Engineering Yarrowia lipolytica to Produce Glycoproteins Homogeneously Modified with the Universal Man3GlcNAc2 N-Glycan Core

Yarrowia lipolytica is a dimorphic yeast that efficiently secretes various heterologous proteins and is classified as “generally recognized as safe.” Therefore, it is an attractive protein production host. However, yeasts modify glycoproteins with non-human high mannose-type N-glycans. These structures reduce the protein half-life in vivo and can be immunogenic in man. Here, we describe how we genetically engineered N-glycan biosynthesis in Yarrowia lipolytica so that it produces Man3GlcNAc2 structures on its glycoproteins. We obtained unprecedented levels of homogeneity of this glycanstructure. This is the ideal starting point for building human-like sugars. Disruption of the ALG3 gene resulted in modification of proteins mainly with Man5GlcNAc2 and GlcMan5GlcNAc2 glycans, and to a lesser extent with Glc2Man5GlcNAc2 glycans. To avoid underoccupancy of glycosylation sites, we concomitantly overexpressed ALG6. We also explored several approaches to remove the terminal glucose residues, which hamper further humanization of N-glycosylation; overexpression of the heterodimeric Apergillus niger glucosidase II proved to be the most effective approach. Finally, we overexpressed an α-1,2-mannosidase to obtain Man3GlcNAc2 structures, which are substrates for the synthesis of complex-type glycans. The final Yarrowia lipolytica strain produces proteins glycosylated with the trimannosyl core N-glycan (Man3GlcNAc2), which is the common core of all complex-type N-glycans. All these glycans can be constructed on the obtained trimannosyl N-glycan using either in vivo or in vitro modification with the appropriate glycosyltransferases. The results demonstrate the high potential of Yarrowia lipolytica to be developed as an efficient expression system for the production of glycoproteins with humanized glycans

Engineering the yeast <it>Yarrowia lipolytica</it> for the production of therapeutic proteins homogeneously glycosylated with Man₈GlcNAc₂ and Man₅GlcNAc₂

<p>Abstract</p> <p>Background</p> <p>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. <it>Yarrowia lipolytica</it> is a suitable host that is generally regarded as safe (GRAS). Yeasts, however, modify their glycoproteins with heterogeneous glycans containing mainly mannoses, which complicates downstream processing and often interferes with protein function in man. Our aim was to glyco-engineer <it>Y. lipolytica</it> to abolish the heterogeneous, yeast-specific glycosylation and to obtain homogeneous human high-mannose type glycosylation.</p> <p>Results</p> <p>We engineered <it>Y. lipolytica</it> to produce homogeneous human-type terminal-mannose glycosylated proteins, <it>i.e.</it> glycosylated with Man₈GlcNAc₂ or Man₅GlcNAc₂. First, we inactivated the yeast-specific Golgi α-1,6-mannosyltransferases <it>Yl</it>Och1p and <it>Yl</it>Mnn9p; the former inactivation yielded a strain producing homogeneous Man₈GlcNAc₂ glycoproteins. We tested this strain by expressing glucocerebrosidase and found that the hypermannosylation-related heterogeneity was eliminated. Furthermore, detailed analysis of N-glycans showed that <it>Yl</it>Och1p and <it>Yl</it>Mnn9p, despite some initial uncertainty about their function, are most likely the α-1,6-mannosyltransferases responsible for the addition of the first and second mannose residue, respectively, to the glycan backbone. Second, introduction of an ER-retained α-1,2-mannosidase yielded a strain producing proteins homogeneously glycosylated with Man₅GlcNAc₂. The use of the endogenous LIP2pre signal sequence and codon optimization greatly improved the efficiency of this enzyme.</p> <p>Conclusions</p> <p>We generated a <it>Y. lipolytica</it> expression platform for the production of heterologous glycoproteins that are homogenously glycosylated with either Man₈GlcNAc₂ or Man₅GlcNAc₂ N-glycans. This platform expands the utility of <it>Y. lipolytica</it> as a heterologous expression host and makes it possible to produce glycoproteins with homogeneously glycosylated N-glycans of the human high-mannose-type, which greatly broadens the application scope of these glycoproteins.</p

Engineering of glycosylation in yeast and other fungi: current state and perspectives

With the increasing demand for recombinant proteins and glycoproteins, research on hosts for producing these proteins is focusing increasingly on more cost-effective expression systems. Yeasts and other fungi are promising alternatives because they provide easy and cheap systems that can perform eukaryotic post-translational modifications. Unfortunately, yeasts and other fungi modify their glycoproteins with heterogeneous high-mannose glycan structures, which is often detrimental to a therapeutic protein’s pharmacokinetic behavior and can reduce the efficiency of downstream processing. This problem can be solved by engineering the glycosylation pathways to produce homogeneous and, if so desired, human-like glycan structures. In this review, we provide an overview of the most significant recently reported approaches for engineering the glycosylation pathways in yeasts and fungi

DSA-FACE analysis of engineered <i>Y. lipolytica</i> strains.

<p>A, oligomaltose reference. B–K, N-glycans derived from different sources: B, bovine RNaseB reference; C, MTLY60 wild type strain; D, <i>alg3</i> knock-out strain; E, <i>alg3</i> mutant strain overexpressing Alg6p. F–J, the <i>alg3</i> mutant strain overexpressing Alg6p engineered with: F, <i>Y. lipolytica</i> GIIα; G, <i>Y. lipolytica</i> GIIα HDEL-tagged; H, both α and β subunits of <i>Y. lipolytica</i> GII; I, the HDEL-tagged <i>A. niger</i> GIIα; J, both α and β subunits of <i>A. niger</i> GII. K, The latter strain engineered with an HDEL-tagged <i>T. reesei</i> α-1,2-mannosidase. This fully engineered strain produces glycoproteins with more than 85% trimannosyl core N-glycans.</p

<i>T. brucei</i> GII and mutanase tested as engineering approach.

<p>(<b>A</b>) <b>The dual N-glycosylation system in </b><b><i>T. brucei</i></b><b>.</b> Both Man₉GlcNAc₂ and Man₅GlcNAc₂ can be transferred to proteins. Next, these proteins are reglucosylated and deglucosylated in the folding cycle by glucosyltransferase and GII, respectively. (<b>B</b>) <b>DSA-FACE analysis of reference N-glycans and N-glycans derived from strains engineered with </b><b><i>T. brucei</i></b><b> GII or treated with mutanase.</b> A, Oligomaltose reference. B, N-glycans from RNaseB reference. C, N-glycans from the <i>alg3</i> mutant strain overexpressing Alg6p. D-F, N-glycan from the <i>alg3</i> mutant strain overexpressing Alg6p and engineered in different ways: D, engineered with <i>T. brucei</i> GII; E, engineered with <i>T. brucei</i> GII with HDEL tag; F, engineered with <i>T. brucei</i> GII with HDEL tag and pre-lip2 signal. G, N-glycans derived from the <i>alg3</i> mutant strain overexpressing Alg6p treated with mutanase.</p

Primers used in this study.

<p>Restriction sites in ‘’ refer to overhanging parts of them.</p

<i>Y. lipolytica</i> strains used in this study.

<p><i>Y. lipolytica</i> strains used in this study.</p

N-glycosylation and engineering thereof in yeast. (A) N-glycosylation in wild type yeast and (B) The approach used to engineer the yeast specific pathway.

<p>A: Standard N-glycosylation pathway in the ER. The early steps in N-glycosylation start with the synthesis of a dolichol-linked Man₅GlcNAc₂ glycan precursor that flips to the ER lumen, where it is further elongated with mannoses starting with the activity of Alg3p mannosyltransferase. The resulting dolichol-linked Man₉GlcNAc₂ precursor is then also glucosylated starting with the activity of Alg6p glucosyltransferase. When complete, the Glc₃Man₉GlcNAc₂ glycan is transferred <i>en bloc</i> to the nascent polypeptide chain. These glycans are then subjected to a protein folding quality control process involving de-glucosylation by glucosidases I and II (GI, GII) and re-glucosylation glucosyltransferase. B: The engineering strategies used to obtain a <i>Y. lipolytica</i> strain that produces glycoproteins homogeneously modified with the trimannosyl core N-glycan (Man₃GlcNAc₂). First, <i>ALG3</i> was knocked out (1), then Alg6p was overexpressed (2), then GII was overexpressed (3), and finally α-1,2-mannosidase was overexpressed (4). Conforming to the representation proposed by the Consortium for Functional Glycomics Nomenclature Committee, the green and blue spheres represent mannose (Man) and glucose (Glc), respectively, and blue squares represent N-acetylglucosamine residues (GlcNAc). C: Man₃GlcNAc₂-glycans can be further modified to any complex-type N-glycan structure using a combination of glycosyl-transferases, either <i>in vitro</i> or <i>in vivo</i>.</p

Identification of N-glycans by exoglycosidase digestion and DSA-FACE analysis.

<p>A: Oligomaltose reference. B, N-glycans from RNaseB reference. C–G, N-glycans from different strains: C, MTLY60 wild type strain; D, <i>alg3</i> knock-out strain; E, The same as D but treated with α-1,2-mannosidase; F, The same as D but treated with JB α-mannosidase; G, The same as D but treated with glucosidase II. The N-glycan structures in the <i>alg3</i> knock-out strain are consistent with Man₅GlcNAc₂, GlcMan₅GlcNAc₂ and Glc₂Man₅GlcNAc₂.</p