Lidia Maria Pepe de Moraes

Bioethanol from lignocelluloses: Status and perspectives in Brazil.

The National Alcohol Program--PróAlcool, created by the government of Brazil in 1975 resulted less dependency on fossil fuels. The addition of 25% ethanol to gasoline reduced the import of 550 million barrels oil and also reduced the emission CO(2) by 110 million tons. Today, 44% of the Brazilian energy matrix is renewable and 13.5% is derived from sugarcane. Brazil has a land area of 851 million hectares, of which 54% are preserved, including the Amazon forest (350 million hectares). From the land available for agriculture (340 million hectares), only 0.9% is occupied by sugarcane as energy crop, showing a great expansion potential. Studies have shown that in the coming years, ethanol yield per hectare of sugarcane, which presently is 6000 L/ha, could reach 10,000 L/ha, if 50% of the produced bagasse would be converted to ethanol. This article describes the efforts of different Brazilian institutions and research groups on second generation bioethanol production, especially from sugarcane bagasse.

Transcriptome characterization of the dimorphic and pathogenic fungus Paracoccidioides brasiliensis by EST analysis

Paracoccidioides brasiliensis is a pathogenic fungus that undergoes a temperature‐dependent cell morphology change from mycelium (22° C) to yeast (36° C). It is assumed that this morphological transition correlates with the infection of the human host. Our goal was to identify genes expressed in the mycelium (M) and yeast (Y) forms by EST sequencing in order to generate a partial map of the fungus transcriptome. Individual EST sequences were clustered by the CAP3 program and annotated using Blastx similarity analysis and InterPro Scan. Three different databases, GenBank nr, COG (clusters of orthologous groups) and GO (gene ontology) were used for annotation. A total of 3938 (Y = 1654 and M = 2274) ESTs were sequenced and clustered into 597 contigs and 1563 singlets, making up a total of 2160 genes, which possibly represent one‐quarter of the complete gene repertoire in P. brasiliensis. From this total, 1040 were successfully annotated and 894 could be classified in 18 functional COG categories as follows: cellular metabolism (44%); information storage and processing (25%); cellular processes—cell division, posttranslational modifications, among others (19%); and genes of unknown functions (12%). Computer analysis enabled us to identify some genes potentially involved in the dimorphic transition and drug resistance. Furthermore, computer subtraction analysis revealed several genes possibly expressed in stage‐specific forms of P. brasiliensis. Further analysis of these genes may provide new insights into the pathology and differentiation of P. brasiliensis. All EST sequences have been deposited in GenBank under Accession Nos CA580326–CA584263. Copyright

Biochemical characterization of α-amylase from the yeast Cryptococcus flavus

During our screening of amylolytic microorganisms from Brazilian fruits, we isolated a yeast strain classified as Cryptococcus flavus. When grown on starch-containing medium this strain exhibited the highest amylase production after 24 h of cultivation. The extracellular amylase from C. flavus was purified from the culture broth by a single step using chromatography on a Sephacryl S-100 column. The enzyme was purified 16.14-fold with a yield of 50.21% of the total activity. The purified enzyme was a glycoprotein with an apparent molecular mass of 75 and 84.5 kDa as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and gel filtration, respectively. The enzyme lost approximately 50% of the molecular mass after treatment with glycosidases. The major end products of starch, amylose, amylopectin, pullulan and glycogen were maltose and maltotriose. The Km value for the pure enzyme was 0.056 mg ml−1 with soluble starch as the substrate. Enzyme activity was optimal at pH 5.5 and 50°C. The enzyme retained 90% of the activity after incubation at 50°C for 60 min and was inhibited by Cu2+, Fe2+ and Hg2+.

Xylose Fermentation by Saccharomyces cerevisiae: Challenges and Prospects

Many years have passed since the first genetically modified Saccharomyces cerevisiae strains capable of fermenting xylose were obtained with the promise of an environmentally sustainable solution for the conversion of the abundant lignocellulosic biomass to ethanol. Several challenges emerged from these first experiences, most of them related to solving redox imbalances, discovering new pathways for xylose utilization, modulation of the expression of genes of the non-oxidative pentose phosphate pathway, and reduction of xylitol formation. Strategies on evolutionary engineering were used to improve fermentation kinetics, but the resulting strains were still far from industrial application. Lignocellulosic hydrolysates proved to have different inhibitors derived from lignin and sugar degradation, along with significant amounts of acetic acid, intrinsically related with biomass deconstruction. This, associated with pH, temperature, high ethanol, and other stress fluctuations presented on large scale fermentations led the search for yeasts with more robust backgrounds, like industrial strains, as engineering targets. Some promising yeasts were obtained both from studies of stress tolerance genes and adaptation on hydrolysates. Since fermentation times on mixed-substrate hydrolysates were still not cost-effective, the more selective search for new or engineered sugar transporters for xylose are still the focus of many recent studies. These challenges, as well as under-appreciated process strategies, will be discussed in this review.

Deciphering the magainin resistance process of Escherichia coli strains in light of the cytosolic proteome.

ABSTRACT Antimicrobial peptides (AMPs) are effective antibiotic agents commonly found in plants, animals, and microorganisms, and they have been suggested as the future of antimicrobial chemotherapies. It is vital to understand the molecular details that define the mechanism of action of resistance to AMPs for a rational planning of the next antibiotic generation and also to shed some light on the complex AMP mechanism of action. Here, the antibiotic resistance of Escherichia coli ATCC 8739 to magainin I was evaluated in the cytosolic subproteome. Magainin-resistant strains were selected after 10 subsequent spreads at subinhibitory concentrations of magainin I (37.5 mg · liter−1), and their cytosolic proteomes were further compared to those of magainin-susceptible strains through two-dimensional electrophoresis analysis. As a result, 41 differentially expressed proteins were detected by in silico analysis and further identified by tandem mass spectrometry de novo sequencing. Functional categorization indicated an intense metabolic response mainly in energy and nitrogen uptake, stress response, amino acid conversion, and cell wall thickness. Indeed, data reported here show that resistance to cationic antimicrobial peptides possesses a greater molecular complexity than previously supposed, resulting in cell commitment to several metabolic pathways.

Molecular epidemiology and antimicrobial susceptibility of Enterococci recovered from Brazilian intensive care units

We studied the antimicrobial resistance and the molecular epidemiology of 99 enterococcal surveillance isolates from two hospitals of Brasilia, Brazil. Conventional biochemical tests were used to identify the enterococcal species and the disk diffusion method was used to determine their resistance profiles. Enterococcus faecalis (76%) and E. faecium (9%) were the most prevalent species. No enterococci showed the vanA or vanB vancomycin resistance phenotypes or genotypes. Only the intrinsically resistant species E. gallinarum (n=2) and E. casseliflavus (n=3) harbored the vancomycin-resistance genes vanC1 and vanC2/3, respectively. We found E. faecalis isolates with high-level resistance to gentamicin (22%) and streptomycin (8%) and both E. faecalis and E. faecium isolates with resistance to more than two antimicrobials (84% and 67%, respectively). Nine E. faecalis isolates (12%) were resistant to ampicillin; the minimal inhibitory concentration (MIC) values were 16 microg/mL (n=6) and 32 microg/mL (n=3). Among these ampicillin-resistant E. faecalis, seven were also resistant to gentamicin, ciprofloxacin, rifampin, penicillin, chloramphenicol, tetracycline and erythromycin. Pulsed-field gel electrophoresis classified those isolates in three different genotypes, suggesting dissemination of genetically related ampicillin-resistant E. faecalis strains among different patients.

Molecular characterization of the 3-phosphoglycerate kinase gene (PGK1) from the methylotrophic yeast Pichia pastoris

We report the cloning of the 3‐phosphoglycerate kinase gene (PGK1) from the methylotrophic yeast Pichia pastoris by a PCR approach. The coding sequence of the PGK1 gene comprises 1251 bp with the potential to encode a polypeptide of 416 amino acid residues, which shows high identity to homologous proteins from other yeasts. The promoter region of this gene (PPGK1) contains regulatory cis‐elements found in other PGK1 genes, such as TATA box, CT‐rich block and a heat shock element. In the 3′ downstream region we identified a tripartite element 5′‐TAG–TAGT–TTT‐3′, which is supposed to be important for transcription termination. As in other yeasts, the PGK1 gene from P. pastoris is present as a single‐copy gene. Northern blot analysis revealed that the gene is transcribed as a 1.5 kb mRNA; when cells are grown on glucose the levels of this mRNA are increased two‐fold in comparison to cells grown on glycerol. The transcriptional regulation of this gene by the carbon source was further confirmed when the α‐amylase gene from Bacillus subtilis was placed under the control of PPGK1: higher levels of expression were obtained when cells were grown on glucose as compared to glycerol and methanol. Preliminary results related to the strength of PPGK1 show that it represents a potential alternative to constitutive heterologous expression in P. pastoris. The sequence of the gene has been deposited in GenBank under Accession No. AY288296. Copyright

Purification and some properties of an α-amylase glucoamylase fusion protein from Saccharomyces cerevisiae

A fusion gene containing the Bacillus subtilis α-amylase gene and Aspergillus awamori glucoamylase cDNA was expressed in Saccharomyces cerevisiae. The resulting bifunctional fusion protein having both α-amylase and glucoamylase activities secreted into the culture medium was purified to apparent homogeneity by affinity chromatography and gel filtration on Sephadex G-100. The enzyme had an apparent molecular mass of 150 kDa and showed an optimum pH and temperature of 6.0 and 60 °C, respectively. The main hydrolysis products from soluble starch were glucose and maltose.

Biochemical and Structural Characterization of Amy1: An Alpha-Amylase from Cryptococcus flavus Expressed in Saccharomyces cerevisiae

An extracellular alpha-amylase (Amy1) whose gene from Cryptococcus flavus was previously expressed in Saccharomyces cerevisiae was purified to homogeneity (67 kDa) by ion-exchange and molecular exclusion chromatography. The enzyme was activated by NH(4) (+) and inhibited by Cu(+2) and Hg(+2). Significant biochemical and structural discrepancies between wild-type and recombinant α-amylase with respect to K(m) values, enzyme specificity, and secondary structure content were found. Far-UV CD spectra analysis at pH 7.0 revealed the high thermal stability of both proteins and the difference in folding pattern of Amy1 compared with wild-type amylase from C. flavus, which reflected in decrease (10-fold) of enzymatic activity of recombinant protein. Despite the differences, the highest activity of Amy1 towards soluble starch, amylopectin, and amylase, in contrast with the lowest activity of Amy1(w), points to this protein as being of paramount biotechnological importance with many applications ranging from food industry to the production of biofuels.

Genetic characterization and construction of an auxotrophic strain of Saccharomyces cerevisiae JP1, a Brazilian industrial yeast strain for bioethanol production

Used for millennia to produce beverages and food, Saccharomyces cerevisiae also became a workhorse in the production of biofuels, most notably bioethanol. Yeast strains have acquired distinct characteristics that are the result of evolutionary adaptation to the stresses of industrial ethanol production. JP1 is a dominant industrial S. cerevisiae strain isolated from a sugarcane mill and is becoming increasingly popular for bioethanol production in Brazil. In this work, we carried out the genetic characterization of this strain and developed a set of tools to permit its genetic manipulation. Using flow cytometry, mating type, and sporulation analysis, we verified that JP1 is diploid and homothallic. Vectors with dominant selective markers for G418, hygromycin B, zeocin, and ρ-fluoro-dl-phenylalanine were used to successfully transform JP1 cells. Also, an auxotrophic ura3 mutant strain of JP1 was created by gene disruption using integration cassettes with dominant markers flanked by loxP sites. Marker excision was accomplished by the Cre/loxP system. The resulting auxotrophic strain was successfully transformed with an episomal vector that allowed green fluorescent protein expression.