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| + | <section class="article p-t-30 p-b-54"> |
| + | <h1 class="content-header">Engineering</h1> |
| + | |
| + | <section> |
| + | <h1 class="title blue">Background </h1> |
| + | <p>Alcohol (ethanol) is an important chemical in the fields of food, medicine, and biofuels. The |
| + | traditional fermentation method uses corn or tapioca starch as raw material to produce alcohol, |
| + | which is obtained by rectification after fermentation by <i>Saccharomyces cerevisia</i>e. The raw |
| + | materials of corn or tapioca starch must be pretreated by crushing first, and then α-amylase |
| + | (liquefaction enzyme) is added to hydrolyze the α-1,4-glucosidic bond in the starch under high |
| + | temperature conditions to cut the starch into short-chain pastes of varying lengths. The |
| + | liquefaction enzyme needs to be added exogenously. If <i>Saccharomyces cerevisiae</i> can express |
| + | the liquefaction enzyme and/or saccharification enzyme genes heterologously, so that the yeast can |
| + | hydrolyze starch and produce sugar by itself, the enzymes can be reduced. Add the amount to reduce |
| + | costs and improve efficiency.</p> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title blue"></h1> |
| + | <p>We considered to express the glucoamylase gene GA derived from <i>Saccharomycopsis fibuligera</i> in |
| + | <I>Saccharomyces cerevisiae</I>. In order to test the effect of gene expression time-specificity on |
| + | the alcohol production of strains fermented starch liquefaction mash, codon optimized GA were |
| + | controlled by 3 different promoters. The three promotors are the constitutive promoter TEF1, the |
| + | glucose-inducible promoter HXT7, and the glucose-repressive promoter ICL1. The transcription of GA |
| + | gene also controlled by strong terminator CYC1. </p> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title blue">Build </h1> |
| + | <p>The Golden gate modular one-step non-marking cloning construction method was used to construct |
| + | plasmids pYES2-TGC, pYES2-HGC and pYES2-IGC (Figure 1). The recombinant plasmids were transformed |
| + | into <i>Saccharomyces cerevisiae</i>. </p> |
| + | <div class="img-container m-t-18"> |
| + | <img src="https://static.igem.org/mediawiki/2021/4/40/T--Fujian_United--img_engineering_1.jpg" alt="" style="width: 70%;"> |
| + | <span class="figure">Figure 1. Schematic map of Glucoamylase expression plasmids. pYES2-ctl is control plasmid.</span> |
| + | </div> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title blue">Test </h1> |
| + | <p>In the test, the glucoamylase activity of the culture supernatant of the obtained strains and the |
| + | ability of fermented corn starch to reduce enzymes to produce alcohol are tested, and the income is |
| + | calculated. The GA activity of <i>S. cerevisiae</i> strains harboring various plasmids was |
| + | determined using the glucoamylase activity assay kit. As shown in Figure 2, the control stain (left |
| + | column) exhibited no GA activity. The strains harboring pYES2-TGC and pYES2-HGC plasmids, which |
| + | means the GA’s expression was driven by the constitutive promoter TEF1 and glucose inducible |
| + | promoter HXT7, showed almost the same and high GA activity. However, the strain in which the |
| + | expression of GA was under the glucose repressive promoter ICL1, exhibited a very low GA activity, |
| + | this is because the substrate of the glucoamylase assay kit is glucose, the expression of the GA |
| + | cassette was inhibited.</p> |
| + | <div class="img-container m-t-18"> |
| + | <img src="https://static.igem.org/mediawiki/2021/4/40/T--Fujian_United--img_engineering_2.jpg" alt="" style="width: 60%;"> |
| + | <span class="figure">Figure 2. S. cerevisiae strains harboring various plasmids glucoamylase activity determination.</span> |
| + | </div> |
| + | <p>**Statistical significance between indicated strains by Student's t test, p < 0.01. ns, not |
| + | significant. Data represent the means of two independent colonies.</p> |
| + | <p>To verify the GA secretion capacity of the GA-expressing <i>S. cerevisiae</i> strains, we measured |
| + | the |
| + | glucose concentration inside the cell during the corn starch fermentation process. As shown in |
| + | Figure 3A, at the initial stage (0 h), when the GA was added during the “starch-to-glucose” process, |
| + | higher contents of glucose were detected than the process without GA addition. This is because GA |
| + | can degrade starch to make more glucose. Along with the fermentation process, the glucose |
| + | concentration in the pYES2-ctl containing strain decreased obviously (Figure 3B), due to the without |
| + | more glucose production, glucose was utilized by the strain. However, the pYES2-TGC and pYES2-HGC |
| + | containing stains showed higher glucose concentration without GA addition, this is because the GA |
| + | produced by the strains could hydrolysis starch to prepare glucose, in other words, the |
| + | GA-expressing strains could reduce the enzyme usage. In contrast to this, when the GA was added in |
| + | the medium preparation process, all the strains showed almost the same glucose concentration at 24 |
| + | h. </p> |
| + | <div class="img-container m-t-18"> |
| + | <img src="https://static.igem.org/mediawiki/2021/6/61/T--Fujian_United--img_engineering_3.jpg" alt="" style="width: 60%;"> |
| + | <span class="figure">Figure 3. Glucose concentration inside the cell during the GA-expressing S. cerevisiae strains corn starch fermentation process.</span> |
| + | </div> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title blue">Learn</h1> |
| + | <p>Genetically engineered yeast can produce glucoamylase enzymes. The GA-expressing strains showed a |
| + | comparable glucose production capacity with the GA addition in the medium. </p> |
| + | </section> |
| + | </section> |
| + | </div> |
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