Engineering
Background
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 Saccharomyces cerevisiae. 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 Saccharomyces cerevisiae 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.
We considered to express the glucoamylase gene GA derived from Saccharomycopsis fibuligera in Saccharomyces cerevisiae. 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.
Build
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 Saccharomyces cerevisiae.
Figure 1. Schematic map of Glucoamylase expression plasmids. pYES2-ctl is control plasmid.
Test
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 S. cerevisiae 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.
Figure 2. S. cerevisiae strains harboring various plasmids glucoamylase activity determination.
**Statistical significance between indicated strains, p < 0.01. ns, not significant. Data represent the means of two independent colonies.
To verify the GA secretion capacity of the GA-expressing S. cerevisiae 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.
Figure 3. Glucose concentration inside the cell during the GA-expressing S. cerevisiae strains corn starch fermentation process.
Learn
Genetically engineered yeast can produce glucoamylase enzymes. The GA-expressing strains showed a comparable glucose production capacity with the GA addition in the medium.