Microbial production of mogrol and mogrosides
The present invention provides host cells and methods for making mogrol glycosides, including Mogroside V (Mog.V), Mogroside VI (Mog.VI), Iso-Mogroside V (Isomog.V), siamenoside, and glycosylation products that are minor products in Siraitia grosvenorii . The invention provides engineered enzymes and engineered host cells for producing mogrol glycosylation products, such as Mog.V, Mog.VI, and Isomog.V, at high purity and/or yield. The present technology further provides methods of making products containing mogrol glycosides, such as Mog.V, Mog.VI, and Isomog.V, including food products, beverages, oral care products, sweeteners, and flavoring products.
1 . A method for making mogrol or mogroside, comprising:
providing a recombinant microbial host cell expressing a heterologous enzyme pathway catalyzing the conversion of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to mogrol or mogroside, the heterologous enzyme pathway comprising:
a farnesyl diphosphate synthase (FPPS),
a squalene synthase (SQS),
a squalene epoxidase (SQE) catalyzing the formation of 2,3;22,23-dioxidosqualene from squalene,
a triterpene cyclase (TTC) catalyzing the formation of 24,25-epoxycucurbitadienol from 2,3;22,23-dioxidosqualene,
an epoxide hydrolase (EPH) catalyzing the formation of 24,25-dihydroxycucurbitadienol from 24,25-epoxycucurbitadienol, and
a cytochrome P450 enzyme catalyzing the formation of mogrol from 24,25-dihydroxycucurbitadienol;
wherein the cytochrome P450 enzyme comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 171; and
culturing the host cell under conditions for producing the mogrol or mogroside.
2 . The method of claim 1 , wherein the squalene epoxidase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 39.
3 . The method of claim 1 , wherein the SQS comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOS: 2 to 16, 166, and 167.
4 . The method of claim 1 , wherein the TTC comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOS: 40, 191, 192, and 193.
5 . The method of claim 4 , wherein the TTC comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 40.
6 . The method of claim 1 , wherein the heterologous enzyme pathway comprises at least one TTC that comprises an amino acid sequence that is at least 90% identical to one of SEQ ID NO: 191, SEQ ID NO: 192, and SEQ ID NO: 193.
7 . The method of claim 1 , wherein the EPH comprises an amino acid sequence that is at least 90% identical to one of: SEQ ID NOS: 189, 58, 184, 185, 187, 188, 190, and 212.
8 . The method of claim 1 , wherein the cytochrome P450 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 171.
9 . The method of claim 1 , wherein the heterologous enzyme pathway further comprises one or more uridine diphosphate-dependent glycosyltransferase (UGT) enzymes, thereby producing one or more mogrol glycosides.
10 . The method of claim 9 , wherein the one or more mogrol glycosides are selected from Mog.II-E, Mog.III, Mog.III-A1, Mog.III-A2, Mog.III, Mog.IV, Mog.IV-A, siamenoside, Mog.V, and Mog.VI.
11 . The method of claim 9 , wherein at least one uridine diphosphate dependent glycosyltransferase (UGT) enzyme comprises an amino acid sequence having at least 90% sequence identity to one of SEQ ID NO: 164, 165, 138, 204 to 211, and 213 to 218.
12 . The method of claim 11 , wherein at least one UGT enzyme further comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 146.
13 . The method of claim 11 , wherein at least one UGT enzyme further comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 202.
14 . The method of claim 11 , wherein the microbial host cell expresses at least three UGT enzymes: a first UGT enzyme catalyzing primary glycosylation at the C24 hydroxyl of mogrol, a second UGT enzyme catalyzing primary glycosylation at the C3 hydroxyl of mogrol, and a third UGT enzyme catalyzing one or more branching glycosylation reactions.
15 . The method of claim 1 , wherein the microbial host cell is a bacterium selected from Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Rhodobacter capsulatus, Rhodobacter sphaeroides, Zymomonas mobilis, Vibrio natriegens , or Pseudomonas putida ; or is a yeast selected from Saccharomyces cerevisiae, Pichia pastoris , and Yarrowia lipolytica.
16 . The method of claim 1 , wherein the mogrol glycoside products are recovered from the extracellular media.
17 . A method for making a product comprising a mogrol glycoside, comprising:
producing a mogrol glycoside in accordance with claim 1 , and incorporating the mogrol glycoside into a product.
18 . A microbial host cell expressing a heterologous enzyme pathway catalyzing the conversion of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to mogrol or mogroside, the heterologous enzyme pathway comprising:
a farnesyl diphosphate synthase (FPPS),
a squalene synthase (SQS),
a squalene epoxidase (SQE) catalyzing the formation of 2,3;22,23-dioxidosqualene from squalene,
a triterpene cyclase (TTC) catalyzing the formation of 24,25-epoxycucurbitadienol from 2,3;22,23-dioxidosqualene,
an epoxide hydrolase (EPH) catalyzing the formation of 24,25-dihydroxycucurbitadienol from 24,25-epoxycucurbitadienol, and
a cytochrome P450 enzyme catalyzing the formation of mogrol from 24,25-dihydroxycucurbitadienol;
wherein the cytochrome P450 enzyme comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 171.
19 . The method of claim 1 , wherein the microbial host cell further comprises a cytochrome P450 reductase enzyme.
20 . The method of claim 18 , wherein the microbial host cell further comprises a cytochrome P450 reductase enzyme.