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

Described herein are devices and methods for increasing the production of steviol glycosides, which have industrial and economic value. The steviol glycosides produced by the devices and methods disclosed herein do not require the ultra purification that is common in conventional or commercial methods and do not have a bitter aftertaste, making them better suited as flavor-enhancing additives to food, pharmaceutical, and nutritional supplement products.

The disclosure discloses a method for efficient biosynthesis of Reb D by glycosyltransferase, belonging to the field of biocatalytic synthesis. According to the disclosure, a glycosyltransferase having an activity to catalyze synthesis of Reb D from Reb A is obtained, and a mutant YojK-I241T/G327N with high catalytic activity is obtained through directed evolution. The glycosyltransferase mutant YojK-I241T/G327N and a sucrose synthase AtSuSy derived from Arabidopsis thaliana are used for constructing a coupling reaction to realize efficient catalytic synthesis of Reb D with Reb A as a substrate. The reaction is carried out by using 19.32 g/L (20 mmol/L) of Reb A as the substrate for 15 h to efficiently synthesize 20.59 g/L of Reb D, and the yield of Reb D reaches 91.29%, which provides an efficient and green new pathway for production of Reb D.

The disclosure discloses a method for efficient biosynthesis of Reb D by glycosyltransferase, belonging to the field of biocatalytic synthesis. According to the disclosure, a glycosyltransferase having an activity to catalyze synthesis of Reb D from Reb A is obtained, and a mutant YojK-I241T/G327N with high catalytic activity is obtained through directed evolution. The glycosyltransferase mutant YojK-I241T/G327N and a sucrose synthase AtSuSy derived from Arabidopsis thaliana are used for constructing a coupling reaction to realize efficient catalytic synthesis of Reb D with Reb A as a substrate. The reaction is carried out by using 19.32 g/L (20 mmol/L) of Reb A as the substrate for 15 h to efficiently synthesize 20.59 g/L of Reb D, and the yield of Reb D reaches 91.29%, which provides an efficient and green new pathway for production of Reb D.

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.

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.

The present invention relates to a group of glycosyltransferase, and an application thereof. Specifically, provided is using glycosyltransferase GT29-32, GT29-33, GT29-34, GT29-4, GT29-5, GT29-7, GT29-9, GT29-11, GT29-13, GT29-17, GT29-18, GT29-19, GT29-20, GT29-21, GT29-22, GT29-23, GT29-24, GT29-25, GT29-36, GT29-37, GT29-42, GT29-43, GT29-45, GT29-46, PNUGT29-1, PNUGT29-2, PNUGT29-3, PNUGT29-4, PNUGT29-5, PNUGT29-6, PNUGT29-7, PNUGT29-8, PNUGT29-9, PNUGT29-14, and PNUGT29-15, as well as derived polypeptides thereof to catalyze the first glycosyl at position C-20, the first glycosyl at position C-6, and the first glycosyl at position C-3 of a tetracyclic triterpene compound substrate to elongate a carbohydrate chain, thereby obtaining a catalytic reaction of ginsenoside products such as ginsenoside Rg3, ginsenoside Rd, ginsenoside Rb1, ginsenoside Rb3, saponin DMGG, saponin DMGX, gypenoside LXXV, gypenoside XVII, gypenoside XIII, gypenoside IX, notoginsenoside U, and notoginsenoside R1, notoginsenoside R2, notoginsenoside R3, 3-O-β-(D-xylopyranosyl)-β-(D-glucopyranosyl)-PPD, 3-O-β-(D-xylopyranosyl)-β-(D-glucopyranosyl)-CK, 20-O-Glucosylginsenoside Rf, and Ginsenoside F3. Glycosyltransferase in the present invention can further be applied to construction of artificially synthesized ginsenoside, novel ginsenoside, and derivatives thereof.

The present invention relates to a group of glycosyltransferase, and an application thereof. Specifically, provided is using glycosyltransferase GT29-32, GT29-33, GT29-34, GT29-4, GT29-5, GT29-7, GT29-9, GT29-11, GT29-13, GT29-17, GT29-18, GT29-19, GT29-20, GT29-21, GT29-22, GT29-23, GT29-24, GT29-25, GT29-36, GT29-37, GT29-42, GT29-43, GT29-45, GT29-46, PNUGT29-1, PNUGT29-2, PNUGT29-3, PNUGT29-4, PNUGT29-5, PNUGT29-6, PNUGT29-7, PNUGT29-8, PNUGT29-9, PNUGT29-14, and PNUGT29-15, as well as derived polypeptides thereof to catalyze the first glycosyl at position C-20, the first glycosyl at position C-6, and the first glycosyl at position C-3 of a tetracyclic triterpene compound substrate to elongate a carbohydrate chain, thereby obtaining a catalytic reaction of ginsenoside products such as ginsenoside Rg3, ginsenoside Rd, ginsenoside Rb1, ginsenoside Rb3, saponin DMGG, saponin DMGX, gypenoside LXXV, gypenoside XVII, gypenoside XIII, gypenoside IX, notoginsenoside U, and notoginsenoside R1, notoginsenoside R2, notoginsenoside R3, 3-O-β-(D-xylopyranosyl)-β-(D-glucopyranosyl)-PPD, 3-O-β-(D-xylopyranosyl)-β-(D-glucopyranosyl)-CK, 20-O-Glucosylginsenoside Rf, and Ginsenoside F3. Glycosyltransferase in the present invention can further be applied to construction of artificially synthesized ginsenoside, novel ginsenoside, and derivatives thereof.