Isolation of local strains of the yeast Metschnikowia for biocontrol and lipid production purposes.

The bioprospection of indigenous microorganism strains with biotechnological potential represents a prominent trend. Metschnikowia yeasts exhibit diverse capabilities, such as ethanol reduction in winemaking, biocontrol potential, and lipid production. In this work, local Metschnikowia strains were isolated from different fruits by their ability to produce pulcherrimic acid, a molecule that has been linked to biocontrol activity and that binds iron giving colored colonies. Five strains were selected, each from one of five distinct sources. All of them were identified as M. pulcherrima. All five were able inhibit other yeasts and one M. pulcherrima, called M7, inhibited the growth of Aspergillus nidulans. The selected strains accumulated lipid bodies in stationary phase. Certain non-conventional yeasts like Hanseniaspora vineae are very sensitive to biomass drying, but cell extracts from M. pulcherrima added to the growth media as a source of antioxidant lipids increased their tolerance to drying. All strains isolated showed good stress tolerance (particularly to heat) and have nutrient requirements similar to a commercial M. pulcherrima strain. In addition, the M7 strain had a good growth in sugarcane and beet molasses and behaved like Saccharomyces cerevisiae in a growth medium derived from agricultural waste, a persimmon hydrolysate. Therefore, the isolation of local strains of Metschnikowia able to grow in a variety of substrates is a good source of biocontrol agents.

Heterologous pulcherrimin production in Saccharomyces cerevisiae confers inhibitory activity on Botrytis conidiation.

Pulcherrimin is an iron (III) chelate of pulcherriminic acid that plays a role in antagonistic microbial interactions, iron metabolism, and stress responses. Some bacteria and yeasts produce pulcherriminic acid, but so far, pulcherrimin could not be produced in Saccharomyces cerevisiae. Here, multiple integrations of the Metschnikowia pulcherrima PUL1 and PUL2 genes in the S. cerevisiae genome resulted in red colonies, which indicated pulcherrimin formation. The coloration correlated positively and significantly with the number of PUL1 and PUL2 genes. The presence of pulcherriminic acid was confirmed by mass spectrometry. In vitro competition assays with the plant pathogenic fungus Botrytis caroliana revealed inhibitory activity on conidiation by an engineered, strong pulcherrimin-producing S. cerevisiae strain. We demonstrate that the PUL1 and PUL2 genes from M. pulcherrima, in multiple copies, are sufficient to transfer pulcherrimin production to S. cerevisiae and represent the starting point for engineering and optimizing this biosynthetic pathway in the future.

Genome-scale metabolic models reveal determinants of phenotypic differences in non-Saccharomyces yeasts.

BACKGROUND: Use of alternative non-Saccharomyces yeasts in wine and beer brewing has gained more attention the recent years. This is both due to the desire to obtain a wider variety of flavours in the product and to reduce the final alcohol content. Given the metabolic differences between the yeast species, we wanted to account for some of the differences by using in silico models. RESULTS: We created and studied genome-scale metabolic models of five different non-Saccharomyces species using an automated processes. These were: Metschnikowia pulcherrima, Lachancea thermotolerans, Hanseniaspora osmophila, Torulaspora delbrueckii and Kluyveromyces lactis. Using the models, we predicted that M. pulcherrima, when compared to the other species, conducts more respiration and thus produces less fermentation products, a finding which agrees with experimental data. Complex I of the electron transport chain was to be present in M. pulcherrima, but absent in the others. The predicted importance of Complex I was diminished when we incorporated constraints on the amount of enzymatic protein, as this shifts the metabolism towards fermentation. CONCLUSIONS: Our results suggest that Complex I in the electron transport chain is a key differentiator between Metschnikowia pulcherrima and the other yeasts considered. Yet, more annotations and experimental data have the potential to improve model quality in order to increase fidelity and confidence in these results. Further experiments should be conducted to confirm the in vivo effect of Complex I in M. pulcherrima and its respiratory metabolism.

Fate of carbon in synthetic media fermentations containing Metschnikowia pulcherrima or Meyerozyma guilliermondii in the presence and absence of Saccharomyces cerevisiae.

While sequentially inoculating non-Saccharomyces yeasts with Saccharomyces cerevisiae can lower the alcohol contents of wine, the abilities of these yeasts to utilize/produce ethanol or generate other byproducts remained unclear. Metschnikowia pulcherrima or Meyerozyma guilliermondii were inoculated into media with or without S. cerevisiae to assess byproduct formation. Both species metabolized ethanol in a yeast-nitrogen-base medium but produced the alcohol in a synthetic grape juice medium. In fact, Mt. pulcherrima and My. guilliermondii generated less ethanol per gram of metabolized sugar (0.372 and 0.301 g/g, respectively) compared to S. cerevisiae (0.422 g/g). Sequentially inoculating each non-Saccharomyces species with S. cerevisiae into grape juice media achieved up to 3.0% v/v alcohol reduction compared to S. cerevisiae alone while producing variable glycerol, succinic acid, and acetic acid concentrations. However, neither non-Saccharomyces yeasts released appreciable CO2 under fermentative conditions regardless of incubation temperature. Despite equivalent peak populations, S. cerevisiae produced more biomass (2.98 g/L) than the non-Saccharomyces yeasts while sequential inoculations yielded higher biomass with Mt. pulcherrima (3.97 g/L) but not My. guilliermondii (3.03 g/L). To reduce ethanol concentrations, these non-Saccharomyces species may metabolize ethanol and/or produce less from metabolized sugars compared to S. cerevisiae but also divert carbon towards glycerol, succinic acid, and/or biomass.

Biosynthesis of silver nanoparticles with antimicrobial and anticancer properties using two novel yeasts.

AgNPs are nanomaterials with many potential biomedical applications. In this study, the two novel yeast strains HX-YS and LPP-12Y capable of producing biological silver nanoparticles were isolated. Sequencing of ribosomal DNA-ITS fragments, as well as partial D1/D2 regions of 26S rDNA indicated that the strains are related to species from the genus Metschnikowia. The BioAgNPs produced by HX-YS and LPP-12Y at pH 5.0-6.0 and 26 °C ranged in size from 50 to 500 nm. The antibacterial activities of yeast BioAgNPs against five pathogenic bacteria were determined. The highest antibacterial effect was observed on P. aeruginosa, with additional obvious effects on E. coli ATCC8099 and S. aureus ATCC10231. Additionally, the BioAgNPs showed antiproliferative effects on lung cancer cell lines H1975 and A579, with low toxicity in Beas 2B normal lung cells. Therefore, the AgNPs biosynthesized by HX-YS and LPP-12Y may have potential applications in the treatment of bacterial infections and cancer.

New Isolated Metschnikowia pulcherrima Strains from Apples for Postharvest Biocontrol of Penicillium expansum and Patulin Accumulation.

Wild yeasts isolated from the surface of apples were screened for antagonistic activity against Penicillium expansum, the main producer of the mycotoxin patulin. Three antagonistic yeasts (Y33, Y29 and Y24) from a total of 90 were found to inhibit P. expansum growth. Identification by ITS region sequence and characterization showed that three selected isolates of yeast should be different strains of Metschnikowia pulcherrima. Several concentrations of the selected yeasts were used to study their in vitro antifungal effectivity against P. expansum on Petri dishes (plates with 63.6 cm2 surface) whereas their potential activity on patulin reduction was studied in liquid medium. Finally, the BCA that had the best in vitro antifungal capacity against P. and the best patulin degradation capacity was selected to be assessed directly on apples. All the selected strains demonstrated antifungal activity in vitro but the most efficient was the strain Y29. Isolated strains were able to reduce patulin content in liquid medium, Y29 being the only strain that completely reduced patulin levels within 120 h. The application of Y29 as biocontrol agent on the surface of apples inoculated with P. expansum, inhibited fungal growth and patulin production during storage. Therefore, the results shown that this yeast strain could be used for the reduction of P. expansum and its mycotoxin in apples or apple-based products by adapting the procedure application.

The antagonistic Metschnikowia andauensis produces extracellular enzymes and pulcherrimin, whose production can be promoted by the culture factors.

Biological control against microbial infections has a great potential as an alternative approach instead of fungicidal chemicals, which can cause environmental pollution. The pigment producer Metschnikowia andauensis belongs to the antagonistic yeasts, but details of the mechanism by which it inhibits growth of other microbes are less known. Our results confirmed its antagonistic capacity on other yeast species isolated from fruits or flowers and demonstrated that the antagonistic capacity was well correlated with the size of the red pigmented zone. We have isolated and characterized its red pigment, which proved to be the iron chelating pulcherrimin. Its production was possible even in the presence of 0.05 mg/ml copper sulphate, which is widely used in organic vineyards because of its antimicrobial properties. Production and localisation of the pulcherrimin strongly depended on composition of the media and other culture factors. Glucose, galactose, disaccharides and the presence of pectin or certain amino acids clearly promoted pigment production. Higher temperatures and iron concentration decreased the diameter of red pigmented zones. The effect of pH on pigment production varied depending of whether it was tested in liquid or solid media. In addition, our results suggest that other mechanisms besides the iron depletion of the culture media may contribute to the antagonistic capacity of M. andauensis.

A natural approach, the use of killer toxin produced by Metschnikowia pulcherrima in fresh ground beef patties for shelf life extention.

A novel killer toxin produced by yeast Metschnikowia pulcherrima was purified and added into ready to cook meatballs to enhance their microbial safety and extension of their shelf life. The agent was added into ready to cook meatballs at two different concentrations (1%-K1 and 2%-K2). The results of those two groups were compared to the control group (K0) lacking the killer toxin. Physical, chemical and microbiological analyses were carried out in meat dough and all analyses were repeated at two day intervals during 10 day-storage at +4 °C. Addition of inhibitor compound in meat dough decreased the numbers of total aerobic mesophillic bacteria, yeast and molds and lactic acid bacteria. Staphylococci/Micrococci, coliform bacteria and total psychrotrophic bacterial counts of the samples were determined as well. Results showed that all indicators of microbial deterioration were found to be higher in K1 group than K2 group, revealing that there was an inverse correlation between the concentration of killer toxin and the number of microorganisms causing spoilage. In addition to 1 log decrease in the number of microorganisms in toxin added groups, the high TBARS values of the control group also showed the effectiveness of the toxin. Toxic effect analysis results showed that the killer toxin had no toxic effect on L929 mouse fibroblast cells after 24h exposure.

Features of iron accumulation at high concentration in pulcherrimin-producing Metschnikowia yeast biomass.

In previous studies it was found that the antimicrobial properties of pulcherrimin-producing Metschnikowia species are related to the formation of a red pigment-pulcherrimin and sequestration of free iron from their growth medium. For strains of Metschnikowia pulcherrima, M. sinensis, M. shaxiensis, and M. fructicola, at a high, ≈80 mg/kg, elemental Fe concentration in agar growth media we observed the essentially different (metal luster, non-glossy rust like, and colored) yeast biomass coatings. For the studied strains the optical and scanning electron microscopies showed the increased formation of chlamydospores that accumulate a red pigment-insoluble pulcherrimin rich in iron. The chlamydospore formation and decay depended on the iron concentration. In this study pulcherrimin in biomass of the selected Metschnikowia strains was detected by Mössbauer spectroscopy. At ≈80 mg/kg elemental Fe concentration the Mössbauer spectra of biomass of the studied strains were almost identical to these of purified pulcherrimin. Iron in pulcherrimin reached ≈1% of biomass by weight which is very high in comparison with elemental Fe percentage in growth medium and is not necessary for yeast growth. The pulcherrimin in biomass was also observed by Mössbauer spectroscopy at lower, ≈5 mg/kg, elemental Fe concentration. Through chemical binding of iron pulcherrimin sequestrates the soluble Fe in the growth media. However, at high Fe concentrations, the chemical and biochemical processes lead to the pulcherrimin accumulation in biomass chlamydospores. When soluble iron is sequestrated or removed from the growth media in this way, it becomes inaccessible for other microorganisms.

Impact of Sequential Inoculation with the Non-Saccharomyces T. delbrueckii and M. pulcherrima Combined with Saccharomyces cerevisiae Strains on Chemicals and Sensory Profile of Rosé Wines.

Controlled inoculations of non-Saccharomyces yeasts are becoming increasingly used to produce high-quality wines due to their enological potential. In this study, we evaluated the impact of sequential inoculation with the commercial non-Saccharomyces yeasts (Torulaspora delbrueckii and Metschnikowia pulcherrima) in combination with Saccharomyces cerevisiae on the chemical and sensory profile of rosé wines. Sequential inoculation with T. delbrueckii produced wines with an overall reduction in esters, mainly explained by the lower concentrations of ethyl esters of medium-chain fatty acids and isoamyl acetate. The lower ester concentrations of these wines were related to a reduction in fruity descriptors. An increase was observed, however, in other minor esters such as cinnamates and ethyl esters of branched acids. Zinc, ethyl isobutyrate, and ethyl dihydrocinnamate were selected as potential markers for this fermentation strategy. Sequential inoculation with M. pulcherrima resulted in rosé wines with an enhanced ester profile, reduced acetaldehyde, and increased anthocyans and tannins. Compared to the control wines fermented with S. cerevisiae, the changes observed in these wines were far subtler, especially for the volatile profile, sensory characteristics, and color parameters, with isobutyl hexanoate and isoamyl butyrate being selected as potential markers.

Separate and combined Hanseniaspora uvarum and Metschnikowia pulcherrima metabolic volatiles are attractive to Drosophila suzukii in the laboratory and field.

Drosophila suzukii flies cause economic losses to fruit crops globally. Previous work shows various Drosophila species are attracted to volatile metabolites produced by individual fruit associated yeast isolates, but fruits naturally harbour a rich diversity of yeast species. Here, we report the relative attractiveness of D. suzukii to yeasts presented individually or in combinations using laboratory preference tests and field trapping data. Laboratory trials revealed four of 12 single yeast isolates were attractive to D. suzukii, of which Metschnikowia pulcherrima and Hanseniaspora uvarum were also attractive in field trials. Four out of 10 yeast combinations involving Candida zemplinina, Pichia pijperi, M. pulcherrima and H. uvarum were attractive in the laboratory. Whilst a combination of M. pulcherrima + H. uvarum trapped the greatest number of D. suzukii in the field, the efficacy of the M. pulcherrima + H. uvarum combination to trap D. suzukii was not significantly greater than traps primed with volatiles from only H. uvarum. While volatiles from isolates of M. pulcherrima and H. uvarum show promise as baits for D. suzukii, further research is needed to ascertain how and why flies are attracted to certain baits to optimise control efficacy.

Impacts of non-Saccharomyces species and aeration on sequential inoculation with Saccharomyces cerevisiae to produce lower alcohol Merlot wines from Washington state.

BACKGROUND: Species of non-Saccharomyces yeasts isolated from Washington vineyards were evaluated for their abilities to reduce alcohol contents of wines. As many of these yeasts benefit from some oxygen, the effect of limited aeration was also studied. RESULTS: Although fermentations of a high sugar Merlot grape must (310 g L-1 ) did not reach dryness, inoculation of Metschnikowia chrysoperlae, Mt. pulcherrima, Meyerozyma guillermondii, Pichia kluyveri, or P. membranifaciens yielded in wines with lower amounts of ethanol without excessive levels of acetic acid. Aeration frequently resulted in wines with less ethanol but with more acetic acid compared to non-aerated fermentations. Inoculation of Mt. pulcherrima or My. guilliermondii into another Merlot grape must that contained a lower initial amount of fermentable sugar (266 g L-1 ) resulted in dry wines that contained less alcohol. CONCLUSIONS: Inoculation of My. guilliermondii or Mt. pulcherrima before primary alcoholic fermentation resulted in wines with reduced alcohol contents without excessive acetic acid production. © 2020 Society of Chemical Industry.

Metschnikowia pulcherrima represses aerobic respiration in Saccharomyces cerevisiae suggesting a direct response to co-cultivation.

The use of non-Saccharomyces species as starter cultures together with Saccharomyces cerevisiae is becoming a common practice in the oenological industry to produce wines that respond to new market demands. In this context, microbial interactions with these non-Saccharomyces species must be considered for a rational design of yeast starter combinations. Previously, transcriptional responses of S. cerevisiae to short-term co-cultivation with Torulaspora delbrueckii, Candida sake, or Hanseniaspora uvarum was compared. An activation of sugar consumption and glycolysis, membrane and cell wall biogenesis, and nitrogen utilization was observed, suggesting a metabolic boost of S. cerevisiae in response to competing yeasts. In the present study, the transcription profile of S. cerevisiae was analyzed after 3 h of cell contact with Metschnikowia pulcherrima. Results show an over-expression of the gluco-fermentative pathway much stronger than with the other species. Moreover, a great repression of the respiration pathway has been found in response to Metschnikowia. Our hypothesis is that there is a direct interaction stress response (DISR) between S. cerevisiae and the other yeast species that, under excess sugar conditions, induces transcription of the hexose transporters, triggering glucose flow to fermentation and inhibiting respiration, leading to an increase in both, metabolic flow and population dynamics.

Impact of changes in wine composition produced by non-Saccharomyces on malolactic fermentation.

Non-Saccharomyces yeasts have increasingly been used in vinification recently. This is particularly true of Torulaspora delbrueckii and Metschnikowia pulcherrima, which are inoculated before S. cerevisiae, to complete a sequential alcoholic fermentation. This paper aims to study the effects of these two non-Saccharomyces yeasts on malolactic fermentation (MLF) carried out by two strains of Oenococcus oeni, under cellar conditions. Oenological parameters, and volatile and phenolic compounds were analysed in wines. The wines were tasted, and the microorganisms identified. In general, non-Saccharomyces created more MLF friendly conditions, largely because of lower concentrations of SO2 and medium chain fatty acids. The most favourable results were observed in wines inoculated with T. delbrueckii, that seemed to promote the development of O. oeni and improve MLF performance.

Basal catalase activity and high glutathione levels influence the performance of non-Saccharomyces active dry wine yeasts.

Non-Saccharomyces wine yeasts are useful tools for producing wines with complex aromas or low ethanol content. Their use in wine would benefit from their production as active dry yeast (ADY) starters to be used as co-inocula alongside S. cerevisiae. Oxidative stress during biomass propagation and dehydration is a key factor in determining ADY performance, as it affects yeast vitality and viability. Several studies have analysed the response of S. cerevisiae to oxidative stress under dehydration conditions, but not so many deal with non-conventional yeasts. In this work, we analysed eight non-Saccharomyces wine yeasts under biomass production conditions and studied oxidative stress parameters and lipid composition. The results revealed wide variability among species in their technological performance during ADY production. Also, for Metschnikowia pulcherrima and Starmerella bacillaris, better performance correlates with high catalase activity and glutathione levels. Our data suggest that non-Saccharomyces wine yeasts with an enhanced oxidative stress response are better suited to grow under ADY production conditions.

Phenolic Compound Profiles in Alcoholic Black Currant Beverages Produced by Fermentation with Saccharomyces and Non-Saccharomyces Yeasts.

Alcoholic beverages with low ethanol contents were produced by fermenting black currant juice with Saccharomyces and non-Saccharomyces yeasts without added sugar. The effects of different yeasts on the phenolic compounds (anthocyanins, hydroxycinnamic acids, flavonols, and flavan-3-ols) and other selected constituents (the ethanol content, residual sugars, organic acids, and color) of the black currants were assessed. Single yeast-fermented beverages had higher ethanol contents (3.84-4.47%, v/v) than those produced by sequential fermentation. In general, the fermentation of black currant juice increased the contents of organic acids and flavonols, whereas anthocyanin contents decreased. All of the fermentations decreased the contents of glycosylated nitrile-containing hydroxycinnamic acids, resulting in higher contents of the corresponding aglycons. Fermentation with Saccharomyces bayanus resulted in lower anthocyanin and organic acid contents compared to the other yeasts. Sequential fermentations with Saccharomyces cerevisiae and Metschnikowia pulcherrima led to the highest total hydroxycinnamic acids and anthocyanins among all of the fermentations.

Lower-alcohol wines produced by Metschnikowia pulcherrima and Saccharomyces cerevisiae co-fermentations: The effect of sequential inoculation timing.

In Latin, 'pulcherrima' is a superlative form of an adjective that translates as beautiful. Apart from being 'the most beautiful' yeast, Metschnikowia pulcherrima has a remarkable potential in production of wines with lower ethanol content. The oenological performance of six M. pulcherrima strains was hereby tested in sequential cultures with Saccharomyces cerevisiae. The best-performing strain MP2 was further characterised in fermentations with different S. cerevisiae inoculation delays in both white grape juice and Chemically Defined Grape Juice Medium (CDGJM). The analysis of main metabolites, undertaken prior to sequential inoculations and upon fermentation completion, highlighted metabolic interactions and carbon sinks other than ethanol in MP2 treatments. Depending on the inoculation delay, MP2 white wines contained between 0.6% and 1.2% (v/v) less ethanol than the S. cerevisiae monoculture, with even larger decreases detected in the CDGJM. The MP2 treatments also contained higher concentrations of TCA cycle by-products (i.e. fumarate and succinate) and glycerol, and lower concentrations of acetic acid. The analysis of volatile compounds showed increased production of acetate esters and higher alcohols in all MP2 wines, alongside other compositional alterations arising from the S. cerevisiae inoculation delay.

Investigations of the mechanisms of interactions between four non-conventional species with Saccharomyces cerevisiae in oenological conditions.

Fermentation by microorganisms is a key step in the production of traditional food products such as bread, cheese, beer and wine. In these fermentative ecosystems, microorganisms interact in various ways, namely competition, predation, commensalism and mutualism. Traditional wine fermentation is a complex microbial process performed by Saccharomyces and non-Saccharomyces (NS) yeast species. To better understand the different interactions occurring within wine fermentation, isolated yeast cultures were compared with mixed co-cultures of one reference strain of S. cerevisiae with one strain of four NS yeast species (Metschnikowia pulcherrima, M. fructicola, Hanseniaspora opuntiae and H. uvarum). In each case, we studied population dynamics, resource consumed and metabolites produced from central carbon metabolism. This phenotyping of competition kinetics allowed us to confirm the main mechanisms of interaction between strains of four NS species. S. cerevisiae competed with H. uvarum and H. opuntiae for resources although both Hanseniaspora species were characterized by a strong mortality either in mono or mixed fermentations. M. pulcherrima and M. fructicola displayed a negative interaction with the S. cerevisiae strain tested, with a decrease in viability in co-culture. Overall, this work highlights the importance of measuring specific cell populations in mixed cultures and their metabolite kinetics to understand yeast-yeast interactions. These results are a first step towards ecological engineering and the rational design of optimal multi-species starter consortia using modeling tools. In particular the originality of this paper is for the first times to highlight the joint-effect of different species population dynamics on glycerol production and also to discuss on the putative role of lipid uptake on the limitation of some non-conventional species growth although interaction processes.

Microbial Co-Occurrence in Floral Nectar Affects Metabolites and Attractiveness to a Generalist Pollinator.

Microbial metabolism can shape cues important for animal attraction in service-resource mutualisms. Resources are frequently colonized by microbial communities, but experimental assessment of animal-microbial interactions often focus on microbial monocultures. Such an approach likely fails to predict effects of microbial assemblages, as microbe-microbe interactions may affect in a non-additive manner microbial metabolism and resulting chemosensory cues. Here, we compared effects of microbial mono- and cocultures on growth of constituent microbes, volatile metabolite production, sugar catabolism, and effects on pollinator foraging across two nectar environments that differed in sugar concentration. Growth in co-culture decreased the abundance of the yeast Metschnikowia reukaufii, but not the bacterium Asaia astilbes. Volatile emissions differed significantly between microbial treatments and with nectar concentration, while sugar concentration was relatively similar among mono- and cocultures. Coculture volatile emission closely resembled an additive combination of monoculture volatiles. Despite differences in microbial growth and chemosensory cues, honey bee feeding did not differ between microbial monocultures and assemblages. Taken together, our results suggest that in some cases, chemical and ecological effects of microbial assemblages are largely predictable from those of component species, but caution that more work is necessary to predict under what circumstances non-additive effects are important.

Efficient production of isomelezitose by a glucosyltransferase activity in Metschnikowia reukaufii cell extracts.

Metschnikowia reukaufii is a widespread yeast able to grow in the plants' floral nectaries, an environment of extreme conditions with sucrose concentrations exceeding 400 g l-1 , which led us into the search for enzymatic activities involved in this sugar use/transformation. New oligosaccharides were produced by transglucosylation processes employing M. reukaufii cell extracts in overload-sucrose reactions. These products were purified and structurally characterized by MS-ESI and NMR techniques. The reaction mixture included new sugars showing a great variety of glycosidic bonds including α-(1→1), α-(1→3) and α-(1→6) linkages. The main product synthesized was the trisaccharide isomelezitose, whose maximum concentration reached 81 g l-1 , the highest amount reported for any unmodified enzyme or microbial extract. In addition, 51 g l-1 of the disaccharide trehalulose was also produced. Both sugars show potential nutraceutical and prebiotic properties. Interestingly, the sugar mixture obtained in the biosynthetic reactions also contained oligosaccharides such as esculose, a rare trisaccharide with no previous NMR structure elucidation, as well as erlose, melezitose and theanderose. All the sugars produced are naturally found in honey. These compounds are of biotechnological interest due to their potential food, cosmeceutical and pharmaceutical applications.