The bark-beetle-associated fungus, endoconidiophora polonica, utilizes the phenolic defense compounds of its host as a carbon source

Norway spruce (Picea abies) is periodically attacked by the bark beetle Ips typographus and its fungal associate, Endoconidiophora polonica, whose infection is thought to be required for successful beetle attack. Norway spruce produces terpenoid resins and phenolics in response to fungal and bark beetle invasion. However, how the fungal associate copes with these chemical defenses is still unclear. In this study, we investigated changes in the phenolic content of Norway spruce bark upon E. polonica infection and the biochemical factors mediating these changes. Although genes encoding the rate-limiting enzymes in Norway spruce stilbene and flavonoid biosynthesis were actively transcribed during fungal infection, there was a significant time-dependent decline of the corresponding metabolites in fungal lesions. In vitro feeding experiments with pure phenolics revealed that E. polonica transforms both stilbenes and flavonoids to muconoid-type ring-cleavage products, which are likely the first steps in the degradation of spruce defenses to substrates that can enter the tricarboxylic acid cycle. Four genes were identified in E. polonica that encode catechol dioxygenases carrying out these reactions. These enzymes catalyze the cleavage of phenolic rings with a vicinal dihydroxyl group to muconoid products accepting a wide range of Norway spruce-produced phenolics as substrates. The expression of these genes and E. polonica utilization of the most abundant spruce phenolics as carbon sources both correlated positively with fungal virulence in several strains. Thus, the pathways for the degradation of phenolic compounds in E. polonica, initiated by catechol dioxygenase action, are important to the infection, growth, and survival of this bark beetle-vectored fungus and may play a major role in the ability of I. typographus to colonize spruce trees.http://www.aspbjournals.orghb2016Genetic

Investigating harbor porpoise (Phocoena phocoena) population differentiation using RAD-tag genotyping by sequencing

The population status of the harbor porpoise ( Phocoena phocoena ) in the Baltic Sea and adjacent regions is still not fully resolved. Here, we present a pilot study using the double digest restriction-site associated DNA sequencing (ddRAD-seq) genotyping- by -sequencing method on specimens from the Baltic Sea, eastern North Sea, Spain and the Black Sea. From a single Illumina lane and a set of 49 individuals, w e obtained around 6000 SNPs. We used these markers to estimate population structure and differentiation, and identified splits between porpoises from the North Sea and the Baltic, and within regions in the Baltic Sea (between the Belt Sea and the Inner Baltic Sea). The SNP analysis confirms population structure elucidated by previous mtDNA/microsatellite studies. We demonstrate the feasibility of SNP analysis on opportunistically sampled cetacean samples, with varying DNA quality, for population diversity and divergence analysis

Data from: High quality whole genome sequence of an abundant Holarctic odontocete, the harbour porpoise (Phocoena phocoena)

The harbour porpoise (Phocoena phocoena) is a highly mobile cetacean found across the Northern hemisphere. It occurs in coastal waters and inhabits basins that vary broadly in salinity, temperature, and food availability. These diverse habitats could drive subtle differentiation among populations, but examination of this would be best conducted with a robust reference genome. Here we report the first harbour porpoise genome, assembled de novo from an individual originating in the Kattegat Sea (Sweden). The genome is one of the most complete cetacean genomes currently available, with a total size of 2.39 Gb and 50% of the total length found in just 34 scaffolds. Using just 122 of the longest scaffolds, we were able to show high levels of synteny with the genome of the domestic cattle (Bos taurus). Our draft annotation comprises 22,154 predicted genes, which we further annotated through matches to the NCBI nucleotide database, GO categorization, and motif prediction. Within the predicted genes we have confirmed the presence of >20 genes or gene families that have been associated with adaptive evolution in other cetaceans. Overall, this genome assembly and draft annotation represent a crucial addition to the genomic resources currently available for the study of porpoises and Phocoenidae evolution, phylogeny, and conservation

Dynamic metabolic modeling of CHO cell metabolism coupled with N-glycosylation in the industrial pharmaceutical production

Cellular metabolism consists of a complex network of biochemical reactions and thus presents a challenging modeling problem. Experimental and modeling work, described in this article is focused on the metabolic pathway of Chinese hamster ovary (CHO) cells, which are the preferred expression system for monoclonal antibody (mAb) protein production. CHO cells are one of the primary hosts for mAbs production, which have extensive applications in multiple fields like biochemistry, biology and medicine. Here, an approach to explain cellular metabolism with in-silico modeling of a microkinetic reaction network is presented and validated with unique experimental results. Experimental data of 25 different fed-batch bioprocesses included the variation of multiple process parameters, such as pH, agitation speed, oxygen and carbon dioxide content, and dissolved oxygen. 151 metabolites were involved in our proposed metabolic network, which consisted of 132 chemical reactions that describe the reaction pathways, and include 25 reactions describing N-glycosylation and additional reactions for the accumulation of the produced glycoforms. Additional 8 reactions are considered for accumulation of the N-glycosylation products in the extracellular environment and 1 reaction to correlate cell degradation. The following pathways were considered: glycolysis, pentose phosphate pathway, nucleotide synthesis, tricarboxylic acid cycle, lipid synthesis, protein synthesis, biomass production, anaplerotic reactions and membrane transport. Our contribution to this field is the comparison of unique experimental data to our model, which is coupled with biomass production and N-glycosylation. The effect of various operational conditions was assessed and their effect on the cell production process. The modeling performed is a complementary tool to experimentation, nevertheless, with the applied modeling procedure, different operational scenarios and fed-batch techniques can be tested without the need for long-term experimental campaigns

Oleic Acid Metabolism <i>via</i> a Conserved Cytochrome P450 System-Mediated ω-Hydroxylation in the Bark Beetle-Associated Fungus <i>Grosmannia clavigera</i>

<div><p>The bark beetle-associated fungus <i>Grosmannia clavigera</i> participates in the large-scale destruction of pine forests. In the tree, it must tolerate saturating levels of toxic conifer defense chemicals (e.g. monoterpenes). The fungus can metabolize some of these compounds through the ß-oxidation pathway and use them as a source of carbon. It also uses carbon from pine triglycerides, where oleic acid is the most common fatty acid. High levels of free fatty acids, however, are toxic and can cause additional stress during host colonization. Fatty acids induce expression of neighboring genes encoding a cytochrome P450 (CYP630B18) and its redox partner, cytochrome P450 reductase (CPR2). The aim of this work was to study the function of this novel P450 system. Using LC/MS, we biochemically characterized CYP630 as a highly specific oleic acid ω-hydroxylase. We explain oleic acid specificity using protein interaction modeling. Our results underscore the importance of ω-oxidation when the main ß-oxidation pathway may be overwhelmed by other substrates such as host terpenoid compounds. Because this CYP-CPR gene cluster is evolutionarily conserved, our work has implications for metabolism studies in other fungi.</p></div

Schematic representation of selective conservation of the CYP630-CPR2 gene cluster in Pezizomycotina.

<p>The presence of the gene cluster found in seven Pezizomycotina classes is given as a fraction of the species that the gene cluster was identified in relative to all the species of a given class whose genomes were searched. Homologs of either gene were not identified outside of Pezizomycotina. The relative orientation of both genes is given (< >—divergent; > <—convergent, << or >>—co-oriented) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120119#pone.0120119.ref047" target="_blank">47</a>]. The phylogeny was modeled after [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120119#pone.0120119.ref048" target="_blank">48</a>].</p

Calculated kinetic parameters for <i>Gs</i>CPR1 and <i>Gs</i>CPR2.

<p>Calculated kinetic parameters for <i>Gs</i>CPR1 and <i>Gs</i>CPR2.</p

Time course of the root mean square (RMS) changes of the heme moiety in <i>Gs</i>CYP630B18 with oleic acid (left) and stearic acid (right) as a substrate.

<p>The protein was constrained for the first 100 ps.</p

Enzyme activity of <i>Gs</i>CPR1 and <i>Gs</i>CPR2.

<p>EU—one enzyme unit of CPR reduces 1.0 μmol oxidized cytochrome c per minute in the presence of 100 mM NADPH at pH 7.8 and 25°C.</p><p>m—membrane-bound</p><p>Positive control—rabbit liver CPR</p><p>Negative control—membrane fraction of yeast strain expressing empty plasmid pYEDP60U</p><p>Enzyme activity of <i>Gs</i>CPR1 and <i>Gs</i>CPR2.</p