Metabolic engineering of Saccharomyces cerevisiae for hydroxytyrosol overproduction directly from glucose

Hydroxytyrosol (HT) is one of the most powerful dietary antioxidants with numerous applications in different areas, including cosmetics, nutraceuticals and food. In the present work, heterologous hydroxylase complex HpaBC from Escherichia coli was integrated into the Saccharomyces cerevisiae genome in multiple copies. HT productivity was increased by redirecting the metabolic flux towards tyrosol synthesis to avoid exogenous tyrosol or tyrosine supplementation. After evaluating the potential of our selected strain as an HT producer from glucose, we adjusted the medium composition for HT production. The combination of the selected modifications in our engineered strain, combined with culture conditions optimization, resulted in a titre of approximately 375 mg l⁻¹ of HT obtained from shake-flask fermentation using a minimal synthetic-defined medium with 160 g l⁻¹ glucose as the sole carbon source. To the best of our knowledge, this is the highest HT concentration produced by an engineered S. cerevisiae strain.     

Experimental annotation of the human pathogen <Emphasis Type="Italic">Candida albicans</Emphasis> coding and noncoding transcribed regions using high-resolution tiling arrays

Background  

Compared to other model organisms and despite the clinical relevance of the pathogenic yeast Candida albicans, no comprehensive analysis has been done to provide experimental support of its in silico-based genome annotation.     

Ultrasound imaging in an experimental model of fatty liver disease and cirrhosis in rats

Background

Atypical scrapie was first identified in Norwegian sheep in 1998 and has subsequently been identified in many countries. Retrospective studies have identified cases predating the initial identification of this form of scrapie, and epidemiological studies have indicated that it does not conform to the behaviour of an infectious disease, giving rise to the hypothesis that it represents spontaneous disease. However, atypical scrapie isolates have been shown to be infectious experimentally, through intracerebral inoculation in transgenic mice and sheep. The first successful challenge of a sheep with 'field' atypical scrapie from an homologous donor sheep was reported in 2007.

Results

This study demonstrates that atypical scrapie has distinct clinical, pathological and biochemical characteristics which are maintained on transmission and sub-passage, and which are distinct from other strains of transmissible spongiform encephalopathies in the same host genotype.

Conclusions

Atypical scrapie is consistently transmissible within AHQ homozygous sheep, and the disease phenotype is preserved on sub-passage.     

Resistance to the cereal cyst nematode (Heterodera avenae Woll.) transferred from the wild grassAegilops ventricosa to hexaploid wheat by a “stepping-stone” procedure

Transfer of resistance toHeterodera avenae, the cereal cyst nematode (CCN), by a stepping-stone procedure from the wild grassAegilops ventricosa to hexaploid wheat has been demonstrated. The number of nematodes per plant was lower, and reached a plateau much earlier, in the resistant introgression line H93-8 (1–2 nematodes per plant) than in the recipient H10-15 wheat (14–16 nematodes per plant). Necrosis (hypersensitive reaction) near the nematode, little cell fusion, and few, often degraded syncytia were observed in infested H93-8 roots, while abundant, well-formed syncytia were present in the susceptible H10-15 wheat. Line H93-8 was highly resistant to the two Spanish populations tested, as well as the four French races (Fr1-Fr4), and the British pathotype Hall, but was susceptible to the Swedish pathotypes HgI and HgIII. Resistance was inherited as though determined by a single quasi-dominant factor in the F₂ generations resulting from crosses of H93-8 with H10-15 and with Loros, a resistant wheat carrying the geneCre1 (syn.Ccn1). The resistance gene in H93-8 (Cre2 orCcn2) is not allelic with respect to that in Loros. RFLPs and other markers, together with the cytogenetical evidence, indicate that theCre2 gene has been integrated into a wheat chromosome without affecting its meiotic pairing ability. Introduction ofCre2 by backcrossing into a commercial wheat backgroud increases grain yield when under challenge by the nematode and is not detrimental in the absence of infestation.     

Oviposition,larval preference,and larval performance in two polyphagous species: does the larva know best?

It is expected that females preferentially oviposit on plant hosts that allow for optimal larval performance. However, this expectation contradicts empirical evidence where adults do not always choose the best host for their descendants. Recent evidence suggests that females’ host selection depends on the number of potential hosts. Females from oligophagous species seem to be able to choose an appropriate host in terms of larval performance, whereas in polyphagous species, adult oviposition preference is not related with larval performance. This suggests that larvae in polyphagous species could be taking a more active role in host selection than their mothers. Here, we evaluated the oviposition preference and the larval preference and performance of two polyphagous species of economic importance, Copitarsia decolora (Guenée) (Lepidoptera: Noctuidae: Cuculliinae) and Peridroma saucia (Hübner) (Lepidoptera: Noctuidae: Noctuinae), on eight species of cultivated plants. In laboratory and greenhouse choice assays, we tested adult preference for oviposition and larval preference at 1 and 24 h. Larval performance was measured in terms of survival to adulthood, length of larval period, and pupal weight. We found that both adult females and larvae actively choose their hosts and that the larval preference toward the hosts is related to the females’ preference in both herbivore species. However, the females and larvae did not preferentially select the host with the best larval performance, indicating that larval performance is not related to female or larval preference and that other selective pressures are influencing the choice of the host plant in these two species.     

High resolution assembly and characterization of genomes of Canadian isolates of Salmonella Enteritidis

Background

There is a need to characterize genomes of the foodborne pathogen, Salmonella enterica serovar Enteritidis (SE) and identify genetic information that could be ultimately deployed for differentiating strains of the organism, a need that is yet to be addressed mainly because of the high degree of clonality of the organism. In an effort to achieve the first characterization of the genomes of SE of Canadian origin, we carried out massively parallel sequencing of the nucleotide sequence of 11 SE isolates obtained from poultry production environments (n = 9), a clam and a chicken, assembled finished genomes and investigated diversity of the SE genome.

Results

The median genome size was 4,678,683 bp. A total of 4,833 chromosomal genes defined the pan genome of our field SE isolates consisting of 4,600 genes present in all the genomes, i.e., core genome, and 233 genes absent in at least one genome (accessory genome). Genome diversity was demonstrable by the presence of 1,360 loci showing single nucleotide polymorphism (SNP) in the core genome which was used to portray the genetic distances by means of a phylogenetic tree for the SE isolates. The accessory genome consisted mostly of previously identified SE prophage sequences as well as two, apparently full- sized, novel prophages namely a 28 kb sequence provisionally designated as SE-OLF-10058 (3) prophage and a 43 kb sequence provisionally designated as SE-OLF-10012 prophage.

Conclusions

The number of SNPs identified in the relatively large core genome of SE is a reflection of substantial diversity that could be exploited for strain differentiation as shown by the development of an informative phylogenetic tree. Prophage sequences can also be exploited for SE strain differentiation and lineage tracking. This work has laid the ground work for further studies to develop a readily adoptable laboratory test for the subtyping of SE.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-713) contains supplementary material, which is available to authorized users.     

Structural characterization and promoter activity analysis of the γ-kafirin gene from sorghum

A genomic clone encoding the γ-kafirin gene from sorghum was isolated and sequenced. A 2938 bp sequenced fragment includes an intronless open reading frame of 636 nucleotides encoding a putative polypeptide of 212 amino acids. Comparison of the deduced amino acid sequence of γ-kafirin with the published sequences of γ-prolamins of maize, and Coix revealed highly conserved domains. The N-terminal region of these proteins contains the conserved hexapeptide PPPVHL, which is repeated eight times in γ-zein, four times in γ-kafirin and three times in γ-coixin. The number of PPPVHL repeats accounts predominantly for the differences in the molecular weights of γ-prolamins. Several putative regulatory sequences common to the γ-kafirin and γ-zein genes were identified in both the 5′ and the 3′ flanking regions. Putative GCN4-like regulatory sequences were found at positions ?192 and ?476 in the 5′ flanking region of γ-kafirin. In the 3′ noncoding region, three putative polyadenylation signals, two AATAAT and one AATGAA, were found at positions + 658, + 716, and + 785, respectively. In order to investigate the role of the putative GCN4-like motifs and other possible cis-acting element(s) of the γ-kafirin promoter, a series of deleted and chimeric promoter constructs were introduced into maize, Coix and sorghum tissues by particle bombardment. Histochemical analysis of β-glucuronidase (GUS) activity in different tissues indicated that the element(s) responsible for tissue specificity is probably located in the 285-bp proximal region of the promoter, while the remaining promoter sequence seems to carry the element(s) responsible for the quantitative response.     

Miniaturized analytical assays in biotechnology

Biotechnology today is a well-established paradigm in many areas of human endeavor, such as the pharmaceutical industry, agriculture, management of the environment and many others. Meanwhile, biology is undergoing a spectacular transition: whereas systematic biology was replaced gradually by molecular biology, the latter is rapidly being transformed into a new systematic era in which entire genomes are being charted by ever more sophisticated analytical techniques.In the wake of this onslaught of data, new fields are germinating, such as bioinformatics in an attempt to find answers to fundamental questions, answers that may be hidden in the massive amounts of data already available today.