Expression of an endo-rhamnogalacturonase from Aspergillus aculeatus enhances release of Arabidopsis transparent mucilage.

Mucilage is a gelatinous and sticky hydrophilic polysaccharide released from epidermal cells of seed coat after the hydration of mature seeds and is composed primarily of unbranched rhamnogalacturonan I (RG-I). In this study, we produced a recombinant endo-RG-I hydrolase from Aspergillus aculeatus (AaRhgA) in the fission yeast Schizosaccharomyces pombe and examined its substrate preference for pyridylaminated (PA) RG-I with the various degrees of polymerization (DP). Recombinant AaRhgA requires PA-RG-I with a DP of 10 or higher for its hydrolase activity. We heterologously expressed the AarhgA gene under the strong constitutive promoter, cauliflower mosaic virus 35S promoter, in Arabidopsis thaliana. In a series of biochemical analyses of each mucilage fraction released from the water-imbibed seeds of the transgenic plants, we found the enhanced deposition of the transparent mucilage layer that existed in the peripheral regions of the adherent mucilage and was not stained with ruthenium red. This study demonstrated the feasibility of manipulating the mucilage organization by heterologous expression of the endo-RG-I hydrolase.

BPG4 regulates chloroplast development and homeostasis by suppressing GLK transcription factors and involving light and brassinosteroid signaling.

Chloroplast development adapts to the environment for performing suitable photosynthesis. Brassinosteroids (BRs), plant steroid hormones, have crucial effects on not only plant growth but also chloroplast development. However, the detailed molecular mechanisms of BR signaling in chloroplast development remain unclear. Here, we identify a regulator of chloroplast development, BPG4, involved in light and BR signaling. BPG4 interacts with GOLDEN2-LIKE (GLK) transcription factors that promote the expression of photosynthesis-associated nuclear genes (PhANGs), and suppresses their activities, thereby causing a decrease in the amounts of chlorophylls and the size of light-harvesting complexes. BPG4 expression is induced by BR deficiency and light, and is regulated by the circadian rhythm. BPG4 deficiency causes increased reactive oxygen species (ROS) generation and damage to photosynthetic activity under excessive high-light conditions. Our findings suggest that BPG4 acts as a chloroplast homeostasis factor by fine-tuning the expression of PhANGs, optimizing chloroplast development, and avoiding ROS generation.

High accumulation of the Man5GlcNAc2 structure by combining N-acetylglucosaminyltransferase I gene suppression and mannosidase I gene overexpression in Nicotiana tabacum SR1.

High accumulation of a single high-mannose glycan structure is important to ensure the quality of therapeutic proteins. We developed a glyco-engineering strategy for ensuring high accumulation of the Man5GlcNAc2 structure by combining N-acetylglucosaminyltransferase I (GnT I) gene suppression and mannosidase I (Man I) gene overexpression. Nicotiana tabacum SR1 was used as the glyco-engineered host owing to the lower risk of pathogenic contamination than that in mammalian cells. We generated three glyco-engineered plant strains (gnt, gnt-MANA1, and gnt-MANA2) with suppression of GnT I or the combined suppression of GnT I and overexpression of Man I A1 or A2. The quantitative reverse transcriptase-PCR analysis showed a higher level of upregulation of Man I expression in gnt-MANA1/A2 plants than in the wild-type plants. Man I activity assay showed that the gnt-MANA1 plants had a higher Man I activity than did the wild-type and gnt-MANA2 plants. N-glycan analysis independently performed on two plants of each plant strain showed that gnt-MANA1 plants had a low abundance of the Man6-9GlcNAc2 structure (2.8%, 7.1%) and high abundance of the Man5GlcNAc2 structure (80.0%, 82.8%) compared with those in the wild-type and gnt plants. These results indicated that GnT I knockdown suppressed further modification of the Man5GlcNAc2 structure, and Man I overexpression enhanced the conversion of Man6-9GlcNAc2 structures to the Man5GlcNAc2 structure. The developed glyco-engineered plants have potential for serving as novel expression hosts for therapeutic proteins.

High-level transient production of a protease-resistant mutant form of human basic fibroblast growth factor in Nicotiana benthamiana leaves.

The human basic fibroblast growth factor (bFGF) is a protein that plays a pivotal role in cellular processes like cell proliferation and development. As a result, it has become an important component in cell culture systems, with applications in biomedical engineering, cosmetics, and research. Alternative production techniques, such as transient production in plants, are becoming a feasible option as the demand continues to grow. High-level bFGF production was achieved in this study employing an optimized Agrobacterium-mediated transient expression system, which yielded about a 3-fold increase in production over a conventional system. This yield was further doubled at about 185 µg g-1 FW using a mutant protease-resistant version that degraded/aggregated at a three-fold slower rate in leaf crude extracts. To achieve a pure product, a two-step purification technique was applied. The capacity of the pure protease-resistant bFGF (PRbFGF) to stimulate cell proliferation was tested and was found to be comparable to that of E. coli-produced bFGF in HepG2 and CHO-K1 cells. Overall, this study demonstrates a high-level transient production system of functional PRbFGF in N. benthamiana leaves as well as an efficient tag-less purification technique of leaf crude extracts.

Galactosylation of cell-surface glycoprotein required for hyphal growth and cell wall integrity in Schizosaccharomyces japonicus.

Schizosaccharomyces japonicus is a dimorphic yeast, transiting between unicellular and hyphal growth. The glycoproteins of fission yeast contain, in addition to mannose (Man), a large number of galactose (Gal) residues. Previously, we reported that the cell-surface O-glycans of S. japonicus comprise mainly tri-saccharides (Gal-Man-Man) as a main component, in contrast to the tetra-saccharides observed in other Schizosaccharomyces species. Here we have investigated the function of cell-surface Gal residues in S. japonicus. Because disruption of gms1+, encoding the UDP-Gal transporter required for galactomannan synthesis, abolishes cell-surface galactosylation in Schizosaccharomyces pombe, we constructed a deletion mutant of the homologous gene in S. japonicus gms1Δ [gms1 (S.j)] and determined the N- and O-linked oligosaccharide structures present on the cell surface. Disruption of gms1 (S.j) resulted in a complete lack of Gal on the cell surface, indicating that Gms1 plays an essential role in supplying UDP-Gal from the cytoplasm to the Golgi lumen. Analytical microscopy of gms1Δ demonstrated that the lack of cell-surface Gal did not affect cell growth or morphology during vegetative growth. However, hyphal development was blocked in gms1Δ, even in the presence of the topoisomerase I inhibitor camptothecin, which is known to induce hyphal differentiation in wild-type S. japonicus. Collectively, these findings show that Gal-containing oligosaccharides are required for cell wall integrity during filamentous growth in S. japonicus.

Establishment of serum-free adapted Chinese hamster ovary cells with double knockout of GDP-mannose-4,6-dehydratase and GDP-fucose transporter.

Although antibodies have attracted attention as next-generation biopharmaceuticals, the costs of purifying the products and of arranging the environment for cell cultivation are high. Therefore, there is a need to increase antibody efficacy and improve product quality as much as possible. Since antibodies are glycoproteins, their glycan structures have been found to affect the function of antibodies. Especially, afucosylation of the N-linked glycan in the Fc region is known to significantly increase antibody-dependent cellular cytotoxicity. In this study, we established a double-mutant ΔGMDΔGFT in which GDP-mannose 4,6-dehydratase and GDP-fucose transporter were knocked out in Chinese hamster ovary cells, a platform for biopharmaceutical protein production. By adapting ΔGMDΔGFT cells to serum-free medium and constructing suspension-cultured cells, we established host CHO cells with no detected fucosylated glycans and succeeded in production of afucosylated antibodies. We also demonstrated that, in culture in the presence of serum, fucosylation occurs due to contamination from serum components. Furthermore, we found that afucosylation of glycans does not affect cell growth after adaptation to serum-free medium as compared to wild-type CHO cells growth and does not significantly affect the expression levels of other endogenous fucose metabolism-related enzyme genes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00501-3.

Biochemical characterization of Arabidopsis clade F polygalacturonase shows a substrate preference toward oligogalacturonic acids.

Polygalacturonases (PGs) hydrolyze α-1,4-linked d-galacturonic acid (GalUA) in polygalacturonic acid. Previously, PG activity in pea seedlings was found in the Golgi apparatus, where pectin biosynthesis occurs. However, the corresponding genes encoding Golgi-localized PG proteins have never been identified in the higher plants. In this study, we cloned the 5 Arabidopsis genes encoding putative membrane-bound PGs from clade F PGs (AtPGFs) as the first step for the discovery of the Golgi-localized PGs. Five AtPGF proteins (AtPGF3, AtPGF6, AtPGF10, AtPGF14 and AtPGF16) were heterologously produced in Schizosaccharomyces pombe. Among these, only the AtPGF10 protein showed in vitro exo-type PG activity toward fluorogenic pyridylaminated-oligogalacturonic acids (PA-OGAs) as a substrate. The optimum PG activity was observed at pH 5.5 and 60°C. The recombinant AtPGF10 protein showed the maximum PG activities toward PA-OGA with 10 degrees of polymerization. The apparent Km values for the PA-OGAs with 7, 11 and 14 degrees of polymerization were 8.0, 22, and 5.9 µM, respectively. This is the first report of the identification and enzymatic characterization of AtPGF10 as PG carrying putative membrane-bound domain.

Overexpression of cell-wall GPI-anchored proteins restores cell growth of N-glycosylation-defective och1 mutants in Schizosaccharomyces pombe.

The glycoproteins of yeast contain a large outer chain on N-linked oligosaccharides; therefore, yeast is not suitable for producing therapeutic glycoproteins for human use. Using a deletion mutant strain of α1,6-mannosyltransferase (och1Δ), we previously produced humanized N-glycans in fission yeast; however, the Schizosaccharomyces pombe och1Δ cells displayed a growth delay even during vegetative growth, resulting in reduced productivity of heterologous proteins. To overcome this problem, here we performed a genome-wide screen for genes that would suppress the growth defect of temperature-sensitive och1Δ cells. Using a genomic library coupled with screening of 18,000 transformants, we identified two genes (pwp1+, SPBC1E8.05), both encoding GPI-anchored proteins, that increased the growth rate of och1Δ cells, lacking the outer chain. We further showed that a high copy number of the genes was needed to improve the growth rate. Mutational analysis of Pwp1p revealed that the GPI-anchored region of Pwp1p is important in attenuating the growth defect. Analysis of disruptants of pwp1+ and SPBC1E8.05 showed that neither gene was essential for cell viability; however, both mutants were sensitive ß-glucanase, suggesting that Pwp1p and the protein encoded by SPBC1E8.05 non-enzymatically support ß-glucan on the cell-surface of S. pombe. Collectively, our work not only sheds light on the functional relationships between GPI-anchored proteins and N-linked oligosaccharides of glycoproteins in S. pombe, but also supports the application of S. pombe to the production of human glycoprotein. KEY POINTS: • We screened for genes that suppress the growth defect of fission yeast och1Δ cells. • Appropriate expression of GPI-anchored proteins alleviates the growth delay of och1Δ cells. • The GPI-anchor domain of Pwp1p is important for suppressing the growth defect of och1Δ cells.

Production of Human Acid-Alpha Glucosidase With a Paucimannose Structure by Glycoengineered Arabidopsis Cell Culture.

Plant cell cultures have emerged as a promising platform for the production of biopharmaceutics due to their cost-effectiveness, safety, ability to control the cultivation, and secrete products into culture medium. However, the use of this platform is hindered by the generation of plant-specific N-glycans, the inability to produce essential N-glycans for cellular delivery of biopharmaceutics, and low productivity. In this study, an alternative acid-alpha glucosidase (GAA) for enzyme replacement therapy of Pompe disease was produced in a glycoengineered Arabidopsis alg3 cell culture. The N-glycan composition of the GAA consisted of a predominantly paucimannosidic structure, Man3GlcNAc2 (M3), without the plant-specific N-glycans. Supplementing the culture medium with NaCl to a final concentration of 50 mM successfully increased GAA production by 3.8-fold. GAA from an NaCl-supplemented culture showed a similar N-glycan profile, indicating that the NaCl supplementation did not affect N-glycosylation. The results of this study highlight the feasibility of using a glycoengineered plant cell culture to produce recombinant proteins for which M3 or mannose receptor-mediated delivery is desired.

Substrate specificities of α1,2- and α1,3-galactosyltransferases and characterization of Gmh1p and Otg1p in Schizosaccharomyces pombe.

In the fission yeast Schizosaccharomyces pombe, α1,2- and α1,3-linked D-galactose (Gal) residues are transferred to N- and O-linked oligosaccharides of glycoproteins by galactosyltransferases. Although the galactomannans are important for cell-cell communication in S. pombe (e.g., in nonsexual aggregation), the mechanisms underlying galactosylation in cells remain unclear. Schizosaccharomyces pombe has 10 galactosyltransferase-related genes: seven belonging to glycosyltransferase (GT) family 34 and three belonging GT family 8. Disruption of all 10 α-galactosyltransferases (strain Δ10GalT) has been shown to result in a complete lack of α-Gal residues. Here, we have investigated the function and substrate specificities of galactosyltransferases in S pombe by using strains expressing single α-galactosyltransferases in the Δ10GalT background. High-performance liquid chromatography (HPLC) analysis of pyridylaminated O-linked oligosaccharides showed that two GT family 34 α1,2-galactosyltransferases (Gma12p and Gmh6p) and two GT family 8 α1,3-galactosyltransferases (Otg2p and Otg3p) are involved in galactosylation of O-linked oligosaccharide. Moreover, 1H-NMR of N-glycans revealed that three GT family 34 α1,2-galactosyltransferases (Gmh1p, Gmh2p and Gmh3p) are required for the galactosylation of N-linked oligosaccharides. Furthermore, HPLC and lectin-blot analysis revealed that Otg1p showed α1,3-galactosyltransferase activity under conditions of co-expression with Gmh6p, indicating that α-1,2-linked galactose is required for the galactosylation activity of Otg1p in S. pombe. In conclusion, eight galactosyltransferases have been shown to have activity in S. pombe with different substrate specificities. These findings will be useful for genetically tailoring the galactosylation of both N- and O-glycans in fission yeast.

Bombyx mori ß1,4-N-acetylgalactosaminyltransferase possesses relaxed donor substrate specificity in N-glycan synthesis.

N-Glycosylation is one of the most important post-translational protein modifications in eukaryotic cells. Although more than 200 N-glycogenes contributing to N-glycan biosynthesis have been identified and characterized, the information on insect N-glycosylation is still limited. Here, focusing on insect N-glycosylation, we characterized Bombyx mori N-acetylgalactosaminyltransferase (BmGalNAcT) participating in complex N-glycan biosynthesis in mammals. BmGalNAcT localized at the Golgi and was ubiquitously expressed in every organ and in the developmental stage of the middle silk gland of fifth instar larvae. Analysis of recombinant BmGalNAcT expressed in Sf9 cells showed that BmGalNAcT transferred GalNAc to non-reducing terminals of GlcNAcß1,2-R with ß1,4-linkage. In addition, BmGalNAcT mediated transfer of galactose and N-acetylglucosamine residues but not transfer of either glucose or glucuronic acid from the UDP-sugar donor substrate to the N-glycan. Despite this tri-functional sugar transfer activity, however, most of the endogenous glycoproteins of insect cells were present without GalNAc, Gal, or GlcNAc residues at the non-reducing terminal of ß1,2-GlcNAc residue(s). Moreover, overexpression of BmGalNAcT in insect cells had no effect on N-acetylgalactosaminylation, galactosylation, or N-acetylglucosaminylation of the major N-glycan during biosynthesis. These results suggested that B. mori has a novel multifunctional glycosyltransferase, but the N-glycosylation is highly and strictly regulated by the endogenous N-glycosylation machineries.

Functional characterization and overexpression of Δ12-desaturase in the oleaginous yeast Rhodotorula toruloides for production of linoleic acid-rich lipids.

Linoleic acid (LA) has garnered much attention due to its potential applications in the oleochemical and nutraceutical industries. The oleaginous yeast Rhodotorula toruloides has outstanding lipogenecity, and is considered a potential alternative to the current plant-based platforms for LA production. Δ12-fatty acid desaturases (Δ12-Fads) are involved in LA synthesis in various fungi and yeasts, but their functions in R. toruloides remain poorly understood. To achieve the production of LA-rich lipids in R. toruloides, we investigated the function of the native Δ12-FAD (RtFAD2). First, the overexpression of RtFAD2 and its co-overexpression with RtFAD1 (encoding R. toruloides Δ9-Fad) and their effects on LA production in R. toruloides were investigated. The function of RtFad2 was confirmed by heterologous expression in Saccharomyces cerevisiae. Overexpression of RtFAD2 significantly elevated the LA contents and titers in the wild-type strain R. toruloides DMKU3-TK16 (TK16) and in a thermotolerant derivative of TK16 (L1-1). Additionally, overexpression of RtFAD2 in R. toruloides strains also increased the lipid titer and content. Overexpression of RtFAD1 was down-regulated in the RtFAD1 and RtFAD2 co-overexpressing strains, suggesting that the elevated LA content may function as a key regulator of RtFAD1 expression to control C18 fatty-acid synthesis in R. toruloides. We characterized the function of RtFAD2 and showed that its overexpression in R. toruloides increased the lipid and LA production. These findings may assist in the rational design of metabolic engineering related to LA or polyunsaturated fatty acid production in R. toruloides.

Golgi localization of glycosyltransferases requires Gpp74p in Schizosaccharomyces pombe.

The majority of Golgi glycosyltransferases are type II membrane proteins with a small cytosolic tail at their N-terminus. Several mechanisms for localizing these glycosyltransferases to the Golgi have been proposed. In Saccharomyces cerevisiae, the phosphatidylinositol-4-phosphate-binding protein ScVps74p interacts with the cytosolic tail of a Golgi glycosyltransferase and contributes to its localization. In this study, we investigated whether a similar mechanism functions in the fission yeast Schizosaccharomyces pombe. First, we identified gpp74+ (GPP34 domain-containing Vps74 homolog protein), a gene encoding the S. pombe homolog of S. cerevisiae Vps74p. Deletion of the gpp74+ gene resulted in the missorting of three Golgi glycosyltransferases, SpOch1p, SpMnn9p, and SpOmh1p, to vacuoles, but not SpAnp1p, indicating Gpp74p is required for targeting some glycosyltransferases to the Golgi apparatus. Gpp74p with an N-terminal GFP-tag localized to both the Golgi apparatus and the cytosol. Golgi localization of Gpp74p was dependent on the phosphatidylinositol 4-kinase SpPik1p. Site-directed mutagenesis of hydrophobic and basic amino acids in the cytosolic tails of SpOch1p and SpMnn9p resulted in their missorting to vacuoles, indicating these cytosolic N-terminal residues are important for localization in the Golgi. Unexpectedly, no prominent alternations in protein glycosylation were observed in S. pombe gpp74Δ cells, probably due to the residual Golgi localization of some SpOch1p and SpMnn9p in these cells. Collectively, these results demonstrate that both Gpp74p-dependent and Gpp74p-independent mechanisms are responsible for the Golgi localization of glycosyltransferases to the Golgi in S. pombe. KEY POINTS: • Gpp74p is involved in the localization of glycosyltransferases to the Golgi. • The cytosolic tails of glycosyltransferases are important for Golgi localization. • Gpp74p localizes to the Golgi in a SpPik1p-dependent manner.

Transglycosylation toward naringenin-7-O-glucoside using an N180H mutant of Coprinopsis cinerea endo-ß-N-acetylglucosaminidase.

Flavonoids are generally glycosylated, and the glycan moieties of flavonoid glycosides are known to greatly affect their physicochemical and biological properties. Thus, the development of a variety of tools for glycan remodeling of flavonoid glycosides is highly desired. An endo-ß-N-acetylglucosaminidase mutant Endo-CC N180H, which is developed as an excellent chemoenzymatic tool for creating sialylglycoproteins, was employed for the glycosylation of flavonoids. Endo-CC N180H transferred the sialyl biantennary glycans from the sialylglyco peptide to pNP-GlcNAc and narigenin-7-O-glucoside. The kinetic parameters of Endo-CC N180H towards SGP and pNP-GlcNAc were determined. Flavonoid glucosides harboring a 1,3-diol structure in the glucose moieties acted as substrates of Endo-CC N180H. We proposed that the sialyl biantennary glycan transfer to the flavonoid by Endo-CC N180H could pave the way for the improvement of the inherent biological functions of the flavonoids and creation of novel flavonoid glycoside derivatives for future human health benefits including foods and drugs.

SpMnn9p and SpAnp1p form a protein complex involved in mannan synthesis in the fission yeast Schizosaccharomyces pombe.

The cell walls of yeast cells possess a large mannan structure mainly comprising of a linear α1,6-linked mannose oligomer on the N-linked glycans. The biosynthesis of the mannan is initiated by ScOch1p α1,6-mannosyltransfease, and elongated by the mannan polymerase complexes M-Pol I and II in the Golgi of Saccharomyces cerevisiae. Here, we functionally characterized SpMnn9 and SpAnp1 proteins in the fission yeast Schizosaccharomyces pombe; these proteins are homologs of S. cerevisiae M-Pol II complex proteins ScMnn9p and ScAnp1p. Cells harboring disruptions in Spmnn9+ and Spanp1+ genes showed slower growth at 37°C and an increased sensitivity to hygromycin B, characteristic of a glycosylation defect. Results obtained from the acid phosphatase assay and high-performance liquid chromatography analysis of N-linked glycans in Spmnn9Δ and Spanp1Δ mutants suggested that the mannan structure in S. pombe is synthesized sequentially by the α-mannosyltransferases in the order of SpOch1p, SpMnn9p and SpAnp1p. Immunoprecipitation and split YFP analyses demonstrated that SpMnn9p and SpAnp1p form the M-Pol-II like complex. Together, these results provided an improved understanding of the mechanism of mannan synthesis by SpMnn9p and SpAnp1p in S. pombe.

Characterization of Bombyx mori N-acetylglucosaminyltransferase II splicing variants.

N-Acetylglucosaminyltransferase II (GNTII), which catalyzes the transfer of N-acetylglucosamine to N-glycans, plays an essential role in the biosynthesis of branched and complex-type N-glycans. Some characteristics of the GNTIIs from various species have been identified, but not all features have been revealed because some insects have GNTII redundancies due to the possession of splicing variants. In this study, we focused on four splicing variants of silkworm Bombyx mori GNTII (BmGNTII) that differ only in the absence or presence of Exon 2, Exon 9 or both, and we characterized the spatiotemporal transcript levels and enzymatic properties of each. Two of the splicing variants, BmGNTII-B and BmGNTII-D, lack Exon 9, and were expressed more highly in silk glands than any other organs. With respect to the enzymatic properties, optimal temperature and pH were similar among the recombinant BmGNTIIs, but the specific activities and temperature stabilities differed according to the presence or absence of Exon 9 in the splicing variants. These results demonstrate that the B. mori genome encodes splicing variants of GNTII with different enzymatic properties.

Ethanol and H2O2 stresses enhance lipid production in an oleaginous Rhodotorula toruloides thermotolerant mutant L1-1.

Stress tolerance is a desired characteristic of yeast strains for industrial applications. Stress tolerance has been well described in Saccharomyces yeasts but has not yet been characterized in oleaginous Rhodotorula yeasts even though they are considered promising platforms for lipid production owing to their outstanding lipogenicity. In a previous study, the thermotolerant strain L1-1 was isolated from R. toruloides DMKU3-TK16 (formerly Rhodosporidium toruloides). In this study, we aimed to further examine the ability of this strain to tolerate other stresses and its lipid productivity under various stress conditions. We found that the L1-1 strain could tolerate not only thermal stress but also oxidative stress (ethanol and H2O2), osmotic stress (glucose) and a cell membrane disturbing reagent (DMSO). Our results also showed that the L1-1 strain exhibited enhanced ability to maintain ROS homeostasis, stronger cell wall strength and increased levels of unsaturated membrane lipids under various stresses. Moreover, we also demonstrated that ethanol-induced stress significantly increased the lipid productivity of the thermotolerant L1-1. The thermotolerant L1-1 was also found to produce a higher lipid titer under the dual ethanol-H2O2 stress than under non-stress conditions. This is the first report to indicate that ethanol stress can induce lipid production in an R. toruloides thermotolerant strain.

Characterization of N- and O-linked galactosylated oligosaccharides from fission yeast species.

The N- and O-linked oligosaccharides from fission yeast Schizosaccharomyces pombe not only contain large amounts of d-mannose (Man) but also contain large amounts of d-galactose (Gal). Although the galactomannans of S. pombe are mainly composed of α1,2- or α1,3-linked Gals, some of the terminal α1,2-linked Gals are found to be linked to pyruvylated ß1,3-linked galactose (PvGal). We have determined the structural characteristics of the N-glycans and O-glycans in three Schizosaccharomyces species (S. japonicus, S. octosporus, and S. cryophilus) using lectin blot, 1H NMR spectroscopy, and size-fractionation high performance liquid chromatography (HPLC), and found that the galactosylation of oligosaccharides was a common feature in fission yeasts. In addition, each of the terminal Galα1,2-, Galß1,3- and non-substituted Man residues exhibited distinct characteristics. A BLAST search of gene databases in Schizosaccharomyces identified genes homologous to pvg1 encoding pyruvyltransferase of S. pombe. These genes, when expressed in an S. pombe pvg1Δ strains, led to the pyruvylation of non-reducing terminal ß-linked Gal, suggesting the biosynthetic pathway of PvGal-containing oligosaccharides is highly conserved in fission yeasts.

Enhancement of sialylation in rIgG in glyco-engineered Chinese hamster ovary cells.

Since about 70% of commercial biopharmaceutical products have been produced in Chinese hamster ovary (CHO) cells, this cell line is undeniably a workhorse for biopharmaceuticals production. Meanwhile, sialic acid terminals were reported to affect anti-inflammatory activity, antibody-dependent cellular cytotoxicity efficacy of IgG antibodies. Taking these findings together, we aimed to establish CHO cell lines that highly produce sialic acid terminals by overexpressing two N-acetylneuraminic acid-based key enzymes, α(2,6)-sialyltransferase and UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase using dihydrofolate reductase/methotrexate gene amplification method. Indeed, the number of total sialic acid terminal glycan structures increased tremendously, by 12-fold compared to the wild type in total protein extracts. With the methotrexate supplementation, a targeted cell line, CHOmt17-100, showed up to 1.4 times more sialylated structures of glycoforms in total proteins. Interestingly, immunoglobulin G, used as the model protein in CHOmt17-100, showed about 53% sialylated structures in its glycoforms. These resultant sialylated glycans exhibited more than approximately 14.5 times increase as compared to that of the wild type. Moreover, the resultant glycan structures mostly had N-acetylneuraminic acid terminals, while N-glycolylneuraminic acid terminal composition remained less than 5% as compared to the wild type. Engineered antibodies derived from CHO cell lines that produce high levels of sialic acid will contribute to the examination of glycoforms' efficacy and usefulness toward bio-better products.

Biosynthetic Pathway of Indole-3-Acetic Acid in Basidiomycetous Yeast Rhodosporidiobolus fluvialis.

IAA biosynthetic pathways in a basidiomycetous yeast, Rhodosporidiobolus fluvialis DMKU-CP293, were investigated. The yeast strain showed tryptophan (Trp)-dependent IAA biosynthesis when grown in tryptophan supplemented mineral salt medium. Gas chromatography-mass spectrometry was used to further identify the pathway intermediates of Trp-dependent IAA biosynthesis. The results indicated that the main intermediates produced by R. fluvialis DMKU-CP293 were tryptamine (TAM), indole-3-acetic acid (IAA), and tryptophol (TOL), whereas indole-3-pyruvic acid (IPA) was not found. However, supplementation of IPA to the culture medium resulted in IAA peak detection by high-performance liquid chromatography analysis of the culture supernatant. Key enzymes of three IAA biosynthetic routes, i.e., IPA, IAM and TAM were investigated to clarify the IAA biosynthetic pathways of R. fluvialis DMKU-CP293. Results indicated that the activities of tryptophan aminotransferase, tryptophan 2-monooxygenase, and tryptophan decarboxylase were observed in cell crude extract. Overall results suggested that IAA biosynthetic in this yeast strain mainly occurred via the IPA route. Nevertheless, IAM and TAM pathway might be involved in R. fluvialis DMKU-CP293.