Method for the biocatalytic cyclization of terpenes and cyclase mutants employable therein

A method for enzymatic or biocatalytic conversions of compounds of general formula IV

• in which
• “a”, “b”, “c” and “d”, in each case independently of one another, represent a single or double C—C bond, with the proviso that cumulative double bonds are excluded;
• and with the following provisos:
• R1 is defined as:
• (1) when “a” is a double bond:
• R1 is selected from the group consisting of:
• oxo (═O), and
• CH—(CH2)n—Z,
• in which n is 0, 1 or 2 and
• Z is OH, CHO;
• C(O)C1-C4-alkyl;
• COOH, C(CH2)—CH═CH2;
• C(OH)(CH3)—CH═CH2;
• C(CH3)═CH—CH═CH2;
• or a radical of the formula C(CH3)═CH—CH2Y
• in which
• Y is OH, CH2OH, COOH, or CH2C(O)CH3;
• or
• (2) when “a” is a single bond:
• R1 is selected from the group consisting of:
• CH3;
• CHO;
• CH2CH2OH;
• CH═CH2;
• CH2C(O)OH;
• CH2CHO;
• and C3H6CH(CH3)CHO;
• and, when “a” is a double bond, it has E or Z configuration;
• R2 and R3 are defined as:
• (1) when “a” and “b” are each a single bond:
• R2 and R3 independently of one another are H, C1-C4-alkyl, or OH, or R2 and R3 together are a methylene (═CH2) or oxo (═O) group;
• or
• (2) when “a” or “b” is a double bond, one of the radicals R2 and R3 is absent and the other of the two radicals is H, C1-C4-alkyl, or OH;
• R4 is H or hydroxy-C1-C4-alkyl;
• R5 and R6 are defined as:
• (1) when “c” is a single bond:
• R5 and R6 are each H, or R5 and R6 together are an oxo (═O) group;
• or
• (2) when “c” is a double bond, one of the radicals R5 and R6 is absent and the other of the two radicals is H;
• R7, R8, and R9 are defined as:
• (1) when “d” is a single bond:
• two of the radicals R7, R8 and R9 in each case independently of one another are H or C1-C4-alkyl, and the other of the radicals is OH;
• or
• (2) when “d” is a double bond, one of the radicals R7, R8 and R9 is absent and the other of the two radicals in each case independently of one another are H or C1-C4-alkyl;
• R10 is H or hydroxy-C1-C6-alkyl or mono- or polyunsaturated C2-C6-alkenyl;
• comprising reacting a compound of the formula IV in stereoisomerically pure form, or a stereoisomer mixture thereof:
• a) using an enzyme mutant with cyclase activity which is a mutant of a wild-type enzyme comprising the amino acid sequence of SEQ ID NO:
• 2 with a mutation at a position corresponding to position F486 of the amino acid sequence of SEQ ID NO:
• 2,
• wherein up to 10% of the amino acid residues in said enzyme mutant are altered relative to SEQ ID NO:
• 2 by deletion, insertion, substitution, addition, inversion, or a combination thereof;
• or
• b) in the presence of a microorganism expressing said enzyme mutant, wherein the microorganism comprises:
• i) at least one nucleic acid sequence coding for said enzyme mutant;
• ii) at least one expression cassette comprising said at least one nucleic acid sequence;
• or
• iii) at least one vector comprising, under the control of at least one regulatory element, said at least one nucleic acid sequence or said at least one expression cassette.