Evolution of substrate recognition sites (SRSs) in cytochromes P450 from Apiaceae exemplified by the CYP71AJ subfamily

Background Large proliferations of cytochrome P450 encoding genes resulting from gene duplications can be termed as ‘blooms’, providing genetic material for the genesis and evolution of biosynthetic pathways. Furanocoumarins are allelochemicals produced by many of the species in Apiaceaous plants belonging to the Apioideae subfamily of Apiaceae and have been described as being involved in the defence reaction against phytophageous insects.[br/] Results A bloom in the cytochromes P450 CYP71AJ subfamily has been identified, showing at least 2 clades and 6 subclades within the CYP71AJ subfamily. Two of the subclades were functionally assigned to the biosynthesis of furanocoumarins. Six substrate recognition sites (SRS1-6) important for the enzymatic conversion were investigated in the described cytochromes P450 and display significant variability within the CYP71AJ subfamily. Homology models underline a significant modification of the accession to the iron atom, which might explain the difference of the substrate specificity between the cytochromes P450 restricted to furanocoumarins as substrates and the orphan CYP71AJ.[br/] Conclusion Two subclades functionally assigned to the biosynthesis of furanocoumarins and four other subclades were identified and shown to be part of two distinct clades within the CYP71AJ subfamily. The subclades show significant variability within their substrate recognition sites between the clades, suggesting different biochemical functions and providing insights into the evolution of cytochrome P450 ‘blooms’ in response to environmental pressures

Transcriptome analysis of Thapsia laciniata rouy provides insights into terpenoid biosynthesis and diversity in apiaceae

Thapsia laciniata Rouy (Apiaceae) produces irregular and regular sesquiterpenoids with thapsane and guaiene carbon skeletons, as found in other Apiaceae species. A transcriptomic analysis utilizing Illumina next-generation sequencing enabled the identification of novel genes involved in the biosynthesis of terpenoids in Thapsia. From 66.78 million HQ paired-end reads obtained from T. laciniata roots, 64.58 million were assembled into 76,565 contigs (N50: 1261 bp). Seventeen contigs were annotated as terpene synthases and five of these were predicted to be sesquiterpene synthases. Of the 67 contigs annotated as cytochromes P450, 18 of these are part of the CYP71 clade that primarily performs hydroxylations of specialized metabolites. Three contigs annotated as aldehyde dehydrogenases grouped phylogenetically with the characterized ALDH1 from Artemisia annua and three contigs annotated as alcohol dehydrogenases grouped with the recently described ADH1 from A. annua. ALDH1 and ADH1 were characterized as part of the artemisinin biosynthesis. We have produced a comprehensive EST dataset for T. laciniata roots, which contains a large sample of the T. laciniata transcriptome. These transcriptome data provide the foundation for future research into the molecular basis for terpenoid biosynthesis in Thapsia and on the evolution of terpenoids in Apiaceae.Damian Paul Drew, Bjørn Dueholm, Corinna Weitzel, Ye Zhang, Christoph W. Sensen and Henrik Toft Simonse

Injection rate of cylinder lubrication oil in large two-stroke marine diesel engines using a common rail lubrication system

This paper investigates a common rail cylinder lubrication system for large two-stroke marine diesel engines using electronically controlled injectors. The system is studied using the Bosch rate of injection measurement technique The common rail injector has a buildup of mass flow of approximately 1 ms as the injector opens until the nozzle is choked from cavitation. Using a highly viscous fluid, the Bosch rate of injection method is able to predict the injected amount with an error of 5% or lower for nearly the entire tested delivery range of 2 mg to 21 mg. Lubrication of cylinder liners and piston rings is a crucial parameter in operating a two-stoke marine diesel engine efficiently. Both over and under lubrication is harmful for the engine, so the ability to accurately dose the cylinder oil is very important. A mass flow build up time of 1 ms promises high accuracy of dosage even down to 2.5 mg per injection. This paves the way for injecting the oil where and when it is needed, which in turn will improve engine performance and lower harmful emissions.Comment: 9 pages, 8 figures, pre-prin