Combined 6-benzylaminopurine and H2O2 stimulate the astaxanthin biosynthesis in Xanthophyllomyces dendrorhous.

Astaxanthin is one of the most attractive carotenoids due to its high antioxidant activity and beneficial biological properties, while Xanthophyllomyces dendrorhous is one of its main microbial sources. Since astaxanthin is synthesized as a response to oxidative stress, several oxidative agents have been evaluated to increase X. dendrorhous astaxanthin yields. However, the extent of the stimulation is determined by the cellular damage caused by the applied oxidative agent. Phytohormones have also been reported as stimulants of astaxanthin biosynthesis acting directly on its metabolic pathway and indirectly promoting cellular resistance to reactive oxygen species. We reasoned that both oxidative agents and phytohormones lead to increased astaxanthin synthesis, but the latter could mitigate the drawbacks of the former. Thus, here, the stimulation on astaxanthin biosynthesis, as well as the cellular and transcriptional responses of wild type X. dendrorhous to phytohormones (6-benzylaminopurine, 6-BAP; abscisic acid, ABA; and indole-3-acetic acid, IAA), and oxidative agents (glutamate, menadione, H2O2, and/or Fe2+) were evaluated as a single or combined treatments. ABA and 6-BAP were the best individual stimulants leading to 2.24- and 2.60-fold astaxanthin biosynthesis increase, respectively. Nevertheless, the effect of combined 6-BAP and H2O2 led to a 3.69-fold astaxanthin synthesis increase (0.127 ± 0.018 mg astaxanthin/g biomass). Moreover, cell viability (> 82.75%) and mitochondrial activity (> 82.2%) remained almost intact in the combined treatment (6-BAP + H2O2) compared to control (< 52.17% cell viability; < 85.3% mitochondrial activity). On the other hand, mRNA levels of hmgR, idi, crtYB, crtR, and crtS, genes of the astaxanthin biosynthetic pathway, increased transiently along X. dendrorhous fermentation due to stimulations assayed in this study. KEY POINTS: • Combined 6-BAP and H2O2 is the best treatment to increase astaxanthin yields in X. dendrorhous. • 6-BAP preserves cell integrity under oxidative H2O2 stress conditions. • 6-BAP and H2O2 increase transcriptional responses of hmgR, idi, and crt family genes transiently.

Physiological and transcriptome analyses of Kluyveromyces marxianus reveal adaptive traits in stress response.

Kluyveromyces marxianus is a non-conventional yeast with outstanding physiological characteristics and a high potential for lignocellulosic ethanol production. However, achieving high ethanol productivity requires overcoming several biotechnological challenges due to the cellular inhibition caused by the inhibitors present in the medium. In this work, K. marxianus SLP1 was adapted to increase its tolerance to a mix of inhibitory compounds using the adaptive laboratory evolution strategy to study the adaptation and stress response mechanisms used by this non-Saccharomyces yeast. The fermentative and physiological parameters demonstrated that the adapted K. marxianus P8 had a better response against the synergistic effects of multiple inhibitors because it reduced the lag phase from 12 to 4 h, increasing the biomass by 40% and improving the volumetric ethanol productivity 16-fold than the parental K. marxianus SLP1. To reveal the effect of adaptation process in P8, transcriptome analysis was carried out; the result showed that the basal gene expression in P8 changed, suggesting the biological capability of K. marxianus to activate the adaptative prediction mechanism. Similarly, we carried out physiologic and transcriptome analyses to reveal the mechanisms involved in the stress response triggered by furfural, the most potent inhibitor in K. marxianus. Stress response studies demonstrated that P8 had a better physiologic response than SLP1, since key genes related to furfural transformation (ALD4 and ALD6) and stress response (STL1) were upregulated. Our study demonstrates the rapid adaptability of K. marxianus to stressful environments, making this yeast a promising candidate to produce lignocellulosic ethanol. KEY POINTS: • K. marxianus was adapted to increase its tolerance to a mix of inhibitory compounds • The basal gene expression of K. marxianus changed after the adaptation process • Adapted K. marxianus showed a better physiological response to stress by inhibitors • Transcriptome analyses revealed key genes involved in the stress response.

The non-Saccharomyces yeast Pichia kluyveri for the production of aromatic volatile compounds in alcoholic fermentation.

Alcoholic fermentation is influenced by yeast strain, culture media, substrate concentration and fermentation conditions, which contribute to taste and aroma. Some non-Saccharomyces yeasts are recognized as volatile compound producers that enrich aromatic profile of alcoholic beverages. In this work, 21 strains of Pichia kluyveri isolated from different fermentative processes and regions were evaluated. A principal component analysis (PCA) showed statistical differences between strains mainly associated with the variety and concentration of the compounds produced. From the PCA, two strains (PK1 and PK8) with the best volatile compound production were selected to evaluate the impact of culture media (M12 medium and Agave tequilana juice), stirring speeds (100 and 250 rpm) and temperatures (20°C, 25°C and 30°C). Increased ester production was observed at 250 rpm. Greatest effect in alcohols and ester production was found with A. tequilana, identifying PK1 as higher alcohol producer, and PK8 as better ester producer. Regarding temperature, PK1 increased ester production with decreased fermentation temperature. PK8 presented maximum levels of ethyl acetate and ethyl dodecanoate at 20°C, and finally isoamyl acetate increased its production at 30°C. Therefore, P. kluyveri strains are of great interest to produce different aromatic profiles that are affected by factors including medium, agitation and temperature.

Metabolic engineering for high yield synthesis of astaxanthin in Xanthophyllomyces dendrorhous.

Astaxanthin is a carotenoid with a number of assets useful for the food, cosmetic and pharmaceutical industries. Nowadays, it is mainly produced by chemical synthesis. However, the process leads to an enantiomeric mixture where the biologically assimilable forms (3R, 3'R or 3S, 3'S) are a minority. Microbial production of (3R, 3'R) astaxanthin by Xanthophyllomyces dendrorhous is an appealing alternative due to its fast growth rate and easy large-scale production. In order to increase X. dendrorhous astaxanthin yields, random mutant strains able to produce from 6 to 10 mg/g dry mass have been generated; nevertheless, they often are unstable. On the other hand, site-directed mutant strains have also been obtained, but they increase only the yield of non-astaxanthin carotenoids. In this review, we insightfully analyze the metabolic carbon flow converging in astaxanthin biosynthesis and, by integrating the biological features of X. dendrorhous with available metabolic, genomic, transcriptomic, and proteomic data, as well as the knowledge gained with random and site-directed mutants that lead to increased carotenoids yield, we propose new metabolic engineering targets to increase astaxanthin biosynthesis.

Preservation of non-Saccharomyces yeasts: Current technologies and challenges.

There is a recent and growing interest in the study and application of non-Saccharomyces yeasts, mainly in fermented foods. Numerous publications and patents show the importance of these yeasts. However, a fundamental issue in studying and applying them is to ensure an appropriate preservation scheme that allows to the non-Saccharomyces yeasts conserve their characteristics and fermentative capabilities by long periods of time. The main objective of this review is to present and analyze the techniques available to preserve these yeasts (by conventional and non-conventional methods), in small or large quantities for laboratory or industrial applications, respectively. Wine fermentation is one of the few industrial applications of non-Saccharomyces yeasts, but the preservation stage has been a major obstacle to achieve a wider application of these yeasts. This review considers the preservation techniques, and clearly defines parameters such as culturability, viability, vitality and robustness. Several conservation strategies published in research articles as well as patents are analyzed, and the advantages and disadvantages of each technique used are discussed. Another important issue during conservation processes is the stress to which yeasts are subjected at the time of preservation (mainly oxidative stress). There is little published information on the subject for non-Saccharomyces yeast, but it is a fundamental point to consider when designing a preservation strategy.

Respiratory capacity of the Kluyveromyces marxianus yeast isolated from the mezcal process during oxidative stress.

During the mezcal fermentation process, yeasts are affected by several stresses that can affect their fermentation capability. These stresses, such as thermal shock, ethanol, osmotic and growth inhibitors are common during fermentation. Cells have improved metabolic systems and they express stress response genes in order to decrease the damage caused during the stress, but to the best of our knowledge, there are no published works exploring the effect of oxidants and prooxidants, such as H2O2 and menadione, during growth. In this article, we describe the behavior of Kluyveromyces marxianus isolated from spontaneous mezcal fermentation during oxidative stress, and compared it with that of Saccharomyces cerevisiae strains that were also obtained from mezcal, using the W303-1A strain as a reference. S. cerevisiae strains showed greater viability after oxidative stress compared with K. marxianus strains. However, when the yeast strains were grown in the presence of oxidants in the media, K. marxianus exhibited a greater ability to grow in menadione than it did in H2O2. Moreover, when K. marxianus SLP1 was grown in a minibioreactor, its behavior when exposed to menadione was different from its behavior with H2O2. The yeast maintained the ability to consume dissolved oxygen during the 4 h subsequent to the addition of menadione, and then stopped respiration. When exposed to H2O2, the yeast stopped consuming oxygen for the following 8 h, but began to consume oxygen when stressors were no longer applied. In conclusion, yeast isolated from spontaneous mezcal fermentation was able to resist oxidative stress for a long period of time.

Use of non-Saccharomyces yeasts in cider fermentation: Importance of the nutrients addition to obtain an efficient fermentation.

The isolation of autochthonous yeast species presents a good strategy to select new microorganisms for developing an adequate inoculum to carry out fermentations and generate representative products of the cider production zone. However, non-Saccharomyces yeasts have been considered to have low capacity to carry out a complete fermentation as Saccharomyces cerevisiae. In this work, five autochthonous yeasts from a cider fermentation process were isolated and identified as Saccharomyces cerevisiae, Kluyveromyces marxianus, Pichia membranaefaciens, P. kluyveri and Zygosaccharomyces rouxii. A series of fermentations were developed at laboratory level, using each species individually and it was observed that only S. cerevisiae was able to finish the process. K. marxianus consumed less than 50% of the sugars; P. kluyveri and Z. rouxii consumed less than 70% and P. membranaefaciens consumed more than 90% but the yield (ethanol produced for sugar consumed (YP/S)) was 0.39. Nevertheless, the addition of magnesium, zinc and nitrogen increased the fermentative capacity of almost all species: K. marxianus, Z. rouxii and P. kluyveri, showed an increase in ethanol production when nutrients were added, obtaining more than 80 g/L of ethanol, and showing that those nutrients are necessary to complete the fermentation. This work describes the potential use of different non-Saccharomyces species to carry out fermentation of apple juice and highlights the importance of certain nutrients to enable an efficient alcoholic fermentation and the generation of desirable volatile compounds for cider production.

Effect of Carbon Sources in Carotenoid Production from Haloarcula sp. M1, Halolamina sp. M3 and Halorubrum sp. M5, Halophilic Archaea Isolated from Sonora Saltern, Mexico.

The isolation and molecular and chemo-taxonomic identification of seventeen halophilic archaea from the Santa Bárbara saltern, Sonora, México, were performed. Eight strains were selected based on pigmentation. Molecular identification revealed that the strains belonged to the Haloarcula, Halolamina and Halorubrum genera. Neutral lipids (quinones) were identified in all strains. Glycolipid S-DGD was found only in Halolamina sp. strain M3; polar phospholipids 2,3-O-phytanyl-sn-glycerol-1-phosphoryl-3-sn-glycerol (PG), 2,3-di-O-phytanyl-sn-glycero-1-phospho-3'-sn-glycerol-1'-methyl phosphate (PGP-Me) and sodium salt 1-(3-sn-phosphatidyl)-rac-glycerol were found in all the strains; and one unidentified glyco-phospholipid in strains M1, M3 and M4. Strains M1, M3 and M5 were selected for further studies based on carotenoid production. The effect of glucose and succinic and glutamic acid on carotenoid production was assessed. In particular, carotenoid production and growth significantly improved in the presence of glucose in strains Haloarcula sp. M1 and Halorubrum sp. M5 but not in Halolamina sp. M3. Glutamic and succinic acid had no effect on carotenoid production, and even was negative for Halorubrum sp. M5. Growth was increased by glutamic and succinic acid on Haloarcula sp. M1 but not in the other strains. This work describes for first time the presence of halophilic archaea in the Santa Bárbara saltern and highlights the differences in the effect of carbon sources on the growth and carotenoid production of haloarchaea.

Oxidative stress and antioxidant response in a thermotolerant yeast.

Stress tolerance is a key attribute that must be considered when using yeast cells for industrial applications. High temperature is one factor that can cause stress in yeast. High environmental temperature in particular may exert a natural selection pressure to evolve yeasts into thermotolerant strains. In the present study, three yeasts (Saccharomyces cerevisiae, MC4, and Kluyveromyces marxianus, OFF1 and SLP1) isolated from hot environments were exposed to increased temperatures and were then compared with a laboratory yeast strain. Their resistance to high temperature, oxidative stress, and antioxidant response were evaluated, along with the fatty acid composition of their cell membranes. The SLP1 strain showed a higher specific growth rate, biomass yield, and biomass volumetric productivity while also showing lower duplication time, reactive oxygen species (ROS) production, and lipid peroxidation. In addition, the SLP1 strain demonstrated more catalase activity after temperature was increased, and this strain also showed membranes enriched in saturated fatty acids. It is concluded that the SLP1 yeast strain is a thermotolerant yeast with less oxidative stress and a greater antioxidant response. Therefore, this strain could be used for fermentation at high temperatures.

Oxidative stress and antioxidant response in a thermotolerant yeast

Abstract Stress tolerance is a key attribute that must be considered when using yeast cells for industrial applications. High temperature is one factor that can cause stress in yeast. High environmental temperature in particular may exert a natural selection pressure to evolve yeasts into thermotolerant strains. In the present study, three yeasts (Saccharomyces cerevisiae, MC4, and Kluyveromyces marxianus, OFF1 and SLP1) isolated from hot environments were exposed to increased temperatures and were then compared with a laboratory yeast strain. Their resistance to high temperature, oxidative stress, and antioxidant response were evaluated, along with the fatty acid composition of their cell membranes. The SLP1 strain showed a higher specific growth rate, biomass yield, and biomass volumetric productivity while also showing lower duplication time, reactive oxygen species (ROS) production, and lipid peroxidation. In addition, the SLP1 strain demonstrated more catalase activity after temperature was increased, and this strain also showed membranes enriched in saturated fatty acids. It is concluded that the SLP1 yeast strain is a thermotolerant yeast with less oxidative stress and a greater antioxidant response. Therefore, this strain could be used for fermentation at high temperatures.