Sustainable Biological Ammonia Production towards a Carbon-Free Society

脱炭素・次世代エネルギーとしてのアンモニアの食品加工廃棄物からのバイオ生産プラットフォーム --脱炭素社会構築へのバイオテクノロジーによる貢献をめざして--. 京都大学プレスリリース. 2021-09-06.A sustainable society was proposed more than 50 years ago. However, it is yet to be realised. For example, the production of ammonia, an important chemical widely used in the agriculture, steel, chemical, textile, and pharmaceutical industries, still depends on fossil fuels. Recently, biological approaches to achieve sustainable ammonia production have been gaining attention. Moreover, unlike chemical methods, biological approaches have a lesser environmental impact because ammonia can be produced under mild conditions of normal temperature and pressure. Therefore, in previous studies, nitrogen fixation by nitrogenase, including enzymatic ammonia production using food waste, has been attempted. Additionally, the production of crops using nitrogen-fixing bacteria has been implemented in the industry as one of the most promising approaches to achieving a sustainable ammonia economy. Thus, in this review, we described previous studies on biological ammonia production and showed the prospects for realising a sustainable society

Development of a yeast cell surface display method using the SpyTag/SpyCatcher system

生体内タンパク質ライゲーションを用いた新規細胞表層ディスプレイ法の開発 --新しい手法によるタンパク質工学の進展--. 京都大学プレスリリース. 2021-05-28.Yeast cell surface display (YSD) has been used to engineer various proteins, including antibodies. Directed evolution, which subjects a gene to iterative rounds of mutagenesis, selection and amplification, is useful for protein engineering. In vivo continuous mutagenesis, which continuously diversifies target genes in the host cell, is a promising tool for accelerating directed evolution. However, combining in vivo continuous evolution and YSD is difficult because mutations in the gene encoding the anchor proteins may inhibit the display of target proteins on the cell surface. In this study, we have developed a modified YSD method that utilises SpyTag/SpyCatcher-based in vivo protein ligation. A nanobody fused with a SpyTag of 16 amino acids and an anchor protein fused with a SpyCatcher of 113 amino acids are encoded by separate gene cassettes and then assembled via isopeptide bond formation. This system achieved a high display efficiency of more than 90%, no intercellular protein ligation events, and the enrichment of target cells by cell sorting. These results suggested that our system demonstrates comparable performance with conventional YSD methods; therefore, it can be an appropriate platform to be integrated with in vivo continuous evolution

STABILITY CRITERIA FOR THE SYSTEM OF DELAY DIFFERENTIAL EQUATIONS AND ITS APPLICATIONS

In this paper, we consider the asymptotic stability for the system of linear delay differential equations. Because of the complicated interactions induced by the delay effects of the system, there are few results of the asymptotic stability for the system of the delay differential equations with multiple delays. Given this fact, we propose the new stability conditions for the system and apply these conditions to some mathematical models for the population dynamics and neural network system described by the system of delay differential equations

Evaluation of a library of loxP variants with a wide range of recombination efficiencies by Cre

Sparse labeling of individual cells is an important approach in neuroscience and many other fields of research. Various methods have been developed to sparsely label only a small population of cells; however, there is no simple and reproducible strategy for managing the probability of sparse labeling at desired levels. Here, we aimed to develop a novel methodology based on the Cre-lox system to regulate sparseness at desired levels, and we purely analyzed cleavage efficiencies of loxP mutants by Cre. We hypothesized that mutations in the loxP sequence reduce the recognition efficiency by Cre, which enables the regulation of the sparseness level of gene expression. In this research, we mutagenized the loxP sequence and analyzed a library of loxP variants. We evaluated more than 1000 mutant loxP sequences, including mutants with reduced excision efficiencies by Cre ranging from 0.51% to 59%. This result suggests that these mutant loxP sequences can be useful in regulating the sparseness of genetic labeling at desired levels