The invention relates to the production of one or more terpenoids through microbial engineering, and relates to the manufacture of products comprising terpenoids.

The invention relates to a process for the preparation of colchicine 1 from colchicoside 2 which comprises enzymatic conversion of colchicoside 2 to 3-O-demethylcolchicine 3, wherein the enzyme used is a cellulase. According to another aspect of the invention, 3-O-demethylcolchicine 3 can be converted to colchicine 1 using an alkylating agent. The invention also relates to a process or enriching the colchicine 1 content of extracts from plants belonging to the Colchicaceae family containing colchicine 1, colchicoside 2 and 3-(9-demethyl colchicine 3, which comprises conversion by means of a colchicoside 2 cellulase to 3-O-demethylcolchicine 3, followed by conversion of 3-O-demethylcolchicine 3 to colchicine 1 using an alkylating agent.

A method of screening for a stevia plant with high rebaudioside M content ratio, that includes detecting from a test stevia plant the presence and/or absence of at least one of (1) and (2), and at least one of (3) to (7): (1) homozygous for the allele wherein position 290 of SEQ ID NO: 1 is T; (2) homozygous for the allele wherein position 33 of SEQ ID NO: 2 is A; (3) homozygous for the allele wherein position 44 of SEQ ID NO: 3 is T; (4) homozygous for the allele wherein position 40 of SEQ ID NO: 4 is T; (5) homozygous for the allele wherein position 48 of SEQ ID NO: 5 is C; (6) homozygous for the allele wherein positions 55-72 of SEQ ID NO: 6 are deleted; (7) homozygous for the allele wherein position 50 of SEQ ID NO: 7 is A.

The disclosure provides a recombinant cell capable of producing a steviol glycoside, wherein the cell comprises a nucleic acid coding for a variant of a parent polypeptide, wherein the variant has steviol glycoside transport mediating activity, wherein the variant comprises an amino acid sequence which, when aligned with the amino acid sequence of the parent polypeptide, comprises at least one modification of the amino acid residue corresponding to any of the amino acids in the amino acid sequence of the parent polypeptide, wherein the variant has an improved ability to produce rebaudioside M and optionally other steviol glycosides extracellularly if compared with the parent polytpeptide when measured under the same conditions.

The disclosure provides a recombinant cell capable of producing a steviol glycoside, wherein the cell comprises a nucleic acid coding for a variant of a parent polypeptide, wherein the variant has steviol glycoside transport mediating activity, wherein the variant comprises an amino acid sequence which, when aligned with the amino acid sequence of the parent polypeptide, comprises at least one modification of the amino acid residue corresponding to any of the amino acids in the amino acid sequence of the parent polypeptide, wherein the variant has an improved ability to produce rebaudioside M and optionally other steviol glycosides extracellularly if compared with the parent polytpeptide when measured under the same conditions.

The purpose of the present invention is to provide a steviol glycoside hexose transferase, and a method for producing a steviol glycoside that contains glucose and/or rhamnose using said enzyme. The present invention provides a steviol glycoside hexose transferase, and a method for producing a steviol glycoside that contains glucose and/or rhamnose using said enzyme. The present invention also provides a transformant into which a steviol glycoside hexose transferase gene has been introduced, and a method for preparing said transformant.

The purpose of the present invention is to provide a steviol glycoside hexose transferase, and a method for producing a steviol glycoside that contains glucose and/or rhamnose using said enzyme. The present invention provides a steviol glycoside hexose transferase, and a method for producing a steviol glycoside that contains glucose and/or rhamnose using said enzyme. The present invention also provides a transformant into which a steviol glycoside hexose transferase gene has been introduced, and a method for preparing said transformant.

Various aspects of the present invention relate to steviol glycoside extraction. A method of treating a Stevia extract that includes steviol glycosides and/or salts thereof, and malonated steviol glycosides and/or salts thereof, includes treating the Stevia extract at a pH greater than 10 to convert at least some of the malonated steviol glycosides and/or salts thereof to non-malonated steviol glycosides and/or salts thereof, to produce a modified Stevia extract. The method includes decreasing the pH of the modified Stevia extract to a pH of less than 9 to provide a pH-adjusted modified Stevia extract. The method also includes recovering the steviol glycosides and/or salts thereof from the pH-adjusted modified Stevia extract, the recovered steviol glycosides and/or salts thereof including the non-malonated steviol glycosides and/or salts thereof.

Various aspects of the present invention relate to steviol glycoside extraction. A method of treating a Stevia extract that includes steviol glycosides and/or salts thereof, and malonated steviol glycosides and/or salts thereof, includes treating the Stevia extract at a pH greater than 10 to convert at least some of the malonated steviol glycosides and/or salts thereof to non-malonated steviol glycosides and/or salts thereof, to produce a modified Stevia extract. The method includes decreasing the pH of the modified Stevia extract to a pH of less than 9 to provide a pH-adjusted modified Stevia extract. The method also includes recovering the steviol glycosides and/or salts thereof from the pH-adjusted modified Stevia extract, the recovered steviol glycosides and/or salts thereof including the non-malonated steviol glycosides and/or salts thereof.

The invention relates to recombinant microorganisms and methods for producing steviol glycosides and steviol glycoside precursors.