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 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mutagenesis combined with 2-deoxyglucose is not a suitable tool to select strains of Papiliotrema laurentii less sensitive to glucose catabolite repression Eduardo Luís Menezes de Almeida, Pâmela Carvalho Lobato, Rafaela Zandonade Ventorim, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4693745/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Dec, 2024 Read the published version in Biologia → Version 1 posted 5 You are reading this latest preprint version Abstract 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. oleaginous yeasts biorefineries carbon catabolite repression 2-deoxyglucose resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Over the last years, lignocellulosic biomasses have gained special interest as raw materials to produce different biobased products such as fuels, food ingredients and oleochemicals. Some oleaginous yeasts are capable of accumulating at least 20% of its dry biomass as lipids from sugars found in lignocellulosic hydrolysates such as glucose, xylose, arabinose, cellobiose, and galactose (Gallego-García et al. 2023 ). Papiliotrema laurentii UFV-1 was previously isolated and selected by our research group due to its capacity to accumulate high lipid contents using xylose as the sole carbon source (Vieira et al., 2020a ). Nevertheless, it displays glucose catabolite repression, that is, glucose is assimilated prior to alternative carbon sources, extending the time of lipid production in culture media containing lignocellulosic hydrolysates. Hence, obtaining strains of P. laurentii less sensitive to carbon catabolite repression and capable of co-assimilating glucose and xylose is mandatory to achieve high volumetric lipid productivity in biorefineries and boost its application in industrial bioprocesses. In wild yeasts that can use xylose as sole carbon source, such as Spathaspora passalidarum , Kluyveromyces marxianus , Scheffersomyces stipitis , and Meyerozyma guilliermondii , xylose is metabolized in the pentose phosphate pathway (PPP) through three reactions: first, xylose is converted to xylitol by xylose reductase, which requires NADPH or NADH; then xylitol is dehydrogenated to xylulose by xylose dehydrogenase, which recycles NAD + to NADH; finally, xylulose is phosphorylated by xylulose kinase, entering the PPP as xylulose-5-phosphate (Nalabothu et al., 2023 ). In most xylose-consuming yeasts, xylose reductase prefers NADPH as cofactor (Ochoa-Chacón et al. 2022 ; Nalabothu et al. 2023 ). 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 ; Kahar et al. 2011 ; Mikumo et al. 2015 ; Yamada and Kosaka 2015 ; Suprayogi et al. 2016; Gao et al. 2019 ; Trichez et al. 2023 ). Most studies have focused on Ascomycota yeast, mainly 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 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 ; McCartney et al. 2014 ; Kayikci and Nielsen 2015 ; Laussel and Léon 2020 ; Schmidt and O’Donnell 2021 ). In this yeast, 2DG is captured by cells and phosphorylated by hexokinase, forming 2-DG-6-P. The absence of the hidroxil group in the C2 of 2DG impairs its isomerization by phosphoglucose isomerase, preventing its use in the next steps of glycolysis. The accumulation of 2DG-6-P in the intracellular medium activates the signaling pathways related to glucose catabolite repression due to its structural similarity to glucose-6-phosphate, which blocks the use of alternative carbon sources and arrests growth (Schmidt and O’Donnell 2021 ). Yeasts that become resistant to 2DG can present modifications in the glucose catabolite repression signaling and can use alternative carbon sources even in the presence of glucose or 2DG, allowing sugar co-assimilation (Rincón et al. 2001 ; Kahar et al. 2011 ; Mikumo et al. 2015 ; Yamada and Kosaka 2015 ; Suprayogi et al. 2016; Gao et al. 2019 ; Trichez et al. 2023 ). However, research on the regulation of carbon catabolite repression is scarce on Basidiomycota yeast and little is known about the proteins involved and the effects of 2DG on the metabolism of these organisms. Therefore, we hypothesized that mutagenesis combined with 2DG selection would be a suitable strategy for selecting strains of P. laurentii UFV-1 less sensitive to glucose catabolite repression. We selected for the first time a strain of Papiliotrema laurentii UFV-1 tolerant to 2DG. Materials and Methods Microorganisms and maintenance The Papiliotrema laurentii UFV-1 strain used in this work belongs to the culture collection of the Microbial Physiology Laboratory, Microbiology Department, Universidade Federal de Viçosa (UFV). The wildtype strain and all mutants obtained are stored at -80°C in Yeast extract (10 g/L) Peptone (20 g/L) (YP) medium with 40% (v/v) glycerol. Culture medium, inoculum preparation, and cell viability We conducted the pre-inoculum cultivations from a single yeast colony from YP plus glucose (20 g/L) and agar (15 g/L) in Erlenmeyer’s flasks of 250 mL with 50 mL of Yeast Nitrogen Base (YNB, 6.7 g/L) medium w/o amino acids at 200 rpm/30°C for 18 h. Before all cultivations, unless stated otherwise, we centrifuged (5000 g/5 min at 4°C) and washed cells twice with peptone water (0.1% w/v) and adjusted the initial optical density at 600 nm (OD 600 ) to about 0.1. We prepared cells suspensions in these conditions to determine the minimum inhibitory concentration of 2DG, survival curve construction, mutagenesis, and for cultivations in glucose and/or xylose. To determine the cell viability, we stained yeast cells with methylene blue (1:1). We counted non-stained cells using a Neubauer chamber under an Olympus BX51 microscope (Olympus Corporation of the Americas, Pennsylvania, USA). When necessary, we diluted cell suspensions before staining. Minimum inhibitory concentration of 2DG, ultraviolet (UV) survival curve, and mutagenesis We determined the minimum inhibitory concentration of 2DG by cultivating P. laurentii on YNB agar medium with 133 mM of xylose (YNBX agar) and different 2DG concentrations in the range of 200–750 µg/mL. We adjusted the washed pre-inoculum to 10 8 viable cells/mL, serially diluted it, then plated the dilutions in YNBX agar plus 2DG. The control comprised cultivations w/o 2DG. The medium containing the minimum inhibitory concentration of 2DG (650 µg/mL) is referred to as YNBX2DG. Next, we constructed the UV exposition survival curve of P. laurentii UFV-1 as follows: first, we adjusted the viable cell concentration to 10 8 cells/mL and serially diluted then. Then, we plated 50 µL of each dilution in YNBX agar and irradiated cells using a 4 W lamp (Mineralight® modelo UVS-11) in aseptic conditions. To reach the mortality rate of about 99% as described by (Winston 2008 ), we tested irradiation periods from 1–10 min and the distance between the UV lamp and plate surface of 10 and 13 cm. We incubated plates at 30°C for 72 h. We conducted all procedures protected from the light to avoid the activation of the photosensitive repair system. After determining the best condition (9 min and 15 s; 10 cm), we conducted mutagenesis. We recovered individual colonies and stored the mutants as described in the Microorganisms and maintenance section. Wildtype strain growth on glucose and/or xylose and Monod model construction To determine the kinetic parameters in media containing glucose and/or xylose as carbon sources, we cultivated the P. laurentii UFV-1 wildtype strain in YNB media plus xylose 20 g/L (133 mM) (YNB20X); glucose 20 g/L (111 mM) (YNB20G); glucose 5 g/L (28 mM) (YNB5G); glucose 5 g/L (28 mM) and xylose 20 g/L (133 mM) (YNB5G20X); and glucose 20 g/L (111 mM) and xylose 20 g/L (133 mM) (YNB20GX). We cultivated cells for 96 h at 30°C and 200 rpm. We monitored the OD 600 hourly in the first 12 h, then at 24, 48, 72 and 96 h. Next, we constructed Monod models to evaluate the affinity of P. laurentii UFV-1 for glucose and xylose. Sugar concentration in YNB media ranged from 1 to 15 mM, which we prepared in volumetric flasks and filtered in 0.22 µm membranes (Millipore). We cultivated cells for 54 h at 30°C and 200 rpm and monitored the OD 600 at 30 min intervals for 12 h then at 24 and 54 h. In all cultivations, we measured the OD 600 in a Beckman Série DU 600 spectrophotometer (Indianapolis, USA). The specific growth rate comprised the angular coefficient from the linear regression of the ln(OD 600 ) vs. time (h) curve in the exponential growth phase. Selection of mutant strains We conducted the selection of mutant P. laurentii UFV-1 strains as described by Mikumo et al. ( 2015 ) with modifications. First, we activated the UV-induced mutant strains and plated them in YNBX2DG agar, followed by incubation at 30°C for 4 d. To screen the most 2DG resistant isolates, we cultivated the colonies formed in these plates in liquid YNBX medium for 18 h at 30°C/200 rpm, washed the cells twice with peptone water (0.1% w/v), adjusted the OD 600 to 0.5, serially diluted, plated in YNBX agar medium with 700 µg of 2DG and incubated them for 4 d at 30°C. Next, we cultivated the isolates that presented growth in 25 mL of YNBX2DG in 125 mL Erlenmeyer’s flasks under agitation (200 rpm) at 30°C for 5 d or until the medium became turbid. Finally, we selected the mutant that presented the highest OD 600 after this cultivation. Furthermore, we cultivated the most promising mutant and the wildtype isolate in YNB plus glucose (10 g/L – 55 mM) and xylose (10 g/L – 66 mM) to evaluate the sugar consumption profile and lipid production. We also assessed their lipid production in SS2 medium plus xylose (30 g/L – 200 mM) with a C:N ratio of 100:1 [(NH 4 ) 2 SO 4 (0.9 g/L), NaCl (0.1 g/L), CaCl 2 (0.1 g/L), MgSO 4 (0.5 g/L), yeast extract (1 g/L)]. We conducted cultivations in 250 mL Erlenmeyer’s flasks with 50 mL of culture medium at 200 rpm/30°C for 48 h. Physiological comparison between wildtype and mutant strains In order to compare the physiological behavior between the wildtype and most promising mutant strain of P. laurentii UFV-1, we cultivated them in YNB containing glucose and/or xylose (both 10 g/L; 111 and 133 mM, respectively,). We monitored cell growth (OD 600 ) each 4 h in the first 24 h, then at 30 and 48 h. The biomass yield (Y x/s ) comprised the angular coefficient of the linear regression of the biomass (g/L) vs. sugar concentration (g/L) plot. The total biomass production is represented by the difference between the final and initial dry biomass expressed in g/L. Moreover, to assess the effect of 2DG in the xylose metabolism of these two P. laurentii strains, we cultivated cells in YNBX2DG under agitation (200 rpm) at 30°C for 76 h with an initial OD 600 of 0.5. During these cultivations, we monitored the OD 600 and the consumption of 2DG and xylose. Determination of sugar consumption profile We analyzed the concentration of sugars (and the sugar analog 2DG) in the culture supernatant by High Performance Liquid Chromatography (HLPC). First, we collected the supernatant by centrifugation (10,000 g for 10 min at 4°C), then filtered it in a 0.22 µm membrane (Allcrom, Brazil). The HLPC conditions were as follows: LCTO-20AT (Shimadzu, Japan); refractive index detection (RID-20A); Aminex HPX-87H column (300 x 7.8 mm) at 45°C; mobile phase: 5 mM sulfuric acid at 0.7 mL/min. We determined the sugar concentration using calibration curves based on external standards of glucose, xylose and 2DG (Sigma-Aldrich, USA). Lipid quantification We quantified the cell lipids as described by Bligh and Dyer ( 1959 ) with modifications by Vieira et al. ( 2020a ). Results Growth of P. laurentii UFV-1 in culture media containing glucose, xylose, or glucose plus xylose To evaluate the affinity of P. laurentii UFV-1 to glucose and xylose, we first estimated its maximum growth rate (µ max ) and saturation constant (K s ) for both sugars based on the Monod model (Fig. 1 ). The yeast presented a higher affinity for glucose, although the Ks and µ max values were close for glucose and xylose. We also observed that the growth rates (Table 1 ) and growth profiles (Fig. 2 ) in culture media with different concentrations of glucose and/or xylose were similar. It should be noted that higher sugar concentrations led to increased biomass production and yield, being the higher values recorded for cultivation in glucose (Table 1 ). In cultivations with only glucose (5 or 20 g/L; 28 or 111 mM) or xylose (20 g/L – 133 mM), most of the sugar was consumed in the first 24 h (Figs. 2 A-C). However, in the presence of both sugars, xylose was only consumed after glucose depletion for both low (5 g/L – 28 mM) and high (20 g/L – 111 mM) initial glucose concentrations (Figs. 2 D and E). Table 1 – Growth parameters of P. laurentii UFV-1 cultivated in YNB medium with glucose and/or xylose. Carbon source Concentration g/L (mM) µ (h − 1 ) Biomass (g/L) Y x/s (g/g) Glucose 5 (28) 0.334 ± 0.027 a 1.628 ± 0.083 d 0.096 ± 0.006 e Glucose 20 (111) 0.327 ± 0.035 a 5.302 ± 0.062 c 0.303 ± 0.005 b Xylose 20 (133) 0.318 ± 0.005 a 4.834 ± 0.489 bc 0.271 ± 0.003 c Glucose + Xylose 5 + 20 (28 + 133) 0.296 ± 0.033 a 6.036 ± 0.571 ab 0.341 ± 0.017 a Glucose + Xylose 20 + 20 (111 + 133) 0.330 ± 0.002 a 6.337 ± 0.220 a 0.223 ± 0.015 d Means followed by the same letter in the same column did not differ statistically ( p > 0.05). Mutagenesis and obtainment of 2DG-resistant strains First, we determined the minimum inhibitory concentration of 2DG for P. laurentii UFV-1 (Figures S1 and S2). Then, we optimized the UV irradiation procedure in varied times and heights (Figures S3 and S4). The best time was 9 min and 15 s, and the best height was 10 cm, which resulted in a mortality rate of about 99% (Figure S5 and Table S1 ). In this condition, we isolated 30 possible mutants, herein called M1 to 30. Among them, 24 could grow in the presence of 650 µg/mL of 2DG and 14 in the presence of 700 µg/mL (Figure S6 and 7). Next, we cultivated these 14 mutants in liquid YNBX2DG media to determine the best candidate (higher OD 600 in the presence of 650 µg/mL) (Figure S8 and Table 2 ). Since M17 presented the higher OD 600 after 5 d (Table 2 ), we selected it for further experiments. Table 2 – Growth of the 14 best mutant strains and wildtype P. laurentii UFV-1 strain on liquid YNB containing xylose (20 g/L – 133 mM) and 2DG (650 µg/mL) at 30°C/200 rpm for 5 d. Strain OD 600 Wildtype 0.0212 M1 0.0062 M2 0.0171 M4 0.0165 M5 0.1535 M6 0.0062 M9 0.2120 M15 0.2000 M16 0.1590 M17 1.1915 M20 0.0102 M23 0.0096 M26 0.0253 M27 0.0068 M30 0.0142 Physiological characterization of the M17 strain compared to the wildtype Since the M17 strain presented the highest growth in the presence of 2DG, we selected it for further physiological characterization. Its growth rate on glucose media did not differ from the wild strain; nevertheless, M17 grew faster in xylose than the wild strain (Table 3 ). Regarding the sugar consumption profile, both strains completely consumed glucose or xylose in the first 20 or 24 h of growth, respectively (Figs. 3 A-D). In the cultivations containing both carbon sources, the growth and sugar consumption did not differ between the strains (Table 3 and Fig. 3 E, F), indicating that, although M17 grows better in xylose than the wildtype, it is not less sensitive to glucose catabolite repression. This result showed that the combination of mutagenesis and selection in media with 2DG might not be a suitable tool to select P. laurentii strains capable of co-assimilating glucose and xylose. Table 3 – Specific growth rate of P. laurentii UFV-1 wildtype and M17 strains in YNB plus glucose and/or xylose (10 g/L) at 30°C and 200 rpm. Strain Glucose (10 g/L – 55 mM) Xylose (10 g/L – 67 mM) Glucose and Xylose (10 g/L – 55 and 67 mM) µ (h − 1 ) µ (h − 1 ) µ (h − 1 )* Wildtype 0.324 ± 0.018 a 0.249 ± 0.003 b 0.262 ± 0.001 a M17 0.319 ± 0.004 a 0.289 ± 0.010 a 0.262 ± 0.003 a *Calculated in the glucose exponential phase. Means followed by the same letter in the same column did not differ statistically ( p > 0.05). Effects of 2DG on xylose metabolism Since the M17 was not less sensitive to glucose catabolite repression, we hypothesized that its mutations might be specific to the repression promoted by 2DG. Thus, we raised two hypotheses: (i) 2DG induces catabolite repression in P. laurentii ; (ii) M17 developed response mechanisms to deal with 2DG that did not affect glucose catabolite repression. To evaluate these hypotheses, we cultivated both strains (wildtype and M17) on YNB media plus xylose and 2DG (Fig. 4 ). The wild strain neither grew nor consumed xylose in the first 24 h, indicating that 2DG repressed the use of the alternative carbon source (xylose). Importantly, the wild strain started growing after excreting the assimilated 2DG back to the extracellular space (Fig. 4 A). Hence, excretion might be one of the mechanisms to cope with 2DG presence in wild P. laurentii strains. In contrast to the wildtype strain, M17 grew and consumed xylose simultaneously with 2DG assimilation (Fig. 4 B); therefore, although glucose catabolite repression was not alleviated, the repression promoted by 2DG was mitigated. It should be noted that the M17 strain seems to incorporate 2DG in its biomass as a detoxification mechanism. Comparison of lipid production between wildtype and M17 strains Since P. laurentii displays potential to produce lipids in lignocellulosic-based biorefineries, we evaluated the effect of mutagenesis and 2DG selection on the oleaginous phenotype in the M17 strain compared to the wildtype in a medium with a high C:N (100:1) ratio and a low nitrogen concentration (0.30 g/L). Although the strains did not differ regarding the following parameters: biomass yield, lipid titer and lipid content; M17 grew slower than the wildtype strain (Table 4 ). This indicates that the mutant strain had its growth rate impaired under nitrogen-limiting conditions; however, the final biomass and lipid accumulation parameters were not affected. Table 4 – Growth and lipid production of P. laurentii UFV-1 wildtype and M17 strains in SS2 medium containing xylose (30 g/L) with C:N = 100:1. Strain µ (h − 1 ) Final biomass (g/L) Y x/s (g/g) Lipid % (w/w) Lipids (g/L) Wildtype 0.227 ± 0.003 a 5.085 ± 0.317 a 0.23 ± 0.02 a 40.72 ± 3.01 a 2.14 ± 0.15 a M17 0.210 ± 0.002 b 4.603 ± 1.394 a 0.30 ± 0.04 a 38.59 ± 0.86 a 1.77 ± 0.51 a Means followed by the same letter in the same column did not differ statistically ( p > 0.05). Discussion Papiliotrema laurentii UFV-1 can assimilate lignocellulosic sugars such as glucose and xylose and convert them into lipids in culture media with high C:N ratios under nitrogen restriction (Vieira et al., 2020a ; Vieira et al., 2020b ). The co-assimilation of glucose and xylose is desirable to reduce the production time and increase volumetric productivity in lignocellulosic-based biorefineries. Thus, we first evaluated the kinetics of glucose and xylose assimilation in P. laurentii UFV-1 and assessed how glucose affects the consumption of xylose in culture media containing both sugars. We showed that the yeast had a higher affinity for glucose than xylose (Fig. 1 ), and that glucose is the preferred carbon source (Fig. 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 first 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. This strain presented the highest growth in culture media containing 650 µg/mL of 2DG, grew faster than the wild strain in YNB media with xylose as the sole carbon source, and preserved the oleaginous phenotype (Tables 2 – 4 ). However, the M17 strain presented the same sugar consumption profile of the wildtype strain in mixed glucose-xylose media (Fig. 3 ), that is, it is still sensitive to glucose catabolite repression. Therefore, although the combination of mutagenesis with 2DG selection led to a strain with improved xylose growth and 2DG resistance, it was not a suitable strategy to select Papiliotrema laurentii mutant strains with relaxed carbon catabolite repression. Next, we assessed how 2DG affected xylose growth of the M17 strain compared to the wildtype strain. We found that the wild strain only resumed growth after exporting the 2DG, previously assimilated, back to the media and poorly consumed xylose (Fig. 4 A). The secretion of 2DG has also been observed in other yeasts (Reference); therefore, it appears to be a common strategy to circumvent its inhibitory effect on yeast growth. In contrast, M17 grew promptly in the presence of 2DG and consumed all the xylose available (Fig. 4 B). Interestingly, M17 did not export 2DG back to the extracellular space; instead, it assimilated the toxic compound, which is likely a detoxification strategy. In Saccharomyces cerevisiae , in which the glucose catabolite repression phenomenon is better described, 2DG is captured by cells and similarly phosphorylated by hexokinase, forming 2-DG-6-P. The absence of the hydroxyl group in the C2 of 2DG impairs its isomerization by phosphoglucose isomerase and, in turn, its use in the next steps of glycolysis, which severely impairs growth. Meanwhile, the structural similarity of 2DG-6-P is enough to its accumulation intracellularly to activate the signaling pathways related to glucose catabolite repression, which blocks the use of alternative carbon sources and arrests growth (Schmidt and O’Donnell 2021 ). Yeast strains tolerant to 2DG can present different characteristics, including hyperactive Snf1 signaling, induction of DOG phosphatases (converts 2DG-6-P back to 2DG for further export), improved production of α-arrestins, and modulation of the expression of sugar transporters with different affinities (Gao et al., 2019 ; Laussel and Léon, 2020 ). Besides, 2DG-6-P can be converted to 6-phospho-2-deoxygluconate by glucose-6-phosphate dehydrogenase, providing NADPH and entering the pentose-phosphate pathway (PPP). Consistent with this, increased flux in the PPP and eritrose-4-phosphate accumulation have been described in the presence of 2DG (Laussel and Léon, 2020 ). Another pathway for incorporating 2DG into yeast biomass is via protein and lipid glycosylation due to its structural similarity to mannose. In some cases, this incorporation can interfere with N-glycosylation and promote protein misfolding and endoplasmic reticulum stress and trigger the unfolded protein response (UPR) (Laussel and Léon, 2020 ). Based on these mechanisms, we hypothesize that the wildtype strain might have increased phosphatase activity since the compound is exported to the medium after response onset in concentrations equal to the beginning of cultivation. On the other hand, the M17 strain, tolerant to 2DG, did not export 2DG and seems to incorporate it on its biomass. It remains elusive if this incorporation occurs via PPP and/or protein/lipid glycosylation, as well as if it triggers the UPR in Papiliotrema laurentii . Besides, the faster growth of M17 on xylose compared to the wildtype strain might also be related to a higher capacity to regenerate the NADPH pool. This regeneration, besides being crucial for xylose conversion to xylitol, would also be useful for a robust response to the stress generated by 2DG, as well as to its incorporation in the biomass via PPP and/or biosynthetic pathways. Nevertheless, the differences in NADPH availability between the two strains require further evaluation. Conclusion In the present study, we combined random mutagenesis and 2DG and selected for the first time mutant strains of Papiliotrema laurentii UFV-1 tolerant to 2DG. The M17 strain stood out and was further characterized compared to the wild strain. Although M17 grew faster in xylose and preserved the oleaginous phenotype, it did not present a relaxed carbon catabolite repression or glucose-xylose co-assimilation. Moreover, we found that the tolerant strain grew readily in the presence of 2DG and xylose, possibly incorporating the toxic compound on its biomass, while the wildtype strain presented an arrested growth and only grew after exporting 2DG back to the media. Carbon catabolite repression and 2DG response mechanisms are poorly studied in Basidiomycota yeast. Future studies applying omics tools, as well as molecular approaches, based on the M17 and wild strains can deepen our understanding of which mechanisms are employed by Papiliotrema laurentii to cope with 2DG stress and provide insights why the resistance phenotype might not be related to a relaxation on glucose catabolite repression. Declarations Conflict of interest statement The authors have no relevant financial or non-financial interests to disclose. Funding statement This study was financed by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). This work is part of the project “INCT Yeasts: Biodiversity, preservation and biotechnological innovation”, funded by CNPq, grant #406564/2022-1. References Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917 Gallego-García M, Susmozas A, Negro MJ, Moreno AD (2023) Challenges and prospects of yeast-based microbial oil production within a biorefinery concept. Microb Cell Fact 22:246. https://doi.org/10.1186/s12934-023-02254-4 Gao M, Ploessl D, Shao Z (2019) Enhancing the Co-utilization of Biomass-Derived Mixed Sugars by Yeasts. Front Microbiol 9:3264. https://doi.org/10.3389/fmicb.2018.03264 Kahar P, Taku K, Tanaka S (2011) Enhancement of xylose uptake in 2-deoxyglucose tolerant mutant of Saccharomyces cerevisiae. J Biosci Bioeng 111:557–563. https://doi.org/10.1016/j.jbiosc.2010.12.020 Kayikci Ö, Nielsen J (2015) Glucose repression in Saccharomyces cerevisiae . FEMS Yeast Res 15:fov068. https://doi.org/10.1093/femsyr/fov068 Laussel C, Léon S (2020) Cellular toxicity of the metabolic inhibitor 2-deoxyglucose and associated resistance mechanisms. Biochem Pharmacol 182:114213. https://doi.org/10.1016/j.bcp.2020.114213 McCartney RR, Chandrashekarappa DG, Zhang BB, Schmidt MC (2014) Genetic Analysis of Resistance and Sensitivity to 2-Deoxyglucose in Saccharomyces cerevisiae . Genetics 198:635–646. https://doi.org/10.1534/genetics.114.169060 Mikumo D, Takaya M, Orikasa Y, Ohwada T (2015) Improved Leavening Ability of a Wild Yeast, Saccharomyces cerevisiae AK46 2-deoxyglucose Resistant Mutant. FSTR 21:623–630. https://doi.org/10.3136/fstr.21.623 Nalabothu RL, Fisher KJ, LaBella AL et al (2023) Codon Optimization Improves the Prediction of Xylose Metabolism from Gene Content in Budding Yeasts. Mol Biol Evol 40:msad111. https://doi.org/10.1093/molbev/msad111 Ochoa-Chacón A, Martinez A, Poggi-Varaldo HM et al (2022) Xylose Metabolism in Bioethanol Production: Saccharomyces cerevisiae vs Non-Saccharomyces Yeasts. Bioenerg Res 15:905–923. https://doi.org/10.1007/s12155-021-10340-x Ralser M, Wamelink MM, Struys EA et al (2008) A catabolic block does not sufficiently explain how 2-deoxy- d -glucose inhibits cell growth. Proc Natl Acad Sci USA 105:17807–17811. https://doi.org/10.1073/pnas.0803090105 Rincón AM, Codón AC, Castrejón F, Benı́tez T (2001) Improved Properties of Baker’s Yeast Mutants Resistant to 2-Deoxy- d -Glucose. Appl Environ Microbiol 67:4279–4285. https://doi.org/10.1128/AEM.67.9.4279-4285.2001 Schmidt MC, O’Donnell AF (2021) Sugarcoating’ 2-deoxyglucose: mechanisms that suppress its toxic effects. Curr Genet 67:107–114. https://doi.org/10.1007/s00294-020-01122-7 Suprayogi, Nurcholis M, Murata M et al (2016) Characteristics of MX4-inserted Mutants that Exhibit 2-Deoxyglucose Resistance in Thermotolerant Yeast. Open Biot J 10:208–222. https://doi.org/10.2174/18740707016100100208 Trichez D, Steindorff AS, De Morais Júnior WG et al (2023) Identification of traits to improve co-assimilation of glucose and xylose by adaptive evolution of Spathaspora passalidarum and Scheffersomyces stipitis yeasts. Appl Microbiol Biotechnol 107:1143–1157. https://doi.org/10.1007/s00253-023-12362-1 Vieira N, Zandonade Ventorim R, de Moura Ferreira MA et al (2020a) Insights into oleaginous phenotype of the yeast Papiliotrema laurentii. Fungal Genet Biol 144:103456. https://doi.org/10.1016/j.fgb.2020.103456 Vieira NM, Dos Santos RCV, Germano VKDC et al (2020b) Isolation of a new Papiliotrema laurentii strain that displays capacity to achieve high lipid content from xylose. 3 Biotech 10:382. https://doi.org/10.1007/s13205-020-02373-4 Winston F (2008) EMS and UV mutagenesis in yeast. Current Protocols in Molecular Biology 1–5. https://doi.org/10.1002/0471142727.mb1303bs82 Yamada M, Kosaka T (2015) A Kluyveromyces marxianus 2-deoxyglucose-resistant mutant with enhanced activity of xylose utilization. Int Microbiol 235–244. https://doi.org/10.2436/20.1501.01.255 Supplementary Files Supplementarymaterials.docx Cite Share Download PDF Status: Published Journal Publication published 04 Dec, 2024 Read the published version in Biologia → Version 1 posted Editorial decision: Major revisions 14 Oct, 2024 Reviewers invited by journal 12 Jul, 2024 Editor invited by journal 12 Jul, 2024 Editor assigned by journal 10 Jul, 2024 First submitted to journal 08 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4693745","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":326287000,"identity":"1f209313-9f1c-487e-ba56-287e250f5493","order_by":0,"name":"Eduardo Luís Menezes de Almeida","email":"","orcid":"","institution":"Universidade Federal de Viçosa: Universidade Federal de Vicosa","correspondingAuthor":false,"prefix":"","firstName":"Eduardo","middleName":"Luís Menezes","lastName":"de Almeida","suffix":""},{"id":326287001,"identity":"038f99f2-24c8-4526-89a3-a65807f96da0","order_by":1,"name":"Pâmela Carvalho Lobato","email":"","orcid":"","institution":"Universidade Federal de Vicosa","correspondingAuthor":false,"prefix":"","firstName":"Pâmela","middleName":"Carvalho","lastName":"Lobato","suffix":""},{"id":326287002,"identity":"b6ff595e-be67-4ea6-9413-70bdca29d462","order_by":2,"name":"Rafaela Zandonade Ventorim","email":"","orcid":"","institution":"Universidade Federal de Viçosa: Universidade Federal de Vicosa","correspondingAuthor":false,"prefix":"","firstName":"Rafaela","middleName":"Zandonade","lastName":"Ventorim","suffix":""},{"id":326287003,"identity":"ce033399-5766-4ad3-83c4-518f0fc47413","order_by":3,"name":"Wendel Batista da Silveira","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIie3PMWvCQBTA8ScPzuVwTgnUr/BCICCI+SpPhGZRsFuhQ80SF93Tb+EHcLgSOJfQOZstgpODbhk69Gjr0MGLo8P9pzt4P3gPwOW60TyYAoi2eTHAPaACbCZkCP6SEARfSc5jw1kToc3yrToSxB2UwcfnepCstCR8XFtI+T7q5QTDDGVIvB9Nfki+v0yiahz5koAFigePFU5W24xRKgvZHiL/yyxmSFKzeklIiwZSycg357cyRA2sCiaNykrichz2FuSZW7Awi22CV0OK3ELu5mVQ1U/9uDtP01Otnrsd3Up3Uwv5y/v/bQYul8vlsvYNBwlKl/jUDP8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7869-8144","institution":"Universidade Federal de Viçosa: Universidade Federal de Vicosa","correspondingAuthor":true,"prefix":"","firstName":"Wendel","middleName":"Batista da","lastName":"Silveira","suffix":""}],"badges":[],"createdAt":"2024-07-05 17:47:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4693745/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4693745/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11756-024-01847-7","type":"published","date":"2024-12-04T15:57:37+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61887203,"identity":"da962dc4-9d8a-41ad-bf8d-407ca7d452dd","added_by":"auto","created_at":"2024-08-06 17:01:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3361441,"visible":true,"origin":"","legend":"\u003cp\u003eMonod model adjustment for \u003cem\u003eP. laurentii\u003c/em\u003e UFV-1 cultivated in YNB medium plus (A) glucose and (B) xylose with concentrations between 1 and 15 mM.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4693745/v1/951a89273efdae3ee9a61a8e.png"},{"id":61887202,"identity":"994a2d14-d7cd-460e-85b5-71530c8af245","added_by":"auto","created_at":"2024-08-06 17:01:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":154816,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth and sugar consumption of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. laurentii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e UFV-1 cultivated in YNB medium containing glucose and/or xylose.\u003c/strong\u003e (A) Xylose (20 g/L – 133 mM); (B) Glucose (20 g/L - 111 mM); (C) Glucose (5g/L – 28 mM); (D) Glucose (5 g/L – 28 mM) and Xylose (20 g/L – 133 mM); and (E) Glucose (20 g/L – 111 mM) and Xylose (20 g/L – 133 mM). White dots represent the ln(OD\u003csub\u003e600\u003c/sub\u003e), black dots represent xylose concentration and black triangles represent glucose concentration. Data are presented as means and standard deviations (n = 3).\u003c/p\u003e","description":"","filename":"Figure28.png","url":"https://assets-eu.researchsquare.com/files/rs-4693745/v1/92d8f515bb5e9c22e186193d.png"},{"id":61887205,"identity":"ecb6ef11-464e-42fc-a88a-3c10081da95d","added_by":"auto","created_at":"2024-08-06 17:01:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1585132,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth and sugar consumption of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. laurentii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e wildtype and M17 cultivated in YNB medium containing glucose and/or xylose.\u003c/strong\u003e (A) Wildtype and (B) M17 on Glucose (10 g/L – 55 mM); (C) Wildtype and (D) M17 on Xylose (10 g/L – 66 mM); (E) Wildtype and (F) M17 on Glucose and Xylose (both 10 g/L - 55 and 67 mM). White dots represent the ln(OD\u003csub\u003e600\u003c/sub\u003e), black dots represent xylose concentration and black triangles represent glucose concentration. Data are presented as means and standard deviations (n = 3).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4693745/v1/8636900af5da58047880ffe0.png"},{"id":61887204,"identity":"b770bd04-13fe-445d-bd8e-1dd9932445ab","added_by":"auto","created_at":"2024-08-06 17:01:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":657369,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth and sugar consumption of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. laurentii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e wildtype and M17 cultivated in YNB medium containing xylose (20 g/L) and 2DG (650 µg/mL).\u003c/strong\u003e (A) Wildtype and (B) M17. White dots represent the ln(OD\u003csub\u003e600\u003c/sub\u003e), black dots represent xylose concentration and black triangles represent 2DG concentration. Data are presented as means and standard deviations (n = 3).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4693745/v1/94c716282ada1b887f88885d.png"},{"id":70965455,"identity":"e9380cd0-49f5-41ea-b175-4fee8211c624","added_by":"auto","created_at":"2024-12-09 16:19:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11496803,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4693745/v1/cedb51fb-cd73-4abb-81d4-8b11a00f9276.pdf"},{"id":61887206,"identity":"83128cf8-ac33-4ee4-8f48-728071f62b57","added_by":"auto","created_at":"2024-08-06 17:01:20","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":9753299,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-4693745/v1/ae1528cd3ca5fc4ce0b2df84.docx"}],"financialInterests":"","formattedTitle":"Mutagenesis combined with 2-deoxyglucose is not a suitable tool to select strains of Papiliotrema laurentii less sensitive to glucose catabolite repression","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOver the last years, lignocellulosic biomasses have gained special interest as raw materials to produce different biobased products such as fuels, food ingredients and oleochemicals. Some oleaginous yeasts are capable of accumulating at least 20% of its dry biomass as lipids from sugars found in lignocellulosic hydrolysates such as glucose, xylose, arabinose, cellobiose, and galactose (Gallego-Garc\u0026iacute;a et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). \u003cem\u003ePapiliotrema laurentii\u003c/em\u003e UFV-1 was previously isolated and selected by our research group due to its capacity to accumulate high lipid contents using xylose as the sole carbon source (Vieira et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e). Nevertheless, it displays glucose catabolite repression, that is, glucose is assimilated prior to alternative carbon sources, extending the time of lipid production in culture media containing lignocellulosic hydrolysates. Hence, obtaining strains of \u003cem\u003eP. laurentii\u003c/em\u003e less sensitive to carbon catabolite repression and capable of co-assimilating glucose and xylose is mandatory to achieve high volumetric lipid productivity in biorefineries and boost its application in industrial bioprocesses.\u003c/p\u003e \u003cp\u003eIn wild yeasts that can use xylose as sole carbon source, such as \u003cem\u003eSpathaspora passalidarum\u003c/em\u003e, \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e, \u003cem\u003eScheffersomyces stipitis\u003c/em\u003e, and \u003cem\u003eMeyerozyma guilliermondii\u003c/em\u003e, xylose is metabolized in the pentose phosphate pathway (PPP) through three reactions: first, xylose is converted to xylitol by xylose reductase, which requires NADPH or NADH; then xylitol is dehydrogenated to xylulose by xylose dehydrogenase, which recycles NAD\u003csup\u003e+\u003c/sup\u003e to NADH; finally, xylulose is phosphorylated by xylulose kinase, entering the PPP as xylulose-5-phosphate (Nalabothu et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In most xylose-consuming yeasts, xylose reductase prefers NADPH as cofactor (Ochoa-Chac\u0026oacute;n et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Nalabothu et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe 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 (Rinc\u0026oacute;n et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Kahar et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Mikumo et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Yamada and Kosaka \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Suprayogi et al. 2016; Gao et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Trichez et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Most studies have focused on Ascomycota yeast, mainly \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e (Kahar et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Mikumo et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Rinc\u0026oacute;n et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e (Suprayogi et al., 2016; Yamada and Kosaka, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and \u003cem\u003eScheffersomyces stipitis\u003c/em\u003e and \u003cem\u003eSpathaspora passalidarum\u003c/em\u003e (Trichez et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Glucose catabolite repression and the effects of 2DG are well described in \u003cem\u003eS. cerevisiae\u003c/em\u003e (Ralser et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; McCartney et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kayikci and Nielsen \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Laussel and L\u0026eacute;on \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Schmidt and O\u0026rsquo;Donnell \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this yeast, 2DG is captured by cells and phosphorylated by hexokinase, forming 2-DG-6-P. The absence of the hidroxil group in the C2 of 2DG impairs its isomerization by phosphoglucose isomerase, preventing its use in the next steps of glycolysis. The accumulation of 2DG-6-P in the intracellular medium activates the signaling pathways related to glucose catabolite repression due to its structural similarity to glucose-6-phosphate, which blocks the use of alternative carbon sources and arrests growth (Schmidt and O\u0026rsquo;Donnell \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eYeasts that become resistant to 2DG can present modifications in the glucose catabolite repression signaling and can use alternative carbon sources even in the presence of glucose or 2DG, allowing sugar co-assimilation (Rinc\u0026oacute;n et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Kahar et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Mikumo et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Yamada and Kosaka \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Suprayogi et al. 2016; Gao et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Trichez et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, research on the regulation of carbon catabolite repression is scarce on Basidiomycota yeast and little is known about the proteins involved and the effects of 2DG on the metabolism of these organisms. Therefore, we hypothesized that mutagenesis combined with 2DG selection would be a suitable strategy for selecting strains of \u003cem\u003eP. laurentii\u003c/em\u003e UFV-1 less sensitive to glucose catabolite repression. We selected for the first time a strain of \u003cem\u003ePapiliotrema laurentii\u003c/em\u003e UFV-1 tolerant to 2DG.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMicroorganisms and maintenance\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ePapiliotrema laurentii\u003c/em\u003e UFV-1 strain used in this work belongs to the culture collection of the Microbial Physiology Laboratory, Microbiology Department, Universidade Federal de Vi\u0026ccedil;osa (UFV). The wildtype strain and all mutants obtained are stored at -80\u0026deg;C in Yeast extract (10 g/L) Peptone (20 g/L) (YP) medium with 40% (v/v) glycerol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCulture medium, inoculum preparation, and cell viability\u003c/h2\u003e \u003cp\u003eWe conducted the pre-inoculum cultivations from a single yeast colony from YP plus glucose (20 g/L) and agar (15 g/L) in Erlenmeyer\u0026rsquo;s flasks of 250 mL with 50 mL of Yeast Nitrogen Base (YNB, 6.7 g/L) medium w/o amino acids at 200 rpm/30\u0026deg;C for 18 h. Before all cultivations, unless stated otherwise, we centrifuged (5000 g/5 min at 4\u0026deg;C) and washed cells twice with peptone water (0.1% w/v) and adjusted the initial optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) to about 0.1. We prepared cells suspensions in these conditions to determine the minimum inhibitory concentration of 2DG, survival curve construction, mutagenesis, and for cultivations in glucose and/or xylose.\u003c/p\u003e \u003cp\u003eTo determine the cell viability, we stained yeast cells with methylene blue (1:1). We counted non-stained cells using a Neubauer chamber under an Olympus BX51 microscope (Olympus Corporation of the Americas, Pennsylvania, USA). When necessary, we diluted cell suspensions before staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMinimum inhibitory concentration of 2DG, ultraviolet (UV) survival curve, and mutagenesis\u003c/h2\u003e \u003cp\u003eWe determined the minimum inhibitory concentration of 2DG by cultivating \u003cem\u003eP. laurentii\u003c/em\u003e on YNB agar medium with 133 mM of xylose (YNBX agar) and different 2DG concentrations in the range of 200\u0026ndash;750 \u0026micro;g/mL. We adjusted the washed pre-inoculum to 10\u003csup\u003e8\u003c/sup\u003e viable cells/mL, serially diluted it, then plated the dilutions in YNBX agar plus 2DG. The control comprised cultivations w/o 2DG. The medium containing the minimum inhibitory concentration of 2DG (650 \u0026micro;g/mL) is referred to as YNBX2DG.\u003c/p\u003e \u003cp\u003eNext, we constructed the UV exposition survival curve of \u003cem\u003eP. laurentii\u003c/em\u003e UFV-1 as follows: first, we adjusted the viable cell concentration to 10\u003csup\u003e8\u003c/sup\u003e cells/mL and serially diluted then. Then, we plated 50 \u0026micro;L of each dilution in YNBX agar and irradiated cells using a 4 W lamp (Mineralight\u0026reg; modelo UVS-11) in aseptic conditions. To reach the mortality rate of about 99% as described by (Winston \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), we tested irradiation periods from 1\u0026ndash;10 min and the distance between the UV lamp and plate surface of 10 and 13 cm. We incubated plates at 30\u0026deg;C for 72 h. We conducted all procedures protected from the light to avoid the activation of the photosensitive repair system. After determining the best condition (9 min and 15 s; 10 cm), we conducted mutagenesis. We recovered individual colonies and stored the mutants as described in the \u003cspan refid=\"Sec3\" class=\"InternalRef\"\u003e\u003cem\u003eMicroorganisms and maintenance\u003c/em\u003e\u003c/span\u003e section.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eWildtype strain growth on glucose and/or xylose and Monod model construction\u003c/h2\u003e \u003cp\u003eTo determine the kinetic parameters in media containing glucose and/or xylose as carbon sources, we cultivated the \u003cem\u003eP. laurentii\u003c/em\u003e UFV-1 wildtype strain in YNB media plus xylose 20 g/L (133 mM) (YNB20X); glucose 20 g/L (111 mM) (YNB20G); glucose 5 g/L (28 mM) (YNB5G); glucose 5 g/L (28 mM) and xylose 20 g/L (133 mM) (YNB5G20X); and glucose 20 g/L (111 mM) and xylose 20 g/L (133 mM) (YNB20GX). We cultivated cells for 96 h at 30\u0026deg;C and 200 rpm. We monitored the OD\u003csub\u003e600\u003c/sub\u003e hourly in the first 12 h, then at 24, 48, 72 and 96 h.\u003c/p\u003e \u003cp\u003eNext, we constructed Monod models to evaluate the affinity of \u003cem\u003eP. laurentii\u003c/em\u003e UFV-1 for glucose and xylose. Sugar concentration in YNB media ranged from 1 to 15 mM, which we prepared in volumetric flasks and filtered in 0.22 \u0026micro;m membranes (Millipore). We cultivated cells for 54 h at 30\u0026deg;C and 200 rpm and monitored the OD\u003csub\u003e600\u003c/sub\u003e at 30 min intervals for 12 h then at 24 and 54 h.\u003c/p\u003e \u003cp\u003eIn all cultivations, we measured the OD\u003csub\u003e600\u003c/sub\u003e in a Beckman S\u0026eacute;rie DU 600 spectrophotometer (Indianapolis, USA). The specific growth rate comprised the angular coefficient from the linear regression of the ln(OD\u003csub\u003e600\u003c/sub\u003e) \u003cem\u003evs.\u003c/em\u003e time (h) curve in the exponential growth phase.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSelection of mutant strains\u003c/h2\u003e \u003cp\u003eWe conducted the selection of mutant \u003cem\u003eP. laurentii\u003c/em\u003e UFV-1 strains as described by Mikumo et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) with modifications. First, we activated the UV-induced mutant strains and plated them in YNBX2DG agar, followed by incubation at 30\u0026deg;C for 4 d. To screen the most 2DG resistant isolates, we cultivated the colonies formed in these plates in liquid YNBX medium for 18 h at 30\u0026deg;C/200 rpm, washed the cells twice with peptone water (0.1% w/v), adjusted the OD\u003csub\u003e600\u003c/sub\u003e to 0.5, serially diluted, plated in YNBX agar medium with 700 \u0026micro;g of 2DG and incubated them for 4 d at 30\u0026deg;C. Next, we cultivated the isolates that presented growth in 25 mL of YNBX2DG in 125 mL Erlenmeyer\u0026rsquo;s flasks under agitation (200 rpm) at 30\u0026deg;C for 5 d or until the medium became turbid. Finally, we selected the mutant that presented the highest OD\u003csub\u003e600\u003c/sub\u003e after this cultivation.\u003c/p\u003e \u003cp\u003eFurthermore, we cultivated the most promising mutant and the wildtype isolate in YNB plus glucose (10 g/L \u0026ndash; 55 mM) and xylose (10 g/L \u0026ndash; 66 mM) to evaluate the sugar consumption profile and lipid production. We also assessed their lipid production in SS2 medium plus xylose (30 g/L \u0026ndash; 200 mM) with a C:N ratio of 100:1 [(NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e (0.9 g/L), NaCl (0.1 g/L), CaCl\u003csub\u003e2\u003c/sub\u003e (0.1 g/L), MgSO\u003csub\u003e4\u003c/sub\u003e (0.5 g/L), yeast extract (1 g/L)]. We conducted cultivations in 250 mL Erlenmeyer\u0026rsquo;s flasks with 50 mL of culture medium at 200 rpm/30\u0026deg;C for 48 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhysiological comparison between wildtype and mutant strains\u003c/h2\u003e \u003cp\u003eIn order to compare the physiological behavior between the wildtype and most promising mutant strain of \u003cem\u003eP. laurentii\u003c/em\u003e UFV-1, we cultivated them in YNB containing glucose and/or xylose (both 10 g/L; 111 and 133 mM, respectively,). We monitored cell growth (OD\u003csub\u003e600\u003c/sub\u003e) each 4 h in the first 24 h, then at 30 and 48 h. The biomass yield (Y\u003csub\u003ex/s\u003c/sub\u003e) comprised the angular coefficient of the linear regression of the biomass (g/L) \u003cem\u003evs.\u003c/em\u003e sugar concentration (g/L) plot. The total biomass production is represented by the difference between the final and initial dry biomass expressed in g/L.\u003c/p\u003e \u003cp\u003eMoreover, to assess the effect of 2DG in the xylose metabolism of these two \u003cem\u003eP. laurentii\u003c/em\u003e strains, we cultivated cells in YNBX2DG under agitation (200 rpm) at 30\u0026deg;C for 76 h with an initial OD\u003csub\u003e600\u003c/sub\u003e of 0.5. During these cultivations, we monitored the OD\u003csub\u003e600\u003c/sub\u003e and the consumption of 2DG and xylose.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of sugar consumption profile\u003c/h2\u003e \u003cp\u003eWe analyzed the concentration of sugars (and the sugar analog 2DG) in the culture supernatant by High Performance Liquid Chromatography (HLPC). First, we collected the supernatant by centrifugation (10,000 g for 10 min at 4\u0026deg;C), then filtered it in a 0.22 \u0026micro;m membrane (Allcrom, Brazil). The HLPC conditions were as follows: LCTO-20AT (Shimadzu, Japan); refractive index detection (RID-20A); Aminex HPX-87H column (300 x 7.8 mm) at 45\u0026deg;C; mobile phase: 5 mM sulfuric acid at 0.7 mL/min. We determined the sugar concentration using calibration curves based on external standards of glucose, xylose and 2DG (Sigma-Aldrich, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eLipid quantification\u003c/h2\u003e \u003cp\u003eWe quantified the cell lipids as described by Bligh and Dyer (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1959\u003c/span\u003e) with modifications by Vieira et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGrowth of P. laurentii UFV-1 in culture media containing glucose, xylose, or glucose plus xylose\u003c/h2\u003e \u003cp\u003eTo evaluate the affinity of \u003cem\u003eP. laurentii\u003c/em\u003e UFV-1 to glucose and xylose, we first estimated its maximum growth rate (\u0026micro;\u003csub\u003emax\u003c/sub\u003e) and saturation constant (K\u003csub\u003es\u003c/sub\u003e) for both sugars based on the Monod model (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The yeast presented a higher affinity for glucose, although the Ks and \u0026micro;\u003csub\u003emax\u003c/sub\u003e values were close for glucose and xylose.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also observed that the growth rates (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and growth profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) in culture media with different concentrations of glucose and/or xylose were similar. It should be noted that higher sugar concentrations led to increased biomass production and yield, being the higher values recorded for cultivation in glucose (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In cultivations with only glucose (5 or 20 g/L; 28 or 111 mM) or xylose (20 g/L \u0026ndash; 133 mM), most of the sugar was consumed in the first 24 h (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). However, in the presence of both sugars, xylose was only consumed after glucose depletion for both low (5 g/L \u0026ndash; 28 mM) and high (20 g/L \u0026ndash; 111 mM) initial glucose concentrations (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and E).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003e\u0026ndash;\u003c/b\u003e Growth parameters of \u003cem\u003eP. laurentii\u003c/em\u003e UFV-1 cultivated in YNB medium with glucose and/or xylose.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCarbon source\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentration g/L (mM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026micro; (h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBiomass (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eY\u003csub\u003ex/s\u003c/sub\u003e (g/g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.334\u0026thinsp;\u0026plusmn;\u0026thinsp;0.027 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.628\u0026thinsp;\u0026plusmn;\u0026thinsp;0.083 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.096\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006 e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20 (111)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.327\u0026thinsp;\u0026plusmn;\u0026thinsp;0.035 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.302\u0026thinsp;\u0026plusmn;\u0026thinsp;0.062 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.303\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005 b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eXylose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20 (133)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.318\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.834\u0026thinsp;\u0026plusmn;\u0026thinsp;0.489 bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.271\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose\u0026thinsp;+\u0026thinsp;Xylose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026thinsp;+\u0026thinsp;20 (28\u0026thinsp;+\u0026thinsp;133)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.296\u0026thinsp;\u0026plusmn;\u0026thinsp;0.033 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.036\u0026thinsp;\u0026plusmn;\u0026thinsp;0.571 ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.341\u0026thinsp;\u0026plusmn;\u0026thinsp;0.017 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose\u0026thinsp;+\u0026thinsp;Xylose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u0026thinsp;+\u0026thinsp;20 (111\u0026thinsp;+\u0026thinsp;133)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.330\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.337\u0026thinsp;\u0026plusmn;\u0026thinsp;0.220 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.223\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015 d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eMeans followed by the same letter in the same column did not differ statistically (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMutagenesis and obtainment of 2DG-resistant strains\u003c/h2\u003e \u003cp\u003eFirst, we determined the minimum inhibitory concentration of 2DG for \u003cem\u003eP. laurentii\u003c/em\u003e UFV-1 (Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2). Then, we optimized the UV irradiation procedure in varied times and heights (Figures S3 and S4). The best time was 9 min and 15 s, and the best height was 10 cm, which resulted in a mortality rate of about 99% (Figure S5 and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In this condition, we isolated 30 possible mutants, herein called M1 to 30. Among them, 24 could grow in the presence of 650 \u0026micro;g/mL of 2DG and 14 in the presence of 700 \u0026micro;g/mL (Figure S6 and 7). Next, we cultivated these 14 mutants in liquid YNBX2DG media to determine the best candidate (higher OD\u003csub\u003e600\u003c/sub\u003e in the presence of 650 \u0026micro;g/mL) (Figure S8 and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Since M17 presented the higher OD\u003csub\u003e600\u003c/sub\u003e after 5 d (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), we selected it for further experiments.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003e\u0026ndash;\u003c/b\u003e Growth of the 14 best mutant strains and wildtype \u003cem\u003eP. laurentii\u003c/em\u003e UFV-1 strain on liquid YNB containing xylose (20 g/L \u0026ndash; 133 mM) and 2DG (650 \u0026micro;g/mL) at 30\u0026deg;C/200 rpm for 5 d.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOD\u003csub\u003e600\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWildtype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0212\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0062\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0171\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0165\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.1535\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0062\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.2120\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.2000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.1590\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.1915\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0102\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0096\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0253\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0068\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0142\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePhysiological characterization of the M17 strain compared to the wildtype\u003c/h2\u003e \u003cp\u003eSince the M17 strain presented the highest growth in the presence of 2DG, we selected it for further physiological characterization. Its growth rate on glucose media did not differ from the wild strain; nevertheless, M17 grew faster in xylose than the wild strain (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Regarding the sugar consumption profile, both strains completely consumed glucose or xylose in the first 20 or 24 h of growth, respectively (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D). In the cultivations containing both carbon sources, the growth and sugar consumption did not differ between the strains (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F), indicating that, although M17 grows better in xylose than the wildtype, it is not less sensitive to glucose catabolite repression. This result showed that the combination of mutagenesis and selection in media with 2DG might not be a suitable tool to select \u003cem\u003eP. laurentii\u003c/em\u003e strains capable of co-assimilating glucose and xylose.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003e\u0026ndash;\u003c/b\u003e Specific growth rate of \u003cem\u003eP. laurentii\u003c/em\u003e UFV-1 wildtype and M17 strains in YNB plus glucose and/or xylose (10 g/L) at 30\u0026deg;C and 200 rpm.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eGlucose\u003c/p\u003e \u003cp\u003e(10 g/L \u0026ndash; 55 mM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eXylose\u003c/p\u003e \u003cp\u003e(10 g/L \u0026ndash; 67 mM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGlucose and Xylose\u003c/p\u003e \u003cp\u003e(10 g/L \u0026ndash; 55 and 67 mM)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u0026micro; (h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026micro; (h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026micro; (h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWildtype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.324\u0026thinsp;\u0026plusmn;\u0026thinsp;0.018 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.249\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.262\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.319\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.289\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.262\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e*Calculated in the glucose exponential phase.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eMeans followed by the same letter in the same column did not differ statistically (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffects of 2DG on xylose metabolism\u003c/h2\u003e \u003cp\u003eSince the M17 was not less sensitive to glucose catabolite repression, we hypothesized that its mutations might be specific to the repression promoted by 2DG. Thus, we raised two hypotheses: (i) 2DG induces catabolite repression in \u003cem\u003eP. laurentii\u003c/em\u003e; (ii) M17 developed response mechanisms to deal with 2DG that did not affect glucose catabolite repression. To evaluate these hypotheses, we cultivated both strains (wildtype and M17) on YNB media plus xylose and 2DG (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe wild strain neither grew nor consumed xylose in the first 24 h, indicating that 2DG repressed the use of the alternative carbon source (xylose). Importantly, the wild strain started growing after excreting the assimilated 2DG back to the extracellular space (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Hence, excretion might be one of the mechanisms to cope with 2DG presence in wild \u003cem\u003eP. laurentii\u003c/em\u003e strains. In contrast to the wildtype strain, M17 grew and consumed xylose simultaneously with 2DG assimilation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB); therefore, although glucose catabolite repression was not alleviated, the repression promoted by 2DG was mitigated. It should be noted that the M17 strain seems to incorporate 2DG in its biomass as a detoxification mechanism.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eComparison of lipid production between wildtype and M17 strains\u003c/h2\u003e \u003cp\u003eSince \u003cem\u003eP. laurentii\u003c/em\u003e displays potential to produce lipids in lignocellulosic-based biorefineries, we evaluated the effect of mutagenesis and 2DG selection on the oleaginous phenotype in the M17 strain compared to the wildtype in a medium with a high C:N (100:1) ratio and a low nitrogen concentration (0.30 g/L). Although the strains did not differ regarding the following parameters: biomass yield, lipid titer and lipid content; M17 grew slower than the wildtype strain (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This indicates that the mutant strain had its growth rate impaired under nitrogen-limiting conditions; however, the final biomass and lipid accumulation parameters were not affected.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003e\u0026ndash;\u003c/b\u003e Growth and lipid production of \u003cem\u003eP. laurentii\u003c/em\u003e UFV-1 wildtype and M17 strains in SS2 medium containing xylose (30 g/L) with C:N\u0026thinsp;=\u0026thinsp;100:1.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u0026micro;\u003c/p\u003e \u003cp\u003e(h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFinal biomass (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eY\u003csub\u003ex/s\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(g/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLipid % (w/w)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eLipids\u003c/p\u003e \u003cp\u003e(g/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWildtype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.227\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.085\u0026thinsp;\u0026plusmn;\u0026thinsp;0.317 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e40.72\u0026thinsp;\u0026plusmn;\u0026thinsp;3.01 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.210\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.603\u0026thinsp;\u0026plusmn;\u0026thinsp;1.394 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e38.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eMeans followed by the same letter in the same column did not differ statistically (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003ePapiliotrema laurentii\u003c/em\u003e UFV-1 can assimilate lignocellulosic sugars such as glucose and xylose and convert them into lipids in culture media with high C:N ratios under nitrogen restriction (Vieira et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e; Vieira et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e). The co-assimilation of glucose and xylose is desirable to reduce the production time and increase volumetric productivity in lignocellulosic-based biorefineries. Thus, we first evaluated the kinetics of glucose and xylose assimilation in \u003cem\u003eP. laurentii\u003c/em\u003e UFV-1 and assessed how glucose affects the consumption of xylose in culture media containing both sugars. We showed that the yeast had a higher affinity for glucose than xylose (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and that glucose is the preferred carbon source (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). 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 [\u003cem\u003ee.g., Saccharomyces cerevisiae\u003c/em\u003e (Kahar et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Mikumo et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Rinc\u0026oacute;n et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e (Suprayogi et al., 2016; Yamada and Kosaka, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e)] and/or with improved assimilation of alternative sugars [\u003cem\u003eScheffersomyces stipitis\u003c/em\u003e and \u003cem\u003eSpathaspora passalidarum\u003c/em\u003e (Trichez et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)], we applied this strategy to select for the first time mutant strains of \u003cem\u003ePapiliotrema laurentii\u003c/em\u003e (Basidiomycota yeast) with these characteristics. Among the 14 mutant strains selected due to their resistance to inhibitory concentrations of 2DG, the M17 stood out. This strain presented the highest growth in culture media containing 650 \u0026micro;g/mL of 2DG, grew faster than the wild strain in YNB media with xylose as the sole carbon source, and preserved the oleaginous phenotype (Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). However, the M17 strain presented the same sugar consumption profile of the wildtype strain in mixed glucose-xylose media (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), that is, it is still sensitive to glucose catabolite repression. Therefore, although the combination of mutagenesis with 2DG selection led to a strain with improved xylose growth and 2DG resistance, it was not a suitable strategy to select \u003cem\u003ePapiliotrema laurentii\u003c/em\u003e mutant strains with relaxed carbon catabolite repression.\u003c/p\u003e \u003cp\u003eNext, we assessed how 2DG affected xylose growth of the M17 strain compared to the wildtype strain. We found that the wild strain only resumed growth after exporting the 2DG, previously assimilated, back to the media and poorly consumed xylose (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The secretion of 2DG has also been observed in other yeasts (Reference); therefore, it appears to be a common strategy to circumvent its inhibitory effect on yeast growth. In contrast, M17 grew promptly in the presence of 2DG and consumed all the xylose available (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Interestingly, M17 did not export 2DG back to the extracellular space; instead, it assimilated the toxic compound, which is likely a detoxification strategy.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, in which the glucose catabolite repression phenomenon is better described, 2DG is captured by cells and similarly phosphorylated by hexokinase, forming 2-DG-6-P. The absence of the hydroxyl group in the C2 of 2DG impairs its isomerization by phosphoglucose isomerase and, in turn, its use in the next steps of glycolysis, which severely impairs growth. Meanwhile, the structural similarity of 2DG-6-P is enough to its accumulation intracellularly to activate the signaling pathways related to glucose catabolite repression, which blocks the use of alternative carbon sources and arrests growth (Schmidt and O\u0026rsquo;Donnell \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Yeast strains tolerant to 2DG can present different characteristics, including hyperactive Snf1 signaling, induction of DOG phosphatases (converts 2DG-6-P back to 2DG for further export), improved production of α-arrestins, and modulation of the expression of sugar transporters with different affinities (Gao et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Laussel and L\u0026eacute;on, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBesides, 2DG-6-P can be converted to 6-phospho-2-deoxygluconate by glucose-6-phosphate dehydrogenase, providing NADPH and entering the pentose-phosphate pathway (PPP). Consistent with this, increased flux in the PPP and eritrose-4-phosphate accumulation have been described in the presence of 2DG (Laussel and L\u0026eacute;on, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Another pathway for incorporating 2DG into yeast biomass is via protein and lipid glycosylation due to its structural similarity to mannose. In some cases, this incorporation can interfere with N-glycosylation and promote protein misfolding and endoplasmic reticulum stress and trigger the unfolded protein response (UPR) (Laussel and L\u0026eacute;on, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Based on these mechanisms, we hypothesize that the wildtype strain might have increased phosphatase activity since the compound is exported to the medium after response onset in concentrations equal to the beginning of cultivation. On the other hand, the M17 strain, tolerant to 2DG, did not export 2DG and seems to incorporate it on its biomass. It remains elusive if this incorporation occurs via PPP and/or protein/lipid glycosylation, as well as if it triggers the UPR in \u003cem\u003ePapiliotrema laurentii\u003c/em\u003e. Besides, the faster growth of M17 on xylose compared to the wildtype strain might also be related to a higher capacity to regenerate the NADPH pool. This regeneration, besides being crucial for xylose conversion to xylitol, would also be useful for a robust response to the stress generated by 2DG, as well as to its incorporation in the biomass via PPP and/or biosynthetic pathways. Nevertheless, the differences in NADPH availability between the two strains require further evaluation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn the present study, we combined random mutagenesis and 2DG and selected for the first time mutant strains of \u003cem\u003ePapiliotrema laurentii\u003c/em\u003e UFV-1 tolerant to 2DG. The M17 strain stood out and was further characterized compared to the wild strain. Although M17 grew faster in xylose and preserved the oleaginous phenotype, it did not present a relaxed carbon catabolite repression or glucose-xylose co-assimilation. Moreover, we found that the tolerant strain grew readily in the presence of 2DG and xylose, possibly incorporating the toxic compound on its biomass, while the wildtype strain presented an arrested growth and only grew after exporting 2DG back to the media. Carbon catabolite repression and 2DG response mechanisms are poorly studied in Basidiomycota yeast. Future studies applying omics tools, as well as molecular approaches, based on the M17 and wild strains can deepen our understanding of which mechanisms are employed by \u003cem\u003ePapiliotrema laurentii\u003c/em\u003e to cope with 2DG stress and provide insights why the resistance phenotype might not be related to a relaxation on glucose catabolite repression.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest statement\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003ch2\u003eFunding statement\u003c/h2\u003e \u003cp\u003eThis study was financed by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior - Brazil (CAPES) - Finance Code 001, Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq), and Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de Minas Gerais (FAPEMIG). This work is part of the project \u0026ldquo;INCT Yeasts: Biodiversity, preservation and biotechnological innovation\u0026rdquo;, funded by CNPq, grant #406564/2022-1.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911\u0026ndash;917\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGallego-Garc\u0026iacute;a M, Susmozas A, Negro MJ, Moreno AD (2023) Challenges and prospects of yeast-based microbial oil production within a biorefinery concept. 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Int Microbiol 235\u0026ndash;244. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2436/20.1501.01.255\u003c/span\u003e\u003cspan address=\"10.2436/20.1501.01.255\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biol","sideBox":"Learn more about [Biologia](http://link.springer.com/journal/11756)","snPcode":"11756","submissionUrl":"https://www.editorialmanager.com/biol/default2.aspx","title":"Biologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"oleaginous yeasts, biorefineries, carbon catabolite repression, 2-deoxyglucose resistance","lastPublishedDoi":"10.21203/rs.3.rs-4693745/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4693745/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAssimilation of major sugars from lignocellulosic biomasses is pivotal for achieving a feasible oil production by oleaginous yeasts in biorefineries. \u003cem\u003ePapiliotrema laurentii\u003c/em\u003e 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 \u003cem\u003eP. laurentii\u003c/em\u003e 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.\u003c/p\u003e","manuscriptTitle":"Mutagenesis combined with 2-deoxyglucose is not a suitable tool to select strains of Papiliotrema laurentii less sensitive to glucose catabolite repression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-06 17:01:15","doi":"10.21203/rs.3.rs-4693745/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2024-10-14T06:27:12+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-12T17:27:04+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Biologia","date":"2024-07-12T13:10:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-10T13:12:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biologia","date":"2024-07-08T09:08:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biol","sideBox":"Learn more about [Biologia](http://link.springer.com/journal/11756)","snPcode":"11756","submissionUrl":"https://www.editorialmanager.com/biol/default2.aspx","title":"Biologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"39828eec-dbc8-4e31-8fec-e6d780758e40","owner":[],"postedDate":"August 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-09T16:15:08+00:00","versionOfRecord":{"articleIdentity":"rs-4693745","link":"https://doi.org/10.1007/s11756-024-01847-7","journal":{"identity":"biologia","isVorOnly":false,"title":"Biologia"},"publishedOn":"2024-12-04 15:57:37","publishedOnDateReadable":"December 4th, 2024"},"versionCreatedAt":"2024-08-06 17:01:15","video":"","vorDoi":"10.1007/s11756-024-01847-7","vorDoiUrl":"https://doi.org/10.1007/s11756-024-01847-7","workflowStages":[]},"version":"v1","identity":"rs-4693745","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4693745","identity":"rs-4693745","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}