Regulation of amino-acid metabolism controls flux to lipid accumulation in <i>Yarrowia lipolytica</i>

Yarrowia lipolytica is a promising microbial cell factory for the production of lipids to be used as fuels and chemicals, but there are few studies on regulation of its metabolism. Here we performed the first integrated data analysis of Y. lipolytica grown in carbon and nitrogen limited chemostat cultures. We first reconstructed a genome-scale metabolic model and used this for integrative analysis of multilevel omics data. Metabolite profiling and lipidomics was used to quantify the cellular physiology, while regulatory changes were measured using RNAseq. Analysis of the data showed that lipid accumulation in Y. lipolytica does not involve transcriptional regulation of lipid metabolism but is associated with regulation of amino-acid biosynthesis, resulting in redirection of carbon flux during nitrogen limitation from amino acids to lipids. Lipid accumulation in Y. lipolytica at nitrogen limitation is similar to the overflow metabolism observed in many other microorganisms, e.g. ethanol production by Sacchromyces cerevisiae at nitrogen limitation

Rapid online buffer exchange for screening of proteins, protein complexes and cell lysates by native mass spectrometry

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is time-consuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes or clarified cell lysates. In the liquid chromatography coupled to mass spectrometry (LC-MS) approach described in this protocol, samples in MS-incompatible conditions are injected onto a short size-exclusion chromatography column. Proteins and protein complexes are separated from small molecule non-volatile buffer components using an aqueous, non-denaturing mobile phase. Eluted proteins and protein complexes are detected by the mass spectrometer after electrospray ionization. Mass spectra can inform regarding protein sample purity and oligomerization, and additional tandem mass spectra can help to further obtain information on protein complex subunits. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization

An electron channeling study of polycrystalline YBa2Cu3Ox

An electron channeling study has been done on large grained YBa2Cu3Ox samples. Selected area channeling patterns (SACP) were used to examine several dozen grains on electropolished surfaces and it was demonstrated that (a) the twin planes observed in polarized optical light microscopy lie parallel to {110} crystal planes, and (b) the long flat sides of high aspect ratio grains are formed by basal planes, and the shorter sides are formed by either (010), (100), or {110} planes. A majority of the large grains examined were found to contain subgrains, misaligned by 0.5°–1° and ranging in size from less than 3 to 20 μm. The origin of the subgrains is not understood

A molecular genetic toolbox for Yarrowia lipolytica

Background: Yarrowia lipolytica is an ascomycete yeast used in biotechnological research for its abilities to secrete high concentrations of proteins and accumulate lipids. Genetic tools have been made in a variety of backgrounds with varying similarity to a comprehensively sequenced strain. Results: We have developed a set of genetic and molecular tools in order to expand capabilities of Y. lipolytica for both biological research and industrial bioengineering applications. In this work, we generated a set of isogenic auxotrophic strains with decreased non-homologous end joining for targeted DNA incorporation. Genome sequencing, assembly, and annotation of this genetic background uncovers previously unidentified genes in Y. lipolytica. To complement these strains, we constructed plasmids with Y. lipolytica-optimized superfolder GFP for targeted overexpression and fluorescent tagging. We used these tools to build the "Yarrowia lipolytica Cell Atlas," a collection of strains with endogenous fluorescently tagged organelles in the same genetic background, in order to define organelle morphology in live cells. Conclusions: These molecular and isogenetic tools are useful for live assessment of organelle-specific protein expression, and for localization of lipid biosynthetic enzymes or other proteins in Y. lipolytica. This work provides the Yarrowia community with tools for cell biology and metabolism research in Y. lipolytica for further development of biofuels and natural products

Period-1 Encodes an ATP-Dependent RNA Helicase that Influences Nutritional Compensation of the Neurospora Circadian Clock

Mutants in the period-1 (prd-1) gene, characterized by a recessive allele, display a reduced growth rate and period lengthening of the developmental cycle controlled by the circadian clock. We refined the genetic location of prd-1 and used whole genome sequencing to find the mutation defining it, confirming the identity of prd-1 by rescuing the mutant circadian phenotype via transformation. PRD-1 is an RNA helicase whose orthologs, DDX5 [DEAD (Asp-Glu-Ala-Asp) Box Helicase 5] and DDX17 in humans and DBP2 (Dead Box Protein 2) in yeast, are implicated in various processes, including transcriptional regulation, elongation, and termination, ribosome biogenesis, and mRNA decay. Although prd-1 mutants display a long period (∼25 h) circadian developmental cycle, they interestingly display a WT period when the core circadian oscillator is tracked using a frq-luciferase transcriptional fusion under conditions of limiting nutritional carbon; the core oscillator in the prd-1 mutant strain runs with a long period under glucose-sufficient conditions. Thus, PRD-1 clearly impacts the circadian oscillator and is not only part of a metabolic oscillator ancillary to the core clock. PRD-1 is an essential protein, and its expression is neither light-regulated nor clock-regulated. However, it is transiently induced by glucose; in the presence of sufficient glucose, PRD-1 is in the nucleus until glucose runs out, which elicits its disappearance from the nucleus. Because circadian period length is carbon concentration-dependent, prd-1 may be formally viewed as a clock mutant with defective nutritional compensation of circadian period length

Discourse and identity in a corpus of lesbian erotica

This article uses corpus linguistic methodologies to explore representations of lesbian desires and identities in a corpus of lesbian erotica from the 1980s and 1990s. We provide a critical examination of the ways in which “lesbian gender,” power, and desire are represented, (re-)produced, and enacted, often in ways that challenge hegemonic discourses of gender and sexuality. By examining word frequencies and collocations, we critically analyze some of the themes, processes, and patterns of representation in the texts. Although rooted in linguistics, we hope this article provides an accessible, interdisciplinary, and timely contribution toward developing understandings of discursive practices surrounding gender and sexuality

Leaf-litter stoichiometry is affected by streamwater phosphorus concentrations and litter type

The stoichiometric ratios of organisms and their food resources can influence C and nutrient dynamics in aquatic ecosystems. Several investigators have quantified linkages between nutrient enrichment and consumer stoichiometry for stream detritivores, but very few have systematically quantified the effect of P enrichment on leaf-litter stoichiometry. Here, we examine the potential stoichiometric changes of 2 species of leaf litter subjected to varying levels of P enrichment in laboratory microcosms and mixed species across a natural P gradient of streams in the Ozark Highlands Region, Arkansas, USA. Leaf-litter %P content increased and C:P ratios decreased with increasing levels of P enrichment and with increasing lability of the leaf species. In the laboratory study, C:P of maple and oak leaves in the control treatment was ,2500, whereas this ratio decreased to 500 and 1000 in the high-P treatments, respectively. Total P (TP) was inversely related to leaf-litter C:P along the natural P gradient of streams in the Ozarks. Our results add to the growing body of information on the potential bottom-up effects of anthropogenic nutrient loading in streams and the influence of water-column nutrients and leaf quality on this response

A versatile toolkit for high throughput functional genomics with Trichoderma reesei

<p>Abstract</p> <p>Background</p> <p>The ascomycete fungus, <it>Trichoderma reesei </it>(anamorph of <it>Hypocrea jecorina</it>), represents a biotechnological workhorse and is currently one of the most proficient cellulase producers. While strain improvement was traditionally accomplished by random mutagenesis, a detailed understanding of cellulase regulation can only be gained using recombinant technologies.</p> <p>Results</p> <p>Aiming at high efficiency and high throughput methods, we present here a construction kit for gene knock out in <it>T. reesei</it>. We provide a primer database for gene deletion using the <it>pyr4, amdS </it>and <it>hph </it>selection markers. For high throughput generation of gene knock outs, we constructed vectors using yeast mediated recombination and then transformed a <it>T. reesei </it>strain deficient in non-homologous end joining (NHEJ) by spore electroporation. This NHEJ-defect was subsequently removed by crossing of mutants with a sexually competent strain derived from the parental strain, QM9414.</p> <p>Conclusions</p> <p>Using this strategy and the materials provided, high throughput gene deletion in <it>T. reesei </it>becomes feasible. Moreover, with the application of sexual development, the NHEJ-defect can be removed efficiently and without the need for additional selection markers. The same advantages apply for the construction of multiple mutants by crossing of strains with different gene deletions, which is now possible with considerably less hands-on time and minimal screening effort compared to a transformation approach. Consequently this toolkit can considerably boost research towards efficient exploitation of the resources of <it>T. reesei </it>for cellulase expression and hence second generation biofuel production.</p