ArticleOne-step transformation of the dimorphic yeast Yarrowia lipolyticaSeptember 1997Applied Microbiology and Biotechnology 48(2):232-5DOI:10.1007/s002530051043SourcePubMedAuthors: D C ChenD C ChenThis person is not on ResearchGate, or hasn t claimed this research yet. Jean-Marie BeckerichJean-Marie BeckerichThis person is not on ResearchGate, or hasn t claimed this research yet. Claude GaillardinFrench National Institute for Agriculture, Food, and Environment (INRAE) Request full-text PDFTo read the full-text of this research, you can request a copy directly from the authors.Request full-textDownload citation Copy link Link copied Request full-text Download citation Copy link Link copiedTo read the full-text of this research, you can request a copy directly from the authors.Citations (134)References (3)AbstractAn efficient one-step transformation method for the dimorphic yeast Yarrowia lipolytica is described. Using cells grown overnight on agar plates, the whole process is carried out within 1 h. The transformant clones could be recovered on selective plates as early as 36-48 h after plating. The efficiency was better than 10(5) transformants/micrograms replicative plasmid DNA. Effects of cell density, dithiothreitol, heat shock, poly(ethylene glycol) 4000 concentration and the wetness of selective plates were investigated. Discover the world s research20+ million members135+ million publications700k+ research projectsJoin for freeNo full-text available To read the full-text of this research, you can request a copy directly from the authors.Request full-text PDFCitations (134)References (3)... Transformation of C. phangngensis strains was based on the protocol developed by Chen, Beckerich and Gaillardin (1997) for Y. lipolytica with some modifications. Briefly, a lawn of cells was grown overnight (16-20 h) on solid YPD, and then yeast cells were dispersed in sterile water to OD 620 ≈ 25. ...... Transformation mix (0.1 mL) and plasmid DNA (2-4 μg) were added to the cell pellet and mixed well by vortexing. The final concentration of reagents in the transformation mix was identical to those described previously (Chen et al. 1997). Integration plasmids were linearized by overnight digestion with AlwNI or XbaI before transformation. ...Engineering Candida phangngensis - an oleaginous yeast from the Yarrowia clade - for enhanced detoxification of lignocellulose-derived inhibitors and lipid overproductionArticleSep 2018FEMS Yeast Res Josh QuartermanPatricia J. Slininger Ronald E. HectorBruce S. DienCandida phangngensis is an ascomycetous yeast and a phylogenetic relative of the industrial workhorse Yarrowia lipolytica. Here, we report that genetic tools already established for use in the latter organism-including promoters, expression vectors, antibiotic resistance genes, a transformation protocol, and the Cre/lox system for marker recycle-can be transferred to the newer member of the Yarrowia clade with little or no need for modifications. Using these tools, we engineered C. phangngensis for improved cellulosic lipid production by introducing two heterologous yeast genes. First, overexpression of Saccharomyces cerevisiae ADH6 enhanced in situ detoxification of aldehyde fermentation inhibitors that are generated during biomass pretreatment (e.g. furfural). Subsequently, Y. lipolytica DGA1 expression boosted lipid accumulation in C. phangngensis by pulling additional carbon flux into the triacylglycerol synthesis pathway. In acid-pretreated switchgrass hydrolysate cultures, the final engineered strain JQCP04 showed a 58% decrease in lag time and a 32% increase in lipid titer as compared to wild-type PT1-17. Furthermore, we expect that this study will generate new interest in the highly oleaginous yeast C. phangngensis, which is closely related to a safe, industrial species, and is shown here to be quite amenable for genetic manipulation.ViewShow abstract... It is made available under a The copyright holder for this preprint (which this version posted May 4, 2020. . https://doi.org/10.1101/2020.05.03.075259 doi: bioRxiv preprint All plasmids constructed were transformed into the Y. lipolytica host strain Po1g ΔLeu using the lithium acetate/single-strand carrier DNA/PEG method (Chen, Beckerich, Gaillardin, 1997). And single fresh Y. lipolytica colonies were picked from YNB selective plates and inoculated into YNB seed media, which were grown at 30 °C for 48 h. ...... Upon sequence verification by Genewiz, the restriction enzyme AvrII, NheI, NotI, ClaI and SalI (Fermentas, Thermo Fisher Scientific) were used to digest these vectors, and the donor DNA fragments were gel purified and assembled into the recipient vector containing previous pathway components in compliance with the YaliBricks subcloning protocol (Wong et al., 2017;Wong, Holdridge, Engel, Xu, 2019). All assembled plasmids were verified by gel digestion and were subsequently transformed into the Y. lipolytica host strain Po1g ΔLeu using the lithium acetate/single-strand carrier DNA/PEG method (Chen et al., 1997). In chromosomal integration process, pYLXP vector assembled with functional genes was linearized by restriction enzyme NotI (Fermentas, Thermo Fisher Scientific). ...Optimizing mevalonate pathway for squalene production in Yarrowia lipolyticaPreprintFull-text availableMay 2020Fang WangLi DengHuan Liu Peng XuSqualene is the gateway molecule for triterpene-based natural products and steroids-based pharmaceuticals. As a super lubricant, it has been used widely in health care industry due to its skin compatibility and thermostability. Squalene is traditionally sourced from shark-hunting or oil plant extraction, which is cost-prohibitive and not sustainable. Reconstitution of squalene biosynthetic pathway in microbial hosts is considered as a promising alternative for cost-efficient and scalable synthesis of squalene. In this work, we reported the engineering of the oleaginous yeast, Y. lipolytica, as a potential host for squalene production. We systematically identified the bottleneck of the pathway and discovered that the native HMG-CoA reductase led to the highest squalene improvement. With the recycling of NADPH from the mannitol cycle, the engineered strain produced about 180.3 mg/l and 188.2 mg/L squalene from glucose or acetate minimal media, respectively. By optimizing the C/N ratio, controlling the media pH and mitigating the acetyl-CoA flux competition from lipogenesis, the engineered strain produced about 502.7 mg/L squalene in shake flaks, a 28-fold increase compared to the parental strain (17.2 mg/L). We also profiled the metabolic byproducts citric acid and mannitol level and observed that they are reincorporated into cell metabolism at the late stage of fermentation. This work may serve as a baseline to harness Y. lipolytica as an oleaginous cell factory for production of squalene or terpene-based chemicals.ViewShow abstract... In addition to the homologous or heterologous gene of interest, targeting components can be optionally included into the ORF, in order to direct the resulting recombinant protein to precise intracellular organelles or to the secretion pathway, either for release into the cultivation medium (vesicular secretion) or for display on the cellular surface (surface display). When classical genetic engineering strategies are used, TUs are carried by either integrative or replicative shuttle vectors, built and propagated in Escherichia coli strains, that are then introduced into Y. lipolytica cells rendered competent using chemical treatments [43,162,210] or electroporation [211,212]. ...... In Y. lipolytica, the pre or prepro regions from either XPR2 or LIP2 genes, encoding the major secreted enzymes, have been used for that purpose since the 1980s, together with XPR2/LIP2 prepro hybrid sequences [19,20,42,247,248]. Up to now, the smaller and efficient XPR2 pre sequence [210] is generally a preferred choice and this secretion signal was selected for the pYLSC1 secretion vector from Yeastern YLEX commercial kit (cf. Section 2.3.2). ...Yarrowia lipolytica Strains and Their Biotechnological Applications: How Natural Biodiversity and Metabolic Engineering Could Contribute to Cell Factories ImprovementArticleFull-text availableJul 2021J. Fungi Catherine MadzakAmong non-conventional yeasts of industrial interest, the dimorphic oleaginous yeast Yarrowia lipolytica appears as one of the most attractive for a large range of white biotechnology applications, from heterologous proteins secretion to cell factories process development. The past, present and potential applications of wild-type, traditionally improved or genetically modified Yarrowia lipolytica strains will be resumed, together with the wide array of molecular tools now available to genetically engineer and metabolically remodel this yeast. The present review will also provide a detailed description of Yarrowia lipolytica strains and highlight the natural biodiversity of this yeast, a subject little touched upon in most previous reviews. This work intends to fill this gap by retracing the genealogy of the main Yarrowia lipolytica strains of industrial interest, by illustrating the search for new genetic backgrounds and by providing data about the main publicly available strains in yeast collections worldwide. At last, it will focus on exemplifying how advances in engineering tools can leverage a better biotechnological exploitation of the natural biodiversity of Yarrowia lipolytica and of other yeasts from the Yarrowia clade.ViewShow abstract... The component cells were mixed with more than 1 μg of DNA fragments and electroporated using a BTX ECM830 Electroporator (Genetronics, San Diego, CA, USA) with the conditions of 800 V, 1000 Ω, and 25 μF. The one-step lithium acetate transformation method described by Chen et al. (1997) was used for the transformation of Y. lipolytica. In brief, 4 mL of stationary cells was harvested and mixed with 100-μL T-buffer (90 μL of 50% polyethylene glycol 3350, 5 μL of 2 M lithium acetate, 5 μL of 2 M DLdithiothreitol), 2 μL of dimethyl sulfoxide, 5 μL salmon sperm DNA, 10 μL DNA fragments, and then incubated at 30°C for 1 h. ...... The yeast Y. lipolytica has been used as a platform organism for production of lipids and non-native chemicals in recent years (Xu et al. 2016Darvishi et al. 2018). Because linear DNAs are generally more easily inserted into the genome than circle DNAs (Chen et al. 1997;Guo et al. 2018), a circle DNA vector containing Cre and a URA3 gene was constructed and used to test the Cre-loxP-based method in Y. lipolytica (Supplemental Fig. S3). The expression of Cre on the circle DNA vector successfully induced the removal of the URA3 gene flanked by two loxP sites, but the circle DNA vectors were not lost during cell reproduction. ...Selectable marker recycling in the nonconventional yeast Xanthophyllomyces dendrorhous by transient expression of Cre on a genetically unstable vectorArticleFull-text availableJan 2019Appl Microbiol BiotechnolNing ZhangJiaxin Li Fuli Li Shi-An WangSelectable marker recycling is a basic technique in bioengineering. However, this technique is usually unavailable in non-model microorganisms. In this study, we proposed a simple and efficient method for selectable marker recycling in the astaxanthin-synthesizing yeast Xanthophyllomyces dendrorhous. This method was based on a Cre-loxP system, in which the transient expression of the Cre recombinase was controlled by a genetically unstable vector independent of episomal plasmids and inducible promoters. The selectable markers in single-gene locus and multigene loci were removed along with the loss of the Cre vector with a ratio of 100% and 29%, respectively. The significance of the method was highlighted by the finding that stable autotrophic mutants were not readily obtained in X. dendrorhous. Comparative studies in X. dendrorhous and the non-homologous end joining dominant yeast Yarrowia lipolytica suggested that the method could be universally used in homologous recombination dominant yeasts.ViewShow abstract... The above mentioned plasmids pSWV-β-amylase, pSWV-GTase, pSWV-βa-GT and pSWV-GT-βa were digested with EcoR I to produce the linear fragments. Referring to the one-step transformation method based on lithium acetate reported by Chen et al. [16] , the linear fragments were used to transform Y. lipolytica. Y. lipolytica CGMCC7326 was activated and cultured on the plate at 30 ℃ for one night. ...One step production of isomalto-oligosaccharides by engineered Yarrowia lipolytica yeast co-displayed β-amylase and α-transglucosidaseArticleFull-text availableJan 2019Dawen Liu Hairong ChengZixin DengIsomalto-oligosaccharides (IMO) have good physiochemical properties and excellent physiological functions to make it widely used in food, medicine, feed, cosmetics and other industries. However, the procedures for industrial production of IMO are complicated. Therefore, it is necessary to develop an economical and easy-to-operate method. The genes encoding for β-amylase and α-transglucosidase were fused and co-displayed on the yeast cell surface of Yarrowia lipolytica which can convert liquefied starch to IMO in one step. The highest IMO purity of 75.3% was obtained using the displayed fusion-enzyme at 50 °C. This method showed potential application in IMO production.ViewShow abstract... Different previously characterized intergenic loci in Y. lipolytica were used to integrate yeast vectors into the genome of the parent strain, as described in Holkenbrink et al. [39]. To perform DNA transformation into Y. lipolytica, the integrative vectors were linearized with FastDigest NotI (Thermo Fisher Scientific, Waltham, MA, USA) and transformed into Y. lipolytica using a lithium-acetate protocol [40]. The transformants were selected on YPD + Hygromycin/Nourseothricin or SC-Ura plates. ...Enhancement of Astaxanthin Biosynthesis in Oleaginous Yeast Yarrowia lipolytica via Microalgal PathwayArticleFull-text availableOct 2019 Larissa TramontinKanchana R. KildegaardSuresh Sudarsan Irina BorodinaAstaxanthin is a high-value red pigment and antioxidant used by pharmaceutical, cosmetics, and food industries. The astaxanthin produced chemically is costly and is not approved for human consumption due to the presence of by-products. The astaxanthin production by natural microalgae requires large open areas and specialized equipment, the process takes a long time, and results in low titers. Recombinant microbial cell factories can be engineered to produce astaxanthin by fermentation in standard equipment. In this work, an oleaginous yeast Yarrowia lipolytica was engineered to produce astaxanthin at high titers in submerged fermentation. First, a platform strain was created with an optimised pathway towards β-carotene. The platform strain produced 331 ± 66 mg/L of β-carotene in small-scale cultivation, with the cellular content of 2.25% of dry cell weight. Next, the genes encoding β-ketolase and β-hydroxylase of bacterial (Paracoccus sp. and Pantoea ananatis) and algal (Haematococcus pluvialis) origins were introduced into the platform strain in different copy numbers. The resulting strains were screened for astaxanthin production, and the best strain, containing algal β-ketolase and β-hydroxylase, resulted in astaxanthin titer of 44 ± 1 mg/L. The same strain was cultivated in controlled bioreactors, and a titer of 285 ± 19 mg/L of astaxanthin was obtained after seven days of fermentation on complex medium with glucose. Our study shows the potential of Y. lipolytica as the cell factory for astaxanthin production.ViewShow abstract... All expression plasmids were linearized by MluI and used to transform Po1g according to the method that has been described by Chen et al. (1992Chen et al. ( , 1997. After incubation on MD solid medium about 48 h at 28 ℃, transformants were further screened on BMSY medium plates supplemented with 10 mL/L glyceryl tributyrate to form clear transparent circle. ...Overexpression of GRAS Rhizomucor miehei Lipase in Yarrowia lipolytica via Optimizing Promoter, Gene Dosage and Fermentation ParametersArticleDec 2019J BIOTECHNOL Qinghua Zhou Liangcheng JiaoYangge Qiao Yan YunjunView... The Yarrowia lipolytica nukmΔ deletion strain used in this study was described previously [26]. All point mutations were generated in E. coli by PCR mutagenesis and then used for transformation of Y. lipolytica strain nukmΔ employing the one step transformation method as described [27]. After transformation into nukmΔ, plasmids were recovered, and the entire open reading frames were re-sequenced to verify the introduced mutations and to exclude others changes in the sequence. ...Mutations in a conserved loop in the PSST subunit of respiratory complex I affect ubiquinone binding and dynamicsArticleFull-text availableJun 2019BBA-BIOENERGETICSOuti Haapanen Ilka Wittig Volker Zickermann Etienne Galemou YogaRespiratory complex I catalyses the reduction of ubiquinone (Q) from NADH coupled to proton pumping across the inner membrane of mitochondria. The electrical charging of the inner mitochondrial membrane drives the synthesis of ATP, which is used to power biochemical reactions of the cell. The recent surge in structural data on complex I from bacteria and mitochondria have contributed to significant understanding of its molecular architecture. However, despite these accomplishments, the role of various subdomains in redox-coupled proton pumping remains entirely unclear. In this work, we have mutated conserved residues in the loop of the PSST subunit that faces the ~30 Å long unique Q-binding tunnel of respiratory complex I. The data show a drastic decrease in Q reductase activity upon mutating several residues despite full assembly of the complex. In-silico modeling and multiple microsecond long molecular dynamics simulations of wild-type and enzyme variants with exchanges of conserved arginine residues revealed remarkable ejection of the bound Q from the site near terminal electron donor N2. Based on experiments and long-time scale molecular simulations, we identify microscopic elements that dynamically control the diffusion of Q and are central to redox-coupled proton pumping in respiratory complex I.ViewShow abstract... Without carrier DNA, this protocol results in less than 20 transformants (Davidow et al., 1985). Later studies have optimized this method by developing a onestep protocol where cells are directly prepared from a YPD plate (Chen et al., 1997), increasing site specific integration by using a strain with a zeta docking platform (Bordes et al., 2007), and adapting this protocol for high-throughput transformation in a 96-well plate (Leplat et al., 2015). Each of these methods reports maximum transformation efficiencies generally ranging from 10 3 -10 4 transformants per µg DNA, but suffer from limited efficiency for site-directed integration due to Y. lipolytica s natural propensity for NHEJ. ...Metabolic engineering in the host Yarrowia lipolyticaArticleJul 2018METAB ENG Ahmad Abdel-MawgoudKelly A. MarkhamClaire M. PalmerHal S. AlperThe nonconventional, oleaginous yeast, Yarrowia lipolytica is rapidly emerging as a valuable host for the production of a variety of both lipid and nonlipid chemical products. While the unique genetics of this organism pose some challenges, many new metabolic engineering tools have emerged to facilitate improved genetic manipulation in this host. This review establishes a case for Y. lipolytica as a premier metabolic engineering host based on innate metabolic capacity, emerging synthetic tools, and engineering examples. The metabolism underlying the lipid accumulation phenotype of this yeast as well as high flux through acyl-CoA precursors and the TCA cycle provide a favorable metabolic environment for expression of relevant heterologous pathways. These properties allow Y. lipolytica to be successfully engineered for the production of both native and nonnative lipid, organic acid, sugar and acetyl-CoA derived products. Finally, this host has unique metabolic pathways enabling growth on a wide range of carbon sources, including waste products. The expansion of carbon sources, together with the improvement of tools as highlighted here, have allowed this nonconventional organism to act as a cellular factory for valuable chemicals and fuels.ViewShow abstract... been described in the transformation protocol of Chen, Beckerich and Gaillardin (1997). While DSM-21 175 grows exclusively in the yeast-like form, DSM-3286 predominantly forms hyphae. ...Golden Gate-based metabolic engineering strategy for wild-type strains of Yarrowia lipolyticaArticleFeb 2019FEMS Microbiol Lett Michael EgermeierMichael Sauer Hans MarxThe yeast Yarrowia lipolytica represents a future microbial cell factory for numerous applications in a bio-based economy. Outstanding feature of this yeast is the metabolic flexibility in utilising various substrates (sugars, fatty acids, glycerol, etc.). The potential of wild-type isolates of Y. lipolytica to convert glycerol into various value-added compounds is attracting attention of academia and industry. However, the already established tools for efficient engineering of the metabolism of Y. lipolytica are often dependent on genetic features like auxotrophic markers. With the present work we want to introduce a new set of vectors for metabolic engineering strategies, including CRISPR/Cas9 technology. The system is based on GoldenMOCS, a recently established rapid Golden Gate cloning strategy applicable in multiple organisms. We could show that our new GoldenMOCS plasmids are suitable for the extrachromosomal overexpression of the gene glycerol kinase (GUT1) in wild-type isolates of Y. lipolytica resulting in enhanced conversion of glycerol to erythritol and citric acid. Moreover, a GoldenMOCS plasmid for CRISPR/Cas9 mediated genome editing has been designed, which facilitates single gene knock-outs with efficiencies between 6% and 25% in strains with genetic wild-type background.ViewShow abstract... One of the more recent, optimized protocols achieves up to 2.2 × 10 3 transformants per μg DNA, but requires large quantities of carrier DNA (Leplat, Nicaud and Rossignol 2015). This dependence on carrier DNA is similar to previously reported chemical transformation methods in this host (Davidow et al. 1985;Barth and Gaillardin 1996;Chen, Beckerich and Gaillardin 1997). Specifically, in the absence of carrier DNA, chemical transformation of Y. lipolytica is barely effective with reported efficiencies of 16 transformants per μg DNA (Davidow et al. 1985) and 20 to 200 transformants per μg DNA (Nicaud, Gaillardin and Pignede 2003). ...High efficiency transformation of Yarrowia lipolytica using electroporationArticleJul 2018FEMS Yeast ResSofia VazquezKelly A. MarkhamHal S. AlperYarrowia lipolytica is an industrial host organism with incredible potential for metabolic engineering. However, the genetic tools and capacities in this host lag behind those of conventional counterparts. In this study, we sought to increase the transformation efficiency of Y. lipolytica by creating a simple protocol using electroporation. Efficiency was increased by optimizing wash buffers, pre-culture growth time, OD600 of competent cells, voltage, competent cell volume, DNA concentration, and recovery time. The outcome of these optimizations led to a simple protocol with maximum linear fragment transformation efficiency of 1.6 × 104 transformants per μg DNA and 2.8 × 104 transformants per μg DNA for episomal plasmid transformation. The protocol presented here is superior to other Y. lipolytica transformation protocols as it requires no lengthy pretreatment and no required carrier DNA to achieve efficiencies on par with, or exceeding, previously reported methods.ViewShow abstract... To screen strains F1 to F5 (screening for high α-farnesene production), we randomly selected about 20-30 transformants from each library, extracted the genomic DNA, and confirmed that every overexpressed gene was inserted into the genome by PCR. DNA fragments were transformed into Y. lipolytica using the lithium acetate method [45]. ...Engineering the oleaginous yeast Yarrowia lipolytica for production of α-farneseneArticleFull-text availableDec 2019BIOTECHNOL BIOFUELSYinghang LiuXin Jiang Zhiyong CuiJin HouBackground: Yarrowia lipolytica, a non-traditional oil yeast, has been widely used as a platform for lipid production. However, the production of other chemicals such as terpenoids in engineered Y. lipolytica is still low. α-Farnesene, a sesquiterpene, can be used in medicine, bioenergy and other fields, and has very high economic value. Here, we used α-farnesene as an example to explore the potential of Y. lipolytica for terpenoid production.Results: We constructed libraries of strains overexpressing mevalonate pathway and α-farnesene synthase genes by non-homologous end-joining (NHEJ) mediated integration into the Y. lipolytica chromosome. First, a mevalonate overproduction strain was selected by overexpressing relevant genes and changing the cofactor specificity. Based on this strain, the downstream α-farnesene synthesis pathway was overexpressed by iterative integration. Culture conditions were also optimized. A strain that produced 25.55 g/L α-farnesene was obtained. This is the highest terpenoid titer reported in Y. lipolytica.Conclusions: Yarrowia lipolytica is a potentially valuable species for terpenoid production, and NHEJ-mediated modular integration is effective for expression library construction and screening of high-producer strains.ViewShow abstract... All plasmids constructed were transformed into the Y. lipolytica host strain Po1g ΔLeu using the lithium acetate/ single-strand carrier DNA/PEG method (Chen et al. 1997). Single colonies were picked for culturing and subsequently harvested the supernatant from flask culture at various time points to perform the luciferase assay using the Nano-Glo Luciferase Assay System kit from Promega (catalog number N1120). ...Understanding lipogenesis by dynamically profiling transcriptional activity of lipogenic promoters in Yarrowia lipolyticaArticleFull-text availableApr 2019Appl Microbiol BiotechnolHuan LiuMonireh MarsafariLi Deng Peng XuLipogenesis is a complicated process involving global transcriptional reprogramming of lipogenic pathways. It is commonly believed that nitrogen starvation triggers a metabolic shift that reroutes carbon flux from Krebs cycles to lipogenesis. In this study, we systematically surveyed and dynamically profiled the transcriptional activity of 22 lipogenic promoters aiming to delineate a picture how nitrogen starvation regulates lipogenesis in Y. lipolytica. These lipogenic promoters drive the expression of critical pathways that are responsible for the generation of reducing equivalents (NADPH), carbon backbones (acetyl-CoA, malonyl-CoA, DHAP, etc.), synthesis and degradation of fatty acids. Specifically, our investigated promoters span across an array of metabolic pathways, including glycolysis, Krebs cycle, pentose phosphate pathway, mannitol cycle, glutamine–GABA cycle, fatty acid and lipid synthesis, glyoxylate, β-oxidation, and POM (pyruvate–oxaloacetate–malate) cycle. Our work provides evidences that mannitol cycle, glutamine–GABA cycle and amino acid degradation, pyruvate oxidation, and acetate assimilation pathways are lipogenesis-related steps involved in generating cytosolic NADPH and acetyl-CoA precursors. This systematic investigation and dynamic profiling of lipogenic promoters may help us better understand lipogenesis, facilitate the formulation of structure-based kinetic models, as well as develop efficient cell factories for fuels and chemical production in oleaginous species.ViewShow abstract... All plasmids and primers used in this study are listed in Table 1 and Table 2. Y. lipolytica Po1f genomes were extracted by the TIANamp Yeast DNA kit (Tiangen, Beijing, China). Transformation of Y. lipolytica was performed using the lithium acetate method (Chen et al. 1997). ...Enhanced itaconic acid production in Yarrowia lipolytica via heterologous expression of a mitochondrial transporter MTTArticleFull-text availableMar 2019Appl Microbiol Biotechnol Chen Zhao Zhao XiangyingJianjun Liu Zhiyong CuiItaconic acid, a promising platform chemical, has been applied in many fields of industrial production. As a potential candidate for itaconic acid production, Yarrowia lipolytica possesses several innate abilities such as the tolerance of low-pH and high-shear stress, fast growth rate, cultivation flexibility, and easy for genetic manipulation. Here, Y. lipolytica Po1f which was tested to show high tolerance to itaconic acid could accumulate itaconic acid (0.363 g/L) by expressing the Aspergillus terreus cis-aconitic acid decarboxylase (CAD). Then, we tried to improve the supply and transport of the immediate precursor cis-aconitic acid by overexpressing a series of genes; these results indicate that overexpression of mitochondrial cis-aconitate transporter MTT is beneficial to the itaconic acid biosynthesis in Y. lipolytica. Further culture optimization enabled 22.03 g/L of itaconic acid to be produced in bioreactors, about 60-fold improvement over the initial titer, which is the highest itaconic acid production achieved at low pH by yeast reported worldwide, to data. This study demonstrates the great potential of Y. lipolytica as an industrial platform for itaconic acid production.ViewShow abstract... Standard recombinant DNA and molecular cloning techniques were used (Silhavy et al., 1984;Ausubel et al., 1988;Sambrook et al., 1989;Amberg et al., 2005). Transformation of Y. lipolytica was performed according to the method of Chen et al. (1997). ...Engineering Two Species of Yeast as Cell Factories for 2’-FucosyllactoseArticleFull-text availableDec 2018METAB ENGKerry HollandsChristopher M. BaronKatharine J. GibsonSteven Cary RothmanOligosaccharides present in human breast milk have been linked to beneficial effects on infant health. Inclusion of these human milk oligosaccharides (HMOs) in infant formula can recapitulate these health benefits. As a result, there is substantial commercial interest in a cost-effective source of HMOs as infant formula ingredients. Here we demonstrate that the yeast species Saccharomyces cerevisiae and Yarrowia lipolytica both can be engineered to produce 2′-fucosyllactose (2′FL), which is the most abundant oligosaccharide in human breast milk, at high titer and productivity. Both yeast species were modified to enable uptake of lactose and synthesis of GDP-fucose – the two precursors of 2′FL – by installing a lactose transporter and enzymes that convert GDP-mannose to GDP-fucose. Production of 2′FL was then enabled by expression of α-1,2-fucosyltransferases from various organisms. By screening candidate transporters from a variety of sources, we identified transporters capable of exporting 2′FL from yeast, which is a key consideration for any biocatalyst for 2′FL production. In particular, we identified CDT2 from Neurospora crassa as a promising target for further engineering to improve 2′FL efflux. Finally, we demonstrated production of 2′FL in fermenters at rates and titers that indicate the potential of engineered S. cerevisiae and Y. lipolytica strains for commercial 2′FL production.ViewShow abstract... Y. lipolytica strain nukmΔ (nukm::LEU2, nugm-Htg2, ndh2i, ura3 − , lys11 − ) 26 lacking the entire nuclear reading frame encoding subunit PSST was transformed with replicative plasmid pUB4 containing either wild-type or site-directed mutant copies of the NUKM gene under the control of its endogenous promoter. Transformation of Y. lipolytica was performed according to the one-step protocol described by Chen et al. 27 . Briefly, 500 µl of an overnight YPD (1% yeast extract, 2% bacto-peptone, and 2% dextrose) culture of nukmΔ cells were spun down, washed with deionized water and resuspended in 100 μl one-step buffer (45%(w/v) PEG 4000, 0.1 M lithium acetate, 0.1 M DTT, 0.25 mg ml −1 ssDNA). ...Reversible decoupling of the proton pumps of mitochondrial complex I by fixing a loop in the ubiquinone reduction pocketArticleSep 2018BBA-BIOENERGETICS Alfredo Cabrera-Orefice Etienne Galemou Yoga Christophe WirthUlrich BrandtView... Yeast genomes were extracted by TIANamp Yeast DNA Kit (TIANGEN, Beijing, China). Transformation of Y. lipolytica was performed using the the Lithium Acetate Method (Chen et al., 1997), and the transformants were selected in YNBG, YNBG-ura, YNBG-casamino acids or YNBG-hyg plates. ...Exploring succinic acid production by engineered Yarrowia lipolytica strains using glucose at low pHArticleFull-text availableAug 2018BIOCHEM ENG JQinglin Yu Zhiyong CuiYaqin Zheng Cuijuan GaoSuccinic acid is an important platform chemical that can be used to produce a broad range of compounds with extensive applications. By applying rational metabolic engineering, we had previously developed a series of innovative succinic acid-producing Yarrowia lipolytica strains that could produce succinic acid effectively using glycerol. In the present study, glucose was used for succinic acid production by these engineered strains, without pH control. Strain PGC11505, in which the gene (YlACH1) of acetyl-CoA hydrolase corresponding for acetic acid overflow was deleted, exhibited the ability to utilize glucose for succinic acid production efficiently. Strain PGC202, with a strengthened reductive carboxylation and oxidative tricarboxylic acid cycle by overexpression of ScPCK and endogenous YlSCS2, yielded 23.8 g/L succinic acid. After fermentation optimization, the succinic acid titer reached to 32.6 g/L in flask cultures. In a bench-top fermenter, this strain produced 53.6 g/L succinic acid with a yield of 0.61 g/g-glucose without pH control. In conclusion, through rational metabolic engineering of Y. lipolytica, we have successfully achieved the high-yield production of succinic acid using glucose as the sole carbon source under natural low pH condition. This new Y. lipolytica strain would be highly useful in industrial processes for the production of succinic acid using low-cost glucose.ViewShow abstract... Y. lipolytica strain nukmΔ (nukm::LEU2, nugm-Htg2, ndh2i, ura3 − , lys11 − ) 26 lacking the entire nuclear reading frame encoding subunit PSST was transformed with replicative plasmid pUB4 containing either wild-type or site-directed mutant copies of the NUKM gene under the control of its endogenous promoter. Transformation of Y. lipolytica was performed according to the one-step protocol described by Chen et al. 27 . Briefly, 500 µl of an overnight YPD (1% yeast extract, 2% bacto-peptone, and 2% dextrose) culture of nukmΔ cells were spun down, washed with deionized water and resuspended in 100 μl one-step buffer (45%(w/v) PEG 4000, 0.1 M lithium acetate, 0.1 M DTT, 0.25 mg ml −1 ssDNA). ...Locking loop movement in the ubiquinone pocket of complex I disengages the proton pumpsArticleFull-text availableOct 2018 Alfredo Cabrera-Orefice Etienne Galemou Yoga Christophe WirthUlrich BrandtComplex I (proton-pumping NADH:ubiquinone oxidoreductase) is the largest enzyme of the mitochondrial respiratory chain and a significant source of reactive oxygen species (ROS). We hypothesized that during energy conversion by complex I, electron transfer onto ubi-quinone triggers the concerted rearrangement of three protein loops of subunits ND1, ND3, and 49-kDa thereby generating the power-stroke driving proton pumping. Here we show that fixing loop TMH1-2 ND3 to the nearby subunit PSST via a disulfide bridge introduced by site-directed mutagenesis reversibly disengages proton pumping without impairing ubiquinone reduction, inhibitor binding or the Active/Deactive transition. The X-ray structure of mutant complex I indicates that the disulfide bridge immobilizes but does not displace the tip of loop TMH1-2 ND3. We conclude that movement of loop TMH1-2 ND3 located at the ubiquinone-binding pocket is required to drive proton pumping corroborating one of the central predictions of our model for the mechanism of energy conversion by complex I proposed earlier.ViewShow abstract... All yeast strains were transformed according to a modification of the method described by Lin-Cereghino et al. (2005), except K. marxianus which was transformed according to a modification of the method of Chen et al. (1997). All yeasts were transformed with all of the vectors described in the previous section. ...Heterologous coexpression of the benzoate-para-hydroxylase CYP53B1 with different cytochrome P450 reductases in various yeastsArticleFull-text availableOct 2018 Chrispian W. Theron Michel Labuschagne Jacobus Albertyn Martie SmitCytochrome P450 monooxygenases (P450) are enzymes with high potential as biocatalysts for industrial applications. Their large‐scale applications are, however, limited by instability and requirement for coproteins and/or expensive cofactors. These problems are largely overcome when whole cells are used as biocatalysts. We previously screened various yeast species heterologously expressing self‐sufficient P450s for their potential as whole‐cell biocatalysts. Most P450s are, however, not self‐sufficient and consist of two or three protein component systems. Therefore, in the present study, we screened different yeast species for coexpression of P450 and P450‐reductase (CPR) partners, using CYP53B1 from Rhodotorula minuta as an exemplary P450. The abilities of three different coexpressed CPR partners to support P450 activity were investigated, two from basidiomycetous origin and one from an ascomycete. The various P450‐CPR combinations were cloned into strains of Saccharomyces cerevisiae, Kluyveromyces marxianus, Hansenula polymorpha, Yarrowia lipolytica and Arxula adeninivorans, using a broad‐range yeast expression vector. The results obtained supported the previous finding that recombinant A. adeninivorans strains perform excellently as whole‐cell biocatalysts. This study also demonstrated for the first time the P450 reductase activity of the CPRs from R. minuta and U. maydis. A very interesting observation was the variation in the supportive activity provided by the different reductase partners tested and demonstrated better P450 activity enhancement by a heterologous CPR compared to its natural partner CPR. This study highlights reductase selection as a critical variable for consideration in the pursuit of optimal P450‐based catalytic systems. The usefulness of A. adeninivorans as both a host for recombinant P450s and whole‐cell biocatalyst was emphasized, supporting earlier findings.ViewShow abstract... All the integrative vectors together with gRNA vectors are listed in Supplementary Table 2. Plasmids and BioBricks were transformed into parental strains by standard lithium acetate protocol (Chen et al., 1997) described in detail elsewhere (Holkenbrink et al., 2017). All the integration vectors were linearized with endonuclease NotI (Thermo Fisher Scientific) and gel purified for yeast transformation. ...Engineering Oleaginous Yeast as the Host for Fermentative Succinic Acid Production From GlucoseArticleFull-text availableNov 2019Mahsa Babaei Aligholi NiaeiKanchana R. Kildegaard Irina BorodinaOleaginous yeast Yarrowia lipolytica is a prospective host for production of succinic acid. The interruption of tricarboxylic acid cycle through succinate dehydrogenase gene (SDH) deletion was reported to result in strains incapable of glucose utilization and this ability had to be restored by chemical mutation or long adaptive laboratory evolution. In this study, a succinate producing strain of Y. lipolytica was engineered by truncating the promoter of SDH1 gene, which resulted in 77% reduction in SDH activity but did not impair the ability of the strain to grow on glucose. The flux toward succinic acid was further improved by overexpressing the genes in the glyoxylate pathway and the oxidative TCA branch, and expressing phosphoenolpyruvate carboxykinase from Actinobacillus succinogenes. A short adaptation on glucose reduced the lag phase of the strain and increased its tolerance to high glucose concentrations. The resulting strain produced 7.8 ± 0.0 g/L succinic acid with a yield of 0.105 g/g glucose in shake flasks without pH control, while mannitol (11.8 ± 0.8 g/L) was the main by-product. Further investigations showed that mannitol accumulation was caused by low pH stress and buffering the fermentation medium eliminated mannitol formation. In a fed-batch bioreactor in mineral medium at pH 5, at which point according to Ka values of succinic acid, the major fraction of product was in acidic form rather than dissociated form, the strain produced 35.3 ± 1.5 g/L succinic acid with 0.26 ± 0.00 g/g glucose yield.ViewShow abstract... 18 µL of desired DNA and 92 µL of transformation mix (80 μL 60% PEG4000, 5 µL 2 M DTT, 5 µL 2 M lithium acetate pH 6, and 2 µL 10 mg/mL single stranded salmon sperm DNA) were added to the cell pellet. The transformation reaction was mixed by vortexing and heat shocked at 39 °C for 1 h [50]. Cells were centrifuged, the supernatant was discarded, and cells were resuspended in 1 mL of a suitable non-selective medium, transferred to culture tubes, and cultured overnight at 30 °C before plating on selective media. ...High-oleate yeast oil without polyunsaturated fatty acidsArticleFull-text availableMay 2018BIOTECHNOL BIOFUELS Vasiliki TsakraklidesAnnapurna KamineniAndrew L. ConsiglioElena E. BrevnovaBackgroundOleate-enriched triacylglycerides are well-suited for lubricant applications that require high oxidative stability. Fatty acid carbon chain length and degree of desaturation are key determinants of triacylglyceride properties and the ability to manipulate fatty acid composition in living organisms is critical to developing a source of bio-based oil tailored to meet specific application requirements. ResultsWe sought to engineer the oleaginous yeast Yarrowia lipolytica for production of high-oleate triacylglyceride oil. We studied the effect of deletions and overexpressions in the fatty acid and triacylglyceride synthesis pathways to identify modifications that increase oleate levels. Oleic acid accumulation in triacylglycerides was promoted by exchanging the native ∆9 fatty acid desaturase and glycerol-3-phosphate acyltransferase with heterologous enzymes, as well as deletion of the Δ12 fatty acid desaturase and expression of a fatty acid elongase. By combining these engineering steps, we eliminated polyunsaturated fatty acids and created a Y. lipolytica strain that accumulates triglycerides with 90% oleate content. ConclusionsHigh-oleate content and lack of polyunsaturates distinguish this triacylglyceride oil from plant and algal derived oils. Its composition renders the oil suitable for applications that require high oxidative stability and further demonstrates the potential of Y. lipolytica as a producer of tailored lipid profiles.ViewShow abstract... The spheroplast, lithium cation, and electroporation transformation methods have been successfully applied to a number of different yeast species. These include Hansenula polymorpha (34,48,102,116), Klyveromyces spp (7,30,34,61), Yamadazyma ohmeri (94), Yarrowia lipolytica (21), and Schwanniomyces occidentalis (26,34,76). ...Genetic Transformation of YeastArticleFull-text availableApr 2001BIOTECHNIQUES R. Daniel GietzRobin A. WoodsView... Transformation of Yarrowia lipolytica was performed using a lithium-acetate based heat shock method previously described by [16]. Briefly, the strain was plated on YPD agar plates and grown at 30˚C for 18h. ...Deletion of MHY1 abolishes hyphae formation in Yarrowia lipolytica without negative effects on stress toleranceArticleFull-text availableApr 2020PLOS ONE Oliver Konzock Joakim NorbeckThere is a need for development of sustainable production processes for production of fats/oils and lipid derived chemicals. The dimorphic oleaginous yeast Yarrowia lipolytica is a promising organism for conversion of biomass hydrolysate to lipids, but in many such processes hyphae formation will be problematic. We have therefore constructed and compared the performance of strains carrying deletions in several published gene targets suggested to abolish hyphae formation (MHY1, HOY1 and CLA4). The MHY1-deletion was the only of the tested strains which did not exhibit hyphae formation under any of the conditions tested. The MHY1-deletion also had a weak positive effect on lipid accumulation without affecting the total fatty acid composition, irrespective of the nitrogen source used. MHY1 has been suggested to constitute a functional homolog of the stress responsive transcription factors MSN2/4 in Saccharomyces cerevisiae, the deletion of which are highly stress sensitive. However, the deletion of MHY1 displayed only minor difference on survival of a range of acute or long term stress and starvation conditions. We conclude that the deletion of MHY1 in Y.lipolytica is a reliable way of abolishing hyphae formation with few detectable negative side effects regarding growth, stress tolerance and lipid accumulation and composition.ViewShow abstract... Finally, glutamyl-1semialdehyde aminotransferase (GSA-AM, encoding by HemL gene) catalyzes glutamyl-1-semialdehyde to produce 5-ALA. [26], which will be randomly integrated into the genome through endogenous non-homologous end joining (NHEJ) repair. Positive transformants were selected on appropriate plates and checked by colony PCR. 10 transformants were randomly picked, and the engineered strains with high 5-ALA production were screened through shaking flasks fermentation. ...Efficient 5-aminolevulinic acid production through reconstructing the metabolic pathway in SDH-deficient Yarrowia lipolyticaArticleFull-text availableJul 2021BIOCHEM ENG J Jinhong Zhang Zhiyong CuiZiwei Zhu Qingsheng Qi5-Aminolevulinic acid (5-ALA) is a precursor for the biosynthesis of tetrapyrrole compounds such as chlorophyll a, heme, and vitamin B12, which has been widely used in the fields of agriculture and medicine. 5-ALA can be biosynthesized through C4 pathway that use succinyl-CoA and glycine as precursor, or through C5 pathway that use glutamate as precursor. Yarrowia lipolytica is an important unconventional yeast and has wide applications in metabolic engineering and industrial biotechnology. It possesses strong metabolic flux through tricarboxylic acid cycle, and prominent acid tolerance, which make it an attractive chassis cell for 5-ALA fermentation. However, there are no reports on the 5-ALA production in Y. lipolytica so far. In this study, a series of 5-ALA producing engineered strains were constructed by using succinate dehydrogenase (SDH)-deficient Y. lipolytica. The related genes of C4 and C5 pathway were co-expressed, and the best engineered strain PGC62-IAL (MatA, xpr2-322, axp-2, leu2-270, ura3-302, ΔSdh5::loxP, ΔAch1::loxP, ScPck, ScHemⅠ, StHemA, EcHemL) for efficient ALA production was obtained. Through optimizing culture conditions, the titer of 5-ALA was 1050 mg/L in shake flask fermentation with glycerol as carbon source. Finally, the 5-ALA titer reached 2216.8 mg/L in the fed-batch fermentation, which is the highest 5-ALA titer achieved using yeast globally. Due to accumulation of the by-products including succinic acid and porphyrin compounds, the 5-ALA yield only reached 0.024 g/g glucose. Nevertheless, Y. lipolytica was engineered to produce 5-ALA for the first time, and indicated that SDH-deficient strains can be used as platform for efficient 5-ALA production.ViewShow abstract... Yeast genomic DNA used for diagnostic PCR reactions was isolated by using the SDS/LiAc protocol [314]. Y. lipolytica transformation was performed with the LiAc method as previously described [480]. Four to eight colonies were re-streaked on selective medium to select for single clones and diagnostic PCRs were performed to verify the correct genotypes. ...Engineering of vitamin and cofactor synthesis in yeastsThesisJul 2021 Thomas PerliInspired by previous research demonstrating how adaptivelaboratory evolution and metabolic engineering could successfully eliminate vitamin B7 dependency and enable biosynthesis of tetrahydrobiopterin to functionally express an opioid producing pathway in S. cerevisiae, the goals of the present study were two-fold: i) investigating whether vitamin prototrophy of S. cerevisiae for all seven class-B vitamins could be achieved, and ii) whether S. cerevisiae could be engineered for synthesis of Molybdenum cofactor, a coenzyme new to S. cerevisiae, whose production in this yeast could potentially enable expression of new enzyme families.ViewShow abstract... The expression plasmids were then digested by NotI to obtain the integrated DNA fragments. These linearized fragments were transformed into Y. lipolytica strains through the lithium acetate method as previously reported [45], to construct the expression library via NHEJ-mediated random genome integration. Positive transformants were selected on appropriate plates and checked by colony PCR. 10 transformants were randomly picked, and the engineered strains with high SA production were screened through shaking flasks fermentation. ...Engineering of Yarrowia lipolytica transporters for high-efficient production of biobased succinic acid from glucoseArticleFull-text availableJun 2021BIOTECHNOL BIOFUELS Zhennan JiangZiwei Zhu Qingsheng Qi Zhiyong CuiBackgroundSuccinic acid (SA) is a crucial metabolic intermediate and platform chemical. Development of biobased processes to achieve sustainable SA production has attracted more and more attention in biotechnology industry. Yarrowia lipolytica has a strong tricarboxylic acid cycle and tolerates low pH conditions, thus making it a potential platform for SA production. However, its SA titers in glucose media remain low.ResultsIn this study, we screened mitochondrial carriers and C4-dicarboxylic acid transporters to enhance SA secretion in Y. lipolytica . PGC62-SYF-Mae strain with efficient growth and SA production was constructed by optimizing SA biosynthetic pathways and expressing the transporter SpMae1. In fed-batch fermentation, this strain produced 101.4 g/L SA with a productivity of 0.70 g/L/h and a yield of 0.37 g/g glucose, which is the highest SA titer achieved using yeast, with glucose as the sole carbon resource.ConclusionOur results indicated that transporter engineering is a powerful strategy to achieve the efficient secretion of SA in Y. lipolytica , which will promote the industrial production of bio-based SA.ViewShow abstract... In addition to the homologous or heterologous gene of interest, targeting components can be optionally included into the ORF, in order to direct the resulting recombinant protein to precise intracellular organelles or to the secretion pathway, either for release into the cultivation medium (vesicular secretion) or for display on the cellular surface (surface display). When classical genetic engineering strategies are used, TUs are carried by either integrative or replicative shuttle vectors, built and propagated in Escherichia coli strains, that are then introduced into Y. lipolytica cells rendered competent using chemical treatments [35,138,173] or electroporation [174,175]. ...Yarrowia lipolytica strains and their biotechnological applications: how natural biodiversity and metabolic engineering could contribute to cell factories improvementPreprintJun 2021 Catherine MadzakAmong non-conventional yeasts of industrial interest, the dimorphic oleaginous yeast Yarrowia lipolytica appears as one of the most attractive for a large range of white biotechnology applications, from heterologous proteins secretion to cell factories process development. The past, present and potential applications of wild type, traditionally improved or genetically modified Yarrowia lipolytica strains will be resumed, together with the wide array of molecular tools now available to genetically engineer and metabolically remodel this yeast. The present review will also provide a detailed description of Yarrowia lipolytica strains and highlight the natural biodiversity of this yeast, a subject little touched upon in most previous reviews. This work intends to fill this gap by retracing the genealogy of the main Yarrowia lipolytica strains of industrial interest, by illustrating the search for new genetic backgrounds and by providing data about the main publicly available strains in yeast collections worldwide. At last, it will focus on exemplifying how advances in engineering tools can leverage a better biotechnological exploitation of the natural biodiversity of Yarrowia lipolytica and of other yeasts from the Yarrowia clade.ViewShow abstract... Transformation of Y. lipolytica was performed using a lithium-acetate based heat shock method previously described [39]. Briefly, the strain was plated on YPD agar plates and grown at 30°C for 18 h. ...Tolerance of Yarrowia lipolytica to inhibitors commonly found in lignocellulosic hydrolysatesArticleFull-text availableMar 2021BMC MICROBIOLSimone Zaghen Oliver Konzock Joakim NorbeckBackgroundLignocellulosic material is a suitable renewable carbon and energy source for microbial cell factories, such as Yarrowia lipolytica . To be accessible for microorganisms, the constituent sugars need to be released in a hydrolysis step, which as a side effect leads to the formation of various inhibitory compounds. However, the effects of these inhibitory compounds on the growth of Y. lipolytica have not been thoroughly investigated.ResultsHere we show the individual and combined effect of six inhibitors from three major inhibitor groups on the growth of Y. lipolytica . We engineered a xylose consuming strain by overexpressing the three native genes XR, XDH, and XK and found that the inhibitor tolerance of Y. lipolytica is similar in glucose and in xylose. Aromatic compounds could be tolerated at high concentrations, while furfural linearly increased the lag phase of the cultivation, and hydroxymethylfurfural only inhibited growth partially. The furfural induced increase in lag phase can be overcome by an increased volume of inoculum. Formic acid only affected growth at concentrations above 25 mM. In a synthetic hydrolysate, formic acid, furfural, and coniferyl aldehyde were identified as the major growth inhibitors.ConclusionWe showed the individual and combined effect of inhibitors found in hydrolysate on the growth of Y. lipolytica . Our study improves understanding of the growth limiting inhibitors found in hydrolysate and enables a more targeted engineering approach to increase the inhibitor tolerance of Y. lipolytica . This will help to improve the usage of Y. lipolytica as a sustainable microbial cell factory.ViewShow abstract... The transformation of constructed plasmids and relevant editing templates for deleting target genes using the CRISPR/Cas9 system was conducted using a standard lithium acetate protocol (Chen et al., 1997). Briefly, fresh cells from YPD plates and DNAs were mixed with a buffer containing 88 µl of 50% (w/v) PEG 4000, 5 µl of 2 M dithiothreitol, 5 µl of lithium acetate (pH 6.0), and 2 µl of single-strand carrier DNA (10 µg/µl) purchased from Thermo Fisher Scientific. ...Application of Random Mutagenesis and Synthetic FadR Promoter for de novo Production of ω-Hydroxy Fatty Acid in Yarrowia lipolyticaArticleFull-text availableFeb 2021 Beom Gi ParkJunyeob KimEun-Jung KimByung-Gee KimAs a means to develop oleaginous biorefinery, Yarrowia lipolytica was utilized to produce ω-hydroxy palmitic acid from glucose using evolutionary metabolic engineering and synthetic FadR promoters for cytochrome P450 (CYP) expression. First, a base strain was constructed to produce free fatty acids (FFAs) from glucose using metabolic engineering strategies. Subsequently, through ethyl methanesulfonate (EMS)-induced random mutagenesis and fluorescence-activated cell sorting (FACS) screening, improved FFA overproducers were screened. Additionally, synthetic promoters containing bacterial FadR binding sequences for CYP expression were designed to respond to the surge of the concentration of FFAs to activate the ω-hydroxylating pathway, resulting in increased transcriptional activity by 14 times from the third day of culture compared to the first day. Then, endogenous alk5 was screened and expressed using the synthetic FadR promoter in the developed strain for the production of ω-hydroxy palmitic acid. By implementing the synthetic FadR promoter, cell growth and production phases could be efficiently decoupled. Finally, in batch fermentation, we demonstrated de novo production of 160 mg/L of ω-hydroxy palmitic acid using FmeN3-TR1-alk5 in nitrogen-limited media. This study presents an excellent example of the production of ω-hydroxy fatty acids using synthetic promoters with bacterial transcriptional regulator (i.e., FadR) binding sequences in oleaginous yeasts.ViewShow abstract... Both the overexpression (random chromosomal integration) and deletion vectors (homologous recombination) were digested by NotI restriction enzyme to release the desired cassette. All Y. lipolytica transformations were performed using the lithium acetate procedure [42]. Positive Y. lipolytica transformants were verified by PCR and gel electrophoresis after genomic DNA extraction. ...Overexpression of Citrate Synthase Increases Isocitric Acid Biosynthesis in the Yeast Yarrowia lipolyticaArticleFull-text availableSep 2020 Piotr Hapeta Magdalena Rakicka Piotr Juszczyk Zbigniew LazarYarrowia lipolytica is a non-conventional yeast producing valuable compounds, such as citric acids, from renewable raw materials. This study investigated the impact of citrate synthase overexpression on the biosynthesis of citric and isocitric acid in Y. lipolytica. Two transformants of Y. lipolytica A101.1.31 strain (efficient citric acid producer), overexpressing CIT1 or CIT2 gene (encoding proteins with citrate synthase activity), were constructed. The results revealed that overexpression of either of these genes enhances citrate synthase activity. Additionally, the cit1 knockout strain was unable to use propionate as the sole carbon source, which proves that CIT1 gene encodes a dual activity protein–citrate and 2-methylcitrate synthase. In the overexpressing mutants, a significant increase in isocitric acid biosynthesis was observed. Both CIT1 and CIT2 overexpressing strains produced citric and isocitric acid from vegetable oil in a ratio close to 1 (CA/ICA ratio for wild-type strain was 4.12).ViewShow abstract... Yarrowia lipolytica (Barth and Gaillardin, 1997) for which good genetic tools are available (Chen et al., 1997, Fickers et al., 2003, Yamane et al., 2008, Blazeck et al., 2011, Wang et al., 2011, Blazeck et al., 2013, Liu et al., 2014. Improvements in lipid production by Y. ...Neutral lipid production by the yeast Debaryomyces hansenii NCYC102 under different stress conditionsThesisNov 2017 Zeena AlwanOleaginous yeasts are very efficient in the accumulation of triacylglycerol, and are expected to be one of the most important feedstocks for the biofuel industry in the future. Lipid content can be enhanced through physiological stress or genetic manipulation. Debaryomyces hansenii NCYC102 was selected from three different yeast species (also including Yarrowia lipolytica NCYC476 and Cryptococcus curvatus NCYC2904) due to the highest neutral lipid content. The growth rate, osmolytes and neutral lipids were measured in cells grown under different concentrations (0, 0.8, 1.6 M) of NaCl. The maximum content of total osmolytes was found in 0.8 M NaCl YM medium. However, the highest level of glycerol was measured in 1.6 M NaCl grown cells. The main osmolytes identified by 1 H NMR spectroscopy were glycerol, arabitol, glucose and trehalose. Debaryomyces. hansenii cells were grown in minimal medium with different carbon/nitrogen ratios using either glucose or glycerol as the sole carbon source along with ammonium sulphate as nitrogen source. Maximal neutral lipid production was observed in 48:0.5 glucose/ammonium sulphate ratio which achieved 1.4-fold increase compared with glycerol-based medium (8 glycerol: 0.25 ammonium sulphate). GC-MS analysis of the transesterified fatty acids showed that palmitic, oleic and stearic acids were the main fatty acids present, under normal and stress conditions (high salt and limited nitrogen source). Deletion of the GUT2 encoding for G3P dehydrogenase increased neutral lipid production up to a 1.4-fold compared to wild type strains. The mutant strains displayed slightly higher cell densities in medium with glucose when compared with wild type strains, while they failed to grow on glycerol as a sole carbon source. Collectively, these results indicate that D. hansenii is a good organism to produce biofuels as it has an intrinsic ability to accumulate neutral lipids and this can be further enhanced by genetic and metabolic engineering. II AcknowledgmentViewShow abstract... All plasmids constructed were transformed into the Y. lipolytica host strain Po1g (Leu−) using the lithium acetate/single-strand carrier DNA/PEG method (Chen et al., 1997). Single fresh Y. lipolytica colonies were picked from YNB selective plates and inoculated into YNB seed media, which were grown at 30°C for 48 h. ...Genetic and bioprocess engineering to improve squalene production in Yarrowia lipolyticaArticleFull-text availableAug 2020BIORESOURCE TECHNOL Fang WangHuan LiuLi Deng Peng XuSqualene is the precursor for triterpene-based natural products and steroids-based drugs. It has been widely used as pharmaceutical intermediates and personal care products. The aim of this work is to test the feasibility of engineering Yarrowia lipolytica as a potential host for squalene production. The bottleneck of the pathway was removed by overexpressing native HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase. With the recycling of NADPH from the mannitol cycle, the engineered strain produced about 180.3 mg/L and 188.2 mg/L squalene from glucose or acetate minimal media. By optimizing the C/N ratio, controlling the media pH and mitigating acetyl-CoA flux competition from lipogenesis, the engineered strain produced 502.7 mg/L squalene, a 28-fold increase over the parental strain (17.2 mg/L). This work may serve as a baseline to harness Y. lipolytica as an oleaginous cell factory for sustainable production of squalene or terpenoids-based chemicals and natural products.ViewShow abstract... The standard protocols of Y. lipolytica transformation by the lithium acetate method were described as previously reported. 63,64 In brief, one milliliter cells was harvested during the exponential growth phase (16−24 h) from 2 mL YPD medium (yeast extract 10 g/L, peptone 20 g/ L, and glucose 20 g/L) in the 14 mL shake tube, and washed twice with 100 mM phosphate buffer (pH 7.0). Then, cells were resuspended in 105 μL transformation solution, containing 90 μL 50% PEG4000, 5 μL lithium acetate (2 M), 5 μL boiled single stand DNA (salmon sperm, denatured) and 5 μL DNA products (including 200−500 ng of plasmids, lined plasmids or DNA fragments), and incubated at 39°C for 1 h, then spread on selected plates. ...Engineering Yarrowia lipolytica as a Chassis for De Novo Synthesis of Five Aromatic-Derived Natural Products and ChemicalsArticleFull-text availableJul 2020 Yang GuJingbo MaYonglian Zhu Peng XuYarrowia lipolytica is a novel microbial chassis to upgrade renewable low-cost carbon feedstocks to high-value commodity chemicals and natural products. In this work, we systematically characterized and removed the rate-limiting steps of the shikimate pathway and achieved de novo synthesis of five aromatic chemicals in Y. lipolytica. We determined that eliminating amino acids formation and engineering feedback-insensitive DAHP synthases are critical steps to mitigate precursor competition and relieve the feedback regulation of shikimate pathway. Further overexpression of heterologous phosphoketolase and deletion of pyruvate kinase provided a sustained metabolic driving force that channels E4P (erythrose 4-phosphate) and PEP (phosphoenolpyruvate) precursors through the shikimate pathway. Precursor competing pathways and byproduct formation pathways were also blocked by inactivating chromosomal genes. To demonstrate the utility of our engineered chassis strain, three natural products, 2-phenylethanol (2-PE), p-coumaric acid and violacein, which were derived from phenylalanine, tyrosine and tryptophan, respectively, were chosen to test the chassis performance. We obtained 2426.22 ± 48.33 mg/L of 2-PE, 593.53 ± 28.75 mg/L of p-coumaric acid, 12.67 ± 2.23 mg/L of resveratrol, 366.30 ± 28.99 mg/L of violacein and 55.12 ± 2.81 mg/L of deoxyviolacein from glucose in shake flask. The 2-PE production represents a 286-fold increase over the initial strain (8.48 ± 0.50 mg/L). Specifically, we obtained the highest 2-PE, violacein and deoxyviolacein titer ever reported from the de novo shikimate pathway in yeast. These results set up a new stage of engineering Y. lipolytica as a sustainable biorefinery chassis strain for de novo synthesis of aromatic compounds with economic values.ViewShow abstract... 18 μL of desired DNA and 92 μL of transformation mix (80 μL 60% PEG4000, 5 μL 2 M DTT, 5 μL 2 M lithium acetate pH 6 and 2 μL 10 mg/mL single stranded salmon sperm DNA) were added to the cell pellet. The transformation reaction was mixed by vortexing and heat shocked at 39 • C for 1 h (Chen, Beckerich and Gaillardin 1997;Friedlander et al. 2016). Cells were centrifuged, the supernatant was discarded, cells were resuspended in 1 mL of non-selective medium (YPD for fad2 or supplemented YPD as described above for ole1), transferred to culture tubes and cultured overnight at 30 • C before plating 100 μL on selective media. ...Promoters for lipogenesis-specific downregulation in Yarrowia lipolyticaArticleFull-text availableJun 2020FEMS Yeast ResShuyan ChenGamuchirai ChifambaAnnapurna Kamineni Vasiliki TsakraklidesYarrowia lipolytica is a non-conventional yeast with potential applications in the biofuel and biochemical industries. It is an oleaginous yeast that accumulates lipids when it encounters nutrient limitation in the presence of excess carbon. Its molecular toolbox includes promoters for robust constitutive expression, regulated expression through the addition of media components, and inducible expression during lipid accumulation. To date, no promoters have been identified that lead to downregulation at the transition from growth to lipid accumulation. We identified four native Y. lipolytica promoters that downregulate the expression of genes at this natural transition. Using the fatty acid desaturase genes FAD2 and OLE1 as reporter genes for these promoters, we correlated repression of desaturase transcript levels with a reduction of desaturated fatty acids at the transition to lipid accumulation. These promoters can restrict to the growth phase an essential or favorable activity that is undesirable during lipid accumulation under traditional fermentation conditions without media additions. This expression pattern results in lipogenesis phase-specific changes that could be useful in applications relating to optimizing lipid yield and composition.ViewShow abstract... Y. lipolytica cells were transformed as described (39). Briefly, cells were grown overnight in YP 1 / 2 D (1% (w/v) yeast extract, 2% (w/v) peptone and 1% (w/v) glucose, at 28 • C, and collected by centrifugation, or alternatively harvested from a fresh YP 1 / 2 D plate. ...Pathogenic mutations in NUBPL affect complex I activity and cold tolerance in the yeast model Yarrowia lipolyticaArticleFull-text availableJul 2018Hum Mol GenetVirginia E. KimonisAndrew E MacleanJanneke BalkComplex I deficiency is a common cause of mitochondrial disease, resulting from mutations in genes encoding structural subunits, assembly factors or defects in mitochondrial gene expression. Advances in genetic diagnostics and sequencing have led to identification of several variants in NUBPL, encoding an assembly factor of complex I, which are potentially pathogenic. To help assign pathogenicity and learn more about the function of NUBPL, amino acid substitutions were recreated in the homologous Ind1 protein of the yeast model Yarrowia lipolytica. Leu102Pro destabilized the Ind1 protein, leading to a null-mutant phenotype. Asp103Tyr, Leu191Phe and Gly285Cys affected complex I assembly to varying degrees, whereas Gly136Asp substitution in Ind1 did not impact on complex I levels nor dNADH:ubiquinone activity. Blue-native PAGE and immunolabelling of the structural subunits NUBM and NUCM revealed that all Ind1 variants accumulated a Q-module intermediate of complex I. In the Ind1 Asp103Tyr variant the matrix arm intermediate was virtually absent, indicating a dominant effect. Dysfunction of Ind1, but not absence of complex I, rendered Y. lipolytica sensitive to cold. The Ind1 Gly285Cys variant was able to support complex I assembly at 28°C, but not at 10°C. Our results indicate that Ind1 is required for progression of assembly from the Q module to the full matrix arm. Cold sensitivity could be developed as a phenotype assay to demonstrate pathogenicity of NUBPL mutations and other complex I defects.ViewShow abstract... For Y. lipolytica transformations, log phase cells were either treated with 50 mM hydroxyurea for 2 h (gene deletions) [41] or were processed directly for transformation (plasmid transformation) [23]. Y. lipolytica competent cells were prepared following a protocol adapted from Chen et al. [42]. Cells were washed with water and resuspended in a volume of water equal to the volume of the wet cell pellet. ...Identification of a Yarrowia lipolytica acetamidase and its use as a yeast genetic markerArticleFull-text availableFeb 2020MICROB CELL FACTMaureen HamiltonKyle MacEwenAndrew L. Consiglio Vasiliki TsakraklidesBackground: Yarrowia lipolytica is an oleaginous yeast that can be genetically engineered to produce lipid and non-lipid biochemicals from a variety of feedstocks. Metabolic engineering of this organism usually requires genetic markers in order to select for modified cells. The potential to combine multiple genetic manipulations depends on the availability of multiple or recyclable selectable markers.Results: We found that Y. lipolytica has the ability to utilize acetamide as the sole nitrogen source suggesting that the genome contains an acetamidase gene. Two potential Y. lipolytica acetamidase gene candidates were identified by homology to the A. nidulans acetamidase amdS. These genes were deleted in the wild-type Y. lipolytica strain YB-392, and deletion strains were evaluated for acetamide utilization. One deletion strain was unable to grow on acetamide and a putative acetamidase gene YlAMD1 was identified. Transformation of YlAMD1 followed by selection on acetamide media and counterselection on fluoroacetamide media showed that YlAMD1 can be used as a recyclable genetic marker in Saccharomyces cerevisiae and Ylamd1Δ Y. lipolytica.Conclusions: These findings add to our understanding of Y. lipolytica nitrogen utilization and expand the set of genetic tools available for engineering this organism, as well as S. cerevisiae.ViewShow abstractEngineering Yarrowia lipolytica for Use in Biotechnological Applications: A Review of Major Achievements and Recent InnovationsArticleFull-text availableAug 2018MOL BIOTECHNOL Catherine MadzakYarrowia lipolytica is an oleaginous saccharomycetous yeast with a long history of industrial use. It aroused interest several decades ago as host for heterologous protein production. Thanks to the development of numerous molecular and genetic tools, Y. lipolytica is now a recognized system for expressing heterologous genes and secreting the corresponding proteins of interest. As genomic and transcriptomic tools increased our basic knowledge on this yeast, we can now envision engineering its metabolic pathways for use as whole-cell factory in various bioconversion processes. Y. lipolytica is currently being developed as a workhorse for biotechnology, notably for single-cell oil production and upgrading of industrial wastes into valuable products. As it becomes more and more difficult to keep up with an ever-increasing literature on Y. lipolytica engineering technology, this article aims to provide basic and actualized knowledge on this research area. The most useful reviews on Y. lipolytica biology, use, and safety will be evoked, together with a resume of the engineering tools available in this yeast. This mini-review will then focus on recently developed tools and engineering strategies, with a particular emphasis on promoter tuning, metabolic pathways assembly, and genome editing technologies.ViewShow abstractPathogenic mutations in NUBPL affect complex I activity and cold tolerance in the yeast model Yarrowia lipolyticaPreprintFull-text availableJun 2018Andrew E MacleanVirginia KimonisJanneke BalkComplex I deficiency is a common cause of mitochondrial disease, resulting from mutations in genes encoding structural subunits, assembly factors or defects in mitochondrial gene expression. Advances in genetic diagnostics and sequencing have led to identification of several variants in NUBPL, an assembly factor of complex I, which are potentially pathogenic. To help assign pathogenicity and learn more about the function of NUBPL, amino acid substitutions were recreated in the homologous Ind1 protein of the yeast model Yarrowia lipolytica . L102P destabilized the Ind1 protein, leading to a null-mutant phenotype. D103Y, L191F and G285C affected complex I assembly to varying degrees, whereas the G138D variant did not impact on complex I levels or dNADH:ubiquinone activity. Blue-native PAGE and immunolabelling of the structural subunits NUBM and NUCM revealed that all Ind1 variants accumulated a Q-module intermediate of complex I. In the D103Y variant the matrix arm intermediate was virtually absent, indicating a dominant effect. Dysfunction of Ind1, but not absence of complex I, rendered Y. lipolytica sensitive to cold. The Ind1 G285C variant was able to support complex I assembly at 28 o C, but not at 10 o C. Our results indicate that Ind1 is required for progression of assembly from the Q module to the full matrix arm. Cold sensitivity could be developed as a phenotype assay to demonstrate pathogenicity of NUBPL mutations and other complex I defects.ViewShow abstractEngineering β-oxidation in Yarrowia lipolytica for methyl ketone productionArticleMay 2018METAB ENG Erik HankoCharles M. Denby Violeta Sànchez i Nogué Jay D KeaslingMedium- and long-chain methyl ketones are fatty acid-derived compounds that can be used as biofuel blending agents, flavors and fragrances. However, their large-scale production from sustainable feedstocks is currently limited due to the lack of robust microbial biocatalysts. The oleaginous yeast Yarrowia lipolytica is a promising biorefinery platform strain for the production of methyl ketones from renewable lignocellulosic biomass due to its natively high flux towards fatty acid biosynthesis. In this study, we report the metabolic engineering of Y. lipolytica to produce long- and very long-chain methyl ketones. Truncation of peroxisomal β-oxidation by chromosomal deletion of pot1 resulted in the biosynthesis of saturated, mono-, and diunsaturated methyl ketones in the C13-C23 range. Additional overexpression and peroxisomal targeting of a heterologous bacterial methyl ketone biosynthesis pathway yielded an initial titer of 151.5mg/L of saturated methyl ketones. Dissolved oxygen concentrations in the cultures were found to substantially impact cell morphology and methyl ketone biosynthesis. Bioreactor cultivation under optimized conditions resulted in a titer of 314.8mg/L of total methyl ketones, representing more than a 6,000-fold increase over the parental strain. This work highlights the potential of Y. lipolytica to serve as chassis organism for the biosynthesis of acyl-thioester derived long- and very long-chain methyl ketones.ViewShow abstractFunctional genomics for the oleaginous yeast Yarrowia lipolyticaArticleMay 2018METAB ENGKurt Patterson James YuJenny Landberg Suzanne SandmeyerOleaginous yeasts are valuable systems for biosustainable production of hydrocarbon-based chemicals. Yarrowia lipolytica is one of the best characterized of these yeast with respect to genome annotation and flux analysis of metabolic processes. Nonetheless, progress is hampered by a dearth of genome-wide tools enabling functional genomics. In order to remedy this deficiency, we developed a library of Y. lipolytica insertion mutants via transposon mutagenesis. The Hermes DNA transposon was expressed to achieve saturation mutagenesis of the genome. Over 534,000 independent insertions were identified by next-generation sequencing. Poisson analysis of insertion density classified ~22% of genes as essential. As expected, most essential genes have homologs in Saccharomyces cerevisiae and Schizosaccharomyces pombe, and the majority of those are also essential. As an obligate aerobe, Y. lipolytica has significantly more respiration - related genes that are classified as essential than do S. cerevisiae and S. pombe. Contributions of non-essential genes to growth in glucose and glycerol carbon sources were assessed and used to evaluate two recent genome-scale models of Y. lipolytica metabolism. Fluorescence-activated cell sorting identified mutants in which lipid accumulation is increased. Our findings provide insights into biosynthetic pathways, compartmentalization of enzymes, and distinct functions of paralogs. This functional genomic analysis of the oleaginous yeast Y. lipolytica provides an important resource for modeling, bioengineering, and design of synthetic minimalized strains of respiratory yeasts.ViewShow abstractLipid production by oleaginous yeastsChapterJan 2021ADV APPL MICROBIOL Atrayee ChattopadhyayMrinal K. MaitiMicrobial lipid production has been studied extensively for years; however, lipid metabolic engineering in many of the extraordinarily high lipid-accumulating yeasts was impeded by inadequate understanding of the metabolic pathways including regulatory mechanisms defining their oleaginicity and the limited genetic tools available. The aim of this review is to highlight the prominent oleaginous yeast genera, emphasizing their oleaginous characteristics, in conjunction with diverse other features such as cheap carbon source utilization, withstanding the effect of inhibitory compounds, commercially favorable fatty acid composition—all supporting their future development as economically viable lipid feedstock. The unique aspects of metabolism attributing to their oleaginicity are accentuated in the pretext of outlining the various strategies successfully implemented to improve the production of lipid and lipid-derived metabolites. A large number of in silico data generated on the lipid accumulation in certain oleaginous yeasts have been carefully curated, as suggestive evidences in line with the exceptional oleaginicity of these organisms. The different genetic elements developed in these yeasts to execute such strategies have been scrupulously inspected, underlining the major types of newly-found and synthetically constructed promoters, transcription terminators, and selection markers. Additionally, there is a plethora of advanced genetic toolboxes and techniques described, which have been successfully used in oleaginous yeasts in the recent years, promoting homologous recombination, genome editing, DNA assembly, and transformation at remarkable efficiencies. They can accelerate and effectively guide the rational designing of system-wide metabolic engineering approaches pinpointing the key targets for developing industrially suitable yeast strains.ViewShow abstractHomology-independent genome integration enables rapid library construction for enzyme expression and pathway optimization in Yarrowia lipolytica: CUI et al.ArticleFull-text availableNov 2018BIOTECHNOL BIOENGXin JiangHuihui ZhengJin Hou Zhiyong CuiYarrowia lipolytica is an important oleaginous industrial microorganism used to produce biofuels and other value‐added compounds. Although several genetic engineering tools have been developed for Y. lipolytica, there is no efficient method for genomic integration of large DNA fragments. In addition, methods for constructing multi‐gene expression libraries for biosynthetic pathway optimization are still lacking in Y. lipolytica. In this study, we demonstrate that multiple and large DNA fragments can be randomly and efficiently integrated into the genome of Y. lipolytica in a homology‐independent manner. This homology‐independent integration generates variation in the chromosomal locations of the inserted fragments and in gene copy numbers, resulting in expression differences in the integrated genes or pathways. Because of these variations, gene expression libraries can be easily created through one‐step integration. As proof of concept, a LIP2 (producing lipase) expression library and a library of multiple genes in the β‐carotene biosynthetic pathway were constructed, and high‐production strains were obtained through library screening. Our work demonstrates the potential of homology‐independent genome integration for library construction, especially for multivariate modular libraries for metabolic pathways in Y. lipolytica, and will facilitate pathway optimization in metabolic engineering applications.This article is protected by copyright. All rights reserved.ViewShow abstractSelection of Heterologous Protein-Producing Strains in Yarrowia lipolytica: 2003–2007 The First Five YearsChapterJan 2019Meth Mol Biol Paul Soudier Macarena LarroudeEwelina Celińska Jean-Marc NicaudYarrowia lipolytica has emerged as an alternative expression system for heterologous protein production and enzyme evolution. Several different expression systems dedicated for this species have been developed, ranging from the simple cloning of expression vectors to recently developed high-throughput methodologies using efficient cloning and assembly such as Gateway and Golden Gate strategies. The latter strategies, due to their modular character, enable multiple vector construction and the construction of expression cassettes containing different genes or a gene under different promoters of various strengths.ViewShow abstractEngineering acetyl-CoA metabolic shortcut for eco-friendly production of polyketides triacetic acid lactone in Yarrowia lipolyticaPreprintFull-text availableApr 2019Fang WangHuan LiuMonireh Marsafari Peng XuAcetyl-CoA is the central metabolic node connecting glycolysis, Krebs cycle and fatty acids synthase. Plant-derived polyketides, are assembled from acetyl-CoA and malonyl-CoA, represent a large family of biological compounds with diversified bioactivity. Harnessing microbial bioconversion is considered as a feasible approach to large-scale production of polyketides from renewable feedstocks. Most of the current polyketide production platform relied on the lengthy glycolytic steps to provide acetyl-CoA, which inherently suffers from complex regulation with metabolically-costly cofactor/ATP requirements. Using the simplest polyketide triacetic acid lactone (TAL) as a target molecule, we demonstrate that acetate uptake pathway in oleaginous yeast (Yarrowia lipolytica) could function as an acetyl-CoA shortcut to achieve metabolic optimality in producing polyketides. We identified the metabolic bottlenecks to rewire acetate utilization for efficient TAL production in Y. lipolytica, including generation of the driving force for acetyl-CoA, malonyl-CoA and NADPH. The engineered strain, with the overexpression of endogenous acetyl-CoA carboxylase (ACC1), malic enzyme (MAE1) and a bacteria-derived cytosolic pyruvate dehydrogenase (PDH), affords robust TAL production with titer up to 4.76 g/L from industrial glacier acetic acid in shake flasks, representing 8.5-times improvement over the parental strain. The acetate-to-TAL conversion ratio (0.149 g/g) reaches 31.9% of the theoretical maximum yield. The carbon flux through this acetyl-CoA metabolic shortcut exceeds the carbon flux afforded by the native acetyl-CoA pathways. Potentially, acetic acid could be manufactured in large-quantity at low-cost from Syngas fermentation or heterogenous catalysis (methanol carbonylation). This alternative carbon sources present a metabolic advantage over glucose to unleash intrinsic pathway limitations and achieve high carbon conversion efficiency and cost-efficiency. This work also highlights that low-cost acetic acid could be sustainably upgraded to high-value polyketides by oleaginous yeast species in an eco-friendly and cost-efficient manner.ViewShow abstractConstruction of wild-type Yarrowia lipolytica IMUFRJ 50682 auxotrophic mutants using dual CRISPR/Cas9 strategy for novel biotechnological approachesArticleJun 2020ENZYME MICROB TECH Camilla Souza Bernardo Dias Ribeiro Maria Alice Z. Coelho Jean-Marc NicaudYarrowia lipolytica IMUFRJ 50,682 is a Brazilian wild-type strain with potential application in bioconversion processes which can be improved through synthetic biology. In this study, we focused on a combinatorial dual cleavage CRISPR/Cas9-mediated for construction of irreversible auxotrophic mutants IMUFRJ 50,682, which genomic information is not available, thought paired sgRNAs targeting upstream and downstream sites of URA3 gene. The disruption efficiency ranged from 5 to 28 % for sgRNAs combinations closer to URA3’s start and stop codon and the auxotrophic mutants lost about 970 bp containing all coding sequence, validating this method for genomic edition of wild-type strains. In addition, we introduced a fluorescent phenotype and achieved cloning rates varying from 80 to 100 %. The ura3Δ strains IMUFRJ 50,682 were also engineered for β-carotene synthesis as proof of concept. Carotenoid-producing strains exhibited a similar growth profile compared to the wild-type strain and were able to synthesized 30.54–50.06 mg/L (up to 4.8 mg/g DCW) of β-carotene in YPD and YNB flask cultures, indicating a promisor future of the auxotrophic mutants IMUFRJ 50,682 as a chassis for production of novel value-added chemicals.ViewShow abstractProduction of Functional gamma-Linolenic Acid (GLA) by Expression of Fungal Delta12- and Delta6-Desaturase Genes in the Oleaginous Yeast: Yarrowia lipolyticaChapterApr 2009Lu-Te ChuangYing-Hsuan Chen Catherine Madzak Jean-Marc NicaudViewDroplet-Based Microfluidic High-Throughput Screening of Enzyme Mutant Libraries Secreted by Yarrowia lipolyticaChapterApr 2021Meth Mol Biol Thomas Beneyton Tristan RossignolYarrowia lipolytica has emerged as an attractive solution for screening enzyme activities thanks to the numerous tools available for heterologous protein production and its strong secretory ability. Nowadays, activity screening for improved enzymes mostly relies on the evaluation of independent clones in microtiter plates. However, even with highly robotized screening facilities, the relatively low throughput and high cost of the technology do not enable the screening of large diversities, which significantly reduce the probability of isolating improved variants. Droplet-based microfluidics is an emerging technology that allows the high-throughput and individual picoliter droplets manipulation and sorting based on enzymatic substrate fluorescence. This technology is an attractive alternative to microtiter plate screenings with higher throughputs and drastic reduction of working volume and cost. Here, we present a droplet-based microfluidic platform for the screening of libraries expressed in the yeast Y. lipolytica, from the generation of a random mutagenesis library of a heterologous enzyme and its expression in Y. lipolytica to the droplet-based microfluidic procedures composed of cell encapsulation and growth and activity screening or sorting of improved clones.ViewShow abstractEasyCloneYALI: Toolbox for CRISPR-Mediated Integrations and Deletions in Yarrowia lipolyticaChapterApr 2021Meth Mol BiolJonathan DahlinCarina Holkenbrink Irina BorodinaIn order to unlock the full potential of Yarrowia lipolytica, as model organism and production host, simple and reliable tools for genome engineering are essential. In this chapter, the practical details of working with the EasyCloneYALI Toolbox are described.Highlights of the EasyCloneYALI Toolbox are high genome editing efficiencies, multiplexed Cas9-mediated knockouts, targeted genomic integrations into characterized intergenic loci, as well as streamlined and convenient cloning for both marker-based and marker-free integrative expression vectors.ViewShow abstractHarness Yarrowia lipolytica to Make Small Molecule ProductsChapterJun 2021Kang ZhouGregory StephanopoulosYarrowia lipolytica is an oleaginous yeast and is now well known for its extraordinary ability of producing lipids from glucose and organic acids. It first gained attention in industrial biotechnology during the 1960s for its potential use in producing single cell proteins from alkanes. Through studying its nutrient requirement, Y. lipolytica was found to accumulate organic acids under nutrient limitation conditions. These discoveries led to the development of large‐scale processes for producing some organic acids from sugars and alkanes. In the past ten years, the number of products made by Y. lipolytica fermentation has exploded thanks to engineering of its metabolism. This chapter briefly describes the use of Y. lipolytica in industrial biotechnology (Section) and the key genetic engineering tools available for engineering of this species (Section). Then basic metabolic engineering principles frequently used with this species are discussed through ∼10 examples, which can be categorized based on the family of their products: short‐chain organic acids (Section), triacylglycerol (Section), and other molecules derived from acetyl‐CoA (Section, including three examples covering polyunsaturated fatty acids, polyketides, and isoprenoids). The chapter concludes with a short discussion on opportunities and challenges that may be faced by Y. lipolytica metabolic engineers (Section).ViewShow abstractEngineering acetyl-CoA metabolic shortcut for eco-friendly production of polyketides triacetic acid lactone in Yarrowia lipolyticaArticleAug 2019METAB ENGHuan LiuMonireh MarsafariFang Wang Peng XuAcetyl-CoA is the central metabolic node connecting glycolysis, Krebs cycle and fatty acids synthase. Plant-derived polyketides, are assembled from acetyl-CoA and malonyl-CoA, represent a large family of biological compounds with diversified bioactivity. Harnessing microbial bioconversion is considered as a feasible approach to large-scale production of polyketides from renewable feedstocks. Most of the current polyketide production platform relied on the lengthy glycolytic steps to provide acetyl-CoA, which inherently suffers from complex regulation with metabolically-costly cofactor/ATP requirements. Using the simplest polyketide triacetic acid lactone (TAL) as a testbed molecule, we demonstrate that acetate uptake pathway in oleaginous yeast (Yarrowia lipolytica) could function as an acetyl-CoA shortcut to achieve metabolic optimality in producing polyketides. We identified the metabolic bottlenecks to rewire acetate utilization for efficient TAL production in Y. lipolytica, including generation of the driving force for acetyl-CoA, malonyl-CoA and NADPH. The engineered strain, with the overexpression of endogenous acetyl-CoA carboxylase (ACC1), malic enzyme (MAE1) and a bacteria-derived cytosolic pyruvate dehydrogenase (PDH), affords robust TAL production with titer up to 4.76 g/L from industrial glacier acetic acid in shake flasks, representing 8.5-times improvement over the parental strain. The acetate-to-TAL conversion ratio (0.149 g/g) reaches 31.9% of the theoretical maximum yield. The carbon flux through this acetyl-CoA metabolic shortcut exceeds the carbon flux afforded by the native glycolytic pathways. Potentially, acetic acid could be manufactured in large-quantity at low-cost from Syngas fermentation or heterogenous catalysis (methanol carbonylation). This alternative carbon sources present a metabolic advantage over glucose to unleash intrinsic pathway limitations and achieve high carbon conversion efficiency and cost-efficiency. This work also highlights that low-cost acetic acid could be sustainably upgraded to high-value polyketides by oleaginous yeast species in an eco-friendly and cost-efficient manner.ViewShow abstractShow moreRapid colony transformation of Saccharomyces cerevisiaeArticleFull-text availableMay 1991NUCLEIC ACIDS RES Rohan T BakerViewThe TSR1 Gene of Yarrowia lipolytica Is Involved in the Signal Recognition Particle-dependent Translocation Pathway of Secretory ProteinsArticleFull-text availableOct 1996J BIOL CHEMChoukri Ben MamounJean-Marie Beckerich Claude GaillardinWe have isolated suppressors (tsr1 to tsr5) of the thermosensitive growth of the scr2.II-13 mutation, which affects the stability of the signal recognition particle. The growth of these mutants is largely affectedin the SCR2 context at 34°C. We have studied the synthesis and secretion of an alkaline extracellular protease (AEP) in both wild-typeand tsr1-1(SCR2+) thermosensitive mutant strains. Pulse-chase labeling and immunoprecipitation of this protein showed that the level of AEPprecursors in the tsr1-1(SCR2+) strain is 70% less than in the wild-type strain under conditions where the global protein synthesis is practically unaffected.This defect was observed as early as 10 min after the shift to nonpermissive temperature. In neither strain was there anyeffect on the kinetics of secretion, and no cytoplasmic accumulation was detected. We have cloned the TSR1 gene by complementing the thermosensitive phenotype of a tsr1-1(SCR2+) mutant. Analysis of the TSR1 DNA sequence revealed an open reading frame of 1383 base pairs, encoding a serine-rich protein of 461 amino acids with anamino-terminal signal peptide, and a membrane-spanning domain of 20 amino acids that could act as a stop transfer signal toensure membrane localization of Tsr1p. Two homologues of the TSR1 gene were identified in Saccharomyces cerevisiae (YHC8) and Hansenula polymorpha (YLU2). Disruption of the TSR1 gene revealed that it is an essential single-copy gene. The TSR1 gene encodes a single mRNA of 1.5 kilobase pairs. The study of the synthesis and secretion of AEP in the complemented tsr1-1(SCR2+,TSR1+) strain revealed that the TSR1 gene ensures complete recovery of the synthesis defect and thus could encode an important component of the endoplasmic reticulummembrane involved in the early steps of the signal recognition particle-dependent translocation pathway.ViewShow abstractThe yeast Yarrowia lipolytica has two, functional, signal recognition particle 7S RNA genesArticleApr 1990CURR GENETFeng HeDebbie Yaver jean-marie Beckerich Claude GaillardinCells containing a deletion of either the SCR1 or SCR2 genes, which code for the 7SL RNA component of the signal recognition particle (SRP) homologue, were found to be viable. Two independent approaches demonstrated that cells containing deletions of both genes were inviale. Therefore, Yarrowia lipolytica contains two (and only two) functional 7SL RNA genes.ViewShow abstractRecommended publicationsDiscover moreSponsored contentINRAE is hiring 10 research scientists - Call for research projects (CRCN)October 2020INRAE is hiring researchers who have already shown their ability to produce research of excellence under supervision, attested by high-level publications. Candidates must be prepared to work independently and propose an ambitious research project in INRAE’s main areas of research: Agriculture,...View postSponsored contentINRAE is hiring 45 Scientists through open competitions and offering permanent positions.January 2020On January 1, 2020, INRA (French National Institute for Agronomical Research) and IRSTEA (National Research Institute of Science and Technology for Environment and Agriculture) merged to become the National Research Institute for Agriculture, Food and the Environment – INRAE.In 2020, INRAE...View postArticleGenetic changes in yeast cells cotransformed with mitochondrial DNA and plasmid YEp13 are not elicit...December 1982 · Biochemical and Biophysical Research CommunicationsSam W. WooAnthony W. Linnane Phillip Nagleystrain DC5-E2 that lacks mtDNA () can be cotransformed with a mixture of mtDNA and the plasmid YEp13 (LEU2/2μ/pBR322) to produce Leu+ transormants which, on being mated to tester strains, generate respiratory competent diploids (such events are denoted marker rescue). In this work strain DC5-E2 was transformed with recombinant molecules consisting of a mtDNA segment including the gene inserted ... [Show full abstract] into YEp13. The Leu+ transformants made with the recombinant plasmids were unable to rescue testers carrying mutations in the region, in contrast to Leu+ cotransformants made with mixtures of YEp13 and mtDNA. We conclude that marker rescue events occur as a result of interactions between the mtDNA of the tester and the mtDNA sequences introduced by transformation. Such interactions cannot occur when the latter mtDNA is forced to replicate in covalent association with YEp13, probably in the nucleus.Read moreArticleExpression of invertase activity in Yarrowia lipolytica and its use as a selective markerOctober 1989 · Current Genetics Jean-Marc Nicaud Claude Gaillardin Emmanuelle FabreFew selective markers are available for the transformation of the industrial yeast Yarrowia lipolytica, and those that are require the use of specialized hosts (e.g., auxotrophs, antibiotic sensitive). To enable the transformation of any Y. lipolytica strain, we used the property that Y. lipolytica cannot use sucrose as a sole carbon source. We have constructed a gene fusion where the ... [Show full abstract] Saccharomyces cerevisiae SUC2 gene is placed under the control of the promoter and signal sequence of the Y. lipolytica XPR2 gene, which encodes an Alkaline Extracellular Protease (AEP). Strains bearing this fusion express invertase activity and grow on sucrose as a carbon source. The activity follows the same regulation as does the alkaline extracellular protease, is secreted into the periplasm and confers a Suc+ phenotype. It was shown that this chimeric gene could be used as a dominant marker for transformation in a one-step procedure.Read moreDataFull-text availableFigure S3December 2010Danielle H. DubeBin Li Ethan Greenblatt[...] Jennifer KohlerLOC and CAT plasmids are not toxic to yeast. Wild-type yeast (BY4741) were transformed with the indicated plasmids and grown on CSM-Leu-Ura agar plates (A) at 30°C in the presence of 5 mg/L of Congo red or (B) at 37°C in the absence of Congo red. Each row shows ten-fold serial dilutions of the indicated transformant. In the context of this OCH1-expressing strain, the LOC and CAT constructs do not ... [Show full abstract] affect growth.(TIF)View full-textArticleFull-text availablePhysical Factors Affects Bud Formation Pattern in Wild Type Yeast Model (Saccharomyces cerevisiae)August 2013 Olalekan Michael Ogundele Olumuyiwa Ademola Alao Olayinka Idris[...]B A OsoThe pattern of cell division in the yeast models suggests that the process of cell division is not spontaneous but the direction of such cell divisions is predetermined by several physical factors. The study is aimed at investigating the mathematical relationship involved in cell division (in the budding yeast) and how it can be affected by certain physical factors (light and gravity). The ... [Show full abstract] hypothesis was further tested using live yeast models that were sub-cultured in potatoe dextrose agar and kept at room temperature on a microscope stage. The illumination technique was adjusted to reduce the intensity of the incident light. The image was recorded on the computer interface to determine time dependence and effects of light and gravity in determining the direction of division and cell movement in bud formation in the S. Cerevisiae. The angle of budding φN+2 at event N+2, was observed to be dependent on φN and φN+1 as ∆φ = φN-φN+1, where ∆φ = φN+2 and are positive for successive buds.View full-textLooking for the full-text?You can request the full-text of this article directly from the authors on ResearchGate.Request full-textAlready a member? Log in ResearchGate iOS AppGet it from the App Store now.InstallKeep up with your stats and moreAccess scientific knowledge from anywhere orDiscover by subject areaRecruit researchersJoin for freeLoginEmail Tip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? Keep me logged inLog inorContinue with GoogleWelcome back! 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