US 12,077,801 B2
Regioselective hydroxylation of isophorone
Werner Bonrath, Kaiseraugst (CH); Martin Schürmann, Geleen (NL); Michele Tavanti, Manchester (GB); Fabio Parmeggiani, Manchester (GB); Nicholas Turner, Manchester (GB); Andrea Mattevi, Pavia (IT); and Jose Ruben Casstellanos, Pavia (IT)
Assigned to DSM IP ASSETS B.V., Heerlen (NL)
Appl. No. 16/500,497
Filed by DSM IP Assets B.V., Heerlen (NL)
PCT Filed Apr. 6, 2018, PCT No. PCT/EP2018/058897
§ 371(c)(1), (2) Date Oct. 3, 2019,
PCT Pub. No. WO2018/185304, PCT Pub. Date Oct. 11, 2018.
Claims priority of application No. 17165528 (EP), filed on Apr. 7, 2017.
Prior Publication US 2021/0108234 A1, Apr. 15, 2021
Int. Cl. C12P 7/26 (2006.01); C12N 9/02 (2006.01)
CPC C12P 7/26 (2013.01) [C12N 9/0081 (2013.01); C12Y 114/15006 (2013.01)] 12 Claims
OG exemplary drawing
 
1. A biocatalytic process for the conversion of α-isophorone into ketoisophorone (KIP) comprising the enzymatic steps of:
(a) converting α-isophorone into 4-hydroxy-α-isophorone (HIP) via catalytic action of a P450 monooxygenase wherein
1) at least 80% of α-isophorone is converted into HIP;
2) the P450 monooxygenase comprises
i. a first polypeptide having at least 90% identity to SEQ ID NO:1 or
ii. a second polypeptide having at least 62% identity to SEQ ID NO:3, said second polypeptide comprising an F at position 97 according to SEQ ID NO:3 and one or more substitutions at positions 88, 245, and 248 according to SEQ ID NO:3, wherein said one or more substitutions comprise F88W, L245X, and V248X, wherein “X” is any amino acid; and wherein the total turnover number of the second polypeptide is increased by at least 2-fold compared to a reference P450 monooxygenase according to SEQ ID NO: 3; and
(b) converting the HIP from (a) into KIP via catalytic action of an enzyme having alcohol dehydrogenase or carbonyl reductase activity, wherein at least 40% of HIP is converted into KIP.