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Ascomycete Aspergillus oryzae Is an Efficient Expression Host for Production of Basidiomycete Terpenes by Using Genomic DNA Sequences.

ABSTRACT: Basidiomycete fungi are an attractive resource for biologically active natural products for use in pharmaceutically relevant compounds. Recently, genome projects on mushroom fungi have provided a great deal of biosynthetic gene cluster information. However, functional analyses of the gene clusters for natural products were largely unexplored because of the difficulty of cDNA preparation and lack of gene manipulation tools for basidiomycete fungi. To develop a versatile host for basidiomycete genes, we examined gene expression using genomic DNA sequences in the robust ascomycete host Aspergillus oryzae, which is frequently used for the production of metabolites from filamentous fungi. Exhaustive expression of 30 terpene synthase genes from the basidiomycetes Clitopilus pseudo-pinsitus and Stereum hirsutum showed two splicing patterns, i.e., completely spliced cDNAs giving terpenes (15 cases) and mostly spliced cDNAs, indicating that A. oryzae correctly spliced most introns at the predicted positions and lengths. The mostly spliced cDNAs were expressed after PCR-based removal of introns, resulting in the successful production of terpenes (14 cases). During this study, we observed relatively frequent mispredictions in the automated program. Hence, the complementary use of A. oryzae expression and automated prediction will be a powerful tool for genome mining.IMPORTANCE The recent large influx of genome sequences from basidiomycetes, which are prolific producers of bioactive natural products, may provide opportunities to develop novel drug candidates. The development of a reliable expression system is essential for the genome mining of natural products because of the lack of a tractable host for heterologous expression of basidiomycete genes. For this purpose, we applied the ascomycete Aspergillus oryzae system for the direct expression of fungal natural product biosynthetic genes from genomic DNA. Using this system, 29 sesquiterpene synthase genes and diterpene biosynthetic genes for bioactive pleuromutilin were successfully expressed. Together with the use of computational tools for intron prediction, this Aspergillus oryzae system represents a practical method for the production of basidiomycete natural products.

SUBMITTER: Nagamine S

PROVIDER: S-EPMC6643257 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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-TNF? 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 (Fc?R) 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 Fc?RIIIa. Results:Adalimumab was expressed in A. oryzae by the fusion protein system with ?-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 TNF? neutralizing activities of the adalimumab produced by A. oryzae were comparable with those of a commercial product Humira®. No apparent binding with the Fc?RIIIa 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.

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Ascomycete Aspergillus oryzae Is an Efficient Expression Host for Production of Basidiomycete Terpenes by Using Genomic DNA Sequences.

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