A three-years survey of microbial contaminants in industrial hemp inflorescences from two Italian cultivation sites.

BACKGROUND: The use of industrial Cannabis sativa L. for recreational, cosmeceutical, nutraceutical, and medicinal purposes has gained momentum due to its rich content of valuable phytochemicals, such as cannabidiol (CBD) and cannabigerol (CBG). However, there are concerns regarding the risk of microbial contamination in plants grown outside controlled environments. Microbes associated with hemp can be either epiphytes or endophytes and may pose a risk of infectious illness for humans. METHODS: Seven Italian hemp genotypes, including Bernabeo, Carmagnola, Carmaleonte, Codimono, CS, Eletta Campana, and Fibranova, were cultivated in two distinct geographic locations, Catania and Rovigo, for three consecutive years from 2019 to 2021. Total aerobic microbes (TAMC), total combined yeasts/moulds (TYMC), the presence of bile-tolerant Gram-negative bacteria, and the absence of Escherichia coli and Salmonella spp. were evaluated and compared. The main phytocannabinoid content was measured and correlated with microbial contamination. RESULTS: Most samples analyzed in this study did not meet the European Pharmacopoeia microbiological limits. The detection of potential pathogens, such as E. coli and Salmonella spp., in the samples indicates that the use of inflorescences may represent a possible source of infection. Microbial contamination varied among harvesting seasons and production sites, with agroclimatic conditions influencing microbial load and composition. The presence of potentially pathogenic bacteria was less associated with seasonal climate variability and more likely affected by sporadic contamination from external sources. CBD concentration exhibited a negative correlation with bile-tolerant Gram-negative bacteria and total yeasts/moulds levels. Samples with lower CBD content were more contaminated than those with higher CBD levels, suggesting a potential protective effect of this phytochemical on the plant. CONCLUSIONS: The threshing residues (inflorescences, floral bracts, and leaves) of industrial hemp varieties represent a valuable product and a source of beneficial phytochemicals that warrants further exploration. While post-harvest sterilization methods may reduce microbiological risks, they may also degrade heat- and light-sensitive bioactive phytochemicals. The most promising strategy involves implementing best agronomic practices to maintain healthy and uncontaminated cultures. Rigorous monitoring and quality certification protocols are essential to mitigate the microbiological risk associated with the consumption of hemp-derived products.

First Data on the Investigation of Gut Yeasts in Hermit Beetle (Osmoderma barnabita Motschulsky, 1845) Larvae in Lithuania.

In this study, yeasts from the gut of O. barnabita larvae were isolated and molecularly identified. It is worth noting that this research provides the first analysis of the gut yeast community in O. barnabita larvae in Lithuania, which is a significant contribution to the field. Two hermit-like L3-praepupa instars were collected from a decaying oak log in Lithuania. The isolation, morphology, biochemistry, and physiology of the yeast isolates were characterized using standards commonly employed in yeast taxonomy studies. The isolates were identified by sequencing the large subunit (26S) rDNA (D1/D2 domain of the LSU). All gut compartments were colonized by the yeast. A total of 45 yeast strains were obtained from the gut of both O. barnabita larvae, with 23 strains originating from Larva 1, 16 strains from Larva 2, and 6 strains from the galleries. According to our identification results of the 45 yeast strains, most of the species were related to Ascomycota, with most of them belonging to the Saccharomycetales order. Yeasts of the genera Candida, Debaryomyces, Meyerozyma, Priceomyces, Schwanniomyces, Spencermartinsiella, Trichomonascus, and Blastobotrys were present in gut of O. barnabita larvae. Species of the Trichosporonales order represented the Basidiomycota phylum.

Identification of Food Spoilage Fungi Using MALDI-TOF MS: Spectral Database Development and Application to Species Complex.

Fungi, including filamentous fungi and yeasts, are major contributors to global food losses and waste due to their ability to colonize a very large diversity of food raw materials and processed foods throughout the food chain. In addition, numerous fungal species are mycotoxin producers and can also be responsible for opportunistic infections. In recent years, MALDI-TOF MS has emerged as a valuable, rapid and reliable asset for fungal identification in order to ensure food safety and quality. In this context, this study aimed at expanding the VITEK® MS database with food-relevant fungal species and evaluate its performance, with a specific emphasis on species differentiation within species complexes. To this end, a total of 380 yeast and mold strains belonging to 51 genera and 133 species were added into the spectral database including species from five species complexes corresponding to Colletotrichum acutatum, Colletotrichum gloeosporioides, Fusarium dimerum, Mucor circinelloides complexes and Aspergillus series nigri. Database performances were evaluated by cross-validation and external validation using 78 fungal isolates with 96.55% and 90.48% correct identification, respectively. This study also showed the capacity of MALDI-TOF MS to differentiate closely related species within species complexes and further demonstrated the potential of this technique for the routine identification of fungi in an industrial context.

Effect of Hanseniaspora vineae and Saccharomyces cerevisiae co-fermentations on aroma compound production in beer.

In recent years, the boom of the craft beer industry refocused the biotech interest from ethanol production to diversification of beer aroma profiles. This study analyses the fermentative phenotype of a collection of non-conventional yeasts and examines their role in creating new flavours, particularly through co-fermentation with industrial Saccharomyces cerevisiae. High-throughput solid and liquid media fitness screening compared the ability of eight Saccharomyces and four non-Saccharomyces yeast strains to grow in wort. We determined the volatile profile of these yeast strains and found that Hanseniaspora vineae displayed a particularly high production of the desirable aroma compounds ethyl acetate and 2-phenethyl acetate. Given that H. vineae on its own can't ferment maltose and maltotriose, we carried out mixed wort co-fermentations with a S. cerevisiae brewing strain at different ratios. The two yeast strains were able to co-exist throughout the experiment, regardless of their initial inoculum, and the increase in the production of the esters observed in the H. vineae monoculture was maintained, alongside with a high ethanol production. Moreover, different inoculum ratios yielded different aroma profiles: the 50/50 S. cerevisiae/H. vineae ratio produced a more balanced profile, while the 10/90 ratio generated stronger floral aromas. Our findings show the potential of using different yeasts and different inoculum combinations to tailor the final aroma, thus offering new possibilities for a broader range of beer flavours and styles.

Codon usage bias in yeasts and its correlation with gene expression, growth temperature, and protein structure.

Codon usage bias (CUB) has been described in viruses, prokaryotes, and eukaryotes and has been linked to several cellular and environmental factors, such as the organism's growth temperature, gene expression levels, and regulation of protein synthesis and folding. Most of the studies in this area have been conducted in bacteria and higher eukaryotes, in some cases with different results. In this study, a comparative analysis of CUB in yeasts isolated from cold and template environments was performed in order to evaluate the correlation of CUB with yeast optimal temperature of growth (OTG), gene expression levels, cellular function, and structure of encoded proteins. Among the main findings, highly expressed ORFs tend to have a more similar CUB within and between yeasts, and a direct correlation between codons ending in C and expression level was generally found. A low correspondence between CUB and OTG was observed, with an inverse correlation for some codons ending in C. The clustering of yeasts based on their CUB partially aligns with their OTG, being more consistent for yeasts with lower OTG. In most yeasts, the abundance of preferred codons was generally lower at the 5' end of ORFs, higher in segments encoding beta strand, lower in segments encoding extracellular and transmembrane regions, and higher in "translation" and "energy metabolism" pathways, especially in highly expressed ORFs. Based on our findings, it is suggested that the abundance and distribution of preferred and non-preferred codons along mRNAs contribute to proper protein folding and functionality by regulating protein synthesis rates, becoming a more important factor under conditions that require faster protein synthesis in yeasts.

The Effect of Sucrose and Yeast Extract on Total Phenolic, Flavonoid, and Anthocyanin of Lactic-Acid-Fermented Mangosteen Fruit Peel (Garcinia mangostana L.).

Objectives: This study aimed to determine the most suitable concentration of sucrose and yeast extract (SYE) and its impact on the levels of total phenol, flavonoid, and anthocyanin (TPFA) for lactic acid fermentation in mangosteen fruit peel. Materials and Methods: In this study, the primary components were mangosteen fruit peel, SYE, and lactic acid bacteria starter. The experimental design was conducted using the Factorial Design method. The colorimetric method was used to determine the total phenol (Folin-Ciocalteu reagent) and total flavonoid (AlCl3 reagent). In addition, the differential pH method was used to determine the total anthocyanins using KCl and the CH3COONa reagent. Results: The addition of SYE during the fermentation of mangosteen fruit peel significantly increased the concentrations of TPFA compared with the control (p value of 0.0001). The high sucrose concentration and low yeast extract produced the highest TPFA levels in mangosteen rind fermentation. Conclusion: The use of SYE affects the levels of TPFA in lactic acid-fermented mangosteen fruit peel, with the most suitable concentrations obtained using sucrose (45 g/L) and yeast extract (2.5 g/L).

Culturable yeast community associated with grape must and honey bees sampled from apiaries located in the vineyards.

AIM: In this study, we investigated culturable yeast community, present in grape must sampled from vineyards with apiaries on the borders, and in honey bees collected in these apiaries. METHODS AND RESULTS: To this aim, yeasts isolated from spontaneously fermented grapes randomly collected in two vineyards (P1 and P2) with apiaries on the borders (A1 and A2) were compared to those isolated from spontaneously fermented grapes collected from a vineyard without apiary (P4). At the same time, yeast community was analyzed on bees collected in each apiary placed in the vineyards, in comparison to yeasts isolated from an apiary (A3) located far from the vineyards. The analysis was performed for two consecutive years (2021 and 2022). The isolated yeasts were identified by restriction analysis of amplified ITS region, followed by sequencing of ITS fragment.Our research showed that the presence of apiaries seems to increase yeast counts of grape must, in particular of Saccharomyces cerevisiae; furthermore, the permanence of apiaries in the vineyards allowed the recovering of these yeasts also from bees. CONCLUSIONS: Our findings seem to corroborate the role of bees as vectors and reservoirs of oenologically relevant yeasts, such as a source of non-conventional yeasts with potential biotechnological applications.

The 'Erlenmeter': a low-cost, open-source turbidimeter for no-sampling phenotyping of microorganism growth.

This work presents a low-cost, open-source turbidimeter, the 'Erlenmeter', designed to monitor the growth of microorganisms in batch cultures. It is easy to build, based exclusively on inexpensive off-the-shelf electronic components and 3D-printed parts. The Erlenmeter allows measuring the optical density of cultures on standard Erlenmeyer flasks without the need to open the flasks to collect aliquots, ensuring speed, minimal use of consumables, and elimination of the risk of contamination. These features make it particularly well-suited not just for routine research assays but also for experimental teaching. Here we illustrate the use of the Erlenmeter turbidimeter to record the growth of the microalga Phaeodactylum tricornutum, of the bacterium Escherichia coli, and of the yeast Saccharomyces cerevisiae, model organisms that are widely used in research and teaching. The Erlenmeter allows a detailed characterization of the growth curves of all organisms, confirming its usefulness for studying microbial populations dynamics both for research purposes and in classroom settings.

Identification and Antifungal susceptibility profile of uncommon yeast species at Fattouma Bourguiba university hospital in Tunisia.

Despite severe impact of uncommon yeast fungal infections and the pressing need for more research on the topic, there are still few studies available on the identification, epidemiology, and susceptibility profile of those pathogens. The aims of the current study were to define the profile of uncommon yeast species at Fattouma Bourguiba university hospital using phenotypic, molecular and proteomic methods and to study their antifungal susceptibility profile. Pre-identified uncommon yeast species were collected from 2018 to 2021. These isolates were further identified using phenotypic methods (ID32C® system and Vitek2® YST), matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and sequencing. The antifungal susceptibility profile was studied using the reference CLSI broth microdilution method. In total, 30 strains were collected during the study period. Referring to the sequencing, the most isolated uncommon species were Saprochaete capitata, Candida lusitaniae, Candida kefyr, Candida inconspicua and Candida guilliermondii. Ninety % of isolates were correctly identified by MALDI-TOF MS compared to 76.7% and 63.3% by ID32® C and VITEK® 2 YST respectively. The isolated species showed variable responses to antifungals. Candida guilliermondii showed increased azole minimum inhibitory concentrations. Misidentification of uncommon yeast species was common using commercial phenotypic methods. The high percentage of concordance of MALDI-TOF results with sequencing highlights its high performance and usefulness as a routine diagnosis tool.

There is still little information on the epidemiology of uncommon emergent yeasts although their implication in severe diseases mainly invasive infections. Thus, the importance of an accurate identification and antifungal susceptibility testing for a better monitoring of related infections.

Oligomeric Structure of Yeast and Other Invertases Governs Specificity.

Invertases, or ß-fructofuranosidases, are metabolic enzymes widely distributed among plants and microorganisms that hydrolyze sucrose and release fructose from various substrates. Invertase was one of the earliest discovered enzymes, first investigated in the mid-nineteenth century, becoming a classical model used in the primary biochemical studies on protein synthesis, activity, and the secretion of glycoproteins. However, it was not until 20 years ago that a member of this family of enzymes was structurally characterized, showing a bimodular arrangement with a ß-propeller catalytic domain, and a ß-sandwich domain with unknown function. Since then, many studies on related plant and fungal enzymes have revealed them as basically monomeric. By contrast, all yeast enzymes in this family that have been characterized so far have shown sophisticated oligomeric structures mediated by the non-catalytic domain, which is also involved in substrate binding, and how this assembly determines the particular specificity of each enzyme. In this chapter, we will review the available structures of yeast invertases to elucidate the mechanism regulating oligomer formation and compare them with other reported dimeric invertases in which the oligomeric assembly has no apparent functional implications. In addition, recent work on a new family of invertases with absolute specificity for the α-(1,2)-bond of sucrose found in cyanobacteria and plant invertases is highlighted.

Detection of Hanseniaspora opuntiae in anovaginal samples of pregnant women in Rio de Janeiro, Brazil-a case report.

In this study, we report the first isolation of Hanseniaspora opuntiae obtained from four pregnant women in Brazil. Clinical isolates were obtained from four samples taken between 35 and 37 gestational weeks, as part of the routine antenatal care for maternal colonization screening for Streptococcus agalactiae group B. The patients were immunocompetent, with two of them diagnosed with gestational diabetes mellitus. Species identification was performed by MALDI-TOF MS and rDNA sequencing. While Hanseniaspora species have not traditionally been considered a typical opportunist pathogen, our findings emphasize the importance of investigating and screening for Hanseniaspora in pregnant populations, highlighting H. opuntiae as a potential agent of human infections.

Exploring the Efficacy of Using Geotrichum fermentans, Rhodotorula rubra, Kluyveromyce marxiamus, Clay Minerals, and Walnut Nutshells for Mycotoxin Remediation.

The aim of this study was to evaluate the effectiveness of nine different biological compounds to reduce mycotoxins concentrations. The hypothesis of this study was that a static in vitro gastrointestinal tract model, as an initial screening tool, can be used to simulate the efficacy of Geotrichum fermentans, Rhodotorula rubra, Kluyveromyce marxiamus yeast cell walls and their polysaccharides, red and white clay minerals, and walnuts nutshells claiming to detoxify AFB1, ZEA, DON, and T-2 toxin mycotoxins. Mycotoxin concentrations were analyzed using high-performance liquid chromatography (HPLC) with fluorescent (FLD) and ultraviolet detectors (UV). The greatest effects on reducing mycotoxin concentrations were determined as follows: for AFB1, inserted G. fermentans cell wall polysaccharides and walnut nutshells; for ZEA, inserted R. rubra and G. fermentans cell walls and red clay minerals; for DON, R. rubra cell wall polysaccharides and red clay minerals; and for T-2 toxin, R. rubra cell walls, K. marxianus, and G. fermentans cell wall polysaccharides and walnut nutshells. The present study indicated that selected mycotoxin-detoxifying biological compounds can be used to decrease mycotoxin concentrations.

Enhancing Selenium Accumulation in Rhodotorula mucilaginosa Strain 6S Using a Proteomic Approach for Aquafeed Development.

It is known that selenium (Se) is an essential trace element, important for the growth and other biological functions of fish. One of its most important functions is to contribute to the preservation of certain biological components, such as DNA, proteins, and lipids, providing protection against free radicals resulting from normal metabolism. The objective of this study was to evaluate and optimize selenium accumulation in the native yeast Rhodotorula mucilaginosa 6S. Sodium selenite was evaluated at different concentrations (5-10-15-20-30-40 mg/L). Similarly, the effects of different concentrations of nitrogen sources and pH on cell growth and selenium accumulation in the yeast were analyzed. Subsequently, the best cultivation conditions were scaled up to a 2 L reactor with constant aeration, and the proteome of the yeast cultured with and without sodium selenite was evaluated. The optimal conditions for biomass generation and selenium accumulation were found with ammonium chloride and pH 5.5. Incorporating sodium selenite (30 mg/L) during the exponential phase in the bioreactor after 72 h of cultivation resulted in 10 g/L of biomass, with 0.25 mg total Se/g biomass, composed of 25% proteins, 15% lipids, and 0.850 mg total carotenoids/g biomass. The analysis of the proteomes associated with yeast cultivation with and without selenium revealed a total of 1871 proteins. The results obtained showed that the dynamic changes in the proteome, in response to selenium in the experimental medium, are directly related to catalytic activity and oxidoreductase activity in the yeast. R. mucilaginosa 6S could be an alternative for the generation of selenium-rich biomass with a composition of other nutritional compounds also of interest in aquaculture, such as proteins, lipids, and pigments.

Investigation of Freezing and Freeze-Drying for Preserving and Re-Using a Whole Microbial Cheese Community.

Preserving microbial ecosystems obtained from traditional cheese-making processes is crucial to safeguarding the biodiversity of microbial cheese communities and thus ensuring that the high flavor quality of traditional cheeses is maintained. Few protocols have been proposed for the long-term storage of microbial consortia. This work aimed to develop preservation methods to stabilize the entire microbial community in smear-ripened cheese without multiplication or isolation. A simplified microbial community, capable of reproducing the metabolic pattern of cheese maturation, was used in three independent cheese productions. Cheese samples were taken before and after the ripening step, mixed with maltodextrin or saline solution, and subjected to different stabilization conditions including freezing and freeze-drying, followed by 1 month of storage. Microbial survival was quantified using the colony-forming unit assay. Differential scanning calorimetry was used to relate the physical events occurring within the samples to the microbial storage stability. Freezing at -80 °C resulted in the lowest loss of culturability (<0.8 log unit), followed by freezing at -20 °C and freeze-drying. The ripening bacteria appeared as the most sensitive microorganisms within the community. Moreover, a successful cheese production using the best-stabilized community showed the possibility of preserving and re-using an entire microbial community of interest.

Exploring xylose metabolism in non-conventional yeasts: kinetic characterization and product accumulation under different aeration conditions.

d-Xylose is a metabolizable carbon source for several non-Saccharomyces species, but not for native strains of S. cerevisiae. For the potential application of xylose-assimilating yeasts in biotechnological processes, a deeper understanding of pentose catabolism is needed. This work aimed to investigate the traits behind xylose utilization in diverse yeast species. The performance of 9 selected xylose-metabolizing yeast strains was evaluated and compared across 3 oxygenation conditions. Oxygenation diversely impacted growth, xylose consumption, and product accumulation. Xylose utilization by ethanol-producing species such as Spathaspora passalidarum and Scheffersomyces stipitis was less affected by oxygen restriction compared with other xylitol-accumulating species such as Meyerozyma guilliermondii, Naganishia liquefaciens, and Yamadazyma sp., for which increased aeration stimulated xylose assimilation considerably. Spathaspora passalidarum exhibited superior conversion of xylose to ethanol and showed the fastest growth and xylose consumption in all 3 conditions. By performing assays under identical conditions for all selected yeasts, we minimize bias in comparisons, providing valuable insight into xylose metabolism and facilitating the development of robust bioprocesses. ONE-SENTENCE SUMMARY: This work aims to expand the knowledge of xylose utilization in different yeast species, with a focus on how oxygenation impacts xylose assimilation.

Easy-to-use imaging-cytometry assay to analyze chitin patterns in yeasts.

BACKGROUND & AIM: Pathogenic fungi are a major threat to public health, and fungal infections are becoming increasingly common and treatment resistant. Chitin, a component of the fungal cell wall, modifies host immunity and contributes to antifungal resistance. Moreover, chitin content is regulated by chitin synthases and chitinases. However, the specific roles and mechanisms remain unclear. In this study, we developed a cytometric imaging assay to quantify chitin content and identify the distribution of chitin in the yeast cell wall. METHODS: The Candida albicans SC5314 and Nakaseomyces glabratus (ex. C. glabrata) ATCC2001 reference strains, as well as 106 clinical isolates, were used. Chitin content, distribution, and morphological parameters were analysed in 12 yeast species. Moreover, machine learning statistical software was used to evaluate the ability of the cytometric imaging assay to predict yeast species using the values obtained for these parameters. RESULTS: Our imaging-cytometry assay was repeatable, reproducible, and sensitive to variations in chitin content in C. albicans mutants or after antifungal stimulation. The evaluated parameters classified the yeast species into the correct clade with an accuracy of 85 %. CONCLUSION: Our findings demonstrate that this easy-to-use assay is an effective tool for the exploration of chitin content in yeast species.

Exploring factors influencing the levels of biogenic amines in wine and microbiological strategies for controlling their occurrence in winemaking.

Fermented beverages, including wine, can accumulate high concentrations of biogenic amines (BAs), which can pose potential health risks. BAs are produced by various yeasts and lactic acid bacteria (LAB) during winemaking. LAB are the main contributors to the formation of histamine and tyramine, the most toxic and food safety relevant biogenic amines. Numerous factors, ranging from agricultural and oenological practices to sanitation conditions, can contribute to the formation of BAs in wines. Moreover, organic and biodynamic wines impose limitations on the use of common food additives employed to control the proliferation of native and spoilage microorganisms during vinification and storage. To mitigate histamine production, commercial starter cultures incapable of synthesising histamine have been effectively utilised to reduce wine histamine content. Alternative fermentative microorganisms are currently under investigation to enhance the safety, quality, and typicity of wines, including indigenous LAB, non-Saccharomyces yeasts, and BAs degrading strains. Furthermore, exploration of extracts from BAs-degrading microorganisms and their purified enzymes has been undertaken to reduce BAs levels in wines. This review highlights microbial contributors to BAs in wines, factors affecting their growth and BA production, and alternative microorganisms that can degrade or avoid BAs. The aim is to lessen reliance on additives, providing consumers with safer wine choices.

Do flower-colonizing microbes influence floral evolution? A test with fast-cycling Brassica.

Pollinators are thought to be the main drivers of floral evolution. Flowers are also colonized by abundant communities of microbes that can affect the interaction between plants and their pollinators. Very little is known, however, about how flower-colonizing microbes influence floral evolution. Here we performed a six-generation experimental evolution study using fast-cycling Brassica rapa, in which we factorially manipulated the presence of pollinators and flower microbes to determine how pollinators and microbes interact in driving floral evolution. We measured the evolution of six morphological traits, as well as plant mating system and flower attractiveness. Only one of the six traits (flower number) evolved in response to pollinators, while microbes did not drive the evolution of any trait, nor did they interact with pollinators in driving evolution of morphological traits. Moreover, we did not find evidence that pollinators or microbes affected the evolution of flower attractiveness to pollinators. However, we found an interactive effect of pollinators and microbes on the evolution of autonomous selfing, a trait that is expected to evolve in response to pollinator limitation. Overall, we found only weak evidence that microbes mediate floral evolution. However, our ability to detect an interactive effect of pollinators and microbes might have been limited by weak pollinator-mediated selection in our experimental setting. Our results contrast with previous (similar) experimental evolution studies, highlighting the susceptibility of such experiments to drift and to experimental artefacts.

Cultivation of yeasts on liquid digestate to remove organic pollutants and nutrients and for potential application as co-culture with microalgae.

In this study, the growth of yeast and yeast-like fungi in the liquid digestate from vegetable wastes was investigated in order to remove nutrients and organic pollutants, and for their application as co-culture members with green microalgae. The studied yeast strains were characterized for their assimilative and enzymatic profiles as well as temperature requirements. In the first experimental stage, the growth dynamics of each strain were determined, allowing to select the best yeasts for further studies. In the subsequent stage, the ability of selectants to remove organic pollutants was assessed. Different cultivation media containing respectively 1:3, 1:1, 3:1 vol ratio of liquid digestate and the basal minimal medium were used. Among all tested yeast strains, Rhodotorula mucilaginosa DSM 70825 showed the most promising results, demonstrating the highest potential for removing organic substrates and nutrients. Depending on the medium, this strain achieved 50-80% sCOD, 45-60% tVFAs, 21-45% TN, 33-52% PO43- reduction rates. Similar results were obtained for the strain Candida sp. OR687571. The high nutrient and organics removal efficiency by these yeasts could likely be linked to their ability to assimilate xylose (being the main source of carbon in the liquid digestate). In culture media containing liquid digestate, both yeast strains achieved good viability and proliferation potential. In the liquid digestate medium, R. mucilaginosa and Candida sp. showed vitality at the level of 51.5% and 45.0%, respectively. These strains seem to be a good starting material for developing effective digestate treatment strategies involving monocultures and/or consortia with other yeasts or green microalgae.

Customizable and stable multilocus chromosomal integration: a novel glucose-dependent selection system in Aureobasidium spp.

BACKGROUND: Non-conventional yeasts hold significant potential as biorefinery cell factories for microbial bioproduction. Currently, gene editing systems used for these yeasts rely on antibiotic and auxotrophic selection mechanisms. However, the drawbacks of antibiotics, including high costs, environmental concerns, and the dissemination of resistance genes, make them unsuitable for large-scale industrial fermentation. For auxotrophic selection system, the engineered strains harboring auxotrophic marker genes are typically supplemented with complex nutrient-rich components instead of precisely defined synthetic media in large-scale industrial fermentations, thus lack selection pressure to ensure the stability of heterologous metabolic pathways. Therefore, it is a critical to explore alternative selection systems that can be adapted for large-scale industrial fermentation. RESULTS: Here, a novel glucose-dependent selection system was developed in a high pullulan-producing non-conventional strain A. melanogenum P16. The system comprised a glucose-deficient chassis cell Δpfk obtained through the knockout of the phosphofructokinase gene (PFK) and a series of chromosomal integration plasmids carrying a selection marker PFK controlled by different strength promoters. Utilizing the green fluorescent protein gene (GFP) as a reporter gene, this system achieved a 100% positive rate of transformation, and the chromosomal integration numbers of GFP showed an inverse relationship with promoter strength, with a customizable copy number ranging from 2 to 54. More importantly, the chromosomal integration numbers of target genes remained stable during successive inoculation and fermentation process, facilitated simply by using glucose as a cost-effective and environmental-friendly selectable molecule to maintain a constant and rigorous screening pressure. Moreover, this glucose-dependent selection system exhibited no significant effect on cell growth and product synthesis, and the glucose-deficient related selectable marker PFK has universal application potential in non-conventional yeasts. CONCLUSION: Here, we have developed a novel glucose-dependent selection system to achieve customizable and stable multilocus chromosomal integration of target genes. Therefore, this study presents a promising new tool for genetic manipulation and strain enhancement in non-conventional yeasts, particularly tailored for industrial fermentation applications.