	US 12,486,499 B2
	Biocatalytic method for the controlled degradation of terpene compounds
Michel Schalk, Satigny (CH); Fabienne Deguerry, Satigny (CH); Daniel Solis Escalante, Satigny (CH); Pauline Anziani, Satigny (CH); and Sabine Beccucci, Satigny (CH)
Assigned to FIRMENICH SA, Satigny (CH)
Appl. No. 17/596,878
Filed by Firmenich SA, Satigny (CH)
PCT Filed Jul. 8, 2020, PCT No. PCT/EP2020/069217 § 371(c)(1), (2) Date Dec. 20, 2021, PCT Pub. No. WO2021/005097, PCT Pub. Date Jan. 14, 2021.
Claims priority of application No. 19000332 (EP), filed on Jul. 10, 2019; and application No. 19208951 (EP), filed on Nov. 13, 2019.
Prior Publication US 2023/0183761 A1, Jun. 15, 2023
Int. Cl. C12P 7/62 (2022.01); C12N 9/02 (2006.01); C12N 9/18 (2006.01)

CPC C12N 9/18 (2013.01) [C12N 9/0069 (2013.01); C12P 7/62 (2013.01); C12Y 113/00 (2013.01)]

20 Claims

1. A biocatalytic method of preparing a compound of formula IV

wherein

R¹represents H or lower alkyl;

R²represents H, a linear or branched, saturated or unsaturated, optionally substituted hydrocarbyl residue, or a residue Cyc-A-

wherein

Cyc represents an optionally substituted, saturated or unsaturated, mono- or polycyclic hydrocarbyl residue, and

A represents a chemical bond or an optionally substituted, straight chain or branched alkylene bridge;

and

R³represent independently of each other H or lower alkyl;

comprising the steps of

(1) contacting a compound of formula V

wherein

R¹, R²and R³are as defined above; and

R⁴represents H or lower alkyl,

R⁵represents H or lower alkyl,

and wherein said compound of formula V is present in stereoisomerically essentially pure form or as a mixture of stereoisomers,

with a polypeptide having enal-cleaving activity, and optionally

(2) isolating the compound of formula IV as obtained in step (1),

wherein said compound of formula IV is provided in stereoisomerically pure form, or as a mixture of stereoisomers;

wherein said polypeptide having enal-cleaving activity is a polypeptide comprising an amino acid sequence selected from the group consisting of:

a) SCH94-3944 set forth in SEQ ID NO: 34;

b) SCH80-05241 set forth in SEQ ID NO: 38;

c) Pdigit7033 set forth in SEQ ID NO: 42;

d) PitalDUF4334-1 set forth in SEQ ID NO: 46;

e) AspWeDUF4334 set forth in SEQ ID NO: 49;

f) RhoagDUF4334-2 set forth in SEQ ID NO: 53;

g) RhoagDUF4334-3 set forth in SEQ ID NO: 56;

h) RhoagDUF4334-4 set forth in SEQ ID NO: 59;

i) CnecaDUF4334 set forth in SEQ ID NO: 62;

j) Rins-DUF4334 set forth in SEQ ID NO: 69;

k) CgatDUF4334 set forth in SEQ ID NO: 72;

l) GclavDUF4334 set forth in SEQ ID NO: 75;

m) TcurvaDUF4334 set forth in SEQ ID NO:81;

n) PprotDUF4334 set forth in SEQ ID NO: 87; and

o) polypeptides comprising an amino acid sequence that has at least 90% sequence identity to any one of the amino acid sequences of a) to n) and retaining said enzymatic activity of degrading a terpene precursor of formula (V).