Proof of Concept
Overview
Alcohol has been used for thousands of years. The application areas of alcohol penetrate through people’s lives, ranging from cosmetics to medical treatment, and drinks, etc. The earliest evidence of alcoholic beverages has been found dating from 5400-5500 BC. In current society, alcoholic drinks also influence our daily life deeply. As one of the major alcoholic drinks, fruit wines have gradually attracted more and more attention. According to data released by JD. Com (One of the largest E-Commerce platforms in China), there have been over 300 fruit wine sellers on JD. Com in 2019, whose sales have endured over 200% compound increase rate from 2014 to 2019. Therefore, our project focuses on the fruit wine industry, hoping to contribute to optimizing the production process, lower production costs, and enhance fruit wine quality.
The preparation process of traditional fruit wines is expensive and cumbersome. This is probably the greatest efficiency issue facing fruit wine today in the production process. During our research, we discovered that majority of the pectinases available on the market are complex enzymes obtained from Aspergillus fermentation, and the price per 1 kg of pectinase varies from 100-250 RMB. The high cost of pectinase is not conducive to the further development of the food manufacturing industry and other fields.
We want to apply CRISPR-Cas technology to successfully heterologously express endo-pgaA, an endogalacturonase gene from Aspergillus niger SC323, in fructooligosaccharide yeast. So, we can obtain a strain that is capable of both pectin degradation and alcohol fermentation. This will help to explore the development of multifunctional fruit wine yeast and reduce the costs in the production of fruit juice and fruit wine. It will also contribute to the development of food industry production and modern brewing engineering.
Supporting Experiment Results
Yeast strain transformation and positive transformants verification
The constructed CRISPR plasmids and repair template DNA were chemically transformed into the S. cerevisiae strains. The positive transformants were selected against YPD medium supplemented with Nours and hygromycin. The resulting colonies were picked up and cultured. To investigate whether the PgaA gene was integrated into yeast genome, we performed PCR experiments using the upstream and downstream primers complementary to HR-L and HR-R genes, respectively. As shown in Fig. 3A, we obtained specific PCR products with expected size of ~1500 bp. The DNA fragments were then extracted and purified for sequencing. The sequencing results finally confirmed that the PgaA gene was successfully integrated into S. cerevisiae genome (Fig. 1).
Fig. 1 Verification of PgaA containing transformants. (A) Agarose gel electrophoresis of PCR products; (B) DNA sequencing result analysis.
To obtain an optimal culture time, we monitored the growth rate of recombinant S. cerevisiae cells from 2 to 72 h. As shown in Table.1, the OD600 of culture increased from 2 h and reached a plateau at 48 h, indicating that the optimal culture time was 48 h.
2. Pectinase activity assay
The pectinase activities of PgaA were determined using the dinitrosalicylic acid (DNS) colorimetric method. Briefly, in the presence of PgaA, pectin can be degraded into galacturonic acids, which reacts with DNS to form a compound with a maximum absorption at 540 nm. Thus, the activity of PgaA can be calculated by measuring the absorbance of the reactants with a spectrophotometer. For accurate quantification, a standard curve was generated using a series of concentrations of pectinase standards. As shown in Table. 2 and Fig. 4, the concentration of enzyme correlates well with the absorbance detected at 540 nm, applying to the Lambert-Beer law.
Fig. 2 Standard curve of pectinase.
With this standard curve, we next determined the concentration of PgaA from recombinant S. cerevisiae strains. Samples from the culture media, total cell lysates and the soluble portion of cell lysates were collected and subjected to DNS colorimetric assay. As shown in Table. 3, the concentration of PgaA in the culture media of sample -1 and -2 were determined at about 0.034 mg/ml and 0.028 mg/ml, respectively, which were relatively higher than that of cell lysates (0.009 mg/ml and 0.007 mg/ml), suggesting that most of the PgaA proteins were secreted into the culture media. In addition, in the cell lysates of sample 1, we detected ~76% of PgaA in the soluble supernatants, implying that most of the PgaA in cells are soluble. Unexpectedly, the concentration of PgaA in the soluble supernatants of sample 2 was higher than that of total cell lysates, this could be due to experimental mistakes.
The experiment results of Pectinase activity assay indicate that we have successfully obtained a gene-edited yeast that is capable of both pectin degradation and alcohol fermentation. It will serve as a solid foundation for our future research and development of wine yeast. Surely, we know that it is essential to make further experiments to enhance the pectin degradation capacity of our yeast, we have already obtained a good start.
Contact Info
