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| + | <!--内容--> |
| + | <div class="content-middle-outer"> |
| + | <div class="content-middle"> |
| + | <section class="article p-t-30 p-b-54"> |
| + | <h1 class="content-header">Engineering</h1> |
| + | |
| + | <section> |
| + | <h1 class="title">Background </h1> |
| + | <p>Pectin is a kind of high molecular carbohydrate existing in the cell wall of plants. The main chain |
| + | is polymerized by galacturonic acid (PGA), while the side chain is composed of various |
| + | polysaccharides such as galactose, rhamnose, and xylose. In fruit wine production, <i>Saccharomyces |
| + | cerevisiae</i> uses the sugars in fruit juice to ferment to produce alcohol. It is necessary to |
| + | add pectinase to destroy the pectin in the cell wall to increase the juice yield and increase the |
| + | dissolution of aromatic substances such as pigments or terpenes. In addition, pectinase can also |
| + | eliminate the turbidity of fruit wine caused by pectin colloids, so as to ensure the light |
| + | transmittance and stability of fruit wine to the greatest extent. Most of the pectinase currently on |
| + | the market is a composite enzyme obtained from the fermentation of <i>Aspergillus</i>. This |
| + | undoubtedly increases the cost of fruit wine production.</p> |
| + | <p>There have been successful cases of exogenous expression of pectinase. All these achievements aimed |
| + | to obtain high-yielding pectinase production strains, but yeast strains that can both decompose |
| + | pectin and complete alcohol fermentation have never been obtained.</p> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title">Design </h1> |
| + | <p>This project uses the CRISPR-Cas technology to realize the heterologous expression of the cohesive |
| + | galacturonase gene endo-pgaA of <i>Aspergillus niger</i> SC323 in the fruit wine yeast, and obtain a |
| + | yeast |
| + | strain that can degrade pectin. This will help to reduce the cost of fruit juice and fruit wine |
| + | production process. </p> |
| + | <p>A double-plasmid expression system of CRISPR-Cas9 was designed to edit genome of <i>Saccharomyces |
| + | cerevisiae</i> (Figure 1). One plasmid is pHCas9-Nours plasmid expressing Cas protein and another is |
| + | pYES2-gRNA-hyg-MCS plasmid containing sgRNA. First, pHCas9-Nours plasmid was introduced yeast cells, |
| + | and then the pYES2–gRNA-hyg-MCS plasmid and the homology arm for repair into the wine yeast. After |
| + | obtaining the transformant, verify endo-pgaA Whether to replace to the HXK1 gene locus.</p> |
| + | |
| + | <div class="img-container"> |
| + | <img src="https://static.igem.org/mediawiki/2021/2/22/T--Xiamen_City--img_engineering_1.jpg" alt="" style="width: 80%"> |
| + | <span class="figure">Figure 1. Design of integrating endo-pgaA gene in wine yeast using CRISPR-Cas9 technology. P is the promoter (promotor); T is the terminator (terminator); CEP is the Cas effector protein; HR is the homology arm; endo-pgaA is the pectinase gene.</span> |
| + | </div> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title">Build </h1> |
| + | <p>Plasmids pHCas9-Nourse and pYES2-gRNA-hyg-MCS were successfully constructed (Figure 2). The |
| + | integration of endo-pgaA gene in wine yeast genome was verified by DNA sequencing. </p> |
| + | <div class="img-container m-t-18"> |
| + | <img src="https://static.igem.org/mediawiki/2021/f/fe/T--Xiamen_City--img_engineering_2.jpg" alt="" style="width: 80%"> |
| + | <span class="figure">Figure 2. Schematic maps of plasmids pHCas9-Nourse and pYES2-gRNA-hyg-MCS.</span> |
| + | </div> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title">Test </h1> |
| + | <p>Yeast transformants were picked pectinase production. The pectinase activity was determined using DNS |
| + | method (inducer is β-galactose) (Table 1). The specific activity was calculated from a reduce sugar |
| + | standard curve. </p> |
| + | <div class="img-container"> |
| + | <span class="figure">Table 1. Pectinase activity in yeast transformants.</span> |
| + | <img src="https://static.igem.org/mediawiki/2021/c/c9/T--Xiamen_City--img_engineering_3.jpg" alt="" style="width: 100%"> |
| + | </div> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title">Learn </h1> |
| + | <p>In this project, we successfully prepared genetically engineered wine yeast strain which contains |
| + | pectinase in its genome. The pectinase produced from yeast can well degrade pectin into small |
| + | sugars. </p> |
| + | </section> |
| + | </section> |
| + | </div> |
| + | </div> |
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