**3. Genetic engineering tools now available to envision future** *N***glycosylation engineering in microalgae**

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

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

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

microalgae involved only diglucosylated *N*-glycan intermediates.

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

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

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

GlcNAc2 -

mature *N*-glycans on the other hand.

182 Microalgal Biotechnology

available yet.

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 of transformed cells. For example, *P. tricornutum* transformation reached 1 per 106 cells with 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 insertions. Very little is known about these mechanisms in microalgae due to their complexity as reported by Daboussi in 2017 [53].

**The clustered regularly interspaced short palindromic repeats (CRISPR)/cas9** system is the most famous engineered nuclease system of this decade because it is a powerful and precise tool applied in numerous eukaryotic organisms [69]. This system is based on the RNA-guided DNA cleavage defense system from archaea and many bacteria. Indeed, these organisms are able to store bacteriophage DNA fragments along a previous bacteriophage infection in the CRISPR locus, which is formed of DNA repeat sequences spaced by a unique DNA sequence. This system establishes the basis of a bacterial defense as a response to bacteriophage attacks [70]. This defense mechanism has been highlighted for the first time by Pr Emmanuelle Charpentier and her team in 2011 [70, 71]. The CRISPR/Cas9 system has been developed into a simple toolkit based on a custom single guide RNA (sgRNA) that contains a targeting sequence (crRNA sequence) and a cas9 nuclease-recruiting sequence (tracrRNA) [52]. In microalgae, CRISPR/cas9 has been used in *C. reinhardtii* [72]. However, the Cas9 nuclease production seemed to be toxic for the microalga limiting efficiency to obtain genome-modified strains [72]. Two years later, a new assay has been performed in this same microalga using another strategy avoiding toxicity [73]. Indeed, the authors succeeded to generate CRISPR/ cas9-induced NHEJ-mediated knock-in mutant strains in three loci [73]. In the same year, CRISPR/cas9 gene knockout technology has been used in *P. tricornutum* to induce mutant for the *CpSRP43* gene, a member of the chloroplast signal recognition particle pathway. Using

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

**MN system ZFN system TALEN system CRISPR/cas9**

Chimeric endonuclease

Xanthomonas [78] Xanthomonas [78] Bacteria and

Expensive (3000–5000\$) RNA guide and cas9

nuclease

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

185

Archaea [70]

Cheap (500\$)

endonuclease

Toxicity in cells Low Moderate1 Moderate1 Moderate [72]

(4000–7000\$)

Source: https://www.news-medical.net/life-sciences/How-Does-CRISPR-Compare-to-Other-Gene-Editing-Techniques.

Source: http://www.biocompare.com/Editorial-Articles/144186-Genome-Editing-with-CRISPRs-TALENs-and-ZFNs/

**Table 1.** Comparison of four specific genomic tools based on nuclease systems in order to generate genomic-modified

Time investment Very high [52] Very high [64] Moderate Low

Low Moderate Moderate High

No No Yes Yes

Up to 29% [59] Not reported Up to 56% [59] Up to 63% [74]

Chimeric endonuclease Chimeric

*Chlamydomonas reinhardtii Saccharomyces cerevisiae*

System cost2 Not reported Expensive

aspx visited [Accessed: 2017-12-06].

[Accessed: 2017-12-06].

species in microalgae.

[59, 77]

Actor(s) of gene targeting

origin

Nuclease specificity

Mutagenesis frequency in microalgae

Possibility of multiple gene targeting

1

2

Engineered protein

Several researches have recently contributed to demonstrate that particular nucleases could be used for targeting stable modifications by acting like molecular scissors. Among these nucleases, we can quote meganucleases (MNs), zinc finger nucleases (ZFNs), transcriptor activator-like effector nucleases (TALENs) and finally, the famous clustered regularly interspaced short palindromic repeats (CRISPR)/nuclease Cas9 system. These four cited nucleases are described in the following paragraphs.

**Meganuclease** is an engineered endonuclease able to recognize and cleave a long specific DNA sequence from 18 to 30 base pairs. The meganuclease strategy requires to design a homing endonuclease from the LAGLIDADG family especially the I-*CreI* enzyme from *C. reinhardtii* implied in the targeting of interesting gene sequences that need to be modified [58]. This was tested for the first time in 2014 using *P. tricornutum* as a model [59]. In this study, two engineered meganucleases targeting genes involved in the lipid metabolism are allowed to obtain 29% of targeted mutagenesis [59]. Even successful, this strategy is time-consuming as compared to the other alternatives [52].

**Zinc finger nucleases (ZFNs)** are hybrid proteins composed of a restriction enzyme *FokI* with a designed zinc-finger DNA-binding domains [60]. These *FokI* enzymes are inactive in a homodimer conformation [61]. Therefore, cleavage of a typical DNA-target sequence requires to design two different ZFNs for binding to adjacent half-sites of a specific locus. Each designed ZFN is able to recognize a sequence of 9–12 nucleotides in the genome [52]. A set of zinc finger nucleases has been recently used to modify by insertion of template DNA, the *Cop3* gene locus encoding a light-activated channel in *C. reinhardtii* [62]. Moreover, in 2017, the genome editing was reliably performed using the ZFN strategy in order to inhibit and modify nuclear photoreceptor genes in this same microalga [63]. Despite these promising results, the ZFN system is barely used because of its low specificity. Indeed, cleavage of DNA requires both ZFN monomers to recognize a homologous target in the genome in the proper spatial orientation to assemble a functional ZFN [64]. Also, ZFN system is time-consuming implementation [64]. Nowadays, other designed nucleases like TALENs or CRISPR/cas9 are emerging in the scientific community to perform genome editing in microalgae.

**Transcriptional activator-like effector nuclease (TALEN)** system is similar to ZFN because it uses nucleases composed of a restriction enzyme domain fused to a DNA-binding domain (here the TAL effector domain) and a nonspecific DNA cleavage domain *FokI* [65]. TALEN proteins are characterized by a repeated 34-amino acid sequence that recognizes specific DNA sequences [66]. *P. tricornutum* lipid metabolism was recently modified using TALEN [59]. In this study, seven genes involved in this metabolism were modified. Each genome modification had a high frequency reaching up to 56% of colonies with targeted mutagenesis [59]. This genetic engineering allowed creation of a high lipid-producing strain by inactivating a key gene for carbohydrate energy storage [59]. Another team has inactivated successfully the urease gene in *P. tricornutum* with 24% of transformed colonies [67]. In addition, TALEN system has also been used in order to inactivate red/far-red light-sensing *phytochrome* gene of this diatom [68].

**The clustered regularly interspaced short palindromic repeats (CRISPR)/cas9** system is the most famous engineered nuclease system of this decade because it is a powerful and precise tool applied in numerous eukaryotic organisms [69]. This system is based on the RNA-guided DNA cleavage defense system from archaea and many bacteria. Indeed, these organisms are able to store bacteriophage DNA fragments along a previous bacteriophage infection in the CRISPR locus, which is formed of DNA repeat sequences spaced by a unique DNA sequence. This system establishes the basis of a bacterial defense as a response to bacteriophage attacks [70]. This defense mechanism has been highlighted for the first time by Pr Emmanuelle Charpentier and her team in 2011 [70, 71]. The CRISPR/Cas9 system has been developed into a simple toolkit based on a custom single guide RNA (sgRNA) that contains a targeting sequence (crRNA sequence) and a cas9 nuclease-recruiting sequence (tracrRNA) [52]. In microalgae, CRISPR/cas9 has been used in *C. reinhardtii* [72]. However, the Cas9 nuclease production seemed to be toxic for the microalga limiting efficiency to obtain genome-modified strains [72]. Two years later, a new assay has been performed in this same microalga using another strategy avoiding toxicity [73]. Indeed, the authors succeeded to generate CRISPR/ cas9-induced NHEJ-mediated knock-in mutant strains in three loci [73]. In the same year, CRISPR/cas9 gene knockout technology has been used in *P. tricornutum* to induce mutant for the *CpSRP43* gene, a member of the chloroplast signal recognition particle pathway. Using

insertions. Very little is known about these mechanisms in microalgae due to their complexity

Several researches have recently contributed to demonstrate that particular nucleases could be used for targeting stable modifications by acting like molecular scissors. Among these nucleases, we can quote meganucleases (MNs), zinc finger nucleases (ZFNs), transcriptor activator-like effector nucleases (TALENs) and finally, the famous clustered regularly interspaced short palindromic repeats (CRISPR)/nuclease Cas9 system. These four cited nucleases

**Meganuclease** is an engineered endonuclease able to recognize and cleave a long specific DNA sequence from 18 to 30 base pairs. The meganuclease strategy requires to design a homing endonuclease from the LAGLIDADG family especially the I-*CreI* enzyme from *C. reinhardtii* implied in the targeting of interesting gene sequences that need to be modified [58]. This was tested for the first time in 2014 using *P. tricornutum* as a model [59]. In this study, two engineered meganucleases targeting genes involved in the lipid metabolism are allowed to obtain 29% of targeted mutagenesis [59]. Even successful, this strategy is time-consuming

**Zinc finger nucleases (ZFNs)** are hybrid proteins composed of a restriction enzyme *FokI* with a designed zinc-finger DNA-binding domains [60]. These *FokI* enzymes are inactive in a homodimer conformation [61]. Therefore, cleavage of a typical DNA-target sequence requires to design two different ZFNs for binding to adjacent half-sites of a specific locus. Each designed ZFN is able to recognize a sequence of 9–12 nucleotides in the genome [52]. A set of zinc finger nucleases has been recently used to modify by insertion of template DNA, the *Cop3* gene locus encoding a light-activated channel in *C. reinhardtii* [62]. Moreover, in 2017, the genome editing was reliably performed using the ZFN strategy in order to inhibit and modify nuclear photoreceptor genes in this same microalga [63]. Despite these promising results, the ZFN system is barely used because of its low specificity. Indeed, cleavage of DNA requires both ZFN monomers to recognize a homologous target in the genome in the proper spatial orientation to assemble a functional ZFN [64]. Also, ZFN system is time-consuming implementation [64]. Nowadays, other designed nucleases like TALENs or CRISPR/cas9 are

emerging in the scientific community to perform genome editing in microalgae.

**Transcriptional activator-like effector nuclease (TALEN)** system is similar to ZFN because it uses nucleases composed of a restriction enzyme domain fused to a DNA-binding domain (here the TAL effector domain) and a nonspecific DNA cleavage domain *FokI* [65]. TALEN proteins are characterized by a repeated 34-amino acid sequence that recognizes specific DNA sequences [66]. *P. tricornutum* lipid metabolism was recently modified using TALEN [59]. In this study, seven genes involved in this metabolism were modified. Each genome modification had a high frequency reaching up to 56% of colonies with targeted mutagenesis [59]. This genetic engineering allowed creation of a high lipid-producing strain by inactivating a key gene for carbohydrate energy storage [59]. Another team has inactivated successfully the urease gene in *P. tricornutum* with 24% of transformed colonies [67]. In addition, TALEN system has also been used in order to inactivate red/far-red light-sensing *phytochrome*

as reported by Daboussi in 2017 [53].

184 Microalgal Biotechnology

are described in the following paragraphs.

as compared to the other alternatives [52].

gene of this diatom [68].


1 Source: https://www.news-medical.net/life-sciences/How-Does-CRISPR-Compare-to-Other-Gene-Editing-Techniques. aspx visited [Accessed: 2017-12-06].

2 Source: http://www.biocompare.com/Editorial-Articles/144186-Genome-Editing-with-CRISPRs-TALENs-and-ZFNs/ [Accessed: 2017-12-06].

**Table 1.** Comparison of four specific genomic tools based on nuclease systems in order to generate genomic-modified species in microalgae.

this strategy, the authors obtained 31% of mutation efficiency [74]. This team targeted two other genes of the diatom using this technology and obtained from 25 to 63% of mutation level [74]. Adaptability of the CRISPR/Cas9 system has been demonstrated in other diatoms like *Thalassiosira pseudonana* [75] as well as in the heterokont, *Nannochloropsis oceanica* in order to knock out the nitrate reductase activity [76]. In conclusion, CRISPR/cas9 system is a promising technology to generate genome-modified organisms in microalgae. **Table 1** compares this system with the other nuclease systems cited above in terms of their technical characteristics and highlights their advantages and disadvantages.

thankful to the University of Rouen Normandy, the region Normandie and the I.U.F. for their

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

, Narimane Mati-Baouche1

1 Normandie Univ, UniRouen, Laboratoire de Glycobiologie et Matrice Extracellulaire

[1] Sasso S, Pohnert G, Lohr M, Mittag M, Hertweck C. Microalgae in the postgenomic era: A blooming reservoir for new natural products. FEMS Microbiology Reviews. Jul 1,

[2] Spolaore P, Joannis-Cassan C, Duran E, Isambert A. Commercial applications of micro-

[3] Mata TM, Martins AA, Caetano NS. Microalgae for biodiesel production and other applications: A review. Renewable and Sustainable Energy Reviews. Jan 1, 2010;**14**(1):217-232

[4] Cadoret J-P, Garnier M, Saint-Jean B. Microalgae, functional genomics and biotechnol-

[5] León-Bañares R, González-Ballester D, Galván A, Fernández E. Transgenic microalgae

[6] Barrera DJ, Mayfield SP. High-value recombinant protein production in microalgae. In: Emeritus ARPD, Hu Q, editors. Handbook of Microalgal Culture [Internet]. Oxford, UK: John Wiley & Sons, Ltd; 2013. pp. 532-544. Available from: http://onlinelibrary.wiley.com/

[7] Hempel F, Maier UG. Microalgae as solar-powered protein factories. Advances in

algae. Journal of Bioscience and Bioengineering. Feb 1, 2006;**101**(2):87-96

as green cell-factories. Trends in Biotechnology. Jan 1, 2004;**22**(1):45-52

ogy. Advances in Botanical Research. Jan 1, 2012;**64**:285-341

doi/10.1002/9781118567166.ch27/summary

Experimental Medicine and Biology. 2016;**896**:241-262

, Patrice Lerouge1

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

and

187

financial support.

**Author details**

Muriel Bardor1,2\*

**References**

Rodolphe Dumontier1

Végétale, Rouen, France

2012;**36**(4):761-785

**Conflict of interest**

The authors have declared no conflict of interest.

, Alain Mareck1

\*Address all correspondence to: muriel.bardor@univ-rouen.fr

2 I.U.F. (Institut Universitaire de France), Paris Cedex 05, France

#### **3.2. Mutant libraries**

The study of mutants impaired in a glycosidase or a glycosyltransferase implied in the *N*-glycan pathway is of great interest. Indeed, the synthesis of oligosaccharides is a sequential process. Inactivation of an enzyme usually results in the accumulation of its *N*-glycan substrate which enables the step-by-step dissection of the entire pathway. Moreover, mutant phenotyping of the glycosylation pathway allows to investigate to which extent the protein *N*-glycan processing is required for normal growth and development. An indexed and mapped mutant library has been created in *C. reinhardtii* by single random insertional mutagenesis of gene cassettes in 2016 [79]. This library already envisioned to study the function of genes encoding putative glycosyltransferases, glycosidases or even putative translocators in microalgae and to confirm their physiological role from reverse genetic studies.
