Synergistic effects on plant growth under several conditions when PGPR and AMF are coinoculated are reported . In our study, however, the effects of combined inocculation with two PGPRs or with PGPRs and AMF never exceeded the sum of the effects of single inocculation with PGPRs and AMF. This indicates additive effects of PGPRs and AMF rather than clear synergistic effects. Roots of tomato after application of both bacteria strains were not only healthier but also showed a significantly higher colonization by AMF Glomus intraradices, indicating that AMF infection in the soils was suppressed directly by pathogens or indirectly as consequence of poor root development. Azcón and Linderman reported that unidentified PGPR have a strong stimulatory effect on the growth of AMF and increased mycelia growth of G. mosseae spores. P. fluorescens 92rk, alone or co-inoculated with P. fluorescent P190r, increased mycorrhizal colonization of tomato roots by G. mosseae BEG12 . Similarly to the results obtained by Marulanda-Aguirre et al. , where Bacillus megaterium inoculated with G. intraradices showed the highest percentage of mycorrhizal root length of Lactuca sativa plants compared to the single inoculation of G. intraradices. These results suggest that PGPR and AMF might be co-inoculated, at least in soils with a low AMF status,vertical hydroponic to optimize the formation and function of the mycorrhizal symbiosis. Both PGPR and AMF inoculation treatments directly and indirectly improved the nutrients acquisition and allocation to the shoots of tomato plants. The concentrations of P, Mn and Zn in tomato shoots were higher after inoculation with P. sp. ”Proradix®” and B. amyloliquefaciens FZB42 when compared to the untreated control. The ability of P. fluorescens and AMF to promote plant growth by improved nutrient acquisition and suppression of soil borne pathogens is well documented.
Both functions may promote plant growth but by different mechanisms. AMF facilitated mineral and water uptake, and increased the defense against soil borne pathogens . PGPRs induced the release of plant growth regulators . Siddique reported that Pseudomonas spp. can synthesize certain enzymes that can modulate plant hormone levels, might limit the available iron via siderophore production and can also kill pathogens by production of certain antibiotics. Our study confirmed that, B. amyloliquefaciens FZB42 can act as a PGPR, as described by De Freitas et al. , Kokalis-Burelle et al. , Kishore et al. and Marulanda-Aguirre et al. . Phae et al. reported that B. subtilis NB22 significantly reduce the occurrence of crown and root rot disease of tomato. Another aspect in the present study was to test if the mixtures of different bacteria species improve the control against FORL compared to one bacterium species alone. Our results did not confirm Pierson and Weller and Schisler et al. who proposed a strategy to increase the efficacy and the consistency of disease control by mixed application of antagonistic microorganisms with different modes of actions. Cordier et al. stated that dual or multiple inoculations of beneficial microorganisms can be neutral, positive or negative depending on the inoculants used. However, our study showed that combined application of two PGPRs improved tomato growth and suppressed FORL to the same extent as single application or further increases P shoot concentrations. Our results are in agreement with studies by Raupach et al. , Pierson and Weller and Duffy et al. , all of which demonstrated that certain mixtures of PGPR were significantly suppressive to cucumber pathogens and take-all disease. Different mechanisms of action for different PGPR strains may explain why combinations of P. sp. ”Proradix®” and Bacillus amyloliquefaciens FZB42 suppress disease similar to inoculation with single strains. Sung and Chung demonstrated that chitinase-producing Streptomyces spp. and B. cereus isolates used in combination with antibiotic-producing P. fluorescens and Burkholderia cepacia isolates had a synergistic effect on the suppression of rice sheath blight and Szczech and Dyśko who reported that among tested bacterial inoculations, only mixture of the bacteria B125 and PT42 tended to affect positively the growth of the plants and to reduce the density of Fusarium spp. in the rhizosphere of tomato plants.
These results indicate that the consistency of biocontrol agents in suppression of soil borne pathogens influenced by many factors, i.e. bacterial strains, soil borne pathogen species, species of plant, etc. Protein expression in plant systems has the potential to provide a safe, cost-effective, and scalable method to meet the increasing need for therapeutic protein production. Plant-based expression offers several advantages to the bio-pharmaceutical industry, including decreased cost of production, scalability, lack of susceptibility to mammalian pathogens, elimination of animal- or human-sourced raw materials, and the production of complex proteins with post-translational modifications such as N-glycosylation. For many therapeutic proteins, N-glycosylation is essential for protein folding, oligomerization, quality control, enzyme activity, ligand interactions, localization, and trafficking. Despite its potential, a possible barrier to the commercialization of plant-made glyco protein drugs is the difference between the N-glycan structures of human and plants. Of particular concern are plant-specific structures contained in complex type N-glycans, namely, α1,3 core fucose, β1,2 bisecting xylose, and the Lewis a epitope. Even though there is no definitive proof of adverse effects from plant-specific glycan structures, the presence of nonhuman glycans could potentially cause unwanted immunogenicity in humans, and the lack of sialic acid termination may lead to reduced blood circulatory half-life. A change in glycan structure could also potentially alter the protein’s structure or accessibility of its epitopes and, consequently, its function. Therefore, to ensure the efficacy of a plant-made bio-similar therapeutic, it is important that the N-glycans are compatible with both the protein’s function and the human immune system. Several strategies exist to modify a glyco protein’s N-glycan structures in planta, such as glycoengineering of the host cells using CRISPR/Cas9 genome editing to knock outβ-xylosyltransferase genes and α-fucosyl transferase genes and RNA interference technology to down regulate XylT and FucT genes, targeting of the protein to specific organelles, addition of compounds to alter the function of glycan-modifying enzymes, and in vitro glycan remodeling using chemoenzymatic reactions. In this work, we utilize kifunensine, a potent and highly specific inhibitor of α-mannosidase I in both plant and animal cells resulting in production of glycoproteins containing predominantly Man8GlcNAc2 and Man9GlcNAc2 structures, to a rice cell suspension culture grown in a bioreactor to inhibit α-mannosidase I activity. More than a few studies of kifunensine treatment in whole Nicotiana benthamiana plants successfully produced predominant Man9 structure glycoproteins; however, the study of kifunensine treatment in plant cell suspension cultures, including transgenic rice cell suspensions, is very limited.
In transgenic rice cell suspensions treated with 5 µM kifunensine cultivated in shake flasks, the productivity of a target glycoprotein, acid α-glucosidase , was significantly lower than the control, but the relative abundance of high-mannose structure GAA increased by 65% compared to the control. Here, we report the effects of kifunensine treatment on production and N-glycosylation of a glycoprotein conducted in a bioreactor. The culture media, method of cultivation, degree of glycosylation, and multi-merization of the product were different from the study by Choi et al.. Rice is generally recognized as safe by the FDA, and the rice alpha amylase 3D promoter, what is vertical farming a metabolically-regulated strong promoter, used in this study was well studied. In addition, semicontinuous bioreactor operations of transgenic rice cell suspensions proved the stability and robustness of transgenic rice cells under the RAmy3D promoter system for long-term recombinant protein production. In this study, we use a transgenic rice cell suspension culture to produce recombinant human butyrylcholinesterase , a bioscavenger hydrolase enzyme that can be used as a therapeutic and prophylactic treatment to counter organophosphorus nerve agents, as a model glycoprotein. Human BChE is a tetrameric glycoprotein with four identical 69-kDa monomers containing nine N-glycosylation sites per each monomer, with its activity, stability, and blood circulatory half-life highly dependent on the presence and structure of these glycans. The production of recombinant human BChE in transgenic rice cell suspension cultures is controlled by the RAmy3D promoter that is highly activated in the absence of sugar. Like other glycoproteins, N-glycosylation of a nascent rrBChE starts in the ER by co- or post-translational transfer of Glc3Man9GlcNAc2 from a dolichol lipid carrier onto Asn-X-Ser/Thr residues, where X is any amino acid except Pro. Since our rrBChE gene construct contains the RAmy3D signal peptide, a glycosylated rrBChE follows the secretory pathway for secretion, involving removal of glucose and mannose residues and addition of new sugar residues and N-acetylglucosamine , leading to complex-type N-glycans of rrBChE, as previously reported.Our goal in this study is to investigate the effects of kifunensine on N-glycosylation modification and the production of rrBChE in a transgenic rice cell culture bioreactor. By adding kifunensine to the medium during bioreactor cultivation at the end of the growth phase and throughout the induction phase, we demonstrate the production and N-glycosylation pattern of rrBChE in culture medium and within the cell aggregates .As shown in Figure 2a, the overall production level of active rrBChE increased by up to 80 µg/g FW, at least 1.5-fold higher than recently reported, using the same two-stage cultures. Table 2 also shows a significant improvement in the volumetric productivity and specific productivity . The increase in total active rrBChE, volumetric productivity, and specific productivity may be the result of increasing sugar-free medium concentration by 1.25 times and decreasing the bioreactor working volume during the media exchange by 1.25 times.Our hypothesis is that the rate of mannose trimming by ER and/or Golgi α-mannosidase I is faster than the rate of diffusion of kifunensine from cell aggregates to individual cells and from individual cells to Golgi and ER compartments.Although the concentration of rrBChE of 7.5 mg/L in the transgenic rice cell suspension culture in this study was significantly lower than the concentration of recombinant BChE of 1–5 g/L from the milk of transgenic goats, it is important to consider the long development time required between gene transfer and lactation for transgenic goats. There is limited quantitative information on production costs for rBChE from the milk of transgenic goats. However, since mammalian production systems can harbor and propagate human pathogens, regulatory requirements for the characterization of the transgenic founder, as well as feeding, housing, health monitoring, genetic stability assessment, and regulated disposal of ex-producer animals, could increase the production costs of human therapeutics in transgenic animals compared with plant-based systems. The expression vector design, cloning, transformation and selection of the callus, and media components were previously described. In brief, rice calli derived from Oryza sativa cv. Taipei 309 embryo/scutellum were co-cultured with Agrobacterium tumefaciens containing the binary vector with the RAmy3D promoter, codon-optimized human BChE gene to express in rice, the RAmy3D signal peptide, and the RAmy3D terminator. After eight rounds of screening starting with more than 300 transformation events, a stable transgenic rice cell line “9-2” was established, which was previously used in other studies as well as this study. The inoculum cultures were grown in 250 mL sugar-rich media in 1 L-shake flasks for 6 days in an Innova 4000 incubator/shaker at 140 rpm and 27 ◦C in the dark.Combined shake flasks described in Section 3.1 were inoculated at about 20% v/vin a 5 L stirred-tank bioreactor equipped with a pitched blade impeller and containing 2.5 L of sterile NB + S added through the head plate inoculation port inside a biosafety cabinet. Bioreactor conditions were controlled at 27 ◦C, 75 rpmagitation speed, 40% dissolved oxygen of air saturation , and 0.2 vvm of the overall mixed gas flow rate. The oxygen uptake rate was measured by the change in DO level in the culture when aeration is stopped but with continued agitation. The culture pH was monitored but not controlled. The cultures grown in the glass bioreactor were exposed to ambient light. Freshly prepared kifunensine solution was added to the bioreactor at 5 µM final concentration in the bioreactor medium at day 6 of cultivation, 24 h before the media exchange. At day 7 of cultivation, media exchange was performed to replace spent sugar-rich medium with sugar-free medium using the same method as previously described. Kifunensine solution was added at day 0 , day 2, and day 4 following induction, assuming 0 µM of kifunensine in the bulk medium prior to each kifunensine addition.