A green strategy to produce potential substitute resource for bear bile using engineered Saccharomyces cerevisiae

Lina Jin; Li Yang; Shujuan Zhao; Zhengtao Wang

doi:10.1186/s40643-022-00517-3

A green strategy to produce potential substitute resource for bear bile using engineered Saccharomyces cerevisiae

Jin, Lina; Yang, Li; Zhao, Shujuan; Wang, Zhengtao 2022-03-27 00:00:00 Background: Bear bile powder is a precious natural material characterized by high content of tauroursodeoxycholic acid ( TUDCA) at a ratio of 1.00–1.50 to taurochenodeoxycholic acid ( TCDCA). Results: In this study, we use the crude enzymes from engineered Saccharomyces cerevisiae to directionally convert TCDCA from chicken bile powder to TUDCA at the committed ratio in vitro. This S. cerevisiae strain was modified with heterologous 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenase (7β-HSDH) genes. S. cerevisiae host and HSDH gene combinatorial optimization and response surface methodology was applied to get the best engineered strain and the optimal biotransformation condition, respectively, under which 10.99 ± 0.16 g/L of powder products containing 36.73 ± 6.68% of TUDCA and 28.22 ± 6.05% of TCDCA were obtained using 12.00 g/L of chicken bile powder as substrate. Conclusion: This study provides a healthy and environmentally friendly way to produce potential alternative resource for bear bile powder from cheap and readily available chicken bile powder, and also gives a reference for the green manufacturing of other rare and endangered animal-derived valuable resource. Keywords: Tauroursodeoxycholic acid, Hydroxysteroid dehydrogenase, Biotransformation, Saccharomyces cerevisiae Introduction by implanting a duct or making an artificial fistula in the Bear bile powder is a kind of precious material that has liver of the bears (Feng et al. 2009). These methods will been used as medicine and healthcare supplementary seriously endanger the health of bears, thus exploring thousands of years ago in Asia area (Yang et al. 2008; substitute resources is of great significance to meet the Feng et al. 2009). It has multiple pharmacological activi- demands of bear bile powder as medicinal and healthcare ties and can be used to treat gallstones, cholecystitis, fatty material. liver and other hepatobiliary diseases (Feng et al. 2009). Bile acids (BAs), a group of steroids with C-17 side At present, bear bile powder is mainly from the drainage chains, are the principal bioactive ingredients of bear bile bear bile extracted from the farmed living bears including powder, among which ursodesoxycholic acid (UDCA) Selenarctos thibetanus (Asiatic black bear) or Ursus arc- or its physiologically active form tauroursodeoxycholic tos (brown bear) with “Free-dripping Fistula Technique” acid (TUDCA) takes a high proportion and is considered as distinct and character of bear bile from other animal bile (Ferrandi et al. 2012; Eggert et al. 2014; Zheng et al. *Correspondence: zhaoshujuan@126.com; zhaoshujuan@shutcm.edu.cn 2017). The authentication and standard quality work The SATCM Key Laboratory for New Resources & Quality Evaluation of Chinese Medicine, The MOE Key Laboratory for Standardization from 20 batches of drainage bear bile powder samples in of Chinese Medicines and Shanghai Key Laboratory of Compound our lab revealed that the average content of TUDCA was Chinese Medicines, Institute of Chinese Materia Medica, Shanghai 26.50%, and the ratio of TUDCA to TCDCA (TUDCA/ University of Traditional Chinese Medicine, Shanghai 201203, People’s Republic of China TCDCA) was from 1.00: 1.00 to 1.50: 1.00 (Wang et al. © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Jin et al. Bioresources and Bioprocessing (2022) 9:32 Page 2 of 10 2018), which could be regarded as the characteristics of et al. 2018). Here, taking the advantage of S. cerevisiae bear bile powder. and the bi-directional catalytic properties of HSDH, we Till now, the synthesized TUDCA and UDCA are the created an efficient way to produce the potential substi - only acceptable substitutes for bear bile because it has tute for bear bile powder using chicken bile powder as similar bioactivities as bear bile, including neuroprotec- raw material through the engineered S. cerevisiae. To our tive action, promoting pancreatic survival and function, knowledge, this is the first report of applying engineered and reducing gallstone formation (Lu et al. 2021; Rosa et S. cerevisiae to make products having certain amount al. 2021; Zangerolamo et al. 2021). There are several of TUDCA and TCDCA equivalent to that in bear bile methods to produce UDCA or TUDCA such as chemical powder. synthesis, whole-cell biocatalysts, and the chemo-enzy- matic method (Momose et al. 1997; Liu et al. 2013; Zheng Materials and methods et al. 2015). All these methods required pure and rare Chemicals and reagents compounds as precursors or substrates, which increased Chicken bile powder (containing 64.78 ± 0.30% of the production costs and limited the industrial applica- TCDCA) were kindly provided by Shanghai Kaibao Phar- tion. In addition, bile acids other than TUDCA or UDCA maceutica Co. Ltd., China. Resin D101 was purchased also have biological activities. For example, TCDCA had from Wuhan Weiqiboxin Biotechnology Co. Ltd., China. anti-inflammatory effect, could stimulate intestinal cell β-NAD and NADP Na was purchased from Coolaber proliferation, protect against apoptotic cell death, and Science & Technology (Beijing, China). All the other alleviate pulmonary fibrosis (Toledo et al. 2004; Zhou chemicals in this study were from Sinopharm Chemical et al. 2013; Li et al. 2019). Therefore, it would be more Reagent Co. Ltd., China. feasible to explore alternatives based on both the thera- peutic effects and the specific chemical properties of nat - Gene selection, codon optimization and synthesis ural bear bile powder. Two 7α-HSDH and two 7β-HSDH genes originating Different from bear bile, poultry bile such as chicken from Clostridium sardiniense, E. coli, and Ruminococ- bile is a cheap and easily available resource. It contains cus gnavus (GenBank accession numbers JN191345, high amount of TCDCA but no TUDCA. Chemically, D10497, and KF052988) were selected to construct four TUDCA is the epimer of TCDCA at C-7 hydroxyl. Bac- combinations as our previous work (Shi et al. 2017). terial 7α-hydroxysteroid dehydrogenase (7α-HSDH) and Briefly, the coding region of these genes was codon opti - 7β-hydroxysteroid dehydrogenase (7β-HSDH) have both mized and synthesized by Life Technologies (Shanghai, syn syn oxidative and reductive activities and can interconvert China), named as Ca7α-HSDH , Ec7α-HSDH , Ca7β- + + syn syn TCDCA and TUDCA coupling with NA D or NA DP HSDH , and Rg7β-HSDH (GenBank accession num- as co-factor (Yoshimoto et al. 1991; Ferrandi et al. 2012; bers KY178305-KY178308) referred to as α1, α2, β1, and Lee et al. 2013; Song et al. 2017) (Fig. 1). We had con- β2, respectively. structed an engineered Escherichia coli with 7α-HSDH and 7β-HSDH genes which could convert a certain pro- Microbial strains, plasmids, and expression vector portion of TCDCA to TUDCA using chicken bile pow- construction der as substrates. Two S. cerevisiae strains W303-1a (MATa; ade2-1; ura3- As a safe and health microbial organism with clear 1; his3-11; trp1-1; leu2-3; leu2-112; can1-100) and CEN. genetic background, Saccharomyces cerevisiae has been PK2-1C (MATa; his3D1; leu2-3_112; ura3-52; trp1-289; widely used in food and pharmaceutical industry (Lian MAL2-8c; SUC2) were used as the hosts to expression NH(CH ) SO H NH(CH ) SO H NH(CH ) SO H 2 2 3 2 2 3 2 2 3 O O NAD(P)+ NAD(P)H NAD(P)+ NAD(P)H HO OH HO O HO OH T-7-KLCA TUDCA TCDCA Fig.1 Reaction catalyzed by 7α-HSDH and 7β-HSDH Jin et al. Bioresources and Bioprocessing (2022) 9:32 Page 3 of 10 syn the 7α-HSDH and 7β-HSDH genes. E. coli DH5α was amount of crude enzyme solution and chicken bile used as hosts for middle expression vectors. Yeast shut- powder. For strain screening and biotransformation tle plasmids pRS424 and pRS426 were separately used condition optimization, the reaction was carried out in syn syn to carry 7α-HSDH and 7β-HSDH expression cas- 1 mL reaction mixture (pH 6.5) with 0.10 g/L of NADP syn settes assembled from P , 7α-HSDH and T , or Na , 150 µL of crude enzyme solution, and 4.80 g/L of PGK1 PGI 2 syn P , 7β-HSDH and T , respectively. A total of chicken bile powder at 30 °C for 6 h, refer to our pre- TDH3 PDC1 four yeast expression vectors were generated, namely vious work (Shi et al. 2017; Xu et al. 2019). For prod- pRS424-P -α1-T , pRS424-P -α2-T , pRS425- uct preparation, the reaction mixture (pH 7.0) was PGK1 PGI PGK1 PGI P -β1-T , and pRS425-P -β2-T . One 1 L containing 170 mL of crude enzyme solution and TDH3 PDC1 TDH3 PDC1 syn syn 7α-HSDH combined with one 7β-HSDH expres- 12.00 g/L of chicken bile powder and biotransforma- sion vectors were co-introduced into S. cerevisiae strains tion condition was 25 °C for 5.23 h. After incubation, W303-1a and CEN.PK2-1C, yielding eight engineered the reaction was stopped by keeping in boiling water yeast strains, named W303a-α1β1, W303a-α1β2, W303a- for 5 min, followed by centrifugation. The supernatant α2β1, W303a-α2β2, CEN-α1β1, CEN-α1β2, CEN-α2β1, was either analyzed by HPLC or made to powder prod- and CEN-α2β2. uct. Concentration of TUDCA and the conversion effi - ciency was used to evaluate the production capacity of engineered S. cerevisiae strains. In this study, the con- Cultivation of the engineered S. cerevisiae strains version efficiency was indicated as the TUDCA yield The engineered yeast cells were cultured in flasks con - − − ◦ calculating according to Eq. (1) as followed: taining SD-Trp -Ura liquid media at 30 C, 220 rpm on a horizontal shaker. For engineered strain screening, a Conversion eﬃciency single colony of each strain was inoculated in 25 mL of Total amount of TUDCA in products g liquid media and cultured for 24 h. Then the yeast cell = × 100%. Total amount of TCDCA in substrates g cultures were sub-cultured in 400 mL of liquid media at (1) a proportion of 1:20 and grew for another 24 h. For bio- transformation condition optimization, another scale-up sub-culture process was carried out in 8 L of liquid media using the first sub-cultures as seed cells at the same ratio In vitro biotransformation condition optimization for the third 24 h. The yeast cell cultures were collected Based on the catalytic properties of the original 7α-HSDH to get the cells for crude enzyme preparation. and 7β-HSDH, and our findings on E. coli (Shi et al. 2017; Xu et al. 2019), substrate concentration, pH value, tempera- Crude enzyme preparation ture, and the fermentation time also have influence on the The engineered S. cerevisiae cells were collected by cen - substrate conversion efficiency. Meanwhile, 7α-HSDH and trifugation. The cells were washed with sterile water and 7β-HSDH are NAD(P) -dependent enzymes. The supply of phosphate buffer saline (PBS, 100 mM) twice, and resus - NADP Na in the reaction mixture would also have effect on pended in appropriate volume of PBS. Cells were lysed the enzyme catalytic activity thereby affecting the conver - either by 10 cycles of vortex-ice bath (vortex 30 s, ice bath sion efficiency. Taken all these factors into account, in vitro keeping for 30 s) after adding certain amount glass beads biotransformation conditions were optimized through (diameter: 424–600 μm), or by homogenization under employing single factor exploration and response surface 600–1000 bar. The lysed cell mixture was centrifuged at methodology (RSM) by setting a certain range of each factor. 4 °C, 12,000 rpm for 5–10 min, and the supernatant that The effects of substrate concentration, ranging from was the crude enzyme solution was transferred into new 2.40 g/L to 40.00 g/L, and pH value, covering 6.0 to 7.5, tubes. The crude enzyme powder was obtained by dry - on the conversion were separately investigated. The ing in a freeze dryer. For engineered yeast strain screen- influence of temperature, incubation time, and the con - ing, 10 mL of cell cultures were finally resuspended in centration of NADP N a was evaluated using Behnken 150 μL of PBS. For biotransformation condition optimi- Design (BBD) of RSM. These three independent factors, zation, 8 L of cell cultures were finally resuspended in respectively, designed as factor A, B, C were investigated 200 mL of PBS, and the concentration of total proteins at three different levels. Factor A was divided into 20 °C, was measured with Braford method and calculated as 30 °C, and 40 °C, factor B was divided into 1 h, 5 h, and 1.08 ± 0.02 mg/mL. 9 h, and factor C was divided into 0.05 g/L, 0.10 g/L, and 0.15 g/L with five repetitions at the central point using In vitro biotransformation of TUDCA the ratio of TUDCA/TCDCA as response. The data are The biotransformation of TUDCA was conducted in a analyzed by using Design-Expert.V8.0.6.1 software. For reaction mixture comprising 100 mM of PBS, a certain Jin et al. Bioresources and Bioprocessing (2022) 9:32 Page 4 of 10 RSM experiment, 12.00 g/L of substrates and pH 7.0 was Detector (CAD) was employed using acetonitrile and used according to results of single factor exploration. water (containing 0.3% formic acid and 5 mM ammo- nium acetate) as the mobile phase at a flow rate of Preparation of powder products 0.6 mL/min. HPLC gradient elution program was total Isometric resin D101 was used to purify the conversion 45 min with acetonitrile proportion from 20 to 90%. products. To do that, the supernatant of the reaction mixture was passed through pre-treated isometric resin D101. The impurities on D101 resin were firstly removed Results by deionized water until the eluent was colorless. Then High productive engineered S. cerevisiae strain screening the resin was washed with 95% ethanol to get the eluent To screen a high productive engineered yeast strain, until colorless. The eluent was mixed and concentrated crude enzymes from the eight engineered S. cerevi- through rotary evaporation at 50 °C and then filtered siae strains were incubated with 4.80 g/L of chicken through 0.45 μm organic filters. The filtrate was evapo - bile powder in PBS at 30 °C. After incubation for 6 h, rated by keeping in water bath at 50 °C to form extrac- only two strains, W303a-α1β2 and CEN-α2β2, pro- tum and then dried to constant weight in vacuum drying duced TUDCA (Fig. 2a). The conversion efficiency of oven at 50 °C. After that, the dried solid was taken out the two strains was 51.0% and 56.9% (Fig. 2a) and the and crushed into powder that is the products. ratio of TUDCA to TCDCA was 1.37 and 1.12, respec- tively. Meanwhile, there was a little of tauro-7-ketone Bile acid analysis lithocholic acid (T-7 K-LCA) intermediate built up For conversion efficiency assay, the supernatant was in all those constructs. The conversion efficiency of directly filtered through a 0.22 μm filter and supplied to crude enzymes from these two strains was further the ThermoUltiMate3000 HPLC machine. For prepared analyzed to select a more practicable one by swap- product assay, methanol was added to resolve the dried ping the biotransformation condition to 25 °C for 3 h, bile acids and filtered through a 0.22 μm filter before sup - 6 h and 9 h. As shown in Fig. 2b, the conversion effi - plied to HPLC machine. The concentration of individual ciency of CEN-α2β2 was 50.62 ± 5.45%, 48.44 ± 4.79%, bile acid was determined according to the curve of each and 54.18 ± 3.44% and the ratio of TUDCA to TCDCA authentic standard. was 1.05 ± 0.23, 0.97 ± 0.18, and 1.22 ± 0.17, respec- tively; whereas that of W303a-α1β2 was below 40% and HPLC condition 0.2, respectively. These results indicated that α2β2 has HPLC was performed according to our previous work higher enzymatic catalytic capability than α1β2, possi- (Xu et al. 2019). Briefly, a ThermoUltiMate3000 HPLC ble due to the enzymatic activity of α2 was more flex - machine equipped with Agilent Poroshell 120 EC-C18 ible than that of α1 under the given conditions. It also column (2.7 μm,4.6 mm × 150 mm) and Corona Ultra TUDCA TUDCA T-7-KLCA T-7-KLCA 4 100 TCDCA TCDCA 5 70 Conversion eﬃciency Conversion eﬃciency 3 4 0 0 0 0 3h 6h 9h 3h 6h 9h W303a-α1β2 CEN-α2β2 Fig. 2 Results of high productive engineered Saccharomyces cerevisiae screening. a Results of eight engineered yeast strains and two control yeast strains under 30 °C, pH 6.5 for 6 h. b Results of W303a-α1β2 and CEN-α2β2 under 25 °C, pH 6.5 Bile a cid concentraon ( g/L) Conversion eﬃciency (%) Bile a cid concentraon ( g/L) Conversion eﬃciency (%) Jin et al. Bioresources and Bioprocessing (2022) 9:32 Page 5 of 10 Evaluation of experimental design of Box–Behnken design indicated that incubation for 9 h was better. Therefore, of RSM CEN-α2β2 was selected in next experiments. The results of RSM of 17 runs from Box–Behnken design (BBD) experiments to study the effects of the three inde - Eec ff t of substrate concentration on the biotransformation pendent factors, temperature (factor A), incubation time of TUDCA (factor B), and the concentration of NADP N a (factor C) To screen an optimal substrate concentration, the reac- on the ratio of TUDCA/TCDCA was loaded into Design- tion mixture was incubated at 30 °C, pH 6.5 for 9 h. Expert.V8.0.6.1 software for regression analysis (Table 1). As shown in Fig. 3a, the conversion efficiency ranged The quadratic polynomial regression equation describing from 66.59 ± 0.93%, 60.22 ± 0.18%, 57.31 ± 0.71%, the response value and independent variables is obtained 44.32 ± 1.63%, 27.76 ± 1.64% to 17.01 ± 0.71% and the as below: ratio of TUDCA/TCDCA was 2.31 ± 0.03, 1.63 ± 0.01, 1.39 ± 0.04, 0.81 ± 0.06, 0.39 ± 0.03, and 0.21 ± 0.01, when feeding with 2.40 g/L, 4.80 g/L, 8.00 g/L, 16.00 g/L, 24.00 g/L, and 40.00 g/L of substrate, respectively. This Table 1 Experimental designs and the results of the Box– result suggested that the conversion efficiency decreased Behnken design with the increase of substrate concentration under the No. A-Temperature B-Incubation C-NADP Na TUDCA/TCDCA given conditions, and 8.00–16.00 g/L of chicken bile 2 (°C) time (h) (mg) powder was good for in vitro biotransformation. 1 30.00 9.00 0.15 1.28 Eec ff t of pH value on the biotransformation of TUDCA 2 20.00 1.00 0.10 0.36 The effect of pH value on TUDCA formation was 3 30.00 5.00 0.10 0.94 assessed by setting the incubation at 25 °C or 30 °C for 4 30.00 5.00 0.10 1.09 9 h and feeding 16.00 g/L of chicken bile powder. As 5 30.00 1.00 0.05 0.33 shown in Fig. 3b, with the increase of pH value, the yield 6 30.00 5.00 0.10 0.88 of TUDCA increased at first and then decreased under 7 40.00 1.00 0.10 0.34 both temperatures. When pH value was 7.0, the con- 8 40.00 5.00 0.15 0.72 version efficiency was the highest, 43.05 ± 4.40% and 9 30.00 5.00 0.10 1.02 50.77 ± 7.34%, and the ratio of TUDCA to TCDCA was 10 30.00 1.00 0.15 0.48 0.77 ± 0.14 and 1.08 ± 0.32 at 25 °C and 30 °C, respec- 11 20.00 5.00 0.05 0.74 tively. These results indicated that 30 °C combined with 12 30.00 9.00 0.05 0.86 pH 7.0 was the best condition for the biotransformation, 13 40.00 5.00 0.05 0.35 followed by 30 °C with pH 7.5, and 25 °C with pH 7.0, 14 40.00 9.00 0.10 0.38 when incubation for 9 h. 15 30.00 5.00 0.10 1.12 16 20.00 9.00 0.10 1.22 17 20.00 5.00 0.15 1.14 TUDCA TUDCA T-7-KLCA T-7-KLCA TCDCA TCDCA 12 80 25 80 Conversion eﬃciency Conversion eﬃciency 15 6 40 0 0 0 0 2.4 g/L4.8 g/L8 g/L16 g/L24 g/L40 g/L 25°C 30°C Fig. 3 Results of substrate concentration and pH value on the biotransformation of TUDCA. a Results of substrate concentration when incubation at 30 °CC, pH6.5 for 9 h. b Results of pH value when incubation for 9 h Bile a cid concentraon ( g/L) Conversion eﬃciency (%) Bile a cid concentraon ( g/L) Conversion eﬃciency (%) Jin et al. Bioresources and Bioprocessing (2022) 9:32 Page 6 of 10 that the smaller the p value, the more dominant the cor- TUDCA responding influencing factor. The p value of C is 0.002, TCDCA far less than 0.01, indicating that NADP Na also plays a = +1.01 − 0.21*A + 0.28*B highly important role in the conversion. The order of the + 0.17*C − 0.20*A*B factors influencing the conversion rate was incubation − 7.500E − 003*A*C time > temperature > NADP Na . + 0.068*B*C − 0.22*A*A The significance of the interaction can be reflected by the (2) characteristics of the contour map of the response surface − 0.22*B*B − 0.055*C*C . analysis diagram. When the contour map is oval, the inter- Analysis of variance showed that the F value of the action is significant; when it is round, it is not significant. model was 20.66, p < 0.001, indicating that the model Therefore, the importance of the interaction among AB, reached a very significant level. There was no signifi - AC and BC could be intuitively observed. As indicated in cant difference in the Lack of fit of the model (p > 0.05). Fig. 4, the interaction between temperature and incubation It means that the non-experimental factors had little time is the most obvious, which means that the change of influence on the experimental results. And the experi - temperature significantly affects the incubation time, and mental error was small, which could accurately explain vice versa. the influence of experimental factors on the response According to the results of RSM (Table 3), when three value. There was a big difference between R = 0.9637 factors (temperature, incubation time, and NADP Na con- and R = 0.7270 in the regression model. It showed pre centration) were considered together by setting the pH at that the error range of TUDCA/TCDCA predicted by 7.0 and using 12.00 g/L of substrates, the optimal condition the model was large. Adeq-Precision was 12.821 greater was 25 °C for 5.23 h with 0.08 g/L of NADP Na . The selected than 4, which indicated that the model could be used for conditions were evaluated by performing three repetitions of prediction. The model adjusted R-square is 0.9197, which biotransformation in a 1 mL reaction mixture, and the aver- indicated that the model covers the reason of 91.97% age value of TUDCA/TCDCA was 1.12, which was close to response value change. the predicted value of response surface optimization design. This result confirmed that the optimization model predicted Results of Box–Behnken design for incubation condition the experimental results well. optimization The p values of variables A and B were less than 0.001 Preparation and chemical analysis of products (Table 2), which means that factors A and B were Using the selected optimal condition selected from extremely significant, indicating that both the incubation RSM, 10.99 ± 0.16 g/L of powder product was obtained time and temperature have significant influence on the when incubation of the crude enzymes from CEN-α2β2 biotransformation of TUDCA, according to the principle with 12.00 g/L of chicken bile powder in 1 L of reaction Table 2 Analysis of variance results of regression simulation Source Sum of squares Degree of freedom Mean square F-value p-value Model 1.83 9 0.20 20.66 0.0003 A-Temperature 0.35 1 0.35 35.46 0.0006 B-Incubation time 0.62 1 0.62 63.22 < 0.0001 C-NADP Na 0.22 1 0.22 22.83 0.0020 AB 0.17 1 0.17 17.10 0.0044 AC 2.250E-004 1 2.250E−004 0.023 0.8840 BC 0.018 1 0.018 1.85 0.2156 A 0.20 1 0.20 20.26 0.0028 B 0.20 1 0.20 20.26 0.0028 C 0.013 1 0.013 1.30 0.2925 Residual 0.069 7 9.832E−003 Lack of fit 0.028 3 9.475E−003 0.94 0.5009 Pure error 0.040 4 0.010 Total 1.90 16 Jin et al. Bioresources and Bioprocessing (2022) 9:32 Page 7 of 10 Fig. 4 Results of response surface methodology for TUDCA/TCDCA mixture by addition of 0.08 g/L of NADP N a at 25 °C Discussion for 5.23 h. This powder product contains 36.73 ± 6.68% Previously, we had engineered an E. coli strain with of TUDCA, and 28.22 ± 6.05% (Table 4). The ratio of 7α-HSDH and 7β-HSDH genes that could directional TUDCA to TCDCA was 1.30:1.00. The typical HPLC pro - convert TCDCA to a certain ratio of TUDCA. Our study files of different samples are exhibited in Fig. 5. As shown, using E. coli as host cell demonstrated that all the four the profile of TUDCA and TCDCA in the biotransforma - combinations of 7α-HSDH and 7β-HSDH, α1β1, α1β2, tion products was very close to that in the natural bear α2β1, and α2β2, have the capability to convert TCDCA bile. Yet, the products also contained 2.76 ± 1.24% of to TUDCA, and fermentation condition has important tauro-7-keto lithocholic acid (T-7-KLCA), 3.15 ± 0.36% effect on the conversion efficiency (Shi et al. 2017; Xu of taurocholic acid (TCA), and 6.34 ± 2.18% of taurourso- et al. 2019). cholic acid (TUCA) (Table 4; Fig. 5). Different from E. coli, S. cerevisiae does not produce endotoxin and is regarded as one of the most ideal and Jin et al. Bioresources and Bioprocessing (2022) 9:32 Page 8 of 10 Table 3 Response surface prediction of top ten reaction conditions No. Temperature (°C) Incubation time NADP Na (mg) TUDCA/TCDCA Desirability (h) 1 25.00 5.23 0.08 1.00 0.58 Selected 2 24.92 5.23 0.08 1.00 0.58 3 25.08 5.21 0.08 1.00 0.58 4 24.83 5.22 0.08 1.00 0.58 5 25.18 5.20 0.08 1.00 0.58 6 25.28 5.21 0.08 1.00 0.58 7 25.23 5.25 0.08 1.00 0.58 8 25.20 5.27 0.08 1.00 0.58 9 25.44 5.21 0.08 1.00 0.58 10 25.40 5.25 0.08 1.00 0.58 The top 10 of 32 solutions were listed and the selected one was highlighted in bold Table 4 Bile acids in the products Substrates/products (%) TUDCA TCDCA T-7-KLCA TCA TUCA Chicken bile powder(CBP) 0.00 65.52 0.00 10.21 0.00 Products 36.73 ± 6.68 28.22 ± 6.05 2.76 ± 1.24 3.15 ± 0.36 6.34 ± 2.18 safe microorganisms for the production of food and et al. 2013). Balanced reaction conditions are required medicinal products. Another advantage of this study is to achieve directional biotransformation with a specific the use of crude enzymes and in vitro biotransformation. ratio of TUDCA to TCDCA. Our results suggested that It separates the bioconversion process with the yeast cell substrate concentration, reaction time, temperature, the growth, which facilitates the optimization of the active pH value and the NADP Na concentration have inte- protein expression and transformation processes, sepa- grated impact on the biotransformation. For example, rately, thereby increasing the productivity for industrial when 16.00 g/L of substrates and 0.100 g/L of NADP N a application. Besides, the batch preparation of crude was used, incubation of the reaction mixture at 30 °C enzymes saves the enzyme purification process, so it is for 9 h was good (Fig. 3b). When 12.00 g/L of substrates more environmentally friendly and green. The disadvan - was used, incubation at 25 °C for 5.23 h with 0.08 g/L of tage is that NADP N a co-factor should be supplemented NADP Na led to the best result (Table 3). 2 2 in vitro reaction mixture. It is worth mentioning that there are two types of In this study, CEN-α2β2 is the best combination for 7α-HSDH in nature: one is dependent on NA D , the TUDCA biotransformation under the given conditions other is dependent on NA DP (Huang et al. 2019). (Fig. 2), which indicates that S. cerevisiae CEN.PK2-1C is According to the literatures (Yoshimoto et al. 1991; Fer- better than w303-1a as host to express active 7α-HSDH randi et al. 2012; Lee et al. 2013), 7α-HSDH from E. coli and 7β-HSDH enzymes. Both CEN.PK2 and W303 are (α2) and C. sardiniense (α1), respectively, used NAD commonly used haploid strains for bioengineering with and NADP as co-factor for enzymatic activity assay. We good sporulation efficiency, and CEN.PK2 has a faster had separately supplemented β-NAD and NADP Na growth rate with doubling times of about 80 min for hap- for in vitro biotransformation containing both 7α-HSDH loid strains (Rogowska-Wrzesinska et al. 2001; Bruder and 7β-HSDH enzymes, but only NADP Na worked et al. 2016). This fast growth feature is possibly one of well. So, NADP Na instead of β-NAD was used in the reasons why α2 and β2 expressed in CEN.PK2-1C is this study. It is possible that Ec7α-HSDH could also use more suitable for TUDCA formation.NADP Na as co-factor in vitro. Both 7α-HSDH and 7β-HSDH have oxidative and In addition, we found S. cerevisiae cell does not allow reductive properties, and their oxidation or reduction the main bile acids of chicken bile to pass through its activity is affected by environmental conditions such membrane system freely, although it was reported that as temperature and the pH value of the reaction mix- lithocholic bile acid (LCA) could enter S. cerevisiae ture (Yoshimoto et al. 1991; Ferrandi et al. 2012; Lee and accumulated in mitochondria (Beach et al. 2015). Jin et al. Bioresources and Bioprocessing (2022) 9:32 Page 9 of 10 Fig. 5 Typical HPLC profiles of bile acids in different samples. a Reference standards. b Natural bear bile powder. c Chicken bile powder (CBP). d Biotransformation products from CBP. Compound 1: tauroursocholic acid ( TUCA). Compound 2: tauroursodeoxycholic acid ( TUDCA). Compound 3: tauro-7-keto lithocholic acid ( T-7-KLCA). Compound 4: taurocholic acid ( TCA). Compound 5: taurochenodeoxycholic acid ( TCDCA) Glucanase, NaCl, and Tween 80 had been supplemented may reduce the T-7-KLCA intermediate, as reported into the media to improve the permeability of cell mem- for 7α-HSDH (Huang et al. 2019). brane, but only Tween 80 had a little effect, resulting In conclusion, through optimizing the S. cerevisiae in the production of small amount of TUDCA. This host and gene combinations, and in vitro biotrans- rendered the currently engineered yeast cell unable to formation conditions, we have created a green way directly acts as a whole-cell factory for TUDCA biotrans- to make use of cheap and easily available chicken bile formation. Further modification of yeast cell membrane powder to produce potential substitute resource for system will be a potential strategy if whole-cell factories bear bile powder. In order to obtain artificial bear bile are employed in the future, or using other generally rec- that closely matches the chemical composition of natu- ognized as safe (GRAS) strains such as Corynebacterium ral bear bile, other engineering such as enzyme directed glutamicum (Fang et al. 2014) as host cells, from the per- evolution will be helpful. spective of food and medicinal product safety. Another issue should be mentioned is that T-7-KLCA, Abbreviations TCA, and TUCA were present in the biotransformation 7α-HSDH: 7α-Hydroxysteroid dehydrogenase; 7β-HSDH: 7β-Hydroxysteroid products (Table 4; Fig. 5d). TCA was originated from dehydrogenase; BAs: Bile acids; BBD: Box–Behnken design; CAD: Corona Ultra Detector; GRAS: Generally recognized as safe; LCA: Lithocholic bile acid; PBS: chicken bile (Fig. 5c) and TUCA was the product of Phosphate buffer saline; RSM: Response surface methodology; T-7-KLCA: TCA catalyzed by 7α-HSDH and 7β-HSDH. Removal of Tauro-7-keto lithocholic acid; TCA : Taurocholic acid; TCDCA: Taurochenodeoxy- the OH at C12 of TCA would yield TCDCA, a process cholic acid; TUCA : Tauroursocholic acid; TUDCA: Tauroursodeoxycholic acid. possibly catalyzed by 12α-hydroxysteroid dehydroge- Acknowledgements nase (Tonin and Arends 2018). T-7-KLCA is the inter- We greatly appreciate Professor Zhibi Hu and Mrs Jiyan Zhou, Shanghai mediate in the biosynthesis of TUDCA from TCDCA University of Traditional Chinese Medicine, for their valuable comments and suggestions, and the company of Shanghai Kaibao Pharmaceutica Co. Ltd., (Fig. 1). Directed evolution of 7α-HSDH and 7β-HSDH China, for providing chicken bile and the standard reference of bile acids. to enhance the substrate activity and product tolerance Jin et al. Bioresources and Bioprocessing (2022) 9:32 Page 10 of 10 Authors’ contributions Lian J, Mishra S, Zhao H (2018) Recent advances in metabolic engineering of LJ performed the core experiments and wrote the original manuscript. LY Saccharomyces cerevisiae: new tools and their applications. Metab Eng supervised the chemical analysis, acquisition the funding, and revised the 50:85–108. https:// doi. org/ 10. 1016/j. ymben. 2018. 04. 011 manuscript. SZ conceptualized the project, acquisition the funding, and Liu L, Braun M, Gebhardt G et al (2013) One-step synthesis of 12-ketour- revised the manuscript. ZW conceptualized and supervised the project. All sodeoxycholic acid from dehydrocholic acid using a multienzymatic authors read and approved the final manuscript. system. Appl Microbiol Biotechnol 97:633–639. https:// doi. org/ 10. 1007/ s00253- 012- 4340-5 Funding Lu Q, Jiang Z, Wang Q et al (2021) The effect of tauroursodeoxycholic acid This work was supported by the National Science and Technology (S&T ) Major ( TUDCA) and gut microbiota on murine gallbladder stone formation. Ann Special Projects (No. 2017ZX09309006) and Natural Science Foundation of Hepatol 23:100289. https:// doi. org/ 10. 1016/j. aohep. 2020. 100289 Shanghai (No. 21ZR1463000). Momose T, Tsubaki T, Iida T, Nambara T (1997) An improved synthesis of taurine- and glycine-conjugated bile acids. Lipids 32:775–778. https:// doi. Availability of data and materialsorg/ 10. 1007/ s11745- 997- 0099-8 The data generated and/or analyzed during this study are available from the Rogowska-Wrzesinska A, Larsen PM, Blomberg A et al (2001) Comparison of corresponding author on reasonable request. the proteomes of three yeast wild type strains: CEN.PK2, FY1679 and W303. Comp Funct Genomics 2:207–225. https:// doi. org/ 10. 1002/ cfg. 94 Rosa LRO, Vettorazzi JF, Zangerolamo L et al (2021) TUDCA receptors and their Declarations role on pancreatic beta cells. Prog Biophys Mol Biol. https:// doi. org/ 10. 1016/j. pbiom olbio. 2021. 09. 003 Ethics approval and consent to participate Shi J, Wang J, Yu L et al (2017) Rapidly directional biotransformation of tauro- Not applicable. ursodeoxycholic acid through engineered Escherichia coli. J Ind Microbiol Biotechnol 44:1073–1082. https:// doi. org/ 10. 1007/ s10295- 017- 1935-y Consent for publication Song C, Wang B, Tan J et al (2017) Discovery of tauroursodeoxycholic acid bio- The authors approved the consent for publishing the manuscript. transformation enzymes from the gut microbiome of black bears using metagenomics. Sci Rep 7:1–8. https:// doi. org/ 10. 1038/ srep4 5495 Competing interests Toledo A, Yamaguchi J, Wang JY et al (2004) Taurodeoxycholate stimulates The authors declare that they have no competing interests. intestinal cell proliferation and protects against apoptotic cell death through activation of NF-κB. Dig Dis Sci 49:1664–1671. https:// doi. org/ 10. Received: 27 December 2021 Accepted: 7 March 2022 1023/B: DDAS. 00000 43383. 96077. 99 Tonin F, Arends IWCE (2018) Latest development in the synthesis of ursode- oxycholic acid (UDCA): a critical review. Beilstein J Org Chem 14:470–483. https:// doi. org/ 10. 3762/ bjoc. 14. 33 Wang J, Xiong A-Z, Cheng R-R et al (2018) Systematical analysis of multiple References components in drainage bear bile powder from different sources. China Beach A, Richard VR, Bourque S et al (2015) Lithocholic bile acid accumulated J Chin Mater Med 43:2326–2332. https:// doi. org/ 10. 19540/j. cnki. cjcmm. in yeast mitochondria orchestrates a development of an anti-aging cel- 20180 125. 001 lular pattern by causing age-related changes in cellular proteome. Cell Xu Y, Yang L, Zhao S, Wang Z (2019) Large-scale production of tauroursodeoxy- Cycle 14:1643–1656. https:// doi. org/ 10. 1080/ 15384 101. 2015. 10264 93 cholic acid products through fermentation optimization of engineered Bruder S, Reifenrath M, Thomik T et al (2016) Parallelised online biomass Escherichia coli cell factory. Microb Cell Fact 18:34. https:// doi. org/ 10. monitoring in shake flasks enables efficient strain and carbon source 1186/ s12934- 019- 1076-2 dependent growth characterisation of Saccharomyces cerevisiae. Microb Yang L, Xiong A, He Y et al (2008) Bile acids metabonomic study on the C Cl - Cell Fact 15:1–14. https:// doi. org/ 10. 1186/ s12934- 016- 0526-3 and alpha-naphthylisothiocyanate-induced animal models: quantitative Eggert T, Bakonyi D, Hummel W (2014) Enzymatic routes for the synthesis of analysis of 22 bile acids by ultraperformance liquid chromatography- ursodeoxycholic acid. J Biotechnol 191:11–21. https:// doi. org/ 10. 1016/j. mass spectrometry. Chem Res Toxicol 21:2280–2288. https:// doi. org/ 10. jbiot ec. 2014. 08. 006 1021/ tx800 225q Fang X, Duan RS, Yang HY, Liu JF (2014) Hyaluronic acid production by genetic Yoshimoto T, Higashi H, Kanatani A et al (1991) Cloning and sequencing of the modified GRAS strains. Adv Mater Res 950:13–17. https:// doi. org/ 10. 4028/ 7 alpha-hydroxysteroid dehydrogenase gene from Escherichia coli HB101 www. scien tific. net/ AMR. 950. 13 and characterization of the expressed enzyme. J Bacteriol 173:2173–2179 Feng Y, Siu K, Wang N et al (2009) Bear bile: dilemma of traditional medicinal Zangerolamo L, Vettorazzi JF, Rosa LRO et al (2021) The bile acid TUDCA and use and animal protection. J Ethnobiol Ethnomed 5:2. https:// doi. org/ 10. neurodegenerative disorders: an overview. Life Sci 272:119252. https:// 1186/ 1746- 4269-5-2 doi. org/ 10. 1016/j. lfs. 2021. 119252 Ferrandi EE, Bertolesi GM, Polentini F et al (2012) In search of sustainable Zheng MM, Wang RF, Li CX, Xu JH (2015) Two-step enzymatic synthesis of chemical processes: cloning, recombinant expression, and functional ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from characterization of the 7α- and 7β-hydroxysteroid dehydrogenases from Ruminococcus torques. Process Biochem 50:598–604. https:// doi. org/ 10. Clostridium absonum. Appl Microbiol Biotechnol 95:1221–1233. https:// 1016/j. procb io. 2014. 12. 026 doi. org/ 10. 1007/ s00253- 011- 3798-x Zheng MM, Chen KC, Wang RF et al (2017) Engineering 7β-Hydroxysteroid Huang B, Zhao Q, Zhou JH, Xu G (2019) Enhanced activity and substrate dehydrogenase for enhanced ursodeoxycholic acid production by mul- tolerance of 7α-hydroxysteroid dehydrogenase by directed evolution for tiobjective directed evolution. J Agric Food Chem 65:1178–1185. https:// 7-ketolithocholic acid production. Appl Microbiol Biotechnol. https:// doi. doi. org/ 10. 1021/ acs. jafc. 6b054 28 org/ 10. 1007/ s00253- 019- 09668-4 Zhou C, Shi Y, Li J et al (2013) The effects of taurochenodeoxycholic acid in Lee J-Y, Arai H, Nakamura Y et al (2013) Contribution of the 7β-hydroxysteroid preventing pulmonary fibrosis in mice. Pak J Pharm Sci 26:761–765 dehydrogenase from Ruminococcus gnavus N53 to ursodeoxycholic acid formation in the human colon. J Lipid Res 54:3062–3069. https:// doi. org/ Publisher’s Note 10. 1194/ jlr. M0398 34 Springer Nature remains neutral with regard to jurisdictional claims in pub- Li L, Liu C, Mao W et al (2019) Taurochenodeoxycholic acid inhibited AP-1 lished maps and institutional affiliations. activation via stimulating glucocorticoid receptor. Molecules 24:1–10. https:// doi. org/ 10. 3390/ molec ules2 42445 13 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioresources and Bioprocessing Springer Journals http://www.deepdyve.com/lp/springer-journals/a-green-strategy-to-produce-potential-substitute-resource-for-bear-HExy8X5wz0

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Publisher: Springer Journals
Copyright: Copyright © The Author(s) 2022
eISSN: 2197-4365
DOI: 10.1186/s40643-022-00517-3
Publisher site: See Article on Publisher Site

Abstract

Background: Bear bile powder is a precious natural material characterized by high content of tauroursodeoxycholic acid ( TUDCA) at a ratio of 1.00–1.50 to taurochenodeoxycholic acid ( TCDCA). Results: In this study, we use the crude enzymes from engineered Saccharomyces cerevisiae to directionally convert TCDCA from chicken bile powder to TUDCA at the committed ratio in vitro. This S. cerevisiae strain was modified with heterologous 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenase (7β-HSDH) genes. S. cerevisiae host and HSDH gene combinatorial optimization and response surface methodology was applied to get the best engineered strain and the optimal biotransformation condition, respectively, under which 10.99 ± 0.16 g/L of powder products containing 36.73 ± 6.68% of TUDCA and 28.22 ± 6.05% of TCDCA were obtained using 12.00 g/L of chicken bile powder as substrate. Conclusion: This study provides a healthy and environmentally friendly way to produce potential alternative resource for bear bile powder from cheap and readily available chicken bile powder, and also gives a reference for the green manufacturing of other rare and endangered animal-derived valuable resource. Keywords: Tauroursodeoxycholic acid, Hydroxysteroid dehydrogenase, Biotransformation, Saccharomyces cerevisiae Introduction by implanting a duct or making an artificial fistula in the Bear bile powder is a kind of precious material that has liver of the bears (Feng et al. 2009). These methods will been used as medicine and healthcare supplementary seriously endanger the health of bears, thus exploring thousands of years ago in Asia area (Yang et al. 2008; substitute resources is of great significance to meet the Feng et al. 2009). It has multiple pharmacological activi- demands of bear bile powder as medicinal and healthcare ties and can be used to treat gallstones, cholecystitis, fatty material. liver and other hepatobiliary diseases (Feng et al. 2009). Bile acids (BAs), a group of steroids with C-17 side At present, bear bile powder is mainly from the drainage chains, are the principal bioactive ingredients of bear bile bear bile extracted from the farmed living bears including powder, among which ursodesoxycholic acid (UDCA) Selenarctos thibetanus (Asiatic black bear) or Ursus arc- or its physiologically active form tauroursodeoxycholic tos (brown bear) with “Free-dripping Fistula Technique” acid (TUDCA) takes a high proportion and is considered as distinct and character of bear bile from other animal bile (Ferrandi et al. 2012; Eggert et al. 2014; Zheng et al. *Correspondence: zhaoshujuan@126.com; zhaoshujuan@shutcm.edu.cn 2017). The authentication and standard quality work The SATCM Key Laboratory for New Resources & Quality Evaluation of Chinese Medicine, The MOE Key Laboratory for Standardization from 20 batches of drainage bear bile powder samples in of Chinese Medicines and Shanghai Key Laboratory of Compound our lab revealed that the average content of TUDCA was Chinese Medicines, Institute of Chinese Materia Medica, Shanghai 26.50%, and the ratio of TUDCA to TCDCA (TUDCA/ University of Traditional Chinese Medicine, Shanghai 201203, People’s Republic of China TCDCA) was from 1.00: 1.00 to 1.50: 1.00 (Wang et al. © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Jin et al. Bioresources and Bioprocessing (2022) 9:32 Page 2 of 10 2018), which could be regarded as the characteristics of et al. 2018). Here, taking the advantage of S. cerevisiae bear bile powder. and the bi-directional catalytic properties of HSDH, we Till now, the synthesized TUDCA and UDCA are the created an efficient way to produce the potential substi - only acceptable substitutes for bear bile because it has tute for bear bile powder using chicken bile powder as similar bioactivities as bear bile, including neuroprotec- raw material through the engineered S. cerevisiae. To our tive action, promoting pancreatic survival and function, knowledge, this is the first report of applying engineered and reducing gallstone formation (Lu et al. 2021; Rosa et S. cerevisiae to make products having certain amount al. 2021; Zangerolamo et al. 2021). There are several of TUDCA and TCDCA equivalent to that in bear bile methods to produce UDCA or TUDCA such as chemical powder. synthesis, whole-cell biocatalysts, and the chemo-enzy- matic method (Momose et al. 1997; Liu et al. 2013; Zheng Materials and methods et al. 2015). All these methods required pure and rare Chemicals and reagents compounds as precursors or substrates, which increased Chicken bile powder (containing 64.78 ± 0.30% of the production costs and limited the industrial applica- TCDCA) were kindly provided by Shanghai Kaibao Phar- tion. In addition, bile acids other than TUDCA or UDCA maceutica Co. Ltd., China. Resin D101 was purchased also have biological activities. For example, TCDCA had from Wuhan Weiqiboxin Biotechnology Co. Ltd., China. anti-inflammatory effect, could stimulate intestinal cell β-NAD and NADP Na was purchased from Coolaber proliferation, protect against apoptotic cell death, and Science & Technology (Beijing, China). All the other alleviate pulmonary fibrosis (Toledo et al. 2004; Zhou chemicals in this study were from Sinopharm Chemical et al. 2013; Li et al. 2019). Therefore, it would be more Reagent Co. Ltd., China. feasible to explore alternatives based on both the thera- peutic effects and the specific chemical properties of nat - Gene selection, codon optimization and synthesis ural bear bile powder. Two 7α-HSDH and two 7β-HSDH genes originating Different from bear bile, poultry bile such as chicken from Clostridium sardiniense, E. coli, and Ruminococ- bile is a cheap and easily available resource. It contains cus gnavus (GenBank accession numbers JN191345, high amount of TCDCA but no TUDCA. Chemically, D10497, and KF052988) were selected to construct four TUDCA is the epimer of TCDCA at C-7 hydroxyl. Bac- combinations as our previous work (Shi et al. 2017). terial 7α-hydroxysteroid dehydrogenase (7α-HSDH) and Briefly, the coding region of these genes was codon opti - 7β-hydroxysteroid dehydrogenase (7β-HSDH) have both mized and synthesized by Life Technologies (Shanghai, syn syn oxidative and reductive activities and can interconvert China), named as Ca7α-HSDH , Ec7α-HSDH , Ca7β- + + syn syn TCDCA and TUDCA coupling with NA D or NA DP HSDH , and Rg7β-HSDH (GenBank accession num- as co-factor (Yoshimoto et al. 1991; Ferrandi et al. 2012; bers KY178305-KY178308) referred to as α1, α2, β1, and Lee et al. 2013; Song et al. 2017) (Fig. 1). We had con- β2, respectively. structed an engineered Escherichia coli with 7α-HSDH and 7β-HSDH genes which could convert a certain pro- Microbial strains, plasmids, and expression vector portion of TCDCA to TUDCA using chicken bile pow- construction der as substrates. Two S. cerevisiae strains W303-1a (MATa; ade2-1; ura3- As a safe and health microbial organism with clear 1; his3-11; trp1-1; leu2-3; leu2-112; can1-100) and CEN. genetic background, Saccharomyces cerevisiae has been PK2-1C (MATa; his3D1; leu2-3_112; ura3-52; trp1-289; widely used in food and pharmaceutical industry (Lian MAL2-8c; SUC2) were used as the hosts to expression NH(CH ) SO H NH(CH ) SO H NH(CH ) SO H 2 2 3 2 2 3 2 2 3 O O NAD(P)+ NAD(P)H NAD(P)+ NAD(P)H HO OH HO O HO OH T-7-KLCA TUDCA TCDCA Fig.1 Reaction catalyzed by 7α-HSDH and 7β-HSDH Jin et al. Bioresources and Bioprocessing (2022) 9:32 Page 3 of 10 syn the 7α-HSDH and 7β-HSDH genes. E. coli DH5α was amount of crude enzyme solution and chicken bile used as hosts for middle expression vectors. Yeast shut- powder. For strain screening and biotransformation tle plasmids pRS424 and pRS426 were separately used condition optimization, the reaction was carried out in syn syn to carry 7α-HSDH and 7β-HSDH expression cas- 1 mL reaction mixture (pH 6.5) with 0.10 g/L of NADP syn settes assembled from P , 7α-HSDH and T , or Na , 150 µL of crude enzyme solution, and 4.80 g/L of PGK1 PGI 2 syn P , 7β-HSDH and T , respectively. A total of chicken bile powder at 30 °C for 6 h, refer to our pre- TDH3 PDC1 four yeast expression vectors were generated, namely vious work (Shi et al. 2017; Xu et al. 2019). For prod- pRS424-P -α1-T , pRS424-P -α2-T , pRS425- uct preparation, the reaction mixture (pH 7.0) was PGK1 PGI PGK1 PGI P -β1-T , and pRS425-P -β2-T . One 1 L containing 170 mL of crude enzyme solution and TDH3 PDC1 TDH3 PDC1 syn syn 7α-HSDH combined with one 7β-HSDH expres- 12.00 g/L of chicken bile powder and biotransforma- sion vectors were co-introduced into S. cerevisiae strains tion condition was 25 °C for 5.23 h. After incubation, W303-1a and CEN.PK2-1C, yielding eight engineered the reaction was stopped by keeping in boiling water yeast strains, named W303a-α1β1, W303a-α1β2, W303a- for 5 min, followed by centrifugation. The supernatant α2β1, W303a-α2β2, CEN-α1β1, CEN-α1β2, CEN-α2β1, was either analyzed by HPLC or made to powder prod- and CEN-α2β2. uct. Concentration of TUDCA and the conversion effi - ciency was used to evaluate the production capacity of engineered S. cerevisiae strains. In this study, the con- Cultivation of the engineered S. cerevisiae strains version efficiency was indicated as the TUDCA yield The engineered yeast cells were cultured in flasks con - − − ◦ calculating according to Eq. (1) as followed: taining SD-Trp -Ura liquid media at 30 C, 220 rpm on a horizontal shaker. For engineered strain screening, a Conversion eﬃciency single colony of each strain was inoculated in 25 mL of Total amount of TUDCA in products g liquid media and cultured for 24 h. Then the yeast cell = × 100%. Total amount of TCDCA in substrates g cultures were sub-cultured in 400 mL of liquid media at (1) a proportion of 1:20 and grew for another 24 h. For bio- transformation condition optimization, another scale-up sub-culture process was carried out in 8 L of liquid media using the first sub-cultures as seed cells at the same ratio In vitro biotransformation condition optimization for the third 24 h. The yeast cell cultures were collected Based on the catalytic properties of the original 7α-HSDH to get the cells for crude enzyme preparation. and 7β-HSDH, and our findings on E. coli (Shi et al. 2017; Xu et al. 2019), substrate concentration, pH value, tempera- Crude enzyme preparation ture, and the fermentation time also have influence on the The engineered S. cerevisiae cells were collected by cen - substrate conversion efficiency. Meanwhile, 7α-HSDH and trifugation. The cells were washed with sterile water and 7β-HSDH are NAD(P) -dependent enzymes. The supply of phosphate buffer saline (PBS, 100 mM) twice, and resus - NADP Na in the reaction mixture would also have effect on pended in appropriate volume of PBS. Cells were lysed the enzyme catalytic activity thereby affecting the conver - either by 10 cycles of vortex-ice bath (vortex 30 s, ice bath sion efficiency. Taken all these factors into account, in vitro keeping for 30 s) after adding certain amount glass beads biotransformation conditions were optimized through (diameter: 424–600 μm), or by homogenization under employing single factor exploration and response surface 600–1000 bar. The lysed cell mixture was centrifuged at methodology (RSM) by setting a certain range of each factor. 4 °C, 12,000 rpm for 5–10 min, and the supernatant that The effects of substrate concentration, ranging from was the crude enzyme solution was transferred into new 2.40 g/L to 40.00 g/L, and pH value, covering 6.0 to 7.5, tubes. The crude enzyme powder was obtained by dry - on the conversion were separately investigated. The ing in a freeze dryer. For engineered yeast strain screen- influence of temperature, incubation time, and the con - ing, 10 mL of cell cultures were finally resuspended in centration of NADP N a was evaluated using Behnken 150 μL of PBS. For biotransformation condition optimi- Design (BBD) of RSM. These three independent factors, zation, 8 L of cell cultures were finally resuspended in respectively, designed as factor A, B, C were investigated 200 mL of PBS, and the concentration of total proteins at three different levels. Factor A was divided into 20 °C, was measured with Braford method and calculated as 30 °C, and 40 °C, factor B was divided into 1 h, 5 h, and 1.08 ± 0.02 mg/mL. 9 h, and factor C was divided into 0.05 g/L, 0.10 g/L, and 0.15 g/L with five repetitions at the central point using In vitro biotransformation of TUDCA the ratio of TUDCA/TCDCA as response. The data are The biotransformation of TUDCA was conducted in a analyzed by using Design-Expert.V8.0.6.1 software. For reaction mixture comprising 100 mM of PBS, a certain Jin et al. Bioresources and Bioprocessing (2022) 9:32 Page 4 of 10 RSM experiment, 12.00 g/L of substrates and pH 7.0 was Detector (CAD) was employed using acetonitrile and used according to results of single factor exploration. water (containing 0.3% formic acid and 5 mM ammo- nium acetate) as the mobile phase at a flow rate of Preparation of powder products 0.6 mL/min. HPLC gradient elution program was total Isometric resin D101 was used to purify the conversion 45 min with acetonitrile proportion from 20 to 90%. products. To do that, the supernatant of the reaction mixture was passed through pre-treated isometric resin D101. The impurities on D101 resin were firstly removed Results by deionized water until the eluent was colorless. Then High productive engineered S. cerevisiae strain screening the resin was washed with 95% ethanol to get the eluent To screen a high productive engineered yeast strain, until colorless. The eluent was mixed and concentrated crude enzymes from the eight engineered S. cerevi- through rotary evaporation at 50 °C and then filtered siae strains were incubated with 4.80 g/L of chicken through 0.45 μm organic filters. The filtrate was evapo - bile powder in PBS at 30 °C. After incubation for 6 h, rated by keeping in water bath at 50 °C to form extrac- only two strains, W303a-α1β2 and CEN-α2β2, pro- tum and then dried to constant weight in vacuum drying duced TUDCA (Fig. 2a). The conversion efficiency of oven at 50 °C. After that, the dried solid was taken out the two strains was 51.0% and 56.9% (Fig. 2a) and the and crushed into powder that is the products. ratio of TUDCA to TCDCA was 1.37 and 1.12, respec- tively. Meanwhile, there was a little of tauro-7-ketone Bile acid analysis lithocholic acid (T-7 K-LCA) intermediate built up For conversion efficiency assay, the supernatant was in all those constructs. The conversion efficiency of directly filtered through a 0.22 μm filter and supplied to crude enzymes from these two strains was further the ThermoUltiMate3000 HPLC machine. For prepared analyzed to select a more practicable one by swap- product assay, methanol was added to resolve the dried ping the biotransformation condition to 25 °C for 3 h, bile acids and filtered through a 0.22 μm filter before sup - 6 h and 9 h. As shown in Fig. 2b, the conversion effi - plied to HPLC machine. The concentration of individual ciency of CEN-α2β2 was 50.62 ± 5.45%, 48.44 ± 4.79%, bile acid was determined according to the curve of each and 54.18 ± 3.44% and the ratio of TUDCA to TCDCA authentic standard. was 1.05 ± 0.23, 0.97 ± 0.18, and 1.22 ± 0.17, respec- tively; whereas that of W303a-α1β2 was below 40% and HPLC condition 0.2, respectively. These results indicated that α2β2 has HPLC was performed according to our previous work higher enzymatic catalytic capability than α1β2, possi- (Xu et al. 2019). Briefly, a ThermoUltiMate3000 HPLC ble due to the enzymatic activity of α2 was more flex - machine equipped with Agilent Poroshell 120 EC-C18 ible than that of α1 under the given conditions. It also column (2.7 μm,4.6 mm × 150 mm) and Corona Ultra TUDCA TUDCA T-7-KLCA T-7-KLCA 4 100 TCDCA TCDCA 5 70 Conversion eﬃciency Conversion eﬃciency 3 4 0 0 0 0 3h 6h 9h 3h 6h 9h W303a-α1β2 CEN-α2β2 Fig. 2 Results of high productive engineered Saccharomyces cerevisiae screening. a Results of eight engineered yeast strains and two control yeast strains under 30 °C, pH 6.5 for 6 h. b Results of W303a-α1β2 and CEN-α2β2 under 25 °C, pH 6.5 Bile a cid concentraon ( g/L) Conversion eﬃciency (%) Bile a cid concentraon ( g/L) Conversion eﬃciency (%) Jin et al. Bioresources and Bioprocessing (2022) 9:32 Page 5 of 10 Evaluation of experimental design of Box–Behnken design indicated that incubation for 9 h was better. Therefore, of RSM CEN-α2β2 was selected in next experiments. The results of RSM of 17 runs from Box–Behnken design (BBD) experiments to study the effects of the three inde - Eec ff t of substrate concentration on the biotransformation pendent factors, temperature (factor A), incubation time of TUDCA (factor B), and the concentration of NADP N a (factor C) To screen an optimal substrate concentration, the reac- on the ratio of TUDCA/TCDCA was loaded into Design- tion mixture was incubated at 30 °C, pH 6.5 for 9 h. Expert.V8.0.6.1 software for regression analysis (Table 1). As shown in Fig. 3a, the conversion efficiency ranged The quadratic polynomial regression equation describing from 66.59 ± 0.93%, 60.22 ± 0.18%, 57.31 ± 0.71%, the response value and independent variables is obtained 44.32 ± 1.63%, 27.76 ± 1.64% to 17.01 ± 0.71% and the as below: ratio of TUDCA/TCDCA was 2.31 ± 0.03, 1.63 ± 0.01, 1.39 ± 0.04, 0.81 ± 0.06, 0.39 ± 0.03, and 0.21 ± 0.01, when feeding with 2.40 g/L, 4.80 g/L, 8.00 g/L, 16.00 g/L, 24.00 g/L, and 40.00 g/L of substrate, respectively. This Table 1 Experimental designs and the results of the Box– result suggested that the conversion efficiency decreased Behnken design with the increase of substrate concentration under the No. A-Temperature B-Incubation C-NADP Na TUDCA/TCDCA given conditions, and 8.00–16.00 g/L of chicken bile 2 (°C) time (h) (mg) powder was good for in vitro biotransformation. 1 30.00 9.00 0.15 1.28 Eec ff t of pH value on the biotransformation of TUDCA 2 20.00 1.00 0.10 0.36 The effect of pH value on TUDCA formation was 3 30.00 5.00 0.10 0.94 assessed by setting the incubation at 25 °C or 30 °C for 4 30.00 5.00 0.10 1.09 9 h and feeding 16.00 g/L of chicken bile powder. As 5 30.00 1.00 0.05 0.33 shown in Fig. 3b, with the increase of pH value, the yield 6 30.00 5.00 0.10 0.88 of TUDCA increased at first and then decreased under 7 40.00 1.00 0.10 0.34 both temperatures. When pH value was 7.0, the con- 8 40.00 5.00 0.15 0.72 version efficiency was the highest, 43.05 ± 4.40% and 9 30.00 5.00 0.10 1.02 50.77 ± 7.34%, and the ratio of TUDCA to TCDCA was 10 30.00 1.00 0.15 0.48 0.77 ± 0.14 and 1.08 ± 0.32 at 25 °C and 30 °C, respec- 11 20.00 5.00 0.05 0.74 tively. These results indicated that 30 °C combined with 12 30.00 9.00 0.05 0.86 pH 7.0 was the best condition for the biotransformation, 13 40.00 5.00 0.05 0.35 followed by 30 °C with pH 7.5, and 25 °C with pH 7.0, 14 40.00 9.00 0.10 0.38 when incubation for 9 h. 15 30.00 5.00 0.10 1.12 16 20.00 9.00 0.10 1.22 17 20.00 5.00 0.15 1.14 TUDCA TUDCA T-7-KLCA T-7-KLCA TCDCA TCDCA 12 80 25 80 Conversion eﬃciency Conversion eﬃciency 15 6 40 0 0 0 0 2.4 g/L4.8 g/L8 g/L16 g/L24 g/L40 g/L 25°C 30°C Fig. 3 Results of substrate concentration and pH value on the biotransformation of TUDCA. a Results of substrate concentration when incubation at 30 °CC, pH6.5 for 9 h. b Results of pH value when incubation for 9 h Bile a cid concentraon ( g/L) Conversion eﬃciency (%) Bile a cid concentraon ( g/L) Conversion eﬃciency (%) Jin et al. Bioresources and Bioprocessing (2022) 9:32 Page 6 of 10 that the smaller the p value, the more dominant the cor- TUDCA responding influencing factor. The p value of C is 0.002, TCDCA far less than 0.01, indicating that NADP Na also plays a = +1.01 − 0.21*A + 0.28*B highly important role in the conversion. The order of the + 0.17*C − 0.20*A*B factors influencing the conversion rate was incubation − 7.500E − 003*A*C time > temperature > NADP Na . + 0.068*B*C − 0.22*A*A The significance of the interaction can be reflected by the (2) characteristics of the contour map of the response surface − 0.22*B*B − 0.055*C*C . analysis diagram. When the contour map is oval, the inter- Analysis of variance showed that the F value of the action is significant; when it is round, it is not significant. model was 20.66, p < 0.001, indicating that the model Therefore, the importance of the interaction among AB, reached a very significant level. There was no signifi - AC and BC could be intuitively observed. As indicated in cant difference in the Lack of fit of the model (p > 0.05). Fig. 4, the interaction between temperature and incubation It means that the non-experimental factors had little time is the most obvious, which means that the change of influence on the experimental results. And the experi - temperature significantly affects the incubation time, and mental error was small, which could accurately explain vice versa. the influence of experimental factors on the response According to the results of RSM (Table 3), when three value. There was a big difference between R = 0.9637 factors (temperature, incubation time, and NADP Na con- and R = 0.7270 in the regression model. It showed pre centration) were considered together by setting the pH at that the error range of TUDCA/TCDCA predicted by 7.0 and using 12.00 g/L of substrates, the optimal condition the model was large. Adeq-Precision was 12.821 greater was 25 °C for 5.23 h with 0.08 g/L of NADP Na . The selected than 4, which indicated that the model could be used for conditions were evaluated by performing three repetitions of prediction. The model adjusted R-square is 0.9197, which biotransformation in a 1 mL reaction mixture, and the aver- indicated that the model covers the reason of 91.97% age value of TUDCA/TCDCA was 1.12, which was close to response value change. the predicted value of response surface optimization design. This result confirmed that the optimization model predicted Results of Box–Behnken design for incubation condition the experimental results well. optimization The p values of variables A and B were less than 0.001 Preparation and chemical analysis of products (Table 2), which means that factors A and B were Using the selected optimal condition selected from extremely significant, indicating that both the incubation RSM, 10.99 ± 0.16 g/L of powder product was obtained time and temperature have significant influence on the when incubation of the crude enzymes from CEN-α2β2 biotransformation of TUDCA, according to the principle with 12.00 g/L of chicken bile powder in 1 L of reaction Table 2 Analysis of variance results of regression simulation Source Sum of squares Degree of freedom Mean square F-value p-value Model 1.83 9 0.20 20.66 0.0003 A-Temperature 0.35 1 0.35 35.46 0.0006 B-Incubation time 0.62 1 0.62 63.22 < 0.0001 C-NADP Na 0.22 1 0.22 22.83 0.0020 AB 0.17 1 0.17 17.10 0.0044 AC 2.250E-004 1 2.250E−004 0.023 0.8840 BC 0.018 1 0.018 1.85 0.2156 A 0.20 1 0.20 20.26 0.0028 B 0.20 1 0.20 20.26 0.0028 C 0.013 1 0.013 1.30 0.2925 Residual 0.069 7 9.832E−003 Lack of fit 0.028 3 9.475E−003 0.94 0.5009 Pure error 0.040 4 0.010 Total 1.90 16 Jin et al. Bioresources and Bioprocessing (2022) 9:32 Page 7 of 10 Fig. 4 Results of response surface methodology for TUDCA/TCDCA mixture by addition of 0.08 g/L of NADP N a at 25 °C Discussion for 5.23 h. This powder product contains 36.73 ± 6.68% Previously, we had engineered an E. coli strain with of TUDCA, and 28.22 ± 6.05% (Table 4). The ratio of 7α-HSDH and 7β-HSDH genes that could directional TUDCA to TCDCA was 1.30:1.00. The typical HPLC pro - convert TCDCA to a certain ratio of TUDCA. Our study files of different samples are exhibited in Fig. 5. As shown, using E. coli as host cell demonstrated that all the four the profile of TUDCA and TCDCA in the biotransforma - combinations of 7α-HSDH and 7β-HSDH, α1β1, α1β2, tion products was very close to that in the natural bear α2β1, and α2β2, have the capability to convert TCDCA bile. Yet, the products also contained 2.76 ± 1.24% of to TUDCA, and fermentation condition has important tauro-7-keto lithocholic acid (T-7-KLCA), 3.15 ± 0.36% effect on the conversion efficiency (Shi et al. 2017; Xu of taurocholic acid (TCA), and 6.34 ± 2.18% of taurourso- et al. 2019). cholic acid (TUCA) (Table 4; Fig. 5). Different from E. coli, S. cerevisiae does not produce endotoxin and is regarded as one of the most ideal and Jin et al. Bioresources and Bioprocessing (2022) 9:32 Page 8 of 10 Table 3 Response surface prediction of top ten reaction conditions No. Temperature (°C) Incubation time NADP Na (mg) TUDCA/TCDCA Desirability (h) 1 25.00 5.23 0.08 1.00 0.58 Selected 2 24.92 5.23 0.08 1.00 0.58 3 25.08 5.21 0.08 1.00 0.58 4 24.83 5.22 0.08 1.00 0.58 5 25.18 5.20 0.08 1.00 0.58 6 25.28 5.21 0.08 1.00 0.58 7 25.23 5.25 0.08 1.00 0.58 8 25.20 5.27 0.08 1.00 0.58 9 25.44 5.21 0.08 1.00 0.58 10 25.40 5.25 0.08 1.00 0.58 The top 10 of 32 solutions were listed and the selected one was highlighted in bold Table 4 Bile acids in the products Substrates/products (%) TUDCA TCDCA T-7-KLCA TCA TUCA Chicken bile powder(CBP) 0.00 65.52 0.00 10.21 0.00 Products 36.73 ± 6.68 28.22 ± 6.05 2.76 ± 1.24 3.15 ± 0.36 6.34 ± 2.18 safe microorganisms for the production of food and et al. 2013). Balanced reaction conditions are required medicinal products. Another advantage of this study is to achieve directional biotransformation with a specific the use of crude enzymes and in vitro biotransformation. ratio of TUDCA to TCDCA. Our results suggested that It separates the bioconversion process with the yeast cell substrate concentration, reaction time, temperature, the growth, which facilitates the optimization of the active pH value and the NADP Na concentration have inte- protein expression and transformation processes, sepa- grated impact on the biotransformation. For example, rately, thereby increasing the productivity for industrial when 16.00 g/L of substrates and 0.100 g/L of NADP N a application. Besides, the batch preparation of crude was used, incubation of the reaction mixture at 30 °C enzymes saves the enzyme purification process, so it is for 9 h was good (Fig. 3b). When 12.00 g/L of substrates more environmentally friendly and green. The disadvan - was used, incubation at 25 °C for 5.23 h with 0.08 g/L of tage is that NADP N a co-factor should be supplemented NADP Na led to the best result (Table 3). 2 2 in vitro reaction mixture. It is worth mentioning that there are two types of In this study, CEN-α2β2 is the best combination for 7α-HSDH in nature: one is dependent on NA D , the TUDCA biotransformation under the given conditions other is dependent on NA DP (Huang et al. 2019). (Fig. 2), which indicates that S. cerevisiae CEN.PK2-1C is According to the literatures (Yoshimoto et al. 1991; Fer- better than w303-1a as host to express active 7α-HSDH randi et al. 2012; Lee et al. 2013), 7α-HSDH from E. coli and 7β-HSDH enzymes. Both CEN.PK2 and W303 are (α2) and C. sardiniense (α1), respectively, used NAD commonly used haploid strains for bioengineering with and NADP as co-factor for enzymatic activity assay. We good sporulation efficiency, and CEN.PK2 has a faster had separately supplemented β-NAD and NADP Na growth rate with doubling times of about 80 min for hap- for in vitro biotransformation containing both 7α-HSDH loid strains (Rogowska-Wrzesinska et al. 2001; Bruder and 7β-HSDH enzymes, but only NADP Na worked et al. 2016). This fast growth feature is possibly one of well. So, NADP Na instead of β-NAD was used in the reasons why α2 and β2 expressed in CEN.PK2-1C is this study. It is possible that Ec7α-HSDH could also use more suitable for TUDCA formation.NADP Na as co-factor in vitro. Both 7α-HSDH and 7β-HSDH have oxidative and In addition, we found S. cerevisiae cell does not allow reductive properties, and their oxidation or reduction the main bile acids of chicken bile to pass through its activity is affected by environmental conditions such membrane system freely, although it was reported that as temperature and the pH value of the reaction mix- lithocholic bile acid (LCA) could enter S. cerevisiae ture (Yoshimoto et al. 1991; Ferrandi et al. 2012; Lee and accumulated in mitochondria (Beach et al. 2015). Jin et al. Bioresources and Bioprocessing (2022) 9:32 Page 9 of 10 Fig. 5 Typical HPLC profiles of bile acids in different samples. a Reference standards. b Natural bear bile powder. c Chicken bile powder (CBP). d Biotransformation products from CBP. Compound 1: tauroursocholic acid ( TUCA). Compound 2: tauroursodeoxycholic acid ( TUDCA). Compound 3: tauro-7-keto lithocholic acid ( T-7-KLCA). Compound 4: taurocholic acid ( TCA). Compound 5: taurochenodeoxycholic acid ( TCDCA) Glucanase, NaCl, and Tween 80 had been supplemented may reduce the T-7-KLCA intermediate, as reported into the media to improve the permeability of cell mem- for 7α-HSDH (Huang et al. 2019). brane, but only Tween 80 had a little effect, resulting In conclusion, through optimizing the S. cerevisiae in the production of small amount of TUDCA. This host and gene combinations, and in vitro biotrans- rendered the currently engineered yeast cell unable to formation conditions, we have created a green way directly acts as a whole-cell factory for TUDCA biotrans- to make use of cheap and easily available chicken bile formation. Further modification of yeast cell membrane powder to produce potential substitute resource for system will be a potential strategy if whole-cell factories bear bile powder. In order to obtain artificial bear bile are employed in the future, or using other generally rec- that closely matches the chemical composition of natu- ognized as safe (GRAS) strains such as Corynebacterium ral bear bile, other engineering such as enzyme directed glutamicum (Fang et al. 2014) as host cells, from the per- evolution will be helpful. spective of food and medicinal product safety. Another issue should be mentioned is that T-7-KLCA, Abbreviations TCA, and TUCA were present in the biotransformation 7α-HSDH: 7α-Hydroxysteroid dehydrogenase; 7β-HSDH: 7β-Hydroxysteroid products (Table 4; Fig. 5d). TCA was originated from dehydrogenase; BAs: Bile acids; BBD: Box–Behnken design; CAD: Corona Ultra Detector; GRAS: Generally recognized as safe; LCA: Lithocholic bile acid; PBS: chicken bile (Fig. 5c) and TUCA was the product of Phosphate buffer saline; RSM: Response surface methodology; T-7-KLCA: TCA catalyzed by 7α-HSDH and 7β-HSDH. Removal of Tauro-7-keto lithocholic acid; TCA : Taurocholic acid; TCDCA: Taurochenodeoxy- the OH at C12 of TCA would yield TCDCA, a process cholic acid; TUCA : Tauroursocholic acid; TUDCA: Tauroursodeoxycholic acid. possibly catalyzed by 12α-hydroxysteroid dehydroge- Acknowledgements nase (Tonin and Arends 2018). T-7-KLCA is the inter- We greatly appreciate Professor Zhibi Hu and Mrs Jiyan Zhou, Shanghai mediate in the biosynthesis of TUDCA from TCDCA University of Traditional Chinese Medicine, for their valuable comments and suggestions, and the company of Shanghai Kaibao Pharmaceutica Co. Ltd., (Fig. 1). Directed evolution of 7α-HSDH and 7β-HSDH China, for providing chicken bile and the standard reference of bile acids. to enhance the substrate activity and product tolerance Jin et al. Bioresources and Bioprocessing (2022) 9:32 Page 10 of 10 Authors’ contributions Lian J, Mishra S, Zhao H (2018) Recent advances in metabolic engineering of LJ performed the core experiments and wrote the original manuscript. LY Saccharomyces cerevisiae: new tools and their applications. Metab Eng supervised the chemical analysis, acquisition the funding, and revised the 50:85–108. https:// doi. org/ 10. 1016/j. ymben. 2018. 04. 011 manuscript. SZ conceptualized the project, acquisition the funding, and Liu L, Braun M, Gebhardt G et al (2013) One-step synthesis of 12-ketour- revised the manuscript. ZW conceptualized and supervised the project. All sodeoxycholic acid from dehydrocholic acid using a multienzymatic authors read and approved the final manuscript. system. Appl Microbiol Biotechnol 97:633–639. https:// doi. org/ 10. 1007/ s00253- 012- 4340-5 Funding Lu Q, Jiang Z, Wang Q et al (2021) The effect of tauroursodeoxycholic acid This work was supported by the National Science and Technology (S&T ) Major ( TUDCA) and gut microbiota on murine gallbladder stone formation. Ann Special Projects (No. 2017ZX09309006) and Natural Science Foundation of Hepatol 23:100289. https:// doi. org/ 10. 1016/j. aohep. 2020. 100289 Shanghai (No. 21ZR1463000). Momose T, Tsubaki T, Iida T, Nambara T (1997) An improved synthesis of taurine- and glycine-conjugated bile acids. Lipids 32:775–778. https:// doi. Availability of data and materialsorg/ 10. 1007/ s11745- 997- 0099-8 The data generated and/or analyzed during this study are available from the Rogowska-Wrzesinska A, Larsen PM, Blomberg A et al (2001) Comparison of corresponding author on reasonable request. the proteomes of three yeast wild type strains: CEN.PK2, FY1679 and W303. Comp Funct Genomics 2:207–225. https:// doi. org/ 10. 1002/ cfg. 94 Rosa LRO, Vettorazzi JF, Zangerolamo L et al (2021) TUDCA receptors and their Declarations role on pancreatic beta cells. Prog Biophys Mol Biol. https:// doi. org/ 10. 1016/j. pbiom olbio. 2021. 09. 003 Ethics approval and consent to participate Shi J, Wang J, Yu L et al (2017) Rapidly directional biotransformation of tauro- Not applicable. ursodeoxycholic acid through engineered Escherichia coli. J Ind Microbiol Biotechnol 44:1073–1082. https:// doi. org/ 10. 1007/ s10295- 017- 1935-y Consent for publication Song C, Wang B, Tan J et al (2017) Discovery of tauroursodeoxycholic acid bio- The authors approved the consent for publishing the manuscript. transformation enzymes from the gut microbiome of black bears using metagenomics. 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Journal

Bioresources and Bioprocessing – Springer Journals

Published: Mar 27, 2022

Keywords: Tauroursodeoxycholic acid; Hydroxysteroid dehydrogenase; Biotransformation; Saccharomyces cerevisiae

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A green strategy to produce potential substitute resource for bear bile using engineered Saccharomyces cerevisiae

A green strategy to produce potential substitute resource for bear bile using engineered Saccharomyces cerevisiae

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A green strategy to produce potential substitute resource for bear bile using engineered Saccharomyces cerevisiae

A green strategy to produce potential substitute resource for bear bile using engineered Saccharomyces cerevisiae

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