Molecular analysis of a Saccharomyces cerevisiae mutant with improved ability to utilize xylose shows enhanced expression of proteins involved in transport, initial xylose metabolism, and the pentose phosphate pathway.

Differences between the recombinant xylose-utilizing Saccharomyces cerevisiae strain TMB 3399 and the mutant strain TMB 3400, derived from TMB 3399 and displaying improved ability to utilize xylose, were investigated by using genome-wide expression analysis, physiological characterization, and biochemical assays. Samples for analysis were withdrawn from chemostat cultures. The characteristics of S. cerevisiae TMB 3399 and TMB 3400 grown on glucose and on a mixture of glucose and xylose, as well as of S. cerevisiae TMB 3400 grown on only xylose, were investigated. The strains were cultivated under chemostat conditions at a dilution rate of 0.1 h-1, with feeds consisting of a defined mineral medium supplemented with 10 g of glucose liter-1, 10 g of glucose plus 10 g of xylose liter-1 or, for S. cerevisiae TMB 3400, 20 g of xylose liter-1. S. cerevisiae TMB 3400 consumed 31% more xylose of a feed containing both glucose and xylose than S. cerevisiae TMB 3399. The biomass yields for S. cerevisiae TMB 3400 were 0.46 g of biomass g of consumed carbohydrate-1 on glucose and 0.43 g of biomass g of consumed carbohydrate-1 on xylose. A Ks value of 33 mM for xylose was obtained for S. cerevisiae TMB 3400. In general, the percentage error was <20% between duplicate microarray experiments originating from independent fermentation experiments. Microarray analysis showed higher expression in S. cerevisiae TMB 3400 than in S. cerevisiae TMB 3399 for (i) HXT5, encoding a hexose transporter; (ii) XKS1, encoding xylulokinase, an enzyme involved in one of the initial steps of xylose utilization; and (iii) SOL3, GND1, TAL1, and TKL1, encoding enzymes in the pentose phosphate pathway. In addition, the transcriptional regulators encoded by YCR020C, YBR083W, and YPR199C were expressed differently in the two strains. Xylose utilization was, however, not affected in strains in which YCR020C was overexpressed or deleted. The higher expression of XKS1 in S. cerevisiae TMB 3400 than in TMB 3399 correlated with higher specific xylulokinase activity in the cell extracts. The specific activity of xylose reductase and xylitol dehydrogenase was also higher for S. cerevisiae TMB 3400 than for TMB 3399, both on glucose and on the mixture of glucose and xylose

The STF2p Hydrophilin from Saccharomyces cerevisiae Is Required for Dehydration Stress Tolerance

The yeast Saccharomyces cerevisiae is able to overcome cell dehydration; cell metabolic activity is arrested during this period but restarts after rehydration. The yeast genes encoding hydrophilin proteins were characterised to determine their roles in the dehydration-resistant phenotype, and STF2p was found to be a hydrophilin that is essential for survival after the desiccation-rehydration process. Deletion of STF2 promotes the production of reactive oxygen species and apoptotic cell death during stress conditions, whereas the overexpression of STF2, whose gene product localises to the cytoplasm, results in a reduction in ROS production upon oxidative stress as the result of the antioxidant capacity of the STF2p protein

Bioengineering beans for phosphate-deficient soils in southern Africa

NatuurwetenskappeBotaniePlease help us populate SUNScholar with the post print version of this article. It can be e-mailed to: [email protected]

Bioengineering beans for phosphate-deficient soils in southern Africa

CITATION: Viktor, A., Cordero-Otero, R. & Valentine, A. 2003. Bioengineering beans for phosphate-deficient soils in southern Africa. South African Journal of Science, 99(11-12):509-511.The original publication is available at https://journals.co.zaAPPROXIMATELY EIGHT SPECIES OF SOUTHern African legumes are currently used as sustainable food crops. Biotechnology has the potential to improve the productivity of growing these plants by small-scale farmers who cannot afford sufficient phosphate fertilizer to optimize their nitrogen fixation and hence conversion to edible protein. The metabolic adaptations that enable legumes to fix atmospheric nitrogen are currently being investigated by our group for the purpose of genetic modification to enhance crop yields. Until now, attempts at modifying host plants or symbiotic bacteria have not significantly enhanced N2 fixation. We propose, instead, to bioengineer the key enzymes that control the mechanisms involved in protein formation. This may lead to enhanced seed protein content, which would be of advantage to poor communities that rely on this source of food. We postulate that misregulation of phosphoenolpyruvate carboxylase (PEPc) could be exploited by biotechnology to improve N2 fixation and protein content. We have found that, as distinct from their roots, legume nodules are under permanent phosphate stress, even during optimal phosphate supply to the host plant, implying that the development of phosphate stress may engage different forms of PEPc to ensure continued nodule functioning.https://journals.co.za/content/sajsci/99/11-12/EJC97570Publisher's versio

Two upstream activation sequences control the expression of the XPR2 gene in the yeast Yarrowia lipolytica.

We have initiated a study of the promoter region of the alkaline extracellular protease gene (XPR2) from Yarrowia lipolytica to identify upstream sequences possibly involved in carbon, nitrogen, and peptone control of XPR2 expression. Deletion analysis showed that the TATA box and two major upstream activation sequences (UASs) were essential for promoter activity under conditions of repression or full induction. Within the distal UAS (UAS1), in vivo footprinting studies with dimethyl sulfate (DMS) identified two sequences similar to Saccharomyces cerevisiae GCN4 (-800 to -792)- and TUF/RAP1 (-790 to -778)-binding sites and two sequences which partially overlap a repeated sequence (-778 to -771 and -720 to -713) similar to the CAR1 upstream repression sequence of S. cerevisiae. Oligonucleotides carrying the TUF/RAP1-like-binding site and adjacent downstream nucleotides restored full transcriptional activity of a UAS1-deleted promoter. Within the proximal UAS (UAS2), a directly repeated decameric sequence (-146 to -137 and -136 to -127) was protected against DMS in vivo. Sequences identical to the ABF1-binding site of S. cerevisiae (-121 to -109) or similar to the GCN4-binding site (-113 to -105) were not clearly protected from DMS in vivo. An oligomer (-150 to -106) carrying these three sequences, inserted into a UAS2-deleted promoter, increased the transcriptional activity. The results from footprints under different physiological conditions suggested that protein binding to both UASs was constitutive. Deletion of both UASs greatly reduced XPR2 expression without abolishing its regulation. Our results strongly suggest that these UASs are targets for transcriptional factors required for assisting specific regulatory proteins

Development of a screening method for the identification of a novel Saccharomyces cerevisiae mutant over-expressing Trichoderma reesei cellobiohydrolase II

In a previous study we showed that the fusion of the cellulose-binding domain (CBD2) from Trichoderma reesei cellobiohydrolase II to a β-glucosidase (BGL1) enzyme from Saccharomycopsis fibuligera significantly hindered its expression and secretion in Saccharomyces cerevisiae. This suggests that the possible low secretion of heterologous cellulolytic enzymes in S. cerevisiae could be attributed to the presence of a cellulose-binding domain (CBD) in these enzymes. The aim of this study was to increase the extracellular production of the chimeric CBD2-BGL1 enzyme (designated CBGL1) in S. cerevisiae. To achieve this, CBGL1 was used as a reporter enzyme for screening mutagenised S. cerevisiae strains with increased ability to secrete CBD-associated enzymes such as cellulolytic enzymes. A mutant strain of S. cerevisiae, WM91-CBGL1, which exhibited up to 200 U L-1 of total activity, was isolated. Such activity was approximately threefold more than that of the parental host strain. Seventy-five per cent of the activity was detected in the extracellular medium. The mutant strain transformed with the T. reesei CBH2 gene produced up to threefold more cellobiohydrolase enzyme than the parental strain, but with 50% of the total activity retained intracellularly. The cellobiohydrolase enzymes from the parent and mutant strains were partially purified and the characteristic properties analysed.8 page(s