerobic crenarchaeon that thrives in acidic volcanic pools. In this study, we have purified and characterized a thermostable -galactosidase from cell extracts of S. solfataricus P2 grown on the trisaccharide raffinose. The enzyme, designated GalS, is highly specific for -linked galactosides, which are optimally hydrolyzed at pH 5 and 90°C. The protein consists of 74.7-kDa subunits and has been identified as the gene product of open reading frame Sso3127. Its primary sequence is most related to plant enzymes of glycoside hydrolase family 36, which are involved in the synthesis and degradation of raffinose and stachyose. Both the galS gene from S. solfataricus P2 and an orthologous gene from Sulfolobus tokodaii have been cloned and functionally expressed in Escherichia coli, and their activity was confirmed. At present, these Sulfolobus enzymes not only constitute a distinct type of thermostable -galactosidases within glycoside hydrolase clan D but also represent the first members from the Archaea.
Fasciola hepatica procathepsin L3 protein expressed by a baculovirus recombinant can partly protect rats against fasciolosis
Reszka, N. ; Cornelissen, J.B.W.J. ; Harmsen, M.M. ; Bree, J. de; Boersma, W.J.A. ; Rijsewijk, F.A.M. - \ 2005
Vaccine 23 (2005)23. - ISSN 0264-410X - p. 2987 - 2993.
cathepsin-l proteinases - saccharomyces-cerevisiae - functional expression - liver fluke - vaccines - sheep - identification - vaccination - ruminants - immunity
Fasciola hepatica juveniles express immunodominant cathepsin L proteins, which are mainly found in their immature, procathepsin form. A gene encoding such a procathepsin L (FheCL3) was expressed by a baculovirus recombinant and by Saccharomyces cerevisiae. The glycosylated FheCL3 proteins obtained by both systems were used in a vaccination/challenge experiment in rats. Both antigens evoked similar antibody responses, but only the baculovirus expressed FheCL3 caused a significant protection against the number of liver flukes (52% protection, P = 0.01), whereas the S. cerevisiae expressed FheCL3 did not. In a second experiment in rats, deglycosylated versions of both antigens were used, but this did not improve their efficacies.
Volatile science? Metabolic engineering of terpenoids in plants
Aharoni, A. ; Jongsma, M.A. ; Bouwmeester, H.J. - \ 2005
Trends in Plant Science 10 (2005)12. - ISSN 1360-1385 - p. 594 - 602.
transgenic arabidopsis plants - linalool synthase gene - isoprenoid biosynthesis - essential oil - monoterpene biosynthesis - diphosphate synthase - s-linalool - functional expression - menthofuran synthase - plastidial pathways
Terpenoids are important for plant survival and also possess biological properties that are beneficial to humans. Here, we describe the state of the art in terpenoid metabolic engineering, showing that significant progress has been made over the past few years. Subcellular targeting of enzymes has demonstrated that terpenoid precursors in subcellular compartments are not as strictly separated as previously thought and that multistep pathway engineering is feasible, even across cell compartments. These engineered plants show that insect behavior is influenced by terpenoids. In the future, we expect rapid progress in the engineering of terpenoid production in plants. In addition to commercial applications, such transgenic plants should increase our understanding of the biological relevance of these volatile secondary metabolites
Differences in intensity and specificity of hypersensitive response induction in Nicotiana spp. by INF1, INF2A, and INF2B of Phytophthora infestans
Huitema, E. ; Vleeshouwers, V.G.A.A. ; Cakir, C. ; Kamoun, S. ; Govers, F. - \ 2005
Molecular Plant-Microbe Interactions 18 (2005)3. - ISSN 0894-0282 - p. 183 - 193.
plant-disease resistance - fungal elicitor cryptogein - sterol carrier protein - potato-virus-x - nonhost resistance - pathogen phytophthora - gene-expression - functional expression - molecular-genetics - defense responses
Elicitins form a family of structurally related proteins that induce the hypersensitive response (HR) in plants, particularly Nicotiana spp. The elicitin family is composed of several classes. Most species of the plant-pathogenic oomycete genus Phytophthora produce the well-characterized 10-kDa canonical elicitins (class I), such as INF1 of the potato and tomato pathogen Phytophthora infestans. Two genes, inf2A and inf2B, encoding a distinct class (class III) of elicitin-like proteins, also occur in P. infestans. Unlike secreted class I elicitins, class III elicitins are thought to be cell-surface-anchored polypeptides. Molecular characterization of the inf2 genes indicated that they are widespread in Phytophthora spp. and occur as a small gene family. In addition, Southern blot and Northern blot hybridizations using gene-specific probes showed that inf2A and inf2B genes and transcripts can be detected in 17 different P. infestans isolates. Functional secreted expression in plant cells of the elicitin domain of the inf1 and inf2 genes was conducted using a binary Potato virus X (PVX) vector (agroinfection) and Agrobacterium tumefaciens transient transformation assays (agroinfiltration), and resulted in HR-like necrotic symptoms and induction of defense response genes in tobacco. However, comparative analyses of elicitor activity of INF1, INF2A, and INF2B revealed significant differences in intensity, specificity, and consistency of HR induction. Whereas INF1 induced the HR in Nicotiana benthamiana, INF2A induced weak symptoms and INF2B induced no symptoms on this plant. Nonetheless, similar to INF1, HR induction by INF2A in N. benthamiana required the ubiquitin ligase-associated protein SGT1. Overall, these results suggest that variation in the resistance of Nicotiana spp. to P. infestans is shadowed by variation in the response to INF elicitins. The ability of tobacco, but not N. benthamiana, to respond to INF2B could explain differences in resistance to P. infestans observed for these two species.
Molecular cloning of two Arabidopsis UDP-galactose transporters by complementation of a deficient Chinese hamster ovary cell line
Bakker, H. ; Routier, F. ; Oelmann, S. ; Jordi, W.J.R.M. ; Lommen, A. ; Gerardy-Schahn, R. ; Bosch, H.J. - \ 2005
Glycobiology 15 (2005)2. - ISSN 0959-6658 - p. 193 - 201.
cmp-sialic acid - nucleotide-sugar transporters - gdp-mannose transporter - expression cloning - n-acetylgalactosamine - golgi-apparatus - gene family - 5'-phosphosulfate transporter - functional expression - carbohydrate epitope
Nucleotide-sugar transporters (NSTs) form a family of structurally related transmembrane proteins that transport nucleotide-sugars from the cytoplasm to the endoplasmic reticulum and Golgi lumen. In these organelles, activated sugars are substrates for various glycosyltransferases involved in oligo- and polysaccharide biosynthesis. The Arabidopsis thaliana genome contains more than 40 members of this transporter gene family, of which only a few are functionally characterized. In this study, two Arabidopsis UDP-galactose transporter cDNAs (UDP-GalT1 and UDP-GalT2) are isolated by expression cloning using a Chinese hamster ovary cell line (CHO-Lec8) deficient in UDP-galactose transport. The isolated genes show only 21% identity to each other and very limited sequence identity with human and yeast UDP-galactose transporters and other NSTs. Despite this low overall identity, the two proteins clearly belong to the same gene family. Besides complementing Lec8 cells, the two NSTs are shown to transport exclusively UDP-galactose by an in vitro NST assay. The most homologous proteins with known function are plant transporters that locate in the inner chloroplast membrane and transport triose-phosphate, phosphoenolpyruvate, glucose-6-phosphate, and xylulose 5-phosphate. Also, the latter proteins are members of the same family, which therefore has been named the NST/triose-phosphate transporter family
Expression of poly-3-(R)-hydroxyalkanoate (PHA) polymerase and acyl-CoA-transacylase in plastids of transgenic potato leads to the synthesis of a hydrophobic polymer, presumably medium-chain-length PHAs
Romano, A. ; Plas, L.H.W. van der; Witholt, B. ; Eggink, G. ; Mooibroek, A. - \ 2005
Planta 220 (2005)3. - ISSN 0032-0935 - p. 455 - 464.
nonrelated carbon-sources - unsaturated fatty-acids - beta-oxidation - pseudomonas-aeruginosa - polyhydroxyalkanoic acid - fluorescent pseudomonads - functional expression - biosynthetic-pathway - particle bombardment - plants
Medium-chain-length poly-3-(R)-hydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters. The minimum gene-set for the accumulation of mcl-PHAs from de novo fatty acid biosynthesis has been identified in prokaryotes [B. Rehm et al. (1998) J. Biol Chem 273:24044–24051] as consisting of the Pha-C1 polymerase and the ACP-CoA-transacylase. In this paper, the synthesis of mcl-PHAs has been attempted in transgenic potato (Solanum tuberosum L.) using the same set of genes that were introduced into potato by particle bombardment. Polymer contents of transgenic lines were analysed by gas chromatography and by a new simple method employing a size-exclusion filter column. The expression of the Pha-C1 polymerase and the ACP-CoA-transacylase in the plastids of transgenic potato led to the synthesis of a hydrophobic polymer composed of mcl-hydroxy-fatty acids with carbon chain lengths ranging from C-6 to C-12 in leaves of the selected transgenic lines. We strongly suggest that the polymer observed consists of mcl-PHAs and that this report establishes for the first time a possible route for the production of mcl-PHAs from de novo fatty acid biosynthesis in plants.
Gain and loss of fruit flavour compounds produced by wild and cultivated strawberry species
Aharoni, A. ; Giri, A.P. ; Verstappen, F.W.A. ; Bertea, C.M. ; Sevenier, R.E. ; Sun, Z. ; Jongsma, M.A. ; Schwab, W. ; Bouwmeester, H.J. - \ 2004
The Plant Cell 16 (2004). - ISSN 1040-4651 - p. 3110 - 3131.
messenger-rna accumulation - arabidopsis-thaliana - fragaria-vesca - functional expression - alkaloid biosynthesis - gene-expression - cdna isolation - lithospermum-erythrorhizon - alcohol-dehydrogenase - secondary metabolites
The blends of flavor compounds produced by fruits serve as biological perfumes used to attract living creatures, including humans. They include hundreds of metabolites and vary in their characteristic fruit flavor composition. The molecular mechanisms by which fruit flavor and aroma compounds are gained and lost during evolution and domestication are largely unknown. Here, we report on processes that may have been responsible for the evolution of diversity in strawberry (Fragaria spp) fruit flavor components. Whereas the terpenoid profile of cultivated strawberry species is dominated by the monoterpene linalool and the sesquiterpene nerolidol, fruit of wild strawberry species emit mainly olefinic monoterpenes and myrtenyl acetate, which are not found in the cultivated species. We used cDNA microarray analysis to identify the F. ananassa Nerolidol Synthase1 (FaNES1) gene in cultivated strawberry and showed that the recombinant FaNES1 enzyme produced in Escherichia coli cells is capable of generating both linalool and nerolidol when supplied with geranyl diphosphate (GPP) or farnesyl diphosphate (FPP), respectively. Characterization of additional genes that are very similar to FaNES1 from both the wild and cultivated strawberry species (FaNES2 and F. vesca NES1) showed that only FaNES1 is exclusively present and highly expressed in the fruit of cultivated (octaploid) varieties. It encodes a protein truncated at its N terminus. Green fluorescent protein localization experiments suggest that a change in subcellular localization led to the FaNES1 enzyme encountering both GPP and FPP, allowing it to produce linalool and nerolidol. Conversely, an insertional mutation affected the expression of a terpene synthase gene that differs from that in the cultivated species (termed F. ananassa Pinene Synthase). It encodes an enzyme capable of catalyzing the biosynthesis of the typical wild species monoterpenes, such as alpha-pinene and beta-myrcene, and caused the loss of these compounds in the cultivated strawberries. The loss of alpha-pinene also further influenced the fruit flavor profile because it was no longer available as a substrate for the production of the downstream compounds myrtenol and myrtenyl acetate. This phenomenon was demonstrated by cloning and characterizing a cytochrome P450 gene (Pinene Hydroxylase) that encodes the enzyme catalyzing the C10 hydroxylation of alpha-pinene to myrtenol. The findings shed light on the molecular evolutionary mechanisms resulting in different flavor profiles that are eventually selected for in domesticated species.
Recognition of Cladosporium fulvum Ecp2 elicitor by non-host Nicotiana spp. is mediated by a single dominant gene that is not homologous to known Cf-genes
Kock, M.J.D. de; Iskandar, H.M. ; Brandwagt, B.F. ; Laugé, R. ; Wit, P.J.G.M. de; Lindhout, W.H. - \ 2004
Molecular Plant Pathology 5 (2004)5. - ISSN 1464-6722 - p. 397 - 408.
cultured parsley cells - tobacco mosaic-virus - f-sp lycopersici - disease resistance - confers resistance - short arm - phytophthora-infestans - oligopeptide elicitor - pathogen phytophthora - functional expression
Cladosporium fulvum is a fungal pathogen of tomato that grows exclusively in the intercellular spaces of leaves. Ecp2 is one of the elicitor proteins that is secreted by C. fulvum and is specifically recognized by tomato plants containing the resistance gene Cf-Ecp2. Recognition is followed by a hypersensitive response (HR) resulting in resistance. HR-associated recognition of Ecp2 has been observed in Nicotiana paniculata, N. sylvestris, N. tabacum and N. undulata that are non-host plants of C. fulvum. Absence of Ecp2-recognition did not lead to growth of C. fulvum on Nicotiana plants. We show that HR-associated recognition of Ecp2 is mediated by a single dominant gene in N. paniculata. However, based on PCR and hybridization analysis this gene is not homologous to known Cf-genes
Identification of a novel Fasciola hepatica cathepsin L protease containing protective epitopes within the propeptide
Harmsen, M.M. ; Cornelissen, J.B.W.J. ; Buys-Bergen, W.E.C.M. ; Boersma, W.J.A. ; Jeurissen, S.H.M. ; Milligen, F.J. - \ 2004
International Journal for Parasitology 34 (2004)6. - ISSN 0020-7519 - p. 675 - 682.
newly excysted juveniles - saccharomyces-cerevisiae - asparaginyl endopeptidase - functional expression - cysteine proteinases - liver fluke - in-vitro - immunity - vaccines - hemoglobin
Cathepsin L (CL)-like proteases are important candidate vaccine antigens for protection against helminth infections. We previously identified an immunogenic 32 kDa protein specifically present in newly excysted juveniles (NEJs) of Fasciola hepatica. Here we show by N-terminal protein sequencing that this protein represents a CL-like protease still containing the propeptide. Two cDNAs encoding this CL were subsequently isolated from NEJs by RT-PCR. The predicted amino acid sequences of these cDNAs showed approximately 70% sequence homology to both CL1 and CL2 sequences isolated from adult stage F. hepatica and are, therefore, referred to as CL3. The CL3 clones encoded asparagine at position PI of the propeptide cleavage site, suggesting a dependence on asparaginyl endopeptidases for maturation. Recombinant expression of a CL3 cDNA in Saccharomyces cerevisiae resulted in secretion of the proenzyme form. The propeptide of CL-like proteins was predicted to contain important B-cell epitopes. To determine the contribution of the propeptide to protective immunity, rats were vaccinated with Keyhole Limpet Haemocyanin-conjugated synthetic peptides encoding these putative B-cell epitopes derived from the CL1 or CL3 sequence. A subsequent challenge infection resulted in a significant (P <0.05) reduction of fluke load compared to adjuvant controls. We conclude that the propeptide of CL3 plays an important role in inducing immunity against F. hepatica infection. (C) 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
Isolation and molecular characterization of cathepsin L-like cysteine protease cDNAs from Western flower thrips (Frankliniella occidentalis)
Kuipers, A.G.J. ; Jongsma, M.A. - \ 2004
Comparative Biochemistry and Physiology. B, Biochemistry and Molecular Biology 139 (2004)1. - ISSN 1096-4959 - p. 65 - 75.
l-like enzyme - drosophila-melanogaster - cell-line - proteinase-inhibitors - functional expression - pichia-pastoris - gene family - cloning - beetle - purification
Cysteine proteases are predominant in thrips guts (TGs) and, therefore, a suitable target for selecting effective protease inhibitors against western flower thrips (Frankliniella occidentalis). We report the isolation of four full-length cysteine protease cDNA clones from thrips in a two-step PCR approach with degenerate oligonucleotides designed on conserved cathepsin L domains. At the deduced amino acid level, the clones possessed all functional and structural characteristics of cathepsin L, and showed high mutual identity and strong similarity with cathepsin L-like cysteine proteases from other insects and arthropods. Southern analysis indicated that a family of four closely related and 10 12 less-related genes encode the cathepsin L-like cysteine proteases in the thrips genome. Partial sequencing of genomic DNA demonstrated the presence of three introns in the coding DNA.
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.
Terpenoid Metabolism in Wild-Type and Transgenic Arabidopsis Plants
Aharoni, A. ; Giri, A.P. ; Deuerlein, S. ; Griepink, F.C. ; Kogel, W.J. de; Verstappen, F.W.A. ; Verhoeven, H.A. ; Jongsma, M.A. ; Schwab, W. ; Bouwmeester, H.J. - \ 2003
The Plant Cell 15 (2003)12. - ISSN 1040-4651 - p. 2866 - 2884.
sesquiterpene cyclase gene - linalool synthase gene - functional expression - monoterpene biosynthesis - isoprenoid biosynthesis - diphosphate synthase - cdna isolation - s-linalool - glandular trichomes - thaliana
Volatile components, such as terpenoids, are emitted from aerial parts of plants and play a major role in the interaction between plants and their environment. Analysis of the composition and emission pattern of volatiles in the model plant Arabidopsis showed that a range of volatile components are released, primarily from flowers. Most of the volatiles detected were monoterpenes and sesquiterpenes, which in contrast to other volatiles showed a diurnal emission pattern. The active terpenoid metabolism in wild-type Arabidopsis provoked us to conduct an additional set of experiments in which transgenic Arabidopsis overexpressing two different terpene synthases were generated. Leaves of transgenic plants constitutively expressing a dual linalool/nerolidol synthase in the plastids (FaNES1) produced linalool and its glycosylated and hydroxylated derivatives. The sum of glycosylated components was in some of the transgenic lines up to 40- to 60-fold higher than the sum of the corresponding free alcohols. Surprisingly, we also detected the production and emission of nerolidol, albeit at a low level, suggesting that a small pool of its precursor farnesyl diphosphate is present in the plastids. Transgenic lines with strong transgene expression showed growth retardation, possibly as a result of the depletion of isoprenoid precursors in the plastids. In dual-choice assays with Myzus persicae, the FaNES1-expressing lines significantly repelled the aphids. Overexpression of a typical cytosolic sesquiterpene synthase resulted in the production of only trace amounts of the expected sesquiterpene, suggesting tight control of the cytosolic pool of farnesyl diphosphate, the precursor for sesquiterpenoid biosynthesis. This study further demonstrates the value of Arabidopsis for studies of the biosynthesis and ecological role of terpenoids and provides new insights into their metabolism in wild-type and transgenic plants
Biotransformation of limonene by bacteria, fungi, yeasts, and plants
Duetz, W.A. ; Bouwmeester, H.J. ; Beilen, J.B. ; Witholt, B. - \ 2003
Applied Microbiology and Biotechnology 61 (2003). - ISSN 0175-7598 - p. 269 - 277.
microbiological transformations - bacillus-stearothermophilus - pseudomonas-putida - soil pseudomonad - functional expression - degradation pathway - escherichia-coli - selenium dioxide - perillic acid - bioconversion
The past 5 years have seen significant progress in the field of limonene biotransformation, especially with regard to the regiospecificity of microbial biocatalysts. Whereas earlier only regiospecific biocatalysts for the 1,2 position (limonene-1,2-diol) and the 8-position (¿±-terpineol) were available, recent reports describe microbial biocatalysts specifically hydroxylating the 3-position (isopiperitenol), 6-position (carveol and carvone), and 7-position (perillyl alcohol, perillylaaldehyde, and perillic acid). The present review also includes the considerable progress made in the characterization of plant P-450 limonene hydroxylases and the cloning of the encoding genes.