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

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

The invention provides methods for making steviol glycosides, including RebM and glycosylation products that are minor products in stevia leaves, and provides enzymes, encoding polynucleotides, and host cells for use in these methods. The invention provides engineered enzymes and engineered host cells for producing steviol glycosylation products, such as RebM, at high purity and/or yield. The invention further provides methods of making products containing steviol glycosides, such as RebM, including food products, beverages, oral care products, sweeteners, and flavoring products.

The present invention relates to a method for increasing the degree of glycosylation of a composition comprising steviol glycosides, which method comprises:

a. contacting said composition comprising steviol glycosides with a recombinant microorganism, a cell free extract derived from such a microorganism or an enzyme preparation derived from either thereof; and b. thereby to increase the degree of glycosylation of the composition comprising steviol glycosides, wherein the recombinant microorganism comprises one or more nucleotide sequence(s) encoding: a polypeptide having ent-copalyl pyrophosphate synthase activity;a polypeptide having ent-Kaurene synthase activity; a polypeptide having ent-Kaurene oxidase activity; a polypeptide having kaurenoic acid 13-hydroxylase activity; and one or more polypeptides having UDP-glucosyltransferase activity whereby expression of the nucleotide sequence(s) confer(s) on the microorganism the ability to produce at least one steviol glycoside. The present invention also relates to a composition comprising steviol glycosides obtainable by such a method.

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

The present invention relates a variant polypeptide having kaurenoic acid 13-hydroxylase activity, which variant polypeptide comprises an amino acid sequence which, when aligned with a kaurenoic acid 13-hydroxylase comprising the sequence set out in SEQ ID NO: 1, comprises at least one substitution of an amino acid residue corresponding to any of amino acids 72, 85, 108, 127, 129, 141, 172, 195, 196, 197, 199, 226, 236, 291, 302, 361 or 464, said positions being defined with reference to SEQ ID NO: 1 and wherein the variant has one or more modified properties as compared with a reference polypeptide having kaurenoic acid 13-hydroxylase activity. A variant polypeptide of the invention may be used in a recombinant host for the production of steviol or a steviol glycoside.

Provided herein are compositions and methods for improved production of steviol glycosides in a host cell. In some embodiments, the host cell is genetically modified to comprise a heterologous nucleotide sequence encoding a Stevia rebaudiana kaurenoic acid hydroxylase. In some embodiments, the host cell further comprises one or more heterologous nucleotide sequence encoding further enzymes of a pathway capable of producing one or more steviol glycosides in the host cell. The compositions and methods described herein provide an efficient route for the heterologous production of steviol glycosides, including but not limited to, rebaudioside D and rebaudioside M.

The present invention relates a polypeptide having kaurenoic acid 13-hydroxylase activity, which polypeptide comprises an amino acid sequence which, when aligned with a kaurenoic acid 13-hydroxylase comprising the sequence set out in SEQ ID NO: 1 or SEQ ID NO: 3, comprises at least one substitution of an amino acid corresponding to any of amino acids at positions 136, 248, 336 or 403, said positions being defined with reference to SEQ ID NO: 1 or SEQ ID NO: 3 and wherein the polypeptide has one or more modified properties as compared with a reference polypeptide having kaurenoic acid 13-hydroxylase activity. A polypeptide of the invention may be used in a recombinant host for the production of steviol or a steviol glycoside.

The present invention provides engineered cells and methods for making high purity steviol glycosides, including RebM. In some aspects, the present invention provides host cells, such as bacterial cells (including but not limited to E. coli), that are engineered to overexpress and/or delete or inactivate one or more steviol glycoside transport proteins. The bacterial cells selectively export RebM, or other specific combination of steviol glycosides, out of the cell to increase productivity and reduce production costs associated with downstream purification. Non-target steviol glycosides are not transported to the extracellular medium in significant amounts.

The invention provides methods for making steviol glycosides, including RebM and glycosylation products that are minor products in stevia leaves, and provides enzymes, encoding polynucleotides, and host cells for use in these methods. The invention provides engineered enzymes and engineered host cells for producing steviol glycosylation products, such as RebM, at high purity and/or yield. The invention further provides methods of making products containing steviol glycosides, such as RebM, including food products, beverages, oral care products, sweeteners, and flavoring products.