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 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 present invention relates to methods for producing oxygenated terpenoids. Polynucleotides, derivative enzymes, and host cells for use in such methods are also provided.

The present disclosure relates to methods for producing oxygenated terpenoids, and preparation of compositions and formulations thereof. Polynucleotides, derivative enzymes, and host cells for use in such methods are also provided.

The present invention relates a variant polypeptide having geranylgeranyl pyrophosphate synthase activity, which variant polypeptide comprises an amino acid sequence which, when aligned with a geranylgeranyl pyrophosphate synthase 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 at positions 92, 100 or 235 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 geranylgeranyl pyrophosphate synthase activity. A variant polypeptide of the invention may be used in a recombinant host for the production of steviol or a steviol glycoside.

The present invention relates to a method for the production of a diterpene or a glycosylated diterpene, which method comprises: a. fermenting a recombinant microorganism of the genus Yarrowia in a suitable fermentation medium at a temperature of about 29 C. or higher, wherein the 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; and a polypeptide having kaurenoic acid 13-hydroxylase activity and whereby expression of the nucleotide sequence(s) confer(s) on the microorganism the ability to produce at least steviol; and b. recovering the diterpene or glycosylated diterpene.

The present invention relates to a method for the production of a diterpene or a glycosylated diterpene, which method comprises: a. fermenting a recombinant microorganism of the genus Yarrowia in a suitable fermentation medium at a temperature of about 29 C. or higher, wherein the 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; and a polypeptide having kaurenoic acid 13-hydroxylase activity and whereby expression of the nucleotide sequence(s) confer(s) on the microorganism the ability to produce at least steviol; and b. recovering the diterpene or glycosylated diterpene.

The present disclosure in various aspects provides engineered enzymes and encoding polynucleotides, as well as host cells and methods for making rotundone and other terpenoids. For example, in various aspects, the invention provides engineered ?-Guaiene Synthase (?GS) and Guaiene Oxidase (GO) enzymes that increase biosynthesis of rotundone from farnesyl diphosphate, and in certain embodiments substantially reduce biosynthesis of side products such as ?-Bulnesene or oxygenated side products. In still other aspects, the invention provides engineered terpene synthase enzymes for directing biosynthesis toward a desired product, to thereby improve product profiles and/or product titers from terpene synthase reactions.

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.