João R. M. Almeida | EMBRAPA - Empresa Brasileira de Pesquisa Agropecuária (original) (raw)

Papers by João R. M. Almeida

Archives of Biochemistry and Biophysics, 2007

The biosynthesis of flavonoids and proanthocyanidins was studied in cultivated strawberry (Fragar... more The biosynthesis of flavonoids and proanthocyanidins was studied in cultivated strawberry (Fragaria · ananassa) by combining biochemical and molecular approaches. Chemical analyses showed that ripe strawberries accumulate high amounts of pelargonidin-derived anthocyanins, and a larger pool of 3 0 ,4 0 -hydroxylated proanthocyanidins. Activities and properties of major recombinant enzymes were demonstrated by means of in vitro assays, with special emphasis on specificity for the biologically relevant 4 0 -and 3 0 ,4 0 -hydroxylated compounds. Only leucoanthocyanidin reductase showed a strict specificity for the 3 0 ,4 0 -hydroxylated leucocyanidin, while other enzymes accepted either hydroxylated substrate with different relative activity rates. The structure of late flavonoid pathway genes, leading to the synthesis of major compounds in ripe fruits, was elucidated. Complex developmental and spatial expression patterns were shown for phenylpropanoid and flavonoid genes in fruits throughout ripening as well as in leaves, petals and roots. Presented results elucidate key steps in the biosynthesis of strawberry flavonoid end products.

Biocontrol, 2009

Understanding food web interactions in native or agricultural ecosystems is an important step tow... more Understanding food web interactions in native or agricultural ecosystems is an important step towards establishing sustainable pest management strategies. While the role of generalist predators as biological control agents is increasingly appreciated, the study of trophic interactions between individual predator species and their prey provides practical difficulties. Recently, different approaches have been suggested to determine prey items from predator guts using molecular methods. Macrolophus caliginosus is a generalist predator active in herbaceous agro-ecosystems. We developed a system to identify the DNA of its prey after ingestion, using Myzus persicae as a model. Esterase (MpEST) and cytochrome oxidase I (MpCOI) genes were targeted in the aphid, while M. caliginosus COI gene was used as control for predator DNA. Real time PCR proved to be specific and sensitive enough to detect prey DNA upon ingestion after feeding experiments. The system provided a linear amplification response with only 10 fg of prey genomic DNA as template. The detection system of MpCOI gene was more sensitive than MpEST, while the detection period was similar for both genes. Possibilities for using the system in ecological and biosafety studies with regard to sustainable pest management are discussed.

Biotechnology and Bioengineering, 2008

The development of tolerant microorganisms is needed for the efficient fermentation of inhibitory... more The development of tolerant microorganisms is needed for the efficient fermentation of inhibitory lignocellulose hydrolysates. In the current work, the fermentation performance of six selected strains of Saccharomyces cerevisiae in dilute-acid spruce hydrolysate was compared using two different modes of fermentation; either single pulse addition of hydrolysate to exponentially growing cells or continuous feeding of the same amount of hydrolysate in a controlled fed-batch fermentation was made. All strains performed better in fed-batch mode than when all hydrolysate was added at once. However, the difference between strain performances varied significantly in the two fermentation modes. Large differences were observed between strains during the fed-batch experiments in the in vitro ability to reduce the furan compounds furfural and 5-hydroxymethyl furfural (HMF). A common feature among the strains was the induction of NADPH-coupled reduction of furfural and HMF, with the exception of strain CBS 8066. This strain also performed relatively poorly in both batch and fed-batch fermentations. Strain TMB3000-previously isolated from spent sulphite liquor fermentation-was by far the most efficient strain with respect to specific fermentation rate in both pulse addition and fed-batch mode. This strain was the only strain showing a significant constitutive NADH-coupled in vitro reduction of HMF. The ability to induce NADPH-coupled reduction together with the level of the apparently constitutive NADHcoupled reduction appeared to be key factors for selecting a suitable strain for fed-batch conversion of lignocellulose hydrolysate.

Applied Microbiology and Biotechnology, 2008

Saccharomyces cerevisiae alcohol dehydrogenases responsible for NADH-, and NADPH-specific reducti... more Saccharomyces cerevisiae alcohol dehydrogenases responsible for NADH-, and NADPH-specific reduction of the furaldehydes 5-hydroxymethyl-furfural (HMF) and furfural have previously been identified. In the present study, strains overexpressing the corresponding genes (mut-ADH1 and ADH6), together with a control strain, were compared in defined medium for anaerobic fermentation of glucose in the presence and absence of HMF. All strains showed a similar fermentation pattern in the absence of HMF. In the presence of HMF, the strain overexpressing ADH6 showed the highest HMF reduction rate and the highest specific ethanol productivity, followed by the strain overexpressing mut-ADH1. This correlated with in vitro HMF reduction capacity observed in the ADH6 overexpressing strain. Acetate and glycerol yields per biomass increased considerably in the ADH6 strain. In the other two strains, only the overall acetate yield per biomass was affected. When compared in batch fermentation of spruce hydrolysate, strains overexpressing ADH6 and mut-ADH1 had five times higher HMF uptake rate than the control strain and improved specific ethanol productivity. Overall, our results demonstrate that (1) the cofactor usage in the HMF reduction affects the product distribution, and (2) increased HMF reduction activity results in increased specific ethanol productivity in defined mineral medium and in spruce hydrolysate.

Applied Microbiology and Biotechnology, 2009

Industrial Saccharomyces cerevisiae strains able to utilize xylose have been constructed by overe... more Industrial Saccharomyces cerevisiae strains able to utilize xylose have been constructed by overexpression of XYL1 and XYL2 genes encoding the NADPH-preferring xylose reductase (XR) and the NAD+-dependent xylitol dehydrogenase (XDH), respectively, from Pichia stipitis. However, the use of different co-factors by XR and XDH leads to NAD+ deficiency followed by xylitol excretion and reduced product yield. The furaldehydes 5-hydroxymethyl-furfural (HMF) and furfural inhibit yeast metabolism, prolong the lag phase, and reduce the ethanol productivity. Recently, genes encoding furaldehyde reductases were identified and their overexpression was shown to improve S. cerevisiae growth and fermentation rate in HMF containing media and in lignocellulosic hydrolysate. In the current study, we constructed a xylose-consuming S. cerevisiae strain using the XR/XDH pathway from P. stipitis. Then, the genes encoding the NADH- and the NADPH-dependent HMF reductases, ADH1-S110P-Y295C and ADH6, respectively, were individually overexpressed in this background. The performance of these strains, which differed in their co-factor usage for HMF reduction, was evaluated under anaerobic conditions in batch fermentation in absence or in presence of HMF. In anaerobic continuous culture, carbon fluxes were obtained for simultaneous xylose consumption and HMF reduction. Our results show that the co-factor used for HMF reduction primarily influenced formation of products other than ethanol, and that NADH-dependent HMF reduction influenced product formation more than NADPH-dependent HMF reduction. In particular, NADH-dependent HMF reduction contributed to carbon conservation so that biomass was produced at the expense of xylitol and glycerol formation.

Applied Microbiology and Biotechnology, 2009

There is a growing awareness that lignocellulose will be a major raw material for production of b... more There is a growing awareness that lignocellulose will be a major raw material for production of both fuel and chemicals in the coming decades—most likely through various fermentation routes. Considerable attention has been given to the problem of finding efficient means of separating the major constituents in lignocellulose (i.e., lignin, hemicellulose, and cellulose) and to efficiently hydrolyze the carbohydrate parts into sugars. In these processes, by-products will inevitably form to some extent, and these will have to be dealt with in the ensuing microbial processes. One group of compounds in this category is the furaldehydes. 2-Furaldehyde (furfural) and substituted 2-furaldehydes—most importantly 5-hydroxymethyl-2-furaldehyde—are the dominant inhibitory compounds found in lignocellulosic hydrolyzates. The furaldehydes are known to have biological effects and act as inhibitors in fermentation processes. The effects of these compounds will therefore have to be considered in the design of biotechnological processes using lignocellulose. In this short review, we take a look at known metabolic effects, as well as strategies to overcome problems in biotechnological applications caused by furaldehydes.

Bioresource Technology, 2009

A microplate screening method was used to assess anaerobic growth of 12 Saccharomyces cerevisiae ... more A microplate screening method was used to assess anaerobic growth of 12 Saccharomyces cerevisiae strains in barley straw, spruce and wheat straw hydrolysate. The assay demonstrated significant differences in inhibitor tolerance among the strains. In addition, growth inhibition by the three hydrolysates differed so that wheat hydrolysate supported growth up to 70%, while barley hydrolysate only supported growth up to 50%, with dilute-acid spruce hydrolysate taking an intermediate position.

Yeast, 2006

The fermentation of lignocellulose hydrolysates by Saccharomyces cerevisiae for fuel ethanol prod... more The fermentation of lignocellulose hydrolysates by Saccharomyces cerevisiae for fuel ethanol production is inhibited by 5-hydroxymethyl furfural (HMF), a furan derivative which is formed during the hydrolysis of lignocellulosic materials. The inhibition can be avoided if the yeast strain used in the fermentation has the ability to reduce HMF to 5-hydroxymethylfurfuryl alcohol. To enable the identification of enzyme(s) responsible for HMF conversion in S. cerevisiae, microarray analyses of two strains with different abilities to convert HMF were performed. Based on the expression data, a subset of 15 reductase genes was chosen to be further examined using an overexpression strain collection. Three candidate genes were cloned from two different strains, TMB3000 and the laboratory strain CEN.PK 113-5D, and overexpressed using a strong promoter in the strain CEN.PK 113-5D. Strains overexpressing ADH6 had increased HMF conversion activity in cell-free crude extracts with both NADPH and NADH as co-factors. In vitro activities were recorded of 8 mU/mg with NADH as co-factor and as high as 1200 mU/mg for the NADPH-coupled reduction. Yeast strains overexpressing ADH6 also had a substantially higher in vivo conversion rate of HMF in both aerobic and anaerobic cultures, showing that the overexpression indeed conveyed the desired increased reduction capacity. Copyright © 2006 John Wiley & Sons, Ltd.

Yeast, 2008

We report on the identification and characterization of a mutated alcohol dehydrogenase 1 from th... more We report on the identification and characterization of a mutated alcohol dehydrogenase 1 from the industrial Saccharomyces cerevisiae strain TMB3000 that mediates the NADH-dependent reduction of 5-hydroxymethylfurfural (HMF) to 2,5-bis-hydroxymethylfuran. The co-factor preference distinguished this alcohol dehydrogenase from the previously reported NADPH-dependent S. cerevisiae HMF alcohol dehydrogenase Adh6. The amino acid sequence revealed three novel mutations (S109P, L116S and Y294C) that were all predicted at the vicinity of the substrate binding site, which could explain the unusual substrate specificity. Increased biomass production and HMF conversion rate were achieved in a CEN.PK S. cerevisiae strain overexpressing the mutated ADH1 gene. Copyright © 2008 John Wiley & Sons, Ltd.

Analytical Chemistry, 2009

In this work, mediated amperometry was used to evaluate whether differences in intracellular nico... more In this work, mediated amperometry was used to evaluate whether differences in intracellular nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) level could be observed between a genetically modified Saccharomyces cerevisiae strain, engineered for NAD-PH dependent 5-hydroxymethyl-2-furaldehyde (HMF) reduction, and its control strain. Cells overexpressing the alcohol dehydrogenase 6 gene (ADH6 strain) and cells carrying the corresponding control plasmid (control strain) were each immobilized on Au-microelectrodes. The real-time dynamics of NAD(P)H availability in the two strains, preincubated with HMF, was probed using the menadione-ferricyanide double mediator system. A lower intracellular NADPH level as the consequence of more effective HMF reduction was observed for the ADH6 strain both with and without added glucose, which increases the overall cellular NADPH level. The mediated amperometric signal during real-time monitoring of the concentration dependent HMF reduction in living cells could be translated into the cellular enzyme kinetic parameters: K M,cell app , V MAX , k cat,cell , and k cat,cell /K M,cell app . The results indicated that the overexpression of the ADH6 gene gave a 68% decrease in K M,cell app and 42% increase in V MAX , resulting in a 4-fold increase in k cat,cell /K M,cell app . These results demonstrate that the mediated amperometric method is useful for monitoring the short-term dynamics of NAD(P)H variations and determining cellular enzyme kinetic parameters in S. cerevisiae cells.

Archives of Biochemistry and Biophysics, 2007

The biosynthesis of flavonoids and proanthocyanidins was studied in cultivated strawberry (Fragar... more The biosynthesis of flavonoids and proanthocyanidins was studied in cultivated strawberry (Fragaria · ananassa) by combining biochemical and molecular approaches. Chemical analyses showed that ripe strawberries accumulate high amounts of pelargonidin-derived anthocyanins, and a larger pool of 3 0 ,4 0 -hydroxylated proanthocyanidins. Activities and properties of major recombinant enzymes were demonstrated by means of in vitro assays, with special emphasis on specificity for the biologically relevant 4 0 -and 3 0 ,4 0 -hydroxylated compounds. Only leucoanthocyanidin reductase showed a strict specificity for the 3 0 ,4 0 -hydroxylated leucocyanidin, while other enzymes accepted either hydroxylated substrate with different relative activity rates. The structure of late flavonoid pathway genes, leading to the synthesis of major compounds in ripe fruits, was elucidated. Complex developmental and spatial expression patterns were shown for phenylpropanoid and flavonoid genes in fruits throughout ripening as well as in leaves, petals and roots. Presented results elucidate key steps in the biosynthesis of strawberry flavonoid end products.

Biocontrol, 2009

Understanding food web interactions in native or agricultural ecosystems is an important step tow... more Understanding food web interactions in native or agricultural ecosystems is an important step towards establishing sustainable pest management strategies. While the role of generalist predators as biological control agents is increasingly appreciated, the study of trophic interactions between individual predator species and their prey provides practical difficulties. Recently, different approaches have been suggested to determine prey items from predator guts using molecular methods. Macrolophus caliginosus is a generalist predator active in herbaceous agro-ecosystems. We developed a system to identify the DNA of its prey after ingestion, using Myzus persicae as a model. Esterase (MpEST) and cytochrome oxidase I (MpCOI) genes were targeted in the aphid, while M. caliginosus COI gene was used as control for predator DNA. Real time PCR proved to be specific and sensitive enough to detect prey DNA upon ingestion after feeding experiments. The system provided a linear amplification response with only 10 fg of prey genomic DNA as template. The detection system of MpCOI gene was more sensitive than MpEST, while the detection period was similar for both genes. Possibilities for using the system in ecological and biosafety studies with regard to sustainable pest management are discussed.

Biotechnology and Bioengineering, 2008

The development of tolerant microorganisms is needed for the efficient fermentation of inhibitory... more The development of tolerant microorganisms is needed for the efficient fermentation of inhibitory lignocellulose hydrolysates. In the current work, the fermentation performance of six selected strains of Saccharomyces cerevisiae in dilute-acid spruce hydrolysate was compared using two different modes of fermentation; either single pulse addition of hydrolysate to exponentially growing cells or continuous feeding of the same amount of hydrolysate in a controlled fed-batch fermentation was made. All strains performed better in fed-batch mode than when all hydrolysate was added at once. However, the difference between strain performances varied significantly in the two fermentation modes. Large differences were observed between strains during the fed-batch experiments in the in vitro ability to reduce the furan compounds furfural and 5-hydroxymethyl furfural (HMF). A common feature among the strains was the induction of NADPH-coupled reduction of furfural and HMF, with the exception of strain CBS 8066. This strain also performed relatively poorly in both batch and fed-batch fermentations. Strain TMB3000-previously isolated from spent sulphite liquor fermentation-was by far the most efficient strain with respect to specific fermentation rate in both pulse addition and fed-batch mode. This strain was the only strain showing a significant constitutive NADH-coupled in vitro reduction of HMF. The ability to induce NADPH-coupled reduction together with the level of the apparently constitutive NADHcoupled reduction appeared to be key factors for selecting a suitable strain for fed-batch conversion of lignocellulose hydrolysate.

Applied Microbiology and Biotechnology, 2008

Saccharomyces cerevisiae alcohol dehydrogenases responsible for NADH-, and NADPH-specific reducti... more Saccharomyces cerevisiae alcohol dehydrogenases responsible for NADH-, and NADPH-specific reduction of the furaldehydes 5-hydroxymethyl-furfural (HMF) and furfural have previously been identified. In the present study, strains overexpressing the corresponding genes (mut-ADH1 and ADH6), together with a control strain, were compared in defined medium for anaerobic fermentation of glucose in the presence and absence of HMF. All strains showed a similar fermentation pattern in the absence of HMF. In the presence of HMF, the strain overexpressing ADH6 showed the highest HMF reduction rate and the highest specific ethanol productivity, followed by the strain overexpressing mut-ADH1. This correlated with in vitro HMF reduction capacity observed in the ADH6 overexpressing strain. Acetate and glycerol yields per biomass increased considerably in the ADH6 strain. In the other two strains, only the overall acetate yield per biomass was affected. When compared in batch fermentation of spruce hydrolysate, strains overexpressing ADH6 and mut-ADH1 had five times higher HMF uptake rate than the control strain and improved specific ethanol productivity. Overall, our results demonstrate that (1) the cofactor usage in the HMF reduction affects the product distribution, and (2) increased HMF reduction activity results in increased specific ethanol productivity in defined mineral medium and in spruce hydrolysate.

Applied Microbiology and Biotechnology, 2009

Industrial Saccharomyces cerevisiae strains able to utilize xylose have been constructed by overe... more Industrial Saccharomyces cerevisiae strains able to utilize xylose have been constructed by overexpression of XYL1 and XYL2 genes encoding the NADPH-preferring xylose reductase (XR) and the NAD+-dependent xylitol dehydrogenase (XDH), respectively, from Pichia stipitis. However, the use of different co-factors by XR and XDH leads to NAD+ deficiency followed by xylitol excretion and reduced product yield. The furaldehydes 5-hydroxymethyl-furfural (HMF) and furfural inhibit yeast metabolism, prolong the lag phase, and reduce the ethanol productivity. Recently, genes encoding furaldehyde reductases were identified and their overexpression was shown to improve S. cerevisiae growth and fermentation rate in HMF containing media and in lignocellulosic hydrolysate. In the current study, we constructed a xylose-consuming S. cerevisiae strain using the XR/XDH pathway from P. stipitis. Then, the genes encoding the NADH- and the NADPH-dependent HMF reductases, ADH1-S110P-Y295C and ADH6, respectively, were individually overexpressed in this background. The performance of these strains, which differed in their co-factor usage for HMF reduction, was evaluated under anaerobic conditions in batch fermentation in absence or in presence of HMF. In anaerobic continuous culture, carbon fluxes were obtained for simultaneous xylose consumption and HMF reduction. Our results show that the co-factor used for HMF reduction primarily influenced formation of products other than ethanol, and that NADH-dependent HMF reduction influenced product formation more than NADPH-dependent HMF reduction. In particular, NADH-dependent HMF reduction contributed to carbon conservation so that biomass was produced at the expense of xylitol and glycerol formation.

Applied Microbiology and Biotechnology, 2009

There is a growing awareness that lignocellulose will be a major raw material for production of b... more There is a growing awareness that lignocellulose will be a major raw material for production of both fuel and chemicals in the coming decades—most likely through various fermentation routes. Considerable attention has been given to the problem of finding efficient means of separating the major constituents in lignocellulose (i.e., lignin, hemicellulose, and cellulose) and to efficiently hydrolyze the carbohydrate parts into sugars. In these processes, by-products will inevitably form to some extent, and these will have to be dealt with in the ensuing microbial processes. One group of compounds in this category is the furaldehydes. 2-Furaldehyde (furfural) and substituted 2-furaldehydes—most importantly 5-hydroxymethyl-2-furaldehyde—are the dominant inhibitory compounds found in lignocellulosic hydrolyzates. The furaldehydes are known to have biological effects and act as inhibitors in fermentation processes. The effects of these compounds will therefore have to be considered in the design of biotechnological processes using lignocellulose. In this short review, we take a look at known metabolic effects, as well as strategies to overcome problems in biotechnological applications caused by furaldehydes.

Bioresource Technology, 2009

A microplate screening method was used to assess anaerobic growth of 12 Saccharomyces cerevisiae ... more A microplate screening method was used to assess anaerobic growth of 12 Saccharomyces cerevisiae strains in barley straw, spruce and wheat straw hydrolysate. The assay demonstrated significant differences in inhibitor tolerance among the strains. In addition, growth inhibition by the three hydrolysates differed so that wheat hydrolysate supported growth up to 70%, while barley hydrolysate only supported growth up to 50%, with dilute-acid spruce hydrolysate taking an intermediate position.

Yeast, 2006

The fermentation of lignocellulose hydrolysates by Saccharomyces cerevisiae for fuel ethanol prod... more The fermentation of lignocellulose hydrolysates by Saccharomyces cerevisiae for fuel ethanol production is inhibited by 5-hydroxymethyl furfural (HMF), a furan derivative which is formed during the hydrolysis of lignocellulosic materials. The inhibition can be avoided if the yeast strain used in the fermentation has the ability to reduce HMF to 5-hydroxymethylfurfuryl alcohol. To enable the identification of enzyme(s) responsible for HMF conversion in S. cerevisiae, microarray analyses of two strains with different abilities to convert HMF were performed. Based on the expression data, a subset of 15 reductase genes was chosen to be further examined using an overexpression strain collection. Three candidate genes were cloned from two different strains, TMB3000 and the laboratory strain CEN.PK 113-5D, and overexpressed using a strong promoter in the strain CEN.PK 113-5D. Strains overexpressing ADH6 had increased HMF conversion activity in cell-free crude extracts with both NADPH and NADH as co-factors. In vitro activities were recorded of 8 mU/mg with NADH as co-factor and as high as 1200 mU/mg for the NADPH-coupled reduction. Yeast strains overexpressing ADH6 also had a substantially higher in vivo conversion rate of HMF in both aerobic and anaerobic cultures, showing that the overexpression indeed conveyed the desired increased reduction capacity. Copyright © 2006 John Wiley & Sons, Ltd.

Yeast, 2008

We report on the identification and characterization of a mutated alcohol dehydrogenase 1 from th... more We report on the identification and characterization of a mutated alcohol dehydrogenase 1 from the industrial Saccharomyces cerevisiae strain TMB3000 that mediates the NADH-dependent reduction of 5-hydroxymethylfurfural (HMF) to 2,5-bis-hydroxymethylfuran. The co-factor preference distinguished this alcohol dehydrogenase from the previously reported NADPH-dependent S. cerevisiae HMF alcohol dehydrogenase Adh6. The amino acid sequence revealed three novel mutations (S109P, L116S and Y294C) that were all predicted at the vicinity of the substrate binding site, which could explain the unusual substrate specificity. Increased biomass production and HMF conversion rate were achieved in a CEN.PK S. cerevisiae strain overexpressing the mutated ADH1 gene. Copyright © 2008 John Wiley & Sons, Ltd.

Analytical Chemistry, 2009

In this work, mediated amperometry was used to evaluate whether differences in intracellular nico... more In this work, mediated amperometry was used to evaluate whether differences in intracellular nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) level could be observed between a genetically modified Saccharomyces cerevisiae strain, engineered for NAD-PH dependent 5-hydroxymethyl-2-furaldehyde (HMF) reduction, and its control strain. Cells overexpressing the alcohol dehydrogenase 6 gene (ADH6 strain) and cells carrying the corresponding control plasmid (control strain) were each immobilized on Au-microelectrodes. The real-time dynamics of NAD(P)H availability in the two strains, preincubated with HMF, was probed using the menadione-ferricyanide double mediator system. A lower intracellular NADPH level as the consequence of more effective HMF reduction was observed for the ADH6 strain both with and without added glucose, which increases the overall cellular NADPH level. The mediated amperometric signal during real-time monitoring of the concentration dependent HMF reduction in living cells could be translated into the cellular enzyme kinetic parameters: K M,cell app , V MAX , k cat,cell , and k cat,cell /K M,cell app . The results indicated that the overexpression of the ADH6 gene gave a 68% decrease in K M,cell app and 42% increase in V MAX , resulting in a 4-fold increase in k cat,cell /K M,cell app . These results demonstrate that the mediated amperometric method is useful for monitoring the short-term dynamics of NAD(P)H variations and determining cellular enzyme kinetic parameters in S. cerevisiae cells.