Supplementary materials

Rapidly directional biotransformation of tauroursodeoxycholic acid through engineered Escherichia coli

JieShi1, JieWang1, Lu Yu1,2, Li Yang1, ShujuanZhao1*, ZhengtaoWang1*

1 The SATCM Key Laboratory for New Resources & Quality Evaluation of Chinese Medicine, The MOE Key Laboratory for Standardization of Chinese Medicines and Shanghai Key Laboratory of Complex Prescription, Institute of Chinese MateriaMedica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

2 Department of Traditional Chinese MateriaMedica, Shenyang Pharmaceutical University, Shenyang 110016, China

* Correspondence authors.

Shujuan Zhao

Tel: +8621-51322576, Fax: +8621-51322495, E-mail: , .

Zhengtao Wang

Tel: +8621-51322507, Fax: +8621-51322519, E-mail:.

Table S1. Bacterium strains and vectors used in this study.

Number / Strain or vector / Properties / Reference
S1 / Escherichia coliDH5α / F−, φ80dlacZΔM15, Δ(lacZYA-argF)
U169, recA1, endA1, hsdR17(rk−, mk+), phoA, supE44λ−, thi−1, gyrA96, relA1 / Novagen
S2 / E.coliBL21 Star™ (DE3) / F−ompT gal dcm rne131 lonhsdSB (rB_mB_) λ(DE3) / Invitrogen
1 / pMD / ColE1ori, AmpR / TaKaRa
2 / pETM6 / ColE1ori, AmpR / (Xu et al. 2012)
3 / pETM6-α1 / pETM6 carryingsynthesized Clostridium sardiniense(C. absonum) 7α-hydroxysteroiddehydrogenase gene (Ca7αsyn) / This study.
4 / pETM6-α2 / pETM6 carrying synthesizedEscherichia coli7α-hydroxysteroiddehydrogenase (Ec7αsyn) / This study.
5 / pETM6-β1 / pETM6 carrying synthesizedClostridium sardiniense(C. absonum) 7β-hydroxysteroiddehydrogenase (Ca7βsyn) / This study.
6 / pETM6-β2 / pETM6 carrying synthesizedRuminococcusgnavus7β-hydroxysteroiddehydrogenase (Rg7βsyn) / This study.
7 / pα1β1 / pETM6 carrying Ca7αsyn-Ca7βsyn / This study.
8 / pα1β2 / pETM6 carrying Ca7αsyn-Rg7βsyn / This study.
9 / pα2β1 / pETM6 carryingEc7αsyn-Ca7βsyn / This study.
10 / pα2β1 / pETM6 carrying Ec7αsyn- Rg7βsyn / This study.


FigureS1Typical map of TUDCAePathBrick construct


FigureS2Typical growth curve of engineered E.coliBL21star(DE3) in LB broth


Figure S3Pre-evaluation of oxygen effect on the biotransformationof TUDCA

Fermentation was carried out in 50mL tubes (more oxygen) containing 1 mL of 1xM9 media compared with that in 15mL tubes (less oxygen). Data of 15mL tubes were from triplicates and those of 50mL tubes were from one sample.


FigureS4Effect of temperature and fermentation period on the biotransformation of TUDCA

a,c. Titer of principal bile acids under different conditions. b. Conversion efficiency of TUDCA.

Fermentation was carried out in 50 mL tubes containing 3 mL of PBS-M9 media.


Figure S5 Scale-up fermentation results of BL-pα1β2 under the optimal conditions.

a.Titer of bile acids in the fermentation broth. b. Conversion efficiency of TUDCA.

Fermentation was carried out in 100 mL flasks containing 10 mL of PBS-M9 media.


FigureS6 Typical HPLC profile of fermentation results of BL21-pα1β2

X-axis stands for the retention time. Y-axis represents the signal response strength.

Fermentation was performed at 25℃for 6 hrs in 10 mL of PBS-M9 media using 2.83g/L of TCDCA as substrate with 100 mL of flask. Compound 1, 3, and 4 were identified as TUDCA, T-7K-LCA and TCDCA by comparing with the reference standards and LC MS.Compound 2 was identified as taurocholate acid (TCA) by LC-MS, which was present in the chicken bile powder.