Unlocking the genome of the non-sourdough Kazachstania humilis MAW1: insights into inhibitory factors and phenotypic properties.

BACKGROUND: Ascomycetous budding yeasts are ubiquitous environmental microorganisms important in food production and medicine. Due to recent intensive genomic research, the taxonomy of yeast is becoming more organized based on the identification of monophyletic taxa. This includes genera important to humans, such as Kazachstania. Until now, Kazachstania humilis (previously Candida humilis) was regarded as a sourdough-specific yeast. In addition, any antibacterial activity has not been associated with this species. RESULTS: Previously, we isolated a yeast strain that impaired bio-hydrogen production in a dark fermentation bioreactor and inhibited the growth of Gram-positive and Gram-negative bacteria. Here, using next generation sequencing technologies, we sequenced the genome of this strain named K. humilis MAW1. This is the first genome of a K. humilis isolate not originating from a fermented food. We used novel phylogenetic approach employing the 18 S-ITS-D1-D2 region to show the placement of the K. humilis MAW1 among other members of the Kazachstania genus. This strain was examined by global phenotypic profiling, including carbon sources utilized and the influence of stress conditions on growth. Using the well-recognized bacterial model Escherichia coli AB1157, we show that K. humilis MAW1 cultivated in an acidic medium inhibits bacterial growth by the disturbance of cell division, manifested by filament formation. To gain a greater understanding of the inhibitory effect of K. humilis MAW1, we selected 23 yeast proteins with recognized toxic activity against bacteria and used them for Blast searches of the K. humilis MAW1 genome assembly. The resulting panel of genes present in the K. humilis MAW1 genome included those encoding the 1,3-ß-glucan glycosidase and the 1,3-ß-glucan synthesis inhibitor that might disturb the bacterial cell envelope structures. CONCLUSIONS: We characterized a non-sourdough-derived strain of K. humilis, including its genome sequence and physiological aspects. The MAW1, together with other K. humilis strains, shows the new organization of the mating-type locus. The revealed here pH-dependent ability to inhibit bacterial growth has not been previously recognized in this species. Our study contributes to the building of genome sequence-based classification systems; better understanding of K.humilis as a cell factory in fermentation processes and exploring bacteria-yeast interactions in microbial communities.

Monitoring the yeasts ecology and volatiles profile throughout the spontaneous fermentation of Taggiasca cv. table olives through culture-dependent and independent methods.

Taggiasca table olives are typical of Liguria, a Northwestern Italian region, produced with a spontaneous fermentation carried out by placing the raw drupes directly into brine with a salt concentration of 8-12 % w/v. Such concentrations limit the development of unwanted microbes and favor the growth of yeasts. This process usually lasts up to 8 months. Yeasts are found throughout the entire fermentation process and they are mainly involved in the production of volatile organic compounds, which strongly impact the quality of the final product. The aim of this study was to evaluate the dynamics of autochthonous yeasts in brines and olives in a spontaneous process with no lye pre-treatment or addition of acids in the fermenting brine with 10 % NaCl (w/v) in two batches during 2021 harvest. Three hundred seventy-three yeast colonies were isolated, characterized by rep-PCR and identified by the D1/D2 region of the 26S rRNA gene sequencing. Mycobiota was also studied by 26S rRNA gene metataxonomics, while metabolome was assessed through GC-MS analysis. Traditional culture-dependent methods showed the dominance of Candida diddensiae, Wickerhamomyces anomalus, Pichia membranifaciens and Aureobasidium pullulans, with differences in species distribution between batches, sampling time and type of sample (olives/brines). Amplicon-based sequencing confirmed the dominance of W. anomalus in batch 1 throughout the entire fermentation, while Cyteromyces nyonsensis and Aureobasidium spp. were most abundant in the fermentation in batch 2. Volatilome results were analyzed and correlated to the mycobiota data, confirming differences between fermentation stages. Given the high appreciation for this traditional food, this study helps elucidate the mycobiota associated to Taggiasca cv. table olives and its relationship with the quality of the final product.

Recombinant monoclonal antibody production in yeasts: Challenges and considerations.

Monoclonal antibodies (mAbs) are laboratory-based engineered protein molecules with a monovalent affinity or multivalent avidity towards a specific target or antigen, which can mimic natural antibodies that are produced in the human immune systems to fight against detrimental pathogens. The recombinant mAb is one of the most effective classes of biopharmaceuticals produced in vitro by cloning and expressing synthetic antibody genes in a suitable host. Yeast is one of the potential hosts among others for the successful production of recombinant mAbs. However, there are very few yeast-derived mAbs that got the approval of the regulatory agencies for direct use for treatment purposes. Certain challenges encountered by yeasts for recombinant antibody productions need to be overcome and a few considerations related to antibody structure, host engineering, and culturing strategies should be followed for the improved production of mAbs in yeasts. In this review, the drawbacks related to the metabolic burden of the host, culturing conditions including induction mechanism and secretion efficiency, solubility and stability, downstream processing, and the pharmacokinetic behavior of the antibody are discussed, which will help in developing the yeast hosts for the efficient production of recombinant mAbs.

Physiological and morphological plasticity in response to nitrogen availability of a yeast widely distributed in the open ocean.

Yeasts are prevalent in the open ocean, yet we have limited understanding of their ecophysiological adaptations, including their response to nitrogen availability, which can have a major role in determining the ecological potential of other planktonic microbes. In this study, we characterized the nitrogen uptake capabilities and growth responses of marine-occurring yeasts. Yeast isolates from the North Atlantic Ocean were screened for growth on diverse nitrogen substrates, and across a concentration gradient of three environmentally relevant nitrogen substrates: nitrate, ammonium, and urea. Three strains grew with enriched nitrate while two did not, demonstrating that nitrate utilization is present but not universal in marine yeasts, consistent with existing knowledge of nonmarine yeast strains. Naganishia diffluens MBA_F0213 modified the key functional trait of cell size in response to nitrogen concentration, suggesting yeast cell morphology changes along chemical gradients in the marine environment. Meta-analysis of the reference DNA barcode in public databases revealed that the genus Naganishia has a global ocean distribution, strengthening the environmental applicability of the culture-based observations. This study provides novel quantitative understanding of the ecophysiological and morphological responses of marine-derived yeasts to variable nitrogen availability in vitro, providing insight into the functional ecology of yeasts within pelagic open ocean environments.

Back to the future: Comparing yeast as an outmoded artificial tracer for simulating microbial transport in karst aquifer systems to more modern approaches.

Bacterial contamination of karst groundwater is a major concern for public health. Artificial tracing studies are crucial for establishing links between locations where pollutants can rapidly reach the aquifer systems and subsequent receptors, as well as for enhanced understanding of pollutant transport. However, widely used solute artificial tracers do not always move through the subsurface in the same manner as particles and microorganisms, hence may not be ideal proxies for predicting movement of bacterial contaminants. This study evaluates whether a historically used microbial tracer (yeast) which is readily available, inexpensive, and environmentally friendly, but usually overlooked in modern karst hydrogeological studies due to challenges associated with its detection and quantification in the past, can reemerge as a valuable tracer using the latest technology for its detection. Two field-based studies on separate karst systems were carried out during low-flow conditions using a portable particle counter along with flow cytometry measurements to monitor the recovery of the yeast at the springs. Soluble fluorescent dyes were also injected simultaneously with the yeast for comparison of transport dynamics. On one tracer test, through a karst conduit of much higher velocities, the injected yeast and fluorescent dye arrived at the same time at the spring, in comparison to the tracer test on a conduit system with lower groundwater velocities in which the yeast particles were detected before the dye at the sampling site. Both a portable particle counter and flow cytometry successfully detected yeast during both tests, thereby demonstrating the applicability of this tracer with contemporary instrumentation. Even though no significant advantages of flow cytometry over the portable counter system can be reported on the basis of the presented results, this study has shown that flow cytometry can be successfully used to detect and quantify introduced microbial tracers in karst environments with extremely high precision.

Advances in recombinant protease production: current state and perspectives.

Proteases, enzymes that catalyze the hydrolysis of peptide bonds in proteins, are important in the food industry, biotechnology, and medical fields. With increasing demand for proteases, there is a growing emphasis on enhancing their expression and production through microbial systems. However, proteases' native hosts often fall short in high-level expression and compatibility with downstream applications. As a result, the recombinant production of proteases has become a significant focus, offering a solution to these challenges. This review presents an overview of the current state of protease production in prokaryotic and eukaryotic expression systems, highlighting key findings and trends. In prokaryotic systems, the Bacillus spp. is the predominant host for proteinase expression. Yeasts are commonly used in eukaryotic systems. Recent advancements in protease engineering over the past five years, including rational design and directed evolution, are also highlighted. By exploring the progress in both expression systems and engineering techniques, this review provides a detailed understanding of the current landscape of recombinant protease research and its prospects for future advancements.

Recovery and characterization of ß-glucosidase-producing non-Saccharomyces yeasts from the fermentation broth of Vitis labruscana Baily × Vitis vinifera L. for investigation of their fermentation characteristics.

The present study focuses on investigating 60 strains of yeast isolated from the natural fermentation broth of Vitis labruscana Baily × Vitis vinifera L. These strains underwent screening using lysine culture medium and esculin culture medium, resulting in the identification of 27 local non-Saccharomyces yeast strains exhibiting high ß-glucosidase production. Subsequent analysis of their fermentation characteristics led to the selection of four superior strains (Z-6, Z-11, Z-25, and Z-58) with excellent ß-glucosidase production and fermentation performance. Notably, these selected strains displayed a dark coloration on esculin medium and exhibited robust gas production during Duchenne tubules' fermentation test. Furthermore, all four non-Saccharomyces yeast strains demonstrated normal growth under specific conditions including SO2 mass concentration ranging from 0.1 to 0.3 g/L, temperature between 25 and 30 °C, glucose mass concentration ranging from 200 to 400 g/L, and ethanol concentration at approximately 4%. Molecular biology identification confirmed that all selected strains belonged to Pichia kudriavzevii species which holds great potential for wine production.

Agro-ecosystem of honeybees as source for native probiotic yeasts.

Probiotic microorganisms are used to improve the health and wellness of people and the research on this topic is of current relevance and interest. Fifty-five yeasts, coming from honeybee's ecosystem and belonging to Candida, Debaryomyces, Hanseniaspora, Lachancea, Metschnikowia, Meyerozyma, Starmerella and Zygosacchromyces genera and related different species, were evaluated for the probiotic traits. The resistance to gastrointestinal conditions, auto-aggregation, cell surface hydrophobicity or biofilm formation abilities as well as antimicrobial activity against common human pathogenic bacteria were evaluated. The safety analysis of strains was also carried out to exclude any possible negative effect on the consumer's health. The influence of proteinase treatment of living yeasts and their adhesion to Caco-2 cells were also evaluated. The greatest selection occurred in the first step of survival at the acidic pH and in the presence of bile salts, where more than 50% of the strains were unable to survive. Equally discriminating was the protease test which allowed the survival of only 27 strains belonging to the species Hanseniaspora guilliermondii, Hanseniaspora uvarum, Metschnikowia pulcherrima, Metschnikowia ziziphicola, Meyerozyma caribbica, Meyerozyma guilliermondii, Pichia kluyveri, Pichia kudriavzevii and Pichia terricola. An integrated analysis of the results obtained allowed the detection of seven yeast strains with probiotic aptitudes, all belonging to the Meyerozyma genus, of which three belonging to M. guillermondii and four belonging to M. caribbica species.

Microbial metabolism of caffeine and potential applications in bioremediation.

With increasing global consumption of caffeine-rich products, such as coffee, tea, and energy drinks, there is also an increase in urban and processing waste full of residual caffeine with limited disposal options. This waste caffeine has been found to leach into the surrounding environment where it poses a threat to microorganisms, insects, small animals, and entire ecosystems. Growing interest in harnessing this environmental contaminant has led to the discovery of 79 bacterial strains, eight yeast strains, and 32 fungal strains capable of metabolizing caffeine by N-demethylation and/or C-8 oxidation. Recently observed promiscuity of caffeine-degrading enzymes in vivo has opened up the possibility of engineering bacterial strains capable of producing a wide variety of caffeine derivatives from a renewable resource. These engineered strains can be used to reduce the negative environmental impact of leached caffeine-rich waste through bioremediation efforts supplemented by our increasing understanding of new techniques such as cell immobilization. Here, we compile all of the known caffeine-degrading microbial strains, discuss their metabolism and related enzymology, and investigate their potential application in bioremediation.

Japanese sake making using wild yeasts isolated from natural environments.

Saccharomyces cerevisiae is one of the most important microorganisms for the food industry, including Japanese sake, beer, wine, bread, and other products. For sake making, Kyokai sake yeast strains are considered one of the best sake yeast strains because these strains possess fermentation properties that are suitable for the quality of sake required. In recent years, the momentum for the development of unique sake, which is distinct from conventional sake, has grown, and there is now a demand to develop unique sake yeasts that have different sake making properties than Kyokai sake yeast strains. In this minireview, we focus on "wild yeasts," which inhabit natural environments, and introduce basic research on the wild yeasts for sake making, such as their genetic and sake fermentation aspects. Finally, we also discuss the molecular breeding of wild yeast strains for sake fermentation and the possibility for sake making using wild yeasts.

Isolation and characterization of Candida tropicalis B: a promising yeast strain for biodegradation of petroleum oil in marine environments.

The increasing interest in environmental protection laws has compelled companies to regulate the disposal of waste organic materials. Despite efforts to explore alternative energy sources, the world remains heavily dependent on crude petroleum oil and its derivatives. The expansion of the petroleum industry has significant implications for human and environmental well-being. Bioremediation, employing living microorganisms, presents a promising approach to mitigate the harmful effects of organic hydrocarbons derived from petroleum. This study aimed to isolate and purify local yeast strains from oil-contaminated marine water samples capable of aerobically degrading crude petroleum oils and utilizing them as sole carbon and energy sources. One yeast strain (isolate B) identified as Candida tropicalis demonstrated high potential for biodegrading petroleum oil in seawater. Physiological characterization revealed the strain's ability to thrive across a wide pH range (4-11) with optimal growth at pH 4, as well as tolerate salt concentrations ranging from 1 to 12%. The presence of glucose and yeast extract in the growth medium significantly enhanced the strain's biomass formation and biodegradation capacity. Scanning electron microscopy indicated that the yeast cell diameter varied based on the medium composition, further emphasizing the importance of organic nitrogenous sources for initial growth. Furthermore, the yeast strain exhibited remarkable capabilities in degrading various aliphatic and aromatic hydrocarbons, with a notable preference for naphthalene and phenol at 500 and 1000 mg/l, naphthalene removal reached 97.4% and 98.6%, and phenol removal reached 79.48% and 52.79%, respectively. Optimization experiments using multi-factorial sequential designs highlighted the influential role of oil concentration on the bioremediation efficiency of Candida tropicalis strain B. Moreover, immobilized yeast cells on thin wood chips demonstrated enhanced crude oil degradation compared to thick wood chips, likely due to increased surface area for cell attachment. These findings contribute to our understanding of the potential of Candida tropicalis for petroleum oil bioremediation in marine environments, paving the way for sustainable approaches to address oil pollution.

Research progress on carotenoid production by Rhodosporidium toruloides.

Carotenoids are natural lipophilic pigments, which have been proven to provide significant health benefits to humans, relying on their capacity to efficiently scavenge singlet oxygen and peroxyl radicals as antioxidants. Strains belonging to the genus Rhodosporidium represent a heterogeneous group known for a number of phenotypic traits including accumulation of carotenoids and lipids and tolerance to heavy metals and oxidative stress. As a representative of these yeasts, Rhodosporidium toruloides naturally produces carotenoids with high antioxidant activity and grows on a wide variety of carbon sources. As a result, R. toruloides is a promising host for the efficient production of more value-added lipophilic compound carotenoids, e.g., torulene and torularhodin. This review provides a comprehensive summary of the research progress on carotenoid biosynthesis in R. toruloides, focusing on the understanding of biosynthetic pathways and the regulation of key enzymes and genes involved in the process. Moreover, the relationship between the accumulation of carotenoids and lipid biosynthesis, as well as the stress from diverse abiotic factors, has also been discussed for the first time. Finally, several feasible strategies have been proposed to promote carotenoid production by R. toruloides. It is possible that R. toruloides may become a critical strain in the production of carotenoids or high-value terpenoids by genetic technologies and optimal fermentation processes. KEY POINTS: • Biosynthetic pathway and its regulation of carotenoids in Rhodosporidium toruloides were concluded • Stimulation of abiotic factors for carotenoid biosynthesis in R. toruloides was summarized • Feasible strategies for increasing carotenoid production by R. toruloides were proposed.

How do the carbon and nitrogen sources affect the synthesis of ß-(1,3/1,6)-glucan, its structure and the susceptibility of Candida utilis yeast cells to immunolabelling with ß-(1,3)-glucan monoclonal antibodies?

BACKGROUND: The need to limit antibiotic therapy due to the spreading resistance of pathogenic microorganisms to these medicinal substances stimulates research on new therapeutic agents, including the treatment and prevention of animal diseases. This is one of the goals of the European Green Deal and the Farm-To-Fork strategy. Yeast biomass with an appropriate composition and exposure of cell wall polysaccharides could constitute a functional feed additive in precision animal nutrition, naturally stimulating the immune system to fight infections. RESULTS: The results of the research carried out in this study showed that the composition of Candida utilis ATCC 9950 yeast biomass differed depending on growth medium, considering especially the content of ß-(1,3/1,6)-glucan, α-glucan, and trehalose. The highest ß-(1,3/1,6)-glucan content was observed after cultivation in deproteinated potato juice water (DPJW) as a nitrogen source and glycerol as a carbon source. Isolation of the polysaccharide from yeast biomass confirmed the highest yield of ß-(1,3/1,6)-glucan after cultivation in indicated medium. The differences in the susceptibility of ß-(1,3)-glucan localized in cells to interaction with specific ß-(1,3)-glucan antibody was noted depending on the culture conditions. The polymer in cells from the DPJW supplemented with glycerol and galactose were labelled with monoclonal antibodies with highest intensity, interestingly being less susceptible to such an interaction after cell multiplication in medium with glycerol as carbon source and yeast extract plus peptone as a nitrogen source. CONCLUSIONS: Obtained results confirmed differences in the structure of the ß-(1,3/1,6)-glucan polymers considering side-chain length and branching frequency, as well as in quantity of ß-(1,3)- and ß-(1,6)-chains, however, no visible relationship was observed between the structural characteristics of the isolated polymers and its susceptibility to immunolabeling in whole cells. Presumably, other outer surface components and molecules can mask, shield, protect, or hide epitopes from antibodies. ß-(1,3)-Glucan was more intensely recognized by monoclonal antibody in cells with lower trehalose and glycogen content. This suggests the need to cultivate yeast biomass under appropriate conditions to fulfil possible therapeutic functions. However, our in vitro findings should be confirmed in further studies using tissue or animal models.

Lentils protein isolate as a fermenting substrate for the production of bioactive peptides by lactic acid bacteria and neglected yeast species.

In the current trend where plant-based foods are preferred over animal-based foods, pulses represent an alternative source of protein but also of bioactive peptides (BPs). We investigated the pattern of protein hydrolysis during fermentation of red lentils protein isolate (RLPI) with various lactic acid bacteria and yeast strains. Hanseniaspora uvarum SY1 and Fructilactobacillus sanfranciscensis E10 were the most proteolytic microorganisms. H. uvarum SY1 led to the highest antiradical, angiotensin-converting enzyme-inhibitory and antifungal activities, as found in low molecular weight water soluble extracts (LMW-WSE). The 2039 peptide sequences identified by LMW-WSE were screened using BIOPEP UWM database, and 36 sequences matched with known BPs. Fermentation of RLPI by lactic acid bacteria and yeasts generated 12 peptides undetected in raw RLPI. Besides, H. uvarum SY1 led to the highest abundance (peak areas) of BPs, in particular with antioxidant and ACE-inhibitory activities. The amino acid sequences LVR and LVL, identified in the fermented RLPI, represent novel findings, as they were detected for the first time in substrates subjected to microbial fermentation. KVI, another BP highly characteristic of RLPI-SY1, was previously observed only in dried bonito. 44 novel potential BPs, worthy of further characterization, were correlated with antifungal activity.

Mechanism of carbohydrate and protein conversion during sourdough fermentation: An analysis based on representative Chinese sourdough microbiota.

Sourdough fermentation is attracting growing attention because of its positive effects on properties of leavened baked good. However, the changes in dough features and the mechanisms behind them are not well understood, which limits its widespread use. In this study, we assessed the effects of representative lactic acid bacteria in sourdough monoculture or co-culture with yeasts on dough characteristics. Physicochemical analysis identified increased proteolysis and enhanced nutritional properties of co-culture groups. However, a reduction in organic acids contents of co-culture groups compared to monoculture was detected, and this effect was not limited by the yeast species. The RNA sequencing further demonstrated that the presence of yeast enhanced the protein metabolic activity of lactic acid bacteria, while decreased its organic acid biosynthetic activity. Moreover, the proteomic analysis revealed that endogenous metabolic proteins of flour, such as pyruvate kinase, glucosyltransferase and pyruvate dehydrogenase play a key role in carbohydrate metabolism during fermentation. This study uncovered the influence of typical microorganisms and endogenous enzymes on dough characteristics based on different aspects. Bacteria-mediated consumption of proteins and increased proteolysis in co-culture groups may underlie the improved digestibility and nutritional effects of sourdough fermented products, which provides an important basis for nutrient fortified bread making with multi-strain leavening agent.

Succinic acid production by wine yeasts and the influence of GABA and glutamic acid.

As a consequence of alcoholic fermentation (AF) in wine, several compounds are released by yeasts, and some of them are linked to the general quality and mouthfeel perceptions in wine. However, others, such as succinic acid, act as inhibitors, mainly of malolactic fermentation. Succinic acid is produced by non-Saccharomyces and Saccharomyces yeasts during the initial stages of AF, and the presence of some amino acids such as γ-aminobutyric acid (GABA) and glutamic acid can increase the concentration of succinic acid. However, the influence of these amino acids on succinic acid production has been studied very little to date. In this work, we studied the production of succinic acid by different strains of non-Saccharomyces and Saccharomyces yeasts during AF in synthetic must, and the influence of the addition of GABA or glutamic acid or a combination of both. The results showed that succinic acid can be produced by non-Saccharomyces yeasts with values in the range of 0.2-0.4 g/L. Moreover, the addition of GABA or glutamic acid can increase the concentration of succinic acid produced by some strains to almost 100 mg/L more than the control, while other strains produce less. Consequently, higher succinic acid production by non-Saccharomyces yeast in coinoculated fermentations with S. cerevisiae strains could represent a risk of inhibiting Oenococcus oeni and therefore the MLF.

A transcriptome profile of Wickerhamomyces anomalus incubated with chitosan revealed dynamic changes in gene expression and metabolic pathways.

Previous studies have shown that Wickerhamomyces anomalus can control postharvest diseases of fruits and incubation of the yeast with chitosan can improve its efficiency. In this study, transcriptome study was conducted to determine molecular mechanisms involved in the yeast-chitosan interaction. The bioinformatics analysis of the RNA-seq data confirmed that incubating W. anomalus with 1 % chitosan for 24 h significantly altered the expression of differential genes involved in yeast metabolic and cellular activities. Genes involved in ethyl acetate production, reactive oxygen species regulation, cell wall reinforcement, stress resistance, and signalling were all significantly up-regulated. Pathways which have significant role in the yeast growth and reproduction, energy production, cellular homeostasis, signal transduction, catalytic, and antioxidant activities were significantly enriched. In general, incubation of the yeast with chitosan genes metabolic pathways which are important for the yeast survival, adaptation, and reproduction. Molecular studies are important in providing fundamental theoretical foundation for the practical application of antagonistic yeasts for future uses. As a result, this research will be an input for use of the antagonistic yeast as microbial or biochemical pesticides instead of synthetic chemicals which have both health and environmental effects.

Whole-genome sequence and characterization of a marine red yeast, Rhodosporidium sphaerocarpum GDMCC 60679, featuring the assimilation of ammonia nitrogen.

A marine red yeast, Rhodosporidium sphaerocarpum, is generally used for the production of lipids and carotenoids. In a previous study, we demonstrated that a marine-derived R. sphaerocarpum GDMCC 60679 can efficiently remove ammonia nitrogen and exhibit multiple probiotic functions for shrimp, Litopenaeus vannamei. Here, we performed a genome assembly of the strain GDMCC 60679 using a combination of the data from Illumina PE and PacBio CLR reads. The genome has a size of 18.03 Mb and consists of 32 contigs with an N50 length of 1,074,774 bp and GC content of 63 %. The genome was predicted to contain 6092 protein-coding genes, 5962 of which were functionally annotated. Metabolic pathways responsible for the ammonia assimilation and the synthesis of lipids and carotenoids were particularly examined to explore and characterize genes contributing to these functions. Whole-genome sequence and annotation of the strain lays a foundation to reveal the molecular mechanism of its prominent biological functions and will facilitate us to further expand new applications of yeasts in Rhodosporidium.

Selection of yeast strains in naturally fermented cured meat as promising starter cultures for fermented cured beef, a traditional fermented meat product of northern China.

BACKGROUND: Fermented meat products are meat products with a unique flavor, color, and texture as well as an extended shelf life under natural or artificially controlled conditions. Microorganisms or enzymes are used to ferment the raw meat so that it undergoes a series of biochemical and physical changes. Common fermentation strains are lactic acid bacteria, yeasts, staphylococci, molds, and so forth. Studies on the inhibitory effect of yeast fermentation strain on N-nitrosamines in fermented meat products have not been reported. Two excellent yeast starters were identified to solve the problem of nitrosamines in fermented meat products. RESULTS: Meyerozyma guilliermondii and Debaryomyces hansenii led to weak acid production, strong resistance to NaCl and NaNO2 , and high tolerance to low acidic conditions. The inoculated fermented beef exhibited decreased lightness, moisture content, water activity, pH, protein content, nitrite content, and N-nitrosamine content in comparison with the control group fermented bacon. M. guilliermondii had a better effect, reducing pH from 5.69 to 5.41, protein content from 254.24 to 221.92 g·kg-1 , nitrite content from 28.61 to 25.33 mg·kg-1 and N-nitrosamine by 18.97%, and giving the fermented beef the desired meat color, mouthfeel, odor, taste, and tissue quality. CONCLUSION: In this study, two strains of yeast fermenters that can degrade N-nitrosamine precursors were identified, which to some extent solves the problem of the high risk of generating nitrosamines such as N-nitrosodiethylamine (NDEA) by processing fermented meat products with nitrites as precursors. These two strains are likely to be used as starter cultures for fermented meat products. © 2023 Society of Chemical Industry.

Enhancement of novel Endo-polygalacturonase expression in Rhodotorula mucilaginosa PY18: insights from mutagenesis and molecular docking.

Pectinase is a particular type of enzyme that can break down pectin compounds and is extensively utilised in the agricultural field. In this study, twenty yeast isolates were isolated and assayed for pectinase activity. Molecular identification by PCR amplification and sequencing of internal transcribed spacer (ITS) regions of isolate no. 18 had the highest pectinase activity of 46.35 U/mg, was identified as Rhodotorula mucilaginosa PY18, and was submitted under accession no. (OM275426) in NCBI. Rhodotorula mucilaginosa PY18 was further enhanced through sequential mutagenesis, resulting in a mutant designated as Rhodotorula mucilaginosa E54 with a specific activity of 114.2 U/mg. Using Response Surface Methodology (RSM), the best culture conditions for the pectinase-producing yeast mutant Rhodotorula mucilaginosa E54 were pH 5, 72-h incubation, 2.5% xylose, and 2.5% malt extract, with a pectinase-specific activity of 156.55 U/mg. Then, the obtained sequences of the endo-polygalacturonase PGI gene from Rhodotorula mucilaginosa PY18 and mutant Rhodotorula mucilaginosa E54 were isolated for the first time, sequenced, and submitted to NCBI accession numbers OQ283005 and OQ283006, respectively. The modelled 3D structure of the endo-PGI enzyme (485 residues) was validated using Ramachandran's plot, which showed 87.71, 85.56, and 91.57% in the most favourable region for template Rhodotorula mucilaginosa KR, strain Rhodotorula mucilaginosa PY18, and mutant Rhodotorula mucilaginosa E54, respectively. In molecular docking studies, the results of template Rhodotorula mucilaginosa KR endo-PG1 showed an interaction with an affinity score of - 6.0, - 5.9, and - 5.6 kcal/mol for active sites 1, 2, and 3, respectively. Rhodotorula mucilaginosa PY18 endo-PG1 showed an interaction affinity with a score of - 5.8, - 6.0, and - 5.0 kcal/mol for active sites 1, 2, and 3, respectively. Mutant Rhodotorula mucilaginosa E54 endo-PG1 showed an interaction affinity of - 5.6, - 5.5, - 5.5 and - 5.4 kcal/mol for active sites 1, 2, and 3, respectively. The endo-PGI genes of both the yeast strain Rhodotorula mucilaginosa PY18 and mutant Rhodotorula mucilaginosa E54 were successfully cloned and expressed in E. coli DH5α, showing significantly higher endo-PG1 activity, which recorded 94.57 and 153.10 U/mg for recombinant Rhodotorula mucilaginosa pGEM-PGI-PY18 and recombinant mutant Rhotorula pGEM-PGI-E54, respectively.