Production of C20, C30 and C40 terpenes in the engineered phototrophic bacterium Rhodobacter capsulatus.

Terpenes constitute one of the largest groups of secondary metabolites that are used, for example, as food-additives, fragrances or pharmaceuticals. Due to the formation of an intracytoplasmic membrane system and an efficient intrinsic tetraterpene pathway, the phototrophic α-proteobacterium Rhodobacter capsulatus offers favorable properties for the production of hydrophobic terpenes. However, research efforts have largely focused on sesquiterpene production. Recently, we have developed modular tools allowing to engineer the biosynthesis of terpene precursors. These tools were now applied to boost the biosynthesis of the diterpene casbene, the triterpene squalene and the tetraterpene ß-carotene in R. capsulatus SB1003. Selected enzymes of the intrinsic isoprenoid pathway and the heterologous mevalonate (MVA) pathway were co-expressed together with the respective terpene synthases in various combinations. Remarkably, co-expression of genes ispA, idi and dxs enhanced the synthesis of casbene and ß-carotene. In contrast, co-expression of precursor biosynthetic genes with the squalene synthase from Arabidopsis thaliana reduced squalene titers. Therefore, we further employed four alternative pro- and eukaryotic squalene synthases. Here, the synthase from Methylococcus capsulatus enabled highest product levels of 90 mg/L squalene upon co-expression with ispA. In summary, we demonstrate the applicability of R. capsulatus for the heterologous production of diverse terpene classes and provide relevant insights for further development of such platforms.

Detection of sCD21 in Human Blood Using ELISA is Influenced by Ethylenediaminetetraacetic Acid (EDTA) Containing Blood Collection Tubes.

BACKGROUND: CD21 is a 145 kDa membrane glycoprotein expressed mainly on B-cells and follicular dendritic cells. It is involved in activation, survival and proliferation of B-cells. CD21 can be cleaved to give soluble CD21 (sCD21), which is constantly shed in healthy people whereas the level of sCD21 is low in patients suffering from various autoimmune diseases. METHODS: Blood was collected from 12 healthy donors into Heparin, EDTA and gel-separation tubes. Furthermore, plasma from 6 healthy donors was combined and EDTA or EGTA was subsequently added to analyze its influence on sCD21 levels measured by ELISA. RESULTS: Differences in sCD21 levels were observed dependent on blood collection tubes used. sCD21 levels measured by ELISA were significantly higher if heparin/gel containing tubes were used compared to EDTA blood collection tubes. Upon addition of EDTA/EGTA to plasma drawn using Heparin blood collection tubes, sCD21 levels decrease to amounts found in EDTA-containing blood collection tubes suggesting calcium as the responsible factor rather than magnesium. CONCLUSIONS: EDTA influences sCD21 levels determined by ELISA and therefore sCD21 measurements should be carried out using one type of blood collection tube throughout the experiment/medical examination.