Endogenous xylose pathway in Saccharomyces cerevisiae

The baker's yeast Saccharomyces cerevisiae is generally classified as a non-xylose-utilizing organism. We found that S. cerevisiae can grow on D-xylose when only the endogenous genes GRE3 (YHR104w), coding for a nonspecific aldose reductase, and XYL2 (YLR070c, ScXYL2), coding for a xylitol dehydrogenase (XDH), are overexpressed under endogenous promoters. In nontransformed S. cerevisiae strains, XDH activity was significantly higher in the presence of xylose, but xylose reductase (XR) activity was not affected by the choice of carbon source. The expression of SOR1, encoding a sorbitol dehydrogenase, was elevated in the presence of xylose as were the genes encoding transketolase and transaldolase. An S. cerevisiae strain carrying the XR and XDH enzymes from the xylose-utilizing yeast Pichia stipitis grew more quickly and accumulated less xylitol than did the strain overexpressing the endogenous enzymes. Overexpression of the GRE3 and ScXYL2 genes in the S. cerevisiae CEN.PK2 strain resulted in a growth rate of 0.01 g of cell dry mass liter(-1) h(-1) and a xylitol yield of 55% when xylose was the main carbon source.     

Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials.

Plant cell wall proteins called expansins are thought to disrupt hydrogen bonding between cell wall polysaccharides without hydrolyzing them. We describe here a novel gene with sequence similarity to plant expansins, isolated from the cellulolytic fungus Trichoderma reesei. The protein named swollenin has an N-terminal fungal type cellulose binding domain connected by a linker region to the expansin-like domain. The protein also contains regions similar to mammalian fibronectin type III repeats, found for the first time in a fungal protein. The swollenin gene is regulated in a largely similar manner as the T. reesei cellulase genes. The biological role of SWOI was studied by disrupting the swo1 gene from T. reesei. The disruption had no apparent effect on the growth rate on glucose or on different cellulosic carbon sources. Non-stringent Southern hybridization of Trichoderma genomic DNA with swo1 showed the presence of other swollenin-like genes, which could substitute for the loss of SWOI in the disruptant. The swollenin gene was expressed in yeast and Aspergillus niger var. awamori. Activity assays on cotton fibers and filter paper were performed with concentrated SWOI-containing yeast supernatant that disrupted the structure of the cotton fibers without detectable formation of reducing sugars. It also weakened filter paper as assayed by an extensometer. The SWOI protein was purified from A. niger var. awamori culture supernatant and used in an activity assay with Valonia cell walls. It disrupted the structure of the cell walls without producing detectable amounts of reducing sugars.     

Identification in the mold Hypocrea jecorina of the first fungal D-galacturonic acid reductase

A d-galacturonic acid reductase and the corresponding gene were identified from the mold Hypocrea jecorina (Trichoderma reesei). We hypothesize that the enzyme is part of a fungal d-galacturonic acid catabolic pathway which has not been described previously and which is distinctly different from the bacterial pathway. H. jecorina grown on d-galacturonic acid exhibits an NADPH-dependent d-galacturonic acid reductase activity. This activity is absent when the mold is grown on other carbon sources. The d-galacturonic acid reductase was purified, and tryptic digests of the purified protein were sequenced. The open reading frame of the corresponding gene was then cloned from a cDNA library. The open reading frame was functionally expressed in the yeast Saccharomyces cerevisiae. A histidine-tagged protein was purified, and the enzyme kinetics were characterized. The enzyme converts in a reversible reaction from d-galacturonic acid and NADPH to l-galactonic acid and NADP. The enzyme also exhibits activity with d-glucuronic acid and dl-glyceraldehyde.     

Xylose chemostat isolates of Saccharomyces cerevisiae show altered metabolite and enzyme levels compared with xylose, glucose, and ethanol metabolism of the original strain

The efficient conversion of xylose-containing biomass hydrolysate by the ethanologenic yeast Saccharomyces cerevisiae to useful chemicals such as ethanol still remains elusive, despite significant efforts in both strain and process development. This study focused on the recovery and characterization of xylose chemostat isolates of a S. cerevisiae strain that overexpresses xylose reductase- and xylitol dehydrogenase-encoding genes from Pichia stipitis and the gene encoding the endogenous xylulokinase. The isolates were recovered from aerobic chemostat cultivations on xylose as the sole or main carbon source. Under aerobic conditions, on minimal medium with 30 g l^–1 xylose, the growth rate of the chemostat isolates was 3-fold higher than that of the original strain (0.15 h^–1 vs 0.05 h^–1). In a detailed characterization comparing the metabolism of the isolates with the metabolism of xylose, glucose, and ethanol in the original strain, the isolates showed improved properties in the assumed bottlenecks of xylose metabolism. The xylose uptake rate was increased almost 2-fold. Activities of the key enzymes in the pentose phosphate pathway (transketolase, transaldolase) increased 2-fold while the concentrations of their substrates (pentose 5-phosphates, sedoheptulose 7-phosphate) decreased correspondingly. Under anaerobic conditions, on minimal medium with 45 g l^–1 xylose, the ethanol productivity (in terms of cell dry weight; CDW) of one of the isolates increased from 0.012 g g^–1 CDW h^–1 to 0.017 g g^–1 CDW h^–1 and the yield from 0.09 g g^–1 xylose to 0.14 g g^–1 xylose, respectively.     

Evolution of clostridia and streptomycetes in full-scale composting facilities and pilot drums equipped with on-line temperature monitoring and aeration

The evolution of sporulating bacteria in full-scale composting facilities with online temperature monitoring has been poorly studied, although organic matter recycling increases. We analysed Clostridium perfringens and sulphite-reducing clostridia (SRC) by cultivation, and streptomycetes by real-time PCR in five full-scale, temperature-monitored and aerated composting processes, and two pilot-scale drum composters. Facilities composted woodchips, sawdust, peat, or bark amended sludge or source-separated biowaste. Streptomycetes genes of 0.21-110 × 10⁷ copies/g feed increased fast to 0.019-33 × 10⁹ copies/g, and then were equal or decreased. SRC of 0.06-2.2 × 10⁷ cfu/g feed decreased to 0-600 cfu/g, with re-growth in two facilities. End products were clean of C. perfringens, detected in sludge composts. Although processes contained large quantities of spore-forming bacteria, in the best facilities end products had the high quality. Temperature (>55 °C, >2d) was not related to the end compost quality, but relations between waste and bulking agent qualities, aeration, and processing time should be better controlled.     

Transformation system for Hypocrea jecorina (Trichoderma reesei) that favors homologous integration and employs reusable bidirectionally selectable markers

Hypocrea jecorina is an industrially important filamentous fungus due to its effective production of hydrolytic enzymes. It has received increasing interest because of its ability to convert lignocellulosic biomass to monomeric sugars, which can be converted into biofuels or platform chemicals. Genetic engineering of strains is a highly important means of meeting the requirements of tailor-made applications. Therefore, we report the development of a transformation system that allows highly efficient gene targeting by using a tmus53 (human LIG4 homolog) deletion strain. Moreover, it permits the unlimited reuse of the same marker by employing a Cre/loxP-based excision system. Both marker insertion and marker excision can be positively selected for by combining resistance to hygromycin B and loss of sensitivity to fluoroacetamide. Finally, the marker pyr4, also positively selectable for insertion and loss, can be used to remove the cre gene.     

Endoplasmic reticulum remains continuous and undergoes sheet-to-tubule transformation during cell division in mammalian cells

The endoplasmic reticulum (ER) is a multifaceted cellular organelle both structurally and functionally, and its cell cycle–dependent morphological changes are poorly understood. Our quantitative confocal and EM analyses show that the ER undergoes dramatic reorganization during cell division in cultured mammalian cells as mitotic ER profiles become shorter and more branched. 3D modeling by electron tomography reveals that the abundant interphase structures, sheets, are lost and subsequently transform into a branched tubular network that remains continuous. This is confirmed by observing the most prominent ER subdomain, the nuclear envelope (NE). A NE marker protein spreads to the mitotic ER tubules, although it does not show a homogenous distribution within the network. We mimicked the mitotic ER reorganization using puromycin to strip the membrane-bound ribosomes from the interphase ER corresponding to the observed loss of ribosomes normally occurring during mitosis. We propose that the structural changes in mitotic ER are linked to ribosomal action on the ER membranes.