Large-scale production of tauroursodeoxycholic acid products through fermentation optimization of engineered Escherichia coli cell factory.

BACKGROUND: Bear bile powder is a valuable medicinal material characterized by high content of tauroursodeoxycholic acid (TUDCA) at a certain ratio to taurochenodeoxycholic acid (TCDCA). We had created an engineered E. coli harboring two-step bidirectional oxidative and reductive enzyme-catalyzing pathway that could rapidly convert TCDCA to TUDCA at a specific percentage in shake flasks. RESULTS: We reported here the large-scale production of TUDCA containing products by balancing the bidirectional reactions through optimizing fermentation process of the engineered E. coli in fermenters. The fermentation medium was firstly optimized based on M9 medium using response surface methodology, leading to a glycerol and yeast extract modified M9-GY medium benefits for both cell growth and product conversion efficiency. Then isopropylthio-ß-galactoside induction and fed-stock stage was successively optimized. Finally, a special deep-tank static process was developed to promote the conversion from TCDCA to TUDCA. Applying the optimal condition, fermentation was performed by separately supplementing 30 g refined chicken bile powder and 35 g crude chicken bile powder as substrates, resulting in 29.35 ± 2.83 g and 30.78 ± 3.04 g powder products containing 35.85 ± 3.85% and 27.14 ± 4.23% of TUDCA at a ratio of 1.49 ± 0.14 and 1.55 ± 0.19 to TCDCA, respectively, after purification and evaporation of the fermentation broth. The recovery yield was 92.84 ± 4.21% and 91.83 ± 2.56%, respectively. CONCLUSION: This study provided a practical and environment friendly industrialized process for producing artificial substitute of bear bile powder from cheap and readily available chicken bile powder using engineered E. coli microbial cell factory. It also put forward an interesting deep-tank static process to promote the enzyme-catalyzing reactions toward target compounds in synthetic biology-based fermentation.

Effect of tamoxifen and raloxifene on the conjugation of bile acids with taurine and glycine in ovariectomized rats.

The selective estrogen receptor modulators tamoxifen and raloxifene represent a major therapeutic advance for clinical practice. Unlike estrogens, which are uniformly agonists, tamoxifen and raloxifene exert selective agonistic or antagonistic effects on various estrogen target tissues. The aim of the study was to evaluate the influence of tamoxifen and raloxifene on the conversion of cholesterol to bile acids in estrogen deficiency in rats. The study included 40 female Wistar rats. The animals were divided into four groups: sham operated control, ovariectomized control, ovariectomized rats treated with tamoxifen ovariectomized rats treated with raloxifene. After 42 days of drug administration, bile was collected under anesthesia after administration of radioactive 4-(14)C cholesterol. Bile was assayed for concentration of (14)C bile acids conjugated with taurine and glycine after thin-layer chromatography separation by the use of isotopic technique. In rats treated with tamoxifen and raloxifene, the statistically significant increase in concentration of bile acids conjugated with glycine was observed as compared to ovariectomized animals from control group. Moreover, in rats treated with tamoxifen the concentration of bile acids conjugated with taurine significantly increased. The results of the present study suggest that tamoxifen and raloxifene increase the concentrations of conjugated bile acids in bile.

Feedback regulation of bile acid synthesis in the rat by dietary vs. intravenous cholate or taurocholate.

The regulation of bile acid synthesis was studied (i) in intact or colectomized rats receiving cholate or taurocholate as a dietary supplement and (ii) in experiments using chow-fed animals with a graded intravenous or intraduodenal taurocholate infusion. After the 2-week diet period a bile fistula was established and rates of taurocholate, tauromuricholate and taurochenodeoxycholate secretion were quantitated by high-performance liquid chromatography. During the infusion experiments taurocholate production was calculated from the difference in specific activity of [14C]taurocholate between infusate and bile, whereas tauromuricholate and taurochenodeoxycholate synthesis was derived directly from their secretion rates after pool depletion. Both the 0.5% cholate and taurocholate diet suppressed tauromuricholate and taurochenodeoxycholate secretion nearly totally, but only cholate led to a prolonged inhibition taurocholate synthesis. The diets stimulated total bile acid secretion and expanded the total bile acid pool size 2- to 3-fold, but they also prompted a dramatic increase in the biliary secretion of taurodeoxycholate. In contrast, colectomized animals did not secrete taurodeoxycholate following the cholate diet and, despite a comparable increase in bile acid pool size, tauromuricholate and taurochenodeoxycholate secretion was inhibited to a lesser extent. In addition, the rate of bile acid secretion and synthesis was significantly enhanced when compared to that of intact rats. To determine whether taurocholate affected bile acid synthesis directly, the bile acid was infused intravenously or intraduodenally at varying rates up to 300 mumoles per kg per hr for 54 hr, i.e. a rate exceeding normal total bile acid secretion in these acute bile fistula animals nearly 3-fold.(ABSTRACT TRUNCATED AT 250 WORDS)