Results

Sequence verification:

The first work we completed was to determine the sequence information to be used this year. Our astragalin synthesis pathway contains a total of three enzymes, and we searched many homologous proteins in different medicinal plants. After discussion with the gene synthesis company, we selected three sequences suitable for E. coli expression: F3H from Citrus sinensis, FLS from Citrus unshiu, UGT from Arabidopsis thaliana. And then we asked them to synthesize these three sequences.

Expression test for UGT

1. Verification of the sequence. The sequence we submitted here was come from (Citrus sinensis, Citrus unshiu). In order to get better field, we has done codon optimization for E.coli exprThe three enzymes F3H, FLS and UGT used in this year were 41.36 kD, 39.41 kD and 52.04 kD, respectively. Considering that for E.coli, the higher the molecular weight of the protein, the more difficult it is to express. Therefore, we first detected the largest enzyme UGT. We constructed His-ATUGT +EGFP expression vector, and after induced expression in E.coli BL21 by IPTG, we successfully saw the expression product of UGT gene.
At the same time, we also tested whether there were differences in the expression level under different induction times. According to our results, UGT expression at 12 h was higher than that at 36 h. We proposed that this may be due to the large number of bacteria at 36 h and the high survival pressure, which is not conducive to protein expression.

Expression test for F3H and FLS

When we detected the expression of UGT gene, we accidentally selected a vector with GFP tag. We found that both UGT expression bands and GFP expression bands were seen on SDS-PAGE, which suggested that we could construct the two genes on the same vector.
Therefore, we constructed the first two enzymes of the astragalin production pathway, F3H and FLS, on the same vector. after transforming to E.coli and IPTG induction, we lysed the bacteria for SDS-PAGE and Coomassie brilliant blue staining at last.
This result was in line with our expectation. We detected the expression bands of F3H and FLS enzymes respectively, which indicated that all enzymes in the astragalin production pathway could be expressed in E.coli.
Besides, in keeping with the previous conclusion, the expression level of F3H and FLS after 12 h induction were slightly higher than that with 36 h induction.

Construction for the whole astragalin production pathway

After verifying the expression of each enzyme, we tried to construct the three enzymes on the same plasmid. In order to better express each enzyme, we tried to connect the smaller F3H and FLS with a linker to form a fusion protein, and the larger UGT was expressed separately. Therefore, CSF3H-CuFLS +AtUGT expression vector was constructed.
We took this plasmid and transformed into to E.coli, under proper IPTG concentration induced for 12 h or 36 h, we lysed the bacteria for SDS-PAGE and Coomassie brilliant blue staining. The results showed that we indeed obtained a fusion protein of about 80 kD and a UGT of about 50 kD. Therefore, we constructed a complete astragalin synthesis pathway on this vector.

Astragalin production test

Based on the previous experimental results, we constructed a plasmid for astragalin synthesis and successfully expressed it in E.coli. However, does E.coli generate astragalin?
Since astragalin is a colorless substance, we felt confused to identify the product. Later, we asked relevant experts and carried out HPLC experiments under the advice of experts. We could clearly saw that a peak appeared at 8.164 min. This is consistent with the peak of the standard product astragalin at 8.15 min, which proves that our E.coli is indeed capable of producing astragalin.