Results
1. Glucoamylase expression plasmids construction
Fig.1 Glucoamylase expression plasmids in this project
Glucoamylase (GA) is one of the key enzymes during the starch to glucose process. A genetically engineered GA expressing Saccharomyces cerevisiae strain is strongly required to save the enzyme cost in starch based bioethanol industry.
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).
Fig. 2 Construction of Saccharomycopsis fibuligera derived glucoamylase expressing plasmids
The Golden gate cloning method was used to prepare the GA-expressing plasmids. The basic parts of the plasmids such as the pYES2 backbone, GA coding sequence, promoters, and terminators were all amplified successfully firstly (Fig. 2 A and B), then the multiplex GA expression cassettes were assembled using the overlap PCR method, the resulting parts were cut with SapI restriction enzyme and ligated with the backbone, which was cut with the same enzyme. The primer pairs YZ-IGEM-1/CYC1t-R were set to verify the positive colonies (Fig. 2C). We chose 24 colonies to verify whether the plasmids were correct or not using the colony PCR, the positive rate for the plasmids pYES2-TGC, pYES2-HGC, and pYES2-IGC were 11/24, 17/24, and 17/24, respectively. Two or three positive colonies were sequenced to verify further.
2. GA plasmids transformation
Fig. 3 GA-expressing plasmids transformation and positive S. cerevisiae transformants selection using 50 μg/mL (A) and 350 μg/mL (B) hygromycin. Top left, pYES2-ctl. Top right, pYES2-TGC. Bottom left, pYES2-HGC. Bottom right, pYES2-IGC.
The constructed GA-expressing plasmids were chemically transformed into the S. cerevisiae CEN.PK2-1C strain, the positive transformants were selected against a relatively low concentration of hygromycin (50 μg/mL) firstly to ensure colony growth. As shown in Fig. 1A, plasmids pYES2-ctl, pYES2-TGC, pYES2-HGC, and pYES2-IGC seemed all transformed into the CEN.PK2-1C strain. Then, we streaked colonies from the low concentration hygromycin plates onto the high concentration hygromycin (350 μg/mL) plates again to verify the positive transformants.
Fig. 4 Verification of the plasmids pYES2-ctl (A), pYES2-TGC (B), pYES2-HGC and pYES2-IGC (C) transformation via colony PCR in CEN.PK2-1C strain.
The colonies which grew on the high concentration hygromycin plates were subjected to colony PCR to verify the plasmids transformation again. From Fig. 4 we can see that positive bands implied the plasmids transformation successfully.
Glucoamylase activity assay
Fig. 5 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.
The GA activity of S. cerevisiae strains harboring various plasmids was determined using the glucoamylase activity assay kit. As shown in Fig. 5, 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.
Fermentation test
Fig. 6 Glucose concentration inside the cell during the GA-expressing S. cerevisiae strains corn starch fermentation process.
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 Fig. 6A, 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. 6B), 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. Overall, when the GA was removed from the medium preparation, the GA-expressing strains showed a comparable glucose production capacity with the GA addition situation.