Issatchenkia Orientalis Research Articles

The arrangement of dual-species biofilms of Candida albicans and Issatchenkia orientalis can be modified by the medium: effect of Voriconazole

Both Candida albicans and Issatchenkia orientalis have been isolated from different types of infections over the years. They have the ability to form communities of microorganisms known as biofilms. It has been demonstrated that the medium employed in studies may affect the biofilm development. The aim of this study was to investigate the arrangement of dual-species biofilms of C. albicans and I. orientalis cultivated on either RPMI-1640 or Sabouraud Dextrose Broth (SDB), as well as the inhibitory effect of Voriconazole (VRC). For the experiments performed, ATCC strains were used, and yeast-mixed suspensions were inoculated in 96-well plates with either RPMI-1640 or SDB, in the presence or absence of VRC. The results were observed by counting the number of CFU obtained from scraping off the biofilms produced and plating the content on CHROMagar Candida medium. It was observed that for all conditions tested the medium chosen affected the arrangement of dual-species biofilms: when RPMI-1640 was used, there was a prevalence of C. albicans, while the opposite was noted when SDB was used. It could be suggested that the medium and environment could regulate interactions between both yeast species, including the response to different antifungal drugs.

Influence of an indigenous yeast, CECA, from the Ningxia wine region of China, on the fungal and bacterial dynamics and function during Cabernet Sauvignon wine fermentation.

Saccharomyces cerevisiae CECA was a potential indigenous Chinese wine yeast that can produce aroma and flavor in Cabernet Sauvignon wines. High-throughput sequencing combined with metabolite analysis was applied to analyze the effects of CECA inoculation on the native microbial community interaction and metabolism during Cabernet Sauvignon wine fermentation. Fermentations were performed with three different inoculant strategies: spontaneous fermentation without inoculation, inoculation with CECA after grape must sterilization, and direct inoculation of CECA. Results showed that the diversity of bacteria (P = 0.033) is more sensitive to CECA inoculation than fungi (P = 0.563). In addition, CECA inoculation altered the species composition of core microorganisms (relative abundance >1%) and the keystone species (accounting for the top 1% of the most important interactions), as well as of the biomarkers (linear discriminant analysis > 3.0, P < 0.05). Furthermore, the inoculation could change the cluster of metabolites, and these differential metabolite sets were correlated with four fungal taxa of Issatchenkia, Issatchenkia orientalis, Saccharomycetales, Saccharomycetes and two bacterial taxa of Pantoea, Tatumella ptyseos, were significantly correlated. Inoculated fermentation also altered the correlation between dominant microorganisms and aroma compounds, giving Cabernet Sauvignon wines more herbal, floral, fruity, and cheesy aromas. Saccharomyces cerevisiae CECA and dimethyl dicarbonate (DMDC) inhibition treatments significantly altered the microbial community structure of Cabernet Sauvignon wines, which in turn affected the microbial-metabolite correlation. These findings will help winemakers to control the microbial dynamics and functions during wine fermentation, and be more widely used in regional typical wine fermentations. © 2024 Society of Chemical Industry.

Inhibitor Tolerance Capacity of Pichia kudriavzevii NBRC1279 and NBRC1664

The thermotolerant yeast Pichia kudriavzevii (previously known as Issatchenkia orientalis), can produce ethanol from a variety of carbon sources and grows at around 45 °C. Thus, this yeast is considered a useful biocatalyst for producing ethanol from lignocellulose through simultaneous saccharification and fermentation (SSF). SSF has several advantages, such as a simplified manufacturing process, ease of operation and reduced energy input. Using P. kudriavzevii NBRC1279 and NBRC1664, we previously succeeded in producing ethanol through SSF; however, the extent to which inhibitors by-produced from lignocellulose hydrolysis affect the growth and ethanol productivity of the two strains remains to be investigated. In this study, to better understand the inhibitor tolerance capacity of the two strains, spot assay, growth experiment, real-time quantitative PCR (RT-qPCR) analysis and multiple sequence alignment analysis were carried out. When P. kudriavzevii NBRC1279 and NBRC1664, as well as Saccharomyces cerevisiae BY4742 as a control, were cultured on SCD plates containing 17% ethanol, 42 mM furfural, 56 mM 5-hydroxymethylfurfural (HMF) or 10 mM vanillin, only P. kudriavzevii NBRC1664 was able to grow under all conditions. Moreover, the inhibitor tolerance capacity of P. kudriavzevii NBRC1664 was greater than those of other strains using SCD medium containing the same concentrations of various inhibitors. When an RT-qPCR analysis of seven gene sequences from aldehyde dehydrogenase and the aldehyde dehydrogenase family protein (ADHF) was performed using P. kudriavzevii NBRC1664 cultivated in the presence of 56 mM HMF, ADHF1 and ADHF2 were up-regulated in the early logarithmic growth phase. Moreover, a multiple sequence alignment of the amino acid sequences of ADHF1, ADHF2 and the known ADH suggested that ADHF1 and ADHF2 may catalyze the reversible NAD+-dependent oxidation of HMF. Our data may be useful for future studies on the metabolic engineering of more useful strains for ethanol production from lignocellulose.

Self-Buffering system for Cost-Effective production of lactic acid from glucose and xylose using Acid-Tolerant Issatchenkia orientalis

This study presents a cost-effective strategy for producing organic acids from glucose and xylose using the acid-tolerant yeast, Issatchenkia orientalis. I. orientalis was engineered to produce lactic acid from xylose, and the resulting strain, SD108XL, successfully converted sorghum hydrolysates into lactic acid. In order to enable low-pH fermentation, a self-buffering strategy, where the lactic acid generated by the SD108XL strain during fermentation served as a buffer, was developed. As a result, the SD108 strain produced 67 g/L of lactic acid from 73 g/L of glucose and 40 g/L of xylose, simulating a sugar composition of sorghum biomass hydrolysates. Moreover, techno-economic analysis underscored the efficiency of the self-buffering strategy in streamlining the downstream process, thereby reducing production costs. These results demonstrate the potential of I. orientalis as a platform strain for the cost-effective production of organic acids from cellulosic hydrolysates.

Strain-specific features of Pleurotus ostreatus growth in vitro and some of its biological activities

BackgroundThe production of Pleurotus ostreatus mycelium as a promising object for use in food and other industries is hampered by a lack of information about the strain-specificity of this fungus mycelium growth and its acquisition of various biological activities. Therefore, this research aimed to investigate mycelial growth of different P. ostreatus strains on varies solid and liquid media as well as to evaluate strains antagonistic, antibacterial, antiradical scavenging activities, and total phenolic content.ResultsPotato Dextrose Agar medium was suitable for all strains except P. ostreatus strain 2460. The best growth rate of P. ostreatus 2462 strain on solid culture media was 15.0 ± 0.8 mm/day, and mycelia best growth on liquid culture media—36.5 ± 0.2 g/l. P. ostreatus strains 551 and 1685 were more susceptible to positive effect of plant growth regulators Ivin, Methyur and Kamethur. Using of nutrient media based on combination of natural waste (amaranth flour cake and wheat germ, wheat bran, broken vermicelli and crumbs) has been increased the yield of P. ostreatus strains mycelium by 2.2–2.9 times compared to the control. All used P. ostreatus strains displayed strong antagonistic activity in co-cultivation with Aspergillus niger, Candida albicans, Issatchenkia orientalis, Fusarium poae, Microdochium nivale in dual-culture assay. P. ostreatus 2462 EtOAc mycelial extract good inhibited growth of Escherichia coli (17.0 ± 0.9 mm) while P. ostreatus 2460 suppressed Staphylococcus aureus growth (21.5 ± 0.5 mm) by agar well diffusion method. The highest radical scavenging effect displayed both mycelial extracts (EtOH and EtOAc) of P. ostreatus 1685 (61 and 56%) by DPPH assay as well as high phenolic content (7.17 and 6.73 mg GAE/g) by the Folin-Ciocalteu’s method. The maximal total phenol content (7.52 mg GAE/g) demonstrated of P. ostreatus 2461 EtOH extract.ConclusionsIt is found that the growth, antibacterial, antiradical scavenging activity as well as total phenolic content were dependent on studied P. ostreatus strains in contrast to antagonistic activity. The proposed culture mediums of natural waste could be an alternative to commercial mediums for the production mycelial biomass of P. ostreatus strains.

A transcriptomic atlas of acute stress response to low pH in multiple Issatchenkia orientalis strains.

Issatchenkia orientalis is a promising industrial chassis to produce biofuels and bioproducts due to its high tolerance to multiple environmental stresses such as low pH, heat, and other chemicals otherwise toxic for the most widely used microbes. Yet, little is known about specific mechanisms of such tolerance in this organism, hindering our ability to engineer this species to produce valuable biochemicals. Here, we report a comprehensive study of the mechanisms of acidic tolerance in this species via transcriptome profiling across variable pH for 12 different strains with different phenotypes. We found multiple regulatory mechanisms involved in tolerance to low pH in different strains of I. orientalis, marking potential targets for future gene editing and perturbation experiments.

An end-to-end pipeline for succinic acid production at an industrially relevant scale using Issatchenkia orientalis

Microbial production of succinic acid (SA) at an industrially relevant scale has been hindered by high downstream processing costs arising from neutral pH fermentation for over three decades. Here, we metabolically engineer the acid-tolerant yeast Issatchenkia orientalis for SA production, attaining the highest titers in sugar-based media at low pH (pH 3) in fed-batch fermentations, i.e. 109.5 g/L in minimal medium and 104.6 g/L in sugarcane juice medium. We further perform batch fermentation using sugarcane juice medium in a pilot-scale fermenter (300×) and achieve 63.1 g/L of SA, which can be directly crystallized with a yield of 64.0%. Finally, we simulate an end-to-end low-pH SA production pipeline, and techno-economic analysis and life cycle assessment indicate our process is financially viable and can reduce greenhouse gas emissions by 34–90% relative to fossil-based production processes. We expect I. orientalis can serve as a general industrial platform for production of organic acids.

The bovine nasal fungal community and associations with bovine respiratory disease.

Effective identification and treatment of bovine respiratory disease (BRD) is an ongoing health and economic issue for the dairy and beef cattle industries. Bacteria pathogens Pasteurellamultocida, Mycoplasmabovis, Mannheimia haemolytica, and Histophilus somni and the virus Bovine herpesvirus-1 (BHV-1), Bovine parainfluenza-3 virus (BPIV-3), Bovine respiratory syncytial virus (BRSV), Bovine adenovirus 3 (BAdV3), bovine coronavirus (BoCV) and Bovine viral diarrhea virus (BVDV) have commonly been identified in BRD cattle; however, no studies have investigated the fungal community and how it may also relate to BRD. The objective of this study was to understand if the nasal mycobiome differs between a BRD-affected (n = 56) and visually healthy (n = 73) Holstein steers. Fungal nasal community was determined by using Internal Transcribed Spacer (ITS) sequencing. The phyla, Ascomycota and Basidiomycota, and the genera, Trichosporon and Issatchenkia, were the most abundant among all animals, regardless of health status. We identified differences between healthy and BRD animals in abundance of Trichosporon and Issatchenkia orientalis at a sub-species level that could be a potential indicator of BRD. No differences were observed in the nasal fungal alpha and beta diversity between BRD and healthy animals. However, the fungal community structure was affected based on season, specifically when comparing samples collected in the summer to the winter season. We then performed a random forest model, based on the fungal community and abundance of the BRD-pathobionts (qPCR data generated from a previous study using the same animals), to classify healthy and BRD animals and determine the agreement with visual diagnosis. Classification of BRD or healthy animals using ITS sequencing was low and agreed with the visual diagnosis with an accuracy of 51.9%. A portion of the ITS-predicted BRD animals were not predicted based on the abundance of BRD pathobionts. Lastly, fungal and bacterial co-occurrence were more common in BRD animals than healthy animals. The results from this novel study provide a baseline understanding of the fungal diversity and composition in the nasal cavity of BRD and healthy animals, upon which future interaction studies, including other nasal microbiome members to further understand and accurately diagnose BRD, can be designed.

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.

Yeast diversity in pit mud and related volatile compounds in fermented grains of chinese strong-flavour liquor

Chinese strong-flavour liquor is produced via a traditional solid-state fermentation strategy facilitated by live microorganisms in pit mud-based cellars. For the present analysis, pit mud samples from different spatial locations within fermentation cellars were collected, and the yeast communities therein were assessed via culture-based and denaturing gradient gel electrophoresis (DGGE) approaches. These analyses revealed significant differences in the composition of yeast communities present in different layers of pit mud. In total, 29 different yeast species were detected, and principal component analyses revealed clear differences in microbial diversity in pit mud samples taken from different cellar locations. Culture-dependent strategies similarly detected 20 different yeast species in these samples. However, while Geotrichum silvicola, Torulaspora delbrueckii, Hanseniaspora uvarum, Saturnispora silvae, Issatchenkia orientalis, Candida mucifera, Kazachstania barnettii, Cyberlindnera jadinii, Hanseniaspora spp., Alternaria tenuissima, Cryptococcus laurentii, Metschnikowia spp., and Rhodotorula dairenensis were detected via a PCR-DGGE approach, they were not detectable in culture-dependent analyses. In contrast, culture-based approaches led to the identification of Schizosaccharomyces pombe and Debaryomyces hansenii in these pit mud samples, whereas they were not detected using DGGE fingerprints profiles. An additional HS-SPME-GC-MS-based analysis of the volatile compounds present in fermented grains samples led to the identification of 66 such compounds, with the highest levels of volatile acids, esters, and alcohols being detected in fermented grains from lower layer samples. A canonical correspondence analysis (CCA) suggested they were significant correlations between pit mud yeast communities and associated volatile compounds in fermented grains.

Application of Clostridium butyricum, Rummeliibacillus suwonensis, and Issatchenkia orientalis for Nongxiangxing baijiu fermentation: Improves the microbial communities and flavor of upper fermented grain

Ethyl hexanoate and ethyl butyrate are essential to the flavor compounds in Nongxiangxing baijiu, but low levels of these two esters in upper fermented grains (FG) decreases the quality of upper distilled baijiu, representing the main challenge in Nongxiangxing baijiu production. This paper enhanced fermentation by inoculating functional Clostridium butyricum, Rummeliibacillus suwonensis, and Issatchenkia orientalis strains into upper FG. The results showed that the ethyl butyrate content in the upper FG increased significantly and the content of ethyl hexanoate did improve from the results of many determinations. High-throughput sequencing indicated that the dominant phyla in the FG were Firmicutes, Actinobacteriota, Proteobacteria, Ascomycota, and Basidiomycota. The canonical correspondence analysis (CCA) and person correlation network revealed the relationship between the microbial community, physicochemical environment, and flavor compounds. The temperature, oxygen, and acidity were closely related to the microbial community, while most flavor compounds were positively correlated with Caldicoprobacter, Caproiciproducens, Delftia, Hydrogenispora, Thermoactinomyces, Issatchenkia Bacillus, and Aspergillus. These results helped improve the quality of Nongxiangxing baijiu.

POTENCY OF YEAST AS A BIOCONTROL AGENT OF OCHRATOXIN A-PRODUCING FUNGI AND ITS EFFECT ON ARABICA COFFEE TASTE

Biocontrol agents can be used to control mycotoxigenic fungi, which include different species of yeast. The objectives of this research were to select yeast isolates that can inhibit the growth of ochratoxin A (OTA)-producing fungi (Aspergillus ochraceus BIO 37310) and to increase the taste of Arabica coffee processed using wet and semi-wet methods. Twenty-two yeast isolates (KA, KA2, KB, KB2, KC, KD, Endomyces decipiens BIO 131215, E. fibuliger BIO 132216, BIO 132217, BIO 13218, BIO 132219, BIO 132220, Candida krusei (= Issatchenkia orientalis) BIO 211285, BIO 211286, BIO 211287, BIO 211288, BIO 211289, BIO 211290, BIO 211291, Saccharomyces cerevisiae BIO 341363, BIO 341364, and BIO 341365) were screened for their antagonistic property against A. ochraceus BIO 37310 in vitro using well (dip) test method. The results showed that C. krusei (BIO 211287, BIO 211288, and BIO 211289) inhibited A. ochraceus BIO 37310. In vivo the highest yeast population was found in coffee beans processed using a semi-wet method inoculated with C. krusei BIO 211288 (46,222 ± 9,576 cfu/g), which was not significantly different from that of the coffee beans inoculated with C. krusei BIO 211287 (36,333 ± 14,000 cfu/g). The three yeast isolates were also able to grow either in coffee beans processed using wet or semi-wet methods inoculated with A. ochraceus BIO 37310 and each yeast isolate. Interaction between the three yeast isolates and A. ochraceus BIO 37310 resulted in E-type interaction, i.e. the fungus was not able to grow anymore, while the yeasts grew further. The total cupping scores of coffee beans inoculated with the three yeast isolates were higher than those of coffee beans uninoculated and inoculated with commercial lactic acid bacteria. The three yeast isolates could be used as biocontrol agents of A. ochraceus BIO 37310 and increase the sensorial quality of coffee beverages.

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.

Diversity and enzymatic activity of the microbiota isolated from compost based on restaurant waste and yard trimmings

IntroductionThe bad management of organic waste negatively affects environmental quality and composting has been a viable recycling alternative. Microorganisms are responsible for waste degradation during the composting process and, consequently, for transforming this waste into natural fertilizer. This work aimed to analyze and identify the biodiversity of yeasts and filamentous fungi throughout a composting process based on organic residues under different treatments (commercial inoculum, non-commercial inoculum, and control treatment) and to investigate the enzymatic activity of these microorganisms.MethodsMicroorganisms were isolated and identified from samples at 0, 5, 10, 20, 40, 60, and 120 days. Filamentous fungi were identified according to their macroscopic and microscopic characteristics, and yeasts were identified by sequencing the 18S rDNA region. All identified strains were evaluated for ligninolytic, cellulolytic, hemicellulolytic, amylolytic, pectinolytic, proteolytic, lipolytic, and ammonification. During the composting phases, the filamentous fungi were higher than the yeast population.Results and discussionAt the beginning of the process, a higher species diversity was observed, and the population of yeasts and filamentous fungi was, on average, 6.50 log CFU g−1. The microbial communities were similar throughout the process in the two inoculated treatments, which showed more significant microbial activity, diversity, and efficiency in the transformation of organic matter, and consequently, advantages in terms of the final product quality compared to the control treatment. The yeasts Pichia kudriavzevii, Pichia farinosa, Issatchenkia orientalis, and the filamentous fungi of the genus Aspergillus spp. proved to have high biotechnological value and could be used as starter cultures to accelerate the composting process.

Improving the chemical and sensory characteristics of red and white wines with pectinase-producing non-Saccharomyces yeasts.

This study examined the influence of pectinase-producing non-Saccharomyces yeasts on the chemical and sensory attributes of red and white wines with added pectin. Merlot and Chardonnay wines were produced with or without a mixture of pectinase-producing non-Saccharomyces yeasts (Cryptococcus adeliensis, Issatchenkia orientalis, and Pichia kluyveri) added to the must prior to alcoholic fermentation conducted by a commercial strain of Saccharomyces cerevisiae. To ensure sufficient substrate was present, varying concentrations of apple pectin (up to 1.25 g/L for red wines and 1.00 g/L for white wine) were added at the start of fermentation. After bottling, trained panelists (n = 10) analyzed these wines for aroma, flavor, taste, and mouthfeel attributes. For both wines, significant interactions were noted between the presence of non-Saccharomyces yeasts and pectin addition which affected pH, titratable acidity, and concentrations of D-galacturonic acid. While no significant sensory differences were observed among the red wines, limited changes were noted for white wines. However, a strong positive correlation was found between the D-galacturonic acid and buttery aroma for Chardonnay and with flavor for Merlot. Increasing D-galacturonic acid concentrations, through utilization of non-Saccharomyces yeasts, may improve the wine quality as a buttery aroma is often associated with high-quality Chardonnay. For both red and white wines, the utilization of these particular non-Saccharomyces yeasts significantly influenced chemical properties but yielded minor sensory changes without any faults. PRACTICAL APPLICATION: With the recent trend to reduce alcohol content in commercial wines, the interest in non-Saccharomyces yeasts has grown. This study showed that the addition of non-Saccharomyces yeasts, perhaps due to their pectinase activity, influenced the chemical characteristics of red and white wines with limited sensory differences, making these yeasts a useful tool for winemakers to modify wine properties.

Cas9-Based Metabolic Engineering of Issatchenkia orientalis for Enhanced Utilization of Cellulosic Hydrolysates.

Issatchenkia orientalis, exhibiting high tolerance against harsh environmental conditions, is a promising metabolic engineering host for producing fuels and chemicals from cellulosic hydrolysates containing fermentation inhibitors under acidic conditions. Although genetic tools for I. orientalis exist, they require auxotrophic mutants so that the selection of a host strain is limited. We developed a drug resistance gene (cloNAT)-based genome-editing method for engineering any I. orientalis strains and engineered I. orientalis strains isolated from various sources for xylose fermentation. Specifically, xylose reductase, xylitol dehydrogenase, and xylulokinase from Scheffersomyces stipitis were integrated into an intended chromosomal locus in four I. orientalis strains (SD108, IO21, IO45, and IO46) through Cas9-based genome editing. The resulting strains (SD108X, IO21X, IO45X, and IO46X) efficiently produced ethanol from cellulosic and hemicellulosic hydrolysates even though the pH adjustment and nitrogen source were not provided. As they presented different fermenting capacities, selection of a host I. orientalis strain was crucial for producing fuels and chemicals using cellulosic hydrolysates.

Evaluation of Different Molecular Markers for Genotyping Non-Saccharomyces Wine Yeast Species

Wine quality is determined by the particular yeast strains prevailing at various stages of fermentation. Therefore, the ability to make an easy, fast, and unambiguous discrimination of yeasts at the strain level is of great importance. Here, the tandem repeat-tRNA (TRtRNA) method with the 5GAC or ISSR-MB primer sets and random amplified polymorphic DNA (RAPD) analysis with (GTG)3, R5, and RF2 oligonucleotides were tested on various non-Saccharomyces wine yeast species. The TRtRNA-PCR employing ISSR-MB showed the highest capacity in discriminating Lachancea thermotolerans and Metschnikowia pulcherrima isolates. RAPD with RF2 was the most efficient method in resolving Starmerella bacillaris isolates, although it produced few polymorphic bands. RAPD with R5 showed the highest capacity to discriminate among the Issatchenkia orientalis, Hanseniaspora guilliermondii, and Pichia anomala isolates. RAPD with either R5 or RF2 exhibited the highest ability to discriminate among the Torulaspora delbrueckii isolates. RAPD with (GTG)3 was the most discriminating method for the H. uvarum isolates. Here we concluded that both TRtRNA-PCR and RAPD-PCR offer rapid means for typing non-Saccharomyces species. However, each method performs better for a given species when paired with a particular primer set. The present results can be useful in wine research for the fast fingerprinting of non-Saccharomyces yeasts.

Glucose feeds the tricarboxylic acid cycle via excreted ethanol in fermenting yeast.

Ethanol and lactate are typical waste products of glucose fermentation. In mammals, glucose is catabolized by glycolysis into circulating lactate, which is broadly used throughout the body as a carbohydrate fuel. Individual cells can both uptake and excrete lactate, uncoupling glycolysis from glucose oxidation. Here we show that similar uncoupling occurs in budding yeast batch cultures of Saccharomyces cerevisiae and Issatchenkia orientalis. Even in fermenting S. cerevisiae that is net releasing ethanol, media 13C-ethanol rapidly enters and is oxidized to acetaldehyde and acetyl-CoA. This is evident in exogenous ethanol being a major source of both cytosolic and mitochondrial acetyl units. 2H-tracing reveals that ethanol is also a major source of both NADH and NADPH high-energy electrons, and this role is augmented under oxidative stress conditions. Thus, uncoupling of glycolysis from the oxidation of glucose-derived carbon via rapidly reversible reactions is a conserved feature of eukaryotic metabolism.

Eco-friendly production of thermostable, halotolerant and pH wide range biosurfactant by Issatchenkia orientalis UCP 1603

Microbial surfactants are amphiphilic molecules with attractive industrial prospects due to their advantages over commonly commercialized chemical surfactants, such as low toxicity, high biocompatibility and biodegradability, and efficiency at extreme conditions. However, the large-scale production of biosurfactants still becomes uncompetitive due to the low yields and onerous costs, being considered the use of renewable substrates as a viable strategy. In this sense, the present study aimed to investigate the sustainable production of biosurfactant by the yeast Issatchenkia orientalis UCP 1603 in 2 L of salt-based medium supplemented with 7.5% cassava wastewater, 5% corn steep liquor and 1% post-frying soybean oil. Fermentation was carried out in 2.8 L-Fernbach flasks for 72 h, at 28°C and 150 rpm, and reduction in surface tension to 30.1 mN/m was verified. The highest yield of the biosurfactant produced (4.02 g/L) was observed after extraction with 70% ethanol (2:1, v/v) and the isolated biosurfactant reduced the surface tension of the medium to 28.7 mN/m, the interfacial tension against n-hexadecane to 16.6 mN/m and has a CMC of 800 mg/L. The biocompound showed anionic and polymeric nature and did not present toxicity against cabbage (Brassica oleracea var. capitata) seeds. The stability in the range of pH 4-10, salinity 5-25% and temperature 5-100°C, evidenced a highly stable biosurfactant, with promising potential of application in several industrial activities or environmental processes in adverse conditions.

Examining organic acid production potential and growth-coupled strategies in Issatchenkia orientalis using constraint-based modeling.

Growth-coupling product formation can facilitate strain stability by aligning industrial objectives with biological fitness. Organic acids make up many building block chemicals that can be produced from sugars obtainable from renewable biomass. Issatchenkia orientalis is a yeast strain tolerant to acidic conditions and is thus a promising host for industrial production of organic acids. Here, we use constraint-based methods to assess the potential of computationally designing growth-coupled production strains for I. orientalis that produce 22 different organic acids under aerobic or microaerobic conditions. We explore native and engineered pathways using glucose or xylose as the carbon substrates as proxy constituents of hydrolyzed biomass. We identified growth-coupled production strategies for 37 of the substrate-product pairs, with 15 pairs achieving production for any growth rate. We systematically assess the strain design solutions and categorize the underlying principles involved.