Kazuhito Fujiyama

Some functions of flavonoid glycosides are determined by the glycan structure or enhanced by increasing the attached sugars. Therefore, engineering the glycan moiety can modify the functions of the flavonoid. We attempted to construct a biotransformation method for producing a flavonoid diglycoside, naringin (NRGI) as a model target, using engineered yeast. Naringenin (NRG) glycosylation is initiated by the regiospecific 7-O-glucosyltransferase (7-O-GlcT) to form naringenin-7-O-glucoside (N7G), followed by further rhamnosylation via the branching-type α-1,2-rhamnosyltransferase (1,2RhaT), producing NRGI as the final product. In this study, we introduced three α-1,2-rhamnosyl-glucoside synthesis-related enzymes—-O-GlcT from Arabidopsis thaliana (AtGT-2), 1,2RhaT from Citrus maxima (Cm1,2RhaT), and UDP-Rha synthase from A. thaliana (AtRHM2)—into the fission yeast Schizosaccharomyces pombe. To improve the titer of NRGI, we examined the effects of biotransformation medium composition, initial cell concentration, and cell-permeabilizing reagents. Consequently, we successfully constructed a biotransformation method for producing a flavonoid diglycoside from the aglycone via sequential glycosylation using a single recombinant yeast. Using the optimized biotransformation method, we produced 23.1±1.1 mg l-1 of NRGI, 8.0±0.4 % molar conversion, from 136 mg l-1 of NRG in 24 h cultivation. This study demonstrated the first example of flavonoid O-diglycoside production in engineered S. pombe via sequential glycosylation by uridine diphosphate sugar-dependent glycosyltransferases (UGTs) under optimized production conditions.

Key message Vector minimization, combined with optimized infiltration buffer conditions, enhances recombinant protein expression in Nicotiana benthamiana , offering practical improvements in plant-based biotechnology. Abstract Nicotiana benthamiana is a widely utilized host for transient expression of recombinant proteins, offering a rapid and flexible system for biotechnology applications. However, achieving high-protein output remains a significant challenge. In this study, we explored how vector minimization and infiltration buffer optimization influence recombinant protein expression. We systematically reduced the size of the commonly used pBI121 vector by eliminating non-essential sequences, generating pBI121Δlac and modified pBI121 (mpBI121) constructs. Using ꞵ-glucuronidase (GUS) and acid sphingomyelinase (ASM) as model proteins, we found that vector minimization significantly enhanced both mRNA and protein expression. The mpBI121 backbone improved GUS and ASM mRNA expression levels by 2.02-fold and 2.05-fold, respectively, versus pBI121. Correspondingly, protein levels increased by 1.76-fold and 1.75-fold. Optimization of infiltration buffer—together with co-expression of the silencing suppressor p19, along with acetosyringone, and ascorbic acid—further improved protein output, producing the highest expression of both proteins. These findings demonstrate that both vector minimization and optimized agroinfiltration conditions enhance recombinant protein expression in plant systems, advancing plant-based biotechnology platforms.

Insect cells are attractive hosts for biopharmaceutical production due to their high productivity and mammalian-like post-translational modifications. However, their insect-specific N -glycans differ from mammalian types, thereby reducing product desirability. Here, with an emphasis on engineering N -glycosylation in insect cells, we aimed to develop a more tractable Spodoptera frugiperda Sf9 cell platform as a practical alternative to insect-based systems for engineering the production of tri-antennary N -glycans. A database search revealed that silkworm possesses N -acetylglucosaminyltransferase IV (GNTIV), a putative glycosyltransferase essential for tri-antennary N -glycan synthesis; however, it was not functional in vitro. Then, human GNTIV was introduced into insect cells, resulting in the production of small amounts of tri-antennary N -glycans. This suggested the need for additional factors to efficiently biosynthesize tri-antennary N -glycans. Subsequently, additional insect-derived glycosyltransferases, such as active GNTI and/or GNTII, were co-expressed with GNTIV. Co-expression of three N -acetylglucosaminyltransferases effectively led to the increased biosynthesis of tri-antennary N -glycans. On the other hand, trimming of the N -glycan structure was also observed due to the action of one or more endogenous glycosylhydrolases, which hydrolyze the terminal N -acetylglucosamine residue in insect cells. These facts indicate that effective tri-antennary N -glycan biosynthesis in insect cells requires not only the introduction of exogenous glycosyltransferases but also the knockdown or knockout of endogenous glycosylhydrolase(s).

Plants are promising next‐generation hosts for recombinant protein production; however, major challenges remain with regard to enhancing the efficiency of downstream processing, particularly in the removal of cellular residues and purification of the expressed proteins. Strategies to overcome these limitations include targeting expressed recombinant proteins within a specific organelle or directing their secretion into the extracellular space, thereby facilitating purification by collecting the target matrix. In this study, we focused on protein secretion mechanisms and identified two pathogenesis‐related proteins, glucan endo‐1,3‐β‐glucosidase (GN) and chitinase 8 (Chi8), which accumulated in the apoplast washing fluid following Agrobacterium infiltration of Nicotiana benthamiana leaves. Both proteins contained signal peptides (SPs), SP _GN and SP _Chi8 , respectively. Although the intracellular accumulation of GFP was comparable regardless of the expression level, fusion with either SP _GN or SP _Chi8 resulted in GFP accumulation within the apoplast. In contrast, in N. benthamiana , a mammalian‐derived SP was less effective in facilitating GFP secretion than the plant‐derived SPs. Additionally, replacing the SP of the mammalian‐derived protein β‐glucocerebrosidase (GCase) with SP _GN or SP _Chi8 enhanced the secretion of GCase into the apoplast, indicating their applicability in protein production. Moreover, SP _GN and SP _Chi8 directed the expressed proteins into the culture medium of N. benthamiana suspension cells. These results indicate that SP _GN and SP _Chi8 function as effective secretion signals and highlight the potential application of endogenous SPs for enhancing recombinant protein production in plants.

Key message Tag-free, functional human acid sphingomyelinase was successfully produced in a plant-based system. Apoplastic wash fluid extraction improved downstream processing, and a two-step ion exchange chromatography enabled purification of plant-derived ASM. Abstract Acid sphingomyelinase (ASM) converts sphingomyelin into phosphocholine and ceramide, a process essential for various cellular functions. Given the relevance of ASM to human health and its potential as a therapeutic enzyme, the development of efficient recombinant production systems is of significant interest in biotechnology. We here developed a plant-based expression system for producing human ASM and targeted major limitations related to its purification. The purification was improved in two ways: by engineering a truncated ASM with a plant-derived secretion signal peptide and by utilizing apoplastic wash fluid extraction to improve the purification process. Recombinant ASM was produced in N. benthamiana as a functional protein using an Agrobacterium-mediated transient expression system. The recombinant ASM was then purified using a two-step ion exchange chromatography method, ensuring high purity. After purification, the ASM yield reached approximately 3.5 mg per kg of fresh leaf weight, with a yield of 49.14% and a 21.2-fold purification enhancement. The purified enzyme exhibited a specific activity of 128.18 ± 4.18 mU/mg, confirming that the plant-derived ASM was functionally active. This work represents the first successful production of human ASM in plants, along with the development of an optimized purification method. This achievement marks a significant step forward in overcoming the challenges associated with producing and purifying recombinant proteins in plant-based expression systems, paving the way for future therapeutic applications.

N ‐Glycosylation critically influences the efficacy, safety and pharmacokinetic properties of biopharmaceuticals. Plant expression platforms offer multiple advantages for the production of N ‐glycosylated proteins, but their use is impeded by the presence of plant‐specific N ‐glycan epitopes, which raise concerns of possible immunogenicity to humans. In this study, N ‐glycoengineered Nicotiana benthamiana plants that produce more homogeneous N ‐glycans without plant‐specific epitopes were generated using multiplex CRISPR/Cas9 genome editing. To achieve this N ‐glycosylation modification, ALG3 and GNTI, which function in N ‐glycosylation processes in the ER and Golgi, respectively, were characterised, and single‐ and double‐knockout mutant plants were generated. Comprehensive N ‐glycan profiling revealed that while the ALG3 ‐knockout plant line, alg3 , maintained predominantly plant‐specific N ‐glycans with altered mannose content, the GNTI ‐knockout line, gntI , produced exclusively high‐mannose‐type N ‐glycans. Notably, the alg3gntI double‐knockout mutants yielded highly uniform trimannosidic N ‐glycans. To validate our N ‐glycoengineering approach, we expressed two model biopharmaceuticals, Varlilumab (anti‐CD27 antibody) and β‐glucocerebrosidase (GCase), in wild‐type and mutant plants. While the antibodies expressed in alg3 and alg3gntI showed a certain level of glucosylated endoplasmic reticulum‐type N ‐glycan, with increased non‐ N ‐glycosylated heavy chains, GCase exhibited a more consistent N‐ glycosylation profile, reflecting the engineered N ‐glycosylation pathway. Our findings provide valuable insights into N ‐glycan biosynthesis in N. benthamiana and demonstrate the potential of targeted N ‐glycoengineering for producing biopharmaceuticals with more homogeneous mannose‐type N ‐glycan profiles.

Lactadherin is a major protein in the milk fat globule membrane (MFGM) and is associated with anti-rotaviral effect. Although lactadherin level in breast milk is generally quantified using enzyme-linked immunosorbent assay (ELISA), the many contaminants in the milk samples raise concerns of low accuracy. The aim of this study was to develop a high accuracy method of absolute quantification for breast milk lactadherin using a proteomics-based technique. The developed method uses a full-length expressed stable isotope-labelled protein from the wheat germ cell-free expression system with in-gel digestion and showed much higher accuracy (97.0%–109.9%,) than ELISA (27.2%–31.6%). The developed method revealed lactadherin levels in Japanese breast milk (1 month of postpartum 7.2 [2.2–25.7] mg/100 ml, 6 months of postpartum: 3.8 [1.2–22.8] mg/100 ml). This study has shown that a proteomics approach using a full-length expressed stable isotope-labelled protein with in-gel digestion achieves high accuracy quantification for breast milk lactadherin.

A complete iron deficiency in iron-sensitive oleaginous yeast showed insufficient biomass, resulting in a lower lipid amount, although lipid accumulation was greater compared to deficiency in other ions. In this study, the effect of functional iron deficiency on lipid production on Rhodotorula toruloides NBRC 0559 was examined. Two supplements, an iron-added (growth) supplement and an iron-free (lipid-producing) supplement were tested for detecting functional iron deficiency. The addition of iron-added supplement increased the biomass by 1.5-fold. Furthermore, the addition of iron-free supplement stimulated the growth of R. toruloides NBRC 0559 without loss of biomass (indeed, the biomass increased 1.2-fold) while also resulting in a deficiency of the iron needed for improved growth. Through iron-free supplement, the functional iron starvation effect resulted in improved lipid yield (1.7-fold) and an improved ratio of oleic acid (1.2-fold), which is considered an appropriate material for biodiesel, compared to the non-supplement-treated medium. Moreover, functional iron deficiency led to a 3.4-fold increase in the oleic acid rate compared to when all iron was completely removed from the medium. This study presents the effects and importance of iron in improving biomass and lipid production through the functional iron deficiency.

N-glycan engineering has dramatically evolved for the development and quality control of recombinant antibodies. Fc region of IgG contains two N-glycans whose galactose terminals on Fc-glycan have been shown to increase the stability of CH2 domain and improve effector functions. Nicotiana benthamiana has become one of the most attractive production systems for therapeutic antibodies. In this study, Varlilumab, a CD27-targeting monoclonal antibody, was transiently produced in fresh leaves of soil-grown and hydroponic-grown N. benthamiana, resulted in the yield of 174 and 618 µg/gram, respectively. However, the IgG produced in wild-type N. benthamiana lacked the terminal galactose residues in its N-glycan. Therefore, N-glycan engineering was applied to fine-tune recombinant antibodies produced in plant platforms. We further co-expressed IgG together with murine β1,4-galactosyltransferase (β1,4-GALT) to modify plant N-glycan with β1,4-linked Gal residue(s) and Arabidopsis thaliana β1,3-galactosylatransferase (β1,3-GALT) to improve galactosylation. The co-expression of IgG with each of GALTs successfully resulted in modification of N-glycan structures on the plant-produced IgG. Notably, IgG co-expressed with murine β1,4-GALT in soil-grown N. benthamiana had 42.5% of N-glycans variants having galactose (Gal) residues at the non-reducing terminus and 55.3% of that in hydroponic-grown N. benthamiana plants. Concomitantly, N-glycan profile analysis of IgG co-expressed with β1,3-GALT demonstrated that there was an increased efficiency of galactosylation and an enhancement in the formation of Lewis a structure in plant-derived antibodies. Taken together, our findings show that the first plant-derived Varlilumab was successfully produced with biantennary β1,4-galactosylated N-glycan structures.

Microbial lipids are considered promising and environmentally friendly substitutes for fossil fuels and plant-derived oils. They alleviate the depletion of limited petroleum storage and the decrement of arable lands resulting from the greenhouse effect. Microbial lipids derived from oleaginous yeasts provide fatty acid profiles similar to plant-derived oils, which are considered as sustainable and alternative feedstocks for use in the biofuel, cosmetics, and food industries. Rhodotorula toruloides is an intriguing oleaginous yeast strain that can accumulate more than 70% of its dry biomass as lipid content. It can utilize a wide range of substrates, including low-cost sugars and industrial waste. It is also robust against various industrial inhibitors. However, precise control of the fatty acid profile of the lipids produced by R. toruloides is essential for broadening its biotechnological applications. This mini-review describes recent progress in identifying fatty synthesis pathways and consolidated strategies used for specific fatty acid-rich lipid production via metabolic engineering, strain domestication. In addition, this mini-review summarized the effects of culture conditions on fatty acid profiles in R. toruloides. The perspectives and constraints of harnessing R. toruloides for tailored lipid production are also discussed in this mini-review. Graphical abstract