Proof of Concept
Overview
Glucoamylase (GA) is one of the key enzymes during the starch to glucose process. Our product is Glucoamylase-in Saccharomyces cerevisiae. With it, the ethanol manufacturer can reduce a great amount of glucoamylase input, thus, lower the cost and increase efficiency. To make our product, we design to construct a plasmid that expresses the gene of secreting glucoamylase, and insert it into the Saccharomyces cerevisiae. While reducing the additional glucoamylase needed during ethanol production, the fermentation performance stays the same. In terms of constructing the plasmid, To express the glucoamylase gene under different strength promoters, we designed three plasmids together with a control plasmid pYES2-ctl (Fig. 1A), that is, GA is expressed under constitutive promoter TEF1 (Fig. 1B), GA is expressed under glucose inducible promoter HXT7 (Fig. 1C), and GA is expressed under glucose repression promoter ICL1 (Fig. 1D).
To assess the performance of the Saccharomyces cerevisiae with our constructed plasmids, we carried out different tests. Firstly, we tested the enzymatic activity of the glucoamylase the Saccharomyces cerevisiae secreted by the glucoamylase activity assay kit and the fermentation tests were also conducted to verify the GA secretion capacity by measuring the yielded glucose and ethanol.
Fig.1 Glucoamylase expression plasmids in this project
Experimental Support
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Glucoamylase activity
Since we have obtained the Glucoamylase-in Saccharomyces cerevisiae harboring various plasmids, we need to ensure that our strains could perform the expected. To verify the glucoamylase activity, we used the glucoamylase activity assay kit and the result is given below.
Fig 2. S. cerevisiae strains harboring various plasmids glucoamylase activity determination.
Though 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.
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.
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Fermentation performance
Considering the potential of our Glucoamylase-in Saccharomyces cerevisiae to be applied in the alcohol industry, we decided to conduct several mini fermentation processes and analyze their fermentation performance by measuring the amount of glucose and alcohol.
Fig 3. Glucose concentration inside the cell during the GA-expressing S. cerevisiae strains corn starch fermentation process.
As shown above, 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 (Fig. 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.
Fig. 4 Alcohol production ability of 4 glucoamylase-in Saccharomyces cerevisiae against enzyme concentration and fermentation hours
We built the model to predict the optimal condition for alcohol production of 4 glucoamylase-in Saccharomyces cerevisiae (Fig. 4). Below are the conclusions:
For A strain, the highest value of alcohol concentration is 0.1993 g/L when enzyme concentration is given 0 and the fermentation time is 24 hours;
For B strain, the highest value of alcohol concentration is 0.1991 g/L when enzyme concentration is given 0.01 and the fermentation time is 22 hours;
For C strain, the highest value of alcohol concentration is 0.2049 g/L when enzyme concentration is given 0 and the fermentation time is 13.5 hours;
For D strain, the highest value of alcohol concentration is 0.2018 g/L when enzyme concentration is given 0.56 and the fermentation time is 13 hours.
Overall consideration, we strongly recommend yeast C for alcohol industry use which requires fewer fermentation hours, less additional enzyme but higher yield.