Perspectives for Glyco-Engineering of Recombinant Biopharmaceuticals from Microalgae

Abstract

Microalgae exhibit great potential for recombinant therapeutic protein production, due to lower production costs, immunity to human pathogens, and advanced genetic toolkits. However, a fundamental aspect to consider for recombinant biopharmaceutical production is the presence of correct post-translational modifications. Multiple recent studies focusing on glycosylation in microalgae have revealed unique species-specific patterns absent in humans. Glycosylation is particularly important for protein function and is directly responsible for recombinant biopharmaceutical immunogenicity. Therefore, it is necessary to fully characterise this key feature in microalgae before these organisms can be established as industrially relevant microbial biofactories. Here, we review the work done to date on production of recombinant biopharmaceuticals in microalgae, experimental and computational evidence for N- and O-glycosylation in diverse microalgal groups, established approaches for glyco-engineering, and perspectives for their application in microalgal systems. The insights from this review may be applied to future glyco-engineering attempts to humanize recombinant therapeutic proteins and to potentially obtain cheaper, fully functional biopharmaceuticals from microalgae.

Keywords: algae; glycosylation; recombinant protein.

Figure 1

A schematic of N-glycosylation patterns found in (A) humans, (B) established biofactories including yeasts, plants, CHO cells, and (C) specific N-glycosylation patterns in microalgae. Glycans shown for (A) humans and (B) established biofactories are only illustrative and do not represent the totality of possible glycoforms obtained from these organisms. Differences in specificity of yeast, CHO, plant, and microalgal Golgi glycosyltransferases and glycosidases lead to variations in the final glycosylation profiles compared to humans. Consequently, glycans N-linked to recombinant proteins produced in these biofactories differ from native human proteins, necessitating glycan engineering to produce efficient and safe biopharmaceuticals in these alternative host systems. Green circle = Mannose. Blue circle = Glucose. Yellow circle = Galactose. Blue square = GlcNAc. White diamond = Neu5Gc. Me = Methylated residue. Fuchsia diamond = Sialic acid. Purple star = Xylose. Red triangle = Fucose. α-Man I = α-mannosidase I. GnT I = N-acetylglucosaminyltransferase I. α-Man II = α-mannosidase II. GnT II = N-acetylglucosaminyltransferase II.

Figure 2

A comparison of the different O-glycosylation patterns among humans, CHO cells, yeasts, plants, and microalgae. Experimental evidence for O-glycosylation in microalgae is limited to C. reinhardtii [124]. Native C. reinhardtii proteins possess a (Hyp-O-Ara-Ara) core and methylated residues, characteristics that differ significantly from human O-glycosylation patterns. Ser/Thr = serine or threonine. Hyp = hydroxyproline. Green circle = Mannose. Yellow circle = Galactose. Orange pentagon = Arabinose. Blue square = GlcNAc. Me = Methylated residue. Purple star = Xylose. Red triangle = Fucose.

Figure 3

Hypothetical presence of protein N-glycosylation and O-glycosylation enzymes in the representative microalgal species, P. purpureum (PP), P. tricornutum (PT), Nannochloropsis gaditana (NG), C. reinhardtii (CR), B. braunii (BB), and C. vulgaris (CV), compared with glycosylation enzymes from H. sapiens and A. thaliana. Enzymes are classified as present (dark blue), potentially present (light blue) or missing (grey).

Figure 4

A schematic of different glyco-engineering strategies; (A) protein engineering and (B) cell engineering. Protein glyco-engineering approaches (A) can target (1) the recombinant DNA sequence (rDNA) [137], (2) the sub-cellular location of the biopharmaceutical [112], and (3) the glycosylation pattern of the translated protein [138,139]. Cell glyco-engineering strategies (B) can modify the activity of glycosylation enzymes by (1) random genetic insertion [140], (2) targeted gene knock-in or knock-out [141,142,143,144,145], and (3) inhibitor interference [138]. Green circle = Mannose. Blue circle = Glucose. Yellow circle = Galactose. Blue square = GlcNAc. Fuchsia diamond = Sialic acid. Yellow star = Xylose. Red triangle = Fucose.