Optimal trade-off between boosted tolerance and growth fitness during adaptive evolution of yeast to ethanol shocks.

BACKGROUND: The selection of Saccharomyces cerevisiae strains with higher alcohol tolerance can potentially increase the industrial production of ethanol fuel. However, the design of selection protocols to obtain bioethanol yeasts with higher alcohol tolerance poses the challenge of improving industrial strains that are already robust to high ethanol levels. Furthermore, yeasts subjected to mutagenesis and selection, or laboratory evolution, often present adaptation trade-offs wherein higher stress tolerance is attained at the expense of growth and fermentation performance. Although these undesirable side effects are often associated with acute selection regimes, the utility of using harsh ethanol treatments to obtain robust ethanologenic yeasts still has not been fully investigated. RESULTS: We conducted an adaptive laboratory evolution by challenging four populations (P1-P4) of the Brazilian bioethanol yeast, Saccharomyces cerevisiae PE-2_H4, through 68-82 cycles of 2-h ethanol shocks (19-30% v/v) and outgrowths. Colonies isolated from the final evolved populations (P1c-P4c) were subjected to whole-genome sequencing, revealing mutations in genes enriched for the cAMP/PKA and trehalose degradation pathways. Fitness analyses of the isolated clones P1c-P3c and reverse-engineered strains demonstrated that mutations were primarily selected for cell viability under ethanol stress, at the cost of decreased growth rates in cultures with or without ethanol. Under this selection regime for stress survival, the population P4 evolved a protective snowflake phenotype resulting from BUD3 disruption. Despite marked adaptation trade-offs, the combination of reverse-engineered mutations cyr1A1474T/usv1Δ conferred 5.46% higher fitness than the parental PE-2_H4 for propagation in 8% (v/v) ethanol, with only a 1.07% fitness cost in a culture medium without alcohol. The cyr1A1474T/usv1Δ strain and evolved P1c displayed robust fermentations of sugarcane molasses using cell recycling and sulfuric acid treatments, mimicking Brazilian bioethanol production. CONCLUSIONS: Our study combined genomic, mutational, and fitness analyses to understand the genetic underpinnings of yeast evolution to ethanol shocks. Although fitness analyses revealed that most evolved mutations impose a cost for cell propagation, combination of key mutations cyr1A1474T/usv1Δ endowed yeasts with higher tolerance for growth in the presence of ethanol. Moreover, alleles selected for acute stress survival comprising the P1c genotype conferred stress tolerance and optimal performance under conditions simulating the Brazilian industrial ethanol production.

The joint action of yeast eisosomes and membraneless organelles in response to ethanol stress.

Elevated ethanol concentrations in yeast affect the plasma membrane. The plasma membrane in yeast has many lipid-protein complexes, such as Pma1 (MCP), Can1 (MCC), and the eisosome complex. We investigated the response of eisosomes, MCPs, and membraneless structures to ethanol stress. We found a correlation between ethanol stress and proton flux with quick acidification of the medium. Moreover, ethanol stress influences the symporter expression in stressed cells. We also suggest that acute stress from ethanol leads to increases in eisosome size and SG number: we hypothesized that eisosomes may protect APC symporters and accumulate an mRNA decay protein in ethanol-stressed cells. Our findings suggest that the joint action of these factors may provide a protective effect on cells under ethanol stress.

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

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

Ethanol stress responses in Kluyveromyces marxianus: current knowledge and perspectives.

The rising concern with the emission of greenhouse gases has boosted new incentives for biofuels production, which are less polluting than fossil fuels. Special attention has been given to the second-generation ethanol, as it is produced from abundant feedstocks which do not compete with food production, such as lignocellulosic biomass and whey. Kluyveromyces marxianus stands out in second-generation ethanol production due to its capacity of assimilating lactose, the sugar found in whey, and tolerating high temperatures used in simultaneous saccharification processes. Nonetheless, contrary to Saccharomyces cerevisiae, K. marxianus does not tolerate high ethanol concentrations. Ethanol causes a broad range of toxic effects on yeasts, acting on cell membrane and proteins, as well as inducing the generation of reactive oxygen species (ROS). The ethanol stress responses are not fully understood, mainly in non-conventional yeasts such as K. marxianus. Indeed, many molecular responses to ethanol stress are still inferred from S. cerevisiae. As such, a better understanding of the ethanol stress responses in K. marxianus may provide the basis for improving its use in the biofuel industry. Additionally, the selection of ethanol-tolerant strains by metabolic engineering is useful to provide strains with improved capacity to withstand stressful conditions, as well as to obtain new insights about the ethanol stress responses. Key points • It is still not totally clear why K. marxianus is less tolerant to ethanol than S. cerevisiae. • Understanding the ethanol stress response in K. marxianus is pivotal for improving its application in the biofuel industry. • The Metabolic engineering is expected to improve the ethanol tolerance in K. marxianus.

Modeling the Ethanol Tolerance of the Probiotic Yeast Saccharomyces cerevisiae var. boulardii CNCM I-745 for its Possible Use in a Functional Beer.

Saccharomyces yeasts are able to ferment simple sugars to generate levels of ethanol that are toxic to other yeasts and bacteria. The tolerance to ethanol of different yeasts depends also on the incubation temperature. In this study, the ethanol stress responses of S. cerevisiae and the probiotic yeast S. boulardii CNCM I-745 were evaluated at two temperatures. The growth kinetics parameters were obtained by fitting the Baranyi and Roberts model to the experimental data. The four-parameter logistic Hill equation was used to describe the ethanol tolerance of the yeasts at the temperatures of 28 and 37 °C. Adequate determination coefficients were obtained (R2 > 0.91) in all cases. S. boulardii grown at 28 °C was selected as the yeast with the best ethanol tolerance (6-8%) for use in the elaboration of functional craft beers.

Assessment of ethanol tolerance of Kluyveromyces marxianus CCT 7735 selected by adaptive laboratory evolution.

Kluyveromyces marxianus CCT 7735 shows potential for producing ethanol from lactose; however, its low ethanol tolerance is a drawback for its industrial application. The first aim of this study was to obtain four ethanol-tolerant K. marxianus CCT 7735 strains (ETS1, ETS2, ETS3, and ETS4) by adaptive laboratory evolution. The second aim was to select among them the strain that stood out and to evaluate metabolic changes associated with the improved ethanol tolerance in this strain. The ETS4 was selected for displaying a specific growth rate higher than the parental strain under ethanol stress (122%) and specific ethanol production rate (0.26 g/g/h) higher than those presented by the ETS1 (0.22 g/g/h), ETS2 (0.17 g/g/h), and ETS3 (0.17 g/g/h) under non-stress condition. Further analyses were performed with the ETS4 in comparison with its parental strain in order to characterize metabolic changes. Accumulation of valine and metabolites of the citric acid cycle (isocitric acid, citric acid, and cis-aconitic acid) was observed only in the ETS4 subjected to ethanol stress. Their accumulation in this strain may have been important to increase ethanol tolerance. Furthermore, the contents of fatty acid methyl esters and ergosterol were higher in the ETS4 than in the parental strain. These differences likely contributed to enhance ethanol tolerance in the ETS4. KEY POINTS: • K. marxianus ethanol-tolerant strains were selected by adaptive laboratory evolution. • Valine and metabolites of the TCA cycle were accumulated in the ETS4. • High contents of fatty acids and ergosterol contributed to enhance ethanol tolerance.

Physiological effects of overexpressed sigma factors on fermentative stress response of Zymomonas mobilis.

Zymomonas mobilis is a bacterium of industrial interest due to its high ethanol productivity and high tolerance to stresses. Although the physiological parameters of fermentation are well characterized, there are few studies on the molecular mechanisms that regulate the response to fermentative stress. Z. mobilis ZM4 presents five different sigma factors identified in the genome annotation, but the absence of sigma 38 leads to the questioning of which sigma factors are responsible for its mechanism of fermentative stress response. Thus, in this study, factors sigma 32 and sigma 24, traditionally related to heat shock, were tested for their influence on the response to osmotic and ethanol stress. The overexpression of these sigma factors in Z. mobilis ZM4 strain confirmed that both are associated with heat shock response, as described in other bacteria. Moreover, sigma 32 has also a role in the adaptation to osmotic stress, increasing both growth rate and glucose influx rate. The same strain that overexpresses sigma 32 also showed a decrease in ethanol tolerance, suggesting an antagonism between these two mechanisms. It was not possible to conclude if sigma 24 really affects ethanol tolerance in Z. mobilis, but the overexpression of this sigma factor led to a decrease in ethanol productivity.

The isc gene cluster expression ethanol tolerance associated improves its ethanol production by organic acids flux redirection in the ethanologenic Escherichia coli KO11 strain.

Fossil fuels consumption impacts the greenhouse gas emissions. Biofuels are considered as alternative renewable energy sources to reduce the fossil fuels dependency. Bioethanol produced by recombinant microorganisms is a widely suggested alternative to increase the yield in fermentation processes. However, ethanol and acetate accumulation under the fermentation process had been described as important stressors for the metabolic capabilities of the microorganisms, stopping the fermentation process and affecting the ethanol yield. Ethanol tolerance is a determining factor in the improvement of fermentative properties of microorganisms; however understanding of ethanol tolerance is limited. The engineered Escherichia coli KO11 strain has been studied in detail and used as an ethanologenic bacteria model. The strain is capable of using glucose and xylose for an efficient ethanol yield. In the current work, the effect of the iron-sulfur cluster (ISC) over-expression in the KO11 strain, on its tolerance and ethanol yield, was evaluated. Fatty acids profiles of membrane phospholipids in the E. coli KO11 were modified under ethanol addition, but not due to the hscA mutation. The hscA mutation provoked a decrease in ethanol tolerance in the Kmp strain when was grown with 2% ethanol, in comparison to KO11 parent strain. Ethanol tolerance was improved in the mutant Kmp complemented with the recombinant isc gene cluster (pJC10 plasmid) from LD50 2.16% to LD50 3.8% ethanol. In batch fermentation on 1 L bioreactor using mineral medium with glucose (120 g/L), the KO11 strain showed ethanol production efficiencies of ~ 76.9%, while the hscA mutant (Kmp) ~ 75.4% and the transformed strain Kmp(pJC10) showed ~ 92.4% efficiency. Ethanol amount increase in the engineered Kmp(pJC10) strain was correlated with less organic acids (such as acetate and lactate) production in the fermentation medium (2.3 g/L), compared to that in the KO11 (17.05 g/L) and the Kmp (16.62 g/L). Alcohol dehydrogenase (ADH) activity was increased ~ 350% in the transformed Kmp(pJC10) strain, whereas in the Kmp mutant, the phosphoglycerate kinase (PGK), pyruvate kinase (PYK), and ADH activities were diminished, comparing to KO11. The results suggest that the isc system over-expression in the ethanologenic E. coli KO11 strain, increases ethanol yield mainly by improving ethanol tolerance and ADH activity, and by redirecting the metabolic flux from acetate synthesis to ethanol.

Phenotypic and genomic analysis of Zymomonas mobilis ZM4 mutants with enhanced ethanol tolerance.

Zymomonas mobilis ZM4 is an ethanol-producing microbe that is constitutively tolerant to this solvent. For a better understanding of the ethanol tolerance phenomenon we obtained and characterized two ZM4 mutants (ER79ap and ER79ag) with higher ethanol tolerance than the wild-type. Mutants were evaluated in different ethanol concentrations and this analysis showed that mutant ER79ap was more tolerant and had a better performance in terms of cell viability, than the wild-type strain and ER79ag mutant. Genotyping of the mutant strains showed that both carry non-synonymous mutations in clpP and spoT/relA genes. A third non-synonymous mutation was found only in strain ER79ap, in the clpB gene. Considering that ER79ap has the best tolerance to added ethanol, the mutant alleles of this strain were evaluated in ZM4 and here we show that while all of them contribute to ethanol tolerance, mutation within spoT/relA gene seems to be the most important.

Over-expression of Isu1p and Jac1p increases the ethanol tolerance and yield by superoxide and iron homeostasis mechanism in an engineered Saccharomyces cerevisiae yeast.

The ethanol stress response in ethanologenic yeast during fermentation involves the swishing of several adaptation mechanisms. In Saccharomyces cerevisiae, the Jac1p and Isu1p proteins constitute the scaffold system for the Fe-S cluster assembly. This study was performed using the over-expression of the Jac1p and Isu1p in the industrially utilized S. cerevisiae UMArn3 strain, with the objective of improving the Fe-S assembly/recycling, and thus counteracting the toxic effects of ethanol stress during fermentation. The UMArn3 yeast was transformed with both the JAC1-His and ISU1-His genes-plasmid contained. The Jac1p and Isu1p His-tagged proteins over-expression in the engineered yeasts was confirmed by immunodetection, rendering increases in ethanol tolerance level from a DL50 = ~ 4.5% ethanol (v/v) to DL50 = ~ 8.2% ethanol (v/v), and survival up 90% at 15% ethanol (v/v) comparing to ~ 50% survival in the control strain. Fermentation by the engineered yeasts showed that the ethanol production was increased, producing 15-20% more ethanol than the control yeast. The decrease of ROS and free-iron accumulation was observed in the engineered yeasts under ethanol stress condition. The results indicate that Jac1p and Isu1p over-expression in the S. cerevisiae UMArn3.3 yeast increased its ethanol tolerance level and ethanol production by a mechanism that involves ROS and iron homeostasis related to the biogenesis/recycling of Fe-S clusters dependent proteins.

Osmo-, thermo- and ethanol- tolerances of Saccharomyces cerevisiae S1

Saccharomyces cerevisiae S1, which is a locally isolated and improved strain showed viability at 40, 45 and 50ºC and produced ethanol at 40, 43 and 45ºC. When the cells were given heat shock at 45ºC for 30min and grown at 40ºC, 100% viability was observed for 60h, and addition of 200gl-1 ethanol has led to complete cell death at 30h. Heat shock given at 45ºC (for 30min) has improved the tolerance to temperature induced ethanol shock leading to 37% viability at 30h. when the cells were subjected to ethanol (200gl-1 for 30 min) and osmotic shock (sorbitol 300gl-1), trehalose contents in the cells were increased. The heat shocked cells showed better viability in presence of added ethanol. Soy flour supplementation has improved the viability of S. cerevisiae S1 to 80% in presence of 100gl-1 added ethanol and to 60% in presence of 300gl-1 sorbitol. In presence of sorbitol (200gl-1) and ethanol (50gl-1) at 40ºC, 46% viability was retained by S. cerevisiae S1 at 48h and it was improved to 80% by soy flour supplementation.

Osmo-, Thermo- and Ethanol- Tolerances of Saccharomyces cerevisiae S1.

Saccharomyces cerevisiae S1, which is a locally isolated and improved strain showed viability at 40, 45 and 50°C and produced ethanol at 40, 43 and 45°C. When the cells were given heat shock at 45°C for 30min and grown at 40°C, 100% viability was observed for 60h, and addition of 200gL(-1) ethanol has led to complete cell death at 30h. Heat shock given at 45°C (for 30min) has improved the tolerance to temperature induced ethanol shock leading to 37% viability at 30h. When the cells were subjected to ethanol (200gL(-1) for 30 min) and osmotic shock (sorbitol 300gL(-1)), trehalose contents in the cells were increased. The heat shocked cells showed better viability in presence of added ethanol. Soy flour supplementation has improved the viability of S. cerevisiae S1 to 80% in presence of 100gL(-1) added ethanol and to 60% in presence of 300gL(-1)sorbitol. In presence of sorbitol (200gL(-1)) and ethanol (50gL(-1)) at 40°C, 46% viability was retained by S. cerevisiae S1 at 48h and it was improved to 80% by soy flour supplementation.

Osmo-, thermo- and ethanol- tolerances of Saccharomyces cerevisiae S1

Saccharomyces cerevisiae S1, which is a locally isolated and improved strain showed viability at 40, 45 and 50ºC and produced ethanol at 40, 43 and 45ºC. When the cells were given heat shock at 45ºC for 30min and grown at 40ºC, 100% viability was observed for 60h, and addition of 200gl-1 ethanol has led to complete cell death at 30h. Heat shock given at 45ºC (for 30min) has improved the tolerance to temperature induced ethanol shock leading to 37% viability at 30h. when the cells were subjected to ethanol (200gl-1 for 30 min) and osmotic shock (sorbitol 300gl-1), trehalose contents in the cells were increased. The heat shocked cells showed better viability in presence of added ethanol. Soy flour supplementation has improved the viability of S. cerevisiae S1 to 80% in presence of 100gl-1 added ethanol and to 60% in presence of 300gl-1 sorbitol. In presence of sorbitol (200gl-1) and ethanol (50gl-1) at 40ºC, 46% viability was retained by S. cerevisiae S1 at 48h and it was improved to 80% by soy flour supplementation.

Alcohol dehydrogenase activities and ethanol tolerance in Anastrepha (Diptera, Tephritidae) fruit-fly species and their hybrids.

The ADH (alcohol dehydrogenase) system is one of the earliest known models of molecular evolution, and is still the most studied in Drosophila. Herein, we studied this model in the genus Anastrepha (Diptera, Tephritidae). Due to the remarkable advantages it presents, it is possible to cross species with different Adh genotypes and with different phenotype traits related to ethanol tolerance. The two species studied here each have a different number of Adh gene copies, whereby crosses generate polymorphisms in gene number and in composition of the genetic background. We measured certain traits related to ethanol metabolism and tolerance. ADH specific enzyme activity presented gene by environment interactions, and the larval protein content showed an additive pattern of inheritance, whilst ADH enzyme activity per larva presented a complex behavior that may be explained by epistatic effects. Regression models suggest that there are heritable factors acting on ethanol tolerance, which may be related to enzymatic activity of the ADHs and to larval mass, although a pronounced environmental effect on ethanol tolerance was also observed. By using these data, we speculated on the mechanisms of ethanol tolerance and its inheritance as well as of associated traits.

Alcohol dehydrogenase activities and ethanol tolerance in Anastrepha (Diptera, Tephritidae) fruit-fly species and their hybrids

The ADH (alcohol dehydrogenase) system is one of the earliest known models of molecular evolution, and is still the most studied in Drosophila. Herein, we studied this model in the genus Anastrepha (Diptera, Tephritidae). Due to the remarkable advantages it presents, it is possible to cross species with different Adh genotypes and with different phenotype traits related to ethanol tolerance. The two species studied here each have a different number of Adh gene copies, whereby crosses generate polymorphisms in gene number and in composition of the genetic background. We measured certain traits related to ethanol metabolism and tolerance. ADH specific enzyme activity presented gene by environment interactions, and the larval protein content showed an additive pattern of inheritance, whilst ADH enzyme activity per larva presented a complex behavior that may be explained by epistatic effects. Regression models suggest that there are heritable factors acting on ethanol tolerance, which may be related to enzymatic activity of the ADHs and to larval mass, although a pronounced environmental effect on ethanol tolerance was also observed. By using these data, we speculated on the mechanisms of ethanol tolerance and its inheritance as well as of associated traits.

The time course of ethanol tolerance: associative learning

The effect of different contextual stimuli on different ethanol-induced internal states was investigated during the time course of both the hypothermic effect of the drug and of drug tolerance. Minimitters were surgically implanted in 16 Wistar rats to assess changes in their body temperature under the effect of ethanol. Rat groups were submitted to ethanol or saline trials every other day. The animals were divided into two groups, one receiving a constant dose (CD) of ethanol injected intraperitoneally, and the other receiving increasing doses (ID) during the 10 training sessions. During the ethanol training sessions, conditioned stimuli A (tone) and B (buzzer) were presented at "state +" (35 min after drug injection) and "state -" (170 min after drug injection), respectively. Conditioned stimuli C (bip) and D (white noise) were presented at moments equivalent to stimuli A and B, respectively, but during the saline training sessions. All stimuli lasted 15 min. The CD group, but not the ID group, developed tolerance to the hypothermic effect of ethanol. Stimulus A (associated with drug "state +") induced hyperthermia with saline injection in the ID group. Stimulus B (associated with drug "state -") reduced ethanol tolerance in the CD group and modulated the hypothermic effect of the drug in the ID group. These results indicate that contextual stimuli acquire modulatory conditioned properties that are associated with the time course of both the action of the drug and the development of drug tolerance.