Interconnected pores on the walls of a polymeric honeycomb monolith structure created by the unidirectional freezing of a binary polymer solution

Interconnected submicron pores were created on the walls of a honeycomb monolith structure by the unidirectional freezing of a binary polymer solution. Agglomerated globules of polyethylene glycol (PEG) in a binary solution of polystyrene (PS) and PEG in 1, 4-dioxane solvent were frozen unidirectionally in a liquid nitrogen bath. Removing the frozen solvent and the agglomerated globules of PEG by freeze-drying and leaching, respectively, enabled us to create interconnected pores in the PS walls. The combination of PS and PEG was effective in creating interconnected pores in the walls because PS and PEG are poorly soluble in one another. The higher freezing rate and lower PEG weight fraction of the binary solution effectively reduced the pore size in the microtube walls

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

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

Peptide-Catalyzed Diastereo- and Enantioselective Cyclopropanation of Aromatic α,β-Unsaturated Aldehydes

Highly diastereo- and enantioselective cyclopropanation of aromatic α,β-unsaturated aldehydes was achieved using a resin-supported peptide catalyst under aqueous conditions. In the peptide sequence, the residue possessing an oxygen atom with the appropriate length of the side chain was essential for attaining good diastereoselectivity

Biosynthetic Study on Antihypercholesterolemic Agent Phomoidride: General Biogenesis of Fungal Dimeric Anhydrides

To elucidate the general biosynthetic pathway of fungal dimeric anhydrides, a gene cluster for the biosynthesis of the antihy-percholesterolemic agent phomoidride was identified by heterologous expression of candidate genes encoding the highly reducing polyketide synthase, alkylcitrate synthase (ACS), and alkylcitrate dehydratase (ACDH). An <i>in vitro</i> analysis of ACS and ACDH revealed that they give rise to anhydride monomers. Based on the established monomer biosynthesis, we propose a general biogenesis of dimeric anhydrides involving a single donor unit and four acceptor units