Exploring xylose metabolism in non-conventional yeasts: kinetic characterization and product accumulation under different aeration conditions.

D-xylose is a metabolizable carbon source for several non-Saccharomyces species, but not for native strains of S. cerevisiae. For the potential application of xylose-assimilating yeasts in biotechnological processes, a deeper understanding of pentose catabolism is needed. This work aimed to investigate the traits behind xylose utilization in diverse yeast species.The performance of nine selected xylose-metabolizing yeast strains was evaluated and compared across three oxygenation conditions. Oxygenation diversely impacted growth, xylose consumption and product accumulation. Xylose utilization by ethanol-producing species such as Spathaspora passalidarum and Scheffersomyces stipitis was less affected by oxygen restriction than other xylitol-accumulating species such as Meyerozyma guilliermondii, Naganishia liquefaciens and Yamadazyma sp., for which increased aeration stimulated xylose assimilation considerably. S. passalidarum exhibited superior conversion of xylose to ethanol and showed the fastest growth and xylose consumption in all three conditions. By performing assays under identical conditions for all selected yeasts, we minimize bias in comparisons, providing valuable insight into xylose metabolism and facilitating the development of robust bioprocesses.

Delta subclass HD-Zip proteins and a B-3 AP2/ERF transcription factor interact with promoter elements required for expression of the Arabidopsis cytochrome c oxidase 5b-1 gene.

We have identified transcription factors that interact with a promoter region involved in expression of the Arabidopsis thaliana COX5b-1 gene, which encodes an isoform of the cytochrome c oxidase zinc binding subunit. Elements with the core sequence ATCATT, involved in induction by sugars, are recognized both in vitro and in one-hybrid assays in yeast by HD-Zip proteins from the delta subclass and, though less efficiently, by the trihelix transcription factor GT-3b. DistalB-like elements (CCACTTG), required for induction by abscisic acid (ABA), interact with ESE1, a member of the B-3 subgroup of AP2/ERF transcription factors. The HD-Zip protein Athb-21 and ESE1 are able to interact in yeast two-hybrid assays with the ABA responsive element binding factor AREB2/ABF4, which binds to a G-box absolutely required for expression of the COX5b-1 gene. Overexpression of the identified transcription factors in plants produces an increase in COX5b-1 transcript levels. Moreover, these factors are able to induce the expression of a reporter gene located in plants under the control of the relevant COX5b-1 promoter regions required for expression. Analysis of promoter regions of COX5b genes from different plant species suggests that the identified transcription factors were recruited for the regulation of COX5b gene expression at different stages during the evolution of dicot plants.

PhDSeeker: Pheromone-Directed Seeker for metabolic pathways.

Manually finding relationship networks among compounds can be a hard and time-consuming task. However, this process is fundamental when looking for a metabolic pathway that explains how multiple compounds are related, to identify relevant pathways in organisms, filling gaps on metabolic networks, or when new mechanisms for the synthesis of important compounds are sought. Here, we present PhDSeeker, a new tool for the automatic search of metabolic pathways. This tool is able to relate simultaneously several compounds. Furthermore, its flexibility allows it to be easily configured for addressing a wide range of situations. Solutions found are provided not only in plain text but also as interactive representations that can be analyzed in a web browser. Source code is available at https://github.com/sinc-lab/phdseeker. A web service is also available at https://sinc.unl.edu.ar/web-demo/phds/. Several fully documented study cases, including their settings and solutions files, are also provided as Supplementary Material.

A segment containing a G-box and an ACGT motif confers differential expression characteristics and responses to the Arabidopsis Cytc-2 gene, encoding an isoform of cytochrome c.

Sequences required for the expression of Cytc-2 (At4g10040), one of two cytochrome c genes from Arabidopsis thaliana, were characterized using plants transformed with deleted and mutagenized promoter fragments fused to gus. These studies indicated that a region containing a G-box and an ACGT motif is essential for expression. Mutation of the ACGT motif causes a complete loss of expression, while mutation of the G-box causes decreased expression in aerial parts and abolishes expression in roots and induction by environmental factors. Upstream located site II elements are required for maximal expression, mainly in reproductive tissues, and maximal induction by different factors. One-hybrid screenings allowed the identification of transcription factors from the bZIP and bHLH families that interact mainly with the G-box. Four of these factors were able to bind to the Cytc-2 promoter in vitro and in transactivation assays in Arabidopsis. Analysis of available microarray data indicated that the bZIP transcription factors share expression characteristics with the Cytc-2 gene, suggesting that they act as mediators of its response to tissue-specific, environmental, and metabolic conditions. Site II elements interact with a TCP family protein and may co-ordinate the expression of the Cytc-2 gene with that of other respiratory chain components. A model is proposed for the evolution of the Cytc-2 gene through the incorporation of a segment containing a G-box and an ACGT motif into an ancestral gene that contained site II elements. This may have reduced the importance of site II elements for basal expression and conferred new responses to environmental factors.

Identification of regulatory elements involved in expression and induction by sucrose and UV-B light of the Arabidopsis thaliana COX5b-2 gene, encoding an isoform of cytochrome c oxidase subunit 5b.

The promoter sequences required for expression of the Arabidopsis thaliana COX5b-2 gene, encoding an isoform of cytochrome c oxidase subunit 5b, were analyzed using plants transformed with deleted and mutagenized forms of the promoter fused to gus. A 1000-bp promoter fragment produces expression in root and shoot meristems, leaf and cotyledon tips, and anthers. Deletion analysis indicated the presence of positive and negative regulatory elements. A regulatory element located between -660 and -620 from the translation start site was identified as a G-box by mutagenic analysis. Mutation of the G-box, that is present within the coding region of the preceding gene in the genome, increases expression of COX5b-2 in cotyledon and leaf lamina and abolishes induction by ultraviolet-B (UV-B) light, which presumably acts through the removal of an inhibitory factor. Identified positive regulatory elements include a site II element (TGGGCC), a related element with the sequence TGGGTC and four initiator elements (YTCANTYY) that completely abolish expression when mutated in combination. Site II elements are also involved in the response to sucrose. The results imply that the COX5b-2 gene has retained expression characteristics presented by most respiratory chain component genes, but its expression mechanisms have diverged from those employed by COX5b-1, the other gene encoding cytochrome c oxidase subunit 5b in Arabidopsis.

Feasiblity of Bioethanol Production from Cider Waste.

Wastewater from cider factories (losses during transfers, products discarded due to quality policies, and products returned from the market) exhibit a Chemical Oxygen Demand greater than 170,000 mg O2/l, mainly due to the ethanol content and carbohydrates that are added to obtain the finished product. These effluents can represent up to 10% of the volume of cider produced, and they must be treated to meet environmental regulations. In this work, a process was developed, based on alcoholic fermentation of the available carbohydrates present in ciders. The impact of inhibitors at different pH, size and reuse of inoculums and different nutrient supplementation on the ethanol yield were evaluated. The use of a 0.5 g/l yeast inoculum and corn steep water as the nutrient source allowed for depletion of the sugars in less than 48 h, which increased the content of ethanol to more than 70 g/l.

Conserved homeodomain cysteines confer redox sensitivity and influence the DNA binding properties of plant class III HD-Zip proteins.

The role of four cysteines present within the homeodomain of the homeodomain-leucine zipper (HD-Zip) class III protein Athb-9 has been studied. DNA binding by the Athb-9 HD-Zip domain was only observed after incubation in the presence of reducing agents or the thioredoxin system, suggesting that the protein is sensitive to redox conditions. A similar behavior was observed for proteins that show the same binding specificity of Athb-9 present in nuclear extracts. The use of single and double mutants indicated that two out of three of the cysteines at positions 23, 38 and 42 are required for redox sensitivity, while Cys58 is not involved. A role of Cys23 and Cys58 in determining the DNA binding efficiency and specificity, respectively, of the reduced Athb-9 HD-Zip domain was also evident from these studies. It can be postulated that redox conditions may modulate the function of Athb-9 in plant development. Sequence conservation suggests that the results can be extended to all HD-Zip III transcription factors.

Optimization of a low-cost defined medium for alcoholic fermentation--a case study for potential application in bioethanol production from industrial wastewaters.

In bioethanol production processes, the media composition has an impact on product concentration, yields and the overall process economics. The main purpose of this research was to develop a low-cost mineral-based supplement for successful alcoholic fermentation in an attempt to provide an economically feasible alternative to produce bioethanol from novel sources, for example, sugary industrial wastewaters. Statistical experimental designs were used to select essential nutrients for yeast fermentation, and its optimal concentrations were estimated by Response Surface Methodology. Fermentations were performed on synthetic media inoculated with 2.0 g L(-1) of yeast, and the evolution of biomass, sugar, ethanol, CO2 and glycerol were monitored over time. A mix of salts [10.6 g L(-1) (NH4)2HPO4; 6.4 g L(-1) MgSO4·7H2O and 7.5 mg L(-1) ZnSO4·7H2O] was found to be optimal. It led to the complete fermentation of the sugars in less than 12h with an average ethanol yield of 0.42 g ethanol/g sugar. A general C-balance indicated that no carbonaceous compounds different from biomass, ethanol, CO2 or glycerol were produced in significant amounts in the fermentation process. Similar results were obtained when soft drink wastewaters were tested to evaluate the potential industrial application of this supplement. The ethanol yields were very close to those obtained when yeast extract was used as the supplement, but the optimized mineral-based medium is six times cheaper, which favorably impacts the process economics and makes this supplement more attractive from an industrial viewpoint.

Performance of several Saccharomyces strains for the alcoholic fermentation of sugar-sweetened high-strength wastewaters: Comparative analysis and kinetic modelling.

This work focuses on the performance of ten commercial Saccharomyces yeast strains in the batch alcoholic fermentation of sugars contained in selected industrial wastewaters from the soft drink industry. Fermentation has been applied successfully to treat these effluents prior to their disposal. Although many strains were investigated, similar behaviour was observed between all of the Saccharomyces strains tested. When media were inoculated with 2gL-1 of yeast, all strains were able to completely consume the available sugars in less than 14h. Thus, any of the strains studied in this work could be used in non-conventional wastewater treatment processes based on alcoholic fermentation. However, ethanol production varied between strains, and these differences could be significant from a production point of view. Saccharomyces bayanus produced the most ethanol, with a mean yield of 0.44gethanolgsugarconsumed-1 and an ethanol specific production rate of 5.96gethanol (Lh)-1. As the assayed soft drinks wastewaters contain about 105gsugar/L of fermentable sugars, the concentration of ethanol achieved after the fermentations process was 46.2gethanol/L. A rigorous kinetic modelling methodology was used to model the Saccharomyces bayanus fermentation process. The kinetic model included coupled mass balances and a minimal number of parameters. A simple unstructured model based on the Andrews equation (substrate inhibition) was developed. This model satisfactorily described biomass growth, sugar consumption and bioethanol production. In addition to providing insights into the fermentative performance of potentially relevant strains, this work can facilitate the design of large-scale ethanol production processes that use wastewaters from the sugar-sweetened beverage industry as feedstock.

Wastewater from the soft drinks industry as a source for bioethanol production.

Wastewaters from the soft drinks industry were examined as media for producing bioethanol using yeast-mediated fermentation. Fermentation assays were performed using cola-type, orange and lemon-lime soft drinks and the biomass, sugar and ethanol levels were monitored over time. The effect of the addition of yeast extract was evaluated; the results indicated that 15 g/L is a suitable value for successful fermentation. Depletion of the sugars contained in the soft drinks (10-12% w/v) was achieved in less than 12 h when the medium was inoculated with 2 g/L of Saccharomyces cerevisiae var. Windsor. Ethanol yields were close to the theoretical values. The performance of several kinetic models was evaluated, and their parameters were determined. A model including inhibition by ethanol enabled the best adjustment of the experimental results in all assayed media. Some soft drinks include sodium benzoate in their formulae, the effect of which on yeast metabolism is discussed.

Divergent regulatory mechanisms in the response of respiratory chain component genes to carbohydrates suggests a model for gene evolution after duplication.

The biogenesis of the plant mitochondrial respiratory chain needs the coordinated synthesis and assembly of the products of more than 100 genes located in the nucleus and within the organelle. One of the factors that regulate the expression of nuclear genes is the availability of carbohydrates. This regulation operates at the transcriptional level through elements present in the promoter regions of respiratory chain component genes. Recent studies of the promoters of two Arabidopsis genes that encode subunit 5b of cytochrome c oxidase suggest that these genes use different molecular mechanisms to respond to carbohydrates. A model is postulated in which one of the genes retained ancient expression characteristics while the other one incorporated novel response elements that allowed a progressive divergence of regulatory mechanisms.

Characterization of promoter elements required for expression and induction by sucrose of the Arabidopsis COX5b-1 nuclear gene, encoding the zinc-binding subunit of cytochrome c oxidase.

Arabidopsis COX5b-1 encodes an isoform of the zinc binding subunit 5b of mitochondrial cytochrome c oxidase. A promoter region required for expression and induction by sucrose of this gene was analyzed using plants stably transformed with mutagenized promoter fragments fused to the gus reporter gene. Promoter dependent expression is absolutely dependent on a G-box present at -228 from the translation start site. This element interacts in vitro and in vivo with transcription factors from the bZip family, preferentially with the abscisic acid-responsive element binding factor AREB2/ABF4. A region located upstream of the G-box (-333/-259) contains elements with the core sequence ATCATT and distalB-like sequences (CCACTTG) that are required for expression in vegetative tissues. These sequences bind different sets of proteins present in plant nuclear extracts and participate in induction by sucrose (ATCATT) and abscisic acid (distalB) of the COX5b-1 promoter. We propose that the COX5b-1 promoter has acquired novel regulatory mechanisms during evolution after gene duplication. These novel mechanisms have allowed the diversification of expression patterns, but also the conservation of some responses that, as induction by sucrose, are shared by COX5b-1 and other genes encoding components of the mitochondrial respiratory chain. Conservation of these responses may be a pre-requisite for the successful incorporation of new regulatory elements in this class of genes.

Transcriptional coordination of the biogenesis of the oxidative phosphorylation machinery in plants.

Publicly available microarray experiments were used to analyze Arabidopsis thaliana genes whose expression is correlated with that of nuclear genes encoding components of the oxidative phosphorylation machinery (OxPhos genes). This analysis indicated the existence of coordination in the expression of genes encoding components of the five respiratory complexes. For these genes, preferential expression was observed in anthers and roots, especially in the elongation zone, while reduced or very low relative expression was evident in leaves and mature pollen grains. A global induction of OxPhos genes by carbohydrates, photo-destruction of chloroplasts, inhibition of cellulose synthesis, release from dormancy and germination, among other conditions, was also observed. Cluster analysis of the response of Arabidopsis genes to a set of 15 treatments allowed the identification of DNA motifs, known as site II, that are frequently present in the upstream regions of genes with responses like those of OxPhos genes. Mutagenic analysis of site II motifs in several genes encoding respiratory chain components showed that they actively participate in transcription of these genes. We conclude that an important number of nuclear genes encoding components of the five respiratory complexes show coordinated expression under various circumstances, and that site II elements and the putative proteins that interact with them are, together with as yet unidentified factors, important actors in this coordinated response.

Structure of homeodomain-leucine zipper/DNA complexes studied using hydroxyl radical cleavage of DNA and methylation interference.

Homeodomain-leucine zipper (HD-Zip) proteins, unlike most homeodomain proteins, bind a pseudopalindromic DNA sequence as dimers. We have investigated the structure of the DNA complexes formed by two HD-Zip proteins with different nucleotide preferences at the central position of the binding site using footprinting and interference methods. The results indicate that the respective complexes are not symmetric, with the strand bearing a central purine (top strand) showing higher protection around the central region and the bottom strand protected toward the 3' end. Binding to a sequence with a nonpreferred central base pair produces a decrease in protection in either the top or the bottom strand, depending upon the protein. Modeling studies derived from the complex formed by the monomeric Antennapedia homeodomain with DNA indicate that in the HD-Zip/DNA complex the recognition helix of one of the monomers is displaced within the major groove respective to the other one. This monomer seems to lose contacts with a part of the recognition sequence upon binding to the nonpreferred site. The results show that the structure of the complex formed by HD-Zip proteins with DNA is dependent upon both protein intrinsic characteristics and the nucleotides present at the central position of the recognition sequence.