A C35 Carotenoid Biosynthetic Pathway

Upon coexpression with Erwinia geranylgeranyldiphosphate (GGDP) synthase in Escherichia coli, C30 carotenoid synthase CrtM from Staphylococcus aureus produces novel carotenoids with the asymmetrical C35 backbone. The products of condensation of farnesyldiphosphate and GDP, C35 structures comprise 40 to 60% of total carotenoid accumulated. Carotene desaturases and carotene cyclases from C40 or C30 pathways accepted and converted the C35 substrate, thus creating a C35 carotenoid biosynthetic pathway in E. coli. Directed evolution to modulate desaturase step number, together with combinatorial expression of the desaturase variants with lycopene cyclases, allowed us to produce at least 10 compounds not previously described. This result highlights the plastic and expansible nature of carotenoid pathways and illustrates how combinatorial biosynthesis coupled with directed evolution can rapidly access diverse chemical structures

Fusing enzymes to transcription activator LuxR for the rapid creation of metabolite sensors

Metabolite sensors have been applied for high-throughput screening for improved biosynthetic pathways, as well as for dynamic control of the metabolic networks. Obviously, however, current repertoire of natural sensors covers only a small fraction of the known metabolite. We have been developing the new robust workflow for the rapid creation of metabolite sensors where biosynthetic enzymes can be adopted as the sensory (recognizing) components. Most of the known metabolites act as the direct substrates of some enzymes, and they are recognized and converted by these enzymes in physiologically relevant concentrations. Thus, ever-increasing repertoire of available enzymes is a rich and reliable source of sensory units. We found that the transcription activator LuxR can be fused with various biosynthetic enzymes without losing its function. By adding moderately de-stabilizing mutations, typically by random mutagenesis of the resultant fusion proteins followed by screening a small number (~100) of variants, we could have quickly isolated variants that can activate LuxR-dependent promoter in response to the substrates of the enzymes fused to LuxR. In this presentation, we demonstrate various metabolites can be detected by this manner. Detailed analysis of the thus-obtained fusion proteins indicated that function of LuxR is dependent on the substrate binding-induced stabilization of the enzymes. The biosensors with this mode of action exhibited various unique features. For instance, we found that the sensitivity (EC50) and dynamic range of these sensors to the target metabolites can be flexibly altered by the concentration of homoserine lactones, the cognate ligand of LuxR, in the media. Also, this provides unique opportunity to indirectly visualizing the substrate-binding to the enzyme in high-throughput manner. Indeed, multi-round mutagenesis and screening of the fusion protein of isopentenyl diphosphate isomerase (IDI) with LuxR variant (IDI-LuxR) revealed that many of the mutations that improved sensory performance of IDI-LuxR also elevated the catalytic performance of IDI. Some of such mutations turned out to elevate IDI activity even without fusion partner LuxR. Altogether, by fusing to LuxR, random mutagenesis, and traditional reporter (fluorescence)-based screening, one can not only adopt a variety of biosynthetic enzymes as sensor components but also laboratory evolve their catalytic functions

Development of legal mind in the era of high uncertainty: The approach of systematic connection of social studies between elementary school and junior high school

This study aims to enable students to consider the significance and role of legal education in social life and problems concerning intellectual property right (trademark right), relating them to consumers' rights and duty, and company's social responsibility through lesson practice in elementary and junior high school, and acquire the basic way of thinking such as procedural justice. This experience is considered valuable from the perspective of legal literacy. As the achievement of this study is that considering the actual cases enabled the students to organize their own opinions by comprehending the facts, compare their own opinion with the others and understand the importance of creating a more desirable civil society

Construction of carotenoid biosynthetic pathways using squalene synthase

AbstractThe first committed steps of steroid/hopanoid pathways involve squalene synthase (SQS). Here, we report the Escherichia coli production of diaponeurosporene and diapolycopene, yellow C30 carotenoid pigments, by expressing human SQS and Staphylococcus aureus dehydrosqualene (C30 carotenoid) desaturase (CrtN). We suggest that the carotenoid pigments are synthesized mainly via the desaturation of squalene rather than the direct synthesis of dehydrosqualene through the non-reductive condensation of prenyl diphosphate precursors, indicating the possible existence of a “squalene route” and a “lycopersene route” for C30 and C40 carotenoids, respectively. Additionally, this finding yields a new method of colorimetric screening for the cellular activity of squalene synthases, which are major targets for cholesterol-lowering drugs

Social studies in elementary and junior high consistency type to foster international qualities (2): The point of view of legal literacy

本研究を実践することで，他者の異なる意見に耳を傾け，具体的な根拠を挙げて議論できるようになり，同時に自由や平等，公正といった基本的価値に留意しつつ，関連する法制度や具体的な問題を理解・評価できるようになると考える。本研究は，小中それぞれの授業実践を通して，社会生活の中で生じる様々な考え方の違いをどのように調整することで，よりよい社会を形成することができるのか，事実に基づき，憲法や法律も参考にしつつ，効率・公正の視点から考えさせるものである。この経験は，グローバル社会の中でも希望を持って生き抜くことのできる国際的な資質を育成する上で求められるリーガルリテラシーの視点から見ても，意義あることだと考える。成果としては，具体的な事象を通して考えることで，事実をつかみ，他の意見も考慮に入れながら思考・判断することで，自分の考えを構築し，より望ましい市民社会を形成していくことの重要性を理解することができた。今後は，実践の更なる積み重ねを通じて，児童・生徒がより効果的にリーガルマインドを育てることのできるカリキュラム開発を行っていきたい。Through this research, we can listen to different opinion of others and discuss them with specific grounds to discuss basic values such as freedom, equality, fairness and related legal systems and specific problems it will be able to understand and evaluate. This research, through the practice of elementary and junior high school classes, by adjusting the differences in various ways of thinking arising in social life, it is possible to create a better society by referring to the Constitution and the law, It makes us think from a fair perspective. This experience is also significant from the viewpoint of legal literacy required for nurturing international qualities. As a result, the importance of forming a more desirable civil society by grasping facts by thinking through concrete events, building thoughts while considering and judging while considering the opinions of others, I was able to understand

A nucleoside kinase as a dual selector for genetic switches and circuits

The development of genetic switches and their integrated forms (genetic circuits) with desired specifications/functions is key for success in synthetic biology. Due to the difficulty in rational design, genetic switches and circuits with desirable specifications are mostly obtained by directed evolution. Based on a virus-derived nucleotide kinase as a single-gene dual selector, we constructed a robust, efficient and stringent selection system for genetic switches. This method exhibited unprecedented enrichment efficacy (>30 000-fold) of functional switches from non-functional ones in a single selection cycle. In addition, negative (OFF) selection was exceptionally stringent, allowing the rapid and efficient selection of non-leaky from leaky circuits

Evolution of a Pathway to Novel Long-Chain Carotenoids

Using methods of laboratory evolution to force the C(30) carotenoid synthase CrtM to function as a C(40) synthase, followed by further mutagenesis at functionally important amino acid residues, we have discovered that synthase specificity is controlled at the second (rearrangement) step of the two-step reaction. We used this information to engineer CrtM variants that can synthesize previously unknown C(45) and C(50) carotenoid backbones (mono- and diisopentenylphytoenes) from the appropriate isoprenyldiphosphate precursors. With this ability to produce new backbones in Escherichia coli comes the potential to generate whole series of novel carotenoids by using carotenoid-modifying enzymes, including desaturases, cyclases, hydroxylases, and dioxygenases, from naturally occurring pathways

Method to protect a targeted amino acid residue during random mutagenesis

To generate a random mutant library that is free from mutation at a particular amino acid residue, we replace the codon of interest with a detachable, short DNA sequence containing a BsaXI recognition site. After PCR mutagenesis, this sequence is removed and intramolecular ligation of the sequences flanking the insert regenerates the gene. The three-base cohesive ends for ligation correspond to the codon for the targeted residue and any sequences with mutations at this site will fail to ligate. As a result, only the variants that are free from mutation at this site are in the proper reading frame. In a random library of C(30) carotenoid synthase CrtM, this method was used to exclude readily accessible mutations at position F26, which confer C(40) synthase function. This enabled us to identify two additional mutations, W38C and E180G, which confer the same phenotype but are present in the random library at much lower frequencies