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Metabolic Engineering Jul 2023The robust nature of the non-conventional yeast Issatchenkia orientalis allows it to grow under highly acidic conditions and therefore, has gained increasing interest in...
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.
Topics: CRISPR-Cas Systems; Saccharomyces cerevisiae; Gene Editing; Succinates
PubMed: 37343658
DOI: 10.1016/j.ymben.2023.06.010 -
Mycoses Nov 2023Rapid and accurate yeasts species identification in clinical laboratories is important for appropriate and timely antifungal treatment. We evaluate the performance of...
Rapid and accurate yeasts species identification in clinical laboratories is important for appropriate and timely antifungal treatment. We evaluate the performance of the new medium CHROMagar™ Candida Plus for presumptive identification of yeasts species and MALDI-TOF identification. We identify 303 strains belonging to 60 clinically relevant yeasts species by using the new medium. Presumptive identification was correct at the Candida albicans complex, Candida tropicalis and Pichia kudriavzevii (Candida krusei) species. However, although this medium was able to identify all Candida auris and Candida glabrata strains, other species were misidentified as C. auris or C. glabrata. A total of 215 strains were identified by using MALDI-TOF and evaluated two incubation temperatures (30°C and 37°C) and two incubation times (24 h and 72 h). Most strains (94%; 202/215) were correctly identified at the species (n:190) or complex level (n:12) at both temperatures and incubation times. However, we observed that the time of incubation (24 h vs. 72 h) affects the score values when yeasts are incubated at 37°C, but does not affect score values when yeasts are incubated at 30°C. In conclusion, the new medium has a good performance in the presumptive identification of the C. albicans complex, C. tropicalis and P. kudriavzevii (C. krusei). In addition, this medium is useful for the screening of C. auris and C. glabrata isolates, but identification should be confirmed by other more specific techniques, like MALDI-TOF.
Topics: Humans; Candida; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Culture Media; Yeasts; Candida albicans; Candida glabrata; Candida tropicalis
PubMed: 37518770
DOI: 10.1111/myc.13633 -
BMC Microbiology May 2024Spontaneous fermentation of cereals like millet involves a diverse population of microbes from various sources, including raw materials, processing equipment, fermenting...
Spontaneous fermentation of cereals like millet involves a diverse population of microbes from various sources, including raw materials, processing equipment, fermenting receptacles, and the environment. Here, we present data on the predominant microbial species and their succession at each stage of the Hausa koko production process from five regions of Ghana. The isolates were enumerated using selective media, purified, and phenotypically characterised. The LAB isolates were further characterised by 16S rRNA Sanger sequencing, typed using (GTG) repetitive-PCR, and whole genome sequencing, while 28S rRNA Sanger sequencing was performed for yeast identification. The pH of the millet grains ranged from mean values of 6.02-6.53 to 3.51-3.99 in the final product, depending on the processors. The mean LAB and yeast counts increased during fermentation then fell to final counts of log 2.77-3.95 CFU/g for LAB and log 2.10-2.98 CFU/g for yeast in Hausa koko samples. At the various processing stages, the counts of LAB and yeast revealed significant variations (p < 0.0001). The species of LAB identified in this study were Limosilactobacillus pontis, Pediococcus acidilactici, Limosilactobacillus fermentum, Limosilactobacillus reuteri, Pediococcus pentosaceus, Lacticaseibacillus paracasei, Lactiplantibacillus plantarum, Schleiferilactobacillus harbinensis, and Weissella confusa. The yeasts were Saccharomyces cf. cerevisiae/paradoxus, Saccharomyces cerevisiae, Pichia kudriavzevii, Clavispora lusitaniae and Candida tropicalis. The identification and sequencing of these novel isolates and how they change during the fermentation process will pave the way for future controlled fermentation, safer starter cultures, and identifying optimal stages for starter culture addition or nutritional interventions. These LAB and yeast species are linked to many indigenous African fermented foods, potentially acting as probiotics in some cases. This result serves as the basis for further studies into the technological and probiotic potential of these Hausa koko microorganisms.
Topics: Fermentation; Ghana; Yeasts; Food Microbiology; Fermented Foods; Millets; Lactobacillales; RNA, Ribosomal, 16S; Phylogeny; Hydrogen-Ion Concentration; Edible Grain
PubMed: 38745280
DOI: 10.1186/s12866-024-03317-1 -
Journal of the Science of Food and... Jul 2023The last decade has seen a growing interest in reducing the use of chemical fungicides for postharvest decay control. In the research for new, safe alternatives, the...
BACKGROUND
The last decade has seen a growing interest in reducing the use of chemical fungicides for postharvest decay control. In the research for new, safe alternatives, the combined application of biocontrol agents and passive modified-atmosphere packaging (MAP) has been shown to be a promising strategy to extend fruit quality. Therefore, the aim of this work was to evaluate the effect of the combined application of MAP and two antagonistic yeasts, Metschnikowia pulcherrima L672 and Pichia kudriavzevii PK18, on sweet cherry shelf life.
RESULTS
Microbiological, physico-chemical, and quality fruit analysis from batches treated with antagonistic yeast were compared with a control batch without yeast application and a batch to which fludioxonil (Scholar®) was applied. The composition of the atmosphere and physico-chemical traits showed similar values among the different batches during cold storage. However, interestingly, the combination of MAP with the antagonistic yeasts M. pulcherrima L672 and P. kudriavzevii PK18 increases the control of microbiological spoilage with results comparable to the application of fludioxonil. In addition, these batches experienced a slight decrease in volatile compounds associated with fresh fruit aroma, whereas in the control batch an increase of altered fruit aromas was observed. The same effect of control of spoilage was observed during the shelf life period.
CONCLUSION
These results showed the positive effect of the combination of antagonistic yeasts and MAP, obtaining similar results in terms of control of microbiological spoilage and physico-chemical quality compared with the application of fludioxonil. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
Topics: Prunus avium; Fruit; Yeasts; Atmosphere; Food Packaging
PubMed: 36850053
DOI: 10.1002/jsfa.12532 -
International Journal of Food... Jun 2024Previous investigations proved the potential of Saccharomyces cerevisiae MBELGA62 and Pichia kudriavzevii MBELGA61 as suitable biocontrolling agents against Aspergillus...
Previous investigations proved the potential of Saccharomyces cerevisiae MBELGA62 and Pichia kudriavzevii MBELGA61 as suitable biocontrolling agents against Aspergillus sp. through the production of soluble and volatile bioactive antifungal compounds. The present study delves into those finding by means of the identification of the volatile compounds produced by brewer's strains that demonstrated fungistatic and fungicidal effects against Aspergillus flavus and A. parasiticus when cultured in brewer's wort agar plates. Traditional brewer's yeasts such as S. cerevisiae MBELGA62 and Saccharomyces pastorianus SAFS235 synthetize volatiles that fully inhibited mycelial development for up to 9 days at 30 °C. The non-conventional brewer's strains P. kudriavzevii MBELGA61 and Meyerozyma guilliermondii MUS122 increased the lag phase by >100% and significantly reduced the fungal growth rate by 27.5-43.0% and 15.4-31.4%, respectively. In this context, 2-phenylethanol, 2-phenylethyl acetate and benzyl alcohol were identified as the main antifungal agents involved in Aspergillus sp.'s inhibition.
Topics: Aspergillus; Fermentation; Antifungal Agents; Volatile Organic Compounds; Saccharomyces cerevisiae; Pichia; Phenylethyl Alcohol
PubMed: 38640817
DOI: 10.1016/j.ijfoodmicro.2024.110692 -
Mycopathologia Dec 2023We performed a retrospective survey of non-Candida albicans candidemia in patients with cancer, including those with solid tumors and those with hematological...
We performed a retrospective survey of non-Candida albicans candidemia in patients with cancer, including those with solid tumors and those with hematological malignancies as well as transplants patients both, solid-organ transplant recipients and hematopoietic stem cell transplant recipients. The study was performed at two healthcare centers in New York City and covered the years 2018-2022. A total of 292 patients (318 isolates) were included in the study. In order of frequency, C. glabrata (38%) was the most common species recovered, followed by C. parapsilosis (19.2%), C. tropicalis (12.6%), C. krusei (10.7%), C. lusitaniae (5.7%), and C. guilliermondii (4.4%). Micafungin was the most common antifungal treatment and 18.5% of patients were on antifungal prophylaxis. The 30-day crude mortality was 40%. 4.5% of patients had more than one non-albicans species detected. In conclusion, this study represents one of the largest surveys of non-albicans species in cancer and transplant patients and provides data on the current epidemiology of these Candida species in this patient population.
Topics: Humans; Antifungal Agents; Transplant Recipients; Retrospective Studies; Microbial Sensitivity Tests; Candida; Candidemia; Candida glabrata; Candida parapsilosis; Candida tropicalis; Neoplasms
PubMed: 37365379
DOI: 10.1007/s11046-023-00765-7 -
Oral Oncology Nov 2023Head and neck cancer (HNC) impairs patient immunity and increases susceptibility to oral fungal infections (OFIs). Effectively treating such infections requires accurate...
OBJECTIVES
Head and neck cancer (HNC) impairs patient immunity and increases susceptibility to oral fungal infections (OFIs). Effectively treating such infections requires accurate identification of the causative pathogens. This study aimed to characterize the mycobiota profile of OFIs in HNC patients undergoing radiation treatment (RT).
MATERIALS AND METHODS
A 6-year retrospective analysis of oral mucosal samples from HNC patients with a history of RT and OFIs between 2014 and 2019 was conducted using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) profiling. Samples from the Clinical Microbiology Laboratory at Karolinska University Hospital were evaluated for mycobiota diversity and species co-occurrence patterns in the ongoing-RT and post-RT groups.
RESULTS
A total of 190 oral fungi (88% Candida, 5% Pichia) were isolated from 162 HNC patients receiving RT. In the ongoing-RT group, the emergent non-albicans Candida (NAC) species; F. solani and C. jadinii, were detected for the first time. The dominant pathogens in both ongoing and post-RT groups were C. albicans, C. glabrata, P. kudriavzevii, C. parapsilosis, and C. tropicalis, as shown by Venn analysis. Network analysis revealed greater fungi diversity and multi-species co-occurrence in the ongoing-RT group. C. albicans commonly co-occurred with C. glabrata in both ongoing-RT (21%) and post-RT groups (30%).
CONCLUSION
MALDI-TOF MS identified a wide range of oral fungal species in HNC patients receiving RT. While C. albicans remains the most prevalent OFIs pathogen, multi-species co-occurrence and novel NACs were noted. Understanding the ecological interactions among these causative pathogens could significantly advance the development of effective therapeutics for treating OFIs in HNC patients.
Topics: Humans; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Retrospective Studies; Candida; Mycoses; Head and Neck Neoplasms
PubMed: 37611433
DOI: 10.1016/j.oraloncology.2023.106556 -
Molecules (Basel, Switzerland) Nov 2023species produce different classes of antimicrobial and antioxidant substances: peptides or proteins with different structural compositions and molecular masses and a...
species produce different classes of antimicrobial and antioxidant substances: peptides or proteins with different structural compositions and molecular masses and a broad range of volatile organic compounds (VOCs), some of which may serve as biomarkers for microorganism identification. The aim of this study is the identification of biologically active compounds synthesized by five species using gas chromatography coupled to mass spectrometry (GC-MS). The current study profoundly enhances the knowledge of antibacterial and antioxidant metabolites ensuring the unambiguous identification of VOCs produced by some species, which were isolated from vegetable samples of potato, carrot, and tomato. Phylogenetic and biochemical studies were used to identify the bacterial isolates after culturing. Phylogenetic analysis proved that five bacterial isolates BSS12, BSS13, BSS16, BSS21, and BSS25 showed 99% nucleotide sequence similarities with AS-08, WAB2133, NiuFun, FORT 102, and F3, respectively. The crude extract was prepared from bacterial isolates to assess the antibiotic resistance potency and the antimicrobial potential against various targeted multidrug-resistant strains, including yeast strains such as , , and bacterial strains of , , , , , , group B, , , , , , and . GC-MS analysis of bacterial strains found that VOCs from species come in a variety of chemical forms, such as ketones, alcohols, terpenoids, alkenes, etc. Overall, 69 volatile organic compounds were identified from five species, and all five were found to share different chemical classes of volatile organic components, which have a variety of pharmacological applications. However, eight antibacterial compounds with different concentrations were commonly found in all five species: acetoin, acetic acid, butanoic acid, 2-methyl-, oxime-, methoxy-phenyl, phenol, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, nonanoic acid, and hexadecanoic acid, methyl. The present study has demonstrated that bacterial isolates BSS25, BSS21, and BSS16 display potent inhibitory effects against , while BSS25, BSS21, and BSS13 exhibit the ability to restrain the growth and activity of . Notably, BSS25 and BSS21 are the only isolates that demonstrate substantial inhibitory activity against . This disparity in inhibitory effects could be attributed to the higher concentrations of acetoin in BSS25 and BSS21, whereas BSS16 and BSS13 have relatively elevated levels of butanoic acid, 2-methyl-. Certainly, the presence of acetoin and butanoic acid, 2-methyl-, contributes to the enhanced antibacterial potential of these bacterial strains, in conjunction with other organic volatile compounds and peptides, among other factors. The biology and physiology of can be better understood using these results, which can also be used to create novel biotechnological procedures and applications. Moreover, because of its exceptional ability to synthesize and produce a variety of different antibacterial compounds, species can serve as natural and universal carriers for antibiotic compounds in the form of probiotic cultures and strains to fight different pathogens, including mycobacteria.
Topics: Anti-Bacterial Agents; Gas Chromatography-Mass Spectrometry; Volatile Organic Compounds; Antioxidants; Butyric Acid; Acetoin; Phylogeny; Bacillus; Anti-Infective Agents; Escherichia coli; Bacillus cereus; Enterobacter aerogenes; Peptides; Microbial Sensitivity Tests
PubMed: 38005278
DOI: 10.3390/molecules28227556 -
Microbiology Spectrum Apr 2024Azole drugs are the main therapeutic drugs for invasive fungal infections. However, azole-resistant strains appear repeatedly in the environment, posing a major threat...
Azole drugs are the main therapeutic drugs for invasive fungal infections. However, azole-resistant strains appear repeatedly in the environment, posing a major threat to human health. Several reports have shown that mitochondria are associated with the virulence of pathogenic fungi. However, there are few studies on the mechanisms of mitochondria-mediated azoles resistance. Here, we first performed mitochondrial proteomic analysis on multiple species (, , and ) and analyzed the differentially expressed mitochondrial proteins (DEMPs) between azole-sensitive and azole-resistant species. Subsequently, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, gene ontology analysis, and protein-protein interaction network analysis of DEMPs. Our results showed that a total of 417, 165, and 25 DEMPs were identified in resistant , and , respectively. These DEMPs were enriched in ribosomal biogenesis at cytosol and mitochondria, tricarboxylic acid cycle, glycolysis, transporters, ergosterol, and cell wall mannan biosynthesis. The high activations of these cellular activities, found in and (at low scale), were mostly opposite to those observed in two fermenter species- and . Several transcription factors including Rtg3 were highly produced in resistant that experienced a complex I activation of mitochondrial electron transport chain (ETC). The reduction of mitochondrial-related activities and complex IV/V of ETC in and was companying with the reduced proteins of Tor1, Hog1, and Snf1/Snf4.IMPORTANCE spp. are common organisms that cause a variety of invasive diseases. However, spp. are resistant to azoles, which hinders antifungal therapy. Exploring the drug-resistance mechanism of pathogenic spp. will help improve the prevention and control strategy and discover new targets. Mitochondria, as an important organelle in eukaryotic cells, are closely related to a variety of cellular activities. However, the role of mitochondrial proteins in mediating azole resistance in spp. has not been elucidated. Here, we analyzed the mitochondrial proteins and signaling pathways that mediate azole resistance in spp. to provide ideas and references for solving the problem of azole resistance. Our work may offer new insights into the connection between mitochondria and azoles resistance in pathogenic fungi and highlight the potential clinical value of mitochondrial proteins in the treatment of invasive fungal infections.
Topics: Humans; Candida; Azoles; Antifungal Agents; Proteomics; Drug Resistance, Fungal; Candida albicans; Signal Transduction; Mitochondria; Invasive Fungal Infections; Mitochondrial Proteins; Microbial Sensitivity Tests
PubMed: 38442003
DOI: 10.1128/spectrum.04042-23 -
Nature Communications Oct 2023Microbial production of succinic acid (SA) at an industrially relevant scale has been hindered by high downstream processing costs arising from neutral pH fermentation...
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.
Topics: Succinic Acid; Bioreactors; Fermentation; Pichia
PubMed: 37788990
DOI: 10.1038/s41467-023-41616-9