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

Purification and Comparison of Peroxisomal and Cytosolic Catalases from a Methanol-Grown Yeast, Kloeckera sp. 2201

Catalases were purified from a peroxisome-containing particulate fraction and a cytosolic fraction of methanol-grown Kloeckera sp. 2201 cells after subcellular fractionation. No difference was observed between the enzymes in the behaviours on column chromatographies, molecular mass of the subunits (M, 62, 000 daltons), and terminal amino acids, alanine. In addition, similar patterns were obtained with the peroxisomal and cytosolic enzymes on sodium dodecylsulfate/polyacrylamide slab-gel electrophoresis of the peptide fragments prepared by partial digestion with Staphylococcus aureus V 8 protease and papain. These results indicate that cytosolic catalase, even if functional, essentially has identical properties with the peroxisomal one in spite of the different subcellular distribution

Molecular Breeding of Advanced Microorganisms for Biofuel Production

Large amounts of fossil fuels are consumed every day in spite of increasing environmental problems. To preserve the environment and construct a sustainable society, the use of biofuels derived from different kinds of biomass is being practiced worldwide. Although bioethanol has been largely produced, it commonly requires food crops such as corn and sugar cane as substrates. To develop a sustainable energy supply, cellulosic biomass should be used for bioethanol production instead of grain biomass. For this purpose, cell surface engineering technology is a very promising method. In biobutanol and biodiesel production, engineered host fermentation has attracted much attention; however, this method has many limitations such as low productivity and low solvent tolerance of microorganisms. Despite these problems, biofuels such as bioethanol, biobutanol, and biodiesel are potential energy sources that can help establish a sustainable society

Isolation of the Gene Encoding Yeast Peroxisomal Isocitrate Lyase by a Combination of the Plaque Hybridization with Non-Radioactive Probes and the Amplification of Phages in a Small Scale

A genomic DNA encoding isocitrate lyase, one of peroxisomal enzymes, was successfully isolated from an n-alkane-utilizable yeast genomic library prepared in a λEMBL phage by a combination of the plaque hybridization with a non-radioactive, biotin labeled, cDNA and the amplification of the phages in a small scale. Three clones, partially overlapping, with sizes of about 11, 13 and 16 kbp respectively were finally obtained. The genomic Southern blot analysis using the biotin-labeled probe suggested the presence of one isocitrate lyase gene in the genomic DNA

Expression Cloning of Catalase Genomic Gene : Genomic DNA Expression Library of Candida boidinii in Saccharomyces cerevisiae

The genomic DNA expression library of a methylotrophic yeast, Candida boidinii (Kloeckera sp.) 2201, was prepared in Saccharomyces cerevisiae by the electroporation method. Five transformants harbouring a catalase gene were independently isolated with an anti-C. boidinii catalase antibody from this library. Furthermore, exhibition of catalase activity in these transformants demonstrated that C. boidinii genes could sufficiently function even in S. cerevisiae

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

Rapid preparation of mutated influenza hemagglutinins for influenza virus pandemic prevention

Influenza viruses have periodically caused pandemic due to frequent mutation of viral proteins. Influenza viruses have two major membrane glycoproteins: hemagglutinin (HA) and neuraminidase (NA). Hemagglutinin plays a crucial role in viral entry, while NA is involved in the process of a viral escape. In terms of developing antiviral drugs, HA is a more important target than NA in the prevention of pandemic, since HA is likely to change the host specificity of a virus by acquiring mutations, thereby to increase the risk of pandemic. To characterize mutated HA functions, current approaches require immobilization of purified HA on plastic wells and carriers. These troublesome methods make it difficult to respond to emerging mutations. In order to address this problem, a yeast cell surface engineering approach was investigated. Using this technology, human HAs derived from various H1N1 subtypes were successfully and rapidly displayed on the yeast cell surface. The yeast-displayed HAs exhibited similar abilities to native influenza virus HAs. Using this system, human HAs with 190E and 225G mutations were shown to exhibit altered recognition specificities from human to avian erythrocytes. This system furthermore allowed direct measurement of HA binding abilities without protein purification and immobilization. Coupled with the ease of genetic manipulation, this system allows the simple and comprehensive construction of mutant protein libraries on yeast cell surface, thereby contributing to influenza virus pandemic prevention

Novel application of yeast molecular display system to analysis of protein functions

This review fundamentally covers the functional analysis of proteins and the preparation of modified proteins for the creation of novel functions using a yeast molecular display system as a molecular tool. Yeast molecular display is a genetic technique of producing proteins or peptides on the yeast cell surface, such as the cell wall or cell membrane. In this review, recent studies on the yeast molecular display system in the field of not only applied studies but also fundamental studies are described with illustrations

Elucidation of the recognition mechanisms for hemicellulose and pectin in Clostridium cellulovorans using intracellular quantitative proteome analysis

Clostridium cellulovorans is an anaerobic, cellulolytic bacterium, capable of effectively degrading and metabolizing various types of substrates, including cellulose, hemicellulose (xylan and galactomannan), and pectin. Among Clostridia, this ability to degrade and metabolize a wide range of hemicellulose and pectin substrates is a unique feature; however, the mechanisms are currently unknown. To clarify the mechanisms of hemicelluloses and pectin recognition and metabolism, we carried out a quantitative proteome analysis of C. cellulovorans cultured with these substrates. C. cellulovorans was cultured in the medium of glucose (control), xylan, galactomannan (Locus bean gum, LBG), or pectin for 36 h. Xylan and galactomannan were used to search for the common recognition mechanisms of hemicellulose, and pectin was used to search for unique recognition systems in C. cellulovorans. Using an isobaric tag method and liquid chromatograph/mass spectrometer equipped with a long monolithic silica capillary column, we identified 734 intracellular proteins from all substrates. We performed KEGG analyses and cluster analyses of the resulting proteins. In the KEGG analyses, we found common degradation mechanisms for hemicellulose and pectin. In the cluster analysis corresponding to the genome analysis, we detected substrate-specific clusters that include genes involved in substrate recognition, substrate degradation, and metabolism. Combining the results of the KEGG analyses and cluster analyses, we propose the mechanisms involved in the recognition and metabolism of hemicellulose and pectin in C. cellulovorans

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