1) Preparation of salt resistance enhancing plasmid in budding yeast

When Na+ ions are collected in S. cerevisiae cells, high concentration of Na+ might damage the cells. We have to develop tools which protect S. cerevisiae cells from salt damage. We used two devices here, “compatible solute synthesis” and “chaperon-like protein”. Did you know that a certain group of yeast is working hard in high salt condition, to provide great contribution to tables in the world? They are, Zygosaccharomyces rouxii, soy sauce-brewing yeast. They produce glycerol as compatible solute to counteract osmotic stress. We cloned from Z. rouxii ZrGPD1 (glycerol-3-phosphate dehydrogenase) and ZrFPS1 (glycerol transporter), both of which are important for the glycdrol metabolism in Z. rouxii. We tried to increase salt tolerance of our Swallowmyces cerevisiae by these two genes. Another gene we focuced on is mangrin, a small peptide from mangrove, salt resistant plants. It is a chaperon-like protein and believed that it repairs salt damaged proteins. We decided to test this gene to increase yeast salt tolerance.

2)P reparation of yeast to incorporate Na+

S. cerevisiae has Na+ transporter to remove Na+ from their cytoplasm. The main transporters includes NHA1, ENA1, ENA2, ENA4. When all of these genes were knocked out, the deletion strain shows hypersensitivity against NaCl. To produce yeast strain that uptake more Na+, using homologous recombination system, we knocked-out all of the above genes. Furthermore, we found a protein which is involved in influx of Na+ in plants. It is AtHKT1. By overexpressing AtHKT1, we expect that more Na+ will be collected by the yeast. Another candidate we found is McHKT2. McHKT2 is a Na+ transporter of Mesembryanthemum crystallinum. M. crystallinum, “ice-plant”, is a salt tolerance plant. It is salty. It is reported that McHKT2 is involved the salt compartimentalization in this plant. High concentration Na+ in cytoplasm might damage the cells. To overcome this problem, we will use the salt plants’ salt tolerance system, where Na+ in cytoplasm is sequestered into vacuole by Na+/H+ exchanger. These factors includes Na+/H+ antiporter AtNHXS1 from A. thaliana, SseNHX1, a paralog of AtNHXS1 from salt plant, and a vacuolar protein AVP1 which inclease H+ concentration in vacuole. By enhanceing Na+ influx, by preventing Na+ efflux, and by redirecting cytoplasmic Na+ into vacuoles, we aim to create yeast strain which accumulate more Na+.

3) Reduce the concentration of NaCl in the medium

The goal of our Swallowmyces cerevisiae is not the uptake of Na+. We aim to reduce salt concentration of the water by this device. By combining experimental data and mathematical modeling, we try to optimize our system, to achieve maximum desalination. What will happen when we mix our best strain into high salt-containing media?

4) Development of aggregation system

Even if our device efficiently reduce NaCl concentration in the media, we will never stop our research and development. We try to construct robust safety system for the biocontainment of our genetically modified yeast cells. For this purpose, we selected two gene, SdrG and FgBeta. SdrG is a surface component of Staphylococcus epidermidis. It is known that SdrG tightly binds to a small N-terminus domain of human fibrinogen beta. Reportedly, the binding between SdrG and fibrinogen beta is as strong as a covalent bond. If we express these proteins on the surface of yeast separately, we might see strong coupling of two yeast cells mediated by this interaction. In such a case, as one cell will display multiple handles, we might see a big ball of conjugated yeast cells, when we mix the two “hundle-desplaying” strains.

・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)

SdrGはStaphylococcus epidermidisのsurface componentです。このタンパク質はヒトのfibrinogen beta由来の25アミノ酸という短い配列に非常に強く結合すること、これが生体内へのバイオフィルムの固着に関与していること、などが示されています。これらのタンパク質を別々の酵母でsurface displayにより発現させれば、強い結合力を利用して２細胞の強い結束が実現できるのではないでしょうか？　一つの酵母が複数のハンドルを発現するとすれば、二種類の酵母をブレンドするだけで、酵母は全体で一つの大きな細胞塊になってしまうのでは？　 さて、上の1-4まで、順番にどうなったか、見てみましょう。　→　（リンク）