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N-glycosylation in wild type strains and the approach used to engineer the yeast pathway. (A) Standard N-glycosylation in yeast and mammalian cells. The early N-glycan steps in the ER are strongly conserved between higher and lower eukaryotes until the stage at which folded proteins bearing the eukaryotic common high mannose Man8GlcNAc2 glycan structure (isomer B) leave the ER and enter the Golgi apparatus. At this point, the glycans undergo further species and cell-type specific processing. The pathways in the Golgi complex diverge notably. In higher eukaryotes, the Man8GlcNAc2 structure is further trimmed to Man5GlcNAc2 by mannosidase I and can then be further modified to complex glycans (bottom). In yeast, the Man8GlcNAc2 glycan structure is further elongated with mannoses by mannosyl- and phosphomannosyltransferases, and in some cases this results in hypermannosylation (top). (B) Glyco-engineering in yeast. A knock-out of the OCH1 gene prevents the elongation of Man8GlcNAc2 glycans (isomer B), and upon expression of the HDEL-tagged α-1,2-mannosidase, Man5GlcNAc2 glycans are formed. Conforming to the representation proposed by the Consortium for Functional Glycomics Nomenclature Committee, the green and yellow spheres represent mannose (Man) and galactose (Gal), respectively, blue squares represent N-acetylglucosamine (GlcNAc) residues and the red diamonds represent sialic acid (Sia).
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Background
Protein-based therapeutics represent the fastest growing class of compounds in the pharmaceutical industry. This has created an increasing demand for powerful expression systems. Yeast systems are widely used, convenient and cost-effective. Yarrowia lipolytica is a suitable host that is generally regarded as safe (GRAS). Yeasts, however,...
Citations
... Yeasts are more advantageous among all other hosts due to easy gene manipulation, short generation time, simple medium requirements, and most importantly, they don't release endotoxins. So, yeasts are usually regarded as safe (GRAS) organisms [52,53]. P. pastoris is majorly used for recombinant antibody production. ...
... Yarrowia lipolytica: Y. lipolytica is a Crabtree-negative ascomycete yeast with good protein secretion capacities. Compared to other yeasts, Y. lipolytica lacks α-1,3-mannosyltransferase, a factor that limits the amount of excessive mannosylation of secreted heterologous glycoproteins and constitutes a valuable asset for the production of therapeutic proteins [87]. Y. lipolytica W29 is a wild-type strain with a remarkable characteristically high secretion level of proteins [88]. ...
Polyolefins, including polyethylene (PE), polypropylene (PP) and polystyrene (PS), are widely used plastics in our daily life. The excessive use of plastics and improper handling methods cause considerable pollution in the environment, as well as waste of energy. The biodegradation of polyolefins seems to be an environmentally friendly and low-energy consumption method for plastics degradation. Many strains that could degrade polyolefins have been isolated from the environment. Some enzymes have also been identified with the function of polyolefin degradation. With the development of synthetic biology and metabolic engineering strategies, engineered strains could be used to degrade plastics. This review summarizes the current advances in polyolefin degradation, including isolated and engineered strains, enzymes and related pathways. Furthermore, a novel strategy for polyolefin degradation by artificial microbial consortia is proposed, which would be helpful for the efficient degradation of polyolefin.
... In order to remove the A-branch, expression of HDEL-tagged versions of α-1,2 mannosidase from A. saitoi or of α-1,2 mannosidase from T. reesei were used and yielded glycoproteins predominantly containing the Man 3 GlcNAc 2 glycan. This approach was implemented in P. pastoris, H. polymorpha, Y. lipolytica, A. niger and A. nidulans, respectively, however, was not effective in K. marxiani (Bobrowicz et al., 2004;Kainz et al., 2008;Oh et al., 2008;De Pourcq et al., 2012b;Lee et al., 2020;Anyaogu et al., 2021). Using this strategy, GnTI and GnTII activities are required in the Golgi apparatus. ...
Yeasts are widely used and established production hosts for biopharmaceuticals. Despite of tremendous advances on creating human-type N-glycosylation, N-glycosylated biopharmaceuticals manufactured with yeasts are missing on the market. The N-linked glycans fulfill several purposes. They are essential for the properties of the final protein product for example modulating half-lives or interactions with cellular components. Still, while the protein is being formed in the endoplasmic reticulum, specific glycan intermediates play crucial roles in the folding of or disposal of proteins which failed to fold. Despite of this intricate interplay between glycan intermediates and the cellular machinery, many of the glycoengineering approaches are based on modifications of the N-glycan processing steps in the endoplasmic reticulum (ER). These N-glycans deviate from the canonical structures required for interactions with the lectins of the ER quality control system. In this review we provide a concise overview on the N-glycan biosynthesis, glycan-dependent protein folding and quality control systems and the wide array glycoengineering approaches. Furthermore, we discuss how the current glycoengineering approaches partially or fully by-pass glycan-dependent protein folding mechanisms or create structures that mimic the glycan epitope required for ER associated protein degradation.
... Genomic data from this yeast has revealed the presence of protein families of 16 lipases, 4 esterases, 38 aspartyl proteases, and 10 alkaline proteases [36], indicating the ability to produce a diverse amount of proteins. Among those, lipases have been extensively studied regarding biochemical characterization, culture conditions for production, industrial and in vivo applications [50][51][52]. ...
... Until leaving the endoplasmic reticulum where, as a net result, a 10-residue Man8GlcNAc2 is transferred to the protein [70]. Yeast glycosylation is conserved among other types of eukaryotes (either higher or lower eukaryotes), differing in the Golgi apparatus after the action of glycosyltransferases [52]. However, the N-glycosylation patterns at N113 and N134 of lipase produced by Yarrowia lipolytica, that is, the ones obtained from Lip2 expression, do not seem to be critical for its catalytic activities (retain more activity for longer chain acyl substrates than for p-nitrophenyl butyrate) and secretion [71]. ...
Lipases are enzymes that, in aqueous or non-aqueous media, act on water-insoluble substrates, mainly catalyzing reactions on carboxyl ester bonds, such as hydrolysis, aminolysis, and (trans)esterification. Yarrowia lipolytica is a non-conventional yeast known for secreting lipases and other bioproducts; therefore, it is of great interest in various industrial fields. The production of lipases can be carried on solid-state fermentation (SSF) that utilizes solid substrates in the absence, or near absence, of free water and presents minimal problems with microbial contamination due to the low water contents in the medium. Moreover, SSF offers high volumetric productivity, targets concentrated compounds, high substrate concentration tolerance, and has less wastewater generation. In this sense, the present work provides a temporal evolution perspective regarding the main aspects of lipase production in SSF by Y. lipolytica, focusing on the most relevant aspects and presenting the potential of such an approach.
... Section 2.3.2) and reviewed previously [27]. The production of recombinant proteins for industrial or therapeutical applications represents an important research field that has been extensively reviewed previously [18][19][20][21][22]. The particular need to obtain more human-compatible recombinant glycoproteins for possible use as therapeutic agents has prompted the development of glyco-engineered (aka humanized) Y. lipolytica strains, which will be evoked in Section 3.1.6. ...
... Section 3.1.5). Notably, glyco-engineered strains able to produce glycoproteins homogeneously carrying Man5GlcNAc2 residues were obtained by the deletion of a two yeast-specific mannosyltransferases and heterologous expression of a fungal mannosidase [21]. Similarly, the mannosyltransferase deleted strain was further engineered by overexpression of a glucosyltransferase and heterologous overexpression of a fungal mannosidase and a fungal glucosidase, which generated a strain able to produce glycoproteins homogeneously carrying Man3GlcNAc2 residues, a core structure common to the different mammalian N-glycans that can additionally be modified in vitro to generate any kind of complex-type N-glycan [192]. ...
Among 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.
... This peculiarity, contrasting to the situation in S. cerevisiae and most yeasts for which secretion is mainly post-translational, allows Y. lipolytica to be very efficient in the folding and secretion of large and/or complex heterologous proteins and has contributed to its success as a heterologous production host [17][18][19]. In addition, Y. lipolytica is one of the few yeasts, with Pichia pastoris, which lacks an -1,3-mannosyltransferase, a factor that limits the amount of excessive mannosylation of secreted heterologous glycoproteins and constitutes a valuable asset for the production of therapeutic proteins [20,21]. ...
... Section 2.3.2). The production of recombinant proteins for industrial or therapeutical applications represents an important research field that has been extensively reviewed previously [17][18][19][20][21]. The particular need to obtain more human-compatible recombinant glycoproteins for possible use as therapeutic agents has prompted the development of glyco-engineered (aka humanized) Y. lipolytica strains, which will be evoked in Section 3.1.6. ...
... Section 3.1.5). Notably, glyco-engineered strains able to produce glycoproteins homogeneously carrying Man5GlcNAc2 residues were obtained by the deletion of a two yeast-specific mannosyltransferases and heterologous expression of a fungal mannosidase [20]. Similarly, the mannosyltransferase deleted strain was further engineered by overexpression of a glucosyltransferase and heterologous overexpression of a fungal mannosidase and a fungal glucosidase, which generated a strain able to produce glycoproteins homogeneously carrying Man3GlcNAc2 residues, a core structure common to the different mammalian N-glycans that can additionally be modified in vitro to generate any kind of complex-type N-glycan [161]. ...
Among 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.
... In parallel a group of Belgian laboratories also designed a glycoengineered strain by knocking out both yeast-specific mannosyltransferases and expressing a fungal mannosidase, allowing the production of recombinant proteins carrying homogeneous Man5GlcNAc2 residues (cf. glucan core in Fig. 2; De Pourcq et al., 2012a). Another project from the same authors, in which a mannosyltransferase knocked-out strain was then genetically modified to overexpress a glucosyltransferase and to coexpress fungal mannosidase and glucosidase, led to the production of homogeneous Man3-GlcNAc2 residues, which constitute the common core of all mammalian N-glycans (De Pourcq et al., 2012b). ...
After its first industrial uses, dating back to the 1950s, Yarrowia lipolytica has aroused interest as host for heterologous protein production. This oleaginous yeast has become a recognized expression system, with a remarkable secretion capacity. The development of tools for engineering its metabolic pathways has allowed its use as cell factory in a variety of bioconversion processes, notably single-cell oil and biofuel production and also valorization of industrial wastes into high-value compounds. The more recent development of new DNA assembly methods and of CRISPR genome editing technologies has boosted further the possibilities for Y. lipolytica engineering. Consequently the ever-increasing number of publications makes it now difficult for new players to acquire a general overview on the field. This chapter aims to provide basic knowledge on the subject, to direct novice readers to most useful recent reviews, and to provide an update on recently developed engineering tools and technological applications.
... Because hypermannosylation has been identified as a significant constraint for therapeutic glycoprotein production in wild type yeast cells [173], P. pastoris, S. cerevisiae, H. polymorpha, and Y. lipolytica have all been extensively glycoengineered to reduce this xenoantigenic glycan motif by knocking out genes for α-1,6 mannosyltransferase [174,175]. ...
... Beyond reducing hypermannosylation, yeast cells have been glycoengineered to produce human-like glycosylation through ectopic expression of branching [176], elongation [177], and mannose-trimming enzymes [175], which in many cases have been modified to include Golgi localisation components to ensure that the glycosylation reactions occur in the appropriate sequence [178,179]. ...
Therapeutic monoclonal antibodies (mAbs) are glycoproteins that contain a pair of N-glycosylation sites on their constant fragment and are widely prescribed for the treatment of several types of cancer and autoimmune disorders. Glycosylation is considered a critical quality attribute of mAbs because it is essential to the safety, pharmacokinetics, and pharmacodynamics of these life-saving biopharmaceuticals. High degrees of glycosylation variability have been observed across different production campaigns of the same mAb product and arise from the numerous biological reactions involved in the glycosylation process, their sensitivity to cell culture conditions, and the genetic background of the production host. Due to its influence in defining the quality of mAb products, substantial effort has been made to develop strategies that minimise mAb glycosylation heterogeneity. This chapter recapitulates the progress made towards controlling mAb glycosylation within the context of biopharmaceutical quality assurance. The chapter presents a critical review of the vast number of (i) cellular, (ii) metabolic, and (iii) in vitro glycoengineering strategies that have been developed to enhance the quality of therapeutic mAbs. We conclude by outlining how these strategies can be combined to achieve the complex task of manufacturing homogenous mAb glycoforms that elicit optimal therapeutic outcomes in the clinic.KeywordsN-linked glycosylationGlycoengineeringTherapeutics antibodiesCell EngineeringMetabolic glycoengineering
in vitro glycoengineering
... The strictly aerobic yeast Y. lipolytica is a promising cell factory for production of valuable compounds, such as organic acids [3,4,5], polyhydroxy alcohols [6, 7] and aromas [8]. High protein secretion capacity [9] and FDA awarded GRAS status [10] makes the yeast a potential platform for therapeutic protein production [11]. The most prominent characteristic of Y. lipolytica is, however, its ability to accumulate large amounts of intracellular lipids that can reach up to 20% of cell dry weight in wild-type strains [12]. ...
Yarrowia lipolytica is an important industrial microorganism used for the production of oleochemicals. The design of effective biotechnological processes with this cell factory requires an in-depth knowledge of its metabolism. Here we present a transcriptomic study of Y. lipolytica grown in the presence of glycerol and glucose, and mixture of both at different carbon to nitrogen ratios. It emerged that the transcriptomic landscape of Y. lipolytica is more sensitive to the nitrogen availability than to the utilized carbon source, as evidenced by more genes being differentially expressed in lower carbon to nitrogen ratio. Specifically, expression of hexokinase (HXK1) is significantly susceptible to changes in nitrogen concentrations. High HXK1 expression in low nitrogen seems to impact other genes which are implicated in tricarboxylic acid cycle and erythritol biosynthesis. We further show that expression of HXK1 and two genes belonging to the sugar porter family might be controlled by GATA-like zinc-finger proteins.
... Thus, not only is this a good method for selection, but also this is useful for minimizing contamination in fermentation process. Furthermore, marker rescue and reuse in Y. lipolytica can be realized by the Cre-lox recombination system [31]. An iterative gene integration method was recently created by combining the Cre-lox system with 26 s rDNA region. ...
Yarrowia lipolytica is an oleaginous yeast that has been substantially engineered for production of oleochemicals and drop-in transportation fuels. The unique acetyl-CoA/malonyl-CoA supply mode along with the versatile carbon-utilization pathways makes this yeast a superior host to upgrade low-value carbons into high-value secondary metabolites and fatty acid-based chemicals. The expanded synthetic biology toolkits enabled us to explore a large portfolio of specialized metabolism beyond fatty acids and lipid-based chemicals. In this review, we will summarize the recent advances in genetic, omics, and computational tool development that enables us to streamline the genetic or genomic modification for Y. lipolytica. We will also summarize various metabolic engineering strategies to harness the endogenous acetyl-CoA/malonyl-CoA/HMG-CoA pathway for production of complex oleochemicals, polyols, terpenes, polyketides, and commodity chemicals. We envision that Y. lipolytica will be an excellent microbial chassis to expand nature’s biosynthetic capacity to produce plant secondary metabolites, industrially relevant oleochemicals, agrochemicals, commodity, and specialty chemicals and empower us to build a sustainable biorefinery platform that contributes to the prosperity of a bio-based economy in the future.
