Molecular and physiological characterization of mono- and dicarboxylic acids permeases in the yeast Kluyveromyces lactis

Poster apresentado em XXI International Conference on Yeast Genetics and Molecular Biology, Gotemburgo, Suécia, 7 Jul. - 12 Jul. 200

Transcription profiles of hydrogenases related genes in the cyanobacterium Lyngbya majuscula CCAP 1446/4

<p>Abstract</p> <p>Background</p> <p><it>Lyngbya majuscula </it>CCAP 1446/4 is a N₂-fixing filamentous nonheterocystous strain that contains two NiFe-hydrogenases: an uptake (encoded by <it>hupSL</it>) and a bidirectional enzyme (encoded by <it>hoxEFUYH</it>). The biosynthesis/maturation of NiFe-hydrogenases is a complex process requiring several accessory proteins for e.g. for the incorporation of metals and ligands in the active center (large subunit), and the insertion of the FeS clusters (small subunit). The last step in the maturation of the large subunit is the cleavage of a C-terminal peptide from its precursor by a specific endopeptidase. Subsequently, the mature large and small subunits can assemble forming a functional enzyme.</p> <p>Results</p> <p>In this work we demonstrated that, in <it>L. majuscula</it>, the structural genes encoding the bidirectional hydrogenase are cotranscribed, and that <it>hoxW </it>(the gene encoding its putative specific endopeptidase) is in the same chromosomal region but transcribed from a different promoter. The gene encoding the putative specific uptake hydrogenase endopeptidase, <it>hupW</it>, can be cotranscribed with the structural genes but it has its own promoter. <it>hoxH</it>, <it>hupL</it>, <it>hoxW </it>and <it>hupW </it>transcription was followed in <it>L. majuscula </it>cells grown under N₂-fixing and non-N₂-fixing conditions over a 12 h light/12 h dark cycle. The transcription of <it>hoxH</it>, <it>hoxW </it>and <it>hupW </it>did not vary remarkably in the conditions tested, while the <it>hupL </it>transcript levels are significantly higher under N₂-fixing conditions with a peak occurring in the transition between the light and the dark phase. Furthermore, the putative endopeptidases transcript levels, in particular <it>hoxW</it>, are lower than those of the respective hydrogenase structural genes.</p> <p>Conclusion</p> <p>The data presented here indicate that in <it>L. majuscula </it>the genes encoding the putative hydrogenases specific endopeptidases, <it>hoxW </it>and <it>hupW</it>, are transcribed from their own promoters. Their transcript levels do not vary notably in the conditions tested, suggesting that HoxW and HupW are probably constantly present and available in the cells. These results, together with the fact that the putative endopeptidases transcript levels, in particular for <it>hoxW</it>, are lower than those of the structural genes, imply that the activity of the hydrogenases is mainly correlated to the transcription levels of the structural genes. The analysis of the promoter regions indicates that <it>hupL </it>and <it>hupW </it>might be under the control of different transcription factor(s), while both <it>hoxH </it>and <it>xisH </it>(<it>hoxW</it>) promoters could be under the control of LexA.</p

Continuous production of pectinase by immobilized yeast cells on spent grains

A yeast strain secreting endopolygalacturonase was used in this work to study the possibility of continuous production of this enzyme. It is a feasible and interesting alternative to fungal batch production essentially due to the specificity of the type of pectinase excreted by Kluyveromyces marxianus CCT 3172, to the lower broth viscosity and to the easier downstream operations. In order to increase the reactors’ productivity, a cellulosic carrier obtained from barley spent grains was tested as an immobilization support. Two types of reactors were studied for pectinase production using glucose as a carbon and energy source - a continuous stirred tank reactor (CSTR) and a packed bed reactor (PBR) with recycled flow. The highest value for pectinase volumetric productivity (Pv=0.98 U mlˉ¹ hˉ¹) was achieved in the PBR for D=0.40 hˉ¹, a glucose concentration on the inlet of Sin =20 g lˉ¹, and a biomass load in the support of Xi=0.225 g gˉ¹. The results demonstrate the attractiveness of the packed bed system for pectinase production.Fundação para a Ciência e a Tecnologia (FCT) - BD/18203/98, SFRH/BPD/3541/2000

Use of two different carriers in a packed bed reactor for endopolygalacturonase production by a yeast strain

A packed bed reactor (PBR) design was tested for the purpose of continuous pectinase production with yeasts, as a possible alternative to the traditional batch process using fungal cultures. Two different carriers - a porous glass (Siran) and a cellulosic carrier obtained from spent grains (barley) - were used to immobilize Kluyveromyces marxianus CCT 3172, a yeast strain secreting endopolygalacturonase. To improve cell distribution throughout the column, part of the outflow was recycled. Cell loads of 0.204 and 0.247 g(biomass)/g(carrier) were obtained at the top and bottom of the PBR with spent grains, respectively. Using the PBR with Siran as the immobilization support, 0.071 g(biomass)/g(carrier) was the biomass load at the top of the column while at the bottom a value of 0.147 g(biomass)/g(carrier) was found. The highest value for pectinase volumetric productivity (Pv = 1.68 U/ml h) was achieved in the PBR with Siran for a D = 0.260 h(-1) and a glucose concentration on the inlet of S-in = 40 g/l. Both carriers were suitable for pectinase production. The best results were obtained with a high and uniform biomass concentration in the column, together with high dilution rates and total glucose consumption.Fundação para a Ciência e a Tecnologia (FCT) - BD/18203/98 , SFRH/BPD/3541/2000

Acquisition of flocculation phenotype by Kluyveromyces marxianus when overexpressing GAP1 gene encoding an isoform of glyceraldehyde-3-phosphate dehydrogenase

The use of flocculating yeast strains has been considered as a convenient approach to obtain high cell densities in bioreactors with increasing productivity in continuous operations. In Kluyveromyces marxianus ATTC 10022, the GAP1 gene encodes an isoform of glyceraldehyde-3-phosphate dehydrogenase–p37—that is accumulated in the cell wall and is involved in flocculation. To test the use of p37 as a tool for engineering Kluyveromyces cells to display a flocculation phenotype, K. marxianus CCT 3172 was transformed with an expression vector containing GAP1. This vector is based on the pY37 previously described, harbouring a S11 Kluyveromyces origin of replication, and the expression of GAP1 is under the control of GAL1. Kluyveromyces cells overexpressing GAP1 acquired a flocculent phenotype together with the accumulation of p37 in the cell wall. The results support the use of GAP1 gene as a molecular tool for inducing flocculation.Fundação para a Ciência e a Tecnologia (FCT) - BD/18203/98

Malic acid degradation by indigenous and commercial Saccharomyces cerevisiae wine strains

Malic acid contributes to the acidic taste of wine and is, together with tartaric acid, the most abundant organic acid in wine. Contaminating lactic acid bacteria cause wine spoilage after bottling and may use malic acid as a substrate. It is therefore essential to remove excess malic acid from wines to ensure their physical, biochemical and microbial stability. The aim of this work was to gain insight in the differences regarding malic acid metabolism under fermentative conditions among a collection of 294 indigenous Saccharomyces cerevisiae strains selected from the Vinho Verde Region in comparison to commercial wine yeast strains. All strains were screened regarding ethanol tolerance, capacity to utilize acetic and malic acid and acetic acid as well as H2S production. A remarkable heterogeneity of phenotypical traits was found, and only 5 strains (1.7%) of the 294 isolates showed enhanced malic acid degradation using a selective culture medium. The fermentative profiles of 3 strains (318, 319, and 320) in a synthetic must medium were very similar to the ones observed for the commercial strains QA23 and 71B. Considerable differences were also found among these strains regarding the activity of key enzymes involved in the metabolism of malic acid (malic enzyme, malate dehydrogenase, fumarase).POCTI/BIO/38106/2001.POCI 2010(FEDER/FCT, POCTI/AGR/56102/2004).AGRO (ENOSAFE, Nº 762)

A walk into the luxR regulators of actinobacteria : phylogenomic distribution and functional diversity

LuxR regulators are a widely studied group of bacterial helix-turn-helix (HTH) transcription factors involved in the regulation of many genes coding for important traits at an ecological and medical level. This regulatory family is particularly known by their involvement in quorum-sensing (QS) mechanisms, i.e., in the bacterial ability to communicate through the synthesis and binding of molecular signals. However, these studies have been mainly focused on Gram-negative organisms, and the presence of LuxR regulators in the Gram-positive Actinobacteria phylum is still poorly explored. In this manuscript, the presence of LuxR regulators among Actinobacteria was assayed using a domain-based strategy. A total of 991 proteins having one LuxR domain were identified in 53 genome-sequenced actinobacterial species, of which 59% had an additional domain. In most cases (53%) this domain was REC (receiver domain), suggesting that LuxR regulators in Actinobacteria may either function as single transcription factors or as part of two-component systems. The frequency, distribution and evolutionary stability of each of these sub-families of regulators was analyzed and contextualized regarding the ecological niche occupied by each organism. The results show that the presence of extra-domains in the LuxR-regulators was likely driven by a general need to physically uncouple the signal sensing from the signal transduction. Moreover, the total frequency of LuxR regulators was shown to be dependent on genetic, metabolic and ecological variables. Finally, the functional annotation of the LuxR regulators revealed that the bacterial ecological niche has biased the specialization of these proteins. In the case of pathogens, our results suggest that LuxR regulators can be involved in virulence and are therefore promising targets for future studies in the health-related biotechnology field.Fundação para a Ciência e TecnologiaEuropean Regional Development Fund - COMPETE program and FCT - Fundacão para a Ciência e Tecnologia, with the project FCOMP-01-0124-FEDER-022718 (ref FCT Pest-C/SAU/LA0002/2011). MVM was supported by ‘‘Programa Ciência 2007’’ sponsored by POPH QREN Tipologia 4.2 Promoção do Emprego Cientifico program and co-financed by the European Social Fund and Portuguese national funds from the MCTES. CLS was supported by the FCT fellowship SFRH/BPD/62978/2009

Evaluation of the antioxidant activity of cell extracts from microalgae

A growing market for novel antioxidants obtained from non-expensive sources justifies educated screening of microalgae for their potential antioxidant features. Characterization of the antioxidant profile of 18 species of cyanobacteria (prokaryotic microalgae) and 23 species of (eukaryotic) microalgae is accordingly reported in this paper. The total antioxidant capacity, accounted for by both water- and lipid-soluble antioxidants, was evaluated by the (radical cation) ABTS method. For complementary characterization of cell extracts, a deoxyribose assay was carried out, as well as a bacteriophage P22/Salmonella-mediated approach. The microalga Scenedesmus obliquus strain M2-1 exhibited the highest (p > 0.05) total antioxidant capacity (149 ± 47 AAU) of intracellular extracts. Its scavenger activity correlated well with its protective effects against DNA oxidative damage induced by copper(II)-ascorbic acid; and against decay in bacteriophage infection capacity induced by H2O2. Finally, performance of an Ames test revealed no mutagenic effects of the said extract.info:eu-repo/semantics/publishedVersio

The involvement of sphingolipids in apoptosis induced by acetic acid in yeast

This work was funded by FEDER through the program “Programa Operacional Factores de Competitividade-COMPETE” and by FCT (Fundação para a Ciência e Tecnologia) through the projects Pest-C/BIA/UI4050/2011 and FCOMP-01-0124-FEDER-007047. A.R. was supported by a FCT fellowship (FCOMP-01-0124-FEDER-007047)