Comparison ofSpathaspora passalidarumand recombinantSaccharomyces cerevisiaefor integration of first- and second-generation ethanol production

Abstract

First-generation ethanol (E1G) is based on the fermentation of sugars released from saccharine or starch sources, while second-generation ethanol (E2G) is focused on the fermentation of sugars released from lignocellulosic feedstocks. During the fractionation process to release sugars from hemicelluloses (mainly xylose), some inhibitor compounds are released hindering fermentation. Thus, the biggest challenge of using hemicellulosic hydrolysate is selecting strains and processes able to efficiently ferment xylose and tolerate inhibitors. With the aim of diluting inhibitors, sugarcane molasses (80% of sucrose content) can be mixed to hemicellulosic hydrolysate in an integrated E1G-E2G process. Cofermentations of xylose and sucrose were evaluated for the native xylose consumer Spathaspora passalidarum and a recombinant Saccharomyces cerevisiae strain. The industrial S. cerevisiae strain CAT-1 was modified to overexpress the XYL1, XYL2, XKS1 genes and a mutant ([4-59Δ]HXT1) version of the low-affinity HXT1 permease, generating strain MP-C5H1. Although S. passalidarum showed better results for xylose fermentation, this yeast showed intracellular sucrose hydrolysis and low sucrose consumption in microaerobic conditions. Recombinant S. cerevisiae showed the best performance for cofermentation, and a batch strategy at high cell density in bioreactor achieved unprecedented results of ethanol yield, titer and volumetric productivity in E1G-E2G production process.

Strategies for Efficient Expression of Heterologous Monosaccharide Transporters in Saccharomyces cerevisiae

“…For example, the S. cerevisiae HXT1 transporter has 4 lysine residues at its N-terminal domain that are involved in its ubiquitinylation and endocytosis, and by truncating the whole N-terminal domain (deletion of 56 residues to remove all 4 lysine residues), the stability of the transporter in the plasma membrane is improved [68]. We have recently used this strategy to enhance xylose consumption and fermentation in an industrial S. cerevisiae strain [73], while site-directed mutation of these four residues in the HXT1 transporter was also shown to improve growth in this carbon source [74]. Another example involves one of the few yeast active xylose transporters characterized in S. cerevisiae, the Candida intermedia glucose/xylose H + -symporter, CiGXS1 [75].…”

New Strategies for Efficient Expression of Heterologous Sugar Transporters in Saccharomyces cerevisiae

“…For example, the S. cerevisiae HXT1 transporter has 4 lysine residues at its N-terminal domain involved in its ubiquitinylation and endocytosis, and truncating the whole N-terminal domain (deleting 56 residues to remove all 4 lysine residues) gives a functional transporter that is stable at the plasma membrane [58]. We have used recently this strategy to enhance xylose consumption and fermentation by an industrial S. cerevisiae strain [63], while site-directed mutation of these 4 residues in the HXT1 transporter was also shown to improve cell growth on this carbon source [64]. Another example involves one of the few yeast active xylose transporters characterized in S. cerevisiae, the Candida intermedia CiGXS1 gene encoding for a glucose/xylose H + -symporter [65].…”

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

“…The yeast used was the recombinant S. cerevisiae strain MP-C5H1 (Pereira et al, 2021). As described by Pereira et al (2021), the strain is the industrial yeast S. cerevisiae CAT-1 (Babrzadeh et al, 2012) modified to overexpress the xylose reductase (XR) XYL1 and xylitol dehydrogenase (XDH) XYL2 genes from S. stipitis, and the S. cerevisiae The recombinant strain was kept at −80°C in cryotube containing rich yeast and peptone medium, YP, (10 g L −1 yeast extract and 20 g L −1 peptone) containing 10 g L −1 glucose and 10 g L −1 xylose (YPDX) and 50% (v/v) glycerol (1:1).…”

“…The yeast used was the recombinant S. cerevisiae strain MP-C5H1 (Pereira et al, 2021). As described by Pereira et al (2021), the strain is the industrial yeast S. cerevisiae CAT-1 (Babrzadeh et al, 2012) modified to overexpress the xylose reductase (XR) XYL1 and xylitol dehydrogenase (XDH) XYL2 genes from S. stipitis, and the S. cerevisiae The recombinant strain was kept at −80°C in cryotube containing rich yeast and peptone medium, YP, (10 g L −1 yeast extract and 20 g L −1 peptone) containing 10 g L −1 glucose and 10 g L −1 xylose (YPDX) and 50% (v/v) glycerol (1:1). The yeast reactivation was made in plates containing YPDX medium with 15 g L −1 agar, and incubated at 30°C for 48 h. After growth, one colony was inoculated in a 125 mL Erlenmeyer flask containing 50 mL of rich YP medium containing 10 g L −1 sucrose and 10 g L −1 xylose (YPSX), with the pH adjusted to 5.0 with HCl.…”

“…The yeast reactivation was made in plates containing YPDX medium with 15 g L −1 agar, and incubated at 30°C for 48 h. After growth, one colony was inoculated in a 125 mL Erlenmeyer flask containing 50 mL of rich YP medium containing 10 g L −1 sucrose and 10 g L −1 xylose (YPSX), with the pH adjusted to 5.0 with HCl. The flasks were incubated in shaker (Tecnal TE-424) at 30°C and 200 rpm for 12 h. An aliquot was then transferred (10% v/v) to a 250 mL Erlenmeyer flask containing 90 mL of YPSX media, supplemented with 2.3 g L −1 of urea and 1 g L −1 of MgSO 4 .7H 2 O, and incubated as described above for 24 h (Pereira et al, 2021).…”