Enhancing Key Metabolic Processes Of Catechin Synthesis In Saccharomyces Cerevisiae

Flavan-3-ols and dihydroquercetin are a kind of flavonoids with pharmaceutical values.However,there is a low extraction rate from plants.These years,microbiological biosynthesis of flavonoids has shown more advantages due to the low cost and easy industrialization.This research focused on key metabolic pathway related to the biosynthesis of flavan-3-ols and dihydroquercetin.The engineered Saccharomyces cerevisiae strain E32 that produces naringenin from glucose was further engineered for de novo production of two basic flavan-3-ols.By metabolic regulation and fermentation condition optimization,flavan-3-ols production was further improved.In addition,the intermediate dihydroquercetin is also a valuable flavonoid and the low titer limit the generation of catechin,and the metabolic regulation of its biosynthesis from naringenin was also studied.The strategies presented could be applied for a more efficient production of flavonoids by various microorganisms.The main contents and results are summarized as follows:1)Two strains which can realize the de novo production of afzelechin and catechin were constructed.Through introduction of flavonoid 3-hydroxylase,flavonoid 3’-hydroxylase,dihydroflavonol 4-reductase,and leucoanthocyanidin reductase,de novo production of afzelechin and catechin can be achieved.The combination of Fa DFR from Fragaria x ananassa and Vv LAR from Vitis vinifera was optimal.（GGGGS）₂ and（EAAAK）₂ linkers between DFR and LAR proved optimal for the production of AFZ and CAT,respectively.Optimization of promoters and the enhanced supply of NADPH further increased the production.2)The fermentation condition was further optimized in 5L bioreactor.10 g·L^-1 Ca CO₃was added to main p H,and calcium ion maybe serve as an essential catalytic cofactor to activate the specific enzymes involved in certain metabolic pathways,resulting in redirection of the intracellular carbon and energy flux.Removing tryptone from the feed medium to reduce the cost and optimizing the dissolved oxygen.The result showed that 10%saturation is suitable.By combining the best engineering strategies,the optimum strains produced 500.5 mg·L^-1 AFZ and 321.3 mg·L^-1 CAT,respectively,after fermentation for 90 h in a 5-L bioreactor.3)The metabolic pathway from naringenin to dihydroquercetin were regulated.Synthesis of naringin(500 g·L^-1)to dihydroquercetin(254 mg·L^-1)was achieved by integrating flavonoid3-hydroxylase（F3H）、flavonoid 3’-hydroxylase（F3’H）、cytochrome P450 reductase（CPR）from Silybum marianum at Ty2 multi-copy site of S.cerevisiae’s genome.609 mg·L^-1dihydroquercetin can be obtained when adding 1 g·L^-1 naringenin as substrate.By enlarging the endoplasmic reticulum size,the membrane-bound P450 enzyme was more anchored to the endoplasmic reticulum,and the production of dihydroquercetin was further increased.In this process,we found that too much eriodictyol accumulated.In order to solve this problem,the expression of F3H was optimized and the TCA cycle was reconstructed to realize the coupling between the production and growth of dihydroquercetin.But there was no effect.2.7 g·L^-1dihydroquercetin can be obtained after fermentation for 78 h when adding 5 g·L^-1 naringenin as substrate in a 5-L bioreactor.