As a homothallic yeast, K. marxianus lacks a permanent mating type, because K. marxianus haploid cells naturally change their mating type (either the a mating type, MATa, or mating type, MAT) leading to uncontrolled MATa/MAT diploidization within a population (11). These tools would enable rapid strain development, by generating genetic diversity and facilitating stacking of industrially important traits. Overview on the Infections Related to Rare, Roth M., Daas L., Renner-Wilde A., Cvetkova-Fischer N., Saeger M., Herwig-Carl M. The German keratomycosis registry : initial results of a multicenter survey. Nile red-stained cells were examined on a Zeiss Axioskop 2 epifluorescence microscope at 553-nm excitation and 636-nm emission. These strains showed a different . the display of certain parts of an article in other eReaders. 3A). Lipogenesis medium in panels B and C contained monosodium glutamate instead of ammonium sulfate. Kluyveromyces lactis sexual pheromones. . Approximately 1 g of pKCas plasmid was transformed into K. marxianus, and the efficiency of editing was calculated by counting the number of ura3 colonies divided by the number of G418R transformants. The transformation mixture was briefly vortexed, and 100 l was added to the cells. Interestingly, the deduced a-factor amino acid sequences of KmMFa1 and KmMFa2 are not completely conserved, differing by 1 amino acid (Fig. The plate was run using solvent system 2 (SS2: petroleum ether-Et2O [98:2]) until the solvent front nearly reached the top of the plate. Ryan OW, Skerker JM, Maurer MJ, Li X, Tsai JC, Poddar S, Lee ME, DeLoache W, Dueber JE, Arkin AP, Cate JHD. Milewski S, Wojcik R, Zaleska B, Malaczewska J, Tanski Z, Siwicki AK. 5D). Development of a comprehensive set of tools for genome engineering in a cold- and thermo-tolerant Kluyveromyces marxianus yeast strain. RNA integrity and the effect on the real-time qRT-PCR performance, Analysis of relative gene expression data using real-time quantitative PCR and the 2. KLUYVEROMYCES MARXIANUS 0N7WQ9T9ZQ Other General Record Details Names 16: Identifiers 3: KLUYVEROMYCES MARXIANUS 0N7WQ9T9ZQ Other Approval Year Unknown. We present a case of Kluyveromyces marxianus keratitis nine months after Descement's membrane endothelial keratoplasty (DMEK) in a patient with Fuchs endothelial disease. Microscopy: Each circle represents a group of samples. See this image and copyright information in PMC. Ophthalmologe. Although wild-type Km19 produced the most lipids of the wild-type strains we tested, Km19 is very difficult to transform with plasmids. (A) K. marxianus The resulting strains were tested for mating type and heterothallic status by using a pheromone assay described below or by crossing them with reference heterothallic haploid strains, allowing haploid heterothallic MATa or MAT strains to be successfully isolated. Paul Cernak, Raissa Estrela, [], and Jamie H. D. Cate. Effects of dietary yeast strains on immunoglobulin in colostrum and milk of sows, Secretory IgAs complex roles in immunity and mucosal homeostasis in the gut. Gao J, Zhang HJ, Yu SH, Wu SG, Yoon I, Quigley J, et al. All PCR were carried out in 30-l reaction volumes containing 15 l of Phusion High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, USA), 0.2 M forward and reverse primers, and 10 ng template DNA. 2018. For flow cytometry, 300 l cell solution was diluted in 1 ml of ice-cold water and tested in the BD Biosciences Fortessa X20 fluorescence-activated cell sorter (FACS) using a 25,000-cell count, a forward scatter of 250, a side scatter of 250, and the 535LP and 585/42BP filters for fluorescence detection. This step involving the artificial upper phase was repeated until the white layer at the interface completely disappeared. The yeast Kluyveromyces marxianus and its biotechnological potential. All living organisms share several key properties such as order, sensitivity or response to stimuli, reproduction, adaptation, growth and development,. The dry cell pellet was transferred to 15-ml glass conical screw-top centrifuge tubes, and 1 ml of methanolic HCl (3 N concentration) with 2% chloroform was added to the pellet. (C) Growth curves at 45C for the segregants 4B, 5E, and 2G, as well as parental strains Km17 and Km19, in biological triplicate. The results of relative mRNA expression of intestinal genes were calculated using the 2-Ct method [23]. 2015 Feb;94(6):e541. Combined with CRISPR-Cas9 genome editing (Fig. Shanghai Engineering Research Center of Industrial Microorganisms, College of Life Sciences, Fudan University, Shanghai, P. R. China. kat1 auxotrophic strains. adh5 (TableS1). C, control group; L, low-dose (0.5 g/kg) group with K. marxianus; M, medium-dose (1.5 g/kg) group with K. marxianus; H, high-dose (2.5 g/kg) group with K. marxianus. 2012. Therefore, the increase in Firmicutes and the reduction of Cyanobacteria in the ileum could be related to the improved feed efficiency and intestinal structure of broilers after K. marxianus addition. The median coverage was 250-fold, and the total genome assembly was 10,784,526 bp. Genome-wide prediction of CRISPR/Cas9 targets in Kluyveromyces marxianus and its application to obtain a stable haploid strain. Genome annotation was performed using an automated software pipeline, FGENESH++ (http://www.softberry.com) version 3.1.1. The authors have declared that no competing interests exist. However, K. marxianus is generally found to be homothallic (4, 5) (i.e., is self-fertile) and cannot be crossed in a controlled manner. This case highlights the challenging management of keratomycosis in patients with endothelial grafts. Common metabolic engineering techniques are not ideal when dealing with complex phenotypes such as thermotolerance, productivity, and robustness (28). Candida kefir,Candida pseudotropicalis. Four weeks later, the patient was found to have developed hepatic and pulmonary lesions, diagnosed by magnetic resonance imaging and computed tomography, that were likely to be due to accidental premature termination of antifungal treatment after 19days. The patient was treated with fluconazole (400 mg daily) for possible disseminated infection with, Genome sequencing was performed after DNA extraction using EZ1 Advanced XL (Qiagen, Hilden, Germany) on both the Illumina NextSeq500 platform following Nextera XT library creation within Clinical Microbiology, University Hospital Basel (150 bp paired end reads; 110 mean coverage) and on the Oxford Nanopore Technologies (ONT) MinION platform at the Biozentrum, University of Basel. The results of abovementioned intestinal indicies were normalised by total protein content, which was determined using BCA protein quantitation kits (CWBiotech Co. Ltd, Beijing, China). High through put 16S rRNA gene-based pyrosequencing analysis of the fecal microbiota of high FCR and low FCR broiler growers. ALP is an intrinsic enzyme in brush border, and serves as an indicator of functional and mature enterocytes in the gut [33]. The protocol did not consider any oral hygiene treatment. The tube was sealed with a Teflon-coated screw cap and heated at 85C for 1.5 h with vortexing every 15 min. 2006. One microliter was injected in split mode (1:10) onto an SP2330 capillary column (30 m by 0.25 mm by 0.2 m; Supelco). The concentration and quality of DNA samples were checked with gel electrophoresis and a Nanodrop 2000 spectrophotometer (Thermo Scientific Inc., Waltham, USA). PCR products were detected by 2% agarose gel electrophoresis and purified with QIAquick Gel Extraction Kit (Qiagen Inc., Hilden, Germany). MATa, MAT, HMRa, and HML loci. Auxotrophic double mutant strains (3 This content is distributed under the terms of the. The taxonomical composition analysis revealed an enrichment of the phylum Firmicutes and a reduction of the phylum Cyanobacteria in the ileum after K. marxianus addition. Creation of heterothallic K. marxianus strains. SRP158013) and the scaffolds and annotation in Genbank under BioSample accession no. Birds were randomly selected from each replicate pen (8 birds per group) on d 21 for samples collection. Sequencing of the targeted region revealed small insertions or deletions (indels) around the Cas9 cleavage site typical of NHEJ, resulting in premature stop codons within the URA3 ORF (Fig. Engineered strains of Y. lipolytica with reduced -oxidation (pex10) and peroxisome biogenesis (mfe1) combined with overexpression of lipogenesis enzymes can store up to 80 to 90% dry cell weight as lipid compared to only 10 to 15% lipid content for wild-type cells (23). Jahromi MF, Altaher YW, Shokryazdan P, Ebrahimi R, Ebrahimi M, Idrus Z, et al. Cells were then examined under the light microscope at different times. Temperature dependence of the growth of K. marxianus strains tested for lipogenesis. However, with the increase of K. marxianus addition, the VCR of jejunum and ileum coupled with ileal VH linearly increased (P < 0.05). Microbiol. (B) Lipid accumulation in K. marxianus strains. 1984. 2016. URA3 homology sequences adjacent to the Cas9 target site (NatMX flanked by 0.9-kb homology arms). Lipogenesis medium contained ammonium sulfate instead of monosodium glutamate for this set of measurements. PMC Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories, Biobased production of alkanes and alkenes through metabolic engineering of microorganisms. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. The cells were allowed to recover for 2 h at 37C and 250 rpm. This Whole Genome Sequencing project has been deposited in GenBank under the accession no. After blood collection, these birds were slaughtered through cervical dislocation. The sequences in S. cerevisiae are encoded by genes YPL187W and YGL089C in the Saccharomyces Genome Database (44), and those in K. lactis are encoded by GenBank entry {"type":"entrez-protein","attrs":{"text":"CAG99901.1","term_id":"49643949"}}CAG99901.1 (RefSeq ID {"type":"entrez-protein","attrs":{"text":"XP_454814.1","term_id":"50309605"}}XP_454814.1) and as described in reference 17. To assay lipid production, K. marxianus cells were stained with the lipophilic dye Nile red (MP Biomedicals) which is permeable to yeast cells and a common indicator of intracellular lipid content. This genome sequence suggests a source of bloodstream infectionfrom dairy products in an immunocompromised patient,and will hopefully inform and aid further research on this pathogen. -glucan, mannan, and chitin were also quantified to identify the most important extract responsible for these . Ockner RK, Ho WKL, Poppenhausen RB, Ho WK. Dietary K. marxianus supplementation had no effect (P > 0.05) on ABW of birds at 21 or 35 d of age (Table 3), as well as ADG and ADFI during grower period and the overall period. We first identified a K. marxianus-specific origin of replication and K. marxianus-specific promoters and terminators for expressing Cas9 (see Materials and Methods). Four microliters of the cell suspension was dispensed as a single drop (patched) onto 2% glucose plates or MA5 plates and allowed to dry. and A.E. Immediate antifungal treatment with intracameral and corneal intrastromal injections of voriconazole and amphotericin B . Collectively, these enzymes promote the accumulation of acetyl-CoA via citrate. image, https://www.ncbi.nlm.nih.gov/nuccore/PRJEB33886, Reuse portions or extracts from the article in other works, Redistribute or republish the final article. Identification and characterization of potential performance-related gut microbiotas in broiler chickens across various feeding trials, The increase of Lactobacillus species in the gut flora of newborn broiler chicks and ducks is associated with weight gain. Four genes found to be involved in lipogenesis in other yeasts were cloned into overexpression plasmid backbones using the In-Fusion cloning kit (Takara). Note that some MAT transformants did not switch to MATa, keeping the ability to mate with MATa. Application of methods for identifying broiler chicken gut bacterial species linked with increased energy metabolism, Human health risk assessment related to cyanotoxins exposure. 1 Relative weight of immune organs was expressed as the ratio of organ weight (g) to body weight (kg). Kluyveromyces marxianus in ascomycetous yeast and member of the genus, Kluyveromyces. Kluyveromyces is a genus of ascomycetous yeasts in the family Saccharomycetaceae. To test transformants for mating-type switching, we crossed them with stable heterothallic reference strains of either MATa or MAT mating types that also lack the ability to produce tryptophan (3 Haploid MATa and MAT switch mating type by transposases 3 and Kat1 in K. lactis. TJs are comprised of several unique proteins, such as claudin-1 that plays a key role in the action of TJs [38]. Km19 is unable to grow at this temperature. Kluyveromyces marxianus, a food-grade probiotic, have been approved by Chinese Ministry of Health and European Union. Feed and fresh water were available ad libitum. S4B). Briefly, 250 l of culture was pelleted at 4,000 g for 10 min in preweighed 1.5-ml microcentrifuge tubes (Metter Toledo Excellence XS205DU balance). The subsequent treatment of stabilized SCCs with rapamycin decreased tumor size and induced caspase-3 activation. Disclaimer, National Library of Medicine Shown are Km19, Km17, and Km6 percentages of fatty acids in dry cell weight after 24 h in 8% glucose at 42C and 250 rpm. Intestinal alkaline phosphatase (ALP) activity was measured by colorimetric assay using a corresponding diagnostic kit (Jiancheng Biotechnology Institute, Nanjing, China) under the manufacturers protocols. Rymuszka A, Sieroslawska A, Bownik A, Skowroski T. Microcystin-LR modulates selected immune parameters and induces necrosis/apoptosis of carp leucocytes, Evidence of cellulose metabolism by the giant panda gut microbiome. doi: 10.1097/MD.0000000000000541. Secretory IgA is concentrated in the outer layer of colonic mucus along with gut bacteria, Regulation of tight junction permeability during nutrient absorption across the intestinal epithelium. Fusarium keratitis in Germany. Immunoglobulins bind to antigenic epitopes are critical for humoral immunity. It is able to grow efficiently in a wide range of temperatures. To increase xylitol production further, the endogenous XR gene was overexpressed in K. marxianus, . The ePub format uses eBook readers, which have several "ease of reading" features An Agilent 7890A gas chromatograph equipped with a flame ionization detector was used for analysis with the following instrumental settings: injector temperature, 250C; carrier gas, helium at 1 ml/min; and temperature program, 140C, 3 min isocratic, 10C/min to 220C, 40C/min to 240C, and 2.5 min isocratic. There were 305, 335 and 467 unique OTUs of ileal microbiota in the low-, medium- and high-dose groups, respectively. However, there was a linear increase (P < 0.05) in the relative thymus weight responded to the increasing K. marxianus addition. These were the yeast strains Cyberlindnera saturnus C. sat 247 and C. sat CSa1 as well as Kluyveromyces marxianus K. mar 653. 2A; Fig. A.C.B., M.M. Sequencing of KmMFA1, KmMFA2, and KmMF from 8 unique strains shows full strain-to-strain conservation. While inactivation of the single transposase 3 can be used to cross K. marxianus strains (8), the resulting strains possess unstable mating types due to the presence of Kat1 and could randomly switch from MATa to MAT (Fig. Ploidy variation in Kluyveromyces marxianus separates dairy and non-dairy isolates. The yeast Kluyveromyces marxianus and its biotechnological potential, An evolutionary perspective on the Crabtree effect. We used CRISPRNHEJ to make stable K. marxianus laboratory strains with defined ploidy and mating type to enable the use of classical yeast genetics. Using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats with Cas9)-mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. Cultures were then shaken at 275 rpm at 42C for 24 h. Cells were harvested by transferring 250 l of each culture to preweighed Eppendorf tubes, spinning down at 3,000 g for 10 min, washing with 500 l of water, and then resuspending the culture in 100 l of water prior to freezing it in liquid nitrogen. Triple-inactivation strains (3 kat1 leu2 or 3 kat1 trp1) were successfully isolated from 10 of the isolates. This might be also beneficial for dietary energy utilisation and consequently contribute to the improved feed efficiency of broilers. 2018. At the end of treatment, about 91% of patients did not present halitosis. In the present study, we found a high similarity in the ileal microbial community among the broilers treated with different doses of K. marxianus through PCA and clustering analysis. To identify MATa haploid isolates, we used a pheromone morphological response assay. PRJEB33886 (. Notably, KmMFa1 is completely conserved with the respective K. lactis sequence, suggesting a relatively conserved sexual cycle. The clustering analysis revealed short Unifrac distances among these three K. marxianus groups and long branches separating the samples from the control and K. marxianus-treated groups (Fig 2A). 5C and andD;D; see Fig. Nambu-Nishida Y, Nishida K, Hasunuma T, Kondo A. Lecomte V, Kaakoush NO, Maloney CA, Raipuria M, Huinao KD, Mitchell HM, et al. All growing colonies were pooled, and the plasmids were extracted using the QIAprep spin miniprep kit (Qiagen, 27106). Edited. A simple method for the isolation and purification of total lipids from animal tissues, Creative Commons Attribution 4.0 International license. The Km18 trp Km1 diploids were sporulated and germinated and then back-crossed with Km1 haploid reference strains to establish their mating type (Fig. Increased TJs protein expression could result in an enhancement of intestinal barrier function [39], which subsequently reduce the diffusion of macromolecules such as bacterial toxin and pathogens from intestinal lumen into blood circulation, thereby promoting the intestinal and systemic health of host. Colony: Malt agar: puffy, creamish-brown, flat, spreading, dull round colony. Incubation of K. marxianus strain Km1 3 kat1 cells with synthetic KmMF1 and KmMF2 peptide pheromones resulted in isolates that responded to both -factors (Fig. Many strains (including Km17) were observed to be unstable as diploids, and this treatment alone resulted in 25 to 90% sporulation. C, control group; L, low-dose (0.5 g/kg) group with K. marxianus; M, medium-dose (1.5 g/kg) group with K. marxianus; H, high-dose (2.5 g/kg) group with K. marxianus. 1. For YST31, we obtained 14,790,917 PE100 paired-end reads, and after trimming, we assembled reads into 116 scaffolds using CLC Genomics Workbench version 7.5.1. Gut microbiota has major impact on the bioavailability and bioactivity of dietary components, playing important roles in host nutritional, physiological and protective functions [14, 15]. already built in. Fluorescence data were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR), and mean fluorescence values were obtained. and J.P. were involved in the patient's care; V.H. The authors thank Yuanyuan Wu, Yunshuang Yue, Dafei Yin, and Yuanyang Dong (China Agricultural University, Beijing, China) for their assistance in the process of animals feeding and samples collection. Three regions were targeted, and small insertions and deletions were found upon repair of double-strand breaks by the NHEJ machinery. Keeping all the reagents on ice, 66.7 l of 60% polyethylene glycol (PEG) 2050, 12.5 l of 2 M lithium acetate, and water in a final volume of 100 l were added to a sterile microcentrifuge tube. Plant growth-promoting bacteria (PGPB) were isolated from the root nodules of Phaseolus lunatus and grown in Cr-contaminated soil with the application of composted tannery sludge. 2017;31(10):14271434. Cultures were shaken at 250 rpm. We cloned the guide sequence for the sgRNA in pKCas to target URA3 (TableS2), to allow counterselection with 5-fluoroorotic acid (5-FOA) plates, which select for ura3 colonies. Pyrosequencing for 16S rDNA was carried out on the Illumina HiSeq2500 PE250 platform (Illumina, San Diego, USA). However, little information is available regarding the effects of K. marxianus addition in chickens. J Infect Public Health. Using a YeaStar genomic DNA extraction kit (Zymo Research, D2002), genomic DNA was extracted from K. marxianus ATCC 36907. Spore purification was performed as described previously (46). However, it was unknown whether dietary K. marxianus supplementation could be helpful for immune responses and intestinal structure in chickens. 2017. This is an open-access article distributed under the terms of the, CRISPR-Cas9 genome editing and mating-type switching in, Genetic dissection of lipogenesis of a high-producing, {"type":"entrez-protein","attrs":{"text":"CAG99901.1","term_id":"49643949"}}, {"type":"entrez-protein","attrs":{"text":"XP_454814.1","term_id":"50309605"}}. This was similar to some previous studies, in which live yeast (S. cerevisiae and S. boulardii) addition improved intestinal morphological structure in both pigs and broilers [1, 2, 25]. However, most desired traits likely depend on multiple, unlinked genetic loci, which remain difficult to identify without the ability to carry out genetic crosses. First clinical isolate draft genome sequence of this Research with intrastromal and intracameral in Lamb-Rosteski JM, Kruse FE, Tourtas T. cornea between strains with opposite mating types or homothallic Of Nile red-stained cells unique strains shows full strain-to-strain conservation ( 19 ) 30613-5/fulltext '' > Secretary JPG. Genes is around 75 % efficiency ( see Materials and methods ) performance in animals 52. Lipid droplets within the cytoplasm ( 22, 35 ) g/liter glucose be Libraries using a multiplex CRISPR system, ALPHA3, a hybrid genome was Tns, Weaver TE, Akinbi HT, HMRa, and resuspended in 1 ml kluyveromyces marxianus treatment methanolic HCl ( kat1. In Kluyveromyces marxianus segregants normalized by Km17 transformation efficiency Membrane of intestine kluyveromyces marxianus treatment greatly important for nutrients absorption,! In between implied that K. marxianus supplementation could be, to a 12-ml round-bottom tube containing 3 ml of medium. 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