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

Multiple nucleophilic elbows leading to multiple active sites in a single module esterase from Sorangium cellulosum

The catalytic residues in carbohydrate esterase enzyme families constitute a highly conserved triad: serine, histidine and aspartic acid. This catalytic triad is generally located in a very sharp turn of the protein backbone structure, called the nucleophilic elbow and identified by the consensus sequence GXSXG. An esterase from Sorangium cellulosum Soce56 that contains five nucleophilic elbows was cloned and expressed in Escherichia coli and the function of each nucleophilic elbowed site was characterized. In order to elucidate the function of each nucleophilic elbow, site directed mutagenesis was used to generate variants with deactivated nucleophilic elbows and the functional promiscuity was analyzed. In silica analysis together with enzymological characterization interestingly showed that each nucleophilic elbow formed a local active site with varied substrate specificities and affinities. To our knowledge, this is the first report presenting the role of multiple nucleophilic elbows in the catalytic promiscuity of an esterase. Further structural analysis at protein unit level indicates the new evolutionary trajectories in emerging promiscuous esterases

Supraclavicular recurrence after early breast cancer: a curable condition?

International audienceThe prognosis of ipsilateral supraclavicular lymph node recurrence after early breast cancer appears to be worse than for other loco-regional recurrences, but better than for distant metastases. The purpose of the present study was to investigate the relationship between different types of salvage treatment and primary patient characteristics, treatment response, and survival after supraclavicular recurrence (SR) in a large patient population. From the Danish Breast Cancer Cooperative Group treatment database 1977–2003, 305 patients were identified with SR without distant disease as site of first recurrence. Salvage treatment types as well as other factors were related to response and survival. The median follow-up time for progression after SR was 25 months. Complete remission was 76% among patients receiving excision surgery, 67% with combined loco-regional and systemic therapy, and 48% with systemic therapy alone. Median progression-free survival (PFS) and overall survival was 18 and 29 months, respectively. The 5-year PFS probability was 15%. In univariate analysis, combination salvage therapy, negative nodal status and low malignancy grade were related to longer PFS. In multivariate analysis, salvage therapy and malignancy grade remained independent factors for survival. In conclusion, the prognosis of SR is generally poor. However, it appears to be a curable condition. An independent marker of improved outcome is local and systemic combination salvage treatment, which can be considered

Predição do Consumo Voluntário do Capim-Tanzânia (Panicum maximum, J. cv. tanzânia), sob Pastejo, por Vacas em Lactação, a partir das Características de Degradação Prediction of Tanzania Grass (Panicum Maximum, J.) Voluntary Intake, under Grazing by Lactating Cows, from Degradation Characteristics

O consumo de matéria seca (CMS) do capim tanzânia de 24 vacas lactantes mestiças (HPB x Gir) e Gir, sob pastejo, foi estimado no mês de janeiro de 1998, a partir da relação entre a digestibilidade da MS da forragem e a produção fecal obtida com auxílio do cromo mordente por meio de um modelo não-linear. Os resultados do consumo estimado foram comparados aos consumos preditos por diferentes equações baseadas nos dados de degradabilidade do capim, no rúmen. A pastagem foi manejada com taxa de lotação de dois animais/ha, em sistema de pastejo rotativo com três dias de ocupação do piquete e 39 dias de descanso. Foram utilizadas para predizer o CMS diferentes equações: CMS = -1,19 + 0,035 (a+ b) + 28,5c (1), CMS = -0,822 + 0,0748 (a+ b) + 40,7c (2), CMS = -8,286 + 0,266a + 0,102b +17,696c (3) e CMS = [%FDN na MS]* [consumo de FDN ] / [(1-a-b)/K P +b/(c+ k p)]/24] (4). As equações, em geral, subestimaram o consumo obtido no modelo não-linear (9,6 kg/vaca/dia). Os consumos médios de capim de 6,2 e 6,0 kg MS/vaca/dia obtidas, respectivamente, nas equações de (2) e (4) foram semelhantes entre si e inferiores ao das equações de (1) (12,7 kg/vaca/dia) e (3) (8,1 kg/vaca/dia). A predição do consumo de forrageiras tropicais, sob pastejo, utilizando-se as equações baseadas nas variáveis da degradação in situ, constitui um importante potencial para estas avaliações. Entretanto, mais estudos dessa natureza devem ser realizados para validar o uso destas equações na prática.<br>Dry matter intake (DMI) of Tanzania grass under grazing conditions was estimated using 24 Holstein-Zebu and Gir cows from the in vitro dry matter digestibility of extrusa samples (esophageal fistulated cows) and fecal output. The fecal output was estimated using chromium mordant and a non-linear model. The pasture was managed in a rotational system with three days paddock occupation and 39 days of resting period. The stocking rate was 2.0 cows/ha during the rainy season. Four different equations based on in situ degradation characteristics were used to predict DMI: DMI = -1.19 + 0.035 (a+ b) + 28.5c (1), DMI = -0.822 + 0.0748 (a+ b) + 40.7c (2), DMI = -8.286 + 0.266a + 0.102b +17.696c (3) and DMI = [%FDN MS]* [FDN intake] / [(1-a-b)/K P +b/(c+ k p)]/24] (4). The DMI predicted from the equations, in general, underestimated the results obtained with the non-linear model (9.6 kg DM/cow/day). The DMI data using the equations (2) and (4) (6.2 and 6.0 kg of DM/cow/day) were similar and different from the results obtained using equations (1) (12.7 kg DM/cow/day) and (3) (8.1 kg DM/cow/day). All the DMI were underestimated in relation to results from the non-linear model, except those from equation (1). The prediction of tropical forages intake, under grazing condition using equations based in degradation characteristics constitute an important tool for these evaluations. Eventhough, more studies need to be done to validate these equations in practice