This paper reviews the recent progress made with K. marxianus biotechnology for sustainable production of ethanol, and other products utilizing lignocellulosic sugars. Frameshift mutations in each gene created the desired auxotrophies, and the strains so generated could be targeted with other plasmids to create defined double and triple mutants. (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. Biotechnology; Enzymes; Food; Kluyveromyces marxianus; Metabolites. POLYMER PARTICLES AND USES THEROF: : US14927321: : 2015-10-29: (): US20160175419A1: (): 2016-06-23: : Polybatics Limited; To minimise variations in expression due to copy number, we used centromeric plasmids with a kanMX cassette. Microbiologyopen. (2013). It is the sexual stage of Atelosaccharomyces pseudotropicalis[citation needed] also known as Candida kefyr. One was TSS-seq transcriptome data published alongside its genome (Lertwattanasakul et al., 2015), as well as a small-scale gene expression study using fluorescent reporters of genes involved in carbon metabolism (Suzuki et al., 2015). Glycerol is an ideal co-substrate for xylitol production with Kluyveromyces marxianus. The biological parts collected and characterized here are intended to provide the same functions for K. marxianus in all the relevant categories of YTK parts (Figure 1A). 7, 26472655. As a proof of principle, we transformed K. marxianus with pUCC001 containing a gRNA targeting the LAC4 locus. While a number of gene expression studies have been carried out on K. marxianus strains, few of them have focused on strains with the potential to be synthetic biology chassis, or on have instead focused on explicitly industrial conditions (Gao et al., 2015; Schabort et al., 2016; Diniz et al., 2017). The new cassette was assembled from long oligonucleotides (Integrated DNA Technologies, USA) by annealing them in a thermocycler. doi: 10.1093/nar/21.14.3329, Bergkamp, R. J. M., Bootsman, T. C., Toschka, H. Y., Mooren, A. T. A., Kox, L., Verbakel, J. M. A., et al. Standardised biological parts for the expression and maintenance of genes, and means to assemble them, are a cornerstone of synthetic biology to build new biological systems. Genetic basis of the highly efficient yeast Kluyveromyces marxianus: complete genome sequence and transcriptome analyses. aegilopshas potential to be exploited as a native, abundant raw material for the production of ethanol. Table 3. All data are plotted as the mean s.d. doi: 10.1016/j.jbiotec.2014.04.010, Choo, J. H., Han, C., Kim, J. Y., and Kang, H. A. doi: 10.1007/978-1-4939-0799-1_4, Gombert, A. K., Madeira, J. V., Cerdn, M.-E., and Gonzlez-Siso, M.-I. J Fungi (Basel). Kluyveromyces marxianus in ascomycetous yeast and member of the genus, Kluyveromyces. Colonies with frameshift mutations are then grown overnight in YPD and then passaged twice to fresh YPD cultures to ensure loss of the gRNA plasmid before being preserved. For the experiments carried out here, for the purpose of evaluating integration sites, mVenus was always under the control of the PDC1 promoter (P2) and the INU1 terminator (T1). For Golden Gate assembly the protocol was as follows: 25 cycles of digestion and ligation (2 min at 42C followed by 5 min at 16C), followed by 10 min digestion at 60C and 10 min inactivation at 80C. MBio 9, 116. We acknowledge the help of Amy Bergin and Beth Mulcahy in cloning the parts plasmids. 89, 126136. In total, 8 parts were assembled and transformed into E.coli per plasmid. 2022 Apr 20;10:851768. doi: 10.3389/fbioe.2022.851768. [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. Fermentable oligo-, di-, and monosaccharides and polyols (FODMAPs) are small molecules that are poorly absorbed in the small intestine and rapidly fermented in the large intestine. Alternating the type IIS enzymes between assemblies allows construction of different plasmid levels. Starting from amplified PCR products, parts are cloned into level I plasmids for storage. ALD4pr was chosen from a genome-wide expression study for its low background in glucose and high fold-induction by xylose (Lertwattanasakul et al., 2015). 7, 969984. doi: 10.1016/j.fgb.2015.12.001, Weber, E., Engler, C., Gruetzner, R., Werner, S., and Marillonnet, S. (2011). Careers. Separation of these clades based on the presence of the WGD event explains why, even though the two species are closely related, fundamental differences exist between them. (2015). ACS Synth. Biotechnol. When glucose concentrations become depleted to 6 g/L, the lactose co-transport initiates.[10]. 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 due to its fast growth rate, thermotolerance, and acid tolerance. It remains unclear if the different mutations suppress random integration to the same extent. Natural yeast promoter variants reveal epistasis in the generation of transcriptional-mediated noise and its potential benefit in stressful conditions. The Cre-loxP system produces structural variations, such as deletion, duplication, inversion and translocation, at specific loci and induces chromosomal rearrangements in the genome. Rate the pronunciation difficulty of Kluyveromyces marxianus. Bookshelf [4] The ability for K. marxianus to reduce lactose is useful because of the potential to transform industrial whey waste, a problematic waste product for disposal, into useful biomass for animal feed, food additives or fuel. To decide which NHEJ mutant gave us the highest rate of targeted integration, we tested the integration of a YFP expression cassette at the LAC4 locus in backgrounds that were either wild-type or had either YKU80, DNL4, or NEJ1 previously inactivated by CRISPR/Cas9 (YBL001, KmASR.005, and YBL003, respectively; Table 1). AR and JV carried out the experimental work, interpreted the data and wrote the manuscript. Keywords: Kluyveromyces marxianus and related species are used widely due to their capacity to assimilate lactose, the carbohydrate present in cheese whey, but it can also grow on inulin, a fructose polymer found in some plants, and other simple sugars such as glucose, fructose, and sucrose; therefore, sometimes it is also grown in molasses. It exhibited a 10-fold induction by lactose relative to glucose alone when used to express YFP in NBRC1777. 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. TSA1pr has the strongest fold-induction at 42C and the highest measured fluorescence at high-temperature, whereas that of SSA2pr remains at increasing medium-to-high levels (relative to the constitutive promoters) as temperature increases (Figure 2B). FEMS Yeast Res. RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growth-biased metabolic engineering of, Fluorescence lifetime imaging of NAD(P)H upon oxidative stress in. Table 1. 2022 ). (2012). Environmental isolations have been made from cheese and dairy products. Yeast 27, 2939. doi: 10.1002/mbo3.1290. Microbiol. Level I plasmids correspond to part plasmids. Typically, half of a Golden Gate assembly was used to transform chemically competent E.coli DH5. mBio. doi: 10.1007/s10529-014-1576-4, Curran, K. A., Karim, A. S., Gupta, A., and Alper, H. S. (2013). Cell factory applications of the yeast Kluyveromyces marxianus for the biotechnological production of natural flavour and fragrance molecules. Biotechnol. Microbiol. Rev. Du C, Li Y, Zhao X, Pei X, Yuan W, Bai F, Jiang Y. Appl Microbiol Biotechnol. [8] The ploidy of K. marxianus was originally thought to be a haploid but recent research has shown that many strains used in research and industry are diploid. The site is secure. Nonetheless, the increase in basal expression as seen in the inulinase promoter due to a change in a stronger terminator is enough to halve fold-induction by inulin (Figure 3C). Bioeng. Biotechnol. 18, 116. Ascus: Evanescent. All data are normalised to cell number using OD, and are plotted as the mean s.d. In general, such yeasts have had niche applications in biotechnology but are being developed to be next-generation cell factories. 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). (2018). Recruitment and dissociation of nonhomologous end joining proteins at a DNA double-strand break in Saccharomyces cerevisiae. PLoS ONE 11:e156242. Microbiol. Complementary oligonucleotides comprising the gRNA sequence are designed with 5 and 3 overhangs (5-CGTC-3 and 5-AAAC-3,) on the sense and antisense oligonucleotides respectively, creating sticky ends to be ligated into pUCC001 cut by BsaI. whey, at 307C, pH 4.5, yeast cells were harvested, diluted in 0 . doi: 10.1038/s41598-018-25366-z, Lertwattanasakul, N., Kosaka, T., Hosoyama, A., Suzuki, Y., Rodrussamee, N., Matsutani, M., et al. AMB Express 8, 111. 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). Several constitutive and inducible promoters allow precise expression of a GoI under different conditions specific to K. marxianus, and terminator choice further fine-tunes gene expression. 3 Biotech. 2022 Aug 5;27(15):4981. doi: 10.3390/molecules27154981. Biosynthesis of 2-phenylethanol from glucose with genetically engineered Kluyveromyces marxianus. Two genes, LAC12 and LAC4, allow K. marxianus to absorb and use lactose as a carbon source. Obstacles to such development include inefficient and random native gene targeting (hindering the stable expression of integrated heterologous genes), limited knowledge of its biochemistry and genetics, and a lack of standardised regulatory parts and expression systems on the level of baker's yeast. Assembly of the construct was verified by colony PCR with ASR_K001F and ASR_K005R as primers and subsequent restriction mapping of plasmids from PCR-positive colonies with NotI. The lack of standardised parts, and efficient synthetic biology tools and strategies has limited the scope or sophistication of these efforts. ACS Synth. (1993). The curated and characterised tools we have provided in this kit will serve as a base to efficiently build next-generation cell factories from this alternative yeast. [2] Within this complex, two categories are defined based on the presence in certain taxa of a whole-genome duplication event: the two clades are referred to as pre-Whole Genome Duplication (WGD) and post-WGD. While the promoters we have characterized are largely selected from strain CBS6556, all but one of them had >90% sequence similarity with strain DMKU3-1042 and with strain NBRC1777, where the characterization was carried out. 2022 Sep 19;11(18):2910. doi: 10.3390/foods11182910. [citation needed] Treatment with amphotericin B have been effective against K. marxianus in one case report. Copyright 2019 Rajkumar, Varela, Juergens, Daran and Morrissey. doi: 10.1016/j.biortech.2017.07.116, Liu, J., Martin-Yken, H., Bigey, F., Dequin, S., Franois, J. M., and Capp, J. P. (2015). In the case of the latter, it is admittedly unclear if these extra copies are integrated sequentially at LAC4, or elsewhere in the genome. Epub 2019 Nov 28. Genome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9/gRNA co-expression plasmid. doi: 10.1128/mSphere.00385-16, Obst, U., Lu, T. K., and Sieber, V. (2017). A BsmBI site, and a BsaI site that after digestion will generate overhangs specific to that part type, flank each functional part. PLoS ONE 6:e16765. The contrasting low variation in YFP fluorescence seen in DNL4 and YKU80 mutants suggests that these backgrounds incorporate a single copy of the YFP cassette. [5] This species also ferments inulin, glucose, raffinose, sucrose and lactose into ethanol. [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. Annu. In spite of correct integration, wild-type transformants still had a wide spread of measured fluorescent intensities (Figure 5B). Keywords Kluyveromyces marxianus Saccharomyces cerevisiae ethanol productivity physiology Metabolic engineering a yeast to produce astaxanthin. The .gov means its official. 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. For insertion sites of the first type, we chose the LAC4 locus (I1); insertion there has a visible phenotype that does not affect core metabolism (as opposed to ADE2, for example). [11] This species has been associated with candidemia[12] and has been recovered from catheters. [5] Due to the heat tolerance of K. marxianus, high heat fermentations are feasible, reducing the costs normally expended for cooling as well as the potential for contamination by other fungi or bacteria. (2012). Fifty femto mole of the annealed gRNA insert is then used with 100 ng of pUCC001 in a Golden Gate reaction with BsaI. Genome sequence of the thermotolerant yeast Kluyveromyces marxianus var. In the present study, the kefir yeast Kluyveromyces marxianus 4G5 was used as host for the transformation of form I and form II RubisCO genes derived from the nonsulfur purple bacterium Rhodopseudomonas palustris using the Promoter-based Gene . official website and that any information you provide is encrypted Genome-wide prediction of CRISPR/Cas9 targets in Kluyveromyces marxianus and its application to obtain a stable haploid strain. is also in some commercial preparations of yeast to contribute flavor complexity. 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. To achieve chromosomal rearrangements in Kluyveromyces marxianus, the positions and sequences of centromeres were identified in this species for the first time. Furthermore, all white colonies of the NHEJ-deficient strains had the YFP cassette correctly integrated, as opposed to <20% for the wild-type strain (Table 3). Biochem. 2022 Nov;60:108027. doi: 10.1016/j.biotechadv.2022.108027. An ease of genetic manipulation, along with a wealth of genomic, genetic and biochemical knowledge, have made it able to produce a diverse range of compounds with S. cerevisiae from simple or economical feedstocks. [5][8] K. marxianus is widely used in industry because of its ability to use lactose. Technology valuation of cellulosic ethanol production by Kluyveromyces marxianus CCT 7735 based on the concepts of circular economy was investigated. Cernak P, Estrela R, Poddar S, Skerker JM, Cheng YF, Carlson AK, Chen B, Glynn VM, Furlan M, Ryan OW, Donnelly MK, Arkin AP, Taylor JW, Cate JHD. While thermotolerant, fast-growing and able to use various carbon and nitrogen sources, it broadly has the same nutritional requirements and culture techniques as S. cerevisiae. Please go there to see whether you have right to access the fulltext or not. All assembled part plasmids with their Addgene plasmid IDs are listed in Table S1, and primers to amplify the parts are listed in Table S2. (D) The effect of genetic context on expression levels of the same gene. These were the yeast strains Cyberlindnera saturnus C. sat 247 and C. sat CSa1 as well as Kluyveromyces marxianus K. mar 653. When using CRISPR/Cas9 to edit a site in the genome, a gRNA sequence targeting the gene of interest was first predicted using the sgRNA software (Xie et al., 2014). Epub 2022 Sep 3. Please enable it to take advantage of the complete set of features! 94, 396406. The genes involved in GABA catabolism (UGA1 and UGA2) and anabolism (GAD1) were found in K.. doi: 10.1007/s00253-017-8432-0, Emerson, C. H., and Bertuch, A.
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