**2.** *N***-glycosylation in microalgae**

#### **2.1. General aspects**

*N*-glycosylation is a major post-translational modification of proteins in eukaryotes. Protein *N*-glycosylation first starts by the synthesis of a lipid-linked oligosaccharide formed by transfer of monosaccharides on a dolichol pyrophosphate (PP-Dol) anchored in the membrane of the endoplasmic reticulum (ER) *via* the action of a set of enzymes named asparagine-linked glycosylation (ALG) [22, 23]. The final Glc<sup>3</sup> Man9 GlcNAc2 precursor is transferred *en bloc* by the oligosaccharyltransferase (OST) complex onto the asparagine residues of the consensus Asn-X-Ser/Thr sequences of a protein [22] (**Figure 1**). Alternative consensus sequences, such as Asn-X-Cys and Asn-X-Val, have also been found to be glycosylated in some proteins [24–26].

of recombinant proteins [4–7]. Among different attempts to produce vaccines and biopharmaceuticals in microalgae, the production of monoclonal antibodies (mAbs) represents the most extensive work [7, 8]. Indeed, the first significant effort to produce recombinant mAb fragments was made in the green microalga *Chlamydomonas reinhardtii* with the synthesis and accumulation in its chloroplast of a human single chain antibody directed against the herpes simplex virus glycoprotein D (HSV8-lsc) [8]. Later, a full-length human IgG1 directed against anthrax was produced successfully in the chloroplast of *C. reinhardtii* [9]. The *Chlamydomonas*made mAb was able to bind the anthrax protective antigen 83 (PA83) [9]. In another study, a series of complex chimeric proteins was expressed in the chloroplast of *C. reinhardtii.* Such chimeric proteins were composed of a single chain antibody fragment (*scFv*) targeting the B-cell surface antigen CD22, genetically fused either to the eukaryotic ribosome inactivating protein, gelonin, from *Gelonium multiflorm* [10] or to *Pseudomonas aeruginosa* exotoxin A domains 2 and 3 [11]. These molecules, termed immunotoxins, were encoded by a single gene that produces an antibody-toxin chimeric protein. Such algae-made immunotoxins are able to bind target B cells and efficiently kill them *in vitro* [11]. Full-length mAbs have also been expressed in the diatom *Phaeodactylum tricornutum* through nuclear transformation [12–14]. Those mAbs correspond respectively to a recombinant mAb directed against the nucleoprotein of Marburg virus, a close relative of Ebola virus [14] and to a human IgG1 directed against the Hepatitis B virus Antigen (HBsAg) [12, 13]. The latter has been biochemically characterized in order to check the quality of the diatom-made mAb as well as its *N*-glycosylation profile [15]. Moreover, it has been demonstrated that this glycosylated antibody is able to bind human Fcy receptors

When the production of biopharmaceuticals is considered, their *N*-glycosylation has to be investigated. Indeed, among the biopharmaceuticals that were approved in 2016 and 2017, 96% were glycosylated [17]. The glycosylation of the approved biopharmaceutical represents a critical quality attribute (CQA) that may affect its safety and biological activities [18–20]. In addition, introduction by the expression system of nonhuman epitopes on the recombinant protein may induce immune response after injection to patients [21]. Thus, the *N-*glycosylation of biopharmaceuticals is a real challenge for the commercial production of biopharmaceuticals. The glycosylation state of therapeutic proteins has to be accurately identified and characterized as per the World Health Organization and International Conference on Harmonization Q6B guidelines [17]. Therefore, in the context of developing the microalgae as alternative platforms for the production of biopharmaceuticals, the capability of these unicellular eukaryotic cells to introduce *N*-glycans on their endogenous proteins and on recom-

binant proteins, as well as their regulation, have to be considered and understood.

*N*-glycosylation is a major post-translational modification of proteins in eukaryotes. Protein *N*-glycosylation first starts by the synthesis of a lipid-linked oligosaccharide formed by transfer of monosaccharides on a dolichol pyrophosphate (PP-Dol) anchored in the membrane of

[16], thus suggesting that it could be efficient in human therapy.

**2.** *N***-glycosylation in microalgae**

**2.1. General aspects**

178 Microalgal Biotechnology

**Figure 1.** Comparison of protein *N*-glycosylation pathways in eukaryotes. Biosynthesis steps occurring in the ER are gathered in the box. Mature *N*-glycan structures observed in mammals, plants, insects, yeasts and filamentous fungi are drawn according to [33]. , N-acetylglucosamine; , xylose; , mannose; , fucose; , galactose; , sialic acid; Asn, asparagine; PP-Dol, pyroPhosphate dolichol; FuT, fucosyltransferase; GalT, galactosyltransferase; SiaT, sialyltransferase; XylT, xylosyltransferase; ALG, asparagine-linked glycosylation; OST, oligosaccharyltransferase.

In the ER, neo-synthesized glycoproteins are then submitted to a quality control process through the deglucosylation by glucosidases and reglucosylation by an UDP-glucose: glycoprotein glucosyltransferase (UGGT) of the *N*-glycans. This allows the synthesis of monoglucosylated glycan intermediates that interact with ER-resident chaperones, thus ensuring proper folding of the glycoproteins [27]. When the glycoprotein is correctly folded, α-glucosidase II would finally remove the last glucose residue, and ER-mannosidase will eventually remove one mannose residue that leads to the formation of an oligomannoside Man9/8GlcNAc2 . The quality control events are conserved in eukaryotes because they are crucial for the secretion of well-folded proteins [28]. As a consequence, whatever the expression system used, a recombinant therapeutic protein leaving the ER compartment exhibits a *N*-glycosylation similar to one of the reference proteins with unique oligomannoside Man9/8GlcNAc2 attached to the Asn residue of the *N*-glycosylation consensus site.

After transfer to the Golgi apparatus, oligomannosides resulting from the ER processing are modified by the action of specific mannosidases and glycosyltransferases [29]. These Golgi cell-specific repertoires give rise to various organism-specific oligosaccharides. In most eukaryotes, a *N*-acetylglucosaminyltransferase I (GnT I)-dependent *N*-glycan processing occurs (**Figure 1**). In this pathway, the α-mannosidase I converts Man9/8GlcNAc2 into the branched isomer of Man5 GlcNAc2. Then, actions of GnT I, α-mannosidase II and GnT II, respectively, give rise to the core GlcNAc2 Man3 GlcNAc2 that is common to most eukaryotes [27–31] (**Figure 1**). This core is then decorated by the action of specific glycosyltransferases that differ from one organism to another. This allows the protein to be decorated by organism-specific *N*-glycans that confer to the mature protein *in vivo* bioactivities [32]. It is worth noting that GnT I-independent *N*-glycan processing also occurs in some eukaryotes such as filamentous fungi and yeasts in which *N*-glycosylation in the Golgi apparatus results in the synthesis of high mannose and hypermannose *N*-glycans, respectively (**Figure 1**). As a consequence, in the context of the production of biopharmaceuticals by genetic engineering, such a diversity of mature *N*-linked glycans is a limitation because the expression system used may introduce inappropriate epitopes and heterogeneous glycosylation on the therapeutics and may also fail in introducing glycan sequences that are required for *in vivo* bioactivity of the biopharmaceuticals.

oligomannosides, the structure of Man5

Man9

GlcNAc2

methylation occurring in the ER cannot be ruled out yet [38].

GlcNAc2

to [33]. , N-acetylglucosamine; , xylose; , mannose; , fucose; , galactose; Asn, asparagine; Me, methyl.

*N-*glycan profile from *P. tricornutum* has been described to contain Man3

sequences based on ESI-MSn analyses [40]. Although mature *N*-glycans from *Porphyridium* sp. and *C. reinhardtii* share common structural features, the location of the xylose residues on the *N*-glycan differs between these two microalgae (**Figure 2**). As mature *N*-glycans do not exhibit any terminal GlcNAc residues, they were proposed to result from Golgi xylosylation and *O*-methylation of oligomannosides deriving from the precursor synthesized in the ER in a GnT I-independent processing, even if this needs to be completely elucidated and that

**Figure 2.** Major mature N-linked glycans from the green microalga *Chlamydomonas reinhardtii* and *Botryococcus braunii*, the red microalga *Porphyridium* sp. and the diatom *Phaeodactylum tricornutum*. N-glycan structures are drawn according

Toward Future Engineering of the *N*-Glycosylation Pathways in Microalgae for Optimizing the Production…

*N*-glycans (**Figure 2**) [41]. In contrast to *Porphyridium sp*. and *C. reinhardtii*, these *N*-glycans result from a GnT I-dependent pathway (**Figure 2**) [41]. As evidence, GnT I gene predicted in the *P. tricornutum* genome encodes an enzyme able to restore the maturation of complex-type *N*-glycans in the CHO Lec1 mutant that lacks endogenous GnT I activity [41]. *N*-glycans arising from a GnT I-dependent pathway have also been recently reported in the green microalga *Botryococcus braunii* through a glycoproteomic approach [42]. In contrast to *P. tricornutum*, these *N*-glycans harbor a GlcNAc residue at the nonreducing end as well as mono- and di-*O*-methylations of the core mannose residue. Moreover, this *N*-glycan bearing a terminal GlcNAc resulting from the GnT I activity could be further elongated with an additional hexose or methyl-hexose residue. In addition, proteins from this green microalga also exhibit methylated *N*-linked oligomannosides carrying core fucose and core xylose residues (**Figure 2**) [42]. In support to these biochemical data, protein *N*-glycosylation in microalgae can be drawn on the basis of public genomic databases. Microalgae genomes from different phyla are available

oligomannosides and also minute amount of paucimannosidic fucosylated

was re-evaluated in 2017 as being linear

http://dx.doi.org/10.5772/intechopen.73401

181

GlcNAc2

to

#### **2.2. Protein** *N***-glycosylation in microalgae**

Overall, protein *N*-glycosylation in microalgae received little attention. Few studies, published in the 1990s have demonstrated that proteins secreted by green microalgae carry mainly oligomannosides or xylose-containing *N*-glycans based on affinodetection or enzymatic sequencing [34–36]. More recently, analysis by mass spectrometry of glycans *N*-linked to microalgae endogenous proteins has been reported. First, the 66 kDa cell wall glycoprotein from the red microalga *Porphyridium* sp. has been found to carry Man8 GlcNAc2 and Man9 GlcNAc2 oligomannosides containing 6-*O*-methyl mannose residues and substituted by one or two xylose residues [37, 38] (**Figure 2**). Investigation of *C. reinhardtii* has demonstrated that proteins in this green microalga carry oligomannosides ranging from Man2 GlcNAc2 to Man5 GlcNAc2 as well as Man4 GlcNAc2 and Man5 GlcNAc2 *N*-glycans containing 6-*O*-methyl mannoses and substituted by one or two xylose residues (**Figure 2**) [39]. Initially reported as branched

Toward Future Engineering of the *N*-Glycosylation Pathways in Microalgae for Optimizing the Production… http://dx.doi.org/10.5772/intechopen.73401 181

In the ER, neo-synthesized glycoproteins are then submitted to a quality control process through the deglucosylation by glucosidases and reglucosylation by an UDP-glucose: glycoprotein glucosyltransferase (UGGT) of the *N*-glycans. This allows the synthesis of monoglucosylated glycan intermediates that interact with ER-resident chaperones, thus ensuring proper folding of the glycoproteins [27]. When the glycoprotein is correctly folded, α-glucosidase II would finally remove the last glucose residue, and ER-mannosidase will eventually remove one mannose residue that leads to the formation of an oligomannoside Man9/8GlcNAc2

quality control events are conserved in eukaryotes because they are crucial for the secretion of well-folded proteins [28]. As a consequence, whatever the expression system used, a recombinant therapeutic protein leaving the ER compartment exhibits a *N*-glycosylation similar to

After transfer to the Golgi apparatus, oligomannosides resulting from the ER processing are modified by the action of specific mannosidases and glycosyltransferases [29]. These Golgi cell-specific repertoires give rise to various organism-specific oligosaccharides. In most eukaryotes, a *N*-acetylglucosaminyltransferase I (GnT I)-dependent *N*-glycan processing occurs (**Figure 1**). In this pathway, the α-mannosidase I converts Man9/8GlcNAc2

Man3

[27–31] (**Figure 1**). This core is then decorated by the action of specific glycosyltransferases that differ from one organism to another. This allows the protein to be decorated by organism-specific *N*-glycans that confer to the mature protein *in vivo* bioactivities [32]. It is worth noting that GnT I-independent *N*-glycan processing also occurs in some eukaryotes such as filamentous fungi and yeasts in which *N*-glycosylation in the Golgi apparatus results in the synthesis of high mannose and hypermannose *N*-glycans, respectively (**Figure 1**). As a consequence, in the context of the production of biopharmaceuticals by genetic engineering, such a diversity of mature *N*-linked glycans is a limitation because the expression system used may introduce inappropriate epitopes and heterogeneous glycosylation on the therapeutics and may also fail in introducing glycan sequences that are required for *in vivo* bioactivity of the

Overall, protein *N*-glycosylation in microalgae received little attention. Few studies, published in the 1990s have demonstrated that proteins secreted by green microalgae carry mainly oligomannosides or xylose-containing *N*-glycans based on affinodetection or enzymatic sequencing [34–36]. More recently, analysis by mass spectrometry of glycans *N*-linked to microalgae endogenous proteins has been reported. First, the 66 kDa cell wall glycoprotein from the red

mannosides containing 6-*O*-methyl mannose residues and substituted by one or two xylose residues [37, 38] (**Figure 2**). Investigation of *C. reinhardtii* has demonstrated that proteins in

and substituted by one or two xylose residues (**Figure 2**) [39]. Initially reported as branched

GlcNAc2

GlcNAc2. Then, actions of GnT I, α-mannosidase II and GnT II,

GlcNAc2

GlcNAc2 *N*-glycans containing 6-*O*-methyl mannoses

and Man9

GlcNAc2

GlcNAc2

to Man5

oligo-

GlcNAc2

one of the reference proteins with unique oligomannoside Man9/8GlcNAc2

residue of the *N*-glycosylation consensus site.

respectively, give rise to the core GlcNAc2

**2.2. Protein** *N***-glycosylation in microalgae**

GlcNAc2

microalga *Porphyridium* sp. has been found to carry Man8

this green microalga carry oligomannosides ranging from Man2

and Man5

the branched isomer of Man5

180 Microalgal Biotechnology

biopharmaceuticals.

as well as Man4

. The

into

attached to the Asn

that is common to most eukaryotes

**Figure 2.** Major mature N-linked glycans from the green microalga *Chlamydomonas reinhardtii* and *Botryococcus braunii*, the red microalga *Porphyridium* sp. and the diatom *Phaeodactylum tricornutum*. N-glycan structures are drawn according to [33]. , N-acetylglucosamine; , xylose; , mannose; , fucose; , galactose; Asn, asparagine; Me, methyl.

oligomannosides, the structure of Man5 GlcNAc2 was re-evaluated in 2017 as being linear sequences based on ESI-MSn analyses [40]. Although mature *N*-glycans from *Porphyridium* sp. and *C. reinhardtii* share common structural features, the location of the xylose residues on the *N*-glycan differs between these two microalgae (**Figure 2**). As mature *N*-glycans do not exhibit any terminal GlcNAc residues, they were proposed to result from Golgi xylosylation and *O*-methylation of oligomannosides deriving from the precursor synthesized in the ER in a GnT I-independent processing, even if this needs to be completely elucidated and that methylation occurring in the ER cannot be ruled out yet [38].

*N-*glycan profile from *P. tricornutum* has been described to contain Man3 GlcNAc2 to Man9 GlcNAc2 oligomannosides and also minute amount of paucimannosidic fucosylated *N*-glycans (**Figure 2**) [41]. In contrast to *Porphyridium sp*. and *C. reinhardtii*, these *N*-glycans result from a GnT I-dependent pathway (**Figure 2**) [41]. As evidence, GnT I gene predicted in the *P. tricornutum* genome encodes an enzyme able to restore the maturation of complex-type *N*-glycans in the CHO Lec1 mutant that lacks endogenous GnT I activity [41]. *N*-glycans arising from a GnT I-dependent pathway have also been recently reported in the green microalga *Botryococcus braunii* through a glycoproteomic approach [42]. In contrast to *P. tricornutum*, these *N*-glycans harbor a GlcNAc residue at the nonreducing end as well as mono- and di-*O*-methylations of the core mannose residue. Moreover, this *N*-glycan bearing a terminal GlcNAc resulting from the GnT I activity could be further elongated with an additional hexose or methyl-hexose residue. In addition, proteins from this green microalga also exhibit methylated *N*-linked oligomannosides carrying core fucose and core xylose residues (**Figure 2**) [42].

In support to these biochemical data, protein *N*-glycosylation in microalgae can be drawn on the basis of public genomic databases. Microalgae genomes from different phyla are available to date (https://genome.jgi.doe.gov/pages/tree-of-life.jsf) [4, 43]. Since protein *N*-glycosylation occurs in the ER and the Golgi apparatus, bioinformatics analyses of microalgae genomes must be investigated independently for the two compartments: search for gene encoding proteins involved in the precursor biosynthesis and the ER protein quality control on the one hand, and search for Golgi glycosidases and glycosyltransferases involved in the synthesis of mature *N*-glycans on the other hand.

would be challenging and requiring metabolic engineering of the *N*-glycosylation pathway in microalgae. This will include the inactivation of enzymes that introduce nonhuman glycoepitopes onto *N*-linked glycans and complementation of microalgae with appropriate glycosyltransferases to introduce missing glycan sequences. These strategies have already been successfully carried out for the engineering of the *N*-glycan pathways in plants and yeasts [46, 47]. In addition, the success of the complementation with human glycosyltransferases requires the availability in the Golgi apparatus of appropriate nucleotide-activated sugars [48]. For instance, sialic acids that terminate bi-antennary *N*-glycans in mammals have not been reported in microalgae such as *P. tricornutum* and *Porphyridium* sp. [38, 41]. As well, there is no evidence for the import of GlcNAc in the Golgi apparatus in microalgae exhibiting a GnT I-independent *N*-glycan pathway, even if putative candidates for UDP-GlcNAc transporter have been identified in microalgae such as *C. reinhardtii* [49]. Indeed, the two GlcNAc of the chitobiose unit of *N*-linked glycans are transferred onto the PP-Dol lipid in the cytosolic face of the ER membrane. Currently, metabolic engineering strategies are now feasible due to the recent development of transgene expression and gene inactivation in microalgae as sum-

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**3. Genetic engineering tools now available to envision future** *N***-**

of transformed cells. For example, *P. tricornutum* transformation reached 1 per 106

Classical strategies of genetic engineering involve the modulation of gene expression including overexpression and inactivation by RNA interference [50–52]. The most used engineering methods are based on random insertional mutagenesis obtained by various processes such as conjugation, agitation with glass beads, electroporation, biolistic microparticle bombardment, agrobacterium-mediated transformation or multipulse electroporation. The transformation step is followed by phenotypic selection using antibiotics to generate genome-modified organisms [53]. Those processes present the advantage to be simple and reach a high level

biolistic bombardment system [54]. However, cell-wall-less strains are required for almost all the classical methods quoted above [50, 55]. Furthermore, genetic stability of the mutagenesis obtained after transformation by random insertion depends on microalgae species [53]. For example, a high mutagenesis stability has been shown in *C. reinhardtii* [55]. Unlike, mutagenesis was unstable in *Thalassosiara weissflogii* [56]. More recently, new tools have been developed in order to knock in, knock out, modify, replace, or insert genes. These new genetic engineering tools consist of the action of nucleases effecting their molecular scissor activities in specific loci [52]. A break in the DNA causes activation of DNA repair mechanisms, which can be either the homologous-recombination (HR) or the non-homologous end-joining (NHEJ) [52]. The HR results in sequence modification in the target locus [57]. In the NHEJ process, the two ends of the broken chromosome are stuck together causing small deletions or small insertions [57]. These events confer several modifications of the target gene such as gene inactivations or

cells with

**glycosylation engineering in microalgae**

**3.1. Different tools to generate genome-modified organisms**

marized in Section 3.

Genes encoding subunits of OST, glucosidases, as well as ER-resident UGGT and chaperones are predicted in microalgae genomes suggesting that the process of ER quality control in these unicellular organisms is similar to the one described in other eukaryotic cells [41, 44, 45]. Among these putative ER candidates, only the activity of the α(1,3)-glucosidase, also called glucosidase II, from the red microalga *Porphyridium* sp. has been biochemically confirmed [44]. Most ALG genes are also predicted in microalgae genomes [39, 41, 44] suggesting that the synthesis of the oligosaccharide precursor is overall conserved. However, some of these ALG, that is ALG3, ALG9 and ALG12, are not predicted in *C. reinhardtii* [39, 45]. These ER enzymes are involved in the completion of the biosynthesis of the precursor Man9 GlcNAc2 - PP-Dol, prior to its glucosylation, by addition of mannose residues on the α(1,6)-mannose arm of the core (**Figure 1**). Reinvestigation in *C. reinhardtii* of the structure of oligomannosides and analysis of the ER *N*-glycan precursor [40] confirmed the absence of ALG3, ALG9 and ALG12 activities and the synthesis in this green microalga of linear oligomannoside sequences instead of branched isomer initially proposed in [39]. It is worth noting that in this truncated ER pathway, the presence of the triglucosyl extension is likely sufficient to ensure interaction of the *N*-glycan precursor with chaperones of the ER quality control process. In addition to the lack of the ALG3, ALG9 and ALG12 in *C. reinhardtii*, other microalgae genomes lack genes encoding ALG10 and GCS1, an α(1,2)-glucosidase [44]. Because ALG10 is the α(1, 2)-glucosyltransferase responsible for the addition of the α(1, 2)-glucose residue on the precursor *N*-glycan and GCS1 is responsible for trimming this residue, we hypothesize that the ER quality control in these microalgae involved only diglucosylated *N*-glycan intermediates.

With regard to Golgi *N*-glycosylation events, the presence of GnT I is predicted in some microalgae including haptophytes and cryptophytes, but not in *C. reinhardtii*, *Volvox* and *Ostreococcus* [41, 42]. As mentioned previously, *P. tricornutum* GnT I activity was confirmed by the complementation of CHO Lec 1 mutant cell line [41]. A recent study of *B. braunii* [42] confirmed the involvement of this transferase in this green microalga *N*-glycosylation pathway. Concerning other Golgi enzymes, α-mannosidases (CAZy GH 47) and α(1,3) fucosyltransferases (CAZy GT10) are also predicted in microalgae genomes studied so far [41, 44, 45]. These enzymes are respectively involved in the trimming of mannose residue of oligomannosides and the transfer of fucose on the proximal GlcNAc. These sequences exhibit peptide motifs that were demonstrated to be required for activities of such Golgi enzymes, but, in contrast to GnT I, no biochemical data of their activity and specificity are available yet.

As depicted, protein *N*-glycosylation occurring in microalgae is specific and largely differs from the one described in mammals (**Figures 1** and **2**). Therefore, production in microalgae of biopharmaceuticals exhibiting *N*-glycans compatible with their use in human therapy would be challenging and requiring metabolic engineering of the *N*-glycosylation pathway in microalgae. This will include the inactivation of enzymes that introduce nonhuman glycoepitopes onto *N*-linked glycans and complementation of microalgae with appropriate glycosyltransferases to introduce missing glycan sequences. These strategies have already been successfully carried out for the engineering of the *N*-glycan pathways in plants and yeasts [46, 47]. In addition, the success of the complementation with human glycosyltransferases requires the availability in the Golgi apparatus of appropriate nucleotide-activated sugars [48]. For instance, sialic acids that terminate bi-antennary *N*-glycans in mammals have not been reported in microalgae such as *P. tricornutum* and *Porphyridium* sp. [38, 41]. As well, there is no evidence for the import of GlcNAc in the Golgi apparatus in microalgae exhibiting a GnT I-independent *N*-glycan pathway, even if putative candidates for UDP-GlcNAc transporter have been identified in microalgae such as *C. reinhardtii* [49]. Indeed, the two GlcNAc of the chitobiose unit of *N*-linked glycans are transferred onto the PP-Dol lipid in the cytosolic face of the ER membrane. Currently, metabolic engineering strategies are now feasible due to the recent development of transgene expression and gene inactivation in microalgae as summarized in Section 3.
