Isolation of xylose-assimilating yeasts and optimization of xylitol production by a new Meyerozyma guilliermondii strain.

Production of xylitol from lignocellulosic biomass is of interest to modern biorefineries, because this biomass should be processed into a spectrum of chemicals (bio-based products) and not only energy. The isolation of new yeast strains capable of efficiently converting xylose into xylitol and withstanding inhibitors released from biomass hydrolysis can contribute to making its production feasible in biorefineries. Forty-three out of 128 yeast strains isolated from the gut of Passalidae beetles were capable of assimilating xylose as the sole carbon source. Meyerozyma guilliermondii UFV-1 was selected due to its ability to grow and ferment D-xylose in a synthetic medium. This yeast assimilated the broad range of sugars present in lignocellulosic biomass hydrolysates, such as xylose, raffinose, cellobiose, rhamnose, arabinose, and glucose. Its optimum growth conditions were pH 8.0 and a temperature of 30 °C. In concentrations of 0.07 mol/L acetic acid, 0.05 mol/L 5-hydroximethylfurfural, and 0.04 mol/L furfural, M. guilliermondii UFV-1 did not grow. Maximum xylitol production in aerobiosis and hypoxia were 51.88 and 27.73 g/L, respectively. Under aerobic condition, xylose concentration and agitation rate were the factors which were statistically significant, while only the agitation rate was significant in hypoxia. We fitted a response surface (RS) that estimated the best agitation rate (113.33 rpm) and xylose concentration (90 g/L) for maximum xylitol production in aerobiosis. Therefore, M. guilliermondii UFV-1 displays potential for being used for xylitol production in biorefineries.

Chemical structure, antiproliferative and antioxidant activities of a cell wall α-d-mannan from yeast Kluyveromyces marxianus.

Cell wall polysaccharides from filamentous fungi and yeasts have been reported as antioxidant and antiproliferative polymers. Thus, we evaluated these activities from cell wall polysaccharides from Kluyveromyces marxianus CCT7735. By using a centrifugal filter, a 203kDa α-d-mannan (KMM-5) was obtained. KMM-5 exhibited no effect on HeLa cells and a weak antiproliferative activity against Hep-G2 cells. In addition, at higher concentrations, it presented a cytotoxicity to the normal cell line, 3T3. However, KMM-5 showed copper- and iron-chelating abilities, the latter of which presented improved activity. By using 2D-NMR COSY, HSQC edited and HMBC experiments, a structure arrangement was proposed. The main chain was formed by 6)-α-d-Manp-(1→6) units substituted at the 2-O-position by non-reducing terminals α-d-Manp-(1→2) and by a branched tetrasaccharide. The latter was formed by an internal 2)-α-d-Manp-(1→2) unit with linked to it a 2,3)-α-d-Manp-(1→2) unit substituted at the 2-O-position by a non-reducing terminal α-d-Manp-(1→2), and at the 3-O-position by a non-reducing terminal α-d-Manp-(1→3). In conclusion, we considered K. marxianus CCT7735 a source of natural and renewable polysaccharides with pharmacological properties.

Applying functional metagenomics to search for novel lignocellulosic enzymes in a microbial consortium derived from a thermophilic composting phase of sugarcane bagasse and cow manure.

Environments where lignocellulosic biomass is naturally decomposed are sources for discovery of new hydrolytic enzymes that can reduce the high cost of enzymatic cocktails for second-generation ethanol production. Metagenomic analysis was applied to discover genes coding carbohydrate-depleting enzymes from a microbial laboratory subculture using a mix of sugarcane bagasse and cow manure in the thermophilic composting phase. From a fosmid library, 182 clones had the ability to hydrolyse carbohydrate. Sequencing of 30 fosmids resulted in 12 contigs encoding 34 putative carbohydrate-active enzymes belonging to 17 glycosyl hydrolase (GH) families. One third of the putative proteins belong to the GH3 family, which includes ß-glucosidase enzymes known to be important in the cellulose-deconstruction process but present with low activity in commercial enzyme preparations. Phylogenetic analysis of the amino acid sequences of seven selected proteins, including three ß-glucosidases, showed low relatedness with protein sequences deposited in databases. These findings highlight microbial consortia obtained from a mixture of decomposing biomass residues, such as sugar cane bagasse and cow manure, as a rich resource of novel enzymes potentially useful in biotechnology for saccharification of lignocellulosic substrate.

Construction of recombinant Kluyveromyces marxianus UFV-3 to express dengue virus type 1 nonstructural protein 1 (NS1).

The yeast Kluyveromyces marxianus is a convenient host for industrial synthesis of biomolecules. However, despite its potential, there are few studies reporting the expression of heterologous proteins using this yeast. Here, we report expression of a dengue virus protein in K. marxianus for the first time. The dengue virus type 1 nonstructural protein 1 (NS1) was integrated into the K. marxianus UFV-3 genome at the LAC4 locus using an adapted integrative vector designed for high-level expression of recombinant protein in Kluyveromyces lactis. The NS1 gene sequence was codon-optimized to increase the level of protein expression in yeast. The synthetic gene was cloned in frame with K. lactis α-mating factor signal peptide, and the recombinant plasmid obtained was used to transform K. marxianus UFV-3 by electroporation. The transformed cells, selected in yeast extract peptone dextrose containing 200 µg mL(-1) Geneticin, were mitotically stable. Analysis of recombinant strains by RT-PCR and protein detection using blot analysis confirmed both transcription and expression of extracellular NS1 polypeptide. After induction with galactose, the NS1 protein was analyzed by sodium dodecyl sulfate-PAGE and immunogenic detection. Protein production was investigated under two conditions: with galactose and biotin pulses at 24-h intervals during 96 h of induction and without galactose and biotin supplementation. Protease activity was not detected in post-growth medium. Our results indicate that recombinant K. marxianus is a good host for the production of dengue virus NS1 protein, which has potential for diagnostic applications.

Signals of aging associated with lower growth rates in Kluyveromyces lactis cultures under nitrogen limitation.

The effects of aging on the specific growth rate of Kluyveromyces lactis cultures, as a function of (NH4)2SO4 concentration, were evaluated. The growth kinetic parameters maximum specific growth rate and saturation constant for (NH4)2SO4 were calculated to be 0.44 h(-1) and 0.15 mmol·L(-1), respectively. Batch cultures were allowed to age for 16 days without influence of cell density or starvation. The specific growth rates of these cultures were determined each day and decreased as the population aged at different nitrogen concentrations. Aging signals (N-acetylglucosamine content of the cell wall, cell dimensions, and apoptosis markers) were measured. Apoptosis markers were detected after 5 days at limiting (NH4)2SO4 concentrations (0.57, 3.80, and 7.60 mmol·L(-1)) but only after 8 days at a nonlimiting (NH4)2SO4 concentration (38.0 mmol·L(-1)). Similarly, continuous cultures of K. lactis performed under nitrogen limitation and, at lower dilution rates, accumulated cells exhibiting aging signals. The results demonstrate that aging affects growth rate and raise the question of whether nitrogen limitation accelerates aging. Because aging is correlated with growth rate, and each dilution rate of the continuous cultures tends to select and accumulate cells with a respective age, cultures growing at lower growth rates can be useful to investigate yeast physiological responses, including aging.

Oligonucleotide primers for specific detection of actinobacterial laccases from superfamilies I and K.

Although many putative laccase-like genes have been assigned to members of the phylum Actinobacteria, few of the related enzymes have been characterized so far. It is noteworthy, however, that this small number of enzymes has presented properties with industrial relevance. This observation, combined with the recognized biotechnological potential and the capability of this phylum to degrade recalcitrant soil polymers, has attracted attention for bioprospective approaches. In the present work, we have designed and tested primers that were specific for detection of sub-groups of laccase-like genes within actinomycetes, which corresponded to the superfamilies I and K from the classification presented by the laccase and multicopper oxidase engineering database. The designed primers have amplified laccase-like gene fragments from actinomycete isolates that were undetectable by primers available from the literature. Furthermore, phylogenetic alignments suggest that some of these fragments may belong to new laccases-like proteins, and thus emphasize the benefits of designing subgroup-specific primers.

Metabolic engineering of Kluyveromyces lactis for L-ascorbic acid (vitamin C) biosynthesis.

BACKGROUND: L-ascorbic acid (L-AA) is naturally synthesized in plants from D-glucose by 10 steps pathway. The pathway branch to synthesize L-galactose, the key intermediate for L-ascorbic acid biosynthesis, has been recently elucidated. Budding yeast produces an 5-carbon ascorbic acid analogue Dehydro-D-arabinono 1,4-lactone (D-DAL), which is synthesized from D-arabinose. Yeast is able to synthesize L-ascorbic acid only if it is cultivated in the presence of one of its precursors: L-galactose, L-galactono 1,4-lactone, or L-gulono 1,4-lactone extracted from plants or animals. To avoid feeding the yeast culture with this "L" enantiomer, we engineered Kluyveromyces lactis with L-galactose biosynthesis pathway genes: GDP-mannose 3,5-epimerase (GME), GDP-L-galactose phosphorylase (VTC2) and L-galactose-1-phosphate phosphatase (VTC4) isolated from Arabidopsis thaliana. RESULTS: Plasmids were constructed and modified such that the cloned plant genes were targeted to the K. lactis LAC4 Locus by homologous recombination and that the expression was associated to the growth on D-galactose or lactose. Upon K. lactis transformation, GME was under the control of the native LAC4 promoter whereas VTC2 and VTC4 were expressed from the S. cerevisiae promoters GPD1 and ADH1 respectively. The expression in K. lactis, of the L-galactose biosynthesis genes was determined by Reverse Transcriptase-PCR and western blotting. The recombinant yeasts were capable to produce about 30 mg.L(-1) of L-ascorbic acid in 48 hours of cultivation when cultured on rich medium with 2% (w/v) D-galactose. We also evaluated the L-AA production culturing recombinant recombinant strains in cheese whey, a waste product during cheese production, as an alternative source of lactose. CONCLUSIONS: This work is the first attempt to engineer K. lactis cells for L-ascorbic acid biosynthesis by a fermentation process without any trace of "L" isomers precursors in the culture medium. We have engineered K. lactis strains capable of converting lactose and D-galactose into L-galactose, by the integration of the genes from the A. thaliana L-galactose pathway. L-galactose is a rare sugar, which is one of the main precursors for L-AA production.

Kinetics of growth and ethanol formation from a mix of glucose/xylose substrate by Kluyveromyces marxianus UFV-3.

The fermentation of both glucose and xylose is important to maximize ethanol yield from renewable biomass feedstocks. In this article, we analyze growth, sugar consumption, and ethanol formation by the yeast Kluyveromyces marxianus UFV-3 using various glucose and xylose concentrations and also under conditions of reduced respiratory activity. In almost all the conditions analyzed, glucose repressed xylose assimilation and xylose consumption began after glucose had been exhausted. A remarkable difference was observed when mixtures of 5 g L(-1) glucose/20 g L(-1) xylose and 20 g L(-1) glucose/20 g L(-1) xylose were used. In the former, the xylose consumption began immediately after the glucose depletion. Indeed, there was no striking diauxic phase, as observed in the latter condition, in which there was an interval of 30 h between glucose depletion and the beginning of xylose consumption. Ethanol production was always higher in a mixture of glucose and xylose than in glucose alone. The highest ethanol concentration (8.65 g L(-1)) and cell mass concentration (4.42 g L(-1)) were achieved after 8 and 74 h, respectively, in a mixture of 20 g L(-1) glucose/20 g L(-1) xylose. When inhibitors of respiration were added to the medium, glucose repression of xylose consumption was alleviated completely and K. marxianus was able to consume xylose and glucose simultaneously.

Thermostability of xylanolytic enzymes produced by Lentinula edodes UFV70

Xylanolytic enzymes produced by Lentinula edodes UFV70, cultivated in eucalyptus sawdust/rice bran medium, were stable at 50, 60 and 65ºC for 21 hours, losing only 15-25% activity. Fungus incubation at 50ºC for 12 hours and at 65ºC for 24 hours increased the amount of xylose produced.

Thermostability of xylanolytic enzymes produced by Lentinula edodes UFV70.

Xylanolytic enzymes produced by Lentinula edodes UFV70, cultivated in eucalyptus sawdust/rice bran medium, were stable at 50, 60 and 65°C for 21 hours, losing only 15-25% activity. Fungus incubation at 50°C for 12 hours and at 65°C for 24 hours increased the amount of xylose produced.

Debaryomyces hansenii UFV-1 intracellular alpha-galactosidase characterization and comparative studies with the extracellular enzyme.

Debaryomyces hansenii cells cultivated on galactose produced extracellular and intracellular alpha-galactosidases, which showed 54.5 and 54.8 kDa molecular mass (MALDI-TOF), 60 and 61 kDa (SDS-PAGE) and 5.15 and 4.15 pI values, respectively. The extracellular and intracellular deglycosylated forms presented 36 and 40 kDa molecular mass, with 40 and 34% carbohydrate content, respectively. The N-terminal sequences of the alpha-galactosidases were identical. Intracellular alpha-galactosidase showed smaller thermostability when compared to the extracellular enzyme. D. hansenii UFV-1 extracellular alpha-galactosidase presented higher kcat than the intracellular enzyme (7.16 vs 3.29 s-1, respectively) for the p-nitrophenyl-alpha-D-galactopyranoside substrate. The Km for hydrolysis of pNPalphaGal, melibiose, stachyose, and raffinose were 0.32, 2.12, 10.8, and 32.8 mM, respectively. The intracellular enzyme was a competitively inhibited by galactose (Ki = 0.70 mM), and it was inactivated by Cu(II) and Ag(I). Enzyme incubation with soy milk for 6 h at 55 degrees C reduced stachyose and raffinose amounts by 100 and 73%, respectively.

Screening of filamentous fungi for production of xylitol from D-xylose

Foram avaliados onze fungos filamentosos para a producão de xilitol em batelada. A producão foi baixa nas condicões de cultivo utilizadas. A máxima, 0,52 g L-1 de xilitol a partir de 11,50 g L-1 de xilose, foi obtida com Penicillium crustosum, com consumo de 76 per center da xilose inicial.

Production of pectin lyase by Penicillium griseoroseum in bioreactors in the absence of inducer

Penicillium griseoroseum foi cultivado em biorreatores em meio mineral suplementado com extrato de levedura e sacarose. As influências das concentrações do inóculo e da fonte de carbono, da aeração e do pH do meio de cultivo sobre a produção de pectina liase (PL), bem como a capacidade de P. griseoroseum em produzir PL quando cultivado em caldo de cana diluído foram avaliadas. A concentração do inóculo não influenciou significativamente a produção de PL. O cultivo do fungo em biorreatores não aerados favoreceu a produção da PL em detrimento aos biorreatores com injeção de ar. Maior produção de PL foi obtida com o cultivo de P. griseoroseum em meio com pH 6,3 - 7,2, adicionado de 60 mM de sacarose. Quando cultivado em caldo de cana diluído, 25per center (v/v), sem suplementação com extrato de levedura, a atividade máxima de PL alcançada foi igual as das condições citadas acima.