Enzymes and methods for fermentative production of monoterpene esters

An alcohol acyl transferase, comprising an amino acid sequence selected from the group consisting of:

• a) an amino acid sequence as shown in any one of the sequences of SEQ ID NO:
• 2, SEQ ID NO:
• 15 or SEQ ID NO:
• 16;
• b) an amino acid sequence having alcohol acyl transferase activity with i) at least 89% sequence identity at the amino acid level with SEQ ID NO:
• 2, or ii) having at least 60% sequence identity with SEQ ID NO:
• 15 or SEQ ID NO:
• 16, having alcohol acyl transferase activity;
• and
• c) a fragment of the amino acid sequence of a) or b),
• wherein the alcohol acyl transferase is capable of esterifying a tertiary monoterpene alcohol such that at least 30% by mass of said tertiary monoterpene alcohol is esterified.

A method for preparing a monoterpene ester, comprising esterifying a monoterpene alcohol to a monoterpene ester, in the presence of

• (i) an alcohol acyl transferase
• a) having an amino acid sequence as shown in any one of the sequences of SEQ ID NO:
• 2, SEQ ID NO:
• 15 or SEQ ID NO:
• 16;
• or
• b) having an amino acid sequence with at least 60% sequence identity at the amino acid level with SEQ ID NO:
• 2, SEQ ID NO:
• 15 or SEQ ID NO:
• 16, having alcohol acyl transferase activity;
• or
• c) a fragment of the amino acid sequence of a) or b), having alcohol acyl transferase activity;
• or
• (ii) an alcohol acyl transferase comprising an amino acid sequence as shown in database accession number XP 006493396 (SEQ ID NO:
• 13) or UNIPROTKB-A0A2H5PUP1 (SEQ ID NO:
• 14), or an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence as shown in database accession number XP 006493396 (SEQ ID NO:
• 13) or UNIPROTKB-A0A2H5PUP1 (SEQ ID NO:
• 14), wherein the alcohol acyl transferase is capable of esterifying a monoterpene alcohol to a monoterpene ester, or
• (iii) an alcohol acyl transferase of (i) or (ii), wherein the amino acid corresponding to the amino acid at position 371 and 372 of SEQ ID NO:
• 2 is not Tryptophan, or
• (iv) an alcohol acyl transferase of (i) or (ii) further comprising at the positions corresponding to the positions of SEQ ID NO:
• 2 indicated in Table 1 any of those amino acids listed in Table 1 for those positions, or at the positions corresponding to the positions of SEQ ID NO:
• 2 indicated in Table 2 those amino acids listed in Table 2, wherein the alcohol acyl transferase is capable of esterifying a monoterpene alcohol to a monoterpene ester;
• (v) any alcohol acyl transferase of i) to iv) that is capable of esterifying a monoterpene alcohol such that at least 30% by mass of said monoterpene alcohol is esterified;
• or
• (vi) any alcohol acyl transferase of i) to v) that is capable of esterifying a monoterpene alcohol such that at least 50 μg of monoterpene ester per minute and per gram of alcohol acyl transferase is produced, at 30° C. and at a pH in the range of 6.0 to 8.5, and under conditions where the substrates are not limiting.

A method for preparing linalyl acetate, comprising esterifying linalool to linalyl acetate, in the presence of

• (i) an alcohol acyl transferase
• a) having an amino acid sequence as shown in any one of the sequences of SEQ ID NO:
• 2, SEQ ID NO:
• 15 or SEQ ID NO:
• 16;
• or
• b) having an amino acid sequence with at least 60% sequence identity at the amino acid level with SEQ ID NO:
• 2, SEQ ID NO:
• 15 or SEQ ID NO:
• 16, having linalool acyl transferase activity;
• or
• c) a fragment of the amino acid sequence of a) or b), having linalool acyl transferase activity;
• or
• (ii) an alcohol acyl transferase comprising an amino acid sequence as shown in database accession number XP 006493396 (SEQ ID NO:
• 13) or UNIPROTKB-A0A2H5PUP1 (SEQ ID NO:
• 14), or an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence as shown in database accession number XP 006493396 (SEQ ID NO:
• 13) or UNIPROTKB-A0A2H5PUP1 (SEQ ID NO:
• 14), wherein the alcohol acyl transferase is capable of esterifying linalool to linalyl acetate, or
• (iii) an alcohol acyl transferase of (i) or (ii), wherein the amino acid corresponding to the amino acid at position 371 and 372 of SEQ ID NO:
• 2 is not Tryptophan, or
• (iv) an alcohol acyl transferase of (i) or (ii) further comprising at the positions corresponding to the positions of SEQ ID NO:
• 2 indicated in Table 1 any of those amino acids listed in Table 1 for those positions, or at the positions corresponding to the positions of SEQ ID NO:
• 2 indicated in Table 2 those amino acids listed in Table 2, wherein the alcohol acyl transferase is capable of esterifying linalool to linalyl acetate;
• or
• (v) any alcohol acyl transferase of i) to iv) that is capable of esterifying linalool such that at least 30% by mass of said linalool is esterified;
• or
• (vi) any alcohol acyl transferase of i) to v) that is capable of esterifying linalool such that at least 50 μg of monoterpene ester per minute and per gram of alcohol acyl transferase is produced, at 30° C. and at a pH in the range of 6.0 to 8.5, and under conditions where the substrates are not limiting.