Fernando Araripe Gonçalves Torres

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.

PCR multiplex for detection of Salmonella Enteritidis, Typhi and Typhimurium and occurrence in poultry meat

The occurrence of foodborne diseases is increasing throughout the world. Bacteria of the genus Salmonella are responsible for food poisoning and, in some cases, may be fatal. The aim of this study was to adapt the multiplex PCR technique (mPCR) on the rapid and direct identification of the presence of Salmonella sp. as well as serotypes Enteritidis, Typhi and Typhimurium in poultry carcasses (n=127) and viscera (n=73). The implementation of the standard technique using positive controls was successfully adapted. The results of Salmonella sp. detection in refrigerated viscera showed that the mPCR was able to detect Salmonella genus in 2.74% of these samples. Traditional microbiological analysis also identified the same positive samples for Salmonella sp. but was not able to differentiate the serotype. The serotype Enteritidis was detected by mPCR in 1.37% of the samples. Our conclusion was that the mPCR was able to detect the presence of these bacteria in a short period of time and enabled the identification of serotype Enteritidis in one of the samples found positive for Salmonella sp.

Effect of acetic acid present in bagasse hydrolysate on the activities of xylose reductase and xylitol dehydrogenase in Candida guilliermondii

The first two steps in xylose metabolism are catalyzed by NAD(P)H-dependent xylose reductase (XR) (EC 1.1.1.21) and NAD(P)-dependent xylitol dehydrogenase (XDH) (EC 1.1.1.9), which lead to xylose→xylitol→xylulose conversion. Xylitol has high commercial value, due to its sweetening and anticariogenic properties, as well as several clinical applications. The acid hydrolysis of sugarcane bagasse allows the separation of a xylose-rich hemicellulosic fraction that can be used as a substrate for Candida guilliermondii to produce xylitol. However, the hydrolysate contains acetic acid, an inhibitor of microbial metabolism. In this study, the effect of acetic acid on the activities of XR and XDH and on xylitol formation by C. guilliermondii were studied. For this purpose, fermentations were carried out in bagasse hydrolysate and in synthetic medium. The activities of XR and XDH were higher in the medium containing acetic acid than in control medium. Moreover, none of the fermentative parameters were significantly altered during cell culture. It was concluded that acetic acid does not interfere with xylitol formation since the increase in XR activity is proportional to XDH activity, leading to a greater production of xylitol and its subsequent conversion to xylulose.

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

Enhanced xylose fermentation and ethanol production by engineered Saccharomyces cerevisiae strain

We have recently demonstrated that heterologous expression of a bacterial xylose isomerase gene (xylA) of Burkholderia cenocepacia enabled a laboratorial Saccharomyces cerevisiae strain to ferment xylose anaerobically, without xylitol accumulation. However, the recombinant yeast fermented xylose slowly. In this study, an evolutionary engineering strategy was applied to improve xylose fermentation by the xylA-expressing yeast strain, which involved sequential batch cultivation on xylose. The resulting yeast strain co-fermented glucose and xylose rapidly and almost simultaneously, exhibiting improved ethanol production and productivity. It was also observed that when cells were grown in a medium containing higher glucose concentrations before being transferred to fermentation medium, higher rates of xylose consumption and ethanol production were obtained, demonstrating that xylose utilization was not regulated by catabolic repression. Results obtained by qPCR demonstrate that the efficiency in xylose fermentation showed by the evolved strain is associated, to the increase in the expression of genes HXT2 and TAL1, which code for a low-affinity hexose transporter and transaldolase, respectively. The ethanol productivity obtained after the introduction of only one genetic modification and the submission to a one-stage process of evolutionary engineering was equivalent to those of strains submitted to extensive metabolic and evolutionary engineering, providing solid basis for future applications of this strategy in industrial strains.

Enzyme Surface Glycosylation in the Solid Phase: Improved Activity and Selectivity of Candida Antarctica Lipase B

Tailor‐made oligosaccharides and polymers were investigated for a specific surface glycosylation of Candida antarctica lipase (fraction B) (CAL‐B) already immobilized on octyl‐Sepharose by interfacial activation. The chemical modification was performed in the N‐terminal amino acid enzyme residue by using low oxidized aldehyde–dextran polymers through a reductive amination. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) indicated that polymer/enzyme conjugates were obtained in all cases. Circular dichroism experiments revealed interesting conformational changes in secondary and tertiary structures of the protein after modification. The formed immobilized glycosylated lipase biocatalysts were more stable, active, and selective toward different substrates than unmodified CAL‐B. These immobilized conjugates were compared with a genetically glycosylated version of CAL‐B expressed in Pichia pastoris immobilized in the same way. Enzyme thermostability was improved after chemical modification with Dextran‐1500 and also by the genetic glycosylation, retaining 90–96 % activity after 24 h at 55 °C. The catalytic activity of CAL‐B was improved by the incorporation of dextran polymers (Mw=1500 or 6000) more than twofold in the hydrolysis of p‐nitrophenylbutyrate and more than threefold in the hydrolysis of methyl mandelate at pH 7. However, the activity of the genetically glycosylated CAL‐B was threefold lower in the hydrolysis of both substrates. The enantioselectivity of CAL‐B increased for all formed bioconjugates, with the Dextran‐1500–CAL‐B conjugate being the most selective in the hydrolysis of racemic methyl mandelate (up to 88.1 % ee at pH 5). This glycosylated CAL‐B also demonstrated the highest synthetic activity in the transesterification of methyl butyrate with glycerol, with 80 % yield of monoglyceryl ester at 100 % conversion compared to 57 % yield obtained with unmodified CAL‐B or other polymer–lipase conjugates.

Functional expression of Burkholderia cenocepacia xylose isomerase in yeast increases ethanol production from a glucose–xylose blend

This study presents results regarding the successful cloning of the bacterial xylose isomerase gene (xylA) of Burkholderia cenocepacia and its functional expression in Saccharomyces cerevisiae. The recombinant yeast showed to be competent to efficiently produce ethanol from both glucose and xylose, which are the main sugars in lignocellulosic hydrolysates. The heterologous expression of the gene xylA enabled a laboratorial yeast strain to ferment xylose anaerobically, improving ethanol production from a fermentation medium containing a glucose-xylose blend similar to that found in sugar cane bagasse hydrolysates. The insertion of xylA caused a 5-fold increase in xylose consumption, and over a 1.5-fold increase in ethanol production and yield, in comparison to that showed by the WT strain, in 24h fermentations, where it was not detected accumulation of xylitol. These findings are encouraging for further studies concerning the expression of B. cenocepacia xylA in an industrial yeast strain.