Three cystein-tagged cellulases co-immobilized on AuNP and Au-MSNP for the hydrolytic degradation of cellulose. The biochemical properties, stabilities, activities and reusability of these co-immobilized systems were compared to those of mixtures of free cellulases.

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Economical production of bioethanol from lignocellulosic biomass still faces many technical limitations. Cost-effective production of fermentable sugars is still not practical for large-scale production of bioethanol due to high costs of lignocellulolytic enzymes. Therefore, plant molecular farming, where plants are used as bioreactors, was developed for the mass production of cell wall degrading enzymes that will help reduce costs. Subcellular targeting is also potentially more suitable for the accumulation of recombinant cellulases. Herein, we generated transgenic tobacco plants (Nicotiana tabacum cv. SR1) that accumulated Thermotoga maritima BglB cellulase, which was driven by the alfalfa RbcsK-1A promoter and contained a small subunit of the rubisco complex transit peptide. The generated transformants possessed high specific BglB activity and did not show any abnormal phenotypes. Furthermore, we genetically engineered the RbcsK-1A promoter (MRbcsK-1A) and fused the amplification promoting sequence (aps) to MRbcsK-1A promoter to obtain high expression of BglB in transgenic plants. AMRsB plant lines with aps-MRbcsK-1A promoter showed the highest specific activity of BglB, and the accumulated BglB protein represented up to 9.3 % of total soluble protein. When BglB was expressed in Arabidopsis and tobacco plants, the maximal production capacity of recombinant BglB was 0.59 and 1.42 mg/g wet weight, respectively. These results suggests that suitable recombinant expression of cellulases in subcellular compartments such as chloroplasts will contribute to the cost-effective production of enzymes, and will serve as the solid foundation for the future commercialization of bioethanol production via plant molecular farming.

The focus of this study was the mechanism of starch accumulation in Chlamydomonas reinhardtii high-starch mutants. Three C. reinhardtii mutants showing high-starch content were generated using gamma irradiation. When grown under nitrogen-deficient conditions, these mutants had more than twice as much starch than a wild-type control. The mechanism of starch over-accumulation in these mutants was studied with comparative transcriptome analysis. In all mutants, induction of phosphoglucomutase 1 (PGM1) expression was detected; PGM1 catalyzes the inter-conversion of glucose 1-phosphate and glucose 6-phosphate in both starch biosynthetic and glycolytic pathway. Interestingly, transcript levels of phosphoglucose isomerase 1 (PGI1), fructose 1,6-bisphosphate aldolase 1 and 2 (FBA1 and FBA2) were down-regulated in all mutants; PGI1, FBA1, and FBA2 act on downstream of glucose 6-phosphate conversion in glycolytic pathway. Therefore, down-regulations of PGI1, FBA1, and FBA2 may lead to accumulation of upstream metabolites, notably glucose 6-phosphate, resulting in induction of PGM1 expression through feed-forward regulation and that PGM1 overexpression caused starch over-accumulation in mutants. These results suggest that PGI1, FBA1, FBA2, and PGM1 correlate with each other in terms of coordinated transcriptional regulation and play central roles for starch over-accumulation in C. reinhardtii.

The aim of this work was to characterize the phot1 mutant of rice during early seedling growth in various light conditions. We isolated the rice T-DNA insertion mutant phot1a-1 and compared it to the Tos17 insertion mutant phot1a-2. When phot1a mutants were grown under WL (100) and BL (40 miccromol m(-2) s(-1)), they demonstrated a considerable reduction in photosynthetic capacity, which included decreased leaf CO(2) uptake and plant growth. Pigment analysis showed no significant difference between wild-type and mutants in the Chl a:b ratios, whereas in the latter, total concentration was reduced (a 2-fold decrease). Carotenoid contents of the mutants were also decreased considerably, implying the involvement of phot1a in pigment degradation. Deletion of phot1a showed higher contents of H(2)O(2) in leaves. Chloroplastic APX and SOD activities were lower in the mutants whereas the activities of cytosolic enzymes were increased. Immunoblotting indicated reduced accumulation of photosystem proteins (D1, D2, CP43, Lhca2, and PsaC) relative to the other light-harvesting complexes in the mutant. We conclude that the defect of Os Phot1a affects degradation of chlorophylls and carotenoids, and under photosynthetically active photon fluxes, mutation of phot1a results in loss of photosynthetic capacity owing to the damage of photosystems caused by elevated H(2)O(2) accumulation, leading to a reduction in plant growth.