Proof of concept
Ultimately, we manage to produce catechin during the fermentation experiment. During the process, we refer the research done by H Li et al.For shake flask fermentation, the recombinant yeast was precultured in 5 mL of minimal medium at 30 ℃for 20 h on a shaker at 230 rpm，then transfer 1ml overnight cultured yeast into 50ml YPD medium( glucose carbon base is switched into galactose carbon base, since the galactose promoters pGAL1 and pGAL7 involved in catechin pathway). After inoculated with the seed broth at 30 ℃ for 16 h with shaking at 230 rpm, we add various concentration naringenin substrate（1mM and 2mM）, sampling at 24h, 48h, 72h for subsequent HPLC testing.
During the HPLC testing, we purchased final and intermediate metabolism products standard, naringenin, taxifolin, eriodictyol and catechin, and used them in the subsequent HPLC tests.
Fig.1 The results of HPLC of naringenin, taxifolin, eriodictyol, catechin standard and sample from fermentation medium
As shown in Fig.1, since we could not buy leucoanthocyanidins (LCD), we only did HPLC of the four intermediates: Catechin, Eriodictyol, Taxifolin and Naringenin. Catechin and other intermediate metabolites were confirmed by comparing the samples with the standard.
Fig.2 A: Standard curve of catechin production; B: The bar chart of yield of catechin in 1mM naringenin;C: The bar chart of yield of catechin in 2mM naringenin
According to the Catechin standard curve (Fig.2 A), we calculated the peak area into the output of related products (the detailed data of intermediate metabolites is not shown). We found that catechin output could be seen obviously after 24h and gradually increased over time, reaching about 0.3mm at 72h(Fig.2 B&C). In addition, there was no significant difference in catechin yield under different substrate concentrations, and we will verify this correlation with more experiments on concentration of substates in the future.
Fig.3 The HPLC results comparison across three different sample
As the limitation of time, we only observe the fermentation experiment until 72h. Our team will monitor the yield of catechin in longer fermentative period. Surprisingly, according to analysis of dozens of samples and corresponding HPLC results, there is certain products been spotted at appearance time of 7min, with numerous accumulations, which highly-possibly caused by leucoanthocyanidins (LCD). Considering the yield of catechin, we deduct that the conversion from leucoanthocyanidins to catechin is the rate limiting step within the pathway of catechin synthesis, causing the accumulation of leucoanthocyanidins.
 H Li, Gao S , Zhang S , et al. Effects of metabolic pathway gene copy numbers on the biosynthesis of (2S)-naringenin in Saccharomyces cerevisiae[J]. Journal of Biotechnology, 2020, 325