Linking Genotype and Phenotype of Saccharomyces cerevisiae Strains Reveals Metabolic Engineering Targets and Leads to Triterpene Hyper-Producers

Background: Metabolic engineering is an attractive approach in order to improve the microbial production of drugs. Triterpenes is a chemically diverse class of compounds and many among them are of interest from a human health perspective. A systematic experimental or computational survey of all feasible gene modifications to determine the genotype yielding the optimal triterpene production phenotype is a laborious and time-consuming process. Methodology/Principal Findings: Based on the recent genome-wide sequencing of Saccharomyces cerevisiae CEN.PK 113-7D and its phenotypic differences with the S288C strain, we implemented a strategy for the construction of a beta-amyrin production platform. The genes Erg8, Erg9 and HFA1 contained non-silent SNPs that were computationally analyzed to evaluate the changes that cause in the respective protein structures. Subsequently, Erg8, Erg9 and HFA1 were correlated with the increased levels of ergosterol and fatty acids in CEN.PK 113-7D and single, double, and triple gene over-expression strains were constructed. Conclusions: The six out of seven gene over-expression constructs had a considerable impact on both ergosterol and beta-amyrin production. In the case of beta-amyrin formation the triple over-expression construct exhibited a nearly 500% increase over the control strain making our metabolic engineering strategy the most successful design of triterpene microbial producers

A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases

Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply

Surveys of rice sold in Canada for aflatoxins, ochratoxin A and fumonisins

Approximately 200 samples of rice (including white, brown, red, black, basmati and jasmine, as well as wild rice) from several different countries, including the United States, Canada, Pakistan, India and Thailand, were analysed for aflatoxins, ochratoxin A (OTA) and fumonisins by separate liquid Chromatographic methods in two different years. The mean concentrations for aflatoxin B1 (AFB1) were 0.19 and 0.17 ng g−1 with respective positive incidences of 56% and 43% (≥ the limit of detection (LOD) of 0.002 ng g−1). Twenty-three samples analysed in the second year also contained aflatoxin B2 (AFB2) at levels ≥LOD of 0.002 ng g−1 The five most contaminated samples in each year contained 1.44–7.14 ng AFB1 g−1 (year 1) and 1.45–3.48 ng AFB1 g−1 (year 2); they were mostly basmati rice from India and Pakistan and black and red rice from Thailand. The average concentrations of ochratoxin A (OTA) were 0.05 and 0.005 ng g−1 in year 1 and year 2, respectively; incidences of samples containing ≥LOD of 0.05 ng g−1 were 43% and 1%, respectively, in the 2 years. All positive OTA results were confirmed by LC-MS/MS. For fumonisins, concentrations of fumonisin B1 (FB1) averaged 4.5 ng g−1 in 15 positive samples (≥0.7 ng g−1) from year 1 (n = 99); fumonisin B2 (FB2) and fumonisin B3 (FB3) were also present (≥1 ng g−1). In the second year there was only one positive sample (14 ng g−1 FB1) out of 100 analysed. All positive FB1 results were confirmed by LC-MS/MS

The human semicircular canal model of galvanic vestibular stimulation

A vector summation model of the action of galvanic stimuli on the semicircular canals has been shown to explain empirical balance and perceptual responses to binaural-bipolar stimuli. However, published data suggest binaural-monopolar stimuli evoke responses that are in the reverse direction of the model prediction. Here, we confirm this by measuring balance responses to binaural-monopolar stimulation as movements of the upper trunk. One explanation for the discrepancy is that the galvanic stimulus might evoke an oppositely directed balance response from the otolith organs that sums with and overrides the semicircular canal response. We tested this hypothesis by measuring sway responses across the full range of head pitch. The results showed some modulation of sway with pitch such that the maximal response occurred with the head in the primary position. However, the effect fell a long way short of that required to reverse the canal sway response. This indicates that the model is incomplete. Here, we examine alterations to the model that could explain both the bipolar and monopolar-evoked behavioural responses. An explanation was sought by remodelling the canal response with more recent data on the orientation of the individual canals. This improved matters but did not reverse the model prediction. However, the model response could be reversed by either rotating the entire labyrinth in the skull or by altering the gains of the individual canals. The most parsimonious solution was to use the more recent canal orientation data coupled with a small increase in posterior canal gain

Low-volume intraplate volcanism in the Early/Middle Jurassic Pacific basin documented by accreted sequences in Costa Rica

Countless seamounts occur on Earth that can provide important constraints on intraplate volcanism and plate tectonics in the oceans, yet their nature and origin remain poorly known due to difficulties in investigating the deep ocean. We present here new lithostratigraphic, age and geochemical data from Lower/Middle Jurassic and Lower Cretaceous sequences in the Santa Rosa accretionary complex, Costa Rica, which offer a valuable opportunity to study a small-sized seamount from a subducted plate segment of the Pacific basin. The seamount is characterized by very unusual lithostratigraphic sequences with sills of potassic alkaline basalt emplaced within thick beds of radiolarite, basaltic breccia and hyaloclastite. An integration of new geochemical, biochronological and geochronological data with lithostratigraphic observations suggests that the seamount formed ~175 Ma ago on thick oceanic crust away from subduction zones and mid-ocean ridges. This seamount travelled ~65 Ma in the Pacific before accretion. It resembles lithologically and compositionally “petit-spot” volcanoes found off Japan, which form in response to plate flexure near subduction zones. Also, the composition of the sills and lava flows in the accreted seamount closely resembles that of potassic alkaline basalts produced by lithosphere cracking along the Line Islands chain. We hypothesize based on these observations, petrological constraints and formation of the accreted seamount coeval with the early stages of development of the Pacific plate that the seamount formed by extraction of small volumes of melt from the base of the lithosphere in response to propagating fractures at the scale of the Pacific basin

The role of pyroxenite in basalt genesis: Melt-PX, a melting parameterization for mantle pyroxenites between 0.9 and 5GPa

Geochemical and isotopic data suggest that the source regions of oceanic basalts may contain pyroxenite in addition to peridotite. In order to incorporate the wide range of compositions and melting behaviors of pyroxenites into mantle melting models, we have developed a new parameterization, Melt-PX, which predicts near-solidus temperatures and extents of melting as a function of temperature and pressure for mantle pyroxenites. We used 183 high-pressure experiments (25 compositions; 0.9–5 GPa; 1150–1675°C) to constrain a model of melt fraction versus temperature from 5% melting up to the disappearance of clinopyroxene for pyroxenites as a function of pressure, temperature, and bulk composition. When applied to the global set of experimental data, our model reproduces the experimental F values with a standard error of estimate of 13% absolute; temperatures at which the pyroxenite is 5% molten are reproduced with a standard error of estimate of 30°C over a temperature range of ~500°C and a pressure range of ~4 GPa. In conjunction with parameterizations of peridotite melting, Melt-PX can be used to model the partial melting of multilithologic mantle sources—including the effects of varying the composition and the modal proportion of pyroxenite in such source regions. Examples of such applications include calculations of isentropic decompression melting of a mixed peridotite + pyroxenite mantle; these show that although the potential temperature of the upwelling mantle plays an important role in defining the extent of magma production, the composition and mass fraction of the pyroxenite also exert strong controls

IgA in the horse: cloning of equine polymeric Ig receptor and J chain and characterization of recombinant forms of equine IgA

As in other mammals, immunoglobulin A (IgA) in the horse has a key role in immune defense. To better dissect equine IgA function, we isolated complementary DNA (cDNA) clones for equine J chain and polymeric Ig receptor (pIgR). When coexpressed with equine IgA, equine J chain promoted efficient IgA polymerization. A truncated version of equine pIgR, equivalent to secretory component, bound with nanomolar affinity to recombinant equine and human dimeric IgA but not with monomeric IgA from either species. Searches of the equine genome localized equine J chain and pIgR to chromosomes 3 and 5, respectively, with J chain and pIgR coding sequence distributed across 4 and 11 exons, respectively. Comparisons of transcriptional regulatory sequences suggest that horse and human pIgR expression is controlled through common regulatory mechanisms that are less conserved in rodents. These studies pave the way for full dissection of equine IgA function and open up possibilities for immune-based treatment of equine diseases