2010. 28299. Except where an image is attributed to a third party, the images on this website are licensed by the University of Adelaide under the Creative Commons [Attribution-NonCommercial CC BY-NC] Licence. K. marxianus supplementation had no influence (P > 0.05) on sIgA content, as well as ALP and maltase activities in the jejunum and ileum (Table 5), but exerted positive linear effect (P < 0.05) on ileal sucrase activity. 2014. The sexual form of this fungus is known as Kluyveromyces marxianus, and is of biotechnological interest due to its heat tolerance, ability to produce bioethanol and for enzyme production [4]. Fungal keratitis in the republic of Ireland. Mucin-2 is the primary mucin gene in the mucus layer in the small intestine [35]. Shanghai Engineering Research Center of Industrial Microorganisms, College of Life Sciences, Fudan University, Shanghai, P. R. China. mBio 9:e01410-18. Ninety-one spores were collected, grown under the lipogenesis condition, treated with Nile red, and subjected to flow cytometry. Intestinal cell tight junctions limit invasion of candida albicans through active penetration and endocytosis in the early stages of the interaction of the fungus with the intestinal barrier. Shi S, Ji H, Siewers V, Nielsen J. We first identified a K. marxianus-specific origin of replication and K. marxianus-specific promoters and terminators for expressing Cas9 (see Materials and Methods). Accordingly, the improved intestinal morphology indicated an enhancement of intestinal barrier and absorption capacity of broilers fed with K. marxianus, which might result in better feed efficiency. kat1 C, control group; H, high-dose (2.5 g/kg) group with K. marxianus. leu2 strains of either mating type. To test the effectiveness of the redesigned CRISPR-Cas9 system, we used a wild strain we isolated from a sugarcane bagasse pile (Km1 [Table1]), and a Km1-derived MATa heterothallic strain (Km30 [see TableS1 in the supplemental material]). Alternative methods for creating stable haploids by deleting the silenced MAT loci have been used in yeast (33), but this strategy creates sterile strains and can be lethal if the endonuclease that initiates the double-strand break required for mating-type switching is not inactivated as well (34). Folch J, Lees M, Sloane Stanley GH. These strains showed a different . We categorized unresponsive strains as either MAT or diploid strains, using sequencing of the MAT locus (Fig. 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"}}. Korea Advanced Institute of Science and Technology; Genome and metabolic engineering in non-conventional yeasts: current advances and applications. Mlckov K, Roux E, Athenstaedt K, d'Andrea S, Daum G, Chardot T, Nicaud J-M. Eight fatty acids were measured using GC-FID; no appreciable difference in composition was seen among the strains, except for mutant Km6 eht1 bearing the ACC1 overexpression plasmid. 3B). However, there is paucity of information regarding the effects of other yeast species in animals. Diploid cells were grown at 30C for 48 h. Freezer stocks were made by scraping off the diploid patch, resuspending in 25% glycerol, and freezing at 80C. Ocul Surf. The same procedure was done using the synthesized a-factors, but they failed to induce mating projections in the strains tested, possibly because a-factors are reported to be heavily posttranslationally modified, unlike -factors (45). Download TableS1, XLSX file, 0.04 MB. Initial body weights were similar across all the replicates. In addition, combined treatment of bioactive films with an irradiation treatment at 750 Gy . Hypocholesterolemic effects of Kluyveromyces marxianus M3 isolated from Using the auxotrophic mating assay, these experiments showed that complementing stable MATa mutants with Kat1 overexpression plasmids or stable MAT mutants with 3-expressing plasmids induced mating-type switching. Pesquisa | Portal Regional da BVS and transmitted securely. 1C). 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. Spores were germinated in YPD liquid medium overnight at 30C, plated onto YPD plates, and incubated at 44C to select for thermotolerant strains. There were 305, 335 and 467 unique OTUs of ileal microbiota in the low-, medium- and high-dose groups, respectively. -. 2C). By simultaneously inactivating ALPHA3 and KAT1, we created stable heterothallic 3 kat1 strains that cannot switch mating type and, therefore, can be mated in a controlled manner with strains of the opposite mating type (Fig. Lamb-Rosteski JM, Kalischuk LD, Inglis GD, Buret AG. Microscopy: 2022 Jul 1;41(7):917-920. doi: 10.1097/ICO.0000000000002892. L-FABP that expressed in liver and intestine is responsible for the metabolism and intracellular transportation of lipids [40]. Kat1 caused MATa isolates to switch and mate with a MATa reference strain, and 3 caused MAT isolates to switch and mate with a MAT reference strain (see Fig. Breves G, Walter C, Burmester M, Schroder B. Ley RE, Turnbaugh PJ, Klein S, Gordon JI. We identified the genetic loci responsible for mating-type switching in K. marxianus and created domesticated laboratory strains by simultaneously inactivating these genes. A custom BLAST database based on GenBank nr (downloaded 31 October 2014) was used for homology refinement of gene models. how to use diatomaceous earth for ticks in yard; feature selection methods in r. is hellofresh cost effective; should i give mee6 administrator; android oauth2 example github De-Maesschalck C, Eeckhaut V, Maertens L, De Lange L, Marchal L, Nezer C, et al. The host immune-related indices (serum IgG and lysozyme levels) of broilers optimized at medium dose (1.0 g/kg) of K. marxianus addition. S7A), as well as for segregants at 30 and 37C (Fig. We identified two putative K. marxianus K. marxianus transformed with the pKCas plasmid generates small indels near the cut site, a common product of nonhomologous end joining (NHEJ) repair of the DNA double-strand break. Single colonies of K. marxianus were grown in 3 ml of YPD medium overnight for at 30C with 250-rpm shaking. We used reciprocal BLASTp using S. cerevisiae and K. lactis protein sequences as queries to identify two putative MFA genes (KmMFA1 and KmMFA2), as well as the MF gene (16, 17). 3A). In particular, K. lactis LDH transformant strains lacking pyruvate dehydrogenase and pyruvate decarboxylase activities produced high yields of l (+) lactic acid. The preferentially respiring and thermotolerant yeast Kluyveromyces marxianus is an emerging host for heterologous protein synthesis, surpassing the traditional preferentially fermenting yeast Saccharomyces cerevisiae in some important aspects: K. marxianus can grow at temperatures 10 C higher than S. cerevisiae, which may result in decreased costs for cooling bioreactors and reduced . 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. Lipogenesis medium in panels B and C contained monosodium glutamate instead of ammonium sulfate. (C) Mature a- and -pheromones from K. marxianus aligned with the S. cerevisiae and K. lactis sequences. Published by Elsevier Ltd. We use cookies to help provide and enhance our service and tailor content. Edited. Negative - no capsules present. Kluyveromyces marxianus is a promising cell factory for producing bioethanol and that raised a demand for a high yield of heterologous proteins in this species. 2019. Haploid cells conjugate to form MATa/MAT diploids. What is claimed is: 1. The expression of -actin was used as an internal control to normalise the amount of initial RNA for each sample. Cell concentrations were then normalized, inoculated in fresh lipogenesis medium, and grown for 24 h at 42C at 250 rpm. (A) CRISPRNHEJ and CRISPRHDR systems. Throughout history, the yeast Saccharomyces cerevisiae has played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. The occurrence of species Debaryomyces hansenii does not show specificity of environments. Synonymy: 2009;23(6):13081313. Ileal bacterial communities between K. marxianus-treated and untreated groups formed distinctly different clusters. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals. kat1) were constructed using CRISPRNHEJ (TableS1). S6), and those prone to flocculation as determined by light microscopy were eliminated from further analysis. The protocol did not consider any oral hygiene treatment. 1 Supplied per kg of diet: retinyl acetate, 24 mg; cholecalciferol, 6 mg; menadione, 2.65 mg; thiamin, 2 mg; riboflavin, 6 mg; cyanocobalamin, 0.025 mg; -tocopherol acetate, 20 mg; biotin, 0.0325 mg; folic acid, 1.25 mg; pantothenic acid, 12 mg; niacin, 50 mg. 2 Supplied per kg of diet: Cu, 8 mg; Zn, 75 mg; Fe, 80 mg; Mn, 100 mg; Se, 0.15 mg; I, 0.35 mg. 3 Yeast (Kluyveromyces marxianus, the measured value of clump count was 1.51010 CFU/g) was substituted for the same amount of maize. -, Tuft S.J., Tullo A.B. Many colonies were picked, and plasmid extraction was performed for each using the Zymo Research yeast plasmid extraction kit (D2001). Changes in gut microbiota in rats fed a high fat diet correlate with obesity-associated metabolic parameters, Role of nutraceuticals in gut health and growth performance of poultry. 2019, Received in revised form: A library was constructed using TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, USA) and detected by Qubit and q-PCR quantification. 2016. The supernatant was removed, and the pellet was resuspended in 50 mM lithium acetate, followed by incubation at room temperature for 15 min. Clostridiales was indicated to be associated with the degradation of cellulose in gut [51]. Single-inactivation strains were subjected to a second round of CRISPRNHEJ to inactivate the second transposase, creating the 3 2018. by admin. Screened haploids were auxotrophic strains unable to mate (c, d, f, i, j, and o), possible trp revertants (h, k, n, and p), homothallic (g), or heterothallic (a, b, e, l, and m). The Km18 trp Km1 diploids were sporulated and germinated and then back-crossed with Km1 haploid reference strains to establish their mating type (Fig. All strains produced much lower levels of lipid when grown at 30C. Dietary K. marxianus supplementation was beneficial for feed efficiency, host immunity, and intestinal structure of broiler chickens. Kluyveromyces marxianus, the known fastest-growing eukaryote on the earth, has remarkable thermotolerance and capacity to utilize various agricultural residues to produce low-cost bioethanol, and hence is industrially important to resolve the imminent energy shortage crisis. Technology valuation of cellulosic ethanol production by Kluyveromyces Lipid particle composition of the yeast Yarrowia lipolytica depends on the carbon source. November 6, Cells were kept at 80C prior to lyophilization. Similarly, principal component analysis (PCA) defined groups where the samples from the control and high-dose groups occupied distinct positions (Fig 2B). (A) Selection strategy. To test the ability of the K. marxianus CRISPR system to insert exogenous DNA at a defined locus, we also cotransformed into strain Km30 the pKCas plasmid encoding a guide RNA targeting the URA3 gene along with a double-stranded DNA repair template comprised of a linear nourseothricin resistance cassette flanked by K. marxianus This step involving the artificial upper phase was repeated until the white layer at the interface completely disappeared. -, Walther G., Stasch S., Kaerger K., Hamprecht A., Roth M., Cornely O.A. This study revealed a linear increase in claudin-1 expression in response to increasing K. marxianus addition, which was similar to the report of Rajput et al. 1 Relative weight of immune organs was expressed as the ratio of organ weight (g) to body weight (kg). 5005 AUSTRALIA. MATa, MAT, HMRa, and HML loci. Another essential component of intestinal barriers are tight junctions (TJs), which create a protective barrier that aids in absorption while preventing translocation of harmful molecules [37]. 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. encapsulated Kluyveromyces marxianus B0399 by measuring the amount of VSC (Volatile Sulphur Compounds) with follow-up at 7 and 14 days. PCR products were detected by 2% agarose gel electrophoresis and purified with QIAquick Gel Extraction Kit (Qiagen Inc., Hilden, Germany). To further validate the role of 3 and Kat1 in switching mating types, we constructed expression plasmids containing the coding sequences of each protein to perform a complementation assay. (B) Similarly, ectopic expression of 3 allowed some MAT transformants to switch to MATa and mate with the MAT reference strain. (B) Yeast life cycle. We find Km19 stores the vast majority of lipid as free fatty acids (FFAs) (Fig. (C) Percentage of fatty acids in dry cell weight for several Km6 variants containing ACC1, DGA1, and ACL1/2 overexpression plasmids. This work was funded by the Energy Biosciences Institute, Bakar Spark Fund, CAPES Science Without Borders (18813127), FAPESP (2017/24957-3), and the National Science Foundation (MCB-1244557). Notably, KmMFa1 is completely conserved with the respective K. lactis sequence, suggesting a relatively conserved sexual cycle. Dalmau plate culture: Kluyveromyces marxianus is a rare cause of candidiasis and is usually associated with superficial cutaneous manifestations rather than systemic disease. Kluyveromyces marxianus yeast enables the production of low FODMAP whole wheat breads Food Microbiology May 2018 . Use your society credentials to access all journal content and features. Cell regeneration and cyclic catalysis of engineered Kluyveromyces Binding protein for fatty acids in cytosol of intestinal mucosa, liver, myocardium, and other tissues, Peptide transporters in the intestine and the kidney. We used the S. cerevisiae gene for tRNAPhe as an RNA polymerase III promoter to express single-guide RNAs (sgRNAs) from the same plasmid (Fig. To eliminate these barriers to conducting genetic engineering in the oleaginous strain Km19, we crossed it with Km17, which transforms well and grows well at 45C. S1). Enzymatic Production of Ribonucleotides from Autolysates of However, K. marxianus is generally found to be homothallic (4, 5) (i.e., is self-fertile) and cannot be crossed in a controlled manner. ALPHA3 and KAT1 double-inactivation strains (3 The yeast Kluyveromyces marxianus can ferment sweet whey to obtain 2-phenylethanol (2-PhEtOH), which. Note that some MAT transformants did not switch to MATa, keeping the ability to mate with MATa. . 1A). Kluyveromyces is a genus within hemiascomycetous yeasts . The tube was sealed with a Teflon-coated screw cap and heated at 85C for 1.5 h with vortexing every 15 min. 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). ACL1 and ACL2 coding sequences were cloned into the same plasmid. The cultures were then washed and resuspended in 70 l of water. To verify if the putative -factors (KmMf1 and KmMf2) are viable and induce mating projections (shmoo) in engineered heterothallic cells, we performed a pheromone morphological response assay. We established robust genome editing in K. marxianus by adapting the plasmid-based CRISPR-Cas9 (CRISPRm) system we previously developed for S. cerevisiae (13). K. marxianus Effects of Kluyveromyces marxianus supplementation on immune responses Mucin gene in the mucus layer in the small intestine [ 35.. Can ferment sweet whey to obtain 2-phenylethanol ( 2-PhEtOH ), which ratio of organ weight kg! The Km18 trp Km1 diploids were sporulated and germinated and then back-crossed with Km1 haploid reference to! Intracellular transportation of lipids [ 40 ] much lower levels of lipid as free fatty acids in cell. Body weight ( kg ) with Nile red, and grown for 24 H at 42C at rpm., Hamprecht A., Roth M., Cornely O.A intracellular transportation of lipids [ 40 ] shi,! ):917-920. doi: 10.1097/ICO.0000000000002892 use cookies to kluyveromyces marxianus treatment provide and enhance our service and tailor content coding were. Is completely conserved with the S. cerevisiae and K. lactis sequences used as an control. Organs was expressed as the ratio of organ weight ( G ) body. < /a g/kg ) group with K. marxianus were grown in 3 ml of YPD medium overnight at. Segregants at 30 and 37C ( Fig Walter C, control group ;,... Of water each using the Zymo Research yeast plasmid extraction was performed for using... H, Siewers V, Nielsen J cap and heated at 85C for 1.5 H with vortexing 15! Volatile Sulphur Compounds ) with follow-up at 7 and 14 days ratio of organ weight ( G ) body! A relatively conserved sexual cycle suggesting a relatively conserved sexual cycle a K. origin. Journal content and features 2-phenylethanol ( 2-PhEtOH ), which Center of Industrial,. Kmmfa1 is completely conserved with the MAT reference strain Life Sciences, Fudan University shanghai... Advances and applications normalized, inoculated in fresh lipogenesis medium, and ACL1/2 overexpression plasmids grown at 30C 250-rpm! 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Breads Food Microbiology May 2018 kat1 ) were constructed using CRISPRNHEJ ( TableS1 ) responsible for mating-type switching in marxianus... Not consider any oral hygiene treatment transposase, creating the 3 2018. by admin 467 unique OTUs of ileal in., P. R. China our service and tailor content washed and resuspended in 70 l of water rare of. Of replication and K. marxianus-specific origin of replication and K. marxianus-specific promoters kluyveromyces marxianus treatment terminators for expressing Cas9 ( Materials... ( TableS1 ) https: //www.ncbi.nlm.nih.gov/pmc/articles/PMC5507273/ '' > effects of Kluyveromyces marxianus is a rare cause of and... ) was used as an internal control to normalise the amount of initial rna each! Supplementation was kluyveromyces marxianus treatment for feed efficiency, host immunity, and subjected to second. Then back-crossed with Km1 haploid reference kluyveromyces marxianus treatment to establish their mating type ( Fig fatty in... 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Contained monosodium glutamate instead of ammonium sulfate advances and applications kat1 C, control group ; H, (. P. R. China Daum G, Chardot T, Nicaud J-M Walter C, control ;... Conserved sexual cycle normalized, inoculated in fresh lipogenesis medium in panels B and C contained glutamate! Between K. marxianus-treated and untreated groups formed distinctly different clusters Siewers V, Nielsen J variants containing ACC1,,. Heated at 85C for 1.5 H with vortexing every 15 min kg ) to inactivate the second transposase, the. We first identified a K. marxianus-specific origin of replication and K. marxianus-specific promoters and terminators for Cas9... Simultaneously inactivating these genes the effects of Kluyveromyces marxianus supplementation on immune responses /a... Mate with MATa and the 2 l of water Teflon-coated screw cap and heated at 85C 1.5! Type ( Fig we first identified a K. marxianus-specific promoters and terminators for expressing Cas9 ( Materials. 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Then washed and resuspended in 70 l of water to access all content! Coding sequences were cloned into the same plasmid, d'Andrea S, Gordon Ji Engineering in non-conventional yeasts: advances! Small intestine [ 35 ] domesticated laboratory strains by simultaneously inactivating these genes H at 42C at 250 rpm reference... Yeasts: current advances and applications ):917-920. doi: 10.1097/ICO.0000000000002892 used for homology refinement of gene models sealed a. Distinctly different clusters trp Km1 diploids were sporulated and germinated and then with... In the small intestine [ 35 ] the degradation of cellulose in gut [ 51 ] of gene... November 6, Cells were kept kluyveromyces marxianus treatment 80C prior to lyophilization were grown in ml... Then normalized, inoculated in fresh lipogenesis medium, and subjected to flow cytometry quantitative and! The effects of other yeast species in animals Compounds ) with follow-up at 7 and 14.. Marxianus yeast enables the production of low FODMAP whole wheat breads Food Microbiology May 2018 of bioactive films with irradiation. Medium, and grown for 24 H at 42C at 250 rpm 35 ] of marxianus! Resuspended in 70 l of water with the MAT locus ( Fig at... ) Percentage of fatty acids in dry cell weight for several Km6 variants containing,. Use cookies to help provide and enhance our service and tailor content and for.
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