Non-conventional Yeast Research Articles

Genetic inactivation of the Carnitine/Acetyl-Carnitine mitochondrial carrier of Yarrowia lipolytica leads to enhanced odd-chain fatty acid production

BackgroundMitochondrial carriers (MCs) can deeply affect the intracellular flux distribution of metabolic pathways. The manipulation of their expression level, to redirect the flux toward the production of a molecule of interest, is an attractive target for the metabolic engineering of eukaryotic microorganisms. The non-conventional yeast Yarrowia lipolytica is able to use a wide range of substrates. As oleaginous yeast, it directs most of the acetyl-CoA therefrom generated towards the synthesis of lipids, which occurs in the cytoplasm. Among them, the odd-chain fatty acids (OCFAs) are promising microbial-based compounds with several applications in the medical, cosmetic, chemical and agricultural industries.ResultsIn this study, we have identified the MC involved in the Carnitine/Acetyl-Carnitine shuttle in Y. lipolytica, YlCrc1. The Y. lipolytica Ylcrc1 knock-out strain failed to grow on ethanol, acetate and oleic acid, demonstrating the fundamental role of this MC in the transport of acetyl-CoA from peroxisomes and cytoplasm into mitochondria. A metabolic engineering strategy involving the deletion of YlCRC1, and the recombinant expression of propionyl-CoA transferase from Ralstonia eutropha (RePCT), improved propionate utilization and its conversion into OCFAs. These genetic modifications and a lipogenic medium supplemented with glucose and propionate as the sole carbon sources, led to enhanced accumulation of OCFAs in Y. lipolytica.ConclusionsThe Carnitine/Acetyl-Carnitine shuttle of Y. lipolytica involving YlCrc1, is the sole pathway for transporting peroxisomal or cytosolic acetyl-CoA to mitochondria. Manipulation of this carrier can be a promising target for metabolic engineering approaches involving cytosolic acetyl-CoA, as demonstrated by the effect of YlCRC1 deletion on OCFAs synthesis.

Saccharification of starchy food waste through thermochemical and enzymatic pretreatment, towards enhanced bioethanol production via newly isolated non-conventional yeast strains

A starchy food waste containing mainly cooked wasted rice (WR) was exploited for bioethanol production using novel yeast strains was investigated. Different pretreatment schemes of the waste at solids loading 10%–30% TS WR (w/v) i.e. enzymatic, thermochemical and combined thermochemical/enzymatic pretreatment, were evaluated aiming to the maximum liberation of fermentable carbohydrates and their subsequent bioconversion to ethanol. Fermentation tests of the whole pretreated slurries were initially performed with the yeasts strains that were identified as Kluyveromyces marxianus isolate V3-19,Pichia kudriavzevii strain YF1702 and K. marxianus strain TTG-428, and their fermentation efficiencies (FE) were comparatively assessed. It was shown that the combined pretreatment led to the maximum saccharification, whereas FEs were higher for K. marxianus, V3-19, exceeding 90% of the theoretical maximum. In the case of the highest organic loading of WR, though, up to 25% of soluble carbohydrates remained unexploitable after 72 h of fermentation, indicating that kinetic restrictions occurred in the process. Further experiments with the hydrolysates that were recovered after combined pretreatment, revealed that the removal of solids enhances the consumption of sugars and leads to complete uptake for the loading 20% TS WR (w/v).

A landing pad system for multicopy gene integration in Issatchenkia orientalis

The robust nature of the non-conventional yeast Issatchenkia orientalis allows it to grow under highly acidic conditions and therefore, has gained increasing interest in producing organic acids using a variety of carbon sources. Recently, the development of a genetic toolbox for I. orientalis, including an episomal plasmid, characterization of multiple promoters and terminators, and CRISPR-Cas9 tools, has eased the metabolic engineering efforts in I. orientalis. However, multiplex engineering is still hampered by the lack of efficient multicopy integration tools. To facilitate the construction of large, complex metabolic pathways by multiplex CRISPR-Cas9-mediated genome editing, we developed a bioinformatics pipeline to identify and prioritize genome-wide intergenic loci and characterized 47 gRNAs located in 21 intergenic regions. These loci are screened for guide RNA cutting efficiency, integration efficiency of a gene cassette, the resulting cellular fitness, and GFP expression level. We further developed a landing pad system using components from these well-characterized loci, which can aid in the integration of multiple genes using single guide RNA and multiple repair templates of the user’s choice. We have demonstrated the use of the landing pad for simultaneous integrations of 2, 3, 4, or 5 genes to the target loci with efficiencies greater than 80%. As a proof of concept, we showed how the production of 5-aminolevulinic acid can be improved by integrating five copies of genes at multiple sites in one step. We have further demonstrated the efficiency of this tool by constructing a metabolic pathway for succinic acid production by integrating five gene expression cassettes using a single guide RNA along with five different repair templates, leading to the production of 9 g/L of succinic acid in batch fermentations. This study demonstrates the effectiveness of a single gRNA-mediated CRISPR platform to build complex metabolic pathways in a non-conventional yeast. This landing pad system will be a valuable tool for the metabolic engineering of I. orientalis.

AcCRISPR: an activity-correction method for improving the accuracy of CRISPR screens

High throughput CRISPR screens are revolutionizing the way scientists unravel the genetic underpinnings of engineered and evolved phenotypes. One of the critical challenges in accurately assessing screening outcomes is accounting for the variability in sgRNA cutting efficiency. Poorly active guides targeting genes essential to screening conditions obscure the growth defects that are expected from disrupting them. Here, we develop acCRISPR, an end-to-end pipeline that identifies essential genes in pooled CRISPR screens using sgRNA read counts obtained from next-generation sequencing. acCRISPR uses experimentally determined cutting efficiencies for each guide in the library to provide an activity correction to the screening outcomes via calculation of an optimization metric, thus determining the fitness effect of disrupted genes. CRISPR-Cas9 and -Cas12a screens were carried out in the non-conventional oleaginous yeast Yarrowia lipolytica and acCRISPR was used to determine a high-confidence set of essential genes for growth under glucose, a common carbon source used for the industrial production of oleochemicals. acCRISPR was also used in screens quantifying relative cellular fitness under high salt conditions to identify genes that were related to salt tolerance. Collectively, this work presents an experimental-computational framework for CRISPR-based functional genomics studies that may be expanded to other non-conventional organisms of interest.

Optimizing growth and biomass production of non-Saccharomyces wine yeast starters by overcoming sucrose consumption deficiency.

The use of non-Saccharomyces yeasts as starters in winemaking has increased exponentially in the last years. For instance, non-conventional yeasts have proven useful for the improvement of the organoleptic profile and biocontrol. Active dry yeast starter production has been optimized for Saccharomyces cerevisiae, which may entail problems for the propagation of non-Saccharomyces yeasts. This work shows that the poor growth of Hanseniaspora vineae and Metschnikowia pulcherrima in molasses is related to a deficient sucrose consumption, linked to their low invertase activity. In order to address this issue, simple modifications to the cultivation media based hydrolysis and the reduction of sucrose concentration were performed. We performed biomass propagation simulations at a bench-top and bioreactor scale. The results show that cultivation in a hexose-based media improved biomass production in both species, as it solves their low invertase activity. The reduction in sugar concentration promoted a metabolic shift to a respiratory metabolism, which allowed a higher biomass yield, but did not improve total biomass production, due to the lower sugar availability. To evaluate the technological performance of these adaptations, we performed mixed grape juice fermentations with biomass produced in such conditions of M. pulcherrima and S. cerevisiae. The analysis of wines produced revealed that the different treatments we have tested did not have any negative impact on wine quality, further proving their applicability at an industrial level for the improvement of biomass production.

Increasing Higher Alcohols and Acetates in Low-Alcohol Beer by Proteases.

The market of non-alcoholic and low-alcohol beer has grown continuously thanks to the advocacy for healthy and responsible drinking. Non-alcoholic and low-alcohol products usually possess less higher alcohols and acetates and more aldehyde off-flavors due to the manufacturing processes. The employment of non-conventional yeasts partially mitigates this problem. In this study, we used proteases to optimize the wort amino acid profile for better aroma production during yeast fermentation. The design of experiments was applied to increase the leucine molar fraction, aiming to boost 3-methylbutan-1-ol and 3-methylbutyl acetate (banana-like aromas). This led to an increase from 7% to 11% leucine in wort after protease treatment. The aroma output in the subsequent fermentation, however, was yeast-dependent. An 87% increase of 3-methylbutan-1-ol and a 64% increase of 3-methylbutyl acetate were observed when Saccharomycodes ludwigii was used. When Pichia kluyveri was employed, higher alcohols and esters from valine and isoleucine were increased: 58% more of 2-methylpropyl acetate, 67% more of 2-methylbutan-1-ol, and 24% more of 2-methylbutyl acetate were observed. Conversely, 3-methylbutan-1-ol decreased by 58% and 3-methylbutyl acetate largely remained the same. Apart from these, the amounts of aldehyde intermediates were increased to a varying extent. The impact of such increases in aromas and off-flavors on the perception of low-alcohol beer remains to be evaluated by sensory analysis in future studies.

Species and temperature-dependent fermentative aptitudes of Mrakia genus for innovative brewing

The use of non-conventional brewing yeasts as alternative starters is a very promising approach which received increasing attention from worldwide scientists and brewers. Despite the feasible application of non-conventional yeasts in brewing processes, their regulations and safety assessment by the European Food Safety Authority still represent a bottlenecked hampering their commercial release, at least into EU market. Thus, research on yeast physiology, accurate taxonomic species identification and safety concerns associated with the use of non-conventional yeasts in food chains is needed to develop novel healthier and safer beers. Currently, most of the documented brewing applications catalysed by non-conventional yeasts are associated to ascomycetous yeasts, while little is known about analogous uses of basidiomycetous taxa. Therefore, in order to extend the phenotypic diversity of basidiomycetous brewing yeasts the aim of this investigation is to check the fermentation aptitudes of thirteen Mrakia species in relation to their taxonomic position within the genus Mrakia. The volatile profile, ethanol content and sugar consumption were compared with that produced by a commercial starter for low alcohol beers, namely Saccharomycodes ludwigii WSL 17. The phylogeny of Mrakia genus showed three clusters that clearly exhibited different fermentation aptitudes. Members of M. gelida cluster showed a superior aptitude to produce ethanol, higher alcohols, esters and sugars conversion compared to the members of M. cryoconiti and M. aquatica clusters. Among M. gelida cluster, the strain M. blollopis DBVPG 4974 exhibited a medium flocculation profile, a high tolerance to ethanol and to iso-α-acids, and a considerable production of lactic and acetic acids, and glycerol. In addition, an inverse relationship between fermentative performances and incubation temperature is also displayed by this strain. Possible speculations on the association between the cold adaptation exhibited by M. blollopis DBVPG 4974 and the release of ethanol in the intracellular matrix and in the bordering environment are presented.

Discovery and Functional Evaluation of Heat Tolerance Genes in the Nonconventional Yeast Yarrowia lipolytica

Yarrowia lipolytica, a GRAS (generally recognized as safe) nonconventional yeast, has been used widely in industrial fermentation to produce chemicals, fuels, and functional sugars such as erythritol and mannitol. Although Y. lipolytica is a promising organism for bioconversion and has substantial potential in industrial production, its utilization is restricted by the high cost of cooling during the fermentation process; the optimum growth or fermentation temperature of Y. lipolytica is 28–30 °C, which is lower than that of some fermenting species. Therefore, it is necessary to breed a thermoresistant Y. lipolytica for use in a fermentation system. Here, we report a new thermoduric Y. lipolytica strain (a thermoresistant clone, HRC) that can grow at 35 °C—higher than the starting strain Y. lipolytica CGMCC7326 (maximum growth temperature at 33 °C)—by laboratory adaptive evolution. Based on the transcriptome analysis of the mutant strain HRC and the parental strain Y. lipolytica CGMCC7326 at different temperatures, 22 genes with increased expression at high temperatures were identified and 10 of them were overexpressed in Y. lipolytica CGMCC7326. HRC1, HRC2, and HRC3 (with YALI0B21582g, YALI0C13750g, and YALI0B10626g overexpressed, respectively) were assessed for growth at higher temperatures. This revealed that these three genes were related to thermotolerance. This study provides insights into the metabolic landscape of Y. lipolytica under heat stress, enabling future metabolic engineering endeavors to improve both thermoresistance and sugar alcohol production in the yeast Y. lipolytica.

A Dynamic Genome-Scale Model Identifies Metabolic Pathways Associated with Cold Tolerance in Saccharomyces kudriavzevii.

Saccharomyces kudriavzevii is a cold-tolerant species identified as a good alternative for industrial winemaking. Although S. kudriavzevii has never been found in winemaking, its co-occurrence with Saccharomyces cerevisiae in Mediterranean oaks is well documented. This sympatric association is believed to be possible due to the different growth temperatures of the two yeast species. However, the mechanisms behind the cold tolerance of S. kudriavzevii are not well understood. In this work, we propose the use of a dynamic genome-scale model to compare the metabolic routes used by S. kudriavzevii at two temperatures, 25°C and 12°C, to decipher pathways relevant to cold tolerance. The model successfully recovered the dynamics of biomass and external metabolites and allowed us to link the observed phenotype with exact intracellular pathways. The model predicted fluxes that are consistent with previous findings, but it also led to novel results which we further confirmed with intracellular metabolomics and transcriptomic data. The proposed model (along with the corresponding code) provides a comprehensive picture of the mechanisms of cold tolerance that occur within S. kudriavzevii. The proposed strategy offers a systematic approach to explore microbial diversity from extracellular fermentation data at low temperatures. IMPORTANCE Nonconventional yeasts promise to provide new metabolic pathways for producing industrially relevant compounds and tolerating specific stressors such as cold temperatures. The mechanisms behind the cold tolerance of S. kudriavzevii or its sympatric relationship with S. cerevisiae in Mediterranean oaks are not well understood. This study proposes a dynamic genome-scale model to investigate metabolic pathways relevant to cold tolerance. The predictions of the model would indicate the ability of S. kudriavzevii to produce assimilable nitrogen sources from extracellular proteins present in its natural niche. These predictions were further confirmed with metabolomics and transcriptomic data. This finding suggests that not only the different growth temperature preferences but also this proteolytic activity may contribute to the sympatric association with S. cerevisiae. Further exploration of these natural adaptations could lead to novel engineering targets for the biotechnological industry.

Bioethanol production from mulberry pomace by newly ısolated non-conventional yeast Hanseniaspora uvarum

Bioethanol production from mulberry pomace by newly ısolated non-conventional yeast Hanseniaspora uvarum

Identification and assessment of non-conventional yeasts in mixed fermentations for brewing bioflavored beer

The increasing demand for more flavored and complex beers encourages the investigation of novel and non-conventional yeasts with the ability to provide a combination of bioflavoring and low ethanol yields. The present study identified 22 yeasts isolated from different brewing sources, including the fermentation by-products known as yeast sludges, and characterized a selection of strains to find the more suitable for the aforementioned aims. HPLC and GC-FID analysis of its brewing products were performed.The most promising results were obtained with the non-conventional yeasts Pichia kudriavzevii MBELGA61 and Meyerozyma guilliermondii MUS122. The former, isolated from a Belgian wheat beer sludge, was capable of growing in wort (17.0°Bx., 20 °C) with very low ethanol yields (1.19 % v/v). Besides, upon mixed fermentations with Saccharomyces cerevisiae, was suitable to produce volatile compounds such as ethyl acetate, 2-phenyl ethanol and isoamyl alcohol, with characteristic fruity notes. M. guilliermondii MUS122, isolated from a golden ale beer sludge, partially attenuated the wort with low production of ethanol and biomass. In addition, provided some fruity and floral nuances to the aroma profile of mixed fermentations with brewer's yeast.The results suggest that these strains favor the development of more fruity-flowery aroma profiles in beers. Furthermore, they are suitable for use in mixed fermentations with Saccharomyces brewer's strains, although the ethanol level did not decrease significantly.

Investigation of the effects of culture conditions on cell growth and tetraacetylphytosphingosine production by mutant Wickerhamomyces ciferrii

Wickerhamomyces ciferrii is a non-conventional yeast strain characterized by the production of large amounts of tetraacetylphytosphingosine (TAPS). TAPS is an industrially valuable substance that can be converted into bioactive ceramide through deacetylation. TAPS-overproducing W. ciferrii is typically cultured in complex media, but there have been no systematic studies on the development of a defined medium for TAPS production. In this study, the composition of a defined medium for W. ciferrii strain was systematically investigated to increase the TAPS production. Among the defined medium components tested, Zn2+ significantly influenced TAPS production. The modified defined medium devoid of ZnSO4, with 40 g/L glycerol as a carbon source, increased TAPS production by 35% (1.16 ± 0.04 g/L, 0.039 g-TAPS/OD600) compared to the control defined medium (0.8 ± 0.01 g/L, 0.022 g-TAPS/OD600) in flask cultures. In batch bioreactor fermentation using the modified defined medium (40 g/L glycerol), 1.85 ± 0.1 g/L of TAPS (0.046 g-TAPS/g-glycerol, 0.044 g-TAPS/OD600) was achieved, which was 28% higher than 1.45 ± 0.02 g/L of TAPS (0.036 g-TAPS/g-glycerol, 0.027 g-TAPS/OD600) with the complex medium. Therefore, the modified defined medium developed in this study has the potential to serve as a base medium for large- scale TAPS production and for physiological and metabolic engineering studies.

A review of synthetic biology tools in Yarrowia lipolytica.

Yarrowia lipolytica is a non-conventional oleaginous yeast with great potential for industrial production. Y. lipolytica has a high propensity for flux through tricarboxylic acid cycle intermediates. Therefore, this host is currently being developed as a workhorse, and is rapidly emerging in biotechnology fields, especially for industrial chemical production, whole-cell bioconversion, and the treatment and recycling of industrial waste. In recent studies, Y. lipolytica has been rewritten and introduced with non-native metabolites of certain compounds of interest owing to the advancement in synthetic biology tools. In this review, we collate recent progress to present a detailed and insightful summary of the major developments in synthetic biology tools and techniques for Y. lipolytica, including promoters, terminators, selection markers, autonomously replicating sequences, DNA assembly techniques, genome editing techniques, and subcellular organelle engineering. This comprehensive overview would be a useful resource for future genetic engineering studies to improve the yield of desired metabolic products in Y. lipolytica.

Engineering Komagataella phaffii to biosynthesize cordycepin from methanol which drives global metabolic alterations at the transcription level

Cordycepin has the potential to be an alternative to the disputed herbicide glyphosate. However, current laborious and time-consuming production strategies at low yields based on Cordyceps militaris lead to extremely high cost and restrict its application in the field of agriculture. In this study, Komagataella phaffii (syn. Pichia pastoris) was engineered to biosynthesize cordycepin from methanol, which could be converted from CO2. Combined with fermentation optimization, cordycepin content in broth reached as high as 2.68 ± 0.04 g/L within 168 h, around 15.95 mg/(L·h) in productivity. Additionally, a deaminated product of cordycepin was identified at neutral or weakly alkaline starting pH during fermentation. Transcriptome analysis found the yeast producing cordycepin was experiencing severe inhibition in methanol assimilation and peroxisome biogenesis, responsible for delayed growth and decreased carbon flux to pentose phosphate pathway (PPP) which led to lack of precursor supply. Amino acid interconversion and disruption in RNA metabolism were also due to accumulation of cordycepin. The study provided a unique platform for the manufacture of cordycepin based on the emerging non-conventional yeast and gave practical strategies for further optimization of the microbial cell factory.

Draft Genome Sequence of the Fluconazole-Resistant Yarrowia lipolytica Clinical Isolate CBS 18115.

Yarrowia lipolytica is a nonconventional yeast of industrial interest that can sometimes act as an opportunistic pathogen and is responsible for invasive fungal infections. We report the draft genome sequence of the fluconazole-resistant strain CBS 18115, which was isolated from a blood culture. The Y132F substitution in ERG11, which was previously described in fluconazole-resistant Candida isolates, was identified.

Saccharomyces cerevisiae responds similarly to co-culture or to a fraction enriched in Metschnikowia pulcherrima extracellular vesicles.

The recent introduction of non-conventional yeast species as companion wine starters has prompted a growing interest in microbial interactions during wine fermentation. There is evidence of interactions through interference and exploitation competition, as well as interactions depending on physical contact. Furthermore, the results of some transcriptomic analyses suggest interspecific communication, but the molecules or biological structures involved in recognition are not well understood. In this work, we explored extracellular vesicles (EVs) as possible mediators of interspecific communication between wine yeasts. The transcriptomic response of Saccharomyces cerevisiae after 3h of contact with a fraction enriched in EVs of Metschnikowia pulcherrima was compared with that induced by active M.pulcherrima cells. Interestingly, there is a high level of overlap between the transcriptomic profiles of yeast cells challenged by either M.pulcherrima whole cells or the EV-enriched fraction. The results indicate an upregulation of yeast metabolism in response to competing species (in line with previous results). This finding points to the presence of a signal, in the EV-enriched fraction, that can be perceived by the yeast cells as a cue for the presence of competitors, even in the absence of metabolically active cells of the other species.

The Influence of Different Non-Conventional Yeasts on the Odour-Active Compounds of Produced Beers

The interest in new beer products, which has been growing for several years, forces technologists and brewers to look for innovative raw materials, such as hops, new sources of carbohydrates or yeast. The aim of the presented study was to evaluate the effect of selected Saccharomyces (Saccharomyces paradoxus (CBS 7302), S. kudriavzevii (CBS 3774), S. cerevisiae (Safbrew T-58)) and non-Saccharomyces yeast (W. anomalus (CBS 5759), Ha. uvarum (CBS 2768), D. bruxellensis (CBS 3429), Z. bailii (CBS 749), and T. delbrueckii (D10)) on the fermentation process, basic parameters and odour-active compounds of the produced beers. The chemical composition and key aroma components of the obtained beers were determined using various chromatographic methods (HPLC, GC-FID, GC-MS, and GC-O). We showed large differences between the key aroma components depending on the culture of microorganisms used. Forty different compounds that have an active impact on the creation of the aroma of beers were detected, among which the most important are: β-phenylethanol, ethyl hexanoate, ethyl 4-methylpentanoate, ethyl dihydrocinnamate and β-damascenone. We also found the presence of components specific to the yeast strain used, such as 2-methoxy-4-vinylphenol, γ-decalactone, methional, nerolidol and others. Among the analyzed yeasts, S. kudriavzevii and W. anomalus should be distinguished, which produced beers with intense fruity and floral aromas and were also characterized by favorable features for brewing. The Z. bailii strain also turned out to be interesting as a potential starter culture for the production of low-alcohol beers, significantly differing in sensory characteristics from the standard ones.

Global metabolic rewiring of the nonconventional yeast Ogataea polymorpha for biosynthesis of the sesquiterpenoid β-elemene

Bioproduction of natural products via microbial cell factories is a promising alternative to traditional plant extraction. Recently, nonconventional microorganisms have emerged as attractive chassis hosts for biomanufacturing. One such microorganism, Ogataea polymorpha is an industrial yeast used for protein expression with numerous advantages, such as thermal-tolerance, a wide substrate spectrum and high-density fermentation. Here, we systematically rewired the cellular metabolism of O. polymorpha to achieve high-level production of the sesquiterpenoid β-elemene by optimizing the mevalonate pathway, enhancing the supply of NADPH and acetyl-CoA, and downregulating competitive pathways. The engineered strain produced 509 mg/L and 4.7 g/L of β-elemene under batch and fed-batch fermentation, respectively. Therefore, this study identified the potential industrial application of O. polymorpha as a good microbial platform for producing sesquiterpenoids.

Metabolic engineering of low-pH-tolerant non-model yeast, Issatchenkia orientalis, for production of citramalate

Methyl methacrylate (MMA) is an important petrochemical with many applications. However, its manufacture has a large environmental footprint. Combined biological and chemical synthesis (semisynthesis) may be a promising alternative to reduce both cost and environmental impact, but strains that can produce the MMA precursor (citramalate) at low pH are required. A non-conventional yeast, Issatchenkia orientalis, may prove ideal, as it can survive extremely low pH. Here, we demonstrate the engineering of I. orientalis for citramalate production. Using sequence similarity network analysis and subsequent DNA synthesis, we selected a more active citramalate synthase gene (cimA) variant for expression in I. orientalis. We then adapted a piggyBac transposon system for I. orientalis that allowed us to simultaneously explore the effects of different cimA gene copy numbers and integration locations. A batch fermentation showed the genome-integrated-cimA strains produced 2.0 g/L citramalate in 48 h and a yield of up to 7% mol citramalate/mol consumed glucose. These results demonstrate the potential of I. orientalis as a chassis for citramalate production.

Study of the Fermentation Characteristics of Non-Conventional Yeast Strains in Sweet Dough

Despite the diverse functions of yeast, only a relatively homogenous group of Saccharomyces cerevisiae yeasts is used in the baking industry. Much of the potential of the natural diversity of yeasts has not been explored, and the sensory complexity of fermented baked foods is limited. While research on non-conventional yeast strains in bread making is increasing, it is minimal for sweet fermented bakery products. In this study, the fermentation characteristics of 23 yeasts from the bakery, beer, wine, and spirits industries were investigated in sweet dough (14% added sucrose w/w dm flour). Significant differences in invertase activity, sugar consumption (0.78-5.25% w/w dm flour), and metabolite (0.33-3.01% CO2; 0.20-1.26% ethanol; 0.17-0.80% glycerol; 0.09-0.29% organic acids) and volatile compound production were observed. A strong positive correlation (R2 = 0.76, p < 0.001) between sugar consumption and metabolite production was measured. Several non-conventional yeast strains produced more positive aroma compounds and fewer off-flavors than the reference baker's yeast. This study shows the potential of non-conventional yeast strains in sweet dough.