Functional production of human antibody by the filamentous fungus Aspergillus oryzae.

BACKGROUND: Monoclonal antibodies (mAbs) as biopharmaceuticals take a pivotal role in the current therapeutic applications. Generally mammalian cell lines, such as those derived from Chinese hamster ovaries (CHO), are used to produce the recombinant antibody. However, there are still concerns about the high cost and the risk of pathogenic contamination when using mammalian cells. Aspergillus oryzae, a filamentous fungus recognized as a GRAS (Generally Regarded As Safe) organism, has an ability to secrete a large amount of proteins into the culture supernatant, and thus the fungus has been used as one of the cost-effective microbial hosts for heterologous protein production. Pursuing this strategy the human anti-TNFα antibody adalimumab, one of the world's best-selling antibodies for the treatment of immune-mediated inflammatory diseases including rheumatoid arthritis, was chosen to produce the full length of mAbs by A. oryzae. Generally, N-glycosylation of the antibody affects immune effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) via binding to the Fc receptor (FcγR) on immune cells. The CRISPR/Cas9 system was used to first delete the Aooch1 gene encoding a key enzyme for the hyper-mannosylation process in fungi to investigate the binding ability of antibody with FcγRIIIa. RESULTS: Adalimumab was expressed in A. oryzae by the fusion protein system with α-amylase AmyB. The full-length adalimumab consisting of two heavy and two light chains was successfully produced in the culture supernatants. Among the producing strains, the highest amount of antibody was obtained from the ten-protease deletion strain (39.7 mg/L). Two-step purifications by Protein A and size-exclusion chromatography were applied to obtain the high purity sample for further analysis. The antigen-binding and TNFα neutralizing activities of the adalimumab produced by A. oryzae were comparable with those of a commercial product Humira®. No apparent binding with the FcγRIIIa was detected with the recombinant adalimumab even by altering the N-glycan structure using the Aooch1 deletion strain, which suggests only a little additional activity of immune effector functions. CONCLUSION: These results demonstrated an alternative low-cost platform for human antibody production by using A. oryzae, possibly offering a reasonable expenditure for patient's welfare.

A novel glucuronoyl esterase from Aspergillus fumigatus-the role of conserved Lys residue in the preference for 4-O-methyl glucuronoyl esters.

Cellulose in plant cell walls is mainly covered by hemicellulose and lignin, and thus efficient removal of these components is thought to be a key step in the optimal utilization of lignocellulose. The recently discovered carbohydrate esterase (CE) 15 family of glucuronoyl esterases (GEs) which cleave the linkages between the free carboxyl group of D-glucuronic acid in hemicellulose and the benzyl groups in lignin residues could contribute to this process. Herein, we report the identification, functional expression, and enzymatic characterization of a GE, AfGE, from the filamentous fungus Aspergillus fumigatus. AfGE was heterologously expressed in Aspergillus oryzae, and the purified enzyme displayed the ability to degrade the synthetic substrates mimicking the ester linkage between hemicellulose and lignin. AfGE is a potentially industrially applicable enzyme due to its characteristic as a thermophilic enzyme with the favorable temperature of 40-50 °C at pH 5. Molecular modeling and site-directed mutagenesis studies of AfGE demonstrated that Lys209 plays an important role in the preference for the substrates containing 4-O-methyl group in the glucopyranose ring.

Functional expression and characterization of a glucuronoyl esterase from the fungus Neurospora crassa: identification of novel consensus sequences containing the catalytic triad.

The complete hydrolysis of lignocellulose requires the actions of a variety of enzymes, including those that cleave the linkage between lignin and hemicellulose. The enzyme glucuronoyl esterase (GE) that constitutes a novel family of carbohydrate esterases, CE15, has been shown to display a unique ability to cleave the ester linkage between lignin alcohols and xylan-bound 4-O-methyl-D-glucuronic acid of hemicellulose. We herein report identification, expression, and functional characterization of a new GE, NcGE, from the filamentous fungus Neurospora crassa. C-terminally c-myc and hexahistidine-tagged NcGE was heterologously expressed in the methylotrophic yeast Pichia pastoris. NcGE purified from the culture supernatant through Ni-NTA and anion exchange chromatographies showed the ability to hydrolyze the substrate 3-(4-methoxyphenyl) propyl methyl 4-O-methyl-α-D-glucopyranosiduronate, which mimics the ester linkage of 4-O-methyl-D-glucuronic acid in lignin-carbohydrate complexes (LCCs). This esterase showed the characteristic of a mesophilic enzyme with the temperature optimum at 40-50°C, and displayed the optimal activity at pH 7 and broad pH stability. Based on the alignment of NcGE with other GEs so far characterized, we propose novel consensus sequences for GEs containing the catalytic triad.