Identification of traits to improve co-assimilation of glucose and xylose by adaptive evolution of Spathaspora passalidarum and Scheffersomyces stipitis yeasts

Trichez, Débora; Steindorff, Andrei Stecca; Júnior, Wilson Galvão de Morais; Vilela, Nathália; Bergmann, Jessica Carvalho; Formighieri, Eduardo Fernandes; Gonçalves, Sílvia Belém; Almeida, J. R. M. de

doi:10.1007/s00253-023-12362-1

Cited by 5 publications

(5 citation statements)

References 62 publications

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“…2). As random mutagenesis and 2DG has been successfully used as a screening toll to select yeast mutant strains, especially Saccharomycotina, less sensitive to glucose catabolite repression [e.g., Saccharomyces cerevisiae (Kahar et al, 2011;Mikumo et al, 2015;Rincón et al, 2001), Kluyveromyces marxianus (Suprayogi et al, 2016;Yamada and Kosaka, 2015)] and/or with improved assimilation of alternative sugars [Scheffersomyces stipitis and Spathaspora passalidarum (Trichez et al, 2023)], we applied this strategy to select for the rst time mutant strains of Papiliotrema laurentii (Basidiomycota yeast) with these characteristics. Among the 14 mutant strains selected due to their resistance to inhibitory concentrations of 2DG, the M17 stood out.…”

Section: Discussionmentioning

confidence: 99%

“…The combination of mutagenesis and 2-deoxyglucose (2DG) screening has been previously used to select yeast strains with relaxed carbon catabolite repression and improved use of alternative carbon sources (Rincón et al 2001 (Suprayogi et al, 2016;Yamada and Kosaka, 2015), and Scheffersomyces stipitis and Spathaspora passalidarum (Trichez et al, 2023). Glucose catabolite repression and the effects of 2DG are well described in S. cerevisiae (Ralser et al 2008 ).…”

Section: Introductionmentioning

confidence: 99%

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Mutagenesis combined with 2-deoxyglucose is not a suitable tool to select strains of Papiliotrema laurentii less sensitive to glucose catabolite repression

de Almeida,

Lobato,

Ventorim

et al. 2024

Preprint

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Assimilation of major sugars from lignocellulosic biomasses is pivotal for achieving a feasible oil production by oleaginous yeasts in biorefineries. Papiliotrema laurentii UFV-1 is an oleaginous yeast capable of converting lignocellulosic sugars such as glucose and xylose into lipids; however, glucose is assimilated before xylose, impairing high volumetric lipid productivity. To circumvent this drawback, we hypothesized that random mutagenesis combined with 2-deoxyglucose (2DG) selection would be a suitable strategy for selecting strains of P. laurentii UFV-1 less sensitive to glucose repression. First, we determined the growth kinetics parameters of the wild strain in minimum medium with glucose and/or xylose. Then, the yeast was subjected to mutagenesis by ultraviolet irradiation, and mutants were selected in a culture medium containing 2DG. Among the 24 selected mutants, the M17 strain stood out due to its capacity to achieve a higher cell density at the 2DG inhibitory concentration. Surprisingly, both M17 and wildtype strains presented the same xylose and glucose consumption profile. Although M17 grew faster in xylose and preserved the oleaginous phenotype, it could not co-assimilate glucose and xylose. Interestingly, the tolerant strain grew assimilating 2DG and xylose simultaneously, likely incorporating 2DG into its biomass. Otherwise, the wild strain presented arrested growth and only grew after exporting 2DG back to the media. Since carbon catabolite repression and 2DG response mechanisms are poorly studied and remains elusive in Basidiomycota yeasts, we provided cues to guide future studies that will allow a better understanding of the mechanisms involved with 2DG resistance in these yeasts.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Mutagenesis combined with 2-deoxyglucose is not a suitable tool to select strains of Papiliotrema laurentii less sensitive to glucose catabolite repression

de Almeida,

Lobato,

Ventorim

et al. 2024

Preprint

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“…One strategy has been to learn from other fungi that consume xylose (but often lack other useful traits found in S. cerevisiae ), and several studies identified genes that, when overexpressed in S. cerevisiae , enable or improve xylose metabolism (e.g., GYC1 , YPR1 ) [ 80 , 81 , 82 ]. Other studies have used rational engineering and directed laboratory evolution to obtain xylose-fermenting S. cerevisiae strains [ 79 , 83 , 84 ].…”

Section: Engineering Yeast For Ethanol Production From Lignocellulosi...mentioning

confidence: 99%

Advances in S. cerevisiae Engineering for Xylose Fermentation and Biofuel Production: Balancing Growth, Metabolism, and Defense

Wagner

Gasch

2023

JoF

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Genetically engineering microorganisms to produce chemicals has changed the industrialized world. The budding yeast Saccharomyces cerevisiae is frequently used in industry due to its genetic tractability and unique metabolic capabilities. S. cerevisiae has been engineered to produce novel compounds from diverse sugars found in lignocellulosic biomass, including pentose sugars, like xylose, not recognized by the organism. Engineering high flux toward novel compounds has proved to be more challenging than anticipated since simply introducing pathway components is often not enough. Several studies show that the rewiring of upstream signaling is required to direct products toward pathways of interest, but doing so can diminish stress tolerance, which is important in industrial conditions. As an example of these challenges, we reviewed S. cerevisiae engineering efforts, enabling anaerobic xylose fermentation as a model system and showcasing the regulatory interplay’s controlling growth, metabolism, and stress defense. Enabling xylose fermentation in S. cerevisiae requires the introduction of several key metabolic enzymes but also regulatory rewiring of three signaling pathways at the intersection of the growth and stress defense responses: the RAS/PKA, Snf1, and high osmolarity glycerol (HOG) pathways. The current studies reviewed here suggest the modulation of global signaling pathways should be adopted into biorefinery microbial engineering pipelines to increase efficient product yields.

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“…Though adaptive laboratory evolution (ALE) has been applied to several studies, especially to S. cerevisiae , for developing industrially preferable strains [ 24 , 28 , 29 ], there are few reports on the use of evolutionary engineering in S. passalidarum and most of them were conducted in S. passalidarum strain NRRL Y-27907 (Table 1 ). As shown in Table 1 , ALE was utilized to enhance S. passalidarum for three main purposes: improved glucose-xylose co-fermentation [ 30 – 32 ], increased tolerance to hydrolysate inhibitors [ 33 , 34 ], and increased fermentation ability [ 35 ]. To improve glucose-xylose co-fermentation, UV mutagenesis or genome shuffling was conducted together with ALE.…”

Section: Introductionmentioning

confidence: 99%

“…Improved co-assimilation of glucose and xylose Spc3 UV mutagenesis and ALE (2-DOG) Spc3 showed a slight improvement in glucose and xylose co-fermentation compared to WT, however, the consumption rate of both sugars was slower than WT. [ 30 ] X2, X5 Genome shuffling between S. cerevisiae and S. passalidarum and ALE (YP medium containing 20 g/l xylose at 40 °C) Under mixed sugars of glucose-xylose condition at 40 °C, X2 and X5 could utilize glucose and xylose faster than WT. Both adapted strains produced ethanol 1.5-fold higher than WT.…”

Section: Introductionmentioning

confidence: 99%

Improving furfural tolerance in a xylose-fermenting yeast Spathaspora passalidarum CMUWF1–2 via adaptive laboratory evolution

Saengphing,

Sattayawat,

Kalawil

et al. 2024

Microb Cell Fact

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Background Spathaspora passalidarum is a yeast with the highly effective capability of fermenting several monosaccharides in lignocellulosic hydrolysates, especially xylose. However, this yeast was shown to be sensitive to furfural released during pretreatment and hydrolysis processes of lignocellulose biomass. We aimed to improve furfural tolerance in a previously isolated S. passalidarum CMUWF1−2, which presented thermotolerance and no detectable glucose repression, via adaptive laboratory evolution (ALE). Results An adapted strain, AF2.5, was obtained from 17 sequential transfers of CMUWF1−2 in YPD broth with gradually increasing furfural concentration. Strain AF2.5 could tolerate higher concentrations of furfural, ethanol and 5-hydroxymethyl furfuraldehyde (HMF) compared with CMUWF1−2 while maintaining the ability to utilize glucose and other sugars simultaneously. Notably, the lag phase of AF2.5 was 2 times shorter than that of CMUWF1−2 in the presence of 2.0 g/l furfural, which allowed the highest ethanol titers to be reached in a shorter period. To investigate more in-depth effects of furfural, intracellular reactive oxygen species (ROS) accumulation was observed and, in the presence of 2.0 g/l furfural, AF2.5 exhibited 3.41 times less ROS accumulation than CMUWF1−2 consistent with the result from nuclear chromatins diffusion, which the cells number of AF2.5 with diffuse chromatins was also 1.41 and 1.24 times less than CMUWF1−2 at 24 and 36 h, respectively. Conclusions An enhanced furfural tolerant strain of S. passalidarum was achieved via ALE techniques, which shows faster and higher ethanol productivity than that of the wild type. Not only furfural tolerance but also ethanol and HMF tolerances were improved.

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Identification of traits to improve co-assimilation of glucose and xylose by adaptive evolution of Spathaspora passalidarum and Scheffersomyces stipitis yeasts

Cited by 5 publications

References 62 publications

Mutagenesis combined with 2-deoxyglucose is not a suitable tool to select strains of Papiliotrema laurentii less sensitive to glucose catabolite repression

Mutagenesis combined with 2-deoxyglucose is not a suitable tool to select strains of Papiliotrema laurentii less sensitive to glucose catabolite repression

Advances in S. cerevisiae Engineering for Xylose Fermentation and Biofuel Production: Balancing Growth, Metabolism, and Defense

Improving furfural tolerance in a xylose-fermenting yeast Spathaspora passalidarum CMUWF1–2 via adaptive laboratory evolution

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