1. Construction of CRISPR expression plasmids
The PgaA is an enzyme that hydrolyzes the α-1,4 glycosidic bonds between galacturonic acid residues present in polygalacturonan in plant cell walls and therefore facilitate plant cell wall breakdown. In the production of fruit wine, pectinase has been used to destroy the pectin in the cell wall in order to improve the juice yield and increase the dissolution of aromatic substances such as pigments or terpenes.
Fig.1 Construction CRISPR expression plasmids. (A) Schematic representation of CRISPR expression vectors; (B) Agarose gel electrophoresis of pHCas9-Nours (lanes 1 and 2) and pYES2–gRNA-hyg-MCS (lanes 3 and 4) plasmids.
We sought to integrate PgaA gene into S. cerevisiae genome through CRISPR technology in order to obtain the yeast strains that not only produce alcohol but can also decompose pectin. To this end, we designed two plasmids expressing Cas9 and gRNA (Fig. 1A), as well as the repair template. The agarose gel electrophoresis results indicated that the plasmids of pHCas9-Nours and pYES2–gRNA-hyg-MCS were extracted from DH5 with high quality and could be used for the following transformation experiments.
2. Construction of the repair template
The repair template DNA containing PgaA gene (Fig. 2A) was generated by the overlap-PCR method. Firstly, the DNA fragments of upstream and downstream homologous regions were amplified with ~20 bp ends overlapping to the PgaA gene, producing ~500 bp PCR products (Fig. 2B). Secondly, the two fragments were annealed to the 5’- and 3’-ends of PgaA. Finally, the annealed products were further amplified using end primers of HR-L and HR-R, resulting in a DNA fragment of 1.5 kbp, which was verified as correct by agarose gel electrophoresis and DNA sequencing (Figs. 2C and 2D).
Fig. 2 Construction of repair template. (A) Schematic representation of repair template; (B) Agarose gel electrophoresis of PCR products; (C) DNA sequencing result analysis.
3. 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 colony PCR using the upstream and downstream primers complementary to HR-L and HR-R genes, respectively. As shown in Fig. 3A, the target PCR products at~1500 bp were then extracted and purified for sequencing. The sequencing results finally confirmed that the PgaA gene was successfully integrated into S. cerevisiae genome (Fig. 3B).
Fig. 3 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 and Fig. 4, the OD600 of culture increased from 2 h and reached a plateau at 48 h, indicating that the optimal culture time was 48 h.
4. 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. 5, the concentration of enzyme correlates well with the absorbance detected at 540 nm, applying to the Lambert-Beer law.
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 samples -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 error. Since we measured the Culture media 2 first which possesses higher concentration of PgaA then the Soluble portion of cell lysate 2, the ultrasonic probe might haven’t been fully cleaned which would result the measurement error about the concentration of PgaA in the Soluble portion of cell lysate 2.