Coupled, Self-Sufficient Biotransformation of Chenodeoxcholic Acid to Ursodeoxycholic Acid and Novel Enzyme Mutants Applicable in said Process

20200407766

16/309580

June 19, 2017

The present invention relates to a coupled biotransformation process of converting chenodeoxycholic acid (CDCA) and related compounds to ursodeoxycholic acid (UDCA) and related compounds. It also relates to the cloning, expression, and biochemical characterization of a novel NADP+-dependent 7α-hydroxysteroid dehydrogenase (7α-HSDH) from Clostridium difficile, cofactor switch mutants thereof, and their application for the oxidation of bile acids. A further aspect of the invention relates to novel NADP-dependent cofactor switch mutants of the NADP+-dependent 7α-HSDH of E. coli and their application for the oxidation of bile acids.

1. A coupled biocatalytic process for preparing an ursodeoxycholic acid (UDCA) compound of the general Formula (1), [chemical expression included] wherein R represents alkyl, H an alkali metal ion or N(R3)4+ wherein residue R3 are the same or different and represent H or alkyl, or wherein group —CO2R is replaced by an acid amide group CONR1R2, wherein R1 and R2 independently of each other represent an alkyl residue; which method comprises a) reacting a chenodeoxycholic acid (CDCA) compound of general Formula (2) [chemical expression included] wherein R has the same meanings as defined above for group —CO2R or is replaced by an acid amide group —CONR1R2, in the presence of a 7α-hydroxysteroid dehydrogenase (7α-HSDH) and a 7ß-hydroxysteroid dehydrogenase (7β-HSDH) and a cofactor selected from NAD+ and NADP+, wherein said 7α-HSDH and said 7β-HSDH have the ability of utilizing the same cofactor system selected from NAD+/NADH and NADP+/NADPH, and wherein a1) said 7α-HSDH catalyzes the oxidation of said CDCA compound of general Formula (2) to the corresponding intermediate 7-ketolithocholic acid (7-KLCA) compound of general Formula (3) [chemical expression included] wherein R is as identified above or wherein group —CO2R or is replaced by an acid amide group —CONR1R2 as defined above, and a2) said 7β-HSDH catalyzes the reduction of said 7-KLCA compound of general Formula (3) as formed in reaction step a1) to said UDCA compound of general Formula (1) and under regeneration of the cofactor consumed in reaction step a1); and b) optionally further purifying the reaction product.

2. The process of claim 1, wherein step a) is performed in the presence of isolated 7α-HSDH and 7β-HSDH enzymes or in the presence of one or more recombinant microorganism functionally expressing said enzymes.

3. The process of claim 1 or 2, wherein said 7α-HSDH and said 7β-HSDH both utilize the cofactor system NAD+/NADH; or wherein said 7α-HSDH and said 7β-HSDH both utilize the cofactor system NADP+/NADPH.

4. The process of one of the preceding claims wherein said 7α-HSDH and said 7β-HSDH both utilize the cofactor system NAD+/NADH; wherein a) said 7α-HSDH is selected from (1) a 7α-HSDH comprising an amino acid sequence according to SEQ ID NO: 37 which is isolated from Escherichia coli, which catalyzes at least the stereospecific enzymatic oxidation of 7α-hydroxysteroids to the corresponding 7-ketosteroids, and a mutant thereof having at least 80% sequence identity to SEQ ID NO: 37 and retaining the ability to utilize NAD+/NADH and the ability to catalyze at least the stereospecific enzymatic oxidation of 7α-hydroxysteroids to the corresponding 7-ketosteroids; and (2) a 7α-HSDH which is a mutant of Clostridium difficile 7α-HSDH with SEQ ID NO: 34 which mutant catalyzes at least the stereospecific enzymatic oxidation of 7α-hydroxysteroids to the corresponding 7-ketosteroids under consumption of NAD+ as cofactor, wherein said 7α-HSDH comprises at least one mutation in a position selected from K16, A37 and R38 of SEQ ID NO: 34 and shows a sequence identity of at least 80% to SEQ ID NO:34 b) said 7β-HSDH is selected from (1) a 7ß-HSDH, which is a mutant of Collinsella aerofaciens 7β-HSDH with SEQ ID NO: 54, which mutant catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7β-hydroxysteroid under consumption of NADH as cofactor, wherein said 7β-HSDH comprises at least one mutation in a position selected from T17, G39, R40, R41 and K44 of SEQ ID NO: 54 and shows a sequence identity of at least 80% to SEQ ID NO:54;

5. The process of one of the claims 1 to 3 wherein said 7α-HSDH and said 7β-HSDH both utilize the cofactor system NADP+/NADPH, wherein a) said 7α-HSDH is selected from (1) a 7α-HSDH comprising an amino acid sequence according to SEQ ID NO: 34, which is isolated from Clostridium difficile, which catalyzes at least the stereospecific enzymatic oxidation of 7α-hydroxysteroids to the corresponding 7-ketosteroids under consumption of NADP+ as cofactor, and a mutant thereof having at least 80% sequence identity to SEQ ID NO: 34 and retaining the ability to utilize NADP+/NADPH and the ability to catalyze at least the stereospecific enzymatic oxidation of 7α-hydroxysteroids to the corresponding 7-ketosteroids; (2) a 7α-HSDH which is a mutant of Escherichia coli 7α-HSDH with SEQ ID NO: 37 which mutant catalyzes at least the stereospecific enzymatic oxidation of 7α-hydroxysteroids to the corresponding 7-ketosteroids under consumption of NADP+ as cofactor, wherein said 7α-HSDH comprises at least one mutation in a position selected from D42 and 143 of SEQ ID NO: 37. and shows a sequence identity of at least 80% to SEQ ID NO: 37. b) said 7β-HSDH is selected from (1) a 7ß-HSDH, comprises an amino acid sequence according to SEQ ID NO: 54, and is isolated from Collinsella aerofaciens which catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7ß-hydroxysteroid under consumption of NADPH as cofactor, and a mutant thereof having at least 80% sequence identity to SEQ ID NO: 54 and retaining the ability to utilize NADP+/NADPH and the ability to catalyze at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7ß-hydroxysteroid; (2) a 7ß-HSDH, which is a mutant of Collinsella aerofaciens 7ß-HSDH with SEQ ID NO: 54, which mutant catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7ß-hydroxysteroid under consumption of NADPH as cofactor, wherein said 7ß-HSDH comprises at least one mutation in a position selected from T17, G39, and R64 of SEQ ID NO: 54 and shows a sequence identity of at least 80% to SEQ ID NO:54; (3) a 7ß-HSDH, comprises an amino acid sequence according to SEQ ID NO: 56, and is isolated from Ruminococcus gnavus which catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7ß-hydroxysteroid under consumption of NADPH as cofactor, and a mutant thereof having at least 80% sequence identity to SEQ ID NO: 56, and retaining the ability to utilize NADP+/NADPH and the ability to catalyze at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7ß-hydroxysteroid.

6. A 7α-HSDH which is a mutant of E. coli 7α-HSDH with SEQ ID NO: 37, which mutant catalyzes at least the stereospecific enzymatic oxidation of 7α-hydroxysteroids to the corresponding 7-ketosteroids under consumption of NADP+ as cofactor, wherein the enzyme comprises at least one mutation at an amino acid sequence position selected from D42 and 143 of SEQ ID NO: 37 and shows a sequence identity of at least 80% to SEQ ID NO:37.

7. The 7α-HSDH of claim 6, wherein the amino acid sequence mutation is selected from single or multiple mutations comprising: a) D42X1 and/or b) I43X2 wherein X1 represents an amino acid residue different from aspartic acid (D) and X2 represents an amino acid residue different from isoleucine (I).

8. 7α-HSDH according to any one of the preceding claims 6 and 7, wherein said mutation is selected from a) the single mutations D42X1 and I43X2 and the b) double mutations D42X1/I43 X2 wherein X1 represents G, A, or V and X2 represents R, H, or K

9. 7α-HSDH according to any one of the claims 6 to 8 which, if compared to 7α-HSDH of SEQ ID NO:37 show the following feature profile: a) an increased specific activity (Vmax [U/mg]) for NADP+ during the enzymatic oxidation of CDCA with NADP+ as cofactor; b) a modified cofactor specificity with regard to NADH and NADPH, c) wherein features a) to b) may be present individually or in any combination.

10. A 7α-HSDH which is isolated from C. difficile and has an amino acid sequence according to SEQ ID NO: 34; or which is a mutant of C. difficile 7α-HSDH with SEQ ID NO: 34, which mutant catalyzes at least the stereospecific enzymatic oxidation of 7α-hydroxysteroids to the corresponding 7-ketosteroids under consumption of NAD+ as cofactor wherein the enzyme mutant comprises a mutation in at least one amino acid position selected from K16, A37 and R38 of SEQ ID NO: 34 and shows a sequence identity of at least 80% to SEQ ID NO:34.

11. The 7α-HSDH of claim 10 wherein the amino acid sequence mutation is selected from single or multiple mutations comprising: a) K16X1 b) A37X2 c) R38X3 wherein X1 represents an amino acid residue different from lysine (K) and X2 represents an amino acid residue different from alanine (A) and X3 represents an amino acid residue different from arginine (R).

12. 7α-HSDH according to any one of the preceding claims 10 and 11, wherein said mutation is selected from a) the single mutations K16X1 A37X2 R38X3 and the b) double mutations K16X1/A37X2 and A37X2/R38X3 wherein X1 represents A, G, or D X2 represents D, or E and X3 represents I.

13. 7α-HSDH according to any one of the claims 10 to 12 which, if compared to 7α-HSDH of SEQ ID NO:34 show the following feature profile: a) an increased specific activity (Vmax [U/mg]) for chenodesoxycholic acid (CDCA) b) an increased specific activity (Vmax [U/mg]) for NAD+ during the enzymatic oxidation of CDCA with NAD+ as co-factor; c) a modified co-factor specificity with regard to NADH and NADPH, d) a reduced or missing substrate inhibition for at least one bile acid, in particular cholic acid (CA) and/or CDCA and/or 7-KLCA. e) wherein features a) to d) may be present individually or in any combination.

14. Nucleotide sequence encoding 7α-HSDH according to any one of the preceding claims 6 to 13.

15. Expression cassette, comprising the control of at least one regulative sequence, at least one nucleotide sequence of claim 14.

16. Expression vector, comprising at least one expression cassette of claim 15.

17. Recombinant microorganism, which carries at least one nucleotide sequence according to claim 14 or at least one expression cassette according to claim 15 or at least one expression vector according to claim 16.

18. The recombinant microorganism according to claim 17, which in addition carries the encoding sequence for at least one further enzyme, selected from further hydroxysteroid dehydrogenases (HSDH) suitable for co-factor regeneration.

19. The recombinant microorganism according to claim 18, which co-expresses a 7α-HSDH and a 7β-HSDH both utilize the cofactor system NAD+/NADH; or which co-expresses a 7α-HSDH and a 7β-HSDH both utilize the cofactor system NADP+/NADPH.

20. The recombinant microorganism according to claim 19, which co-expresses a 7α-HSDH and a 7β-HSDH as defined in one of the claims 4 and 5.

21. Biocatalytic process for the enzymatic or microbial synthesis of 7α-ketosteroids, wherein the corresponding 7-hydroxysteroid in the presence of a 7α-HSDH mutant according to the definition of one of the claims 6 to 13 or in the presence of a recombinant microorganism expressing said 7α-HSDH mutant according to one of the claims 17 to 20 is oxidized and optionally one of the formed reaction products is isolated from the reaction mixture.

22. The process of claim 21, wherein said 7-hydroxysteroid is selected from cholic acid (CA) chenodeoxycholic acid (CDCA), 12-keto-chenodeoxycholic acid (12-keto-CDCA) and, preferably by said 7α-HSDH mutant oxidizable, derivatives thereof, in particular a salt, amide or alkyl ester of the acid

23. The process of claim 21 or 22, wherein the oxidation is performed in the presence and in particular under consumption of NAD+ or NADP+.

24. The process of claim 23, wherein NAD+ or NADP+ as consumed is regenerated by coupling with an NAD+ or NADP+-regenerating enzyme, wherein said enzyme is selected from 7β-HSDHs, alcohol dehydrogenases (ADH) and formiate dehydrogenases (FDH), glucose dehydrogenase (GDH), NADH-dehydrogenases, alcohol dehydrogenases (ADH), glucose-6-phosphate-dehydrogenases (G6PDH), phosphite dehydrogenases (PtDH).

12; 12

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