Marcos Antonio de Morais

Identification and Characterization of Phenylpyruvate Decarboxylase Genes in Saccharomyces cerevisiae

ABSTRACT Catabolism of amino acids via the Ehrlich pathway involves transamination to the corresponding α-keto acids, followed by decarboxylation to an aldehyde and then reduction to an alcohol. Alternatively, the aldehyde may be oxidized to an acid. This pathway is functional in Saccharomyces cerevisiae, since during growth in glucose-limited chemostat cultures with phenylalanine as the sole nitrogen source, phenylethanol and phenylacetate were produced in quantities that accounted for all of the phenylalanine consumed. Our objective was to identify the structural gene(s) required for the decarboxylation of phenylpyruvate to phenylacetaldehyde, the first specific step in the Ehrlich pathway. S. cerevisiae possesses five candidate genes with sequence similarity to genes encoding thiamine diphosphate-dependent decarboxylases that could encode this activity: YDR380w/ARO10, YDL080C/THI3, PDC1, PDC5, and PDC6. Phenylpyruvate decarboxylase activity was present in cultures grown with phenylalanine as the sole nitrogen source but was absent from ammonia-grown cultures. Furthermore, the transcript level of one candidate gene (ARO10) increased 30-fold when phenylalanine replaced ammonia as the sole nitrogen source. Analyses of phenylalanine catabolite production and phenylpyruvate decarboxylase enzyme assays indicated that ARO10 was sufficient to encode phenylpyruvate decarboxylase activity in the absence of the four other candidate genes. There was also an alternative activity with a higher capacity but lower affinity for phenylpyruvate. The candidate gene THI3 did not itself encode an active phenylpyruvate decarboxylase but was required along with one or more pyruvate decarboxylase genes (PDC1, PDC5, and PDC6) for the alternative activity. The Km and Vmax values of the two activities differed, showing that Aro10p is the physiologically relevant phenylpyruvate decarboxylase in wild-type cells. Modifications to this gene could therefore be important for metabolic engineering of the Ehrlich pathway.

Yeast population dynamics of industrial fuel-ethanol fermentation process assessed by PCR-fingerprinting.

Yeast population used in industrial production of fuel-ethanol may vary according to the plant process condition and to the environmental stresses imposed to yeast cells. Therefore, yeast strains isolated from a particular industrial process may be adapted to such conditions and should be used as starter strain instead of less adapted commercial strains. This work reports the use of PCR-fingerprinting method based on microsatellite primer (GTG)5 to characterize the yeast population dynamics along the fermentation period in six distilleries. The results show that indigenous fermenting strains present in the crude substrate can be more adapted to the industrial process than commercial strains. We also identified new strains that dominate the yeast population and were more present either in molasses or sugar cane fermenting distilleries. Those strains were proposed to be used as starters in those industrial processes. This is the first report on the use of molecular markers to discriminate Saccharomyces cerevisiae strains from fuel-ethanol producing process.

Physiological Characterization of the ARO10-Dependent, Broad-Substrate-Specificity 2-Oxo Acid Decarboxylase Activity of Saccharomyces cerevisiae

ABSTRACT Aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK113-7D were grown with different nitrogen sources. Cultures grown with phenylalanine, leucine, or methionine as a nitrogen source contained high levels of the corresponding fusel alcohols and organic acids, indicating activity of the Ehrlich pathway. Also, fusel alcohols derived from the other two amino acids were detected in the supernatant, suggesting the involvement of a common enzyme activity. Transcript level analysis revealed that among the five thiamine-pyrophospate-dependent decarboxylases (PDC1, PDC5, PDC6, ARO10, and THI3), only ARO10 was transcriptionally up-regulated when phenylalanine, leucine, or methionine was used as a nitrogen source compared to growth on ammonia, proline, and asparagine. Moreover, 2-oxo acid decarboxylase activity measured in cell extract from CEN.PK113-7D grown with phenylalanine, methionine, or leucine displayed similar broad-substrate 2-oxo acid decarboxylase activity. Constitutive expression of ARO10 in ethanol-limited chemostat cultures in a strain lacking the five thiamine-pyrophosphate-dependent decarboxylases, grown with ammonia as a nitrogen source, led to a measurable decarboxylase activity with phenylalanine-, leucine-, and methionine-derived 2-oxo acids. Moreover, even with ammonia as the nitrogen source, these cultures produced significant amounts of the corresponding fusel alcohols. Nonetheless, the constitutive expression of ARO10 in an isogenic wild-type strain grown in a glucose-limited chemostat with ammonia did not lead to any 2-oxo acid decarboxylase activity. Furthermore, even when ARO10 was constitutively expressed, growth with phenylalanine as the nitrogen source led to increased decarboxylase activities in cell extracts. The results reported here indicate the involvement of posttranscriptional regulation and/or a second protein in the ARO10-dependent, broad-substrate-specificity decarboxylase activity.

Isolation by genetic and physiological characteristics of a fuel-ethanol fermentative Saccharomyces cerevisiae strain with potential for genetic manipulation

Fuel ethanol fermentation process is a complex environment with an intensive succession of yeast strains. The population stability depends on the use of a well-adapted strain that can fit to a particular industrial plant. This stability helps to keep high level of ethanol yield and it is absolutely required when intending to use recombinant strains. Yeast strains have been previously isolated from different distilleries in Northeast Brazil and clustered in genetic strains by PCR-fingerprinting. In this report we present the isolation and selection of a novel Saccharomyces cerevisiae strain by its high dominance in the yeast population. The new strain, JP1 strain, presented practically the same fermentative capacity and stress tolerance like the most used commercial strains, with advantages of being highly adapted to different industrial units in Northeast Brazil that used sugar cane juice as substrate. Moreover, it presented higher transformation efficiency that pointed out its potential for genetic manipulations. The importance of this strain selection programme for ethanol production is discussed.

Diversity of lactic acid bacteria of the bioethanol process

BackgroundBacteria may compete with yeast for nutrients during bioethanol production process, potentially causing economic losses. This is the first study aiming at the quantification and identification of Lactic Acid Bacteria (LAB) present in the bioethanol industrial processes in different distilleries of Brazil.ResultsA total of 489 LAB isolates were obtained from four distilleries in 2007 and 2008. The abundance of LAB in the fermentation tanks varied between 6.0 × 105 and 8.9 × 108 CFUs/mL. Crude sugar cane juice contained 7.4 × 107 to 6.0 × 108 LAB CFUs. Most of the LAB isolates belonged to the genus Lactobacillus according to rRNA operon enzyme restriction profiles. A variety of Lactobacillus species occurred throughout the bioethanol process, but the most frequently found species towards the end of the harvest season were L. fermentum and L. vini. The different rep-PCR patterns indicate the co-occurrence of distinct populations of the species L. fermentum and L. vini, suggesting a great intraspecific diversity. Representative isolates of both species had the ability to grow in medium containing up to 10% ethanol, suggesting selection of ethanol tolerant bacteria throughout the process.ConclusionsThis study served as a first survey of the LAB diversity in the bioethanol process in Brazil. The abundance and diversity of LAB suggest that they have a significant impact in the bioethanol process.

Five Colletotrichum species are responsible for mango anthracnose in northeastern Brazil

Colletotrichum species are the most important and widespread form of decay affecting mango fruit worldwide. In this study, Colletotrichum species associated with fruit anthracnose isolated from mango in northeastern Brazil were subject to molecular and morphological analyses. The partial sequences of the glyceraldehyde-3-phosphate dehydrogenase gene of 143 Colletotrichum isolates was amplified, as an initial measure of genetic diversity. A subset of 47 isolates, selected to represent the range of genetic diversity and geographic origin, were further sequenced using the partial actin, β-tubulin, calmodulin, glutamine synthetase genes and rDNA-ITS region. The multilocus sequence analysis, together with a critical examination of the phenotypic characters, revealed four previously described species (Colletotrichum asianum, Colletotrichum fructicola, Colletotrichum tropicale and Colletotrichum karstii) and one new species. The new species is introduced as Colletotrichum dianesei and formally described, illustrated and compared with similar taxa. Only C. asianum and C. karstii have previously been reported from mango, while the other species represent the first report associated with the mango fruits worldwide. All species are reported for the first time associated with the mango fruits in Brazil.

The ability to use nitrate confers advantage to Dekkera bruxellensis over S. cerevisiae and can explain its adaptation to industrial fermentation processes

The yeast Dekkera bruxellensis has been regarded as a contamination problem in industrial ethanol production because it can replace the originally inoculated Saccharomyces cerevisiae strains. The present study deals with the influence of nitrate on the relative competitiveness of D. bruxellensis and S. cerevisiae in sugar cane ethanol fermentations. The industrial strain D. bruxellensis GDB 248 showed higher growth rates than S. cerevisiae JP1 strain in mixed ammonia/nitrate media, and nitrate assimilation genes were only slightly repressed by ammonia. These characteristics rendered D. bruxellensis cells with an ability to overcome S. cerevisiae populations in both synthetic medium and in sugar cane juice. The results were corroborated by data from industrial fermentations that showed a correlation between high nitrate concentrations and high D. bruxellensis cell counts. Moreover, the presence of nitrate increased fermentation efficiency of D. bruxellensis cells in anaerobic conditions, which may explain the maintenance of ethanol production in the presence of D. bruxellensis in industrial processes. The presence of high levels of nitrate in sugar cane juice may be due to its inefficient conversion by plant metabolism in certain soil types and could explain the periodical episodes of D. bruxellensis colonization of Brazilian ethanol plants.

Production of extracellular lipase by the phytopathogenic fungus Fusarium solani FS1

A Brazilian strain of Fusarium solani was tested for extracellular lipase production in peptone-olive oil medium. The fungus produced 10,500 U.l-1 of lipase after 72 hours of cultivation at 25oC in shake-flask at 120 rpm in a medium containing 3% (w/v) peptone plus 0.5% (v/v) olive oil. Glucose (1% w/v) was found to inhibit the inductive effect of olive oil. Peptone concentrations below 3% (w/v) resulted in a reduced lipase production while increased olive oil concentration (above 0.5%) did not further stimulate lipase production. The optimum lipase activity was achieved at pH 8.6 and 30oC and a good enzyme stability (80% activity retention) was observed at pH ranging from 7.6 to 8.6, and the activity rapidly dropped at temperatures above 50oC. Lipase activity was stimulated by the addition of n-hexane to the culture medium supernatants, in contrast to incubation with water-soluble solvents.

Endophytic species of Colletotrichum associated with mango in northeastern Brazil

Endophytic species of Colletotrichum associated with Mangifera indica (mango) are poorly understood. In this study, Colletotrichum species were isolated from mango in Pernambuco State, Brazil. There were significant differences in isolation frequencies of Colletotrichum species among sites and plant tissues. Mature leaf blades were colonized by most Colletotrichum isolates at the majority of sites. Partial sequences of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of 97 Colletotrichum isolates were amplified as an initial measure of genetic diversity. Phylogenetic analysis with a subset of 22 isolates were performed based on a multi-locus dataset (ACT, TUB2, CAL, CHS-1, GAPDH, ITS) followed by Apn2/MAT IGS sequence-analysis for isolates within the C. gloeosporioides species complex. Molecular analysis associated with phenotypic characteristics revealed six previously described species [C. asianum, C. cliviae, C. dianesei (syn. C. melanocaulon), C. fructicola, C. karstii and C. tropicale] and one new species. This new species is introduced as C. endomangiferae. All species isolated were pathogenic on mango fruits but varied in their virulence. There was no distribution pattern of species among sites and plant tissues, although C. asianum was the most prevalent species at all sites and in all plant tissues studied. Five previously reported Colletotrichum species causing anthracnose in mango fruits in northeastern Brazil were also recovered as endophytes.

Physiological and molecular analysis of the stress response of Saccharomyces cerevisiae imposed by strong inorganic acid with implication to industrial fermentations

Aims:  This work aimed to identify the molecular mechanism that allows yeast cells to survive at low pH environments such as those of bioethanol fermentation.