The present invention includes systems, methods and compositions for the generation of water-soluble cannabinoids in yeast, and other plant cell suspension cultures as well as novel water-soluble cannabinoid compounds. The present invention also includes compositions of matter that may contain one or more water-soluble cannabinoids.

The present disclosure provides a group of novel uridine diphosphate (UDP)-glycosyltransferases, which are bifunctional C-glycoside arabinosyltransferases and C-glycoside glucosyltransferases. The glycosyltransferases can specifically and efficiently catalyze C-glycoside arabinosylation and glucosylation of dihydrochalcone compounds or 2-hydroxyflavanone compounds, to generate C-glycoside dihydrochalcone or C-glycoside-2-hydroxyflavanone compounds; the C-glycoside-2-hydroxyflavanone compounds are further subjected to a dehydration reaction to form flavone-C-glycoside compounds. The present disclosure also provides an application of the novel UDP-glycosyltransferases to artificially constructed recombinant expression systems to generate C-glycoside dihydrochalcone and flavone-C-glycoside compounds by means of fermentation engineering.

The present disclosure provides a group of novel uridine diphosphate (UDP)-glycosyltransferases, which are bifunctional C-glycoside arabinosyltransferases and C-glycoside glucosyltransferases. The glycosyltransferases can specifically and efficiently catalyze C-glycoside arabinosylation and glucosylation of dihydrochalcone compounds or 2-hydroxyflavanone compounds, to generate C-glycoside dihydrochalcone or C-glycoside-2-hydroxyflavanone compounds; the C-glycoside-2-hydroxyflavanone compounds are further subjected to a dehydration reaction to form flavone-C-glycoside compounds. The present disclosure also provides an application of the novel UDP-glycosyltransferases to artificially constructed recombinant expression systems to generate C-glycoside dihydrochalcone and flavone-C-glycoside compounds by means of fermentation engineering.

Provided is a method for producing 2-pyrone-4,6-dicarboxylic acid (PDC) by culturing a microorganism that produces PDC. The present invention provides a method of producing PDC by culturing a microorganism that produces 2-pyrone-4,6-dicarboxylic acid (PDC), wherein the method comprises: dissolving the starting substance for production of PDC in a buffer solution that contains no alkali metals, and adjusting the pH of a culture solution with a buffer solution that contains no alkali metals.

Described herein are olivetolic acid cyclases (OAC) including non-natural variants capable of forming a 2,4-dihydroxy-6-alkylbenzoic acid from a 3,5,7-trioxoacyl-CoA or a 3,5,7-trioxocarboxylate substrate. In some examples, the non-natural OAC is capable of forming a 2,4-dihydroxy-6-alkylbenzoic acid from a 3,5,7-trioxoacyl-CoA or a 3,5,7-trioxocarboxylate substrate at a greater rate. In some examples, the non-natural OAC has a higher affinity for a 3,5,7-trioxoacyl-CoA or a 3,5,7-trioxocarboxylate substrate, as compared to the wild type OAC. The non-natural OAC can be used with olivetol synthase (OLS) to form the 2,4-dihydroxy-6-alkylbenzoic acid from malonyl-CoA and acyl-CoA through to a 3,5,7-trioxoacyl-CoAintermediate. The non-natural OAC (and OLS) can be expressed in an engineered cell having a pathway to form cannabinoids, which include CBGA, its analogs and derivatives. CBGA can be used for the preparation of cannabigerol (CBG), which can be used in therapeutic compositions.

Described herein are olivetolic acid cyclases (OAC) including non-natural variants capable of forming a 2,4-dihydroxy-6-alkylbenzoic acid from a 3,5,7-trioxoacyl-CoA or a 3,5,7-trioxocarboxylate substrate. In some examples, the non-natural OAC is capable of forming a 2,4-dihydroxy-6-alkylbenzoic acid from a 3,5,7-trioxoacyl-CoA or a 3,5,7-trioxocarboxylate substrate at a greater rate. In some examples, the non-natural OAC has a higher affinity for a 3,5,7-trioxoacyl-CoA or a 3,5,7-trioxocarboxylate substrate, as compared to the wild type OAC. The non-natural OAC can be used with olivetol synthase (OLS) to form the 2,4-dihydroxy-6-alkylbenzoic acid from malonyl-CoA and acyl-CoA through to a 3,5,7-trioxoacyl-CoAintermediate. The non-natural OAC (and OLS) can be expressed in an engineered cell having a pathway to form cannabinoids, which include CBGA, its analogs and derivatives. CBGA can be used for the preparation of cannabigerol (CBG), which can be used in therapeutic compositions.

An isolated or engineered polypeptide, a microorganism comprising a nucleic acid sequence encoded by the polypeptide, and a method for synthesizing a polyphenolic phytochemicals phosphate derivative using the polypeptide or the microorganism are provided. The polypeptide having a homologous protein sequence that is more than 70% identical to the polyphenol phosphorylation synthetase (SEQ ID NO: 13) comprises a conserved domain which sequentially comprises: an ATP-binding domain, which includes active catalytic sites of Lys27, Arg102, and Glu282; a substrate-binding domain, which includes a conserved motif of DDHHFYIDAMLDAKAR (SEQ ID NO: 14), and includes active catalytic sites ofAsp627, His629, and His630; and a phosphorylated histidine catalytic domain, which includes His795 based on SEQ ID NO: 13.

An isolated or engineered polypeptide, a microorganism comprising a nucleic acid sequence encoded by the polypeptide, and a method for synthesizing a polyphenolic phytochemicals phosphate derivative using the polypeptide or the microorganism are provided. The polypeptide having a homologous protein sequence that is more than 70% identical to the polyphenol phosphorylation synthetase (SEQ ID NO: 13) comprises a conserved domain which sequentially comprises: an ATP-binding domain, which includes active catalytic sites of Lys27, Arg102, and Glu282; a substrate-binding domain, which includes a conserved motif of DDHHFYIDAMLDAKAR (SEQ ID NO: 14), and includes active catalytic sites ofAsp627, His629, and His630; and a phosphorylated histidine catalytic domain, which includes His795 based on SEQ ID NO: 13.

Aspects of the disclosure relate to biosynthesis of cannabinoids and cannabinoid precursors in recombinant cells and in vitro.

Aspects of the disclosure relate to biosynthesis of cannabinoids and cannabinoid precursors in recombinant cells and in vitro.