Transcriptome profiling brings new insights into the ethanol stress responses of Spathaspora passalidarum.

Spathaspora passalidarum is a xylose-fermenting microorganism promising for the fermentation of lignocellulosic hydrolysates. This yeast is more sensitive to ethanol than Saccharomyces cerevisiae for unclear reasons. An RNA-seq experiment was performed to identify transcriptional changes in S. passalidarum in response to ethanol and gain insights into this phenotype. The results showed the upregulation of genes associated with translation and the downregulation of genes encoding proteins involved in lipid metabolism, transporters, and enzymes from glycolysis and fermentation pathways. Our results also revealed that genes encoding heat-shock proteins and involved in antioxidant response were upregulated, whereas the osmotic stress response of S. passalidarum appears impaired under ethanol stress. A pseudohyphal morphology of S. passalidarum colonies was observed in response to ethanol stress, which suggests that ethanol induces a misperception of nitrogen availability in the environment. Changes in the yeast fatty acid profile were observed only after 12 h of ethanol exposure, coinciding with the recovery of the yeast xylose consumption ability. These findings suggest that the lack of fast membrane lipid adjustments, the halt in nutrient absorption and cellular metabolism, and the failure to induce the expression of osmotic stress-responsive genes are the main aspects underlying the low ethanol tolerance of S. passalidarum. KEY POINTS: • Ethanol stress halts Spathaspora passalidarum metabolism and fermentation • Genes encoding nutrient transporters showed downregulation under ethanol stress • Ethanol induces a pseudohyphal cell shape, suggesting a misperception of nutrients.

Physiological comparisons among Spathaspora passalidarum, Spathaspora arborariae, and Scheffersomyces stipitis reveal the bottlenecks for their use in the production of second-generation ethanol.

The microbial conversion of pentoses to ethanol is one of the major drawbacks that limits the complete use of lignocellulosic sugars. In this study, we compared the yeast species Spathaspora arborariae, Spathaspora passalidarum, and Sheffersomyces stipitis regarding their potential use for xylose fermentation. Herein, we evaluated the effects of xylose concentration, presence of glucose, and temperature on ethanol production. The inhibitory effects of furfural, hydroxymethylfurfural (HMF), acetic acid, and ethanol were also determined. The highest ethanol yield (0.44 g/g) and productivity (1.02 g/L.h) were obtained using Sp. passalidarum grown in 100 g/L xylose at 32 °C. The rate of xylose consumption was reduced in the presence of glucose for the species tested. Hydroxymethylfurfural did not inhibit the growth of yeasts, whereas furfural extended their lag phase. Acetic acid inhibited the growth and fermentation of all yeasts. Furthermore, we showed that these xylose-fermenting yeasts do not produce ethanol concentrations greater than 4% (v/v), probably due to the inhibitory effects of ethanol on yeast physiology. Our data confirm that among the studied yeasts, Sp. passalidarum is the most promising for xylose fermentation, and the low tolerance to ethanol is an important aspect to be improved to increase its performance for second-generation (2G) ethanol production. Our molecular data showed that this yeast failed to induce the expression of some classical genes involved in ethanol tolerance. These findings suggest that Sp. passalidarum may have not activated a proper response to the stress, impacting its ability to overcome the negative effects of ethanol on the cells.

Five new additions to the genus Spathaspora (Saccharomycetales, Debaryomycetaceae) from southwest China.

Spathaspora is an important genus of d-xylose-fermenting yeasts that are poorly studied in China. During recent yeast collections in Yunnan Province in China, 13 isolates of Spathaspora were obtained from rotting wood and all represent undescribed taxa. Based on morphological and phylogenetic analyses (ITS and nuc 28S), five new species are proposed: Spathaspora elongata, Sp. mengyangensis, Sp. jiuxiensis, Sp. parajiuxiensis and Sp. rosae. Our results indicate a high species diversity of Spathaspora waiting to be discovered in rotting wood from tropical and subtropical southwest China. In addition, the two Candida species, C. jeffriesii and C. materiae, which are members of the Spathaspora clade based on phylogeny, are transferred to Spathaspora as new combinations.

Three new Scheffersomyces species associated with insects and rotting wood in China.

Three species of Scheffersomyces were identified during a diversity study of yeasts. All three are associated with insects and rotting wood in China. Phylogenetic analyses of a genomic dataset combining ITS and nrLSU revealed that these new collections are distinct from known species, thus three new species are introduced i.e. S. jinghongensis, S. paraergatensis, and S. anoplophorae. In our phylogenetic analyses, Scheffersomyces jinghongensis possesses a strong independent lineage and is closely related to S. titanus. S. paraergatensis is closely related to S. ergatensis, while S. anoplophorae is related to S. stambukii. Several differences in physiological traits and molecular data indicate that S. jinghongensis, S. paraergatensis, and S. anoplophorae are three newly identified species.

Candida xylosifermentans sp. nov., a d-xylose-fermenting yeast species isolated in Thailand.

Three strains, representing a novel anamorphic and d-xylose-fermenting yeast species, were isolated from moss (ST-302T), seawater (ST-1169) and peat (DMKU-XE12) collected from the southern part of Thailand. The three strains had identical sequences of the D1/D2 regions of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) regions. Candida flosculorum CBS 10566T and Candida sharkiensis CBS 11368T were the most closely related species with 7.9 % nucleotide substitutions in the D1/D2 regions of the LSU rRNA gene, and 10.3 and 12.6% nucleotide substitutions in the ITS regions, respectively. Phylogenetic analysis based on the concatenated sequences of the ITS and the D1/D2 regions confirmed that the three strains represented a distinct anamorphic species in the Clavispora clade. Therefore, the three strains were described as a novel species, for which we propose the name Candida xylosifermentans sp. nov.

Blastobotrys bombycis sp. nov., a d-xylose-fermenting yeast isolated from the gut of the silkworm larva Bombyx mori.

The gut of insects harbors a yeast community that is still poorly understood. Here, a novel species of the ascomycetous genus Blastobotrys is proposed based on a yeast strain isolated from the larval gut of the silkworm Bombyx mori (Order Lepidoptera). The novel species is closely related to Blastobotrys aristata and Blastobotrys elegans on the basis of the results of molecular phylogenetic analyses. A preliminary screening revealed that it produces 1.5 g l-1 ethanol by fermenting 5 % d-xylose. The novel species, that represents the first report, to our knowledge, of yeast isolation from silkworms, is described as Blastobotrys bombycis sp. nov. (type strain RAAB001T=CBS 15274T=PYCC 8105T=MCC 1427T; MycoBank accession number MB 825095).

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.

Construction and application of multi-host integrative vector system for xylose-fermenting yeast.

The xylose-fermenting yeasts (CTG clade yeasts, e.g. Scheffersomyces stipitis, Spathaspora passalidarum, Candida amazonensis and Candida jeffriesii) have the potential to be superior platforms for the conversion of lignocellulosic hydrolysate into fuel-grade ethanol and other chemical products. Here, a genetic expression system compatible with the genetic coding characteristics of CTG clade yeasts was constructed for use in xylose-fermenting yeasts. The pRACTH-gfpm plasmid based on an 18S rDNA shuttle vector was capable of stable integration into the genomes of a wide range of heterologous hosts. Green fluorescent protein was transformed and functionally expressed in S. stipitis, S. passalidarum, C. jeffriesii, C. amazonensis and Saccharomyces cerevisiae under control of the SpADH1 promoter and SpCYC1 terminator. Finally, the expression system was useful in multiple yeast hosts for construction of the plasmid pRACTH-ldh. Scheffersomyces stipitis, S. passalidarum, C. jeffriesii, C. amazonensis and S. cerevisiae were enabled to produce lactate from glucose or xylose by pRACTH-based expression of a heterologous lactate dehydrogenase. Among them, C. amazonensis (pRACTH-ldh) exhibited the highest lactate fermentation capacity, which reached a maximum of 44 g L-1 of lactate with a yield of 0.85 g lactate/g xylose.

Draft genome sequence of Sugiyamaella xylanicola UFMG-CM-Y1884T, a xylan-degrading yeast species isolated from rotting wood samples in Brazil.

We present the draft genome sequence of the type strain of the yeast Sugiyamaella xylanicola UFMG-CM-Y1884T (= UFMG-CA-32.1T = CBS 12683T), a xylan-degrading species capable of fermenting d-xylose to ethanol. The assembled genome has a size of ~ 13.7 Mb and a GC content of 33.8% and contains 5971 protein-coding genes. We identified 15 genes with significant similarity to the d-xylose reductase gene from several other fungal species. The draft genome assembled from whole-genome shotgun sequencing of the yeast Sugiyamaella xylanicola UFMG-CM-Y1884T (= UFMG-CA-32.1T = CBS 12683T) has been deposited at DDBJ/ENA/GenBank under the accession number MQSX00000000 under version MQSX01000000.