Whole genome sequencing of Saccharomyces cerevisiae: from genotype to phenotype for improved metabolic engineering applications

<p>Abstract</p> <p>Background</p> <p>The need for rapid and efficient microbial cell factory design and construction are possible through the enabling technology, metabolic engineering, which is now being facilitated by systems biology approaches. Metabolic engineering is often complimented by directed evolution, where selective pressure is applied to a partially genetically engineered strain to confer a desirable phenotype. The exact genetic modification or resulting genotype that leads to the improved phenotype is often not identified or understood to enable further metabolic engineering.</p> <p>Results</p> <p>In this work we performed whole genome high-throughput sequencing and annotation can be used to identify single nucleotide polymorphisms (SNPs) between <it>Saccharomyces cerevisiae </it>strains S288c and CEN.PK113-7D. The yeast strain S288c was the first eukaryote sequenced, serving as the reference genome for the <it>Saccharomyces </it>Genome Database, while CEN.PK113-7D is a preferred laboratory strain for industrial biotechnology research. A total of 13,787 high-quality SNPs were detected between both strains (reference strain: S288c). Considering only metabolic genes (782 of 5,596 annotated genes), a total of 219 metabolism specific SNPs are distributed across 158 metabolic genes, with 85 of the SNPs being nonsynonymous (e.g., encoding amino acid modifications). Amongst metabolic SNPs detected, there was pathway enrichment in the galactose uptake pathway (<it>GAL1</it>, <it>GAL10</it>) and ergosterol biosynthetic pathway (<it>ERG8</it>, <it>ERG9</it>). Physiological characterization confirmed a strong deficiency in galactose uptake and metabolism in S288c compared to CEN.PK113-7D, and similarly, ergosterol content in CEN.PK113-7D was significantly higher in both glucose and galactose supplemented cultivations compared to S288c. Furthermore, DNA microarray profiling of S288c and CEN.PK113-7D in both glucose and galactose batch cultures did not provide a clear hypothesis for major phenotypes observed, suggesting that genotype to phenotype correlations are manifested post-transcriptionally or post-translationally either through protein concentration and/or function.</p> <p>Conclusions</p> <p>With an intensifying need for microbial cell factories that produce a wide array of target compounds, whole genome high-throughput sequencing and annotation for SNP detection can aid in better reducing and defining the metabolic landscape. This work demonstrates direct correlations between genotype and phenotype that provides clear and high-probability of success metabolic engineering targets. The genome sequence, annotation, and a SNP viewer of CEN.PK113-7D are deposited at <url>http://www.sysbio.se/cenpk</url>.</p

Identifying Fishes through DNA Barcodes and Microarrays

Background: International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection. Methodology/Principal Findings: This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S), cytochrome b (cyt b), and cytochrome oxidase subunit I (COI) for the identification of 50 European marine fish species by combining techniques of ‘‘DNA barcoding’’ and microarrays. In a DNA barcoding approach, neighbour Joining (NJ) phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the ‘‘position of label’’ effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridisation experiments (.90%) renders the DNA barcoding marker as rather unsuitable for this high-throughput technology. Conclusions/Significance: Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products

Genetic basis of codling moth (Cydia pomonella) resistance to the original isolate of C. pomonella Granulovirus (CpGV-M)

The utilization of insect pest pathogens as biocontrol agents can serve as an effective alternative to chemical pesticides. However, pests can develop resistance to these pathogens, similar to resistance observed with chemicals. Identifying the genetic bases of resistance is a crucial step in comprehending the co-evolution mechanisms between insect hosts and their pathogens, thereby enhancing the durability of these biocontrol methods. Cydia pomonella, a major pest of apple and pear trees found across all continents, can be effectively controlled in commercial orchards through the use of the granulovirus, CpGV. However, since 2005, resistance to the CpGV-M isolate has been reported in Europe, leading to significant challenges in managing C. pomonella in organic orchards. Studies conducted on CPGV-M resistance have revealed that viral replication is blocked in resistant phenotypes, and resistance is linked to a major gene located on the Z sex chromosome. Nevertheless, this major gene appears to be insufficient in explaining resistance to CPGV-M, as other forms of resistance involving autosomal genes have been identified subsequently. The objective of this study was to gain insights into the genetic architecture of C. pomonella resistance to CpGV-M by conducting a genome-wide association study using newly generated whole-genome resequencing data from pools of individuals (Pool-Seq) sampled from 13 susceptible and resistant populations across four regions in France and Italy. Through this analysis, eight candidate regions were identified, including one located on the Z sex chromosome. These eight regions encompass a total of 27 lncRNAs (long non-coding RNAs) and 40 genes, of which three are known to be involved in insects immune pathways

How varying parameters impact insecticide resistance bioassay: An example on the worldwide invasive pest Drosophila suzukii.

Monitoring pesticide resistance is essential for effective and sustainable agricultural practices. Bioassays are the basis for pesticide-resistance testing, but devising a reliable and reproducible method can be challenging because these tests are carried out on living organisms. Here, we investigated five critical parameters and how they affected the evaluation of resistance to the organophosphate phosmet or the pyrethroid lambda-cyhalothrin using a tarsal-contact protocol on Drosophila suzukii, a worldwide invasive pest. Three of the parameters were related to insect biology: (i) sex, (ii) age of the imago (adult stage) and (iii) genetic diversity of the tested population. The two remaining parameters were linked to the experimental setup: (iv) the number of individuals tested per dose and (v) the duration of exposure to the active ingredient. Results showed that response to insecticide differed depending on sex, males being twice as susceptible to phosmet as females. Age principally affected young females' susceptibility to phosmet, because 0-24 hour-old flies were twice as susceptible as 24-48 hour-old and 72-96 hour-old females. Genetic diversity had no observable effect on resistance levels. The precision and accuracy of the median lethal dose (LD50) were greatly affected by the number of individuals tested per dose with a threshold effect. Finally, optimal duration of exposure to the active ingredient was 24 h, as we found an underestimation of mortality when assessed between 1 and 5 h after exposure to lambda-cyhalothrin. None of the main known point mutations on the para sodium channel gene associated with a knockdown effect were observed. Our study demonstrates the importance of calibrating the various parameters of a bioassay to develop a reliable method. It also provides a valuable and transferable protocol for monitoring D. suzukii resistance worldwide

Cross effects of heat stress and three insecticides on the survival of the codling moth Cydia pomonella (L.): Investigating the molecular and biochemical mechanisms

International audienceAs temperature is expected to strongly increase in the future, understanding temperature-mediated toxicity of insecticides is determinant to assess pest management efficiency in a warming world. Investigating molecular and biochemical mechanisms associated with cross mechanisms of temperature and insecticides on pests' tolerance would also be useful in this context. This study aimed to investigate cross effects between temperature and insecticides on the survival of a major pest, the codling moth Cydia pomonella, and their underlying mechanisms. The effect of three insecticidal active ingredients, i.e. chlorantraniliprole, emamectin and spinosad, was assessed at different temperatures on: (i) C. pomonella larval survival; (ii) detoxification enzymes activities (cytochrome P450 multi-function oxygenases, carboxylesterases and glutathione S-transferases) and (iii) genes expression of some detoxification enzymes, heat shock proteins and receptors targeted by the insecticides. We observed a decreased efficiency of emamectin and spinosad at high temperature to control the codling moth while no influence of temperature on chlorantraniliprole efficacy was observed. Detoxification enzymes activities were improved by heat stress alone but not by double stress (temperature + insecticides). Moreover, two detoxification genes (Cyp9A61 and Gst1) were over-expressed by a single stress but not by two stresses while Hsp70 and Cyp6B2 genes may be involved in tolerance to two stresses in C. pomonella. These results confirmed the cross effects of temperature and insecticides on C. pomonella for emamectin and spinosad and provided clues to understand how temperature affects the susceptibility of C. pomonella to insecticides. They illustrate however the complexity of molecular and biochemical responses of individuals facing multiple stresses

Genetic inferences about the population dynamics of codling moth females at a local scale

Estimation of demographic parameters is important for understanding the functioning of natural populations and the underlying ecological and evolutionary processes that may impact their dynamics. Here, we used sibship assignment methods to estimate the local dynamics of codling moth females in eight orchards in a 90-ha domain near Valence, France. Based on full-sib inference among 1,063 genotyped moths, we estimated i) the effective number of females that had offspring, ii) their fertility and iii) the distribution of their oviposition sites within and among orchards. The average number of females in all the orchards increased between the first (~130) and the second (~235) annual generations. The average fertilities of the females were similar at each generation according to the host plant considered (apple, pear, or walnut), but differed between commercial (~10) and non-treated (~25) apple orchards. Females mainly clustered their eggs on contiguous trees along orchard borders, but they also occasionally dispersed their eggs among different orchards independently of the cultivated host plants or the inter-orchard distances (up to 698 m) during the second annual generation. The mean distance between two oviposition sites was 30 m. Sibship estimates of both the effective number of females and the inter-orchard migration rates (~5%) were in agreement with the observed genetic differentiation among the eight orchards (0.00

Regulation of sugar metabolism genes in the N-dependent susceptibility of tomato stems to Botrytis cinerea

Background and Aims: The main soluble sugars are important components of plant defence against pathogens, but the underlying mechanisms are unclear. Upon infection by Botrytis cinerea, the activation of several sugar transporters, from both plant and fungus, illustrates the struggle for carbon resources. In sink tissues, the metabolic use of the sugars mobilized in the synthesis of defence compounds or antifungal barriers is not fully understood.Methods: In this study, the nitrogen-dependent variation of tomato stem susceptibility to B. cinerea was used to examine – before and throughout the course of infection – the transcriptional activity of enzymes involved in sugar metabolism. Under different nitrate nutrition regimes, the expressions of genes that encode the enzymes of sugar metabolism (invertases, sucrose synthases, hexokinases, fructokinases and phosphofructokinases), as well as sugar contents, were measured before inoculation and in asymptomatic tissues surrounding the lesions after inoculation.Key Results: At high nitrogen availability, decreased susceptibility was associated with the over-expression of several genes two days after inoculation: sucrose synthase Sl-SUS1 and Sl-SUS3, cell wall invertase Sl-LIN5 to Sl-LIN9 and some fructokinase and phosphofructokinase genes. By contrast, increased susceptibility corresponded to the early repression of several genes that encode cell wall invertase and sucrose synthase. The evolution of sugar contents was coherent with the gene expressions.Conclusions: The activation of specific genes which encode sucrose synthase is required for enhanced defence. Since the over-expression of fructokinase is also associated with reduced susceptibility, it can be hypothesized that supplemental sucrose cleavage by sucrose synthases is dedicated to the production of cell wall components from UDP-glucose, or to the additional implication of fructose for the synthesis of antimicrobial compounds, or both

Dense collection areas and terrestrial alteration of meteorites in the Atacama Desert

International audienceAbstract In the last 15 years, more than 2700 meteorites have been recovered and officially classified from the Atacama Desert. Although the number of meteorites collected in the Atacama has risen, the physical and climatic properties of the dense collection areas (DCAs) have not been fully characterized. In this article, we compiled the published data of all classified meteorites found in the Atacama Desert to (i) describe the distribution by meteorite groups, (ii) compare the weathering degree of chondrites among different Atacama DCAs and other hot and cold deserts, and (iii) determine the preservation conditions of chondrites in the main Atacama DCAs in relation with the local climatic conditions. The 35 DCAs so far identified in the Atacama Desert are located in three main morphotectonic units: The Coastal Range (CR), Central Depression (CD), and Pre‐Andean Range/Basement. A comparison with reported weathering data from other cold and hot deserts indicates that the mean terrestrial weathering of Atacama chondrites (W1–2), displays less alteration than other hot deserts (W2–3) and resembles the weathering distribution of the Antarctic meteorites (W1–2). The highest abundance of Atacama chondrites with low weathering (≤W2) is localized in the CD (78.8%, N = 1435), which is protected from the coastal fog influence and seasonal rainfalls and displays the oldest surfaces in the Atacama Desert. The morphogenetic classification based on present‐day temperatures and precipitations of the main Atacama DCAs reveals similar regional/subregional climatic conditions in the most productive areas and a truly productive surface for meteorite recovery between 5% and 58% of the quadrangles formally defined for each Atacama DCA. Our morphogenetic classification lacks consideration of some meteorological parameters such as the coastal fog, so it cannot fully explain the differences in weathering patterns among CR chondrites. Future studies of chondrite preservation in the Atacama DCAs should consider other meteorological variables such as relative humidity, specific humidity, or dew point, in combination with exposure ages of meteorites and its surfaces

At least five rhizobial species nodulate Phaseolus vulgaris in a Spanish soil

International audienc