Publications - Cell and Metabolic Biology

Diterpenoids form a diverse group of natural products, many of which are or could become pharmaceuticals or industrial chemicals. The modular character of diterpene biosynthesis and the promiscuity of the enzymes involved make combinatorial biosynthesis a promising approach to generate libraries of diverse diterpenoids. Here, we report on the combinatorial assembly in yeast of ten diterpene synthases producing (+)-copalyldiphosphate-derived backbones and four cytochrome P450 oxygenases (CYPs) in diverse combinations. This resulted in the production of over 200 diterpenoids. Based on literature and chemical database searches, 162 of these compounds can be considered new-to-Nature. The CYPs accepted most substrates they were given but remained regioselective with few exceptions. Our results provide the basis for the systematic exploration of the diterpenoid chemical space in yeast using sequence databases.

The pyrethrum plant, Tanacetum cinerariifolium (Asteraceae) synthesizes a class of compounds called pyrethrins that have strong insecticidal properties but are safe to humans. Class I pyrethrins are esters of the monoterpenoid trans-chrysanthemic acid with one of three jasmonic-acid derived alcohols. We reconstructed the trans-chrysanthemic acid biosynthetic pathway in tomato fruits, which naturally produce high levels of the tetraterpene pigment lycopene, an isoprenoid which shares a common precursor, dimethylallyl diphosphate (DMAPP), with trans-chrysanthemic acid. trans-Chrysanthemic acid biosynthesis in tomato fruit was achieved by expressing the chrysanthemyl diphosphate synthase gene from T. cinerariifolium, encoding the enzyme that uses DMAPP to make trans-chrysanthemol, under the control of the fruit specific promoter PG, as well as an alcohol dehydrogenease (ADH) gene and aldehyde dehydrogenase (ALDH) gene from a wild tomato species, also under the control of the PG promoter. Tomato fruits expressing all three genes had a concentration of trans-chrysanthemic acid that was about 1.7-fold higher (by weight) than the levels of lycopene present in non-transgenic fruit, while the level of lycopene in the transgenic plants was reduced by 68%. Ninety seven percent of the diverted DMAPP was converted to trans-chrysanthemic acid, but 62% of this acid was further glycosylated. We conclude that the tomato fruit is an alternative platform for the biosynthesis of trans-chrysanthemic acid by metabolic engineering.

Among the plant hormones jasmonic acid and related derivatives are known to mediate stress responses and several developmental processes. Biosynthesis, regulation, and metabolism of jasmonic acid in Arabidopsis thaliana are reviewed, including properties of mutants of jasmonate biosynthesis. The individual signalling properties of several jasmonates are described.

Transport processes between plant and fungal cells are key elements in arbuscular mycorrhiza (AM), where H+‐ATPases are considered to be involved in active uptake of nutrients from the symbiotic interface. Genes encoding H+‐ATPases were identified in the genome of Medicago truncatula and three cDNA fragments of the H+‐ATPase gene family (Mtha 1 ‐ 3) were obtained by RT‐PCR using RNA from M. truncatula mycorrhizal roots as template. While Mtha 2 and Mtha 3 appeared to be constitutively expressed in roots and unaffected by AM development, transcripts of Mtha 1 could only be detected in AM tissues and not in controls. Further analyses by RT‐PCR revealed that Mtha 1 transcripts are not detectable in shoots and phosphate availability did not affect RNA accumulation of the gene. Localization of transcripts by in situ hybridization on AM tissues showed that Mtha 1 RNA accumulates only in cells containing fungal arbuscules. This is the first report of arbuscule‐specific induced expression of a plant H+‐ATPase gene in mycorrhizal tissues.

Treatment of barley leaf segments with jasmonic acid methyl ester (JM) leads to the accumulation of a set of newly formed abundant proteins. Among them, the most abun dant protein exhibits a molecular mass of 23 kDa (JIP‐23). Here, data are presented on the occurrence and expression of the lIP‐23 genes in different cultivars of Hordeum vulgare . Southern blot analysis of 80 cultivars revealed the occurrence of 2 to 4 genes coding for JIP‐23 in all cultivars. By means of Northern blot and immunoblot analysis it is shown that some cultivars lack the ex pression of jip‐23 upon treatment of primary leaves with JM as well as upon stress performed by incubation with 1 M sorbitol solution. During germination, however, all tested cultivars ex hibited developmental expression of jip‐23 . The results are dis cussed in terms of possible functions of JIP‐23 in barley.