Provided herein are genetically modified host cells, compositions, and methods for improved production of steviol glycosides. In some embodiments, the host cell is genetically modified to comprise a heterologous nucleic acid expression cassette that expresses an ABC-transporter capable of transporting steviol glycosides to the extracellular space or to the luminal space of an intracellular organelle. 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 host cells, 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.

Recombinant microorganisms, plants, and plant cells are disclosed that have been engineered to express novel recombinant genes encoding steviol biosynthetic enzymes and UDP-glycosyltransferases (UGTs). Such microorganisms, plants, or plant cells can produce steviol or steviol glycosides, e.g., rubusoside or Rebaudioside A, which can be used as natural sweeteners in food products and dietary supplements.

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 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.

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

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.

Recombinant microorganisms, plants, and plant cells are disclosed that have been engineered to express novel recombinant genes encoding steviol biosynthetic enzymes and UDP-glycosyltransferases (UGTs). Such microorganisms plants, or plant cells can produce steviol or steviol glycosides, e.g., rubusoside or Rebaudioside A, which can be used as natural sweeteners in food products and dietary supplements.

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