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.

Adaptive responses of Kluyveromyces marxianus CCT 7735 to 2-phenylethanol stress: Alterations in membrane fatty-acid composition, ergosterol content, exopolysaccharide production and reduction in reactive oxygen species.

2-phenylethanol (2-PE) is a higher aromatic alcohol with a rose-like aroma used in the cosmetic and food industries as a flavoring and displays potential for application as an antifungal. Biotechnological production of 2-PE from yeast is an interesting alternative due to the non-use of toxic compounds and the generation of few by-products. Kluyveromyces marxianus CCT 7735 is a thermotolerant strain capable of producing high 2-PE titers from L-Phenylalanine; however, like other yeast species, its growth has been strongly inhibited by this alcohol. Herein, we aimed to evaluate the effect of 2-PE on cell growth, cell viability, membrane permeability, glucose uptake, metabolism, and morphology in K. marxianus CCT 7735, as well as its adaptive responses. The stress condition was imposed after 4 h of cultivation by adding 3.0 g.L-1 of 2-PE in exponential growing cells. 2-PE stress impaired yeast growth, glucose uptake, fermentative metabolism, membrane permeability, and cell viability. Moreover, the stress condition provoked changes in both morphology and surface roughness. The reactive oxygen species (ROS) increased immediately on exposure to 2-PE. Changes in membrane fatty-acid composition, ergosterol content, exopolysaccharides production, and reduction of the ROS levels appear to be the result of adaptive responses in K. marxianus. Our results provided insights into a better understanding of the effects of 2-PE on K. marxianus and its adaptive responses.

Isolation of xylose-assimilating yeasts and optimization of xylitol production by a new Meyerozyma guilliermondii strain.

Production of xylitol from lignocellulosic biomass is of interest to modern biorefineries, because this biomass should be processed into a spectrum of chemicals (bio-based products) and not only energy. The isolation of new yeast strains capable of efficiently converting xylose into xylitol and withstanding inhibitors released from biomass hydrolysis can contribute to making its production feasible in biorefineries. Forty-three out of 128 yeast strains isolated from the gut of Passalidae beetles were capable of assimilating xylose as the sole carbon source. Meyerozyma guilliermondii UFV-1 was selected due to its ability to grow and ferment D-xylose in a synthetic medium. This yeast assimilated the broad range of sugars present in lignocellulosic biomass hydrolysates, such as xylose, raffinose, cellobiose, rhamnose, arabinose, and glucose. Its optimum growth conditions were pH 8.0 and a temperature of 30 °C. In concentrations of 0.07 mol/L acetic acid, 0.05 mol/L 5-hydroximethylfurfural, and 0.04 mol/L furfural, M. guilliermondii UFV-1 did not grow. Maximum xylitol production in aerobiosis and hypoxia were 51.88 and 27.73 g/L, respectively. Under aerobic condition, xylose concentration and agitation rate were the factors which were statistically significant, while only the agitation rate was significant in hypoxia. We fitted a response surface (RS) that estimated the best agitation rate (113.33 rpm) and xylose concentration (90 g/L) for maximum xylitol production in aerobiosis. Therefore, M. guilliermondii UFV-1 displays potential for being used for xylitol production in biorefineries.

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.

Sugar transport systems in Kluyveromyces marxianus CCT 7735.

The pattern of glucose repression in most Kluyveromyces marxianus strains does not correlate with fermentative behaviour; however, glucose repression and fermentative metabolism appear to be linked to the kinetics of sugar uptake. In this work, we show that lactose transport in K. marxianus CCT 7735 by lactose-grown cells is mediated by a low-affinity H+-sugar symporter. This system is glucose repressed and able to transport galactose with low affinity. We also observed the activity of a distinct lactose transporter in response to raffinose. Regarding glucose uptake, specificities of at least three low-affinity systems rely on the carbon source available in a given growth medium. Interestingly, it was observed only one high-affinity system is able to transport both glucose and galactose. We also showed that K. marxianus CCT 7735 regulates the expression of sugar transport systems in response to glucose availability.

Ethanol stress responses of Kluyveromyces marxianus CCT 7735 revealed by proteomic and metabolomic analyses.

Kluyveromyces marxianus CCT 7735 offers advantages to ethanol production over Saccharomyces cerevisiae, including thermotolerance and the ability to convert lactose to ethanol. However, its growth is impaired at high ethanol concentrations. Herein we report on the protein and intracellular metabolite profiles of K. marxianus at 1 and 4 h under ethanol exposure. The concentration of some amino acids, trehalose and ergosterol were also measured. We observed that proteins and metabolites from carbon pathways and translation were less abundant, mainly at 4 h of ethanol stress. Nevertheless, the concentration of some amino acids and trehalose increased at 8 and 12 h under ethanol stress, indicating an adaptive response. Moreover, our results show that the abundance of proteins and metabolites related to the oxidative stresses responses increased. The results obtained in this study provide insights into understanding the physiological changes in K. marxianus under ethanol stress, indicating possible targets for ethanol tolerant strains construction.

Isolation of a new Papiliotrema laurentii strain that displays capacity to achieve high lipid content from xylose.

In this work, we isolated and selected oleaginous yeasts from rock field soils from two National Parks in Brazil (Caparaó and Serra dos Órgãos) with the potential to accumulate oil from xylose, the main pentose sugar found in lignocellulosic biomass. From the 126 isolates, two were selected based on their lipid contents. They were taxonomically identified as Papiliotrema laurentii (UFV-1 and UFV-2). Of the two, P. laurentii UFV-1 was selected as the best lipid producer. Under unoptimized conditions, lipid production by P. laurentii UFV-1 was higher in glucose than in xylose. To improve its lipid production from xylose, we applied response surface methodology (RSM) with a face-centered central composite design (CCF). We evaluated the effects of agitation rate, initial cell biomass (OD600), carbon/nitrogen ratio (C/N ratio) and pH on lipid production. P. laurentii UFV-1 recorded the highest lipid content, 63.5% (w/w) of the cell dry mass, under the following conditions: C/N ratio = 100:1, pH value = 7.0, initial OD600 = 0.8 and agitation = 300 rpm. Under these optimized conditions, biomass, lipid titer and volumetric lipid productivity were 9.31 g/L, 5.90 g/L and 0.082 g/L.h, respectively. Additionally, we determined the fatty acid composition of P. laurentii UFV-1 as follows: C14:0 (0.5%), C16:0 (28.4-29.4%), C16:1 (0.2%), C18:0 (9.5-11%), C18:1 (58.6-60.5%), and C20:0 (0.7-0.8%). Based on this composition, the predicted properties of biodiesel showed that P. laurentii UFV-1 oil is suitable for use as feedstock in biodiesel production.

Selection of yeasts from bee products for alcoholic beverage production.

The use of appropriate yeast strains allows to better control the fermentation during beverage production. Bee products, especially of stingless bees, are poorly explored as sources of fermenting microorganisms. In this work, yeasts were isolated from honey and pollen from Tetragonisca angustula (Jataí), Nannotrigona testaceicornis (Iraí), Frieseomelitta varia (Marmelada), and honey of Apis mellifera bees and screened according to morphology, growth, and alcohol production. Bee products showed to be potential sources of fermenting microorganisms. From 55 isolates, one was identified as Papiliotrema flavescens, two Rhodotorula mucilaginosa, five Saccharomyces cerevisiae, and nine Starmerella meliponinorum. The S. cerevisiae strains were able to produce ethanol and glycerol at pH 4.0-8.0 and temperature of 10-30 °C, with low or none production of undesirable compounds, such as acetic acid and methanol. These strains are suitable for the production of bioethanol and alcoholic beverages due to their high ethanol production, similar or superior to the commercial strain, and in a broad range of conditions like as 50% (m/v) glucose, 10% (v/v) ethanol, or 500 mg L-1 of sodium metabisulfite.