Construction of mini-chemostats for high-throughput strain characterization.

To achieve large-scale, high-throughput experiments for systems biology research of microorganisms, reliable data from robust cultivation systems are needed. Chemostats are such systems, ensuring reproducibility and quality by providing a stable, well-controlled environment for the cells. However, many of the available chemostat systems require large amounts of media and are complex to set up and expensive to purchase and maintain. To address these concerns, we developed a mini-chemostat (MC) system with 16 reactors, each at a working volume of 40 ml. Sensors measure dissolved oxygen in the reactor, while OD600 is measured in the outflow. We further developed a CO2 and pH sensor array that can be plugged into the outflow of the reactors. The system was used to characterize yeast physiology at four metabolically different conditions: limitations of glucose, both aerobic and anaerobic, nitrogen, and ethanol. The physiology of yeast cells grown at the four different conditions in the MC system was compared with the yeast cells grown in a DASGIP 1 L system using RNAseq analysis. The results show that the MC system provides the same environmental conditions as the DASGIP system and that the MC system is reproducible between different runs. The system is built to be easily scalable with more reactors and to include more sensors, if available. Our study shows that a robust, reproducible chemostat system for high-throughput and large-scale experiments can be built at low costs.

Secondary metabolite comparison of the species within the Heterobasidion annosum s.l. complex.

The metabolite production of the five members of the fungal species complex Heterobasidion annosum s.l., i.e. H. annosum s.s., H. abietinum, H. parviporum, H. occidentale and H. irregulare, was analyzed by LC-HRMS. The five members are described to have differences in host preferences: H. annosum s.s. and H. irregulare are pine infecting species, and H. parviporum, H. occidentale and H. abietinum are non-pine infecting. Principal component analysis (PCA) of the LC-HRMS data showed that samples from the five species could be separated into five groups and in accordance with the differences in host preferences. Twenty-three compounds, important to the observed PCA grouping, were isolated and identified. The main contributor to the separation of the pine infecting species from the non-pine infecting species in PC1 was the benzohydrofuran fomannoxin, which was only detected in the pine infecting species H. annosum s.s. and H. irregulare. These two species were further separated in PC3, and one major contributor here was the sesquiterpene deoxyfomannosin A. The three non-pine infecting species were separated in PC2, by epoxydrimenol that was detected in only two of the species and further in PC4, where a few fomannoxin related compounds were important for the grouping. During the work, three unknown compounds were isolated and described: 3-hydroxy-2-(1,3-dihydroxypropan-2-yl)-2,3-dihydrobenzofuran-5-carbaldehyde, 3-hydroxy-2-(1,2,3-trihydroxypropan-2-yl)-2,3-dihydrobenzofuran-5-carbaldehyde and 3-hydroxy-2,3-dihydrobenzofuran-5-carboxylic acid.

Biosynthesis of fomannoxin in the root rotting pathogen Heterobasidion occidentale.

Fomannoxin is a biologically active benzohydrofuran, which has been suggested to be involved in the pathogenicity of the root rotting fungus Heterobasidion annosum sensu lato. The biosynthesis of fomannoxin was investigated through an isotopic enrichment study utilizing [1-¹³C]glucose as metabolic tracer. ¹³C NMR spectroscopic analysis revealed the labeling pattern and showed that the isoprene building block originates from the mevalonic acid pathway, whereas the aromatic motif is formed via the shikimic acid route by elimination of pyruvate from chorismic acid. A natural product, 4-hydroxy-3-(3-methylbut-2-enyl)benzaldehyde, was isolated and characterized, and was suggested to be a key intermediate in the biosynthesis of fomannoxin and related secondary metabolites previously identified from the H. annosum fungal species complex.

Sesquiterpenes from the conifer root rot pathogen Heterobasidion occidentale.

Investigation of the production of secondary metabolites of Heterobasidion occidentale led to the isolation and identification of six sesquiterpenes (illudolone A and B, illudolactone A and B, deoxyfomannosin A and B) along with the well-known sesquiterpene fomannosin and the previously described benzohydrofuran fomannoxin. The structures and relative configurations of the compounds were determined by 1D and 2D NMR spectroscopic analysis as well as by HRMS. Their absolute configuration and biosynthesis were suggested and discussed in relation to fomannosin. Four compounds showed growth inhibiting activity against several basidiomycetes, Phlebiopsis gigantea, Phanerochaete chrysosporium and H. occidentale, and toxicity towards the moss Physcomitrella patens. In addition, one compound displayed activity against the bacterium Variovorax paradoxus as well as against the ascomycete Fusarium oxysporum.

Insight into trade-off between wood decay and parasitism from the genome of a fungal forest pathogen.

Parasitism and saprotrophic wood decay are two fungal strategies fundamental for succession and nutrient cycling in forest ecosystems. An opportunity to assess the trade-off between these strategies is provided by the forest pathogen and wood decayer Heterobasidion annosum sensu lato. We report the annotated genome sequence and transcript profiling, as well as the quantitative trait loci mapping, of one member of the species complex: H. irregulare. Quantitative trait loci critical for pathogenicity, and rich in transposable elements, orphan and secreted genes, were identified. A wide range of cellulose-degrading enzymes are expressed during wood decay. By contrast, pathogenic interaction between H. irregulare and pine engages fewer carbohydrate-active enzymes, but involves an increase in pectinolytic enzymes, transcription modules for oxidative stress and secondary metabolite production. Our results show a trade-off in terms of constrained carbohydrate decomposition and membrane transport capacity during interaction with living hosts. Our findings establish that saprotrophic wood decay and necrotrophic parasitism involve two distinct, yet overlapping, processes.