Method for producing tertiary β-hydroxy-α-amino acids

1 . A method to make an amino acid having a tertiary alcohol sidechain, the method comprising reacting a pyridoxal-phosphate (PLP)-dependent enzyme selected from the group consisting of an L-threonine aldolase, an L-threonine transaldolase, and a combination thereof, with a ketone substrate and a primary or secondary β-hydroxy α-amino acid for a time, at a temperature, and at a pH wherein the reaction yields a tertiary β-hydroxy α-amino acid.

2 . The method of claim 1 , wherein the PLP-dependent enzyme is an L-threonine aldolase.

3 . The method of claim 2 , wherein the L-threonine aldolase has an amino acid sequence at least 80% identical to SEQ. ID. NO: 1.

4 . The method of claim 2 , wherein the L-threonine aldolase has an amino acid sequence at least 90% identical to SEQ. ID. NO: 1.

5 . The method of claim 2 , wherein the L-threonine aldolase has an amino acid sequence at least 95% identical to SEQ. ID. NO: 1.

6 . The method of claim 3 , wherein the PLP-dependent enzyme is ObiH.

7 . The method of claim 1 , wherein the time is from about 1 hour to about 12 hours, the temperature is from about 30° C. to about 50° C., and the pH is from about 6 to about 8.

8 . The method of claim 1 , further comprising reacting the PLP-dependent enzyme and the ketone substrate in the presence of a reducing system.

9 . The method of claim 1 , wherein the ketone substrate further comprises an electron-withdrawing group.

10 . The method of claim 9 , wherein the electron-withdrawing group is selected from the group consisting of halo, haloalkyl, —NH 3 + , —NO 2 , —CH═CH 2 , —CN, —SO 3 H, —C(═O)OH, —C(═O)H, —C(═O)R, —C(═O)OR, and —NR 3 + , where R is alkyl.

11 . The method of claim 1 , wherein the primary or secondary β-hydroxy α-amino acid is selected from the group consisting of serine, threonine, and 3-phenyl serine.

12 . The method of claim 1 , further comprising reducing aldehyde by-products formed in the reaction.

13 . The method of claim 12 , comprising reducing the aldehyde by-products by contacting them with an alcohol dehydrogenase in the presence of NAD(P)H, wherein the alcohol dehydrogenase reduces at least a portion of the aldehyde by-products, and NAD(P)+ is generated.

14 . The method of claim 13 , wherein the alcohol dehydrogenase has an amino acid sequence at least 80% identical to SEQ. ID. NO: 5.

15 . The method of claim 13 , wherein the alcohol dehydrogenase has an amino acid sequence at least 90% identical to SEQ. ID. NO: 5.

16 . The method of claim 13 , wherein the alcohol dehydrogenase has an amino acid sequence at least 95% identical to SEQ. ID. NO: 5.

17 . The method of claim 13 , further comprising regenerating NAD(P)H from the NAD(P)+ by contacting the NAD(P)+ with a formate dehydrogenase or a glucose dehydrogenase.

18 . The method of claim 17 , wherein the formate dehydrogenase has an amino acid sequence at least 80% identical to SEQ. ID. NO: 7.

19 . The method of claim 17 , wherein the formate dehydrogenase has an amino acid sequence at least 90% identical to SEQ. ID. NO: 7.

20 . The method of claim 17 , wherein the formate dehydrogenase has an amino acid sequence at least 95% identical to SEQ. ID. NO: 7.

21 . The method of claim 13 , wherein the NAD(P)+ is contacted with a glucose dehydrogenase.

22 . The method of claim 21 , wherein the glucose dehydrogenase is derived from Bacillus megaterium or Pseudomonas sp.

23 . The method of claim 1 , wherein the PLP-dependent enzyme is an L-threonine transaldolase.

24 . The method of claim 23 , wherein the L-threonine transaldolase has an amino acid sequence at least 80% identical to SEQ. ID. NO: 3.

25 . The method of claim 23 , wherein the L-threonine transaldolase has an amino acid sequence at least 90% identical to SEQ. ID. NO: 3.

26 . The method of claim 23 , wherein the L-threonine transaldolase has an amino acid sequence at least 95% identical to SEQ. ID. NO: 3.