Browsing by Subject "Biosynthese"

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Molekulare und entwicklungsbiologische Charakterisierung von Schlüsselenzymen der Naturstoffbiosynthese in Drüsenhaaren der Sonnenblume
(2008) Göpfert, Jens C.; Spring, Otmar
Glandular trichomes from anther appendages of sunflower were collected and their RNA was isolated. Sequence comparison with known plant sesquiterpene synthases was used to identify sunflower synthases in RT-PCR reactions. Three enzymes, HaGAS1, HaGAS2 and HaCS with high similarities to already characterized sesquiterpene synthases were identified. Their nucleotide sequences were completely established on the genomic level and as RNA transcripts. The nucleotide sequences as well as the deduced amino acid sequences showed typical characteristics of terpene synthases. In order to characterize the enzymes, the sesquiterpene synthase genes were cloned and expressed in E. coli. In vitro assays with the recombinant enzymes were carried out using the native substrate farnesyldiphosphate. The resulting products were extracted and analysed by GC-MS. They were identified by comparison of data base MS-data and using reference samples under identical analytical conditions. Two expressed enzymes, HaGAS1 and HaGAS2, synthesized germacrene A as a single product. Heterologous in vivo expression of both germacrene A-synthases in S. cerevisiae confirmed the in vitro result, since the analysis of the synthesized product showed a single germacrene A peak. Due to a very low in vitro activity of HaCS, the products of the third synthase could not be directly determined by MS-analysis. Therefore, the enzyme was expressed as a thioredoxin-fusion protein in vivo in transgenic yeast. This attempt resulted in a much higher rate of product yield. Two main and at least six minor products were traced in GC-analysis. They were confirmed as sesquiterpene hydrocarbons by GC-MS analysis. One of the two main products was identified as gamma-cadinene, whereas the second main peak could not be determined conclusively. Among the minor compounds alpha-copaene, alpha-muurolene und beta-caryophyllene were identified. Screening of a H. annuus EST library (established at the Berkeley Center for Synthetic Biology, University of California, Berkeley, USA) from mRNA of trichomes revealed the presence of a cytochrome P450 protein which showed high similarity to an Artemisia annua enzyme involved in artemisinic acid biosynthesis. This enzyme and another similar protein from Lactuca sativa were cloned and coexpressed with the germacrene A-synthase HaGAS2 in yeast. The resulting product was indirectly determined as germacrene A carboxylic acid using GC-MS analysis. These novel cytochrome P450 enzymes from sunflower and lettuce can be characterized as multifunctional germacrene A-monooxygenases. They catalyse a three-step oxidation leading from germacrene A to germacrene A carboxylic acid. This oxidation process represents an essential step towards the biosynthesis of sesquiterpene lactones. Semiquantitative RT-PCR analysis demonstrated that the expression of all three sesquiterpene synthases and the sunflower P450 monooxygenase occurred directly within trichome cells. The expression was highly upregulated during the secretory stage of the capitate glandular trichomes. This developmentally regulated expression was shown for the first time in trichomes. Additionally to sesquiterpene synthase activity in trichomes of anthers and leaves, it also was detected in sunflower roots. In addition, 5-deoxynevadensin was identified as a new constituent of the glandular trichomes of sunflower. This 5-deoxy-flavone is responsible for the bright blue fluorescence of sunflower trichomes detected by fluorescence microscopy. The newly identified component may act as protectant for the STL against UV-degradation.
Neue Cytochrom P450 Enzyme des Sesquiterpenlacton Stoffwechsels der Sonnenblume (Helianthus annuus L.)
(2016) Frey, Maximilian; Spring, Otmar
In the present work additional steps towards the elucidation of the biosynthetic pathway of H. annuus sesquiterpene lactones (STL) were achieved. Firstly candidate sequences were retrieved from a transcriptome database by filtering according to expression pattern and similarity to P450 enzymes known to participate in STL biosynthetic pathways. Open reading frames (ORFs) were obtained using 3´-and 5´-RACE-PCR. Previously described and newly identified candidate genes were then transformed in yeast vectors and expressed in combination with different substrate vectors. A high throughput micro approach was developed that allowed the expression and analysis of many yeast strains at the same time. For the transient expression in N. benthamiana the genes of known and putative enzymes were introduced via Agrobacterium mediated transformation. Using the in planta expression system the complete STL pathway of sunflower to costunolide was reconstructed de novo in a step-by-step approach. Previously described Michael-addition reactions of α-methylene-γ-lactone type STL to the thiol group of cysteine or glutathione in tobacco expression systems could be observed for all STL investigated. Chemically synthesized STL adducts were used as reference for the identification of in planta produced STL adducts. Enzyme characterization was conducted in two different in vivo expression systems, in yeast (Saccharomyces cervisiae) and tobacco (Nicotiana benthamiana). For the investigated biosynthetic pathway, differences between these two expression systems were discussed. Candidate gene M4 showed an unexpected product in yeast (farnesyl-δ-lactone) and led in combination with HaG8H to the production of costunolide. In the plant expression system, germacrene A acid was converted to costunolide by M4 in the absence of HaG8H. In both cases, M4 was involved in the synthesis of costunolide and should therefore be assigned Helianthus annuus costunolide synthase the underlying reaction mechanism should however be investigated more thoroughly. Helianthus annuus costunolide 14-hydroxylase HaC14H (candidate M33) was characterized in yeast and tobacco. A classification into subfamily CYP71CB, together with Tp8878 the Tanacetum parthenium costunolide/parthenolide 3β-hydroxylase is proposed. It was shown that in planta the main product of HaG8H exists most likely as inunolide, which would be the entry point for the biosynthesis of 8-epixanthatine and tomentosine. Candidate S2 from Ikezawa et al. (2011) was found to convert 8β-hydroxy-germacrene A acid to 8β-hydroxy-costunolide (eupatolide) in tobacco, but not in yeast, producing several byproducts. The name Helianthus annuus eupatolide synthase HaES is proposed accordingly. HaES has 47 % amino acid identity to the parthenolide synthase from T. parthenium (TpPTS). A classification into a new CYP71 subfamily is proposed. Two alternative metabolic routes led to 8β-hydroxy-costunolide in the expression studies in tobacco, the underlying mechanisms are discussed. Enzymes involved in STL biosynthesis were expressed in inner tissues of young Helianthus annuus plants; the induction of expression of STL biosynthesis enzymes in leaf primordia correlates with the development of capitate glandular trichomes (CGT) and STL synthesis. HaC14H was found in a chromosomal region in proximity to several P450 enzyme candidates, that share the same subfamily and the expression in capitate glandular trichomes (CGT). Therefore involvement of these enzymes in later steps of the biosynthesis of the elaborate STL structures found in CGT is likely.