Engineering xylose fermentation in an industrial yeast: continuous cultivation as a tool for selecting improved strains.

Production of second-generation ethanol from lignocellulosic residues should be fueling the energy matrix in the near future. Lignocellulosic biomass has received considerable attention as an alternative renewable resource toward reducing the demand for fossil energy sources, contributing to a future sustainable bio-based economy. Fermentation of lignocellulosic hydrolysates poses many scientific and technological challenges as the drawback of Saccharomyces cerevisiae's inability in fermenting pentose sugars (derived from hemicellulose). To overcome the inability of S. cerevisiae to ferment xylose and increase yeast robustness in the presence of inhibitory compound-containing media, the industrial S. cerevisiae strain SA-1 was engineered using CRISPR-Cas9 with the oxidoreductive xylose pathway from Scheffersomyces stipitis (encoded by XYL1, XYL2, and XYL3). The engineered strain was then cultivated in a xylose-limited chemostat under increasing dilution rates (for 64 days) to improve its xylose consumption kinetics under aerobic conditions. The evolved strain (DPY06) and its parental strain (SA-1 XR/XDH) were evaluated under microaerobic in a hemicellulosic hydrolysate-based medium. DPY06 exhibited 35% higher volumetric ethanol productivity compared to its parental strain.

Spathaspora piracicabensis f. a., sp. nov., a D-xylose-fermenting yeast species isolated from rotting wood in Brazil.

Two strains of a novel yeast species were isolated from rotting wood of an ornamental tree (purple quaresmeira, Tibouchina granulosa, Melastomataceae) in an Atlantic Rainforest area in Brazil. Analysis of the sequences of the internal transcribed spacer (ITS-5.8S) region and the D1/D2 domains of the large subunit rRNA gene showed that this species belongs to the Spathaspora clade, and is phylogenetically related to Spathaspora brasiliensis, Candida materiae and Sp. girioi. The novel species ferments D-xylose, producing ethanol, with amounts between 3.37 and 3.48 g L-1 ethanol from 2% D-xylose. Ascospores were not observed from this new species. The name Spathaspora piracicabensis f. a., sp. nov. is proposed to accommodate these isolates. The type strain is UFMG-CM-Y5867T (= CBS 15054T = ESALQ-I54T). The MycoBank number is MB 822,320.

Cyberlindnera xylosilytica sp. nov., a xylitol-producing yeast species isolated from lignocellulosic materials.

Independent surveys of yeasts associated with lignocellulosic-related materials led to the discovery of a novel yeast species belonging to the Cyberlindnera clade (Saccharomycotina, Ascomycota). Analysis of the sequences of the internal transcribed spacer (ITS) region and the D1/D2 domains of the large subunit rRNA gene showed that this species is related to C. japonica, C. maesa and C. easanensis. Six isolates were obtained from different sources, including rotting wood, tree bark and sugar cane filter cake in Brazil, frass from white oak in the USA and decayed leaf in Taiwan. A novel species is suggested to accommodate these isolates, for which the name C. xylosilytica sp. nov. is proposed. The type strain of C. xylosilytica sp. nov. is NRRL YB-2097(T) ( = CBS 13984(T) = UFMG-CM-Y347(T)) and the allotype is UFMG-CM-Y409 ( = CBS 14083). The novel species is heterothallic and complementary mating types are represented by the type and allotype strains. The MycoBank number is MB 811428.

Whole-genome sequencing of the efficient industrial fuel-ethanol fermentative Saccharomyces cerevisiae strain CAT-1.

The Saccharomyces cerevisiae strains widely used for industrial fuel-ethanol production have been developed by selection, but their underlying beneficial genetic polymorphisms remain unknown. Here, we report the draft whole-genome sequence of the S. cerevisiae strain CAT-1, which is a dominant fuel-ethanol fermentative strain from the sugarcane industry in Brazil. Our results indicate that strain CAT-1 is a highly heterozygous diploid yeast strain, and the ~12-Mb genome of CAT-1, when compared with the reference S228c genome, contains ~36,000 homozygous and ~30,000 heterozygous single nucleotide polymorphisms, exhibiting an uneven distribution among chromosomes due to large genomic regions of loss of heterozygosity (LOH). In total, 58 % of the 6,652 predicted protein-coding genes of the CAT-1 genome constitute different alleles when compared with the genes present in the reference S288c genome. The CAT-1 genome contains a reduced number of transposable elements, as well as several gene deletions and duplications, especially at telomeric regions, some correlated with several of the physiological characteristics of this industrial fuel-ethanol strain. Phylogenetic analyses revealed that some genes were likely associated with traits important for bioethanol production. Identifying and characterizing the allelic variations controlling traits relevant to industrial fermentation should provide the basis for a forward genetics approach for developing better fermenting yeast strains.

Draft Genome Sequence of the D-Xylose-Fermenting Yeast Spathaspora arborariae UFMG-HM19.1AT.

The draft genome sequence of the yeast Spathaspora arborariae UFMG-HM19.1A(T) (CBS 11463 = NRRL Y-48658) is presented here. The sequenced genome size is 12.7 Mb, consisting of 41 scaffolds containing a total of 5,625 predicted open reading frames, including many genes encoding enzymes and transporters involved in d-xylose fermentation.

Spectrophotometric flow injection monitoring of sulfide during sugar fermentation.

A spectrophotometric flow injection procedure involving N,N-dimethyl-p-phenylenediamine (DMPD) is applied to the sulfide monitoring of a sugar fermentation by Saccharomyces cerevisiae under laboratory conditions. The gaseous chemical species evolving from the fermentative process, mainly CO(2), are trapped allowing a cleaned sample aliquot to be collected and introduced into the flow injection analyzer. Measurement rate, signal repeatability, detection limit and reagent consumption per measurement were estimated as 150 h(-1), 0.36% (n=20), 0.014 mg L(-1) S and 120 µg DMPD, respectively. The main characteristics of the monitoring record are discussed. The strategy is worthwhile for selecting yeast strain, increasing the industrial ethanol production and improving the quality of wines.

Yeast selection for fuel ethanol production in Brazil.

Brazil is one of the largest ethanol biofuel producers and exporters in the world and its production has increased steadily during the last three decades. The increasing efficiency of Brazilian ethanol plants has been evident due to the many technological contributions. As far as yeast is concerned, few publications are available regarding the industrial fermentation processes in Brazil. The present paper reports on a yeast selection program performed during the last 12 years aimed at selecting Saccharomyces cerevisiae strains suitable for fermentation of sugar cane substrates (cane juice and molasses) with cell recycle, as it is conducted in Brazilian bioethanol plants. As a result, some evidence is presented showing the positive impact of selected yeast strains in increasing ethanol yield and reducing production costs, due to their higher fermentation performance (high ethanol yield, reduced glycerol and foam formation, maintenance of high viability during recycling and very high implantation capability into industrial fermenters). Results also suggest that the great yeast biodiversity found in distillery environments could be an important source of strains. This is because during yeast cell recycling, selective pressure (an adaptive evolution) is imposed on cells, leading to strains with higher tolerance to the stressful conditions of the industrial fermentation.