Engineering 7Î²-Hydroxysteroid Dehydrogenase for Enhanced

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Engineering 7#-hydroxysteroid dehydrogenase for enhanced ursodeoxycholic acid production by multi-objective directed evolution Mingmin Zheng, Kecai Chen, Ru-Feng Wang, Hao Li, Chun-Xiu Li, and Jian-He Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05428 • Publication Date (Web): 24 Jan 2017 Downloaded from http://pubs.acs.org on January 26, 2017

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Journal of Agricultural and Food Chemistry

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jf-2016-05428d, revised

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Engineering

7β-hydroxysteroid

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ursodeoxycholic

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evolution

acid

dehydrogenase

production

by

for

multi-objective

enhanced directed

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Ming-Min Zheng,1 Ke-Cai Chen,1 Ru-Feng Wang,3,4 Hao Li,1 Chun-Xiu Li1,* and

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Jian-He Xu1,2,*

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1

State Key Laboratory of Bioreactor Engineering, and

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Shanghai Collaborative

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Innovation Center for Biomanufacturing Technology, East China University of

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Science and Technology, Shanghai 200237, P.R. China.

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3

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Medicine, Shanghai 201203, P.R. China.

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4

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Interdisciplinary Studies (CBIS), Rensselaer Polytechnic Institute, 110 8th Street,

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Troy, NY 12180, United States

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*Corresponding authors. Tel.: +86-21-6425-2498; Fax: +86-21-6425-0840; E-mails:

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[email protected] (C.X. Li); [email protected] (J.H. Xu).

Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese

Department of Chemical and Biological Engineering, Center for Biotechnology and

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1

ACS Paragon Plus Environment


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Abstract

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Ursodeoxycholic acid (UDCA) is the main active ingredient of natural bear bile

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powder with multiple pharmacological functions. 7β-Hydroxysteroid dehydrogenase

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(HSDH) is a key biocatalyst for the synthesis of UDCA. However, all the 7β-HSDHs

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reported commonly suffer from poor activity and thermostability, resulting in limited

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productivity of UDCA. In this study, a multi-objective directed evolution strategy was

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proposed and applied to improve the activity, thermostability and pH optimum of a

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7β-HSDH. The best variant (V3-1) showed 5.5-fold higher specific activity and 3-fold

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longer half-life than the wild-type. In addition, pH optimum of the variant was shifted

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to a weakly alkaline value. In the cascade reaction, the productivity of UDCA with

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V3-1 increased up to 942 g L−1 d−1, in contrast to 141 g L−1 d−1 with the wild-type.

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Therefore, this study provides a useful strategy for improving the catalytic efficiency

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of a key enzyme which significantly facilitated the bioproduction of UDCA.

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Keywords:

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multi-objective directed evolution; ursodeoxycholic acid

biocatalysis; cascade reaction; 7β-hydroxysteroid dehydrogenase;

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Introduction

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Bear bile powder as a traditional Chinese health food and drug has been used for

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thousands of years. Ursodeoxycholic acid (UDCA) is an important pharmacologically

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active natural product discovered from the bear bile and named by Hammarsten in

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1902. Compared with other endogenous bile acids, UDCA shows much better

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therapeutical performance in the treating gallbladder and liver related diseases.1-3 At

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present, it is the only drug approved by the US Food and Drug Administration (FDA)

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for the treatment of primary biliary cirrhosis. In China most commercial UDCA is still

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obtained from live bears and it is legal. Therefore, alternative and efficient artificial

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synthesis of UDCA is highly desired. Nowadays, UDCA has been successfully

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synthesized by chemical reactions from its epimer chenodeoxycholic acid (CDCA)

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which can be easily obtained from the bile of poultry.4,5 However, complex

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procedures, poor selectivity and low yield greatly limit its industrial production6,7

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More importantly, chemical synthesis inevitably uses organic solvents and/or heavy

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metals that are likely to cause environmental pollution.8,9 Hence, biotechnological

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synthesis of UDCA from CDCA will be an irreversible trend because of its high

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efficiency and relative environmental friendliness.

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In recent years, 7α- and 7β-hydroxysteroid dehydrogenases (HSDHs) were jointly

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used to transform CDCA into UDCA.10-12 However, compared with 7α-HSDHs,

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7β-HSDHs have not been paid substantial attention. In 1991, Yoshimoto et al.13

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reported the cloning and sequencing of a 7α-HSDH gene from Escherichia coli

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HB101, and soon afterwards the crystal structure was obtained.14 But it was not until 3



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20 years later that the first sequence of 7β-HSDH, from Collinsella aerofaciens, was

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reported in 2011.15 7β-HSDHRt identified from Ruminococcus torques ATCC 35915

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by our group is one of the only four 7β-HSDH genes have been described so far. 15-18

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Subsequently, it was innovatively employed in a two-step cascade reaction in

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combination with 7α-HSDH (Scheme 1), resulting in an unprecedented yield of

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UDCA (>98%).18 However, all the reported 7β-HSDHs including 7β-HSDHRt showed

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extremely low activity and poor thermostability compared with 7α-HSDHs which

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limited the productivity of UDCA.13 Moreover, 7β-HSDHs showed their best

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performance under weakly acidic conditions,15-18 whereas a weakly alkaline pH (8.0)

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is preferred to convert CDCA into UDCA for effective coupling of the oxidative and

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reductive reactions. Therefore, an alkaliphilic 7β-HSDH was required and we

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attempted to improve both the activity and thermostability of 7β-HSDHRt under

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weakly alkaline conditions so as to comprehensively enhance the productivity of

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UDCA.

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Various protein engineering methods (e.g. directed evolution, rational design and

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semi-rational design, etc) have been applied to engineer enzymes for further expand

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their industrial application. Directed evolution has emerged as a promising method to

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improve any property of an enzyme, such as to enhance the thermal robustness or

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stereoselectivity, or to expand the substrate scope.19,20 Many mutagenesis techniques

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and strategies have been proposed, the majority of which are based on error-prone

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polymerase chain reaction (epPCR),21 saturation mutagenesis, or DNA shuffling22-26.

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Among these strategies, epPCR has been proved to be the most common strategy for 4


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improving the performance of enzymes effectively although the mutation of

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nucleotide base sometimes has a biased tendency. DNA shuffling generates diversity

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through recombination while it does not introduce new site-mutations into the library.

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To achieve satisfying performance of an enzyme, directed evolution is usually

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combined with a variety of methods in different rounds targeting one certain

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object.27,28 In recent years, simultaneous optimization of two or more parameters has

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gained increasing attention. For example, Li et al. have successfully engineered

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activity, enantioselectivity and thermostability of an epoxide hydrolase at the same

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time.29 However, it is still a challenge in directed evolution to simultaneously

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optimize several objects, as the tradeoff between multiple objectives is common.30

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In this study, multiple techniques including epPCR, DNA shuffling and site directed

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mutagenesis were adopted and combined into a multi-objective directed evolution

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strategy to engineer 7β-HSDHRt by addressing the crucial issues associated with pH

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optimum, activity and thermostability simultaneously (Figure 1). The simple

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combination of several protein engineering methods for one object is common, while

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each technique used in this work took multiple objectives into account. Besides, this

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strategy could be applied to engineering the proteins whose crystal structure has not

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been determined. Consequently, the productivity or space-time yield of UDCA was

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improved by nearly 7 folds using the best mutant in the enzymatic cascade reaction.

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Materials and methods

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Materials

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Chenodeoxycholic

acid

(CDCA),

7-oxo-lithocholic 5


acid

(7-oxo-LCA)

and


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ursodeoxycholic acid (UDCA) were purchased from Shanghai Siyu Chemical

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Technology Co., Ltd. (Shanghai, China). Unless otherwise stated, all other chemicals

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and reagents used in this work were obtained commercially and were of reagent grade.

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Restriction endonucleases (Dpn I, Xho I, EcoR I), rTaq polymerase, PrimeSTAR™

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HS and T4 DNA ligase were all purchased from Takara Biotechnology Co., Ltd

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(Dalian, China). Taq DNA polymerase, KOD polymerase and DNase I were

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purchased from New England Biolabs (Beverley, MA). The expression vector

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pET-28a(+) was purchased from Novagen (Shanghai, China). E. coli BL21 (DE3) was

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used as the cloning and expression host.

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Mutagenesis

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Error-prone PCR: The target enzyme for directed evolution started from the wild

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type 7β-HSDHRt. The methods of cloning and recombinant expression were described

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in our previous report.18 The concentration of MnCl2 added into PCR mixture was 100

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µM to control the desired mutation rate of one to three amino acids. The purified

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PCR-mutated genes were digested with Xho I and EcoR I, and ligated into the

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expression vector pET-28a(+). E. coli BL21(DE3) cells containing the ligation

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plasmids were plated onto LB agar plate which contained 50 µg mL-1 kanamycin.

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About 3000 clones were screened in this round.

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DNA shuffling: In vitro DNA shuffling was conducted as reported previously with

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slight modifications.24.31,32 Eighteen beneficial mutant genes from the epPCR were

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mixed by equal moles for cloning PCR. The purified PCR genes were digested in a

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200 µL reaction solution (100 mM Tris·HCl, 0.1 U DNase I, 20 µg DNA and 10 mM 6


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MnCl2) for 3 min and the 50–200 bp fragments were purified. The PCR reassembly

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reaction mixture contained 5 µL of fragments, 5 µL of 10× buffer, 5 µL of 2 mM

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dNTP mix and 1 µL KOD high-fidelity polymerase in a final volume of 50 µL. The

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PCR product was diluted by 1000-fold and the target genes were amplified via a

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nested PCR using external primers. This PCR product, as the library of target DNA

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shuffling, was cloned into pET-28a(+) and expressed in E. coli BL21(DE3). About

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6000 variants were screened using a 96-well microplate assay and 32 resultant clones

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with improved properties were chosen for rescreening in flasks.

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Site-directed mutagenesis: The reaction mixture of site-directed mutagenesis

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contained 100 ng of template DNA, 0.1 µM of each primer and 1.25 U

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PrimeSTAR™HS in a final volume of 50 µL. The following PCR program was used:

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30 cycles of 98°C for 10 s, 55°C for 15 s, 72°C for 7 min, and a final elongation step

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at 72°C for 10 min. The PCR product was transformed into E. coli BL21(DE3) for

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expression after digested with 10 U Dpn I for 2 h at 37°C.

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High-throughput screening

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Individual clones were picked and cultured in 96-well plates, of which each well

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contained 300 µL LB medium with 50 µg mL-1 kanamycin. After cultivation at 37°C

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overnight, 50 µL of the cell culture were added into 600 µL fresh LB medium with 50

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µg mL-1 kanamycin. Isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added to a

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final concentration of 0.2 mM after cultivation at 37°C for 3 h. The cells were

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harvested by centrifugation after 24 h incubation at 16°C and lysed with 750 mg L-1

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lysozyme and 10 mg L-1 Dnase. The cell lysate was centrifuged at 4000 × g for 10 7



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min, and then an aliquot (50 µL) of the supernatant was used to evaluate the variant’s

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activity by mixing with 150 µL activity assay buffer consisting of 100 mM phosphate

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buffer (pH 8.0), 1 mM 7-oxo-lithocholic acid and 0.2 mM NADPH. The reductive

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activity was monitored at 340 nm for 220 s and 30°C using BioTek Synergy

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microplate reader. The mutants with different activities would show various rates of

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change in optical absorbance at 340 nm. For thermostability measurement, another

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aliquot (50 µL) of supernatant was added into 96-well PCR plates and incubated at

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43°C for 15 min before being chilled at 4°C for 3 min using a 96-well PCR machine.

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The residual activity was assayed as described above.

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Rescreening in shake flasks

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The mutants showing higher activity or thermostability were chosen for rescreening in

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flasks. The activity of cell free extract was determined spectrophotometrically at 340

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nm and 30°C by measuring the oxidation of NAD(P)H. The standard assay mixture (1

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mL) was composed of 0.1 mM NADPH, 1 mM 7-oxo-LCA in 0.1 M phosphate buffer

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(pH 8.0), and 10 µL enzyme. The thermostability of cell free extract was determined

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by assaying the residual activity after incubation at 45°C for 1 h.

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Determination of kinetic parameters

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The method of enzyme purification was described previously.18 A varied

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concentration (0.02–1.00 mM) of substrate 7-oxo-lithocholic acid and 0.2 mM

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NADPH or a varied concentration (5–200 µM) of cofactor NADPH and 1 mM

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7-oxo-lithocholic acid were used for the enzyme activity assay using the standard

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method as described above. The kinetic parameters of the purified variants were 8


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calculated by non-linear fitting using Origin 8.6.33

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Characterization of purified variants

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The pH optimum was determined in the following buffers (final concentration, 100

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mM): sodium citrate (pH 5.0–6.0), phosphate buffer (pH 6.0–8.0) and Tris-HCl (pH

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8.0-9.0). The half-life (t1/2) value of variants was measured by incubating the purified

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enzymes (1.0 mg mL-1) in the phosphate buffer (100 mM, pH 8.0) at 40°C and

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measuring the residual activities at different times.34 The T 15 50

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temperature at which the enzyme retains 50% of its activity after a 15-min incubation,

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was measured by filling one row of a 96-well PCR plate with 50 µL per well of

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purified enzymes at 1 mg mL-1. The 96-well plate was incubated in PCR amplifier at

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temperatures ranging from 40°C to 50°C for 15 min. After cooled on ice, the residual

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activities of heat-treated enzymes were determined as described above.

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Homology modeling and molecular docking

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The structures of 7β-HSDHRt-WT and 7β-HSDHRt-V3-1 were modeled based on a

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7β-HSDH from Collinsella aerofaciens (PDB ID: 5FYD, 76% identity) using

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SWISS-MODEL web server (http://www.swissmodel.expasy.org/) and validated by

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UCLA-DOE LAB-SAVES (http://services.mbi.ucla.edu/SAVES/). AutoDock software

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was used to docking substrate 7-oxo-LCA and NADPH into the structure of

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enzyme.35-37 The distances were measured in PyMOL.

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Enzymatic synthesis of UDCA from 7-oxo-LCA by WT and V3-1

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The reaction for comparing the performance of variants: the reactions were carried out

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at 30°C and 180 rpm in a 10-mL solution containing 100 mM phosphate buffer, pH 9


value, i.e., the


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8.0, 5 mM 7-oxo-LCA, 10 mM glucose, 0.25 mM NADP+, 0.5 U mL−1 GDH (glucose

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dehydroganase) and 4 µg mL−1 of pure V3-1 or WT.

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The reaction for enzymatic synthesis of UDCA: A 10-mL reaction, containing

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7-oxo-LCA (50, 100 or 200 mM), glucose (75, 150 or 300 mM), 3 g L-1 lyophilized

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enzyme powder (cell free extract, CFE) of 7β-HSDHRt (WT or V3-1), 5 g L-1

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lyophilized enzyme powder (CFE) of GDH, 0.5 mM NADP+ and phosphate buffer

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(100 mM, pH 8.0), was carried out at 30°C. The pH was maintained at 8.0 by titration

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with 1.0 M NaOH. The detection of bile acid was performed as described

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previously.18

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Two-step cascade reaction in one-pot using WT or V3-1

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A 10-mL reaction, containing 100 mM CDCA, 150 mM sodium pyruvate, 0.5 mM

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NAD+, 5 g L-1 lyophilized enzyme powder (CFE) of E. coli 7α-HSDH, 5 g L-1

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lyophilized enzyme powder (CFE) of LDH and phosphate buffer (100 mM, pH 8.0),

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was carried out at 30°C. Then the reaction was terminated by boiling for 5 min. After

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cooling, 150 mM glucose, 0.5 mM NADP+, 5 g L-1 lyophilized enzyme powder (CFE)

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of GDH and 3 g L-1 lyophilized enzyme powder (CFE) of V3-1 or WT were added into

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the mixture and the reaction was continued at 30°C. The pH was maintained at 8.0 by

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titration with 1.0 M NaOH. The preparation of 7α-HSDH, LDH and GDH and the

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detection of bile acid were performed as described previously.18

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Results and Discussion

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Multi-objective directed evolution

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In our previous work, the two-step reaction for cascade synthesis of UDCA from

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CDCA contained four enzymes: 7α-HSDH, lactate dehydrogenase (LDH), glucose

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dehydrogenase (GDH) and 7β-HSDHRt.18 Through the characterization of these four

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enzymes, 7β-HSDHRt was identified as the rate-limiting bottleneck (Table S1). By

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means of a multi-objective directed evolution strategy, we aimed to identify essential

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amino acid residues of 7β-HSDHRt that affect activity and thermostability in an

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alkaline environment, and to obtain mutants that favor the efficient synthesis of

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UDCA. Because of the lack of 7β-HSDH crystal structure at the beginning of this

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work, it was difficult to use rational or semi-rational engineering strategies.

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Accordingly, we decided to start with a simple method (i.e., error-prone PCR) that

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was supported by random mutagenesis. Taking into account the desired pH optimum

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for the enzymatic reaction, the subsequent screening was deliberately carried out at

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pH 8.0. Activity enhancement may lead to reducing thermal stability, and vice

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versa.29,30 So we screened the library separately for mutants with either enhanced

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activity or thermostability, resulting in 18 hits with higher activity or better

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thermostability than the WT (Table S2). The variant with the highest activity (V1-1)

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and the most thermostable variant were chosen for purification and determination of

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specific activity and the T15 50 value (Table 1, entries 2 and 3). V1-1 had 3-fold higher

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activity than the WT, but at the cost of obviously decreased thermostability, in

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contrast to the properties of V1-2. The result showed a tradeoff occurred to some

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extent in almost all of the variants in this round.

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Based on the limited success achieved by the method described above, we decided 11



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to adopt a different approach in the second round of evolution. DNA shuffling was

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used to recombine all the 18 beneficial mutations selected from the epPCR library.

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This process allowed the accumulation of multiple mutations and provided a

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cooperative effect of the mutations. As Figure S1 shows, the 32 mutants identified in

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this round are significantly better than the previous variants in terms of activity and

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thermostability. Four best mutants in this round were selected for purification and

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determination of specific activity and T 15 50 , among which V2-1 exhibited the

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highest-activity, V2-4 was the most thermostable variant, and two others (V2-2 and V2-3)

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showed modest performance in both activity and thermostability (Table 1, entries 4 to

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7). The activity of V2-1 was higher than that of V1-1 and the thermostability of V2-4 was

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higher than that of V1-2, although the stability of V2-1 and the activity of V2-4 were still

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poorer. V2-2 and V2-3 showed higher activity while retaining nearly the same level of

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thermostability as the WT, which was a breakthrough in this round of evolution.

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However, these mutants were still not ideal, as we wanted mutants whose activity and

247

stability were simultaneously improved.

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For this purpose, another round of evolution was performed. In directed evolution,

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the “best” mutant from the previous round usually was used as an “anchor” in the next

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round of evolution. This was not appropriate in our case, however, because the best

251

mutant for each target (activity or/and stability) was different. Then, we analyzed all

252

the locations of the 32 mutations chosen in round 2 which had their own advantages

253

in activity or stability, and found that different sites had different frequencies of

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positive mutation. Thus we assumed that the mutated residues, especially those that 12


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occurred with high frequency, should essentially be important for activity or stability.

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By sorting the variants according to their activity and thermostability (Table S3) and

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analyzing the mutation frequency, we found the five most frequent sites in turn as

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potential candidates for saturation mutagenesis: residues Asn240, Thr189, Val207,

259

Ile112 and Val38.

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These five residues were individually subjected to NNK-coded saturation

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mutagenesis experiments. In this way, only 480 transformants must be screened for

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95% coverage.38 Through 96-well-plate screening, the best mutants in each case were

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chosen and subjected to rescreening and sequence determination (Table S4). In the

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terms of Asn240, Thr189 and Val207, the impacts were subtle: the resultant mutants

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showed a significant increase in activity and/or stability. However, the variants at

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positions 38 and 112 showed almost no improvement in activity or stability compared

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with the WT. Thereafter, we combined mutations of these three sites (Asn240, Thr189

268

and Val207) randomly and found that the double-mutant T189V/V207M (named

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variant V3-1) turned out to be the best “compromise” variant (Table S5, Figure S1).

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V3-1 showed more than 5-fold higher activity than the wild-type, along with obviously

271

better thermostability. Consequently, variant V3-1 (T189V/V207M), was selected for

272

further investigation.

273

In summary, the multi-objective directed evolution strategy (Figure 1) is briefly

274

described as follows. 1) Error-prone PCR was employed to discover potential key

275

sites to each object separately (activity or stability). 2) DNA shuffling was used to

276

merge the important properties of mutations from different objects. 3) Site-directed 13



277

mutagenesis was used to further improve both the activity and thermostability under

278

weakly alkaline condition.

279

Performance of variants

280

To gain some insight into the origin of activity improvement, the kinetics of the

281

mutant enzymes were investigated. As listed in Table 1 (entries 1 to 8), the mutants

282

with higher specific activities mainly benefited from increased kcat values while the

283

Km did not change much in comparison with the wild type enzyme. In contrast, in the

284

case of V2-4, whose activity is reduced, the kcat declined while the Km increased by

285

10-fold. The kinetic parameters of mutant V3-1 for the cofactor NADPH have also

286

been determined. From the result, we can find that the binding of the cofactor with the

287

V3-1 is declined compared with WT (Table 1, entries 11 and 12). Compared with WT,

288

the double mutant V3-1 (T189V/V207M) showed a 5.5-fold higher Vmax and an

289

increase of the T1550 value of 2.3°C. To evaluate the thermostability improvement of

290

the mutant, the purified proteins, V3-1 and WT, was examined at 40°C; the half-lives

291

were 28 h and 9.9 h, respectively. In other words, the thermostability of the mutant

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V3-1 was enhanced by nearly 3-fold via directed evolution (Figure S2). Another

293

important parameter of the dehydrogenase that we are interested in is its optimal pH.

294

To shift the pH optimum of the enzyme to suit a weakly alkaline environment, the

295

screening condition of directed evolution mutants was fixed at pH 8.0. As Figure 2

296

shows, the pH optimum of mutant V3-1 was indeed shifted, from pH 6.5 for WT to pH

297

7.5, which is relatively beneficial for enzymatic synthesis of UDCA. This also

298

illustrates that the change in optimum pH partially contributed to the improvement in 14


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activity. It is worth noting that a double mutation led to a triple improvement in

300

catalytic properties: activity, stability and pH optimum.

301

7-Oxo-lithocholic acid (7-oxo-LCA) is the intermediate product of the

302

biotransformation of CDCA to UDCA, and also the substrate of 7β-HSDH. To

303

confirm the superior performance of mutant V3-1 over its parental enzyme, the

304

enzymatic transformation of 7-oxo-LCA to UDCA (5 mM) was compared using the

305

same amount (4 mg L−1 pure enzyme) of wild-type and variant V3-1. As Figure 3

306

shows, full conversion catalyzed by V3-1 was achieved within 10 h, while the WT

307

enzyme with the same load reached just 80% conversion after 36 h. Moreover, the

308

higher thermostability of V3-1 compared with the WT was confirmed by the higher

309

residual activity (Figure 3). It is clear that the catalytic efficiency of V3-1 is superior to

310

that of wild-type 7β-HSDHRt.

311

Retro analysis of random mutations

312

To reveal the roles of sites 189 and 207 respectively in improving the properties of

313

V3-1, two artificially designed single-mutants (VT189V and VV207M) were constructed

314

and subjected to analysis of enzyme properties (Table 1, entries 9 and 10). Kinetic

315

characterization of the variants indicated that the improvement in catalytic activity

316

was mainly due to an increase in kcat and that there is synergy between the two

317

residues. It is also important to note that both single mutants had the same pH

318

optimum as the WT enzyme (Figure S3). That is to say, mutations T189V and

319

V207M synergistically contributed to the pH optimum shift of the V3-1.

320

Very recently, the crystal structure of a 7β-HSDH, from C. aerofaciens, has been 15



321

reported (PDB ID: 5FYD).39 To rationalize the effect of mutations at the molecular

322

level, the structures of 7β-HSDHRt-WT and 7β-HSDHRt-V3-1 were modeled based on

323

the crystal structure of C. aerofaciens 7β-HSDH, which shares ca. 76% identity to

324

7β-HSDHRt. One of the most significant differences between the structures of the

325

wild-type and V3-1 occurs in the substrate loop of the enzyme, as the residue at

326

position 207 is changed from valine to methionine (Figure 4a).39 In addition,

327

7β-HSDHRt is a dimeric dehydrogenase39 whose residues 189 and 207 are located in

328

the interfacial domain where the two monomers interact (Figure 4b). Therefore, the

329

change of amino acids at sites 189 and 207 probably alters the interaction between the

330

two monomers, resulting in the observed improvement in the thermostability of

331

variant V3-1. Site 189 is very close to the substrate 7-oxo-LCA and NADPH, which

332

may partially explain why mutation there affects the activity significantly (Figure 4a).

333

In short, the two residues (189 and 207) play important roles in the crystal structure.

334

To further explore the two key sites, the amino acids at positions 189 and 207 were

335

altered individually to each of the remaining 18 amino acids by site-directed

336

mutagenesis using V3-1 (T189V/V207M) as the template. The residue of 207 was Met

337

when mutating site 189, whereas the residue of 189 was Val when mutating site 207.

338

Figure S4 summarizes the activity and T15 50 values of these purified mutant enzymes,

339

according to classification of the 20 amino acids as nonpolar, polar uncharged, basic

340

or acidic. We found that the activities correlated well with the charge of the amino

341

acid residue at position 189, since all the mutants containing either a positively or

342

negatively charged amino acid , such as T189K/V207M, T189H/V207M, 16


Page 16 of 38

Page 17 of 38


343

T189R/V207M, T189E/V207M, and T189D/V207M, lost their activity completely

344

(Figure S4a). In addition, replacement of Thr189 by a bulky amino acid resulted in

345

significantly reduced activity (Figure S4a). In other words, the 189th site of

346

7β-HSDHRt prefers hydrophobic and small-size amino acids. Even though the trend

347

was less obvious than for site 189, hydrophobic amino acids seem to be more suitable

348

than other amino acids for site 207 such as T189V/V207D and T189V/V207E (Figure

349

S4b).

350

To date, only three 7β-HSDHs other than 7β-HSDHRt, 7β-HSDHCa1 from

351

Clostridium absonum,16 7β-HSDHCa2 from C. aerofaciens15 and 7β-HSDHRg from

352

Ruminococcus gnavus,17 have been cloned. Intrigued by whether or not the functions

353

of the two key sites we identified are highly conserved (Figure S5) among

354

homologous 7β-HSDHs, we decided to build a series of mutants in the other

355

7β-HSDHs by referring to those of 7β-HSDHRt. All the mutants of 7β-HSDHCa2 and

356

7β-HSDHRg showed clearly higher activities, up to 5.5 times those of the respective

357

WTs, and a little better thermostability. However, in 7β-HSDHCa1, only one mutant,

358

7β-HSDHCa1-T189V, exhibited higher thermostability than its wild-type while

359

maintaining a similar activity (Table 2). This is probably because of the relatively low

360

identity between 7β-HSDHCa1 and 7β-HSDHRt (47%), in contrast to 7β-HSDHCa2 and

361

7β-HSDHRg which share 76% identity with 7β-HSDHRt. In general, both mutations

362

T189V and V207M are beneficial to other 7β-HSDHs. Although several recent

363

patents also disclosed some mutations of 7β-HSDHCa2 (G39S/R64E) or 7β-HSDHRg

364

(L3M/T210N) with 2–5-fold higher activities than their parents, they just targeted one 17



365

property (activity) of 7β-HSDH.40,41 To the best of our knowledge, this is the first

366

report on the engineering of 7β-HSDH with successful improvement in multiple

367

properties which are crucial for the enzymatic synthesis of UDCA.

368

Enzymatic synthesis of UDCA by V3-1 or its wild-type

369

To fully assess the productivity of variant V3-1 in comparison with WT for UDCA

370

synthesis, a cascade reaction with high substrate loads was performed. First, the effect

371

of 7-oxo-LCA loads on the conversion was examined. The conversions by mutant and

372

WT were detected respectively when the 7-oxo-LCA load was gradually increased to

373

50, 100, and 200 mM (Figure 5). It is shown that 50 mM 7-oxo-LCA could be

374

completely converted by V3-1 within about 30 min, in contrast to 1.5 h by the WT

375

(Figure 5a). When the 7-oxo-LCA load was increased to 100 mM, the substrate could

376

be completely transformed by V3-1 within 40 min, whereas the conversion by WT

377

reached only 80% after 4.5 h (Figure 5b). The bioconversion could not reach 100%

378

after several hours by either V3-1 or WT when the load of 7-oxo-LCA was further

379

increased up to 200 mM (Figure 5c).

380

Subsequently, the cascade biotransformation of CDCA to UDCA was conducted by

381

the strategy of two-step reactions in one pot (Scheme 1). In the first step, 100 mM of

382

CDCA was transformed to 7-oxo-LCA within 20 min. The complete transformation of

383

the second step took merely 40 min using V3-1, compared with 5 h using an equivalent

384

(3 gCFE L-1) of WT with only 80% conversion. The expression yields of the V3-1

385

variant and WT in cell free extract are on the same level by SDS-PAGE analysis

386

(Figure S6). The space-time yield (or productivity) of UDCA in the two-step cascade 18


Page 18 of 38

Page 19 of 38


387

reaction using V3-1 is nearly 7-fold higher than WT (942 versus 141 g L−1 d−1) (Table

388

3, entries 6 and 7). This productivity is significantly higher than other similar reported

389

bioprocesses (Table 3).42-44 This implies that one liter of the cascade reaction mixture

390

using V3-1 can save 86 bears per year because one bear produces about 4 kg bear bile

391

powder per year.45 Thus the significantly enhanced space-time yield of UDCA can

392

save the lives of more bears.

393

In conclusion, compared with the wild-type enzyme, the best hit variant (V3-1, a

394

double mutant) displayed 5.5-fold higher activity and 3-fold higher stability,

395

accompanied by a desired shift of pH optimum from 6.5 to 7.5. The productivity of

396

UDCA was increased by nearly 7-fold, achieving 942 g L−1 d−1 which is the highest

397

productivity to the best of our knowledge. V3-1 displayed obviously higher

398

transformation efficiency than the wild-type in the multi-enzymatic cascade synthesis

399

of UDCA, which might lay a solid foundation for future study and industrial

400

application.

401

Supporting information

402

Additional experimental results including the overall performance of various mutants

403

from three rounds of directed evolution examined in shake flask culture and other data

404

are available free of charge via the Internet at http://pubs.acs.org.

405

Acknowledgements

406

This work was financially supported by the National Natural Science Foundation of

407

China (Nos. 21276082 & 21536004), Ministry of Science and Technology, P.R.

19



408

China (No. 2011CB710800, 2011AA02A210 and 2012AA022201), and Shanghai

409

Commission of Science and Technology (No. 11431921600).

410

References

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Weinzierl, M.; Pape, R. G.; Sauerbruch, T.; Paumgartner, G. Ursodeoxycholic acid for

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Scheme and figure legends Scheme 1. Multienzymatic cascade synthesis of ursodeoxycholic acid from chenodeoxycholic acid. Figure 1. The multi-objective directed evolution strategy used in this study. Figure 2. Effect of pH on activity of the purified WT 7β-HSDHRt (solid symbols) and the variant V3-1 (open symbols) at 30 °C. Enzyme assay was performed using the standard assay procedure in the following 100 mM buffers: (1) citrate (pH 5.0–6.0, circle symbols); (2) phosphate (pH 6.0–8.0, triangle symbols); (3) Tris–HCl (pH 8.0– 9.5, square symbols). Figure 3. Time courses of enzymatic synthesis of UDCA from 7-oxo-LCA (5 mM) by WT 7β-HSDHRt (solid circle) and variant V3-1 (solid triangle) (4 mg L-1 pure enzyme) at 30 °C. The residual enzyme activity of V3-1 (open triangle) and the WT (open circle) were determined at the same times. Figure 4. Homology modeling and molecular docking of 7β-HSDHRt structure. (a) Alignment of the model of the wild-type enzyme (green) and mutant V3-1 (yellow). The distance between the carbonyl of the substrate 7-oxo-LCA and one of catalytic triad (Tyr156) is 2.1 Å, and that of residue 189 to the nicotinamide of NADPH is 3.0 Å. The catalytic triad (red), 7-oxo-LCA (magenta) and NADPH (cyan) are displayed. (b) 7β-HSDHRt is a symmetrical dimer. The two chains of the protein dimer are colored yellow and green respectively. The structures of 7β-HSDHRt-WT and 7β-HSDHRt-V3-1 were modeled based on 7β-HSDH from Collinsella aerofaciens (PDBID: 5FYD, 76% identity). Figure 5. Time courses of UDCA synthesis by wild-type 7β-HSDHRt (open symbols) 27



or its variant V3-1 (solid symbols) from 7-oxo-LCA with varied loads: (a) 50 mM; (b) 100 mM; (c) 200 mM. The reaction was conducted at 30°C, and the pH was maintained at 8.0 by automatically titrating 1 M NaOH.

28


Page 28 of 38

Page 29 of 38


Table 1. Kinetic parameters and thermostabilities of purified wild-type and mutants of 7β-HSDHRt.

Enzyme

Generation

Mutation sites

Km (µM)

kcat (s−1)

kcat / Km (mM−1 s−1)

Vmax (µmol min−1 mg−1)

T15 50 (°C)

1

WT

—

—

23 ± 1

4.20 ± 0.02

(1.8 ± 0.2) × 102

8.60 ± 0.04

43.0

2

V1-1

1st epPCR

21 ± 2

17.5 ± 0.2

(8.3 ± 0.1) × 102

36.1 ± 0.4

< 37

3

V1-2

1st epPCR

57 ± 4

7.80 ± 0.15

(1.4 ± 0.1) × 102

16.0 ± 0.3

47.0

34 ± 2

31.0 ± 0.4

(9.1 ± 0.1) × 102

64.0 ± 0.9

38.7

Entry

nd

I28V/V38A/I76V/ V207M K44E/T189I G57S/I163V/

4

V2-1

2 DNA shuffling

5

V2-2

2nd DNA shuffling

I112V/N240D

28 ± 5

21.4 ± 0.8

(7.6 ± 0.3) × 102

44.3 ± 1.7

42.4

6

V2-3

2nd DNA shuffling

N240I

23 ± 3

13.4 ± 0.4

(5.8 ± 0.2) × 102

27.7 ± 0.9

43.9

7

V2-4

2nd DNA shuffling

190 ± 20

3.30 ± 0.12

17 ± 0.6

6.70 ± 0.25

47.2

8

V3-1

3rdsite-direct ed mutation

T189V/V207M

40 ± 5

23.0 ± 0.6

(5.8 ± 0.2) × 102

46.8 ± 1.2

45.3

9

VT189V

—

T189V

31 ± 4

20.1 ± 0.6

(6.4 ± 0.2) × 102

41.3 ± 1.3

44.9

10

VV207M

—

V207M

35 ± 3

17.3 ± 0.3

(4.8 ± 0.1) × 102

35.5 ± 0.6

43.3

8.90 ± 0.55

43.0

51.4 ± 3.3

45.3

V207M

I112V/T189I/ N240D

11

WT

a

—

—

7.8 ± 2.2

12

V3-1 a

—

T189V/V207M

47 ± 8

a

a

a

2a

4.30 ± 0.27

(5.5 ± 0.3) × 10

25.1 ±1.6

(5.3 ± 0.3) × 102 a

The Km and kcat values were determined for the cofactor NADPH.

29



Page 30 of 38

Table 2. Improved properties of mutants of three other homologous 7β-HSDHs. Enzyme

Activity (U/mgprot)a

Folds b

T15 50 (°C)

7β-HSDHCa1-WT

15.9 ± 1.2

1.0

37.6

7β-HSDHCa1-T189V

14.9 ± 0.1

0.94

39.7

7β-HSDHCa1-V205M

11.1 ± 0.3

0.70

37.0

7β-HSDHCa1-T189V/V205M

10.1 ± 0.1

0.63

37.0

7β-HSDHCa2-WT

10.7 ± 1.0

1.0

44.4

7β-HSDHCa2-T189V

25.5 ± 1.3

2.4

44.4

7β-HSDHCa2-V207M

29.1± 0.3

2.7

44.7

7β-HSDHCa2-T189V/V207M

58.4 ± 2.7

5.5

44.9

7β-HSDHRg-WT

21.9 ± 0.7

1.0

44.5

7β-HSDHRg-T189V

65.3 ± 1.8

3.0

44.4

7β-HSDHRg-V207M

48.5 ± 3.4

2.2

44.3

7β-HSDHRg-T189V/V207M

41.8 ± 0.9

1.9

45.6

a b

Activity was determined by the standard assay method at pH 8.0. The ratio of activity of mutants and their respective WT.

30


Page 31 of 38


Table 3. Comparison of the biotransformation results of CDCA to UDCA

a b

Entry

[S] (mM)

Time (h)

Conversion (%)

Space-time yield (g L−1 d−1)

Ref.

1

0.50

24

80

0.2

[42]

2

12.5

48

86

2.1

[43]

3

25

24

27

2.6

[12]

4

250

72

>99

33

[44]

5

250

24

>99

98

[41]

6

100

5.3 (0.33 a + 5.0 b)

80

141

This study (WT)

7

100

1.0 (0.33 a + 0.67 b)

99

942

This study (V3-1)

The time it took to completely convert CDCA to 7-oxo-LCA. The time it took to convert 7-oxo-LCA to UDCA.

31



Scheme 1. Multienzymatic cascade synthesis of ursodeoxycholic acid from chenodeoxycholic acid.

32


Page 32 of 38

Page 33 of 38


Figure 1. The multi-objective directed evolution strategy used in this study.

33



120

Relative activity (%)

100 80 60 40 20 0 4.5

5.5

6.5

7.5

8.5

9.5

pH

Figure 2. Effect of pH on activity of the purified WT 7β-HSDHRt (solid symbols) and the variant V3-1 (open symbols) at 30 °C. Enzyme assay was performed using the standard assay procedure in the following 100 mM buffers: (1) citrate (pH 5.0–6.0, circle symbols); (2) phosphate (pH 6.0–8.0, triangle symbols); (3) Tris–HCl (pH 8.0– 9.5, square symbols).

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100

100

80

80

60

60

40

40

20

20

0

Residue activity (%)


Yield of UDCA (%)

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0 0

6

12 18 24 Reaction time (h)

30

36

Figure 3. Time courses of enzymatic synthesis of UDCA from 7-oxo-LCA (5 mM) by WT 7β-HSDHRt (solid circle) and variant V3-1 (solid triangle) (4 mg L-1 pure enzyme) at 30 °C. The residual enzyme activity of V3-1 (open triangle) and the WT (open circle) were determined at the same times.

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(a) 7-oxo-LCA

Substrate loop

NADPH

(b)

Figure 4. Homology modeling and molecular docking of 7β-HSDHRt structure. (a) Alignment of the model of the wild-type enzyme (green) and mutant V3-1 (yellow). The distance between the carbonyl of the substrate 7-oxo-LCA and one of catalytic triad (Tyr156) is 2.1 Å, and that of residue 189 to the nicotinamide of NADPH is 3.0 Å. The catalytic triad (red), 7-oxo-LCA (magenta) and NADPH (cyan) are displayed. (b) 7β-HSDHRt is a symmetrical dimer. The two chains of the protein dimer are colored yellow and green respectively. The structures of 7β-HSDHRt-WT and 7β-HSDHRt-V3-1 were modeled based on 7β-HSDH from Collinsella aerofaciens (PDBID: 5FYD, 76% identity). 36


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(a)

(b)

100

Conversion (%)

Conversion (%)

100 80 60 40 20

80 60 40 20 0

0 0

20

40

60

80

0

100

1

2

3

4

5

Time (h)

Time (min)

Conversion (%)

(c) 60 40 20 0 0

2

4

Time (h)

6

8

Figure 5. Time courses of UDCA synthesis by wild-type 7β-HSDHRt (open symbols) or its variant V3-1 (solid symbols) from 7-oxo-LCA with varied loads: (a) 50 mM; (b) 100 mM; (c) 200 mM. The reaction was conducted at 30°C, and the pH was maintained at 8.0 by automatically titrating 1 M NaOH.

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TOC graphic

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Engineering 7Î²-Hydroxysteroid Dehydrogenase for Enhanced

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