Abstract
BackgroundBear 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.ResultsWe 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-β-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.ConclusionThis 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.
Highlights
Bear bile powder is a valuable medicinal material characterized by high content of tauroursodeoxycholic acid (TUDCA) at a certain ratio to taurochenodeoxycholic acid (TCDCA)
OD600 and conversion efficiency was predicted for the optimized medium by setting three stars for conversion efficiency and one star for OD600 due to the conversion of TUDCA was the primary goal of the study
3.5 h stirring fermentation compared with 3.5 h stirring fermentation alone. f Results of separating cells from fermentation broth on TUDCA
Summary
Bear bile powder is a valuable medicinal material characterized by high content of tauroursodeoxycholic acid (TUDCA) at a certain ratio to taurochenodeoxycholic acid (TCDCA). The basic work of this strategy is to integrate a heterologous pathway in a microbial host cell where balancing the multiple enzyme-catalyzed reactions of the recreated pathway is essential for high production of committed products. A number of technologies have been developed to balance the metabolic flux including structure gene selection and codon optimization [1], gene transcriptional level controlled by varied strength promoters or gene dosage [1, 2], enzyme co-location through fusion protein or co-expressing with synthetic protein scaffolds [3, 4], and microbial co-culture consortium [5, 6]. Bear bile powder is precious traditional medicinal material. It has been used as anti-convulsant drugs for curing hepatic and biliary disorders and other diseases in
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