First- and second-generation integrated process for bioethanol production: Fermentation of molasses diluted with hemicellulose hydrolysate by recombinant Saccharomyces cerevisiae.

The integration of first- (1G) and second-generation (2G) ethanol production by adding sugarcane juice or molasses to lignocellulosic hydrolysates offers the possibility to overcome the problem of inhibitors (acetic acid, furfural, hydroxymethylfurfural and phenolic compounds), and add nutrients (such as salts, sugars and nitrogen sources) to the fermentation medium, allowing the production of higher ethanol titers. In this work, an 1G2G production process was developed with hemicellulosic hydrolysate (HH) from a diluted sulfuric acid pretreatment of sugarcane bagasse and sugarcane molasses. The industrial Saccharomyces cerevisiae CAT-1 was genetically modified for xylose consumption and used for co-fermentation of sucrose, fructose, glucose, and xylose. The fed-batch fermentation with high cell density that mimics an industrial fermentation was performed at bench scale fermenter, achieved high volumetric ethanol productivity of 1.59 g L-1 h-1, 0.39 g g-1 of ethanol yield, and 44.5 g L-1 ethanol titer, and shown that the yeast was able to consume all the sugars present in must simultaneously. With the results, it was possible to establish a mass balance for the global process: from pretreatment to the co-fermentation of molasses and HH, and it was possible to establish an effective integrated process (1G2G) with sugarcane molasses and HH co-fermentation employing a recombinant yeast.

Biochemical analysis of cellobiose catabolism in Candida pseudointermedia strains isolated from rotten wood.

We isolated two Candida pseudointermedia strains from the Atlantic rain forest in Brazil, and analyzed cellobiose metabolization in their cells. After growth in cellobiose medium, both strains had high intracellular ß-glucosidase activity [~ 200 U (g cells)-1 for 200 mM cellobiose and ~ 100 U (g cells)-1 for 2 mM pNPßG] and negligible periplasmic cellobiase activity. During batch fermentation, the strain with the best performance consumed all the available cellobiose in the first 18 h of the assay, producing 2.7 g L-1 of ethanol. Kinetics of its cellobiase activity demonstrated a high-affinity hydrolytic system inside cells, with Km of 12.4 mM. Our data suggest that, unlike other fungal species that hydrolyze cellobiose extracellularly, both analyzed strains transport it to the cytoplasm, where it is then hydrolyzed by high-affinity intracellular ß-glucosidases. We believe this study increases the fund of knowledge regarding yeasts from Brazilian microbiomes.

Strategies for Efficient Expression of Heterologous Monosaccharide Transporters in Saccharomyces cerevisiae.

In previous work, we developed a Saccharomyces cerevisiae strain (DLG-K1) lacking the main monosaccharide transporters (hxt-null) and displaying high xylose reductase, xylitol dehydrogenase and xylulokinase activities. This strain proved to be a useful chassis strain to study new glucose/xylose transporters, as SsXUT1 from Scheffersomyces stipitis. Proteins with high amino acid sequence similarity (78-80%) to SsXUT1 were identified from Spathaspora passalidarum and Spathaspora arborariae genomes. The characterization of these putative transporter genes (SpXUT1 and SaXUT1, respectively) was performed in the same chassis strain. Surprisingly, the cloned genes could not restore the ability to grow in several monosaccharides tested (including glucose and xylose), but after being grown in maltose, the uptake of 14C-glucose and 14C-xylose was detected. While SsXUT1 lacks lysine residues with high ubiquitinylation potential in its N-terminal domain and displays only one in its C-terminal domain, both SpXUT1 and SaXUT1 transporters have several such residues in their C-terminal domains. A truncated version of SpXUT1 gene, deprived of the respective 3'-end, was cloned in DLG-K1 and allowed growth and fermentation in glucose or xylose. In another approach, two arrestins known to be involved in the ubiquitinylation and endocytosis of sugar transporters (ROD1 and ROG3) were knocked out, but only the rog3 mutant allowed a significant improvement of growth and fermentation in glucose when either of the XUT permeases were expressed. Therefore, for the efficient heterologous expression of monosaccharide (e.g., glucose/xylose) transporters in S. cerevisiae, we propose either the removal of lysines involved in ubiquitinylation and endocytosis or the use of chassis strains hampered in the specific mechanism of membrane protein turnover.