Enhancement of Biocontrol Efficacy of Pichia kudriavzevii Induced by Ca Ascorbate against Botrytis cinerea in Cherry Tomato Fruit and the Possible Mechanisms of Action.

This study investigated the effect of Ca ascorbate on the biocontrol efficacy of Pichia kudriavzevii and the possible mechanisms. The results indicated that the biocontrol activity of P. kudriavzevii was significantly enhanced by 0.15 g L-1 of Ca ascorbate, with higher growth rates of yeast cells in vitro and in vivo. The antioxidant enzyme activity in P. kudriavzevii, including catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD), were improved by Ca ascorbate and reached the maximum at 96 h, 96 h, and 72 h, respectively. The expression of the antioxidant enzyme-related genes CAT1 (8.55-fold) and SOD2 (7.26-fold) peaked at 96 h, while PRXIID (2.8-fold) peaked at 48 h, which were similar to the trends of enzyme activities. Compared with the control, 0.15 g L-1 of Ca ascorbate and CaCl2 increased the activity of succinate dehydrogenase in P. kudriavzevii, thereby enhancing the utilization of nutrients by yeast cells, and calcium ascorbate had the strongest effect. The expressions of HXT5, ADH6, PET100p, and Pga62 were significantly higher in the Ca ascorbate treatment than the other groups, and the CaCl2 treatment was also significantly higher than the control. These results indicated that Ca ascorbate can effectively improve the energy metabolism and cell wall synthesis and slow down the senescence of yeast cells. In general, Ca ascorbate can improve the environmental adaptability of P. kudriavzevii and thus improve the biocontrol effect, which is associated with inducing antioxidant enzymes in yeast cells and enhancing energy metabolism and nutrient utilization efficiency to increase nutrient competition with pathogens. IMPORTANCE Antagonistic yeast is a promising way to control postharvest fruit decay because of its safety and broad-spectrum resistance. However, the biocontrol efficacy of yeast is limited by environmental stress, such as oxidative stress. Therefore, the improvement of antioxidant capacity has become a research hot spot in improving the biocontrol efficacy of yeast. The induction of Ca ascorbate on the antioxidant capacity and physiological activity of yeast was studied. The results showed better induction of antioxidant enzyme and physiological activity in yeast by Ca ascorbate for better antioxidant capacity, and Ca2+ also played a synergistic promotion effect, which improved the biocontrol efficacy. These results provide an approach for the research and application of improving the environmental adaptability and biocontrol effectiveness of yeast.

Overexpression of ZmDHN11 could enhance transgenic yeast and tobacco tolerance to osmotic stress.

KEY MESSAGE: Maize group II LEA protein ZmDHN11 could protect protein activity and confer resistance to osmotic stress on transgenic yeast and tobacco. Late embryogenesis abundant (LEA) proteins are widely assumed to play crucial roles in environmental stress tolerance, but their function has remained obscure. Dehydrins are group II LEA proteins, which are highly hydrophilic plant stress proteins. In the present study, a novel group II LEA protein, ZmDHN11, was cloned and identified from maize. The expression of ZmDHN11 was induced by high osmotic stress, low temperature, salinity, and ABA (abscisic acid). The ZmDHN11 protein specifically accumulated in the nuclei and cytosol. Further study indicated that ZmDHN11 is phosphorylated by the casein kinase CKII. ZmDHN11 protected the activity of LDH under water-deficit stress. The overexpression of ZmDHN11 endows transgenic yeast and tobacco with tolerance to osmotic stress.

Pichia kudriavzevii-a potential soil yeast candidate for improving soil physical, chemical and biological properties.

Soil yeasts exhibit an array of beneficial effects to plants viz., plant growth promotion, phosphate solubilization, nitrogen and sulphur oxidation, etc. Yeasts remain as poorly investigated group of microorganisms that represent an abundant and dependable source of bioactive/chemically novel compounds and potential bioinoculants. Hence this study holds the key concept of assessing the performance of soil yeasts with potential plant growth promoting ability in soil quality improvement. Sixteen soil yeast isolates with plant growth promoting traits were assessed for biofilm forming potential and five potential soil yeast isolates were selected and identified through molecular technique. Soil incubation study was performed with these isolates to assess their impact on soil physical, chemical and biological properties. Due to inoculation of soil yeasts, notable changes were observed in soil physical, chemical and biological properties. Among the soil yeast isolates, Pichia kudriavzevii gave better results in soil incubation study.

Bacterial-fungal interactions in the neonatal gut influence asthma outcomes later in life.

Bacterial members of the infant gut microbiota and bacterial-derived short-chain fatty acids (SCFAs) have been shown to be protective against childhood asthma, but a role for the fungal microbiota in asthma etiology remains poorly defined. We recently reported an association between overgrowth of the yeast Pichia kudriavzevii in the gut microbiota of Ecuadorian infants and increased asthma risk. In the present study, we replicated these findings in Canadian infants and investigated a causal association between early life gut fungal dysbiosis and later allergic airway disease (AAD). In a mouse model, we demonstrate that overgrowth of P. kudriavzevii within the neonatal gut exacerbates features of type-2 and -17 inflammation during AAD later in life. We further show that P. kudriavzevii growth and adherence to gut epithelial cells are altered by SCFAs. Collectively, our results underscore the potential for leveraging inter-kingdom interactions when designing putative microbiota-based asthma therapeutics.

Influence of Non-Saccharomyces on Wine Chemistry: A Focus on Aroma-Related Compounds.

Wine fermentation processes are driven by complex microbial systems, which comprise eukaryotic and prokaryotic microorganisms that participate in several biochemical interactions with the must and wine chemicals and modulate the organoleptic properties of wine. Among these, yeasts play a fundamental role, since they carry out the alcoholic fermentation (AF), converting sugars to ethanol and CO2 together with a wide range of volatile organic compounds. The contribution of Saccharomyces cerevisiae, the reference organism associated with AF, has been extensively studied. However, in the last decade, selected non-Saccharomyces strains received considerable commercial and oenological interest due to their specific pro-technological aptitudes and the positive influence on sensory quality. This review aims to highlight the inter-specific variability within the heterogeneous class of non-Saccharomyces in terms of synthesis and release of volatile organic compounds during controlled AF in wine. In particular, we reported findings on the presence of model non-Saccharomyces organisms, including Torulaspora delbrueckii, Hanseniaspora spp,Lachancea thermotolerans, Metschnikowia pulcherrima, Pichia spp. and Candida zemplinina, in combination with S. cerevisiae. The evidence is discussed from both basic and applicative scientific perspective. In particular, the oenological significance in different kind of wines has been underlined.

Effect of Pichia on shaping the fermentation microbial community of sauce-flavor Baijiu.

In spontaneous food fermentation processes, environmental microbiota affects the yield and quality of the fermentation productions. Although the importance of environmental microbiota has been highlighted, the ecological processes that how the environmental microbiota affects the fermentation microbial community are poorly understood. To study the effect of the environmental microbiota on community assembly, the sources of microbiota and the ecological processes of the fermentation were characterized in sauce-flavor Baijiu. Results showed that the process of sauce-flavor Baijiu making could be divided into three phases according to fermentation parameters. Heap fermentation (phase I) was an important period for rapid temperature rise, substrate utilization and production accumulation. The microbial community of heap fermentation was characterized by decrease of diversity and rapid succession of community structure. Virgibacillus, Kroppenstedtia, Bacillus and Oceanobacillus were predominant in the initial heap fermentation, while Lactobacillus was predominant during the later stage. Pichia was the predominant fungal genus during the whole fermentation process. Then, SourceTracker results showed that Daqu provided 95.6% of the bacterial community and 28.10% of the fungal community to heap fermentation, whereas the environments (indoor ground and tools) provided 71.9% of the fungal communities (mainly Pichia) to heap fermentation. Next, the results revealed that the temperature, ethanol and microbial interaction of Pichia synergistically drove the dynamic of the microbial community during the heap fermentation process. Pichia was proved to be the heat-resistant fungi and strong competitor based on growth in different temperature and competition assays in vitro. Finally, the quick succession of heap fermentation microbiota increased the enrichment of volatile flavors such as acids and esters. Our comprehensive methods shows that Pichia, which mainly comes from the environment, can construct the microbial community of Baijiu fermentation, and highlights the importance of environmental microbiota in attempts to control and promote the formation of Baijiu fermentation microbial community. This systematic study of environmental microbiota is valuable for quality control and management during spontaneous fermentation.

Fruit host-dependent fungal communities in the microbiome of wild Queensland fruit fly larvae.

Bactrocera tryoni (Froggatt), the Queensland fruit fly (Qfly), is a highly polyphagous tephritid fly that is widespread in Eastern Australia. Qfly physiology is closely linked with its fungal associates, with particular relationship between Qfly nutrition and yeast or yeast-like fungi. Despite animal-associated fungi typically occurring in multi-species communities, Qfly studies have predominately involved the culture and characterisation of single fungal isolates. Further, only two studies have investigated the fungal communities associated with Qfly, and both have used culture-dependant techniques that overlook non-culturable fungi and hence under-represent, and provide a biased interpretation of, the overall fungal community. In order to explore a potentially hidden fungal diversity and complexity within the Qfly mycobiome, we used culture-independent, high-throughput Illumina sequencing techniques to comprehensively, and holistically characterized the fungal community of Qfly larvae and overcome the culture bias. We collected larvae from a range of fruit hosts along the east coast of Australia, and all had a mycobiome dominated by ascomycetes. The most abundant fungal taxa belonged to the genera Pichia (43%), Candida (20%), Hanseniaspora (10%), Zygosaccharomyces (11%) and Penicillium (7%). We also characterized the fungal communities of fruit hosts, and found a strong degree of overlap between larvae and fruit host communities, suggesting that these communities are intimately inter-connected. Our data suggests that larval fungal communities are acquired from surrounding fruit flesh. It is likely that the physiological benefits of Qfly exposure to fungal communities is primarily due to consumption of these fungi, not through syntrophy/symbiosis between fungi and insect 'host'.

Biocontrol ability and volatile organic compounds production as a putative mode of action of yeast strains isolated from organic grapes and rye grains.

The inhibiting activity of three yeast strains belonging to Pichia kudriavzevii, Pichia occidentalis, and Meyerozyma quilliermondii/Meyerozyma caribbica genera against common plant pathogens representing Mucor spp., Penicillium chrysogenum, Penicillium expansum, Aspergillus flavus, Fusarium cereals, Fusarium poae, as well as Botrytis cinerea genera was investigated. The yeast strains tested had a positive impact on growth inhibition of all target plant pathogens. The degree of inhibition was more than 50% and varied depending on both the yeast antagonist and the mold. Ethyl esters of medium-chain fatty acids, phenylethyl alcohol, and its acetate ester prevailed among the analyzed volatile organic compounds (VOCs) emitted by yeasts in the presence of the target plant pathogens. Due to the method used, assuming no contact between the antagonist and the pathogen, the antagonistic activity of the yeast strains studied resulted mainly from the production of biologically active VOCs. Moreover, the antagonistic activity was not only restricted to a single plant pathogen but effective towards molds of different genera, making the yeast strains studied very useful for potential application in biological control.

Screening antagonistic yeasts against citrus green mold and the possible biocontrol mechanisms of Pichia galeiformis (BAF03).

BACKGROUND: Penicillium digitatum is one of the most important pathogens causing citrus green mold, leading to significant economic losses. Traditionally, synthetic fungicides are used to control diseases. However, the side effects of fungicides should not be ignored. Thus, antagonistic yeasts were proposed to be safe and effective alternatives for managing diseases. Orchards are excellent sources of naturally occurring antagonists against pathogens. Therefore, in the present study, antagonistic yeasts obtained from orchards were screened, and the possible biocontrol mechanisms of the most promising yeast were investigated. RESULTS: Seventy-eight isolates of yeasts (15 species of 10 genera) were obtained from citrus orchards. In in vitro assays, 16 strains showed antifungal activity against Pichia digitatum and 15 strains showed biocontrol potential against green mold on Olinda oranges. Pichia galeiformis (BAF03) exhibited the best antagonistic activity against P. digitatum during 6 days storage at 25 °C and a good antagonistic activity during 29 days at 4 °C. Pichia galeiformis (BAF03) could colonize and amplify quickly in wounded citrus. Scanning electron microscopy results showed that the citrus wound was colonised by the yeast. A total of eight volatile organic compounds (VOCs) were identified by gas chromatography-mass spectrometry The VOCs produced by P. galeiformis (BAF03) efficiently inhibited P. digitatum. CONCLUSION: Pichia galeiformis (BAF03) isolated from a citrus orchard showed potential to control postharvest green mold of citrus. The possible mechanisms of action likely include competition for space and nutrients as well as production of VOCs.

Pichia kudriavzevii retards fungal decay by influencing the fungal community succession during cherry tomato fruit storage.

In this study, P. kudriavzevii was isolated and identified as an effective antagonistic yeast, which could significantly inhibit the rotting rate, weight loss, and delay the color change, with no effect on total soluble solids (TSS), titratable acid (TA), or firmness during cherry tomato storage. High-throughput sequencing was used to survey the effect of P. kudriavzevii on fungal community throughout cold storage. The results showed that the biological succession of predominant pathogens was disrupted by P. kudriavzevii. The abundance of Botrytis and Alternaria was higher in the control than upon P. kudriavzevii treatment at 28 d, but some yeast genera such as Naganishia, Wickerhamomyces, and Cutaneotrichosporon at 14 d, Pichia and Sporidiobolus at 21 d, and Cystofilobasidium at 28 d, had relatively higher abundances in P. kudriavzevii treatments than the control. Oddly, as an antagonist agent, P. kudriavzevii was not the dominant population, indicating that altering the course of succession of the fungal community may be an effective mechanism of antagonistic yeast. Furthermore, the total network correlation analysis of fungal community revealed that the community development was more dependent on similarities in function than on taxonomic relationships.

Yeasts isolated from Brazilian fermented foods in the protection against infection by pathogenic food bacteria.

The consumption of probiotics has increased due to the reported health benefits, mainly in preventing or treating gastrointestinal pathology. This study investigated the antimicrobial capacity of yeasts, Saccharomyces cerevisiae and Pichia kluyveri, previously isolated from fermented foods (indigenous beverage, kefir and cocoa) against the adhesion of foodborne pathogens to Caco-2 cells. Co-aggregation of yeasts with pathogens and were evaluated by quantitative analysis and using scanning electron and laser confocal microscopies. All yeasts strains were able to co-aggregate with the tested pathogens, however, this activity was strain-dependent. The inhibition tests showed that the adhesion of Escherichia coli EPEC, Listeria monocytogenes and Salmonella Enteritidis to Caco-2 was reduced by all the yeasts studied. Most of the evaluated yeasts showed inhibition rates equal to or greater than the commercial probiotic Saccharomyces boulardii. The yeasts were able to reduce up to 50% of the bacterial infection, as observed for CCMA0615 towards EPEC in exclusion assay; CCMA0731, CCMA0732 and CCMA0615 towards L. monocytogenes in exclusion and competition assays; and CCMA0731 in exclusion and CCMA0731, CCMA0732, CCMA0615 in competition assay towards S. Enteritidis. No antimicrobial compounds were produced by the yeasts, showing that competition for nutrients and/or receptors in the intestinal mucosa was the mechanism to bacterial inhibition.

Carbon source requirements for mating and mating-type switching in the methylotrophic yeasts Ogataea (Hansenula) polymorpha and Komagataella phaffii (Pichia pastoris).

The methylotrophic yeasts Ogataea (Hansenula) polymorpha and Komagataella phaffii (Pichia pastoris) have important industrial applications and are models for several biological processes including peroxisome biology and methanol metabolism. We examined the carbon source requirements for mating-type (MAT) switching and mating in both species. Haploid strains of O. polymorpha and K. phaffii are homothallic, and switch MAT by a flip/flop mechanism in which a chromosomal region containing the MAT genes undergoes an inversion. MAT switching is induced by nitrogen starvation in both species and can be detected 4-6 hr after induction. Both switching and mating require a utilizable carbon source that can be either fermentable or nonfermentable. We further observed that although methanol can be used as a sole carbon source in both species, it does not support the induction of MAT switching or mating. Our results provide insight into the nutritional cues that influence entry into sexual processes in methylotrophic yeasts that undergo flip/flop MAT switching.

Mating-type switching and mating-type gene array expression in the methylotrophic yeast Ogataea thermomethanolica TBRC656.

The methylotrophic yeast, Ogataea thermomethanolica TBRC656, is an attractive host organism for heterologous protein production owing to the availability of protein expression vectors and a genome-editing tool. In this study, we focused on mating-type switching and gene expression in order to elucidate its sexual life cycle and establish genetic approaches applicable for the strain. A putative mating-type gene cluster was identified in TBRC656 that is syntenic to the cluster in Ogataea parapolymorpha DL-1 (previously named Hansenula polymorpha). Like DL-1, TBRC656 possesses two mating loci, namely MATa and MATα, and also shows flip-flop mating-type switching. Interestingly, unlike any other methylotrophic yeast, TBRC656 robustly switched mating type during late growth in rich medium (YPD). Under nutrient depletion, mating-type switching was observed within one hour. Transcription from both MATa and MATα mating loci was detected during growth in YPD, and possibly induced upon nitrogen depletion. Gene expression from MATα was detected as a single co-transcript from a three-gene array (α2-α1-a1S). Deletion of a putative a1S ORF at the MATα locus had no observed effect on mating-type switching but demonstrated significant effect on mating-type gene expression at both MATa and MATα loci.

Influence of Pichia pastoris X-33 produced in industrial residues on productive performance, egg quality, immunity, and intestinal morphometry in quails.

This study was conducted in quails to evaluate the probiotic potential of Pichia pastoris X-33, cultivated in parboiled rice effluent supplemented with biodiesel glycerol or in standard medium Yeast Extract-Peptone-Dextrose (YPD). Forty-days-old female quails were divided into three treatments: T1 (Control) received a basal diet without P. pastoris; T2 (Pichia Effluent) received a basal diet supplemented with P. pastoris grown in parboiled rice effluent and biodiesel glycerol, and T3 (Pichia YPD) received a basal diet supplemented with P. pastoris produced in YPD. The birds were vaccinated against Newcastle Disease (NDV), Avian Infectious Bronchitis (IBV), and Gumboro Disease on days 1 and 28. The following parameters were analyzed: performance, egg quality, humoral immune response to the vaccines, organ weight, and intestinal morphometry. P. pastoris grown in YPD increased egg weight (p < 0.05). The lowest liver weight on day 14 was obtained in Pichia Effluent, whereas both P. pastoris supplemented groups had the lowest duodenum weights on day 14. Besides that, livers and duodenums presented no morphological changes in any of the three treatments. Supplementation of P. pastoris modulated the immune system of the birds, increasing anti-IBV, anti-NDV, and anti-Gumboro antibodies levels compared to the Control (p < 0.05). In conclusion, quail's immune response was improved by Pichia pastoris X-33, either it was grown in YPD or industrial residues, and the egg weight increased with Pichia pastoris X-33 grown in YPD, thereby demonstrating to be a promising probiotic for poultry.

Modeling and Regulation of Higher Alcohol Production through the Combined Effects of the C/N Ratio and Microbial Interaction.

Too large of a higher alcohol content has negative effects on the liquor taste and health. Revealing the key microbes and their key driving forces is essential to regulate the higher alcohol content in spontaneous liquor fermentation. Herein, we used high-throughput sequencing associated with a multivariate statistical algorithm to reveal the contributing microbes for higher alcohol production in Chinese light-aroma-type liquor and identified that Saccharomyces and Pichia were the main contributors. In addition, the C/N ratio and microbial interaction were found to significantly affect the production of higher alcohols. Herein, we used response surface methodology to establish a predictive model for higher alcohol production with the regulating factors, and the content of total higher alcohols decreased significantly from 328.80 ± 24.83 to 114.88 ± 5.02 mg/L with the optimized levels of the regulators. This work would facilitate the control of flavor production via regulating microbial communities in food fermentation.

Peroxisome Maintenance Depends on De Novo Peroxisome Formation in Yeast Mutants Defective in Peroxisome Fission and Inheritance.

There is an ongoing debate on how peroxisomes form: by growth and fission of pre-existing peroxisomes or de novo from another membrane. It has been proposed that, in wild type yeast cells, peroxisome fission and careful segregation of the organelles over mother cells and buds is essential for organelle maintenance. Using live cell imaging we observed that cells of the yeast Hansenula polymorpha, lacking the peroxisome fission protein Pex11, still show peroxisome fission and inheritance. Also, in cells of mutants without the peroxisome inheritance protein Inp2 peroxisome segregation can still occur. In contrast, peroxisome fission and inheritance were not observed in cells of a pex11 inp2 double deletion strain. In buds of cells of this double mutant, new organelles likely appear de novo. Growth of pex11 inp2 cells on methanol, a growth substrate that requires functional peroxisomes, is retarded relative to the wild type control. Based on these observations we conclude that in H. polymorpha de novo peroxisome formation is a rescue mechanism, which is less efficient than organelle fission and inheritance to maintain functional peroxisomes.

Improved cadmium resistance and removal capacity in Pichia kudriavzevii A16 by sucrose preincubation.

Removal of heavy metals from food material by growing micro-organisms is limited by the toxicity to cells. In this study, different preincubation treatments were investigated to analyze their effects on cadmium resistance and removal ability of Pichia kudriavzevii A16 and Saccharomyces cerevisiae CICC1211. Sucrose preincubation improved the cadmium resistance of both yeast cells and increased the cadmium-removal rate of P. kudriavzevii A16. An evident decrease of intracellular and cell-surface cadmium accumulation was observed after sucrose preincubation, which may be the primary reason responsible for the improved cadmium resistance. Flow cytometry assay showed that sucrose significantly reduced the production of reactive oxygen species (ROS) and cell death rate of both yeasts under cadmium compared with those normally cultured cells. Under cadmium stress, the content of both protein carbonyls and malonyldialdehyde were also reduced by the addition of sucrose, the results were in accordance with the tendency of ROS, exhibiting a defending function of sucrose. Osmotic regulators as proline and trehalose were increased by sucrose preincubation in P. kudriavzevii A16 in the presence of cadmium. The results suggested that sucrose preincubation could be applied to improve cadmium resistance and removal rate of yeasts.

Induction of signal molecules and expression of functional genes after Pichia pastoris stimulation in Glycyrrhiza uralensis Fisch adventitious roots.

Glycyrrhiza uralensis Fisch is threatened by over-development and consumption, and therefore, in urgent need of protection. Elicitation is considered to be an effective strategy to enhance the secondary metabolites in plant cell and organ cultures. Secondary metabolite, signal molecules, and gene expression in adventitious roots were studied by HPLC-ESI-MSn , commercially available kits and qRT-PCR method, respectively. In the present study, with the addition of linolenic acid, linoleic acid, and Pichia pastoris, the highest concentration of metabolites was achieved by P. pastoris treatment. The contents of total flavonoids (7.16 mg/g) and polysaccharide (149.76 mg/g) peaked at 100 mg/L of P. pastoris, which increased by 3.09-fold and 3.28-fold compared with the control, respectively. However, the highest concentration of glycyrrhizic acid (0.62 mg/g) and glycyrrhetinic acid (0.29 mg/g) were obtained in 200 mg/L of P. pastoris and which were 3.89-fold and 2.42-fold more than the control group, respectively. ESI-MSn analysis indicated that licoricesaponine B2, licoricesapoine G2, licoricesaponine J2, ononin, uralenin, gancaonin C were only identified in the P. pastoris treatment group. Furthermore, P. pastoris also enhanced accumulation of salicylic acid, jasmonic acid, nitric oxide and activities of antioxidant enzymes involved in the plant defense response. In addition, the transcriptional activity of genes involved in glycyrrhizic acid biosynthesis was significantly increased under the treatment of P. pastoris. The results provided a scientific evidence for the further exploitation of G. uralensis adventitious roots and clinical medication. PRACTICAL APPLICATIONS: This study provided an effective strategy to enhance metabolites by Pichia pastoris treatment in adventitious roots of G. uralensis. The data provide a scientific evidence for the further exploitation of G. uralensis adventitious roots and clinical medication.

Gut yeasts do not improve desiccation survival in Drosophila melanogaster.

A healthy gut microbiota generally improves the performance of its insect host. Although the effects can be specific to the species composition of the microbial community, the role of gut microbiota in determining water balance has not been well explored. We used axenic and gnotobiotic (reared with a known microbiota) Drosophila melanogaster to test three hypotheses about the effects of gut yeasts on the water balance of adult flies: 1) that gut yeasts would improve desiccation survival in adult flies; 2) that larval yeasts would improve adult desiccation survival; 3) that the effects would be species-specific, such that yeasts closely associated with D. melanogaster in nature are more likely to be beneficial than those rarely found in association with D. melanogaster. We used Saccharomyces cerevisiae (often used in Drosophila cultures, but rarely associated with D. melanogaster in nature), Lachancea kluyveri (associated with some species of Drosophila, but not D. melanogaster), and Pichia kluyveri (associated with D. melanogaster in nature). Adult inoculation with yeasts had no effect on survival of desiccating conditions. Inoculation with P. kluyveri as larvae did not change desiccation survival in adults; however, rearing with L. kluyveri or S. cerevisiae reduced adult desiccation survival. We conclude that adult inoculation with gut yeasts has no impact on desiccation survival, but that rearing with yeasts can have either no or detrimental effect. The effects appear to be species-specific: P. kluyveri did not have a negative impact on desiccation tolerance, suggesting some level of co-adaptation with D. melanogaster. We note that S. cerevisiae may not be an appropriate species for studying the effects of gut yeasts on D. melanogaster.

Elevated temperatures do not trigger a conserved metabolic network response among thermotolerant yeasts.

BACKGROUND: Thermotolerance is a highly desirable trait of microbial cell factories and has been the focus of extensive research. Yeast usually tolerate only a narrow temperature range and just two species, Kluyveromyces marxianus and Ogataea polymorpha have been described to grow at reasonable rates above 40 °C. However, the complex mechanisms of thermotolerance in yeast impede its full comprehension and the rare physiological data at elevated temperatures has so far not been matched with corresponding metabolic analyses. RESULTS: To elaborate on the metabolic network response to increased fermentation temperatures of up to 49 °C, comprehensive physiological datasets of several Kluyveromyces and Ogataea strains were generated and used for 13C-metabolic flux analyses. While the maximum growth temperature was very similar in all investigated strains, the metabolic network response to elevated temperatures was not conserved among the different species. In fact, metabolic flux distributions were remarkably irresponsive to increasing temperatures in O. polymorpha, while the K. marxianus strains exhibited extensive flux rerouting at elevated temperatures. CONCLUSIONS: While a clear mechanism of thermotolerance is not deducible from the fluxome level alone, the generated data can be valued as a knowledge repository for using temperature to modulate the metabolic activity towards engineering goals.