Identification and Functional Characterization of the Gene Cluster Responsible for Fusaproliferin Biosynthesis in Fusarium proliferatum.

The emerging mycotoxin fusaproliferin is produced by Fusarium proliferatum and other related Fusarium species. Several fungi from other taxonomic groups were also reported to produce fusaproliferin or the deacetylated derivative, known as siccanol or terpestacin. Here, we describe the identification and functional characterization of the Fusarium proliferatum genes encoding the fusaproliferin biosynthetic enzymes: a terpenoid synthase, two cytochrome P450s, a FAD-oxidase and an acetyltransferase. With the exception of one gene encoding a CYP450 (FUP2, FPRN_05484), knock-out mutants of the candidate genes could be generated, and the production of fusaproliferin and intermediates was tested by LC-MS/MS. Inactivation of the FUP1 (FPRN_05485) terpenoid synthase gene led to complete loss of fusaproliferin production. Disruption of a putative FAD-oxidase (FUP4, FPRN_05486) did not only affect oxidation of preterpestacin III to terpestacin, but also of new side products (11-oxo-preterpstacin and terpestacin aldehyde). In the knock-out strains lacking the predicted acetyltransferase (FUP5, FPRN_05487) fusaproliferin was no longer formed, but terpestacin was found at elevated levels. A model for the biosynthesis of fusaproliferin and of novel derivatives found in mutants is presented.

Enhanced Metabolome Coverage and Evaluation of Matrix Effects by the Use of Experimental-Condition-Matched 13C-Labeled Biological Samples in Isotope-Assisted LC-HRMS Metabolomics.

Stable isotope-assisted approaches can improve untargeted liquid chromatography-high resolution mass spectrometry (LC-HRMS) metabolomics studies. Here, we demonstrate at the example of chemically stressed wheat that metabolome-wide internal standardization by globally 13C-labeled metabolite extract (GLMe-IS) of experimental-condition-matched biological samples can help to improve the detection of treatment-relevant metabolites and can aid in the post-acquisition assessment of putative matrix effects in samples obtained upon different treatments. For this, native extracts of toxin- and mock-treated (control) wheat ears were standardized by the addition of uniformly 13C-labeled wheat ear extracts that were cultivated under similar experimental conditions (toxin-treatment and control) and measured with LC-HRMS. The results show that 996 wheat-derived metabolites were detected with the non-condition-matched 13C-labeled metabolite extract, while another 68 were only covered by the experimental-condition-matched GLMe-IS. Additional testing is performed with the assumption that GLMe-IS enables compensation for matrix effects. Although on average no severe matrix differences between both experimental conditions were found, individual metabolites may be affected as is demonstrated by wrong decisions with respect to the classification of significantly altered metabolites. When GLMe-IS was applied to compensate for matrix effects, 272 metabolites showed significantly altered levels between treated and control samples, 42 of which would not have been classified as such without GLMe-IS.

Preparation of uniformly labelled 13C- and 15N-plants using customised growth chambers.

BACKGROUND: Stable isotopically labelled organisms have found wide application in life science research including plant physiology, plant stress and defense as well as metabolism related sciences. Therefore, the reproducible production of plant material enriched with stable isotopes such as 13C and 15N is of considerable interest. A high degree of enrichment (> 96 atom %) with a uniformly distributed isotope (global labelling) is accomplished by a continuous substrate supply during plant growth/cultivation. In the case of plants, 13C-labelling can be achieved by growth in 13CO2(g) atmosphere while global 15N-labelling needs 15N- containing salts in the watering/nutrient solution. Here, we present a method for the preparation of 13C and 15N-labelled plants by the use of closed growth chambers and hydroponic nutrient supply. The method is exemplified with durum wheat. RESULTS: In total, 330 g of globally 13C- and 295 g of 15N-labelled Triticum durum wheat was produced during 87 cultivation days. For this, a total of 3.88 mol of 13CO2(g) and 58 mmol of 15N were consumed. The degree of enrichment was determined by LC-HRMS and ranged between 96 and 98 atom % for 13C and 95-99 atom % for 15N, respectively. Additionally, the isotopically labelled plant extracts were successfully used for metabolome-wide internal standardisation of native T.durum plants. Application of an isotope-assisted LC-HRMS workflow enabled the detection of 652 truly wheat-derived metabolites out of which 143 contain N. CONCLUSION: A reproducible cultivation which makes use of climate chambers and hydroponics was successfully adapted to produce highly enriched, uniformly 13C- and 15N-labelled wheat. The obtained plant material is suitable to be used in all kinds of isotope-assisted research. The described technical equipment and protocol can easily be applied to other plants to produce 13C-enriched biological samples when the necessary specific adaptations e.g. temperature and light regime, as well as nutrient supply are considered. Additionally, the 15N-labelling method can also be carried out under regular glasshouse conditions without the need for customised atmosphere.

Human amniotic membrane as newly identified source of amniotic fluid pulmonary surfactant.

Pulmonary surfactant (PS) reduces surface tension at the air-liquid interface in the alveolar epithelium of the lung, which is required for breathing and for the pulmonary maturity of the developing foetus. However, the origin of PS had never been thoroughly investigated, although it was assumed to be secreted from the foetal developing lung. Human amniotic membrane (hAM), particularly its epithelial cell layer, composes the amniotic sac enclosing the amniotic fluid. In this study, we therefore aimed to investigate a potential contribution of the cellular components of the hAM to pulmonary surfactant found in amniotic fluid. We identified that cells within the native membrane contain lamellar bodies and express all four surfactant proteins as well as ABCA3. Lipidomic profiling by nanoESI - MS/MS revealed the presence of the essential lipid species as found in PS. Also, the biophysical activity of conditioned cell culture supernatant obtained from hAM was tested with captive bubble surfactometry. hAM supernatant showed the ability to reduce surface tension, similar to human PS obtained from bronchoalveolar lavage. This means that hAM produces the essential PS-associated components and can therefore contribute as second potential source of PS in amniotic fluid aside from the foetal lung.