Engineering 7β-Hydroxysteroid Dehydrogenase for Enhanced Ursodeoxycholic Acid Production by Multiobjective Directed Evolution

Zheng, Ming-Min; Chen, Ke-Cai; Wang, Rufeng; Li, Hao; Li, Chun-Xiu; Xu, Jian‐He

doi:10.1021/acs.jafc.6b05428

Cited by 47 publications

(42 citation statements)

References 40 publications

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“…The NADP + -dependent enzyme from Eubacterium aerofaciens shows a significantly lower specific activity [ 74 ] and another NADP + -dependent enzyme was isolated from Ruminococcus gnavus [ 75 ]. In order to increase the activity and stability of 7β-HSDHs, protein engineering studies were carried out, as described in literature by Weuster-Botz et al [ 77 ] and Zheng et al [ 76 ]. Up till now, Xanthomonas maltophilia 7β-HSDH (33 U/mg) represents the only isolated NAD + -dependent enzyme [ 33 ].…”

Section: Reviewmentioning

confidence: 99%

Latest development in the synthesis of ursodeoxycholic acid (UDCA): a critical review

Tonin

Arends

2018

Beilstein J. Org. Chem.

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Ursodeoxycholic acid (UDCA) is a pharmaceutical ingredient widely used in clinics. As bile acid it solubilizes cholesterol gallstones and improves the liver function in case of cholestatic diseases. UDCA can be obtained from cholic acid (CA), which is the most abundant and least expensive bile acid available. The now available chemical routes for the obtainment of UDCA yield about 30% of final product. For these syntheses several protection and deprotection steps requiring toxic and dangerous reagents have to be performed, leading to the production of a series of waste products. In many cases the cholic acid itself first needs to be prepared from its taurinated and glycilated derivatives in the bile, thus adding to the complexity and multitude of steps involved of the synthetic process. For these reasons, several studies have been performed towards the development of microbial transformations or chemoenzymatic procedures for the synthesis of UDCA starting from CA or chenodeoxycholic acid (CDCA). This promising approach led several research groups to focus their attention on the development of biotransformations with non-pathogenic, easy-to-manage microorganisms, and their enzymes. In particular, the enzymatic reactions involved are selective hydrolysis, epimerization of the hydroxy functions (by oxidation and subsequent reduction) and the specific hydroxylation and dehydroxylation of suitable positions in the steroid rings. In this minireview, we critically analyze the state of the art of the production of UDCA by several chemical, chemoenzymatic and enzymatic routes reported, highlighting the bottlenecks of each production step. Particular attention is placed on the precursors availability as well as the substrate loading in the process. Potential new routes and recent developments are discussed, in particular on the employment of flow-reactors. The latter technology allows to develop processes with shorter reaction times and lower costs for the chemical and enzymatic reactions involved.

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Section: Reviewmentioning

confidence: 99%

Latest development in the synthesis of ursodeoxycholic acid (UDCA): a critical review

Tonin

Arends

2018

Beilstein J. Org. Chem.

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“…A comparison of pH optima between immobilized and free enzymes is shown in Figure S4. Both pH optima of the two groups of immobilized enzymes were shifted to more alkaline pH conditions, which was beneficial for the cascade synthesis of UDCA from CDCA, considering the effective coupling of the oxidative and reductive reactions . This shift to alkaline conditions is probably because of chemical bonding between the resin and enzyme.…”

Section: Resultsmentioning

confidence: 99%

“…All other chemicals or reagents used herein were of reagent grade and obtained commercially. The genes of 7α‐HSDH Ec , LDH, 7β‐HSDH Rt ‐M1 , and GDH were prepared as described previously …”

Section: Methodsmentioning

confidence: 99%

“…The initial cascade strategy includes four enzymes in a one‐pot system: 7α‐hydroxysteroid dehydrogenase (7α‐HSDH Ec from Escherichia coli ) and lactate dehydrogenase (LDH) for the first reaction step, and 7β‐hydroxysteroid dehydrogenase (7β‐HSDH Rt ‐M1 ) with glucose dehydrogenase (GDH) for the second reaction step . In the second reaction step, residual 7α‐HSDH Ec from the first step could work together with GDH to convert the intermediate 7‐oxolithocholic acid (7‐oxo‐LCA) back to CDCA.…”

Section: Introductionmentioning

confidence: 99%

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Continuous Production of Ursodeoxycholic Acid by Using Two Cascade Reactors with Co‐immobilized Enzymes

Zheng

Chen

et al. 2017

ChemBioChem

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Ursodeoxycholic acid (UDCA) is an effective drug for the treatment of hepatitis. In this study, 7α-hydroxysteroid dehydrogenase (7α-HSDH) and lactate dehydrogenase (LDH), as well as 7β-hydroxysteroid dehydrogenase (7β-HSDH) and glucose dehydrogenase (GDH), were co-immobilized onto an epoxy-functionalized resin (ES-103) to catalyze the synthesis of UDCA from chenodeoxycholic acid (CDCA). Through optimizing the immobilization pH, time, and loading ratio of enzymes to resin, the specific activities of immobilized LDH-7αHSDH@ES-103 and 7βHSDH-GDH@ES-103 were 43.2 and 25.8 U g , respectively, which were 12- and 516-fold higher than that under the initial immobilization conditions. Continuous production of UDCA from CDCA was subsequently achieved by using immobilized LDH-7αHSDH@ES-103 and 7βHSDH-GDH@ES-103 in two serial packed-bed reactors. The yield of UDCA reached nearly 100 % and lasted for at least 12 h in the packed-bed reactors, which was superior to that of the batchwise reaction. This efficient continuous approach developed herein might provide a feasible route for large-scale biotransformation of CDCA into UDCA.

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“…The detailed methodology and a demonstration of its ability to identify mutants of four proteins with improved fitness have been reported elsewhere, although only one property was tested for each protein (Cadet et al, 2018a;Cadet et al, 2018b). Here, we applied innov'SAR to the problem of multiple parameter optimization, which has been tackled by other researchers, for example, to simultaneously optimize activity and stability (Giver, Gershenson, Freskgard, & Arnold, 1998), enantioselectivity and thermostability (Li, Zhang, Sun, Liu, & Reetz, 2016), thermostability, activity, and stereoselectivity (Acevedo-Rocha et al, 2018), activity and stereoselectivity (Hall, Sarkar, Lee, Munday, & Bell, 2016), or activity, thermostability, and pH range (Zheng et al, 2017). These earlier studies used classical experimental approaches such as iterative saturation mutagenesis (ISM) CASTing, random mutagenesis or recombination, screening, which did not include a machine learning component for fast and efficient screening and selection.…”

Section: Introductionmentioning

confidence: 99%

One‐shot optimization of multiple enzyme parameters: Tailoring glucose oxidase for pH and electron mediators

Ostafe

Fontaine²,

Frank

et al. 2019

Biotech & Bioengineering

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Enzymes are biological catalysts with many industrial applications, but natural enzymes are usually unsuitable for industrial processes because they are not optimized for the process conditions. The properties of enzymes can be improved by directed evolution, which involves multiple rounds of mutagenesis and screening. By using mathematical models to predict the structure–activity relationship of an enzyme, and by defining the optimal combination of mutations in silico, we can significantly reduce the number of bench experiments needed, and hence the time and investment required to develop an optimized product. Here, we applied our innovative sequence–activity relationship methodology (innov'SAR) to improve glucose oxidase activity in the presence of different mediators across a range of pH values. Using this machine learning approach, a predictive model was developed and the optimal combination of mutations was determined, leading to a glucose oxidase mutant (P1) with greater specificity for the mediators ferrocene–methanol (12‐fold) and nitrosoaniline (8‐fold), compared to the wild‐type enzyme, and better performance in three pH‐adjusted buffers. The kcat/KM ratio of P1 increased by up to 121 folds compared to the wild type enzyme at pH 5.5 in the presence of ferrocene methanol.

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Engineering 7β-Hydroxysteroid Dehydrogenase for Enhanced Ursodeoxycholic Acid Production by Multiobjective Directed Evolution

Cited by 47 publications

References 40 publications

Latest development in the synthesis of ursodeoxycholic acid (UDCA): a critical review

Latest development in the synthesis of ursodeoxycholic acid (UDCA): a critical review

Continuous Production of Ursodeoxycholic Acid by Using Two Cascade Reactors with Co‐immobilized Enzymes

One‐shot optimization of multiple enzyme parameters: Tailoring glucose oxidase for pH and electron mediators

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