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The food enzyme leucyl aminopeptidase (EC 3.4.11.1) is produced with the non-genetically modified Aspergillus sp. strain AE-MB by Amano Enzyme Inc. The food enzyme is considered free from viable cells of the production organism. It is intended to be used in five food manufacturing processes: processing of dairy products for the production of (1) flavouring preparations; processing of plant- and fungal-derived products for the production of (2) protein hydrolysates; processing of meat and fish products for the production of (3) protein hydrolysates, (4) modified meat and fish products and processing of (5) yeast and yeast products. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 2.273 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 183 mg TOS/kg bw per day. The calculated margin of exposure for each age group was 135 (infants), 81 (toddlers), 83 (children), 109 (adolescents), 160 (adults) and 184 (the elderly). A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no matches were found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. The safety of the food enzyme could not be established given the derived margins of exposure. Therefore, the Panel concluded that this food enzyme could not be considered safe under the intended conditions of use.

RESUMEN

Sake is a traditional Japanese alcoholic beverage made from rice and water, fermented by the filamentous fungi Aspergillus oryzae and the yeast Saccharomyces cerevisiae. Yeast strains, also called sake yeasts, with high alcohol yield and the ability to produce desired flavor compounds in the sake, have been isolated from the environment for more than a century. Furthermore, numerous methods to breed sake yeasts without genetic modification have been developed. The objectives of breeding include increasing the efficiency of production, improving the aroma and taste, enhancing safety, imparting functional properties, and altering the appearance of sake. With the recent development of molecular biology, the suitable sake brewing characteristics in sake yeasts, and the causes of acquisition of additional phenotypes in bred yeasts have been elucidated genetically. This mini-review summarizes the history and lineage of sake yeasts, their genetic characteristics, the major breeding methods used, and molecular biological analysis of the acquired strains. The data in this review on the metabolic mechanisms of sake yeasts and their genetic profiles will enable the development of future strains with superior phenotypes.

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The food enzyme ß-galactosidase (EC 3.2.1.23) is produced with the non-genetically modified Neobacillus sp. strain AE-LT by Amano Enzyme Inc. The strain is not cytotoxic and does not harbour any known virulence factor or antimicrobial resistance gene. The presence of viable cells of the production strain in the food enzyme could not be excluded, but the likelihood of this being a hazard is considered low. The food enzyme is intended to be used for lactose hydrolysis in milk processing and the manufacture of galacto-oligosaccharides (GOS). The dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 2.971 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,223 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 412. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

RESUMEN

The CRISPR/Cas9 system opens new horizons (M. Adli, Nat Commun, 2018) regarding genetic modifications of living organisms but also as an in vitro tool in laboratory protocols. Therefore, it boosts possibilities in research and future medical treatments. As the controversial claim of genomically edited babies by He Jiankui (Cyranoski D., Nature, 2019) demonstrates, the new gene editing potentials entail ethical discussions. A public or social discussion presupposes not only a theoretical knowledge or understanding of the system, but also profits from direct laboratory experiences showing how easy these techniques can be applied. Introducing numerous students and classes into these emerging techniques in a modern biology classroom depends on a suitable course concept, which fits legal and organizational requirements at the same time. Therefore, we implemented an appropriate hands-on laboratory course for senior high-school students, lasting just 4.5 h. Particularly with regard to European regulations concerning the handling of genetically modified organisms, the constructs and protocols avoid the transfer of Cas9 DNA. This normally mandatory transfer was replaced by in vitro gene-editing. This leads to Cas9 induced gene knock-outs due to frame shifts and/or the excision of DNA fragments in common Escherichia coli (E. coli) plasmids, such as pUC19. This gene knock-out concept covers various steps: In vitro plasmid editing with Cas9, ligation and transformation of E. coli cells with the modified plasmid DNA and finally the spread plating of transformed E. coli cells in order to analyze colonies after overnight incubation. The successful excision of DNA fragments by in vitro Cas9 treatment was determined by subsequent gel electrophoresis.

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Mutations, serving as the raw materials of evolution, have been extensively utilized to increase the chances of engineering molecules or microbes with tailor-made functions. Global and targeted mutagenesis are two main methods of obtaining various mutations, distinguished by the range of action they can cover. While the former one stresses the mining of novel genetic loci within the whole genomic background, targeted mutagenesis performs in a more straightforward manner, bringing evolutionary escape and error catastrophe under control. In this review, we classify the existing techniques of targeted mutagenesis into two categories in terms of whether the diversity is generated in vitro or in vivo, and briefly introduce the mechanisms and applications of them separately. The inherent connections and development trends of the two classes are also discussed to provide an insight into the next generation evolution research.