Goshima T, Tsuji M, Inoue H, Yano S, Hoshino T, Matsushika A. Biosci Biotechnol Biochem. K. marxianus is a respiro-fermentative yeast likely to produce energy by either respiration or fermentation pathways. In addition, fermentations at higher temperatures occur more rapidly, making production much more efficient. (B) Hierarchy of YTK assemblies. (2017). of at least three replicates. For a lactose-inducible promoter, we chose the promoter for the beta-galactosidase LAC4. Biol. Our experiments revealed that inactivating NHEJ resulted in disruption of the LAC4 locus in nearly all transformants, as opposed to only occurring for about ~10% of transformants in wild-type K. marxianus. government site. A collection of biological parts and synthetic biology tools for Kluyveromyces marxianus. A total of four nonalcoholic beers were selected, which were produced with maltose-negative yeasts strains found in a previous study to form particularly fruity flavors during wort fermentation (Methner et al. These were the yeast strains Cyberlindnera saturnus C. sat 247 and C. sat CSa1 as well as Kluyveromyces marxianus K. mar 653. Keywords Kluyveromyces marxianus Saccharomyces cerevisiae ethanol productivity physiology Since lignocellulosic hydrolysates are comprised of diverse monomeric sugars, oligosaccharides and potential metabolism inhibiting compounds, this microorganism can play a pivotal role as it can grow on lignocellulosic hydrolysates coping with vegetal cell wall derived inhibitors. K. marxianus is a respiro-fermentative yeast likely to produce energy by either respiration or fermentation pathways. doi: 10.1038/ncomms15202. As with inactivation by CRISPR/Cas9, correct integration would disrupt the gene and render the yeast unable to break down X-Gal, allowing us to pre-screen colonies by blue/white selection for genotyping (Figure 4B). Transformants were screened by colony PCR with OneTaq Quick-Load DNA polymerase (NEB), using primers in the backbone vector flanking the insert if size permitted, or with one primer binding in the backbone and one in the insert. We observed that over 50% of the hygromycin-resistant colonies did not turn blue when grown on medium containing galactose and X-Gal, as opposed to ~1020% when a deletion cassette is used without CRISPR/Cas9 (Figure 4B, Table 3). Several yeasts, which are eukaryotic microorganisms, have long been used in different industries due to their potential applications, both for fermentation and for the production of specific metabolites. Cell factories can serve as the basis of a new bio-based economy based on the sustainable production of fine chemicals, pharmaceuticals, nutraceuticals, and biofuels from engineered or native microbes. Inactivating any of the key genes involved in NHEJYKU70/80, NEJ1 or DNL4 (Abdel-Banat et al., 2010; Choo et al., 2014; Nambu-Nishida et al., 2017)has been shown to increase targeted integration in K. marxianus by forcing it to use homologous recombination (HR) alone for DNA repair. 8600 Rockville Pike For xylose induction, we cloned and tested the induction of three xylose-inducible promoters: XYL1pr, XYL2pr, and ALD4pr. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fbioe.2019.00097/full#supplementary-material, Abdel-Banat, B. M. A., Nonklang, S., Hoshida, H., and Akada, R. (2010). Kluyveromyces marxianus is one such alternative yeast. Morrissey JP, Etschmann MM, Schrader J, de Billerbeck GM. Microscopy: PGASO: a synthetic biology tool for engineering a cellulolytic yeast. These promoters exhibited a 6-to-10-fold increase in YFP using xylose as a carbon source relative to glucose, with XYL2pr having the strongest fold-induction (Figure 2C). The viability of K. marxianus CIDCA 8154 retained throughout the storage period Kluyveromyces and observed >90% yeast survival after 30-days storage at 20 C. Inclusion of K. marxianus marxianus significantly enhanced the survival rate against the sequential acid-bile treatment (36.3%) as CIDCA 8154 compared to their free cells (8.7%). AR, JV, J-MD, and JM were supported by the CHASSY project which received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. A modular cloning system for standardized assembly of multigene constructs. As a demonstration of more practical applications, we used pUCC001 to rapidly generate defined single, double and triple auxotrophs for uracil, leucine, and histidine (Figures 4C,D; Table 1). Engineering Kluyveromyces marxianus as a Robust Synthetic Biology Platform Host. It is also advantageous to maintain a common standard for assembly to facilitate the exchange of parts between researchers and yeasts as needed. AR and JV carried out the experimental work, interpreted the data and wrote the manuscript. eCollection 2022. (2018). doi: 10.1007/s10529-014-1576-4, Curran, K. A., Karim, A. S., Gupta, A., and Alper, H. S. (2013). Over 30 constitutive and inducible promoters, terminators and centromeres and autonomously replicating sequence (ARS) elements have been selected from existing gene expression data and characterised as part of this collection. Kluyveromyces marxianus; biochemicals; cellulosic ethanol; fermentation; lignocellulosic hydrolysates; synthetic biology. (A) The Kluyveromyces Kit (KmK) provides parts according to the Yeast Toolkit (YTK) standard to express any gene of interest (GoI) under various conditions and expression platforms. 6162, 4447. Biosynthesis of the antibiotic nonribosomal peptide penicillin in baker's yeast. doi: 10.1128/mBio.01410-18. Biofuels 10, 114. Synthetic biology and molecular genetics in non-conventional yeasts: current tools and future advances. They allow the in vitro construction of cloning and expression systems for K. marxianus with the same flexibility one can for S. cerevisiae. [5] This species is considered to be a "crabtree negative fungus", meaning it is unable to convert sugars into ethanol as effectively as crabtree positive taxa such as S. One hundred pico mole oligos are phosphorylated with 1 L of T4 polynucleotide kinase (10U L1, NEB) in a total volume of 10 L and then denatured and annealed. 20fmol of a suitable vector level I plasmid from the Yeast Toolkit [pYTK083; (Lee et al., 2015)] were combined with 40fmol each of the following insert level I plasmids: left and right connectors from the Yeast Toolkit (pYTK002 and pYTK067), mVenus (pYTK034), a kanMX expression marker for G418 resistance, and the appropriate K. marxianus promoters (P1-19), terminators (T1-5), and centromere (typically C1; Table 2). While random integrations of multiple copies of a gene can be advantageous in some biotechnology applications (Lin et al., 2017), it is equally important to integrate single copies of heterologous genes or pathways to rationally construct a cell factory or evaluate different metabolic engineering strategies. The production of ethanol from lignocellulosic biomass by Kluyveromyces marxianus CICC 1727-5 and Spathaspora passalidarum ATCC MYA-4345. doi: 10.1038/s41467-018-03293-x, Diniz, R. H. S., Villada, J. C., Alvim, M. C. T., Vidigal, P. M. P., Vieira, N. M., Lamas-Maceiras, M., et al. A Golden Gate assembly for a level II plasmid was then carried out as described above. POLYMER PARTICLES AND USES THEROF: : US14927321: : 2015-10-29: (): US20160175419A1: (): 2016-06-23: : Polybatics Limited; Microbiol. Interestingly, several of the same orthologous genes in S. cerevisiae also have strong promoters. A BsmBI site, and a BsaI site that after digestion will generate overhangs specific to that part type, flank each functional part. lac4 mutants are unable to convert X-Gal to a blue dye. While metabolically engineering strains it is advantageous to integrate heterologous gene cassettes for stronger and more stable expression. . Epub 2019 Nov 28. [5] These conflicting findings suggest that K. marxianus can exist in vegetative form either as a haploid and a diploid. Constitutive promoters as well (PDC1pr) have a stable output at high temperatures. Biotechnol. However, K. marxianus is unable to complete alcoholic fermentation on its own. When selecting native yeast promoters to use in strain engineering, an important source is gene expression studies under conditions of interest. Zygote: conjugation may immediately proceed sporulation. eCollection 2022 Dec. Front Bioeng Biotechnol. Finally, multiple TU-bearing level II plasmids can be combined to create multi-TU level III plasmids that are either episomal or integrative vectors. Budding yeasts reproduce by developing daughter cells as compared with fission yeast (e.g., Schizosaccharomyces) for which there is a relatively equal division of one cell to form two cells. [8] Alternatively, ascosporogensis can arise directly from diploid cells. When targeting an YFP expression cassette at the LAC4 locus, correct integration in a wild-type strain (as determined by genotyping) does not abolish random integration elsewhere, as seen by the spread in YFP fluorescence measured from five transformed colonies. For part characterization and mutant creation, we used NBRC1777 [Biological Resource Centre, NITE (NBRC), Tokyo, Japan]. The collection includes a number of known centromeres and autonomously replication sequences (ARS). Biotechnol. RG-1 organism. Sci. doi: 10.1126/sciadv.1500248, Wagner, J. M., and Alper, H. S. (2016). To further sidestep this problem, the MoClo standard, based on Golden Gate assembly, allows the efficient hierarchical in vitro assembly of multigene constructs either on episomal or integrative vectors for such purposes (Weber et al., 2011). (A) Terminator choice can change gene expression by a factor of nearly 5 using native terminators, and this range can be even further exchanged if terminators from S. cerevisiae are used. 101, 69696980. A 5 L fermenter at 42C produced 105.22 g/L xylitol using K. marxianus YZJQ016the highest production reported to date from corncob hydrolysate. University of California 2013;77(7):1505-10. doi: 10.1271/bbb.130173. cerevisiae. Biol. A. Kluyveromyces marxianus-specific techniques exist for the efficient in vivo assembly of large multigene constructs (Chang et al., 2012), and in conjunction with CRISPR/Cas9 (Lbs et al., 2017; Nambu-Nishida et al., 2017; Cernak et al., 2018) can allow us to specifically edit a genome, or efficiently target chromosmal integrations. However, insertion sites near essential genes may not always be this accessible; another site we tested near an orthologue of the translation initiation factor TIF1 yielded <10% correct integrations in the wild-type strain and no colonies in an NHEJ-deficient background (data not shown). Construction of efficient centromeric, multicopy and expression vectors for the yeast Kluyveromyces marxianus using homologous elements and the promoter of a purine-cytosine-like permease. 2022 Apr 20;10:851768. doi: 10.3389/fbioe.2022.851768. doi: 10.1007/s11274-015-1899-x, Yang, H., Matsumoto, Y., Trujillo, K. M., Lees-Miller, S. P., Osley, M. A., and Tomkinson, A. E. (2015b). Genet. XKS1 then phosphorylates it to xylulose-5-phosphate, which can then enter the non-oxidative branch of the pentose phosphate pathway. [4] Studies, however, deem it to be crabtree positive which is likely due to strain differences since K. marxianus possesses the necessary genes to be crabtree positive. 2021 Nov 3;9(11):2288. doi: 10.3390/microorganisms9112288. Received: 25 November 2018; Accepted: 16 April 2019; Published: 07 May 2019. Furthermore, as other research has found that the binding of DNL4 to DNA may not require NEJ1 (Wu et al., 2008), and the latter enhances but is not essential for NHEJ entirely (Yang et al., 2015b), this may explain why the inactivation of NEJ1 in YBL003 was insufficient to ensure single-copy gene integration. The BsmBI sites, in turn, are used to clone new parts into the entry vector YTK001 by Golden Gate assembly with that enzyme. Here, multiple TUs are assembled together into a multigene expression or integrative vector, again with BsmBI based on unique overhangs present in the connectors. is also in some commercial preparations of yeast to contribute flavor complexity. Yeast transformation by the LiAc/SS carrier DNA/PEG method. Interestingly, two terminators from the YTKthose for ScADH1 and ScPGK1can change gene expression between them (as measured by YFP fluorescence) by nearly a factor of two in K. marxianus. Disclaimer, National Library of Medicine Nat. Would you like email updates of new search results? Following this, the corresponding promoters were then identified from the CBS6556 genome. and transmitted securely. [15], Last edited on 30 September 2022, at 23:32, "Polymicrobial chronic endophthalmitis diagnosed by culture and molecular technique", "Is an Emerging Pathogen in Patients with Oncohematological Diseases? doi: 10.1093/femsyr/foy012, Kim, T. Y., Lee, S. W., and Oh, M. K. (2014). Also used commercially to produce the lactase enzyme and as a bonding agent for fodder and pet food, and as a source of ribonucleic acid in pharmaceuticals. The methods may include a net balance of cofactor production and consumption. While the wild-type yeast is already in use for producing fragrances and fermented products, the lack of standardised tools for its genetic and metabolic engineering prevent it from being used as a next-generation cell factory for bio-based chemicals. [5][8] K. marxianus is widely used in industry because of its ability to use lactose. Clearly, to move beyond being niche organisms in biotechnology, alternative yeasts require such part collections to make rapid metabolic engineering feasible. Metab. Kluyveromyces, similar to Saccharomyces, is a budding yeast, and this is the most common mode of vegetative reproduction. There is evidence that a diet low in FODMAPs reduces abdominal symptoms in approximately 70% of the patients suffering from irritable bowel syndrome. [5], Colonies of K. marxianus are cream to brown in colour with the occasional pink pigmentation due to production of the iron chelate pigment, pulcherrimin. Bookshelf Clipboard, Search History, and several other advanced features are temporarily unavailable. 40, 309317. Kluyveromyces marxianus as a host for heterologous protein synthesis. (2012). doi: 10.1139/bcb-2016-0001, Feng, J., Jester, B. W., Tinberg, C. E., Mandell, D. J., Antunes, M. S., Chari, R., et al. It generates a wide-ranging specific metabolites and could contribute to a variety of different food and biotechnological industries. Under standard conditions (30C, glucose-rich medium), we provide a broad selection of promoter strengths for gene expression. fermentation by Kluyveromyces marxianus yeast strain. For level II and III plasmids, PCR-positive colonies had their plasmids extracted and digested with NotI to verify the insert size All primers used to genotype strains are listed in Table S3, and maps of all the plasmids in Table S1 are included as Supplementary Material. Please go there to see whether you have right to access the fulltext or not. Front. (March 2020), This page was last edited on 30 September 2022, at 23:32. When plasmids containing type IIS restriction sites that could not be removed, the final digestion and heat inactivation steps were eliminated. This study demonstrated that K. marxianus catabolizes glycerol through the Gut1-Gut2 pathway instead of the previously speculated NADPH-dependent Gcy1-Dak1 pathway using the transient clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9 (CRISPR/Cas9) system. It can natively consume pentose sugars and disaccharides found in agricultural and dairy wastes, making these economic feedstocks for potential cell factories. It was with this goal in mind that we selected and characterized the parts presented in this kit, while maintaining the YTK standard. SgRNAcas9: a software package for designing CRISPR sgRNA and evaluating potential off-target cleavage sites. wildcat mountain difficulty; best bone meal powder for dogs; stix restaurant hours; new york fall foliage 2022; While the terminators included with the original YTK aimed to keep gene expression output roughly constant (Lee et al., 2015), we have provided five native terminators to broaden the range of gene expression our parts can achieve (Figure 3A). From the weakest (REV1pr) to the strongest (PDC1pr) promoters, we can achieve a 40-fold range of gene expression in NBRC1777. doi: 10.1007/s00253-012-4306-7, Lee, M. E., DeLoache, W. C., Cervantes, B., and Dueber, J. E. (2015). Colony: Malt agar: puffy, creamish-brown, flat, spreading, dull round colony. AMB Express 8, 111. (2018). (2014). Sequence differences between promoters for the same gene in different yeast strains can have implications in gene expression, and therefore should be taken into consideration for experimental and industrial applications (Liu et al., 2015; de Paiva et al., 2018); however, at this stage not enough is known about K. marxianus' native transcription factors and regulatory network to functionally dissect our promoter sequences. (C) The diverse carbon source utilization of K. marxianus gives us a unique induction signal for this yeast, as seen by the induction of promoters by lactose, galactose, and xylose. For selecting sites of the second type, we examined existing TSS-seq data (Lertwattanasakul et al., 2015) and found two such sites where the gene clusters were on the same coding strand: one each on chromosome IV and V (I2 and I4) (Figure 5C; Table 2). Yeast ARS elements and centromeres were selected from the literature, and either amplified from genomic DNA or from an appropriate plasmid. The new cassette was assembled from long oligonucleotides (Integrated DNA Technologies, USA) by annealing them in a thermocycler. Assembling TUs includes flanking them with synthetic and directional connector sequences which allow the construction of level III, plasmids. No use, distribution or reproduction is permitted which does not comply with these terms. Characterization of Saccharomyces cerevisiae promoters for heterologous gene expression in Kluyveromyces marxianus. sharing sensitive information, make sure youre on a federal Although Saccharomyces cerevisiae is the most widely used dominant representative in all aspects, many applications of K. marxianus in biotechnology, food and environment have only started to emerge nowadays; some of the most promising applications are reviewed here. Finally, this review provides a discussion of the main challenges and some perspectives for targeted applications of K. marxianus in the modern food technology and applied biotechnology in order to exploit the full potential of this yeast which can be used as a cell factory with great efficiency. Ascus: Evanescent. Elife 4, 123. Here, several K. marxianus strains, isolated from cocoa fermentation, were evaluated for xylose consumption and tolerance towards acetic acid, furfural, and HMF. 8600 Rockville Pike YFP values significantly different from those under baseline conditions (expression using INU1t) are marked with an asterisk (p < 0.05) or a hash (p < 0.001). Appl. Transcriptome analysis of the thermotolerant yeast Kluyveromyces marxianus CCT 7735 under ethanol stress. The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. The site is secure. Nonetheless in vivo assembly as it stands does not eliminate non-specific integrations of incomplete parts of the assembly. FOIA mBio. [9] Due to the ability of K. marxianus to simultaneously utilize lactose and glucose, the prevalence of K. marxianus in industrial settings is high as it decreases production time and increases productivity. Our CRISPR/Cas9 editing plasmid pUCC001 takes advantage of the cross-species pUDP002 system and makes it a more flexible and economical tool by introducing a BsaI cloning site for new gRNA targets (Figure 4A). An official website of the United States government. [5] K. marxianus is highly thermotolerant and able to withstand temperatures up to 45C (113F). 9:3330. doi: 10.3389/fmicb.2018.03330, Vyas, V. K., Barrasa, M. I., and Fink, G. R. (2015). The inset shows the cloning site inserted between the ribozyme units; two BsaI sites allow the construction of new gRNA targets using Golden Gate cloning. Identifying such a background is beneficial to improve the efficiency and specificity of K. marxianus-based in vivo assembly techniques such as PGASO (Chang et al., 2012), and to further define a genotype for a potential future lab strain for K. marxianus. 94, 396406. In our study, K. marxianus XZ1 reduced the disease incidence and patulin content in apple caused by Penicillium expansum at 3 d . All of our inducible promoters selected from expression data exhibited induction under the relevant conditions, though not all to the same extent (Table S6). Kluyvermomyces marxianus's thermotolerance provides a unique induction signal for this yeast's promoters. Extrapolating from research in S. cerevisiae, our findings are given weight by the different roles in NHEJ of the proteins we targeted. (2017). distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 7, 969984. Fermentation: Glucose, Galactose, Sucrose, Raffinose; variable: Maltose, Lactose. A modular toolkit for generating Pichia pastoris secretion libraries. We also foresee the set being expanded by synthetic promoters, engineered promoters (as has been done with INU1pr) and secretion tags (Zhou et al., 2018). In yeasts other than S. cerevisiae, the ability to efficiently target gene integration is hindered by their usage of non-homologous end-joining (NHEJ) as the dominant DNA repair mechanism (Daley et al., 2005). It exhibited a 10-fold induction by lactose relative to glucose alone when used to express YFP in NBRC1777. Bioresouces Technology. As a result, random and incomplete integrations are frequent, and much larger targeting homology sequences are also required (up to 1 kb) compared to S. cerevisiae (50 bp) for a successful integration (Baudin et al., 1993; Choo et al., 2014). Appl. Using gRNA plasmids targeting the K. marxianus orthologues of URA3, HIS3, and LEU2 we created frameshift mutations in these genes leading to loss of function. Would you like email updates of new search results? 19, 8897. However, further optimization of the gRNA expression system is necessary for an optimal K. marxianusspecific multiplexing system. CRISPR-Cas9-enabled genetic disruptions for understanding ethanol and ethyl acetate biosynthesis in Kluyveromyces marxianus. A number of high- and low-copy number origins have also been identified from K. marxianus genomes, or created from minimal elements. In theory, Cas9 and multiple gRNA cassettes could be separately cloned as level II plasmids and then reassembled into a level III multi-TU plasmid. (2014). Growth Sensitivities: grows on 0.01 and 0.1% cycloheximide; variable growth on high glucose and 10% NaCl. This led us to hypothesise that correct integration in a wild-type strain does not preclude random integration. [3] It is also a naturally occurring colonist of plants, including corn. It can tolerate temperatures over 50 C and has the fastest growth rate of any eukaryote [ 15, 16 ]. In the case of inducible promoters, terminator effects are more pronounced under non-inducible conditions. The yeast Kluyveromyces marxianus and its biotechnological potential. doi: 10.1038/s41598-018-25366-z, Lertwattanasakul, N., Kosaka, T., Hosoyama, A., Suzuki, Y., Rodrussamee, N., Matsutani, M., et al. 31, 16411646. This saves the cost and time of cloning the entire gRNA expression cassette for every target as for the original plasmid. Bethesda, MD 20894, Web Policies gRNA targets used in this study are listed in Table S5. 1163, 3344. The yeast Kluyveromyces marxianus CCT 3172, isolated from a cocoa fermentation in Brazil (4, 5), was selected due to its relatively high yields of endopolygalacturonase production and was used. Mol. kluyveromyces marxianus s1.17, obtained by electroporation-mediated genome shuffling between k. marxianus g2-16-1, a cellobiase-producing yeast, and pichia stipitis jcm 10742t, gave a maximum ethanol production level (of 0.86 g/l) from the hydrolysate of dilute sulfuric acid treated sugarcane leaves when treated under aerobic conditions for 72 h, It can survive on a variety of carbon sources under industrially favorable conditions .
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