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 polypeptide 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 polypeptide when measured under the same conditions.

Microorganisms are prolific producers of natural products, a group of molecules that make up the majority of drugs approved by the FDA in the past 35 years. After decades of mining, the low-hanging fruit has been picked and so discovery of drug-like molecules from microorganisms has come to a near-halt. The reason for this lack of productivity is that most biosynthetic pathways that give rise to natural products are not active under typical laboratory growth conditions. These so-called ‘cryptic’ or ‘silent’ pathways are a major source of new bioactive molecules and methods that reliably activate them could have a profound impact on drug discovery. Disclosed herein is a rapid genetics-free method for eliciting and detecting cryptic metabolites using an imaging mass spectrometry-based approach. An organism of choice is challenged with elicitors from a small molecule library. The molecules elicited are then imaged by mass spec, which allows for rapid identification of cryptic metabolites. These are then isolated and characterized. Employing the disclosed approach activated production of cryptic glycopeptides from an actinomycete bacterium. The molecules that result, the keratinimicins and keratinicyclins, are metabolites with important structural features. At least two of these, keratinimicins B and C, are highly bioactive against several pathogenic strains. This approach will allow for rapid activation and identification of cryptic metabolites from diverse microorganisms in the future.

Microorganisms are prolific producers of natural products, a group of molecules that make up the majority of drugs approved by the FDA in the past 35 years. After decades of mining, the low-hanging fruit has been picked and so discovery of drug-like molecules from microorganisms has come to a near-halt. The reason for this lack of productivity is that most biosynthetic pathways that give rise to natural products are not active under typical laboratory growth conditions. These so-called ‘cryptic’ or ‘silent’ pathways are a major source of new bioactive molecules and methods that reliably activate them could have a profound impact on drug discovery. Disclosed herein is a rapid genetics-free method for eliciting and detecting cryptic metabolites using an imaging mass spectrometry-based approach. An organism of choice is challenged with elicitors from a small molecule library. The molecules elicited are then imaged by mass spec, which allows for rapid identification of cryptic metabolites. These are then isolated and characterized. Employing the disclosed approach activated production of cryptic glycopeptides from an actinomycete bacterium. The molecules that result, the keratinimicins and keratinicyclins, are metabolites with important structural features. At least two of these, keratinimicins B and C, are highly bioactive against several pathogenic strains. This approach will allow for rapid activation and identification of cryptic metabolites from diverse microorganisms in the future.

Methods of preparing novel steviol glycosides are described herein. The methods utilize biocatalysts for converting a starting steviol glycoside to a target steviol glycoside. Compositions and consumables comprising said novel steviol glycosides as well as methods of purifying and using said novel steviol glycosides, are also provided.

Methods of preparing novel steviol glycosides are described herein. The methods utilize biocatalysts for converting a starting steviol glycoside to a target steviol glycoside. Compositions and consumables comprising said novel steviol glycosides as well as methods of purifying and using said novel steviol glycosides, are also provided.

Methods of preparing highly purified steviol glycosides, particularly rebaudiosides A, D and M are described. The methods include utilizing recombinant microorganisms for converting various staring compositions to target steviol glycosides. In addition, novel steviol glycosides reb D2, reb M2, and reb I are disclosed, as are methods of preparing the same. The highly purified rebaudiosides are useful as non-caloric sweetener in edible and chewable compositions such as any beverages, confectioneries, bakery products, cookies, and chewing gums.

Methods of preparing highly purified steviol glycosides, particularly rebaudiosides A, D and M are described. The methods include utilizing recombinant microorganisms for converting various staring compositions to target steviol glycosides. In addition, novel steviol glycosides reb D2, reb M2, and reb I are disclosed, as are methods of preparing the same. The highly purified rebaudiosides are useful as non-caloric sweetener in edible and chewable compositions such as any beverages, confectioneries, bakery products, cookies, and chewing gums.

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.