Disclosed are steviol glycosides referred to as rebaudioside V and rebaudioside W. Also disclosed are methods for producing rebaudioside M (Reb M), rebausoside G (Reb G), rebaudioside KA (Reb KA), rebaudioside V (Reb V) and rebaudioside (Reb W).

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 Setaria italica UDP-glycosyltransferase 40087 or its variant UDP-glycosyltransferase. In some embodiments, the host cell is genetically modified to comprise a heterologous nucleotide sequence encoding a UDP-glycosyltransferase sr.UGT_9252778, Bd_UGT10850, and/or Ob_UGT91B1 like. In some embodiments, the host cell further comprises one or more heterologous nucleotide sequence encoding further enzymes of a pathway capable of producing 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.

Provided is a method for preparing Rebaudioside J using an enzymatic method, comprising using rebaudioside A as a substrate, and making the substrate, in the presence of a glycosyl donor, react under the catalysis of a UDP-glycosyltransferase-containing recombinant cell and/or UDP-glycosyltransferase prepared therefrom to generate Rebaudioside J.

The present invention relates, at least in part, to the production of steviol glycoside rebaudioside I through the use of variant UGT enzymes having activity to transfer a glucosyl group from UDP-glucose to rebaudioside A to produce rebaudioside I.

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 recombinant host comprising a recombinant nucleic acid sequence encoding a polypeptide having at least about: a. 85% identity to the amino acid sequence set forth in SEQ ID NO: 1; b. 85% identity to the amino acid sequence set forth in SEQ ID NO: 3; c. 85% identity to the amino acid sequence set forth in SEQ ID NO: 6; d. 85% identity to the amino acid sequence set forth in SEQ ID NO: 9; e. 85% identity to the amino acid sequence set forth in SEQ ID NO: 11; f. 85% identity to the amino acid sequence set forth in SEQ ID NO: 14; g. 85% identity to the amino acid sequence set forth in SEQ ID NO: 17; h. 85% identity to the amino acid sequence set forth in SEQ ID NO: 20; i. 85% identity to the amino acid sequence set forth in SEQ ID NO: 22; or j. 85% identity to the amino acid sequence set forth in SEQ ID NO: 25.

Provided is a method for preparing rebaudioside N using an enzymatic method, comprising using rebaudioside A or rebaudioside J as a substrate, and making the substrate, in the presence of a glycosyl donor, react under the catalysis of a UDP-glycosyl-transferase and/or a UDP-glycosyltransferase-containing recombinant cell to generate rebaudioside N.

The present invention relates, in some aspects, to the production of steviol glycoside rebaudioside D4 through the use of rebaudioside E. In some aspects, the invention relates to mutant CP1 enzymes, mutant HV1 enzymes as well as host cells and methods utilizing such enzymes, such as to produce rebaudioside D4.

The present disclosure relates to the production of steviol glycosides rebaudioside J and rebaudioside N through the use of rebaudioside A as a substrate and a biosynthetic pathway involving various 1,2 RhaT-rhamnosyltransferases.

The present invention relates, at least in part, to the production of steviol glycoside rebaudioside I through the use of variant UGT enzymes having activity to transfer a glucosyl group from UDP-glucose to rebaudioside A to produce rebaudioside I.