Harnessing yeast subcellular compartments for the production of plant terpenoids.

The biologically and commercially important terpenoids are a large and diverse class of natural products that are targets of metabolic engineering. However, in the context of metabolic engineering, the otherwise well-documented spatial subcellular arrangement of metabolic enzyme complexes has been largely overlooked. To boost production of plant sesquiterpenes in yeast, we enhanced flux in the mevalonic acid pathway toward farnesyl diphosphate (FDP) accumulation, and evaluated the possibility of harnessing the mitochondria as an alternative to the cytosol for metabolic engineering. Overall, we achieved 8- and 20-fold improvement in the production of valencene and amorphadiene, respectively, in yeast co-engineered with a truncated and deregulated HMG1, mitochondrion-targeted heterologous FDP synthase and a mitochondrion-targeted sesquiterpene synthase, i.e. valencene or amorphadiene synthase. The prospect of harnessing different subcellular compartments opens new and intriguing possibilities for the metabolic engineering of pathways leading to valuable natural compounds.

Flower proteome: changes in protein spectrum during the advanced stages of rose petal development.

Flowering is a unique and highly programmed process, but hardly anything is known about the developmentally regulated proteome changes in petals. Here, we employed proteomic technologies to study petal development in rose (Rosa hybrida). Using two-dimensional polyacrylamide gel electrophoresis, we generated stage-specific (closed bud, mature flower and flower at anthesis) petal protein maps with ca. 1,000 unique protein spots. Expression analyses of all resolved protein spots revealed that almost 30% of them were stage-specific, with ca. 90 protein spots for each stage. Most of the proteins exhibited differential expression during petal development, whereas only ca. 6% were constitutively expressed. Eighty-two of the resolved proteins were identified by mass spectrometry and annotated. Classification of the annotated proteins into functional groups revealed energy, cell rescue, unknown function (including novel sequences) and metabolism to be the largest classes, together comprising ca. 90% of all identified proteins. Interestingly, a large number of stress-related proteins were identified in developing petals. Analyses of the expression patterns of annotated proteins and their corresponding RNAs confirmed the importance of proteome characterization.

Rose scent: genomics approach to discovering novel floral fragrance-related genes.

For centuries, rose has been the most important crop in the floriculture industry; its economic importance also lies in the use of its petals as a source of natural fragrances. Here, we used genomics approaches to identify novel scent-related genes, using rose flowers from tetraploid scented and nonscented cultivars. An annotated petal EST database of approximately 2100 unique genes from both cultivars was created, and DNA chips were prepared and used for expression analyses of selected clones. Detailed chemical analysis of volatile composition in the two cultivars, together with the identification of secondary metabolism-related genes whose expression coincides with scent production, led to the discovery of several novel flower scent-related candidate genes. The function of some of these genes, including a germacrene D synthase, was biochemically determined using an Escherichia coli expression system. This work demonstrates the advantages of using the high-throughput approaches of genomics to detail traits of interest expressed in a cultivar-specific manner in nonmodel plants. EST sequences were submitted to the GenBank database (accession numbers BQ 103855 to BQ 106728).