The compound (2S)-eriodictyol is an important flavonoid that can be derived from (2S)-naringenin through flavonoid 3′-hydroxylase (F3′H) catalyzation. F3′H is a cytochrome P450 enzyme that requires a cytochrome P450 reductase (CPR) to function. However, P450s have limited applications in industrial scale biosynthesis, owing to their low activity. Here, an efficient SmF3′H and a matched SmCPR were identified from Silybum marianum. To improve the efficiency of SmF3′H, we established a high-throughput detection method for (2S)-eriodictyol, in which the promoter combination of SmF3′H and SmCPR were optimized in Saccharomyces cerevisiae. The results revealed that SmF3′H/SmCPR should be expressed by using promoters with similar and strong expression levels. Furthermore, directed evolution was applied to further improve the efficiency of SmF3′H/SmCPR. With the optimized promoter and mutated combinations SmF3′H D285N /SmCPR I453V , the (2S)-eriodictyol titer was improved to 3.3 g/L, the highest titer in currently available reports. These results indicated that S. cerevisiae is an ideal platform for functional expression of flavonoid related P450 enzymes.
(2S)-Naringenin
is an important flavonoid precursor,
with multiple nutritional and pharmacological activities. Both (2S)-naringenin and other flavonoid production are hindered
by poor water solubility and inhibited cell growth. To address this,
we increased solubility and improved cell growth by partially glycosylating
(2S)-naringenin to naringenin-7-O-glucoside, which
facilitated increased extracellular secretion, by knocking out endogenous
glycosyl hydrolase genes, EXG1 and SPR1, and expressing the glycosyltransferase gene (UGT733C6). Naringenin-7-O-glucoside synthesis was further improved by optimizing
UDP-glucose and shikimate pathways. Then, hydrochloric acid was used
to hydrolyze naringenin-7-O-glucoside to (2S)-naringenin
outside the cell. Thus, our optimized Saccharomyces
cerevisiae strain E32T19 produced 1184.1 mg/L (2S)-naringenin, a 7.9-fold increase on the starting strain.
Therefore. we propose that glycosylation modification is a useful
strategy for the efficient heterologous biosynthesis of (2S)-naringenin in S. cerevisiae.
Quercetin is an essential ingredient in functional foods and nutritional supplements, as well as a promising therapeutic reagent. Also, the green technique to produce quercetin via rutin biotransformation is attractive. Genes encoding two thermostable glycosidases from Dictyoglomus thermophilum were cloned and expressed in Escherichia coli, which were applied in rutin biotransformation to produce highly pure quercetin at a high temperature. The production of biocatalysts were scaled up in a 5-L bioreactor, yielding a several-fold increase in total enzyme activity and a quercetin production of 14.22 ± 0.26 g/L from 30 g/L of rutin. Feeding strategies were optimized to boost biomass and enzyme production, achieving an activity of 104,801.80 ± 161.99 U/L for rhamnosidase and 12,637.23 ± 17.94 U/L for glucosidase, and a quercetin yield of 20.24 ± 0.27 g/L from the complete conversion of rutin. This study proposes a promising approach for producing high-quality quercetin in an industrial setting.
Graphical Abstract
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