RESUMEN
Thraustochytrid, Aurantiochytrium sp., produces various lipids such as polyunsaturated and saturated fatty acids, carotenoids, and other hydrocarbons, which are useful in the fields of health foods, cosmetics, fine chemicals, and biofuels. Lignocellulosic biomass, which is abundant and cheap, is a promising feedstock for producing cheaper bulk and high-value-added products using Aurantiochytrium sp. However, the steam explosion of lignocellulosic biomass for efficient enzymatic saccharification generates substances that inhibit the growth of microorganisms. In this study, the inhibitory activities of these by-products on the growth and lipid production of Aurantiochytrium sp. were investigated. Aurantiochytrium sp. was found to be highly sensitive to furfural and vanillin and moderately sensitive to 5-hydroxymethylfurfural and syringaldehyde. Washing steam-exploded bagasse with water, followed by activated charcoal treatment, significantly reduced furfural, which was a major inhibitory component in the saccharified solution.
Asunto(s)
Saccharum , Estramenopilos , Biocombustibles , Biomasa , Carotenoides , Celulosa , Carbón Orgánico , Ácidos Grasos , Furaldehído , VaporRESUMEN
Thraustochytrid strains belonging to the genus Aurantiochytrium accumulate significant amounts of lipids including polyunsaturated fatty acids and carotenoids and, therefore, are expected to be used for industrial production of various valuable materials. Although various efforts such as chemical mutagenesis and homologous gene recombination have been made to improve lipid productivity of Aurantiochytrium species, low specificity and efficiency in the conventional methods hinder the research progress. Here, we attempted to apply a genome editing technology, the CRISPR-Cas9 system as an alternative molecular breeding technique for Aurantiochytrium species to accelerate the metabolic engineering. The efficiency of specific gene knock-in by the homologous recombination increased more than 10-folds by combining the CRISPR-Cas9 system. As a result of disrupting the genes associated with ß-oxidation of fatty acids by the improved method, the genome edited strains with higher fatty acid productivity were isolated, demonstrating for the first time that the CRISPR-Cas9 system was effective for molecular breeding of the strains in the genus Aurantiochytrium to improve lipid productivity.
Asunto(s)
Ácidos Grasos/biosíntesis , Estramenopilos/metabolismo , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica , Genoma , Ingeniería Metabólica , Mutagénesis , Estramenopilos/genéticaRESUMEN
Small bispecific antibodies (bsAbs) are important therapeutic molecules and represent the first bsAb format approved by the United States Food and Drug Administration. Diabody (Db), a small bsAb format, has four possible domain orders; we previously reported the differences in the expression levels and cancer growth inhibition effects upon rearranging the domain order of this format. However, there have been no comprehensive reports on domain rearrangements of bispecific single-chain Db (scDb) and tandem single-chain Fv (taFv), which are widely used bsAb formats. In this study, we designed all possible domain orders for scDb and taFv (each with eight variants) with identical Fv pairs and individually expressed all 16 variants using Escherichia coli, Pichia pastoris, and Brevibacillus choshinensis. Comprehensive investigations showed that the intrinsic functions of the variants were similar to each other, regardless of the expression host system, but expression levels varied depending on the format as well as on the host cell. Among the 16 variants, we found a promising candidate that exhibited high activity and productivity. Furthermore, we determined that B. choshinensis is an attractive expression host because of its secretory production of recombinant proteins.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Sitios de Unión/inmunología , Proteínas Recombinantes de Fusión/inmunología , Anticuerpos de Cadena Única/inmunología , Anticuerpos Biespecíficos/genética , Sitios de Unión/genética , Brevibacillus/genética , Línea Celular Tumoral , Supervivencia Celular/inmunología , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Variación Genética , Humanos , Pichia/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Anticuerpos de Cadena Única/genéticaRESUMEN
Penicillium strains (n=394) preserved at NBRC (the NITE Biological Resource Center) were compared as to groupings (11 species-clusters) based on phylogeny and the production of bioactive compounds. The strains in two clusters, of which P. chrysogenum and P. citrinum are representative, showed higher rates of positive strains with multi-biological activities.
Asunto(s)
Penicillium/clasificación , Penicillium/metabolismo , Filogenia , Aspergillus/genética , Aspergillus/metabolismo , Bioensayo , Japón , Penicillium/genéticaRESUMEN
Three hundred sixty-six Aspergillus strains preserved at the National Institute of Technology and Evaluation (NITE) were compared as to phylogenetic relationships (11 species-clusters) based on the DNA sequences of the D1/D2 domains of LSU rRNA and ITS regions, including the 5.8S rRNA and biological activities of their secondary metabolites. The results showed relatively well correlation between the phylogenetic distribution and the production of bioactive compounds, especially, antimicrobial activities.