Difference between revisions of "Team:Kyoto/Design"

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<p class="honbun">We tried to enhance halotolerance of yeast in order to make biological devices work even under high salt concentration. For this, we focused on 3 genes, Mangrin, ZrGPD1 and ZrFPS1. Mangrin is derived from a halophyte plant, Mangrove(<i>Bruguiera sexangula</i>), and encodes a shaperon like protein which is already confirmed to express in yeast. It helps proteins exist stably under high salt concentration. A paper has showed that only 71 amino acids of all the sequence is requied for the function, so we used the functional domain.[1]<br>ZrGPD1 and ZrFPS1 is derived from <i>Zygosaccharomyces rouxii</i>. In Japan, it's very popular because used for create soy source. ZrGPD1 encodes the glycerol-3-phosphate dehydrogenase(参考) and related to glycerol synthesis. ZrFPS1 encodes a putative glycerol transporter and inhibit its efflux. Glycerol works as a conpatible solute, so they are expected to work for increase the osmotic torelance and salt one.[2] By introducing these proteins, We tried to expand the range yeast can be addapted. その導入により酵母の適応塩濃度範囲をexpandします。
 
<p class="honbun">We tried to enhance halotolerance of yeast in order to make biological devices work even under high salt concentration. For this, we focused on 3 genes, Mangrin, ZrGPD1 and ZrFPS1. Mangrin is derived from a halophyte plant, Mangrove(<i>Bruguiera sexangula</i>), and encodes a shaperon like protein which is already confirmed to express in yeast. It helps proteins exist stably under high salt concentration. A paper has showed that only 71 amino acids of all the sequence is requied for the function, so we used the functional domain.[1]<br>ZrGPD1 and ZrFPS1 is derived from <i>Zygosaccharomyces rouxii</i>. In Japan, it's very popular because used for create soy source. ZrGPD1 encodes the glycerol-3-phosphate dehydrogenase(参考) and related to glycerol synthesis. ZrFPS1 encodes a putative glycerol transporter and inhibit its efflux. Glycerol works as a conpatible solute, so they are expected to work for increase the osmotic torelance and salt one.[2] By introducing these proteins, We tried to expand the range yeast can be addapted. その導入により酵母の適応塩濃度範囲をexpandします。
 
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Revision as of 14:02, 17 October 2018

Team:Kyoto/Design - 2018.igem.org

Table of contents

高い塩濃度を含む液体から塩を除去する酵母を作るために、われわれは３つのfeatureを酵母に付与することにした。まず、塩濃度の高い環境でも酵母が過剰なダメージを受けないために、耐塩性生物の持つシステムをわれわれの酵母に移植すること。次に、酵母が本来もっているNa+排出システムを破壊して、流入したNa+が外液に戻っていかなくすること。さらに、酵母が細胞質の高いNa+濃度によりダメージを受けないように、細胞内のNa+を積極的に液胞内部へ運ぶポンプを発現していること。この３つを用意することで、細胞内により多くのNa+を蓄積する酵母を作成することを目指した。 さらにわれわれのデバイスの使用用途を拡張するために、biocontainmentのためのハンドルを酵母表面に提示させ、酵母をアグリゲーションさせて回収するシステムを用意した。これら２つを組み合わせることで、より広い環境で脱塩システムを稼働させることができるようになる。

2)塩吸収酵母

私たちは、酵母の細胞膜上、そして液胞膜上のを遺伝子工学的に改変することによりNa+取り込み系の実現を試みました。次のセクションから詳しく記述します。

細胞膜上のNa+輸送に関わるトランスポーターをノックアウトしたり導入したりすることで、細胞膜のNa+透過性を上げ、速度論的にNa+取り込みをimproveします。
・At first, 酵母のネイティブのNa+排出トランスポーターをノックアウトしてNa+の漏出を阻害します。Na+を外部に流出するトランスポーターとして、ENA1,ENA2, ENA5および,NHA1に注目し、以下のノックアウト株の作成をデザインしました。 NHA1Δ、ENA1Δ、NHA1ΔENA1Δ、ENA1,2,5ΔNHA1Δ ENA1, ENA2, and ENA5 array in tandem are nearly identical P-Type ATPases localized on cellular membrane. ENA1 is most characterized and is thought to code a primary membrane Na+-ATPase exporter in S. cerevisiae and contribute to the detoxification of Na+ ion remarkably, so ENA1欠損株は塩感受性を示す事がわかっている。 ゲノムデータベースによると私たちが用いるBY4741、YKO親株がコードするENA1,2,5の遺伝子座は、DBY746/747 and W303.1A/BのENA1-4にあたり、 it is reported that disruption of ENA1-4 genes of S. cerevisiae does not completely eliminate Na+ efflux. [1][2] NHA1 is Na+/H+ antiporter and mediates Na+ efflux through plasma membrane. Its deletion is reported to show salt sensitivity. [3]


・次に、Na+を効率よく内部に取り込むトランスポーターを取り入れることで、Na+をためこませます。そういったトランスポーターとして、シロイヌナズナ由来のAtHKT1,アイスプラント由来のMcHKT2に注目しました。どちらがよりNa+取り込みにおいてパフォーマンスがいいか比較します。 HKT family proteins are high K+ affinity transporters, and AtHKT1 is expressed in xylem of Arabidopsis thaliana. HKT1 from wheatはK+を輸送するが、AtHKT1はそれとは違ってK+の輸送活性はなく、それよりも高いNa+輸送活性を示すことが分かっている。S. cerevisiaeで発現させたとき塩濃度下で生育が阻害されることが確認されており、Na+のため込みに貢献すると考えられる。[4][5] McHK2 is orthologs of AtHKT1 and from Mesembryanthemum crystallinum (ice plant) and thought to be related to cellular Na+ uptake. M.crystallinumはとても強い耐塩性をもっており、McHKT2 has several unique sequence compared to HKT1 ,so it may work better than AtHKT1.[6] (担当、童と仲里さん) (McHKT2はコンストできなかった、と正直にリザルトで書こうと思う)

Na+は様々な酵素の活性を阻害するので(参考)、液胞に隔離させるために、AntiporterNHX1とH+-PpaseAVP1を導入することでNa+取り込み機構を構築します。 NHX1として、シロイヌナズナ由来のAtNHX1をDNAシャッフリングにより活性を高めたAtNHXS1と、2種類の塩生植物のNHX1をDNAシャッフリングしたSseNHX1の２つがあり、どちらがよりよいパフォーマンスをするか選別します。 AtNHXS1

We tried to enhance halotolerance of yeast in order to make biological devices work even under high salt concentration. For this, we focused on 3 genes, Mangrin, ZrGPD1 and ZrFPS1. Mangrin is derived from a halophyte plant, Mangrove(Bruguiera sexangula), and encodes a shaperon like protein which is already confirmed to express in yeast. It helps proteins exist stably under high salt concentration. A paper has showed that only 71 amino acids of all the sequence is requied for the function, so we used the functional domain.[1]
ZrGPD1 and ZrFPS1 is derived from Zygosaccharomyces rouxii. In Japan, it's very popular because used for create soy source. ZrGPD1 encodes the glycerol-3-phosphate dehydrogenase(参考) and related to glycerol synthesis. ZrFPS1 encodes a putative glycerol transporter and inhibit its efflux. Glycerol works as a conpatible solute, so they are expected to work for increase the osmotic torelance and salt one.[2] By introducing these proteins, We tried to expand the range yeast can be addapted. その導入により酵母の適応塩濃度範囲をexpandします。

Figure1:酵母の耐塩性に貢献する３つのタンパク質を表した図。(聞くならmangrin→續さん、Zr:島添君)

4)凝集酵母

私たちの酵母によって塩を回収したあと、酵母を回収しやすくするためそしてバイオセーフティーのためにそれらを凝集させる系の確立を目指しました。そのためにsurface displayを介してSdrG-Fgβ結合という共有結合に匹敵するほど強力なタンパク質間結合の利用に着目しました。SdrG is SD-repeat protein from Staphylococcus epidermidis, and it targets a short peptide sequence of human fibrinogen. それらは下の図のようにin a screw mannweで、FgβがSdrGのN2 domainとN3 domainの間のbinding pocketに入りhydrogen bondを形成して、共有結合なみの結合を形成することが報告されている。Moreover, FgβF3(it contains one extra Phenylalanine) is reported to form a more strong conjugation to SdrG compared to WT Fgβ. So for this system, we decided to use SdrG and FgβF3 connection.

In order to display the proteins on the cell surface, we used sed1 anchoringドメイン which is a kind of GPI anchor and used for yeast surface display. A paper has reported sed1 secretion signal sequence have a good efficiency of surface display. Therefore we designed plasmids by which yeast can display SdrG N2_N3 domain/FgβF3 through the anchoring protein.

(表層提示のイラスト)
Reference
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  • [2] Jos6 A. Miirquez, Ramdn Serrano (1996) Multiple transduction pathways regulate the sodium-extrusion gene PMR2/ENA1 during salt stress in yeast, FEBS Letters Vol.382 Issue1-2 89-92
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