Biosynthesis of Mogroside

Abstract

The present application describes enzymes (e.g., cucurbitacinadienol synthase (CDS), UDP-glycosyltransferase (UGT), C11 hydroxylase, epoxidase (EPH), squalene epoxidase, and / or cytochrome P450 reductase), host cells expressing the enzymes, and methods of producing mogroside precursors, mogroside, and / or mogroside using such host cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application Serial No. 62 / 758,474, filed on November 9, 2018, entitled “Biosynthesis of Mogrosides,” the disclosure of which is incorporated herein by reference in its entirety.

[0003] References to sequence listings submitted as TEXT files via EFS-WEB

[0004] This application contains a sequence listing that has been submitted by EFS-WEB in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on November 9, 2019, named G091970023WO00-SEQ-OMJ.TXT, and is 928 kilobytes in size. Field of the Invention

[0005] The present disclosure relates to the production of mogroside precursors, mogroside, and mogroside in recombinant cells.

[0006] background

[0007] Mogrosides are glycosides of cucurbitane derivatives. Highly sought after as sweeteners and sugar substitutes, mogrosides are synthesized naturally in the fruit of plants, including monk fruit (Siraitia grosvenorii (S. grosvenorii)). Although mogrosides have been implicated in anticancer, antioxidant, and anti-inflammatory properties, characterization of the enzymes specifically involved in mogroside biosynthesis is limited. Furthermore, extraction of mogrosides from the fruit is labor-intensive, and the structural complexity of mogrosides often hinders de novo chemical synthesis.

[0008] Overview

[0009] Aspects of the invention relate to host cells comprising a heterologous polynucleotide encoding a UDP-glycosyltransferase (UGT), wherein the UGT comprises the following regions:

[0010] A region corresponding to residues 83 to 92 of wild-type UGT94-289-1 (SEQ ID NO: 109), wherein the region includes amino acid substitutions relative to residues 83 to 92 of wild-type UGT94-289-1 (SEQ ID NO: 109); and / or a region corresponding to residues 179 to 198 of wild-type UGT94-289-1 (SEQ ID NO: 109), wherein the region includes amino acid substitutions relative to residues 179 to 198 of wild-type UGT94-289-1 (SEQ ID NO: 109); wherein the region includes amino acid substitutions relative to residues 179 to 198 of wild-type UGT94-289-1 (SEQ ID NO: 109); NO:109), the host cell produces at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% more one or more mogrosides in the presence of at least one mogroside precursor.

[0011] In some embodiments, the UGT exhibits at least a 1.3-fold increase in activity (e.g., specific activity) relative to wild-type UGT94-289-1 (SEQ ID NO: 109). In some embodiments, the UGT comprises an amino acid substitution at an amino acid residue in a structural motif corresponding to a structural motif selected from the group consisting of loop 6, alpha helix 3, loop 11, alpha helix 6, loop 12, and alpha helix 7 in wild-type UGT94-289-1 (SEQ ID NO: 109). In some embodiments, the UGT is capable of catalyzing the following transformations: transformation of mogroside to MIA1; transformation of mogroside to MIE1; transformation of MIA1 to MIIA1; transformation of MIE1 to MIIE; transformation of MIIA1 to MIIIA1; transformation of MIA1 to MIIE; transformation of MIIA1 to MIII; transformation of MIIIA1 to simenoside I; transformation of MIIE to MIII; transformation of MIII to simenoside I; transformation of MIIE to MIIIE; and / or transformation of MIIIE to simenoside I. In some embodiments, the UGT is capable of: glycosylation of mogroside at C24; glycosylation of mogroside at C3; branching glycosylation of mogroside at C3; or branching glycosylation of mogroside C24. In some embodiments, the UGT comprises an amino acid replacement at an amino acid residue corresponding to an amino acid residue in wild-type UGT94-289-1 (SEQ ID NO: 109) selected from the group consisting of: H83; T84; T85; N86; P89; L92; Y179; S180; A181; G184; A185; V186; T187; K189; H191; K192; G194; E195; and A198. In some embodiments, the host cell further comprises a heterologous polynucleotide encoding a cucurbitacadienol synthase (CDS).

[0012] Additional aspects of the invention relate to host cells comprising a heterologous polynucleotide encoding a UGT, wherein the UGT comprises the following regions: a region corresponding to residues 83 to 92 of wild-type UGT94-289-1 (SEQ ID NO: 109), wherein the region comprises amino acid substitutions relative to residues 83 to 92 of wild-type UGT94-289-1 (SEQ ID NO: 109); and / or a region corresponding to residues 179 to 198 of wild-type UGT94-289-1 (SEQ ID NO: 109), wherein the region comprises amino acid substitutions relative to residues 179 to 198 of wild-type UGT94-289-1 (SEQ ID NO: 109); wherein the UGT comprises less than 90% identity to SEQ ID NO: 109.

[0013] In some embodiments, the UGT exhibits at least a 1.3-fold increase in activity (e.g., specific activity) relative to wild-type UGT94-289-1 (SEQ ID NO: 109). In some embodiments, the UGT comprises an amino acid substitution at an amino acid residue in a structural motif corresponding to a structural motif selected from the group consisting of loop 6, alpha helix 3, loop 11, alpha helix 6, loop 12, and alpha helix 7 in wild-type UGT94-289-1 (SEQ ID NO: 109). In some embodiments, the UGT is capable of catalyzing: the conversion of mogrosanol to MIA1; the conversion of mogrosanol to MIE1; the conversion of MIA1 to MIIA1; the conversion of MIE1 to MIIE; the conversion of MIIA1 to MIIIA1; the conversion of MIA1 to MIIE; the conversion of MIIA1 to MIII; the conversion of MIIIA1 to simenoside I; the conversion of MIIE to MIII; the conversion of MIII to simenoside I; the conversion of MIIE to MIIIE; and / or the conversion of MIIIE to simenoside I. In some embodiments, the UGT is capable of: glycosylation of mogrosanol at C24; glycosylation of mogroside at C3; branched glycosylated mogroside at C3; or branched glycosylated mogroside C24. In some embodiments, the host cell also includes a heterologous polynucleotide encoding a CDS enzyme.

[0014] Additional aspects of the invention relate to host cells comprising a heterologous polynucleotide encoding a UGT, wherein the UGT comprises an amino acid replacement at a residue corresponding to N143 or L374 of wild-type UGT94-289-1 (SEQ ID NO: 109), wherein the host cell produces at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% more one or more mogrosides in the presence of at least one mogroside precursor relative to a host cell comprising a heterologous polynucleotide encoding wild-type UGT94-289-1 (SEQ ID NO: 109).

[0015] In some embodiments, the UGT exhibits at least a 1.3-fold increase in activity (e.g., specific activity) relative to wild-type UGT94-289-1 (SEQ ID NO: 109). In some embodiments, the UGT comprises an amino acid substitution at an amino acid residue located in a structural motif of the UGT, the structural motif corresponding to a structural motif selected from the group consisting of loop 6, alpha helix 3, loop 11, alpha helix 6, loop 12, and alpha helix 7 in wild-type UGT94-289-1 (SEQ ID NO: 109). In some embodiments, the UGT is capable of catalyzing the following transformations: transformation of mogrosanol to MIA1; transformation of mogrosanol to MIE1; transformation of MIA1 to MIIA1; transformation of MIE1 to MIIE; transformation of MIIA1 to MIIIA1; transformation of MIA1 to MIIE; transformation of MIIA1 to MIII; transformation of MIIIA1 to simenoside I; transformation of MIIE to MIII; transformation of MIII to simenoside I; transformation of MIIE to MIIIE; and / or transformation of MIIIE to simenoside I. In some embodiments, the UGT is capable of: glycosylation of mogrosanol at C24; glycosylation of mogroside at C3; branched glycosylated mogroside at C3; or branched glycosylated mogroside C24. In some embodiments, the host cell further comprises a heterologous polynucleotide encoding a CDS enzyme.

[0016] Additional aspects of the invention relate to host cells comprising a heterologous polynucleotide encoding a UGT, wherein the UGT comprises an amino acid replacement at an amino acid residue in a structural motif corresponding to a structural motif of wild-type UGT94-289-1 (SEQ ID NO: 109) selected from the group consisting of: loop 8; beta sheet 5; loop 10; alpha helix 5; loop 11; loop 2; alpha helix 6; loop 12; alpha helix 1; alpha helix 7; loop 18; alpha helix 14; loop 26; alpha helix 2; loop 6; and alpha helix 3; wherein the host cell produces at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% more one or more mogrosides in the presence of at least one mogroside precursor relative to a host cell comprising a heterologous polynucleotide encoding a UGT that does not comprise the amino acid replacement.

[0017] In some embodiments, the UGT exhibits an increase in activity (e.g., specific activity) of at least 1.3 fold relative to wild-type UGT94-289-1 (SEQ ID NO: 109). In some embodiments, the UGT is capable of catalyzing the following transformations: transformation of mogrosanol to MIA1; transformation of mogrosanol to MIE1; transformation of MIA1 to MIIA1; transformation of MIE1 to MIIE; transformation of MIIA1 to MIIIA1; transformation of MIA1 to MIIE; transformation of MIIA1 to MIII; transformation of MIIIA1 to simenoside I; transformation of MIIE to MIII; transformation of MIII to simenoside I; transformation of MIIE to MIIIE; and / or transformation of MIIIE simenoside I. In some embodiments, the UGT is capable of: glycosylation of mogrosanol at C24; glycosylation of mogroside at C3; branched glycosylated mogroside at C3; or branched glycosylated mogroside C24. In some embodiments, the host cell further comprises a heterologous polynucleotide encoding a CDS enzyme.

[0018] Additional aspects of the invention relate to host cells comprising a heterologous polynucleotide encoding a UGT, wherein the UGT comprises an amino acid substitution at an amino acid residue within 7 angstroms of the catalytic duplex corresponding to H21 / D122 of wild-type UGT94-289-1 (SEQ ID NO: 109), wherein the UGT exhibits at least a 1.3-fold increase in activity (e.g., specific activity) relative to the same UGT not comprising the amino acid substitution.

[0019] Additional aspects of the invention relate to host cells comprising a heterologous polynucleotide encoding a cyclically permuted UDP-glycosyltransferase (UGT), wherein the cyclically permuted UGT comprises: (a) a catalytic dyad; and (b) a cofactor binding site;

[0020] The catalytic dyad is located C-terminal to the cofactor-binding site, and

[0021] Wherein the host cell produces at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% more one or more mogrosides in the presence of at least one mogroside precursor relative to a control host cell comprising a heterologous polynucleotide encoding wild-type UGT94-289-1 (SEQ ID NO: 109).

[0022] In some embodiments, the recursively rearranged UGT comprises a sequence that is at least 90% identical to a sequence in Table 6.

[0023] In some embodiments, the UGT described in this application comprises a sequence that is at least 90% identical to a sequence in Table 3 or Table 7.

[0024] In some embodiments, the UGT comprises an amino acid replacement at an amino acid residue in a structural motif corresponding to a structural motif in wild-type UGT94-289-1 (SEQ ID NO: 109) selected from the group consisting of loop 6, alpha helix 3, loop 11, alpha helix 6, loop 12, and alpha helix 7.

[0025] In some embodiments, the UGT exhibits at least a 1.3-fold increase in activity (e.g., specific activity) relative to wild-type UGT94-289-1 (SEQ ID NO: 109). In some embodiments, the host cell produces at least 40%, 50%, 60%, 70%, 80%, 90%, or 100% more one or more mogrosides in the presence of at least one mogroside precursor relative to a host cell comprising a heterologous polynucleotide encoding a UGT that does not include an amino acid substitution.

[0026] In some embodiments, the UGT is capable of catalyzing the following transformations: transformation of mogrosanol to MIA1; transformation of mogrosanol to MIE1; transformation of MIA1 to MIIA1; transformation of MIE1 to MIIE; transformation of MIIA1 to MIIIA1; transformation of MIA1 to MIIE; transformation of MIIA1 to MIII; transformation of MIIIA1 to simenoside I; transformation of MIIE to MIII; transformation of MIII to simenoside I; transformation of MIIE to MIIIE; and / or transformation of MIIIE to simenoside I. In some embodiments, the UGT is capable of: glycosylation of mogrosanol at C24; glycosylation of mogroside at C3; branched glycosylated mogroside at C3; or branched glycosylated mogroside C24. In some embodiments, the host cell further comprises a heterologous polynucleotide encoding a CDS enzyme.

[0027] In some embodiments, the specific activity of the UGT is at least 1 mmol of glycosylated mogroside target produced per gram of enzyme per hour.

[0028] Additional aspects of the invention relate to host cells comprising a heterologous polynucleotide encoding a UGT, wherein the UGT comprises an amino acid replacement at an amino acid residue corresponding to an amino acid residue in wild-type UGT94-289-1 (SEQ ID NO: 109) selected from the group consisting of: H83; T84; T85; N86; P89; L92; Y179; S180; A181; G184; A185; V186; T187; K189; H191; K192; G194; E195; and A198.

[0029] Additional aspects of the invention relate to host cells comprising a heterologous polynucleotide encoding a UGT, wherein the UGT comprises an amino acid replacement at an amino acid residue corresponding to an amino acid residue in wild-type UGT94-289-1 (SEQ ID NO: 109) selected from the group consisting of: G18; Y19; S123; N47; F124; N143; T144; T145; V149; F276; N355; H373 and L374.

[0030] In some embodiments, the UGT is capable of catalyzing the following transformations: transformation of mogrosanol to MIA1; transformation of mogrosanol to MIE1; transformation of MIA1 to MIIA1; transformation of MIE1 to MIIE; transformation of MIIA1 to MIIIA1; transformation of MIA1 to MIIE; transformation of MIIA1 to MIII; transformation of MIIIA1 to simenoside I; transformation of MIIE to MIII; transformation of MIII to simenoside I; transformation of MIIE to MIIIE; and / or transformation of MIIIE to simenoside I. In some embodiments, the UGT is capable of: glycosylation of mogrosanol at C24; glycosylation of mogroside at C3; branched glycosylated mogroside at C3; or branched glycosylated mogroside C24. In some embodiments, the UGT exhibits an increase in activity (e.g., specific activity) of at least 1.3 times relative to wild-type UGT94-289-1 (SEQ ID NO: 109). In some embodiments, the host cell produces at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% more one or more mogrosides in the presence of at least one mogroside precursor relative to a host cell comprising a heterologous polynucleotide encoding wild-type UGT94-289-1 (SEQ ID NO: 109).

[0031] In some embodiments, Y179 is mutated to glutamic acid, phenylalanine, histidine, isoleucine, lysine, leucine, valine, or tryptophan; S180 is mutated to alanine or valine; A181 is mutated to lysine or threonine; G184 is mutated to alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, or tyrosine; A185 is mutated to cysteine, aspartic acid, glutamic acid, glycine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, or tyrosine. amide, threonine, tryptophan or tyrosine; V186 is mutated to alanine, cysteine, aspartic acid, glutamic acid, glycine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, threonine, tryptophan or tyrosine; T187 is mutated to alanine, cysteine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, arginine, serine, valine, tryptophan or tyrosine; K189 is mutated to alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, leucine 191 is mutated to alanine, cysteine, aspartic acid, glutamic acid, glycine, lysine, methionine, proline, glutamine, serine, threonine, valine, tryptophan, or tyrosine; K192 is mutated to cysteine ​​or phenylalanine; G194 is mutated to aspartic acid, leucine, methionine, asparagine, proline, serine, or tryptophan; E195 is mutated to alanine, isoleucine, lysine, leucine, asparagine, glutamine, serine, threonine, or tyrosine; A198 is mutated to cysteine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, or tyrosine; H83 is mutated to glutamine or tryptophan; T84 is mutated to tyrosine; T85 is mutated to glycine, lysine, proline, serine, or tyrosine; N86 is mutated to alanine, cysteine, glutamic acid, isoleucine, lysine, leucine, serine, tryptophan, or tyrosine; P89 is mutated to methionine or serine; and / or L92 is mutated to histidine or lysine.

[0032] In some embodiments, N143 is mutated to alanine, cysteine, glutamic acid, isoleucine, leucine, methionine, glutamine, serine, threonine, or valine; L374 is mutated to alanine, cysteine, phenylalanine, histidine, methionine, asparagine, glutamine, serine, threonine, valine, tryptophan, or tyrosine; S123 is mutated to alanine, cysteine, glycine, or valine; F124 is mutated to tyrosine; T144 is mutated to alanine, cysteine, asparagine, or proline. or serine; T145 is mutated to alanine, cysteine, glycine, methionine, asparagine, glutamine, or serine; V149 is mutated to cysteine, leucine, or methionine; G18 is mutated to serine; Y19 is mutated to phenylalanine, histidine, leucine, or valine; F276 is mutated to cysteine ​​or glutamine; N355 is mutated to glutamine or serine; H373 is mutated to lysine, leucine, methionine, arginine, valine, or tyrosine; and / or N47 is mutated to glycine.

[0033] In some embodiments, the host cell further comprises a heterologous polynucleotide encoding a CDS enzyme, a C11 hydroxylase, a cytochrome P450 reductase, an epoxidase (EPH), and / or a squalene epoxidase. In some embodiments, the heterologous polynucleotide encoding the CDS is at least 90% identical to SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 12. In some embodiments, the CDS is at least 90% identical to SEQ ID NO: 43, SEQ ID NO: 49, or SEQ ID NO: 52.

[0034] In some embodiments, the activity (e.g., specific activity) of the UGT is at least 1 mmol of glycosylated mogroside target per gram of enzyme per hour. In some embodiments, the cell is a yeast cell, a plant cell, or a bacterial cell. In some embodiments, the cell is a Saccharomyces cerevisiae cell. In some embodiments, the cell is an Escherichia coli (E. coli) cell.

[0035] Another aspect of the invention relates to a method for producing mogrosides, comprising culturing any host cell described herein with at least one mogroside precursor. In some embodiments, the mogroside precursor is selected from mogroside alcohol, MIA1, MIIA1, MIIIA1, MIIE, MIII, and MIIIE. In some embodiments, the mogroside produced is selected from MIA1, MIIA1, MIIIA1, MIIE, MIII, simonoside, and MIIIE.

[0036] Additional aspects of the invention relate to host cells comprising a heterologous polynucleotide encoding a CDS enzyme, wherein the CDS enzyme comprises an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of the sequences in Table 2, and wherein the host cell produces at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% more cucurbitacinadienol compound than the same host cell not expressing the heterologous gene.

[0037] In some embodiments, the cucurbitacinaldehyde compound is 24-25 epoxy-cucurbitacinaldehyde or cucurbitacinaldehyde. In some embodiments, the CDS enzyme includes a leucine at an amino acid residue corresponding to the amino acid residue at position 123 of SEQ ID NO: 73. In some embodiments, the CDS is capable of converting oxidized squalene to a cucurbitacinaldehyde compound. In some embodiments, the oxidized squalene is 2-3-oxidized squalene or 2,3; 22,23-diepoxysqualene. In some embodiments, the CDS enzyme includes a substrate channel and an active-site cavity.

[0038] In some embodiments, the host cell further expresses a heterologous gene encoding a C11 hydroxylase, a cytochrome P450 reductase, an epoxidase (EPH), and / or a squalene epoxidase.

[0039] Another aspect of the invention relates to a method of producing a cucurbitacinaldehyde compound, the method comprising contacting a host cell described in the present application with squalene oxide, thereby producing a cucurbitacinaldehyde compound. In some embodiments, the cucurbitacinaldehyde compound is 24-25 epoxy-cucurbitacinaldehyde or cucurbitacinaldehyde. In some embodiments, the squalene oxide is 2-3-squalene oxide or 2,3;22,23-diepoxysqualene. In some embodiments, the method further comprises isolating the cucurbitacinaldehyde compound.

[0040] Another aspect of the invention relates to a method for producing mogroside or mogroside, the method comprising contacting the host cell described in the present application with squalene oxide, thereby producing mogroside or mogroside. Another aspect of the invention relates to a method for producing mogroside, the method comprising culturing the host cell described in the present application with at least one mogroside precursor.

[0041] Another aspect of the invention relates to a host cell comprising a heterologous polynucleotide encoding a cucurbitacinadienol synthase (CDS), wherein the CDS comprises: a) the motif GX1WASDLGGP (SEQ ID NO: 331), wherein X1 is N or H; b) the motif DX1GWL (SEQ ID NO: 332), wherein X1 is H or Q; and / or c) the motif CWGVCFTYAGW (SEQ ID NO: 333), wherein the CDS does not include the sequence of the Momordica CDS (SEQ ID NO: 73); and wherein the host cell produces at least 10%, 20%, or 30% more cucurbitacinadienol compounds relative to a control, wherein the control is a host cell expressing the Momordica CDS encoded by a polynucleotide corresponding to SEQ ID NO: 33.

[0042] In some embodiments, the motif GX1WASDLGGP (SEQ ID NO:331) is located at a residue in the CDS corresponding to residues 117-126 in SEQ ID NO:73; the motif DX1GWL (SEQ ID NO:332) is located at a residue in the CDS corresponding to residues 479-483 in SEQ ID NO:73, and / or the motif CWGVCFTYAGW (SEQ ID NO:333) is located at a residue in the CDS corresponding to residues 612-622 in SEQ ID NO:73.

[0043] Additional aspects of the invention relate to host cells comprising a heterologous polynucleotide encoding a cucurbitacadienol synthase (CDS), wherein the CDS comprises: a) the motif GHWASDLGGP ((SEQ ID NO: 334); and / or b) the motif DQGWL (SEQ ID NO: 335).

[0044] In some embodiments, the motif GHWASDLGGP (SEQ ID NO:334) is located at a residue in the CDS corresponding to residues 117-126 in SEQ ID NO:73; and / or the motif DQGWL (SEQ ID NO:335) is located at a residue in the CDS corresponding to residues 479-483 in SEQ ID NO:73.

[0045] Additional aspects of the invention relate to host cells comprising a heterologous polynucleotide encoding a cucurbitacadienol synthase (CDS), wherein the CDS comprises: a) the motif GHWANDLGGP (SEQ ID NO: 336); b) the motif DQGWL (SEQ ID NO: 335); and / or c) the motif CWGVCYTYAGW (SEQ ID NO: 337).

[0046] In some embodiments, the motif GHWANDLGGP (SEQ ID NO:336) is located at a residue in the CDS corresponding to residues 117-126 in SEQ ID NO:73; the motif DQGWL (SEQ ID NO:335) is located at a residue in the CDS corresponding to residues 479-483 in SEQ ID NO:73; and / or the motif CWGVCYTYAGW (SEQ ID NO:337) is located at a residue in the CDS corresponding to residues 612-622 in SEQ ID NO:73.

[0047] In some embodiments, the heterologous polynucleotide is at least 90% identical to SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 12. In some embodiments, the CDS is at least 90% identical to SEQ ID NO: 43, SEQ ID NO: 49, or SEQ ID NO: 52. In some embodiments, the heterologous polynucleotide is at least 90% identical to SEQ ID NO: 3. In some embodiments, the CDS is at least 90% identical to SEQ ID NO: 43.

[0048] Additional aspects of the invention relate to host cells comprising a heterologous polynucleotide encoding a cucurbitacinamide synthase (CDS), wherein the heterologous polynucleotide sequence is at least 90% identical to SEQ ID NO:3 and / or the amino acid sequence of the CDS encoded by the heterologous polynucleotide is at least 90% identical to SEQ ID NO:43, wherein the host cell produces a cucurbitacinamide compound.

[0049] In some embodiments, the cucurbitacinaldehyde compound is 24-25 epoxy-cucurbitacinaldehyde or cucurbitacinaldehyde. In some embodiments, the CDS includes a leucine at an amino acid residue corresponding to the amino acid residue at position 123 of SEQ ID NO: 73. In some embodiments, the CDS is capable of converting oxidized squalene to a cucurbitacinaldehyde compound. In some embodiments, the oxidized squalene is 2-3-oxidized squalene or 2,3;22,23-diepoxysqualene. In some embodiments, the CDS enzyme includes a substrate channel and an active site cavity.

[0050] In some embodiments, the host cell further comprises one or more heterologous polynucleotides encoding a UDP-glycosyltransferase (UGT), a C11 hydroxylase, a cytochrome P450 reductase, an epoxidase (EPH), and / or a squalene epoxidase.

[0051] Another aspect of the invention relates to a method for producing a cucurbitacinaldehyde compound, the method comprising contacting any host cell described in the present application with squalene oxide, thereby producing a cucurbitacinaldehyde compound. In some embodiments, the cucurbitacinaldehyde compound is 24-25 epoxy-cucurbitacinaldehyde or cucurbitacinaldehyde. In some embodiments, the squalene oxide is 2-3-squalene oxide or 2,3; 22,23-diepoxysqualene. In some embodiments, the method further comprises isolating the cucurbitacinaldehyde compound. In some embodiments, the host cell is a yeast cell, a plant cell, or a bacterial cell. In some embodiments, the host cell is a Saccharomyces cerevisiae cell. In some embodiments, the host cell is an Escherichia coli cell.

[0052] Each in the limitation of the present invention can encompass various embodiments of the present invention. Therefore, it is contemplated that each in the limitation of the present invention relating to any one element or combination of elements can be included in each aspect of the present invention. Application of the present invention is not limited to the arrangement of the construction details and components shown in the following description or illustrated in the accompanying drawings. The present invention can have other embodiments and can be put into practice or carried out in various ways.

[0053] BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The drawings are not intended to be drawn to scale. The drawings are illustrative only and are not necessary to practice the present disclosure. For clarity, not every component is labeled in every drawing. In the drawings:

[0055] Figures 1A-1D A schematic overview of the putative mogrohol biosynthetic pathway is included. SQS denotes squalene synthase, EPD denotes epoxidase, P450 denotes C11 hydroxylase, EPH denotes epoxidase, and CDS denotes cucurbitacadienol synthase. Figure 1A and Figure 1B The putative biosynthetic pathway for mogrohol is shown. Figure 1C Non-limiting examples of primary UGT activity are shown. Figure 1D Non-limiting examples of secondary UGT activity are shown.

[0056] Figure 2 is a graph showing liquid chromatography-mass spectrometry (LC-MS) spectra of mogroside, mogroside I-A1, mogroside I-E1, mogroside II-A1, mogroside II-A2, mogroside II-E, mogroside III-A1, mogroside III-E, mogroside IV, and simanoside I. An 8-minute LC-MS method was used to distinguish all of these mogroside standards.

[0057] Figure 3A-3BContains a sequence showing the production of mogroside I-A1 ( Figure 3A ) and simanoside I ( Figure 3B ). Two biological replicates of each strain were screened.

[0058] Figures 4A-4B The secondary screen included strains showing hits on a single substrate for the production of mogroside I-A1 from mogroside alcohol. Figure 4A ) and the production of simonoside I from mogroside III-A1 ( Figure 4B ). For each hit strain two biological replicates each with two technical replicates were screened.

[0059] Figure 5 is a schematic representation of the homology model of UGT94-289-1. The catalytic dyad side chains are shown within boxes and positions with activity-enhancing mutations are highlighted in black.

[0060] Figure 6 is a graph showing the proximity of the N-terminus and C-terminus of the UGT94-289-1 homology model.

[0061] Figure 7 It is a schematic diagram showing the cyclic rearrangement of proteins. The original N-terminus and C-terminus of the native protein are connected together directly or through a linker sequence. A new N-terminus and C-terminus are introduced at another site within the protein sequence to generate a sequence / structure variant.

[0062] Figure 8 Depicted are a plot showing a site-specific scoring matrix (PSSM) for residues 50-59 of t85024_N143V (upper panel) and the impact of these potential mutations assessed using the Rosetta energy function (lower panel). In the lower panel, the mutated residues are shown as sticks, and the surrounding atoms are shown as lines.

[0063] Fig. 9 is a schematic diagram depicting a non-limiting example of an alignment between UGT U73C6 (SEQ ID NO: 103) and UGT94-289-1 (SEQ ID NO: 109). The boxes highlight the residues in U73C6 (SEQ ID NO: 103) corresponding to positions 123, 143, and 273 in UGT94-289-1 (SEQ ID NO: 109).

[0064] Fig.10 is a graph showing the relative yields of M, MI and MII compared to MIII, MIV and MV of UGTs generated by cyclic rearrangements.

[0065] Fig.11is a graph showing the relative production of M, MI and MII by putative UGTs compared to MIII, MIV and MV.

[0066] Detailed description

[0067] Mogrosides are widely used as natural sweeteners (e.g., in beverages). However, de novo synthesis and extraction of mogrosides from natural raw materials often involve high production costs and low yields. The present application describes host cells engineered to effectively produce mogroside (or 11,24,25-trihydroxycucurbitacinol), mogrosides, and precursors thereof. The method comprises heterologous expression of cucurbitacinol synthase (CDS), UDP-glycosyltransferase (UGT), C11 hydroxylase, cytochrome P450 reductase, epoxidase (EPH), squalene epoxidase (SQE), or a combination thereof. The present application describes the identification of improved UGT and CDS enzymes for producing mogroside and mogroside. The enzymes and host cells described in the present application can be used to prepare mogroside, mogroside, and precursors thereof.

[0068] Synthesis of Mogroside and Mogroside

[0069] Figure 1A-1B The putative synthesis pathway of mogrohol is shown. The early steps in the pathway involve the conversion of squalene to 2,3-squalene oxide. Figure 1A As shown in , 2,3-oxidosqualene can be first catalyzed to cucurbitacinol and then epoxidized to form 24,25-epoxycucurbitacinol, or 2,3-oxidosqualene can be epoxidized to 2,3,22,23-dioxidosqualene and then cyclized to 24,25-epoxycucurbitacinol. Next, 24,25-epoxycucurbitacinol can be converted to mogroside (aglycone of mogroside), followed by epoxide hydrolysis and then oxidation, or oxidation and then epoxide hydrolysis. Figure 1B As shown in , 2,3-oxidosqualene can first be cyclized to cucurbitacinol, which is then converted to 11-hydroxycucurbitacinol by cytochrome P450 C11 hydroxylase. Cytochrome P450 C11 hydroxylase can then convert 11-hydroxycucurbitacinol to 11-hydroxy-24,25-epoxycucurbitacinol. 11-Hydroxy-24,25-epoxycucurbitacinol can be converted to mogrosanol by epoxidase. C11 hydroxylase and cytochrome P450 reductase (not shown in Figure 1A-1B ) synergistic effect.

[0070] Mogroside can be distinguished from other cucurbitane triterpenes by oxidation at C3, C11, C24 and C25. Glycosylation of mogroside (e.g., at C3 and / or C24) results in the formation of mogrosides.

[0071] The precursor of mogroside includes, but is not limited to, squalene, 2-3-squalene oxide, 2,3,22,23-squalene oxide, cucurbitacinol, 24,25-epoxycucurbitacinol, 11-hydroxycucurbitacinol, 11-hydroxy-24,25-epoxycucurbitacinol, 11-hydroxy-cucurbitacinol, 11-oxy-cucurbitacinol, and 24,25-dihydroxycucurbitacinol. The term "squalene oxide" can be used to refer to 2,3,22,23-diepoxysqualene or 2,3,22,23-squalene oxide. The term "2,3-epoxysqualene" can be used interchangeably with the term "2-3-squalene oxide". As used in the present application, the precursor of mogroside includes a precursor of mogroside, mogroside and mogroside.

[0072] Examples of mogrosides include, but are not limited to, mogroside I-A1 (MIA1), mogroside IE (MIE), mogroside II-A1 (MIIA1), mogroside II-A2 (MIIA2), mogroside III-A1 (MIIIA1), mogroside II-E (MIIE), mogroside III (MIII), simenoside I, mogroside IV, mogroside IVa, isomogroside IV, mogroside III-E (MIIIE), mogroside V, and mogroside VI. In some embodiments, the mogroside produced is simenoside I, which may be referred to as Siam. In some embodiments, the mogroside produced is MIIIE.

[0073] In other embodiments, the mogroside is a compound of Formula I:

[0074]

[0075] In some embodiments, the methods described in this application can be used to produce any compound described in US2019 / 0071705 and incorporated by reference from US2019 / 0071705, including compounds 1-20 disclosed in US2019 / 0071705. In some embodiments, the methods described in this application can be used to produce variants of any compound described in US2019 / 0071705 and incorporated by reference from US2019 / 0071705, including variants of compounds 1-20 disclosed in US2019 / 0071705. For example, variants of compounds described in US2019 / 0071705 can include replacing one or more α-glucosyl linkages in compounds described in US2019 / 0071705 with one or more β-glucosyl linkages. In some embodiments, the variant of the compound described in US2019 / 0071705 includes replacing one or more β-glucosyl linkages in the compound described in US2019 / 0071705 with one or more α-glucosyl linkages. In some embodiments, the variant of the compound described in US2019 / 0071705 is a compound of Formula I shown above.

[0076] Cucurbitacadienol synthase (CDS)

[0077] Aspects of the present disclosure provide a cucurbitacinol synthase (CDS), which can be useful in, for example, the production of a cucurbitacinol compound (such as 24-25-epoxy-cucurbitacinol or cucurbitacinol). The CDS can catalyze the formation of a cucurbitacinol compound (such as 24-25-epoxy-cucurbitacinol or cucurbitacinol) by oxidizing squalene (e.g., 2-3-oxidized squalene or 2,3; 22,23-diepoxysqualene).

[0078] In some embodiments, the CDS enzyme has a leucine residue corresponding to position 123 in SEQ ID NO: 74, which distinguishes the CDS enzyme from other oxidosqualene cyclases (as discussed in Takase et al. Org. Biomol. Chem., 2015, 13, 7331-7336, which is hereby incorporated by reference in its entirety).

[0079] The CDS disclosed herein may include a nucleic acid sequence or an amino acid sequence in Table 2 or a sequence selected from SEQ %, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (including all values ​​therebetween) identical sequences to those of ID NOs: 1-80.

[0080] In some embodiments, the CDS enzyme corresponds to SEQ ID NO:43, SEQ ID NO:52, or SEQ ID NO:49.

[0081] In some embodiments, the polynucleotide sequence encoding the CDS enzyme can be recoded for expression in a specific host cell (including Saccharomyces cerevisiae). In some embodiments, the recoded polynucleotide sequence encoding the CDS enzyme corresponds to SEQ ID NO:34.

[0082] In some embodiments, the polynucleotide sequence encoding the CDS is at least 90% identical to SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 12. In some embodiments, the CDS is at least 90% identical to SEQ ID NO: 43, SEQ ID NO: 49, or SEQ ID NO: 52.

[0083] In some embodiments, the CDS of the present disclosure can use squalene oxide (e.g., 2,3-squalene oxide or 2,3; 22,23-diepoxysqualene) as a substrate. In some embodiments, the CDS of the present disclosure can produce a cucurbitacinol compound (e.g., 24-25 epoxy-cucurbitacinol or cucurbitacinol). In some embodiments, the CDS of the present disclosure catalyzes the formation of a cucurbitacinol compound (e.g., 24-25 epoxy-cucurbitacinol or cucurbitacinol) from squalene oxide (e.g., 2-3-squalene oxide or 2,3; 22,23-diepoxysqualene.

[0084] It should be understood that the activity of a CDS can be measured by any means known to those of ordinary skill in the art. In some embodiments, the activity of a CDS can be measured as the standardized peak area of ​​the cucurbitacanedienol produced. In some embodiments, the activity is measured in arbitrary units. In some embodiments, the activity (such as specific activity) of a CDS of the present disclosure is at least 1.1 times (e.g., at least 1.3 times, at least 1.5 times, at least 1.7 times, at least 1.9 times, at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, or at least 100 times (including all values ​​therebetween) greater than the activity of a control CDS.

[0085] It should be understood that one of ordinary skill in the art will be able to characterize a protein as a CDS enzyme based on structural and / or functional information associated with the protein. For example, in some embodiments, a protein may be characterized as a CDS enzyme based on the function of the protein, such as the ability to produce a cucurbitacinol compound (e.g., 24-25-epoxy-cucurbitacinol or cucurbitacinol) using oxidized squalene (e.g., 2,3-oxidized squalene or 2,3; 22,23-diepoxysqualene) as a substrate. In some embodiments, a protein may be characterized (at least in part) as a CDS enzyme based on the presence of a leucine residue at a position corresponding to position 123 of SEQ ID NO: 73.

[0086] In some embodiments, the CDS comprises the motif GX1WASDLGGP (SEQ ID NO: 331), wherein X1 is N or H. In some embodiments, the motif GX1WASDLGGP (SEQ ID NO: 331) is located in the CDS at residues corresponding to positions 117-126 in SEQ ID NO:73.

[0087] In some embodiments, the CDS includes the motif DX1GWL (SEQ ID NO: 332), wherein X1 is H or Q. In some embodiments, the motif DX1GWL (SEQ ID NO: 332) is located in the CDS at residues corresponding to positions 479-483 in SEQ ID NO: 73.

[0088] In some embodiments, the CDS includes the motif CWGVCFTYAGW (SEQ ID NO: 333). In some embodiments, the motif CWGVCFTYAGW (SEQ ID NO: 333) is located in the CDS at residues corresponding to positions 612-622 in SEQ ID NO: 73.

[0089] In some embodiments, the CDS comprises the motif GHWASDLGGP (SEQ ID NO: 334). In some embodiments, the motif GHWASDLGGP (SEQ ID NO: 334) is located in the CDS at residues corresponding to positions 117-126 in SEQ ID NO: 73. In some embodiments, the CDS comprises the motif DQGWL (SEQ ID NO: 335). In some embodiments, the motif DQGWL (SEQ ID NO: 335) is located in the CDS at residues corresponding to positions 479-483 in SEQ ID NO: 73.

[0090] In some embodiments, the CDS comprises the motif GHWASDLGGP (SEQ ID NO: 334), the motif DQGWL (SEQ ID NO: 335) and / or the motif CWGVCFTYAGW (SEQ ID NO: 333).

[0091] In some embodiments, the CDS includes a leucine at a residue corresponding to position 123 in SEQ ID NO: 73. In some embodiments, the CDS includes a leucine at a residue corresponding to position 483 in SEQ ID NO: 73. In some embodiments, the CDS includes a cysteine ​​at a residue corresponding to position 612 in SEQ ID NO: 73, a glycine at a residue corresponding to position 614 in SEQ ID NO: 73, an alanine at a residue corresponding to position 620 in SEQ ID NO: 73, and / or a glycine at a residue corresponding to position 621 in SEQ ID NO: 73. In some embodiments, the CDS includes a leucine at a residue corresponding to position 123 in SEQ ID NO:73, a leucine at a residue corresponding to position 483 in SEQ ID NO:73, a cysteine ​​at a residue corresponding to position 612 in SEQ ID NO:73, a glycine at a residue corresponding to position 614 in SEQ ID NO:73, an alanine at a residue corresponding to position 620 in SEQ ID NO:73, and / or a glycine at a residue corresponding to position 621 in SEQ ID NO:73.

[0092] In some embodiments, the CDS includes the motif GHWANDLGGP (SEQ ID NO: 336). In some embodiments, the motif GHWANDLGGP (SEQ ID NO: 336) is located in the CDS at residues corresponding to positions 117-126 in SEQ ID NO: 73.

[0093] In some embodiments, the CDS includes the motif DX1GWL (SEQ ID NO: 332). In some embodiments, the motif DX1GWL (SEQ ID NO: 332) is located in the CDS at residues corresponding to positions 479-483 in SEQ ID NO: 73.

[0094] In some embodiments, the CDS includes the motif CWGVCYTYAGW (SEQ ID NO: 337). In some embodiments, the motif CWGVCYTYAGW (SEQ ID NO: 337) is located in the CDS at residues corresponding to positions 612-622 in SEQ ID NO: 73.

[0095] In some embodiments, the CDS includes: the motif GHWANDLGGP (SEQ ID NO:336) located at residues corresponding to positions 117-126 in SEQ ID NO:73; the motif DQGWL (SEQ ID NO:335) located at residues corresponding to positions 479-483 in SEQ ID NO:73; and / or the motif CWGVCYTYAGW (SEQ ID NO:337) located at residues corresponding to positions 612-622 in SEQ ID NO:73.

[0096] In some embodiments, a host cell comprising a heterologous polynucleotide encoding a CDS enzyme produces at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% more cucurbitacadienol compound than the same host cell not expressing the heterologous gene.

[0097] In some embodiments, a host cell comprising a heterologous polynucleotide encoding a CDS enzyme produces at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% more cucurbitacinadienol compounds relative to a control host cell (wherein the control host cell expresses a Monk Fruit CDS encoded by a polynucleotide corresponding to SEQ ID NO: 33).

[0098] Throughout this disclosure, host cells expressing any heterologous polynucleotide described in this application can be compared to control host cells. It should be understood that the control host cell can have the same genetic background as the host cell expressing the specific heterologous polynucleotide sequence, unless the control host cell will express the same specific heterologous polynucleotide sequence.

[0099] In other embodiments, based on the percentage consistency between protein and known CDS enzyme, protein can be characterized as CDS enzyme.For example, protein can be consistent with any sequence in the CDS sequence described in the application or with the sequence of any other CDS enzyme by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (including all numerical values ​​therebetween).In other embodiments, based on the presence of one or more domains related to CDS enzyme in protein, protein can be characterized as CDS enzyme.For example, in certain embodiments, based on the presence of substrate channel and / or active site cavity unique to CDS enzyme as known in the art, protein is characterized as CDS enzyme. In some embodiments, the active site cavity includes residues that serve as the entrance to the channel, helping to exclude water from the cavity. In some embodiments, the active site includes residues that serve as proton donors to initiate epoxidation of the substrate and catalyze the cyclization process.

[0100] In other embodiments, a protein may be characterized as a CDS enzyme based on a comparison of the three-dimensional structure of the protein to the three-dimensional structures of known CDS enzymes. It should be understood that the CDS enzyme may be a synthetic protein.

[0101] UDP-Glycosyltransferase (UGT)

[0102] Aspects of the present disclosure provide UDP-glycosyltransferases (UGTs), which can be useful, for example, in the production of mogrosides (e.g., mogroside I-A1 (MIA1), mogroside IE (MIE), mogroside II-A1 (MIIA1), mogroside II-A2 (MIIA2), mogroside III-A1 (MIIIA1), mogroside II-E (MIIE), mogroside III (MIII), simonoside I, mogroside III-E (MIIIE), mogroside IV, mogroside IVa, isomogroside IV, mogroside V, or mogroside VI).

[0103] As used in this application, "UGT" refers to an enzyme that can catalyze the addition of a glycosyl group from a UTP-sugar to a compound (e.g., mogroside or mogroside alcohol). A UGT can be a primary UGT and / or a secondary UGT. A "primary" UGT refers to a UGT that can catalyze the addition of a glycosyl group at a site on a compound that does not include a glycosyl group. For example, a primary UGT can be capable of adding a glycosyl group to the C3 and / or C24 site of an isoprenoid substrate (e.g., mogroside alcohol). See, e.g. Figure 1CA "secondary" UGT is a UGT that is capable of catalyzing the addition of a sugar moiety to a site on a compound that already includes a sugar moiety. See, e.g. Figure 1D As non-limiting examples, a secondary UGT can add a sugar group to mogroside I-A1 (MIA1), mogroside IE (MIE), mogroside II-A1 (MIIA1), mogroside II-A2 (MIIA2), mogroside III-A1 (MIIIA1), mogroside II-E (MIIE), mogroside III (MIII), simonoside I, mogroside III-E (MIIIE), mogroside IV, mogroside IVa, isomogroside IV, mogroside V, and / or mogroside VI.

[0104] Structurally, UGTs generally include a UDPGT (Prosite: PS00375) domain and a catalytic dyad. As a non-limiting example, one of ordinary skill in the art can identify the catalytic dyad in a UGT by aligning the UGT sequence with UGT94-289-1 (a wild-type UGT sequence from monk fruit Siraitia grosvenorii) and identifying the two residues in the UGT corresponding to histidine 21 (H21) and aspartic acid 122 (D122) of UGT94-289-1.

[0105] The amino acid sequence of UGT94-289-1 is:

[0106] MDAQRGHTTTILMFPWLGYGHLSAFLELAKSLSRRNFHIYFCSTSVNLDAIKPKLPSSSSSDSIQLVELCLPSSPDQLPPHLHTTNALPPHLMPTLHQAFSMAAQHFAAILHTLAPHLLIYDSFQPWAPQLASSLNIPAINFNTTGASVLTRMLHATHYPSSKFPISEFVLHDYWKAMYSAAGGAVTKKDHKIGETLANCLHASCSVILINSFRELEEKYMDYLSVLLNKKVVPVGPLVYEPNQDGEDEGYSSIKNWLDKKEPSSTVFVSFGSEYFPSKEEMEEIAHGLEASEVHFIWVVRFPQGDNTSAIEDALPKGFLERVGERGMVVKGWAPQAKILKHWSTGGFVSHCGWNSVMESMMFGVPIIGVPMHLDQPFNAGLAEEAGVGVEAKRDPDGKIQRDEVAKLIKEVVVEKTREDVRKKAREMSEILRSKGEEKMDEMVAAISLFLKI(SEQ ID NO:109).

[0107] Non-limiting examples of nucleic acid sequences encoding UGT94-289-1 are:

[0108]

[0109] One of ordinary skill in the art will be able to recognize how to determine which amino acid residue in any UGT enzyme corresponds to a specific amino acid residue in UGT94-289-1 (SEQ ID NO: 109) by, for example, aligning the sequences and / or by comparing secondary or tertiary structures.

[0110] In certain embodiments, the UGTs of the present disclosure include one or more structural motifs corresponding to the structural motifs in wild-type UGT94-289-1 (e.g., corresponding to the structural motifs shown in Table 5). In some embodiments, the UGTs include structural motifs corresponding to all of the structural motifs in Table 5. In some embodiments, the UGTs include structural motifs corresponding to some (but not all) of the structural motifs in Table 5. In some embodiments, some structural motifs differ by having different lengths or different helicities. For example, the UGTs of the present disclosure may include an extended form of loop 11, loop 16, loop 20, or a combination thereof in UGT94-289-1. The UGTs of the present disclosure may include a loop having a greater helicity than the helicity of its counterpart in UGT94-289-1 (e.g., loop 11, loop 16, loop 20, or a combination thereof in UGT94-289-1).

[0111] In some embodiments, the UGT is a cyclically rearranged form of a reference UGT. In some embodiments, the UGT comprises a sequence comprising at least two motifs from Table 5 in a different order than the reference UGT. For example, if UGT94-289-1 is used as a reference UGT and comprises a first motif located C-terminal to the second motif, the first motif can be located N-terminal to the second motif in the cyclically rearranged UGT.

[0112] The UGT may include one or more motifs corresponding to one or more motifs selected from loop 1, beta sheet 1, loop 2, alpha helix 1, loop 3, beta sheet 2, loop 4, alpha helix 2, loop 5, beta sheet 3, loop 6, alpha helix 3, loop 7, beta sheet 4, loop 8, alpha helix 4, loop 9, beta sheet 5, loop 10, alpha helix 5, loop 11, alpha helix 6, loop 12, alpha helix 7, loop 13, beta sheet 6, loop 14, alpha helix 8, and loop 15 from Table 5, located at a position corresponding to a beta sheet selected from Table 5. , loop 16, alpha helix 9, loop 17, beta sheet 8, loop 18, alpha helix 10, loop 19, beta sheet 9, alpha helix 11, loop 20, alpha helix 12, loop 21, beta sheet 10, loop 22, alpha helix 13, loop 23, beta sheet 11, loop 24, alpha helix 14, loop 25, beta sheet 12, loop 26, alpha helix 15, loop 27, beta sheet 13, loop 28, alpha helix 16, loop 29, alpha helix 17, loop 30, alpha helix 18, and loop 31.

[0113] In some embodiments, the N-terminal portion of the UGT includes a catalytic site, including a catalytic dyad, and / or a substrate-binding site. In some embodiments, the C-terminal portion of the UGT includes a cofactor-binding site. For example, the N-terminal portion of UGT94-289-1 includes a catalytic dyad corresponding to residues 21 and 122 (e.g., histidine 21 and aspartic acid 122) of wild-type UGT94-289-1. The C-terminal portion of UGT94-289-1 includes a cofactor-binding site.

[0114] Aspects disclosed include UGTs that have been cyclically rearranged. In some embodiments, the N-terminal portion and the C-terminal portion can be reversed in whole or in part in the cyclically rearranged form of the UGT. For example, the C-terminal portion of the cyclically rearranged UGT can include a catalytic site, including a catalytic dyad, and / or a substrate-binding site, while the N-terminal portion can include a cofactor-binding site.

[0115] In some embodiments, the cyclically rearranged form of a UGT comprises a heterologous polynucleotide encoding a UGT, wherein the UGT comprises: a catalytic dyad and a cofactor binding site, wherein the catalytic dyad is located C-terminal to the cofactor-binding site.

[0116] The cyclically rearranged UGTs encompassed by the present disclosure can exhibit different properties than the same UGT that has not undergone cyclic rearrangement. In some embodiments, host cells expressing such cyclically rearranged forms of UGTs produce at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% more one or more mogrosides in the presence of at least one mogroside precursor relative to host cells comprising a heterologous polynucleotide encoding a reference UGT that has not undergone cyclic rearrangement (e.g., wild-type UGT94-289-1 (SEQ ID NO: 109)). In some embodiments, a host cell expressing such a cyclically rearranged form of a UGT produces at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% less one or more mogrosides in the presence of at least one mogroside precursor relative to a host cell comprising a heterologous polynucleotide encoding a non-cyclically rearranged reference UGT (e.g., wild-type UGT94-289-1 (SEQ ID NO: 109)).

[0117] In some embodiments, in the cyclically rearranged form of UGT, the N-terminal portion of UGT includes residues that are normally present in the C-terminal portion of UGT (such as UGT94-289-1). In some embodiments, in the cyclically rearranged form of UGT, the C-terminal portion of UGT includes residues that are normally present in the N-terminal portion of UGT (such as UGT94-289-1).

[0118] In some embodiments, the N-terminal portion of a UGT (e.g., UGT94-289-1) corresponds approximately to a UGT (e.g., UGT94-289-1 (SEQ ID NO:109)) of residues 2-122, residues 2-123, residues 2-124, residues 2-125, residues 2-126, residues 2-127, residues 2-128, residues 2-129, residues 2-130, residues 2-131, residues 2-132, residues 2-133, residues 2-134, residues 2-135, residues 2-136, residues 2-137, residues 2-138, residues 2-139, residues 2-140, residues 2-141, residues 2-142, residues 2-143, residues 2-144, residues 2-145, residues 2-146, residues 2-147, residues 2-148, residues 2-149, residues Base 2-150, residue 2-151, residue 2-152, residue 2-153, residue 2-154, residue 2-155, residue 2-156, residue 2 -157, residue 2-158, residue 2-159, residue 2-160, residue 2-161, residue 2-162, residue 2-163, residue 2-16 4. Residue 2-165, residue 2-166, residue 2-167, residue 2-168, residue 2-169, residue 2-170, residue 2-171, Residue 2-172, Residue 2-173, Residue 2-174, Residue 2-175, Residue 2-176, Residue 2-177, Residue 2-178, Residue 2 -179, residue 2-180, residue 2-181, residue 2-182, residue 2-183, residue 2-184, residue 2-185, residue 2-1 86. Residue 2-187, residue 2-188, residue 2-189, residue 2-190, residue 2-191, residue 2-192, residue 2-193, Residues 2-194, Residues 2-195, Residues 2-196, Residues 2-197, Residues 2-198, Residues 2-199, Residues 2-200, Residues 2-201, residue 2-202, residue 2-203, residue 2-204, residue 2-205, residue 2-206, residue 2-207, residue 2-2 08. Residue 2-209, residue 2-210, residue 2-211, residue 2-212, residue 2-213, residue 2-214, residue 2-215 , residue 2-216, residue 2-217, residue 2-218, residue 2-219, residue 2-220, residue 2-221, residue 2-222, residue 2-223, residue 2-224, residue 2-225, residue 2-226, residue 2-227, residue 2-228, residue 2-229, residue 2-2 30. Residue 2-231, residue 2-232, residue 2-233, residue 2-234, residue 2-235, residue 2-236, residue 2-237,residues 2-238, residues 2-239, residues 2-240, residues 2-241, residues 2-242, residues 2-243, residues 2-244, residues 2-245, residues 2-246, residues 2-247, residues 2-248, residues 2-249, residues 2-250, residues 2-251, or residues 2-252, or the corresponding residues in another UGT.

[0119] In some embodiments, the C-terminal domain of a UGT (e.g., UGT94-289-1) corresponds approximately to UGT (e.g., UGT94-289-1 (SEQ ID NO:109)) residues 123-456, residues 124-456, residues 125-456, residues 126-456, residues 127-456, residues 128-456, residues 129-456, residues 130-456, residues 131-456, residues 132-456, residues 133-456, residues 134-456, residues 135-456, residues 136-456, residues 137-456, residues 138-456, residues 139-456, residues 140-456, residues 141-456, residues 142-456, residues 143-456, residues 144-456, residues 145 -456, residues 146-456, residues 147-456, residues 148-456, residues 149-456, residues 150-456, residue 1 51-456, residues 152-456, residues 153-456, residues 154-456, residues 155-456, residues 156-456, residues 157-456, residues 158-456, residues 159-456, residues 160-456, residues 161-456, residues 162-456, residues Base 163-456, residues 164-456, residues 165-456, residues 166-456, residues 167-456, residues 168-456, Residues 169-456, residues 170-456, residues 171-456, residues 172-456, residues 173-456, residues 174-45 6. Residues 175-456, residues 176-456, residues 177-456, residues 178-456, residues 179-456, residues 180-4 56. Residues 181-456, residues 182-456, residues 183-456, residues 184-456, residues 185-456, residues 186- 456, residues 187-456, residues 188-456, residues 189-456, residues 190-456, residues 191-456, residues 192 -456, residues 193-456, residues 194-456, residues 195-456, residues 196-456, residues 197-456, residue 1 98-456, residues 199-456, residues 200-456, residues 201-456, residues 202-456, residues 203-456, residues 204-456, residues 205-456, residues 206-456, residues 207-456, residues 208-456, residues 209-456, residues Base 210-456, residue 211-456, residue 212-456, residue 213-456, residue 214-456, residue 215-456,Residues 216-456, residues 217-456, residues 218-456, residues 219-456, residues 220-456, residues 221-456, residues 222-456, residues 223-456, residues 224-456, residues 22 5-456, residues 226-456, residues 227-456, residues 228-456, residues 229-456, residues 230-456, residues 231-456, residues 232-456, residues 233-456, residues 234-45 6. residues 235-456, residues 236-456, residues 237-456, residues 238-456, residues 239-456, residues 240-456, residues 241-456, residues 242-456, residues 243-456, residues 244-456, residues 245-456, residues 246-456, residues 247-456, residues 248-456, residues 249-456, residues 250-456, or residues 251-456, or the corresponding residues in another UGT.

[0120] In some embodiments, the UGTs disclosed herein include at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, In some embodiments, the present invention relates to a sequence (e.g., a nucleic acid sequence or an amino acid sequence) that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (including all values ​​therebetween) identical to a sequence (e.g., a nucleic acid sequence or an amino acid sequence).

[0121] In some embodiments, the UGTs of the present disclosure can include amino acid substitutions at amino acid residues corresponding to amino acid residues in wild-type UGT94-289-1 (SEQ ID NO: 109). For example, the amino acid residue containing the substitution can be an amino acid corresponding to an amino acid residue in wild-type UGT94-289-1 (SEQ ID NO: 109) selected from, for example, S123; F124; N143; T144; T145; V149; Y179; G18; S180; A181; G184; A185; V186; T187; K189; Y19; H191; K192; G194; E195; A198; F276; N355; H373; L374; N47; H83; T84; T85; N86; P89; and / or L92. Non-limiting examples of such amino acid substitutions include the following substitutions: S123 can be mutated to alanine, cysteine, glycine or valine, or to any conservative substitution of alanine, cysteine, glycine or valine; F124 can be mutated to tyrosine or to any conservative substitution of tyrosine; N143 can be mutated to alanine, cysteine, glutamic acid, isoleucine, leucine, methionine, glutamine, serine, threonine or valine, or to any conservative substitution of alanine, cysteine, glutamic acid, isoleucine, leucine, methionine, glutamine, serine, threonine or valine; T144 can be mutated to alanine, cysteine, asparagine or proline, or to any conservative substitution of alanine, cysteine, asparagine or proline; T145 can be mutated to alanine, cysteine, glycine, methionine, asparagine or proline. amine, glutamine, or serine, or is mutated to any conservative substitution of alanine, cysteine, glycine, methionine, asparagine, glutamine, or serine; V149 can be mutated to cysteine, leucine, or methionine, or is mutated to any conservative substitution of cysteine, leucine, or methionine; Y179 can be mutated to glutamic acid, phenylalanine, histidine, isoleucine, lysine, leucine, valine, or tryptophan, or is mutated to any conservative substitution of glutamic acid, phenylalanine, histidine, isoleucine, lysine, leucine, valine, or tryptophan; G18 can be mutated to serine or is mutated to any conservative substitution of serine; S180 can be mutated to alanine or valine, or is mutated to any conservative substitution of alanine or valine; A181 can be mutated to lysine or threonine, or is mutated to any conservative substitution of lysine or threonine;G184 can be mutated to alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, or tyrosine, or to any conservative substitution of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, or tyrosine; A185 can be mutated to cysteine, aspartic acid, glutamic acid, glycine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, or tyrosine; V186 can be mutated to alanine, cysteine, aspartic acid, glutamic acid, glycine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, threonine, tryptophan, or tyrosine, or to any conservative substitution of cysteine, aspartic acid, glutamic acid, glycine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, threonine, tryptophan, or tyrosine, or to alanine, cysteine, aspartic acid, glutamic acid, glycine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, threonine, tryptophan, or tyrosine. any conservative substitution of asparagine, proline, glutamine, arginine, threonine, tryptophan, or tyrosine; T187 can be mutated to alanine, cysteine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, arginine, serine, valine, tryptophan, or tyrosine, or mutated to any conservative substitution of alanine, cysteine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, arginine, serine, valine, tryptophan, or tyrosine; K189 can be mutated to alanine, cysteine, valine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine, or is mutated to any conservative substitution of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine; Y19 can be mutated to phenylalanine, histidine, leucine, or valine, or is mutated to any conservative substitution of phenylalanine, histidine, leucine, or valine;H191 can be mutated to alanine, cysteine, aspartic acid, glutamic acid, glycine, lysine, methionine, proline, glutamine, serine, threonine, valine, tryptophan, or tyrosine, or to any conservative substitution of alanine, cysteine, aspartic acid, glutamic acid, glycine, lysine, methionine, proline, glutamine, serine, threonine, valine, tryptophan, or tyrosine; K192 can be mutated to cysteine ​​or phenylalanine, or to any conservative substitution of cysteine ​​or phenylalanine; G194 can be mutated to aspartic acid, leucine, methionine, asparagine, proline, serine, or tryptophan, or or any conservative substitution of aspartic acid, leucine, methionine, asparagine, proline, serine, or tryptophan; E195 can be mutated to alanine, isoleucine, lysine, leucine, asparagine, glutamine, serine, threonine, or tyrosine, or any conservative substitution of alanine, isoleucine, lysine, leucine, asparagine, glutamine, serine, threonine, or tyrosine; A198 can be mutated to cysteine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, or tyrosine. , cysteine, aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, or any conservative substitution of tyrosine; F276 can be mutated to cysteine ​​or glutamine, or mutated to any conservative substitution of cysteine ​​or glutamine; N355 can be mutated to glutamine or serine, or any conservative substitution thereof; H373 can be mutated to lysine, leucine, methionine, arginine, valine, or tyrosine, or mutated to any conservative substitution of lysine, leucine, methionine, arginine, valine, or tyrosine. L374 can be mutated to alanine, cysteine, phenylalanine, histidine, methionine, asparagine, glutamine, serine, threonine, valine, tryptophan, or tyrosine, or to any conservative substitution of alanine, cysteine, phenylalanine, histidine, methionine, asparagine, glutamine, serine, threonine, valine, tryptophan, or tyrosine; N47 can be mutated to glycine or to any conservative substitution of glycine; H83 can be mutated to glutamine or tryptophan, or to any conservative substitution of glutamine or tryptophan; T84 can be mutated to tyrosine or to any conservative substitution of tyrosine;T85 may be mutated to glycine, lysine, proline, serine, or tyrosine, or to any conservative substitution of glycine, lysine, proline, serine, or tyrosine; N86 may be mutated to alanine, cysteine, glutamic acid, isoleucine, lysine, leucine, serine, tryptophan, or tyrosine, or to any conservative substitution of alanine, cysteine, glutamic acid, isoleucine, lysine, leucine, serine, tryptophan, or tyrosine; P89 may be mutated to methionine or serine, or to any conservative substitution of methionine or serine; and / or L92 may be mutated to histidine or lysine, or to any conservative substitution of histidine or lysine.;

[0122] One of ordinary skill in the art will be able to recognize how to determine which amino acid residue of any UGT enzyme corresponds to a particular amino acid residue in UGT94-289-1 (SEQ ID NO: 109) by, for example, aligning the sequence and / or by comparing the secondary structure. As a non-limiting example, Fig. 9 A sequence alignment between UGT94-289-1 (SEQ ID NO: 109) and U73C6 (SEQ ID NO: 103) is provided in (e.g., using Clustal Omega for sequence alignment, see, e.g., Larkin et al., Bioinformatics. 2007 Nov 1; 23(21): 2947-8). For example, in Fig. 9 In the example, the residue in U73C6 (SEQ ID NO: 103) corresponding to position 123 in UGT94-289-1 (SEQ ID NO: 109) is methionine (M). As another non-limiting example, the residue in U73C6 (SEQ ID NO: 103) corresponding to position 143 in UGT94-289-1 (SEQ ID NO: 109) is histidine (H) ( Fig. 9 As another non-limiting example, the residue in U73C6 (SEQ ID NO: 103) corresponding to position 273 in UGT94-289-1 (SEQ ID NO: 109) is phenylalanine (F) ( Fig. 9 ).

[0123] In some embodiments, the UGT comprises amino acid substitutions corresponding to the amino acid substitutions in UGT94-289-1 listed in Table 4.

[0124] The UGTs of the present disclosure may include conservative amino acid substitutions and / or non-conservative amino acid substitutions. In some embodiments, the UGTs of the present disclosure include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 conservative amino acid substitutions. In some embodiments, the UGTs of the present disclosure include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 non-conservative amino acid substitutions. In some embodiments, the conservative or non-conservative amino acid substitutions are not located in the conserved region of the UGT protein. In some embodiments, the conservative or non-conservative amino acid substitutions are not located in the region corresponding to residues 83 to 92 of wild-type UGT94-289-1; residues 179 to 198; residue N143; residue L374; residue H21; or residue D122. One of ordinary skill in the art will be able to test UGTs including conservative and / or non-conservative substitutions to determine whether the conservative and / or non-conservative substitutions affect the activity or function of the UGT.

[0125] In some embodiments, the UGT enzyme contains an amino acid replacement located within 10 angstroms, 9 angstroms, 8 angstroms, 7 angstroms, 6 angstroms, 5 angstroms, 4 angstroms, 3 angstroms, 2 angstroms, or 1 angstrom (including all values ​​therebetween) of the catalytic dyad. The catalytic dyad can correspond to residues 21 and 122 of wild-type UGT94-289-1 (e.g., histidine 21 and aspartic acid 122). It should be understood that one of ordinary skill in the art will be able to recognize how to determine the corresponding positions of the catalytic dyad in any UGT enzyme by, for example, aligning the sequence with UGT94-289-1 (SEQ ID NO: 109) and / or by comparing the secondary structure with UGT94-289-1 (SEQ ID NO: 109).

[0126] In some embodiments, the UGT enzyme contains an amino acid substitution at an amino acid residue located in one or more structural motifs of the UGT. Non-limiting examples of secondary structures in a UGT such as UGT94-289-1 (SEQ ID NO: 109) include: a loop between beta sheet 4 and alpha helix 5; beta sheet 5; a loop between beta sheet 5 and alpha helix 6; alpha helix 6; a loop between alpha helix 6 and alpha helix 7; a loop between beta sheet 1 and alpha helix 1; alpha helix 7; a loop between alpha helix 7 and alpha helix 8; alpha helix 1; alpha helix 8; a loop between beta sheet 8 and alpha helix 13; alpha helix 17; a loop between beta sheet 12 and alpha helix 18; alpha helix 2; a loop between beta sheet 3 and alpha helix 3; alpha helix 3; and a loop between alpha helix 3 and alpha helix 4; loop 8; beta sheet 5; loop 10; alpha helix 5; loop 11; loop 2; alpha helix 6; loop 12; alpha helix 1; alpha helix 7; loop 18; alpha helix 14; loop 26; alpha helix 2; loop 6; and alpha helix 3.

[0127] In some embodiments, the amino acid residue located in loop 8 is the residue corresponding to S123 or F124 in UGT94-289-1 (SEQ ID NO: 109); the amino acid residue located in beta sheet 5 is the residue corresponding to N143 in UGT94-289-1 (SEQ ID NO: 109); the amino acid residue located in loop 10 is the residue corresponding to T144 or T145 in UGT94-289-1 (SEQ ID NO: 109); the amino acid residue located in alpha helix 5 is the residue corresponding to V149 in UGT94-289-1 (SEQ ID NO: 109); the amino acid residue located in loop 11 is the residue corresponding to Y179 in UGT94-289-1 (SEQ ID NO: 109); the amino acid residue located in loop 2 is the residue corresponding to the amino acid residue located in alpha helix 6 is a residue corresponding to S180 or A181 in UGT94-289-1 (SEQ ID NO: 109); the amino acid residue located in loop 12 is a residue corresponding to G184, A185, V186, T187, or K189 in UGT94-289-1 (SEQ ID NO: 109); the amino acid residue located in alpha helix 1 is a residue corresponding to Y19 in UGT94-289-1 (SEQ ID NO: 109); the amino acid residue located in alpha helix 7 is a residue corresponding to H191, K192, G194, E195, or A198 in UGT94-289-1 (SEQ ID NO: 109); the amino acid residue located in loop 18 is a residue corresponding to the amino acid residue located in alpha helix 14 is the residue corresponding to N355 in UGT94-289-1 (SEQ ID NO: 109); the amino acid residue located in loop 26 is the residue corresponding to H373 or L374 in UGT94-289-1 (SEQ ID NO: 109); the amino acid residue located in alpha helix 2 is the residue corresponding to N47 in UGT94-289-1 (SEQ ID NO: 109); the amino acid residue located in loop 6 is the residue corresponding to H83, T84, T85 or N86 in UGT94-289-1 (SEQ ID NO: 109); and / or the amino acid residue located in alpha helix 3 is the residue corresponding to P89 or L92 in UGT94-289-1 (SEQ ID NO: 109).

[0128] In some embodiments, the UGT includes amino acid substitutions (e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8 substitutions) in a region corresponding to residues 83 to 92, residues 179 to 189, residues 1 to 82, residues 93 to 142, residues 144 to 178, residues 199 to 373, or residues 375 to 453 of UGT94-289-1 (SEQ ID NO: 109). In some embodiments, the UGT includes amino acid substitutions at amino acid residues corresponding to amino acid residues selected from N143 and L374 in wild-type UGT94-289-1 (SEQ ID NO: 109). In some embodiments, the residue corresponding to N143 is mutated to a negatively charged R group, a polar uncharged R group, or a non-polar aliphatic R group. In some embodiments, the residue corresponding to L374 is mutated to a non-polar aliphatic R group, a positively charged R group, a polar uncharged R group, or a non-polar aromatic R group. In some embodiments, the UGT includes a region at least 90% identical to residues 83 to 92 of UGT94-289-1 or at least 95% identical to residues 179 to 198 of UGT94-289-1. The UGT can include a region that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical, or 100% identical to residues 83 to 92 of UGT94-289-1. The UGT can include a region that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical, or 100% identical to residues 179 to 198 of UGT94-289-1 (SEQ ID NO: 109).

[0129] In some embodiments, the host cell includes a heterologous polynucleotide encoding a UGT, wherein the UGT includes the following regions: a region corresponding to residues 83 to 92 of wild-type UGT94-289-1 (SEQ ID NO: 109), wherein the region includes amino acid substitutions relative to residues 83 to 92 of wild-type UGT94-289-1 (SEQ ID NO: 109); and / or a region corresponding to residues 179 to 198 of wild-type UGT94-289-1 (SEQ ID NO: 109), wherein the region includes amino acid substitutions relative to residues 179 to 198 of wild-type UGT94-289-1 (SEQ ID NO: 109). It should be understood that the language "amino acid substitution" is not limited to one amino acid substitution, but also encompasses embodiments comprising more than one amino acid substitution. In some embodiments, the host cell includes a heterologous polynucleotide encoding a UGT, wherein the UGT includes a region corresponding to residues 83 to 92 of wild-type UGT94-289-1 (SEQ ID NO: 109), wherein the region includes no more than one amino acid substitution relative to residues 83 to 92 of wild-type UGT94-289-1 (SEQ ID NO: 109); and / or a region corresponding to residues 179 to 198 of wild-type UGT94-289-1 (SEQ ID NO: 109), wherein the region includes no more than one amino acid substitution relative to residues 179 to 198 of wild-type UGT94-289-1 (SEQ ID NO: 109).

[0130] In some embodiments, the UGT comprises an amino acid replacement at an amino acid residue corresponding to an amino acid residue in wild-type UGT94-289-1 (SEQ ID NO: 109) selected from the group consisting of: H83, T84, T85, N86, P89, L92, Y179, S180, A181, G184, A185, V186, T187, K189, H191, K192, G194, E195, or A198.

[0131] In some embodiments, the residue corresponding to H83 is mutated to an amino acid comprising a polar uncharged R group or a nonpolar aromatic R group; the residue corresponding to T84 is mutated to an amino acid comprising a nonpolar aromatic R group; the residue corresponding to T85 is mutated to an amino acid comprising a nonpolar aliphatic R group, a positively charged R group, a polar uncharged R group, or a nonpolar aromatic R group; the residue corresponding to N86 is mutated to an amino acid comprising a nonpolar aliphatic R group, a polar uncharged R group, a negatively charged R group, a positively charged R group, or a nonpolar aromatic R group; the residue corresponding to P89 is mutated to an amino acid comprising a nonpolar aliphatic R group, or a polar uncharged R group; The residue corresponding to L92 is mutated to an amino acid including a positively charged R group; the residue corresponding to Y179 is mutated to an amino acid including a negatively charged R group, a non-polar aromatic R group, a positively charged R group, or a non-polar aliphatic R group; the residue corresponding to S180 is mutated to an amino acid including a non-polar aliphatic R group; the residue corresponding to A181 is mutated to an amino acid including a positively charged R group or a polar uncharged R group; the residue corresponding to G184 is mutated to an amino acid including a non-polar aliphatic R group, a polar uncharged R group, a negatively charged R group, a non-polar aromatic R group, or a positively charged R group; the residue corresponding to A185 is mutated to an amino acid including a The residue corresponding to V186 is mutated to an amino acid including a nonpolar aliphatic R group, a polar uncharged R group, a negatively charged R group, a nonpolar aliphatic R group, a positively charged R group, or a nonpolar aromatic R group; the residue corresponding to T187 is mutated to an amino acid including a nonpolar aliphatic R group, a polar uncharged R group, a negatively charged R group, a positively charged R group, or a nonpolar aromatic R group; the residue corresponding to K189 is mutated to an amino acid including a nonpolar aliphatic R group, a polar uncharged R group, a negatively charged R group, a nonpolar aromatic R group, or a nonpolar aromatic R group. The residue corresponding to H191 is mutated to an amino acid including a nonpolar aliphatic R group, a polar uncharged R group, a negatively charged R group, a positively charged R group, or a nonpolar aromatic R group; the residue corresponding to K192 is mutated to an amino acid including a polar uncharged R group or a nonpolar aromatic R group; the residue corresponding to G194 is mutated to an amino acid including a negatively charged R group, a nonpolar aliphatic R group, a polar uncharged R group, or a nonpolar aromatic R group; the residue corresponding to E195 is mutated to an amino acid including a nonpolar aliphatic R group, a positively charged R group, a polar uncharged R group, or a nonpolar aromatic R group;and / or the residue corresponding to A198 is mutated to an amino acid comprising a polar uncharged R group, a negatively charged R group, a non-polar aromatic R group, a positively charged R, or a non-polar aliphatic R group.;

[0132] In some embodiments, the UGT comprises an amino acid replacement at an amino acid residue corresponding to an amino acid residue selected from N143 and L374 in wild-type UGT94-289-1 (SEQ ID NO: 109). In some embodiments, the residue corresponding to N143 is mutated to a negatively charged R group, a polar uncharged R group, or a non-polar aliphatic R group. In some embodiments, the residue corresponding to L374 is mutated to a non-polar aliphatic R group, a positively charged R group, a polar uncharged R group, or a non-polar aromatic R group.

[0133] The UGT of the present disclosure may be capable of glycosylation of mogroside or mogroside at any oxygen-containing position (e.g., C3, C11, C24, and C25). In some embodiments, the UGT is capable of branched glycosylation (e.g., branched glycosylation of mogroside at C3 or C24).

[0134] Non-limiting examples of substrates suitable for use with the UGTs of the present disclosure include mogroside and mogrosides (e.g., mogroside IA1 (MIA1), mogroside IE (MIE), mogroside II-A1 (MIIA1), mogroside III-A1 (MIIIA1), mogroside II-E (MIIE), mogroside III (MIII), or mogroside III-E (MIIIE), simonoside I).

[0135] In some embodiments, the UGTs of the present disclosure are capable of producing mogroside IA1 (MIA1), mogroside IE (MIE), mogroside II-A1 (MIIA1), mogroside II-A2 (MIIA2), mogroside III-A1 (MIIIA1), mogroside II-E (MIIE), mogroside III (MIII), simonoside I, mogroside III-E (MIIIE), mogroside IV, mogroside IVa, isomogroside IV, and / or mogroside V.

[0136] In some embodiments, the UGT is capable of catalyzing the following transformations: transformation of mogroside to MIA1; transformation of mogroside to MIE1; transformation of MIA1 to MIIA1; transformation of MIE1 to MIIE; transformation of MIIA1 to MIIIA1; transformation of MIA1 to MIIE; transformation of MIIA1 to MIII; transformation of MIIIA1 to simenoside I; transformation of MIIE to MIII; transformation of MIII to simenoside I; transformation of MIIE to MIIE; and / or transformation of MIIIE to simenoside I.

[0137] It should be understood that the activity (e.g., specific activity) of a UGT can be measured by any means known to those of ordinary skill in the art. In some embodiments, the activity (e.g., specific activity) of a UGT (e.g., a variant UGT) can be determined by measuring the amount of glycosylated mogroside produced per unit of enzyme per unit time. For example, the activity (e.g., specific activity) can be measured per gram of enzyme produced per hour of glycosylated mogroside target. Non-limiting examples of methods for measuring activity (e.g., specific activity) are provided in the Examples below. In some embodiments, a UGT (e.g., a variant UGT) of the present disclosure can have an activity (e.g., specific activity) of at least 0.1 mmol (e.g., at least 1 mmol, at least 1.5 mmol, at least 2 mmol, at least 2.5 mmol, at least 3, at least 3.5 mmol, at least 4 mmol, at least 4.5 mmol, at least 5 mmol, at least 10 mmol, including all values ​​therebetween) of glycosylated mogroside target produced per gram of enzyme per hour.

[0138] In some embodiments, the activity (e.g., specific activity) of a UGT of the present disclosure is at least 1.1 times greater (e.g., at least 1.3 times, at least 1.5 times, at least 1.7 times, at least 1.9 times, at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, or at least 100 times (including all values ​​in between) than the activity of a control UGT. In some embodiments, the control UGT is UGT94-289-1 (SEQ ID NO: 109). In some embodiments, for a UGT with an amino acid substitution, the control UGT is the same UGT without the amino acid substitution.

[0139] It should be understood that one of ordinary skill in the art will be able to characterize a protein as a UGT enzyme based on structural and / or functional information associated with the protein. For example, a protein may be characterized as a UGT enzyme based on its function, such as the ability to produce one or more mogrosides in the presence of a mogroside precursor, such as mogrosanol.

[0140] In other embodiments, the protein can be characterized as a UGT enzyme based on the percent consistency between the protein and a known UGT enzyme. For example, the protein can be at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (including all values ​​therebetween) consistent with any sequence in the UGT sequence described in the application. In other embodiments, the protein can be characterized as a UGT enzyme based on the presence of one or more domains associated with the UGT enzyme in the protein. For example, in certain embodiments, the protein is characterized as a UGT enzyme based on the presence of a sugar binding domain and / or a catalytic domain unique to a UGT enzyme known in the art. In certain embodiments, the catalytic domain binds to a substrate to be glycosylated.

[0141] In other embodiments, a protein can be characterized as a UGT enzyme based on a comparison of the three-dimensional structure of the protein to the three-dimensional structure of a known UGT enzyme. For example, a protein can be characterized as a UGT based on the number or location of alpha helical domains, beta pleated domains, etc. It should be understood that a UGT enzyme can be a synthetic protein.

[0142] In some embodiments, the UGT does not include the sequence of SEQ ID NO: 109. In some embodiments, the UGT includes less than 95%, less than 94%, less than 93%, less than 92%, less than 91%, less than 90%, less than 89%, less than 88%, less than 87%, less than 86%, less than 85%, less than 84%, less than 83%, less than 82%, less than 81%, less than 80%, less than 79%, less than 78%, less than 77%, less than 76%, less than 75%, less than 74%, less than 73%, less than 72%, less than 71%, or less than 70% identity to SEQ ID NO: 109.

[0143] C11 Hydroxylase

[0144] Aspects of the present disclosure provide C11 hydroxylases that can be useful, for example, in the production of mogrohol.

[0145] The C11 hydroxylase disclosed herein may include a C11 hydroxylase sequence (e.g., a nucleic acid sequence or an amino acid sequence) in Table 8, a sequence as set forth in SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 129, or SEQ ID NO: 130, or any C11 hydroxylase sequence disclosed in this application or known in the art that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111 In some embodiments, the present invention relates to sequences that are at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical (including all values ​​in between).

[0146] In some embodiments, the C11 hydroxylase of the present disclosure is capable of oxidizing a mogrosanol precursor (e.g., cucurbitacinol, 11-hydroxycucurbitacinol, 24,25-dihydroxy-cucurbitacinol, and / or 24,25-epoxy-cucurbitacinol). In some embodiments, the C11 hydroxylase of the present disclosure catalyzes the formation of mogrosanol.

[0147] It should be understood that the activity (e.g., specific activity) of the C11 hydroxylase can be determined by any means known to those of ordinary skill in the art. In some embodiments, the activity (e.g., specific activity) of the C11 hydroxylase can be measured as the concentration of the mogrosanol precursor or mogrosanol produced per unit of enzyme per unit time. In some embodiments, the C11 hydroxylase of the present disclosure has an activity (e.g., specific activity) of at least 0.0001-0.001 μmol / min / mg, at least 0.001-0.01 μmol / min / mg, at least 0.01-0.1 μmol / min / mg, or at least 0.1-1 μmol / min / mg (including all values ​​therebetween).

[0148] In some embodiments, the activity (e.g., specific activity) of the C11 hydroxylase is at least 1.1 times greater than the activity of a control C11 hydroxylase (e.g., at least 1.3 times, at least 1.5 times, at least 1.7 times, at least 1.9 times, at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 1000 times, or at least 10000 times (including all values ​​in between)).

[0149] Cytochrome P450 Reductase

[0150] Aspects of the present disclosure provide cytochrome P450 reductases, which can be useful, for example, in the production of mogrohol. Cytochrome P450 reductases can also refer to NADPH: methemoglobin oxidoreductase, NADPH: heme protein oxidoreductase, NADPH: P450 oxidoreductase, P450 reductase, POR, CPR, and CYPOR. These reductases can promote C11 hydroxylase activity by catalyzing electron transfer from NADPH to C11 hydroxylase.

[0151] The cytochrome P450 reductases disclosed herein may include at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, Sequences that are at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% (including all values ​​in between) identical.

[0152] In some embodiments, the cytochrome P450 reductase of the present disclosure is capable of promoting the oxidation of a mogrosanol precursor (e.g., cucurbitacinol, 11-hydroxycucurbitacinol, 24,25-dihydroxy-cucurbitacinol, and / or 24,25-epoxy-cucurbitacinol). In some embodiments, the P450 reductase of the present disclosure catalyzes the formation of a mogrosanol precursor or mogrosanol.

[0153] It should be understood that the activity (e.g., specific activity) of cytochrome P450 reductase can be measured by any means known to those of ordinary skill in the art. In some embodiments, the activity (e.g., specific activity) of recombinant cytochrome P450 reductase can be measured as the concentration of mogrosanol precursor or mogrosanol produced per unit of enzyme per unit time in the presence of C11 hydroxylase. In some embodiments, the cytochrome P450 reductase of the present disclosure has an activity (e.g., specific activity) of at least 0.0001-0.001 μmol / min / mg, at least 0.001-0.01 μmol / min / mg, at least 0.01-0.1 μmol / min / mg, or at least 0.1-1 μmol / min / mg (including all values ​​therebetween).

[0154] In some embodiments, the activity (e.g., specific activity) of the cytochrome P450 reductase is at least 1.1 times greater than the activity of a control cytochrome P450 reductase (e.g., at least 1.3 times, at least 1.5 times, at least 1.7 times, at least 1.9 times, at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 1000 times, or at least 10000 times (including all values ​​in between)).

[0155] Cyclooxygenase (EPH)

[0156] Aspects of the present disclosure provide epoxidases (EPHs) that can be useful, for example, in the conversion of 24-25-epoxy-cucurbitacinaldehyde to 24-25-dihydroxy-cucurbitacinaldehyde or in the conversion of 11-hydroxy-24,25-epoxycucurbitacinaldehyde to mogrosanol.EPHs are capable of converting epoxides to two hydroxyl groups.

[0157] The EPHs disclosed herein may include at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, %, at least 97%, at least 98%, at least 99%, or at least 100% identical to the sequences in the invention.

[0158] In some embodiments, the recombinant EPH of the present disclosure is capable of promoting the hydrolysis of the epoxide in the cucurbitacinaldehyde compound (e.g., the hydrolysis of the epoxide in 24-25-epoxy-cucurbitacinaldehyde). In some embodiments, the EPH of the present disclosure catalyzes the formation of a mogrosanol precursor (e.g., 24-25 dihydroxy-cucurbitacinaldehyde).

[0159] It should be understood that the activity (e.g., specific activity) of EPH can be measured by any means known to those of ordinary skill in the art. In some embodiments, the activity (e.g., specific activity) of EPH can be measured as the concentration of the produced mogrosanol precursor (e.g., 24-25 dihydroxy-cucurbitacinadienol) or mogrosanol. In some embodiments, the recombinant EPH of the present disclosure will allow the production of at least 1-100 μg / L, at least 100-1000 μg / L, at least 1-100 mg / L, at least 100-1000 mg / L, at least 1-10 g / L, or at least 10-100 g / L (including all values ​​therebetween).

[0160] In some embodiments, the activity (e.g., specific activity) of the EPH is at least 1.1 times greater than the activity of a control EPH (e.g., at least 1.3 times, at least 1.5 times, at least 1.7 times, at least 1.9 times, at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, or at least 100 times (including all values ​​therebetween)).

[0161] Squalene epoxidase (SQE)

[0162] Aspects of the present disclosure provide squalene epoxidase (SQE) capable of oxidizing squalene (eg, squalene or 2-3-oxidosqualene) to produce squalene epoxide (eg, 2-3-oxidosqualene or 2-3,22-23-diepoxysqualene). SQE may also be referred to as squalene monooxygenase.

[0163] The SQEs disclosed herein may include an SQE sequence (e.g., a nucleic acid sequence or an amino acid sequence) in Table 8, a sequence as listed in SEQ ID NOs: 126-128 or SEQ ID NOs: 142-144, or any SQE sequence disclosed herein or known in the art that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, %, at least 97%, at least 98%, at least 99%, or at least 100% identical to the sequences in the invention.

[0164] In some embodiments, the SQE of the present disclosure is capable of promoting epoxide formation in squalene compounds (e.g., epoxidation in squalene or 2,3-squalene oxide). In some embodiments, the SQE of the present disclosure catalyzes the formation of a mogrosanol precursor (e.g., 2-3-squalene oxide or 2-3,22-23-diepoxysqualene).

[0165] The activity (e.g., specific activity) of the recombinant SQE can be measured as the concentration of the mogrosanol precursor (e.g., 2-3-oxidosqualene or 2-3,22-23-diepoxysqualene) produced per unit of enzyme per unit time. In some embodiments, the SQE of the present disclosure has an activity (e.g., specific activity) of at least 0.0000001 μmol / min / mg (e.g., at least 0.000001 μmol / min / mg, at least 0.00001 μmol / min / mg, at least 0.0001 μmol / min / mg, at least 0.001 μmol / min / mg, at least 0.01 μmol / min / mg, at least 0.1 μmol / min / mg, at least 1 μmol / min / mg, at least 10 μmol / min / mg, or at least 100 μmol / min / mg (including all values ​​therebetween)).

[0166] In some embodiments, the activity (e.g., specific activity) of the SQE is at least 1.1 times greater than the activity of a control SQE (e.g., at least 1.3 times, at least 1.5 times, at least 1.7 times, at least 1.9 times, at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, or at least 100 times (including all values ​​therebetween)).

[0167] Variants

[0168] Aspects of the disclosure relate to polynucleotides encoding any of the described recombinant polypeptides (such as CDS, UGT, C11 hydroxylase, cytochrome P450 reductase, and EPH and SQE enzymes). Variants of the polynucleotide or amino acid sequences described in this application are also encompassed by the disclosure. A variant may share at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, inclusive, of sequence identity with the reference sequence.

[0169] Unless otherwise indicated, the term "sequence identity", as known in the art, refers to the relationship between the sequences of two polypeptides or polynucleotides determined by sequence comparison (alignment). In some embodiments, sequence identity is determined over the entire length of the sequence, while in other embodiments, sequence identity is determined over a region of the sequence.

[0170] Identity can also refer to the degree of sequence relatedness between two sequences as determined by the number of matches between strings of two or more residues (e.g., nucleic acid residues or amino acid residues). Identity measures the percentage of identical matches between the smaller of two or more sequences, with gap alignments (if any) resolved by a specific mathematical model, algorithm or computer program.

[0171] The identity of related polypeptide or nucleic acid sequences can be readily calculated by any method known to those of ordinary skill in the art. The "percent identity" of two sequences (e.g., nucleic acid sequences or amino acid sequences) can be determined, for example, using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, as modified in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993). Such algorithms are incorporated into the algorithm of Altschul et al. and Program (version 2.0) (J. Mol. Biol. 215: 403-10, 1990). Protein search (e.g., using XBLAST program, score = 50, word length = 3 to obtain amino acid sequences homologous to the protein molecules of the present invention). When there is a gap between the two sequences, Gapped (For example, as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997). and Gapped When you use a program, you can use each program (for example, XBL and ) or the parameters may be appropriately adjusted as would be understood by one of ordinary skill in the art.

[0172] Another local alignment technique that can be used is, for example, based on the Smith-Waterman algorithm (Smith, TF & Waterman, MS (1981) "Identification of common molecular subsequences." J. Mol. Biol. 147: 195-197). A general global alignment technique that can be used is, for example, the Needleman–Wunsch algorithm (Needleman, SB & Wunsch, CD (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol. Biol. 48: 443-453), which is based on a dynamic program.

[0173] Recently, a fast optimal global sequence alignment algorithm (FOGSAA) has been developed, which is said to generate a global alignment of nucleic acid and amino acid sequences faster than other optimal global alignment methods (including Needleman-Wunsch algorithm). In some embodiments, the consistency of two polypeptides is determined by comparing two amino acid sequences, calculating the number of consistent amino acids and dividing by the length of one of the amino acid sequences. In some embodiments, the consistency of two nucleic acids is determined by comparing two nucleoside sequences and calculating the number of consistent nucleosides and dividing by the length of one of the nucleic acids.

[0174] For multiple sequence alignment, computer programs including Clustal Omega (Sievers et al., Mol Syst Biol. 2011 Oct 11; 7:539) can be used.

[0175] It should be understood that sequences (including nucleic acid sequences or amino acid sequences) can be found to have a specific percentage identity with a reference sequence (e.g., a sequence disclosed in the present application and / or defined in the claims) using any method known to those of ordinary skill in the art. Different algorithms can produce different percentage identity values ​​for a given set of sequences. The claims of the present application should be understood to cover sequences whose percentage identity with a reference sequence is calculated using default parameters and / or parameters commonly used by those skilled in the art for a given algorithm.

[0176] As used in this application, when a sequence X and a different sequence Y are aligned using an amino acid sequence alignment tool known in the art, a residue (such as a nucleic acid residue or an amino acid residue) in sequence "X" is referred to as corresponding to a position or residue (such as a nucleic acid residue or an amino acid residue) "z" in different sequence "Y" when the residue (such as a nucleic acid residue or an amino acid residue) in sequence "X" is located at the corresponding position of "z" in sequence "Y".

[0177] Variant sequences can be homologous sequences. As used herein, homologous sequences are sequences that share a particular percentage identity (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, At least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (including all values ​​in between) percentage identity) sequence (e.g., nucleic acid sequence or amino acid sequence). Homologous sequences include, but are not limited to, parallel homologous sequences, orthologous sequences, or sequences produced by convergent evolution. Paralogous sequences originate from the duplication of genes within the genome of a species, while orthologous sequences differ after a speciation event. Two different species may evolve independently, but as a result of convergent evolution, each species may include sequences that share a specific percentage identity with sequences from another species.

[0178] In some embodiments, the polypeptide variant (e.g., CDS, UGT, C11 hydroxylase, cytochrome P450 reductase, EPH, or SQE variant) includes a domain that shares a secondary structure (e.g., alpha helix, beta sheet) with a reference polypeptide (e.g., reference CDS, reference UGT, reference C11 hydroxylase, reference cytochrome P450 reductase, reference EPH, or reference SQE). In some embodiments, the polypeptide variant (e.g., CDS, UGT, C11 hydroxylase, cytochrome P450 reductase, EPH, or SQE variant) shares a tertiary structure with a reference polypeptide (e.g., reference CDS, reference UGT, reference C11 hydroxylase, reference cytochrome P450 reductase, reference EPH, or reference SQE). As a non-limiting example, a variant polypeptide may have a low primary sequence identity (e.g., less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% sequence identity) compared to a reference polypeptide, but share one or more secondary structures (e.g., including but not limited to loops, alpha helices, or beta sheets), or have the same tertiary structure as the reference polypeptide. For example, a loop may be located between a beta sheet and an alpha helix, between two alpha helices, or between two beta sheets. Homology simulations can be used to compare two or more tertiary structures.

[0179] Mutation can be performed in the nucleoside sequence by a variety of methods known to those of ordinary skill in the art. For example, it can be performed by PCR directed mutation, according to Kunkel's method (Kunkel, Proc. Nat. Acad. Sci. USA 82: 488-492, 1985) site-directed mutagenesis, by chemical synthesis of genes encoding polypeptides, by gene editing tools, or by insertion (e.g., inserting tags (e.g., HIS tags or GFP tags)). Mutation can include, for example, displacement, deletion, and transposition generated by methods known in the art. The method for generating mutations can be found in references (e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012 or Current Protocols in Molecular Biology, FM Ausubel, et al., eds., John Wiley & Sons, Inc., New York, 2010).

[0180] In some embodiments, the method for generating variants comprises cyclic shuffling (Yu and Lutz, Trends Biotechnol. 2011 Jan; 29(1): 18-25). Non-limiting examples of cyclic shuffling are provided in Example 5 and the accompanying Figure 7 In the cyclic rearrangement, the linear primary sequence of the polypeptide can be cyclized (e.g., by connecting the N-terminus and C-terminus of the sequence) and the polypeptide can be cut ("broken") at different positions. Therefore, as determined by a linear sequence alignment method (e.g., Clustal Omega or BLAST), the linear primary sequence of the new polypeptide can have a low sequence identity (e.g., less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10% or less than 5% (including all values ​​in between)). However, topological analysis of the two proteins can reveal whether the tertiary structures of the two polypeptides are similar or dissimilar. Without being bound by a particular theory, variant polypeptides created by cyclic rearrangement of a reference polypeptide and having a similar tertiary structure to the reference polypeptide can share similar functional properties (e.g., enzymatic activity, enzyme kinetics, substrate specificity or product specificity). In some cases. Cyclic rearrangements can alter secondary, tertiary or quaternary structures and produce enzymes with different functional properties (e.g., increased or decreased enzymatic activity, different substrate specificity or different product specificity). See, e.g., Yu and Lutz, Trends Biotechnol. 2011 Jan; 29(1): 18-25.

[0181] It should be understood that in a protein that has undergone cyclic rearrangement, the linear amino acid sequence of the protein will be different from a reference protein that has not undergone cyclic rearrangement. However, one of ordinary skill in the art will be able to determine which residue in the protein that has undergone cyclic rearrangement corresponds to a residue in the reference protein that has not undergone cyclic rearrangement by, for example, aligning the sequences and detecting conserved motifs, and / or by comparing the structure or predicted structure of the protein (e.g., by homology modeling).

[0182] In some embodiments, the algorithm for determining the percent identity between a sequence of interest and a reference sequence described in the present application illustrates that there is a cyclic rearrangement between the sequence. Methods known in the art can be used (for example, including RASPODOM (Weiner et al., Bioinformatics.2005Apr 1; ​​21 (7): 932-7) to detect the presence of a cyclic rearrangement. In some embodiments, before calculating the percent identity between a sequence of interest and a sequence described in the present application, the presence of a cyclic arrangement is corrected (for example, the domain in at least one sequence is rearranged). The claims of the present application should be understood to encompass the sequence of calculating the percent identity of itself and a reference sequence after considering the cyclic rearrangement of a potential sequence.

[0183] The present disclosure also encompasses functional variants of the recombinant CDS, UGT, C11 hydroxylase, cytochrome P450 reductase, EPH, and squalene epoxidase disclosed in the present application. For example, the functional variant can bind to one or more of the same substrates (e.g., mogroside, mogroside, or a precursor thereof) or produce one or more of the same products (e.g., mogroside, mogroside, or a precursor thereof). Functional variants can be identified using any method known in the art. For example, the algorithm of Karlin and Altschul described above (Proc. Natl. Acad. Sci. USA 87:2264-68, 1990) can be used to identify homologous proteins with known functions.

[0184] Putative functional variants can also be identified by searching for polypeptides with functional annotated domains. Databases (including Pfam (Sonnhammer et al., Proteins. 1997 Jul; 28 (3): 405-20)) can be used to identify polypeptides with specific domains. For example, in some cases, additional CDS enzymes in oxidosqualene cyclase can be identified by searching for polypeptides with a leucine residue corresponding to position 123 of SEQ ID NO: 74. This leucine residue is related to determining the product specificity of the CDS enzyme; mutations in this residue can, for example, result in cycloartenol or parkinol as products. (Takase et al., Org Biomol Chem. 2015 Jul 13 (26): 7331-6).

[0185] Additional UGT enzymes can be identified, for example, by searching for polypeptides having a UDPGT domain (PROSITE Accession No. PS00375).

[0186] Homology simulations can also be used to identify amino acid residues that are susceptible to mutation without affecting function. Non-limiting examples of such methods can include the use of site-specific scoring matrices (PSSMs) and energy minimization schemes (see, e.g., Example 5 below). See, e.g., Stormo et al., Nucleic Acids Res. 1982 May 11; 10(9): 2997-3011.

[0187] The PSSM can be coupled with the calculation of the Rosetta energy function, which determines the difference between the wild type and single point mutants. Without being bound by a particular theory, potential stabilizing mutations are desirable for protein engineering (e.g., generation of functional homologs). In some embodiments, potential stabilizing mutations have a ΔΔG of less than -0.1 (e.g., less than -0.2, less than -0.3, less than -0.35, less than -0.4, less than -0.45, less than -0.5, less than -0.55, less than -0.6, less than -0.65, less than -0.7, less than -0.75, less than -0.8, less than -0.85, less than -0.9, less than -0.95, or less than -1.0) Rosetta energy units (Reu). calc Values. See, e.g., Goldenzweig et al., Mol Cell. 2016 Jul 21; 63(2): 337-346. doi: 10.1016 / j.molcel.2016.06.012.

[0188] In some embodiments, a CDS, UGT, C11 hydroxylase, cytochrome P450 reductase, EPH, or SQE coding sequence includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 , 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more mutations at the sites. In some embodiments, a CDS, UGT, C11 hydroxylase, cytochrome P450 reductase, EPH, or SQE coding sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 , 44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100 or more mutations in a codon. As will be appreciated by those of ordinary skill in the art, due to the degeneracy of the genetic code, mutations within a codon may or may not change the amino acid encoded by the codon. In some embodiments, one or more mutations in a coding sequence do not change the amino acid sequence of the coding sequence relative to the amino acid sequence of a reference polypeptide.

[0189] In some embodiments, one or more mutations in a recombinant CDS, UGT, C11 hydroxylase, cytochrome P450 reductase, EPH, or SQE sequence change the amino acid sequence of the polypeptide relative to the amino acid sequence of a reference polypeptide. In some embodiments, one or more mutations change the amino acid sequence of a recombinant polypeptide relative to the amino acid sequence of a reference polypeptide and change (enhance or decrease) the activity of the polypeptide relative to the reference polypeptide.

[0190] Conventional methods can be used to measure the activity (including specific activity) of any recombinant polypeptide in the recombinant polypeptide described in the application. As a non-limiting example, the activity of the recombinant polypeptide can be determined by measuring the substrate specificity of the recombinant polypeptide, one or more products produced, the concentration of one or more products produced, or a combination thereof. As used in the application, the specific activity of a recombinant polypeptide refers to the amount (e.g., concentration) of a specific product produced by a recombinant polypeptide of a given amount (e.g., concentration) per unit time.

[0191] Those skilled in the art will also recognize that mutations in a recombinant polypeptide coding sequence may result in conservative amino acid substitutions to provide functionally equivalent variants of the aforementioned polypeptides (e.g., variants that retain the activity of the polypeptide). As used in this application, "conservative amino acid substitutions" refer to amino acid substitutions that do not change the relative charge or size characteristics, or functional activity, of the protein to which the amino acid substitution is made.

[0192] In some cases, amino acids are characterized by their R groups (see, e.g., Table 1). For example, amino acids can include non-polar aliphatic R groups, positively charged R groups, negatively charged R groups, non-polar aromatic R groups, or polar uncharged R groups. Non-limiting examples of amino acids including non-polar aliphatic R groups include alanine, glycine, valine, leucine, methionine, and isoleucine. Non-limiting examples of amino acids including positively charged R groups include lysine, arginine, and histidine. Non-limiting examples of amino acids including negatively charged R groups include aspartic acid and glutamic acid. Non-limiting examples of amino acids including non-polar aromatic R groups include phenylalanine, tyrosine, and tryptophan. Non-limiting examples of amino acids including polar uncharged R groups include serine, threonine, cysteine, proline, asparagine, and glutamine.

[0193] Non-limiting examples of functional equivalent variants of polypeptides may include conservative amino acid substitutions in the amino acid sequences of proteins disclosed in the present application. Conservative substitutions of amino acids include substitutions in amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Additional non-limiting examples of conservative amino acid substitutions are provided in Table 1.

[0194] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more residues may be altered when preparing a variant polypeptide. In some embodiments, an amino acid is replaced by a conservative amino acid substitution.

[0195] Table 1. Non-limiting examples of conservative amino acid substitutions

[0196] Original residue R Group Type Conservative amino acid substitution Ala Nonpolar aliphatic R groups Cys, Gly, Ser Arg Positively charged R group His, Lys Asn Polar uncharged R group Asp, Gln, Glu Asp Negatively charged R group Asn, Gln, Glu Cys Polar uncharged R group Ala, Ser Gln Polar uncharged R group Asn, Asp, Glu Glu Negatively charged R group Asn, Asp, Gln Gly Nonpolar aliphatic R groups Ala, Ser His Positively charged R group Arg, Tyr, Trp Ile Nonpolar aliphatic R groups Leu, Met, Val Leu Nonpolar aliphatic R groups Ile, Met, Val Lys Positively charged R group Arg、His Met Nonpolar aliphatic R groups Ile, Leu, Phe, Val Pro Polar uncharged R group Phe Non-polar aromatic R groups Met, Trp, Tyr Ser Polar uncharged R group Ala, Gly, Thr Thr Polar uncharged R group Ala, Asn, Ser Trp Non-polar aromatic R groups His、Phe、Tyr、Met Tyr Non-polar aromatic R groups His、Phe、Trp Val Nonpolar aliphatic R groups Ile, Leu, Met, Thr

[0197] The amino acid substitution in the amino acid sequence of the polypeptide can be carried out by changing the coding sequence of the polypeptide to produce a recombinant polypeptide variant with desired properties and / or activity. Similarly, conservative amino acid substitutions in the amino acid sequence of the polypeptide are usually carried out by changing the coding sequence of a recombinant polypeptide (e.g., UGT, CDS, P450, cytochrome P450 reductase, EPH, or squalene epoxidase) to produce a functional equivalent variant of the polypeptide.

[0198] Expression of nucleic acids in host cells

[0199] Aspects of the present disclosure relate to the recombinant expression of genes encoding enzymes, functional modifications and variants thereof, and related applications thereof.For example, the methods described in the present application can be used to produce mogroside precursors, mogroside and / or mogroside.

[0200] The term "heterologous" is used interchangeably with the terms "exogenous" and "recombinant" for polynucleotides (e.g., polynucleotides including genes), and refers to: a polynucleotide that has been artificially provided to a biological system; a polynucleotide that has been modified within a biological system; or a polynucleotide whose expression or regulation has been manipulated within a biological system. A heterologous polynucleotide introduced into or expressed in a host cell may be a polynucleotide from an organism or species different from the host cell, or may be a synthetic polynucleotide, or may be a polynucleotide that is also endogenously expressed in the same organism or species as the host cell. For example, a polynucleotide endogenously expressed in a host cell may be considered heterologous when it: is not naturally located in the host cell; is recombinantly expressed (stably or transiently) in the host cell; is modified in the host cell; is selectively edited in the host cell; is expressed at a copy number different from the copy number naturally occurring in the host cell; or is expressed in a non-natural manner in the host cell (e.g., by manipulation of a regulatory region that controls the expression of the polynucleotide). In some embodiments, a heterologous polynucleotide is a polynucleotide that is endogenously expressed in a host cell, but whose expression is driven by a promoter that regulates the expression of a non-natural polynucleotide. In other embodiments, a heterologous polynucleotide is a polynucleotide that is endogenously expressed in a host cell and whose expression is driven by a promoter that regulates the expression of a natural polynucleotide, but the promoter or another regulatory region is modified. In some embodiments, the promoter is recombinantly activated or inhibited. For example, gene editing-based techniques can be used to regulate the expression of a polynucleotide (including an endogenous polynucleotide from a promoter (including an endogenous promoter)). See, for example, Chavez et al., Nat Methods. 2016 Jul; 13 (7): 563–567. A heterologous polynucleotide can include a wild-type sequence or a mutant sequence compared to a reference polynucleotide sequence.

[0201] The nucleic acid encoding any recombinant polypeptide (e.g., CDS, UGT, C11 hydroxylase, cytochrome P450 reductase, EPH, or SQE) described in the present application can be incorporated into any suitable vector by any method known in the art. For example, the vector can be an expression vector, including (but not limited to) a viral vector (e.g., a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated viral vector), any vector suitable for transient expression, any vector suitable for constitutive expression, or any vector suitable for inducible expression (e.g., a galactose inducible vector or a doxycycline inducible vector). Non-limiting examples of vectors for expressing recombinant polypeptides (e.g., CDS, UGT, C11 hydroxylase, cytochrome P450 reductase, EPH, or squalene epoxidase) are described in Example 1 below.

[0202] In some embodiments, the carrier replicates autonomously in the cell. The carrier may contain one or more endonuclease restriction sites, which are cut by restriction endonucleases to insert and connect nucleic acids containing genes described in the present application to produce recombinant carriers that can be replicated in cells. The carrier is usually composed of DNA, although RNA carriers are also available. Cloning carriers include (but are not limited to): plasmids, F cosmids (fosmid), phagemids, viral genomes and artificial chromosomes. As used in the present application, the term "expression carrier" or "expression construct" refers to a nucleic acid construct that is recombinantly generated or synthetically generated, with a series of specified nucleic acid elements that allow specific nucleic acids to be transcribed in host cells (such as yeast cells). In some embodiments, the nucleic acid sequence of the gene described in the present application is inserted into the cloning carrier so that it is operably connected to a regulatory sequence and is expressed as an RNA transcript in some embodiments. In some embodiments, the carrier contains one or more markers (such as the selective marker described in the present application) to identify cells transformed or transfected with a recombinant carrier. In some embodiments, the nucleic acid sequence of the gene described in the present application is recoded. Recoding can increase the yield of the gene product by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% (including all values ​​therebetween) relative to the reference sequence that is not recoded.

[0203] A coding sequence and a regulatory sequence are said to be "operably joined" or "operably linked" when they are covalently linked and the expression or transcription of the coding sequence is affected or controlled by the regulatory sequence. A coding sequence and a regulatory sequence are said to be operably linked or linked if the coding sequence is translated into a functional protein, if induction of a promoter in the 5' regulatory sequence permits transcription of the coding sequence, and if the nature of the linkage between the coding sequence and the regulatory sequence does not (1) result in the introduction of a frameshift mutation; (2) interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.

[0204] In some embodiments, the nucleic acid encoding any protein described in the application is controlled by a regulatory sequence (e.g., an enhancer sequence). In some embodiments, the nucleic acid is expressed under the control of a promoter. The promoter can be a natural promoter (e.g., a promoter of a gene in its endogenous environment, which provides normal regulation of gene expression). Alternatively, the promoter can be a promoter different from the natural promoter of the gene, for example, the promoter is different from the promoter of the gene in its endogenous environment. As used in the application, "heterologous promoter" or "recombinant promoter" is a promoter that is not naturally or normally related to the transcription of the DNA sequence operably connected thereto, or is not naturally or normally controlling the promoter of the transcription of the DNA sequence operably connected thereto. In some embodiments, the nucleotide sequence is controlled by a heterologous promoter.

[0205] In some embodiments, the promoter is a eukaryotic promoter. Non-limiting examples of eukaryotic promoters include TDH3, PGK1, PKC1, PDC1, TEF1, TEF2, RPL18B, SSA1, TDH2, PYK1, TPI1, GAL1, GAL10, GAL7, GAL3, GAL2, MET3, MET25, HXT3, HXT7, ACT1, ADH1, ADH2, CUP1-1, ENO2, and SOD1 (see, e.g., Addgene website: blog.addgene.org / plasmids-101-the-promoter-region) as known to those of ordinary skill in the art. In some embodiments, the promoter is a prokaryotic promoter (e.g., a phage promoter or a bacterial promoter). Non-limiting examples of phage promoters include Pls1con, T3, T7, SP6, and PL. Non-limiting examples of bacterial promoters include Pbad, PmgrB, Ptrc2, Plac / ara, Ptac, and Pm.

[0206] In some embodiments, the promoter is an inducible promoter. As used in the present application, "inducible promoter" is a promoter controlled by the presence or absence of a molecule. Non-limiting examples of inducible promoters include chemically regulated promoters and physically regulated promoters. For chemically regulated promoters, transcriptional activity can be regulated by one or more compounds (such as alcohol, tetracycline, galactose, steroids, metals, or other compounds). For physically regulated promoters, transcriptional activity can be regulated by phenomena (such as light or temperature). Non-limiting examples of tetracycline-regulated promoters include anhydrotetracycline (aTc) responsive promoters and other tetracycline-responsive promoter systems (for example, tetracycline repressor (tetR), tetracycline operator sequence (tetO) and tetracycline transactivator fusion protein (tTA)). Non-limiting examples of steroid-regulated promoters include promoters based on rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptor, and promoters from steroid / vitamin A / thyroid receptor superfamily. Non-limiting examples of promoters regulated by metals include promoters derived from metallothionein (proteins that bind and chelate metal ions) genes. Non-limiting examples of promoters regulated by pathogenesis include promoters induced by salicylic acid, ethylene or benzothiadiazole (BTH). Non-limiting examples of temperature / heat-inducible promoters include heat shock promoters. Non-limiting examples of promoters regulated by light include light-responsive promoters from plant cells. In certain embodiments, the inducible promoter is a galactose-inducible promoter. In some embodiments, the inducible promoter is induced by one or more physiological conditions (e.g., pH, temperature, radiation, osmotic pressure, saline gradient, cell surface binding or one or more extrinsic inducers or concentrations of intrinsic inducers). Non-limiting examples of extrinsic inducers or inducers include amino acids and amino acid analogs, carbohydrates and polysaccharides, nucleic acids, protein transcription activators and repressors, cytokines, toxins, petroleum-based compounds, metal-containing compounds, salts, ions, enzyme substrate analogs, hormones or any combination thereof.

[0207] In some embodiments, the promoter is a constitutive promoter. As used in the present application, a "constitutive promoter" refers to an unregulated promoter that allows continuous transcription of a gene. Non-limiting examples of constitutive promoters include TDH3, PGK1, PKC1, PDC1, TEF1, TEF2, RPL18B, SSA1, TDH2, PYK1, TPI1, HXT3, HXT7, ACT1, ADH1, ADH2, ENO2, and SOD1.

[0208] Other inducible or constitutive promoters known to those of ordinary skill in the art are also contemplated herein.

[0209] The exact nature of the regulatory sequences required for gene expression may vary between species or cell types, but generally include 5' non-transcribed sequences and 5' non-translated sequences (such as TATA boxes, capping sequences, CAAT sequences, etc.) that are involved in the initiation of transcription and translation, respectively, as required. In particular, such 5' non-transcribed regulatory sequences will include a promoter region that includes a promoter sequence for transcriptional control of an operably linked gene. The regulatory sequence may also include an enhancer sequence or an upstream activator sequence. The vector disclosed in the present application may include a 5' leader sequence or a 5' signal sequence. The regulatory sequence may also include a terminator sequence. In some embodiments, the terminator sequence marks the end of the gene in the DNA during transcription. The selection and design of one or more appropriate vectors suitable for inducing the expression of one or more genes described in the present application in a host cell is within the ability and judgment of those of ordinary skill in the art.

[0210] Expression vectors containing the necessary elements for expression are commercially available and known to those of ordinary skill in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, 2012).

[0211] In some embodiments, the introduction of a polynucleotide (e.g., a polynucleotide encoding a recombinant polypeptide) into a host cell results in genomic integration of the polynucleotide. In some embodiments, the host cell comprises in its genome at least 1 copy, at least 2 copies, at least 3 copies, at least 4 copies, at least 5 copies, at least 6 copies, at least 7 copies, at least 8 copies, at least 9 copies, at least 10 copies, at least 11 copies, at least 12 copies, at least 13 copies, at least 14 copies, at least 15 copies, at least 16 copies, at least 17 copies, at least 18 copies, at least 19 copies, at least 20 copies, at least 21 copies, at least 22 copies, at least 23 copies, at least 24 copies, at least 25 copies, at least 26 copies, at least 27 copies, at least 28 copies, at least 29 copies, at least 30 copies, at least 31 copies, at least 32 copies, at least 33 copies, at least 34 copies, at least 35 copies, at least 36 copies, at least 37 copies, at least 38 copies, at least 39 copies, at least 40 copies, at least 41 copies, at least 42 copies, at least 43 copies, at least 44 copies, at least 45 copies, at least 46 copies, at least 47 copies, at least 48 copies, at least 49 copies, at least 50 copies, at least 51 copies, at least 52 copies, at least 53 copies, at least 54 copies, at least 55 copies, at least 56 copies, at least 57 copies, at least 58 copies, at least 59 copies, at least 60 copies, at least 61 copies, at least 62 copies, at least 63 copies, at least 64 copies, at least 65 copies, at least 66 copies, at least 67 copies, at least 27 copies, at least 28 copies, at least 29 copies, at least 30 copies, at least 31 copies, at least 32 copies, at least 33 copies, at least 34 copies, at least 35 copies, at least 36 copies, at least 37 copies, at least 38 copies, at least 39 copies, at least 40 copies, at least 41 copies, at least 42 copies, at least 43 copies, at least 44 copies, at least 45 copies, at least 46 copies, at least 47 copies, at least 48 copies, at least 49 copies, at least 50 copies, at least 60 copies, at least 70 copies, at least 80 copies, at least 90 copies, at least 100 copies, or more (including all values ​​in between).

[0212] Host cells

[0213] Any of the proteins or enzymes disclosed herein can be expressed in a host cell. As used in this application, the term "host cell" refers to a cell that can be used to express a polynucleotide (such as a polynucleotide encoding an enzyme for the production of mogroside, mogroside, and its precursors).

[0214] Any suitable host cell (including eukaryotic cells or prokaryotic cells) can be used to produce any recombinant polypeptide disclosed in the present application (including CDS, UGT, C11 hydroxylase, cytochrome P450 reductase, EPH, and SQE). Suitable host cells include (but are not limited to) fungal cells (e.g., yeast cells), bacterial cells (e.g., Escherichia coli cells), algae cells, plant cells, insect cells, and animal cells (including mammalian cells).

[0215] Suitable yeast host cells include, but are not limited to, Candida, Escherichia, Hansenula, Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces, Pichia, Kluyveromyces (e.g., K. lactis), and Yarrowia. In some embodiments, the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae, Saccaromyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia alictaria, Pichia quercuum, Pichia pijperi, Pichia stipites, Pichia methanolica, Pichia tadalafil ... methanolica), Pichia angusta, Kluyveromyces lactis, Candida albicans, or Yarrowia lipolytica.

[0216] In some embodiments, the yeast strain is an industrial polyploid yeast strain. Other non-limiting examples of fungal cells include cells obtained from Aspergillus spp., Penicillium spp., Fusarium spp., Rhizopus spp., Acremonium spp., Neurospora spp., Sordaria spp., Magnaporthe spp., Allomyces spp., Ustilago spp., Botrytis spp., and Trichoderma spp.

[0217] In certain embodiments, the host cell is an algal cell (eg, Chlamydomonas (e.g., C. Reinhardtii and Phormidium (P. sp. ATCC 29409)).

[0218] In other embodiments, the host cell is a prokaryotic cell. Suitable prokaryotic cells include gram-positive bacterial cells, gram-negative bacterial cells, and gram-indeterminate bacterial cells. The host cell can be, but is not limited to, the following species: Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyric acid Butyrivibrio, Buchnera, Campestris, Camplyobacter, Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter, Erwinia, Fusobacterium rium, Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus, Microbacterium rium), Mesorhizobium, Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas,Roseburia, Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus, Synecoccus, Saccharomonospora, Saccharopolyspora, Staphylococcus, Serratia, Salmonella nella), Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula, Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella, Yersinia, and Zymomonas.

[0219] In some embodiments, the bacterial host cell is an Agrobacterium species (e.g., A. radiobacter, A. rhizogenes, A. rubi), an Arthrobacter species (e.g., A. aurescens, A. citreus, A. globformis, A. hydrocarboglutamicus, A. mysorens, A. nicotianae, A. paraffineus, A. protophonniae, A. roseoparaffinus, A. sulfureus, A. ureafaciens), or a Bacillus species (e.g., Bacillus species) (e.g., B. thuringiensis, B. anthracis, B. megaterium, B. subtilis, B. lentus, B. circulans, B. pumilus, B. lautus, B. coagulans, B. brevis, B. firmus, B. alkaophius, B. licheniformis, B. clausii, B. stearothermophilus, B. halodurans, and B. amyloliquefaciens). In particular embodiments, the host cell is an industrial Bacillus strain, including but not limited to Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Bacillus megaterium, Bacillus clausii, Bacillus stearothermum, and Bacillus amyloliquefaciens.In some embodiments, the host cell is an industrial Clostridium species (e.g., C.acetobutylicum, C.tetani E88, C.lituseburense, C.saccharobutylicum, C.perfringens, C.beijerinckii). In some embodiments, the host cell is an industrial Corynebacterium species (e.g., C.glutamicum, C.acetoacidophilum). In some embodiments, the host cell is an industrial Escherichia species (e.g., E. coli). In some embodiments, the host cell is an industrial Erwinia species (e.g., Erwinia uredovora, Erwinia carotovora, Erwinia ananas, Erwinia herbicola, Erwinia punctata, Erwinia terreus). In some embodiments, the host cell is an industrial Pantoea species (e.g., Pantoea citrea, Pantoea agglomerans). In some embodiments, the host cell is an industrial Pseudomonas species (e.g., Pseudomonas putida, Pseudomonas aeruginosa, P. mevalonii). In some embodiments, the host cell is an industrial Streptococcus species (e.g., Streptococcus equisimiles, Streptococcus pyogenes, Streptococcus uberis). In some embodiments, the host cell is an industrial Streptomyces species (e.g., S. ambofaciens, S. achromogenes, S. avermitilis, S. coelicolor, S. aureofaciens, S. aureus, S. fungicidicus, S. griseus, S. lividans). In some embodiments, the host cell is an industrial Zymomonas species (e.g., Zymomonas mobilis, Zymomonas lipolytica).

[0220] The present disclosure is also applicable to a variety of animal cell types, including mammalian cells, such as human (including 293, HeLa, WI38, PER.C6 and Bowes melanoma cells), mouse (including 3T3, NS0, NS1, Sp2 / 0), hamster (CHO, BHK), monkey (COS, FRhL, Vero), and hybridoma cell lines.

[0221] The present disclosure is also suitable for use with a variety of plant cell types.

[0222] As used in this application, the term "cell" may refer to a single cell or a population of cells, such as a population of cells belonging to the same cell line or cell strain. The use of the singular term "cell" should not be interpreted as explicitly referring to a single cell rather than a population of cells.

[0223] The host cell may include genetic modifications relative to the wild-type counterpart. As non-limiting examples, the host cell (e.g., Saccharomyces cerevisiae) may be modified to reduce one or more of the following genes or reduce the activity of one or more of the following genes: hydroxymethylglutaryl-CoA (HMG-CoA) reductase (HMG1), acetyl-CoA C-acetyltransferase (acetoacetyl-CoA thiolase)) (ERG10), 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthetase (ERG13), farnesyl-diphosphate farnesyl transferase (squalene synthase) (ERG9), may be modified to overexpress squalene epoxidase (ERG1), or may be modified to downregulate lanosterol synthase (ERG7). See, e.g., Examples 1 and 2 below.

[0224] Reduction of gene expression and / or gene inactivation can be achieved by any suitable method (including but not limited to deletion of gene, introduction of point mutation into gene, truncation of gene, introduction of insertion sequence into gene, introduction of tag into gene or fusion into gene, or selective editing of gene). For example, a method based on polymerase chain reaction (PCR) (see, e.g., Gardner et al., Methods Mol Biol. 2014; 1205: 45-78) or known gene editing techniques can be used. As a non-limiting example, gene deletion can be performed by gene replacement (e.g., using markers, including selection markers). Genes can also be truncated by using a transposon system (see, e.g., Poussu et al., Nucleic Acids Res. 2005; 33(12): e104).

[0225] Any method known in the art can be used to introduce a vector encoding any recombinant polypeptide described in this application into a suitable host cell. Gietz et al., Yeast transformation can be conducted by the LiAc / SS Carrier DNA / PEG method. Methods Mol Biol. 2006; 313: 107-20 (which is hereby incorporated by reference in its entirety) describes a non-limiting example of a yeast transformation protocol. Host cells can be cultured under any suitable conditions as understood by those of ordinary skill in the art. For example, any culture medium, temperature, and incubation conditions known in the art can be used. For host cells carrying an inducible vector, the cells can be cultured with an appropriate inducible agent to promote expression.

[0226] Any cell disclosed in the present application can be cultivated in any type (complete medium or minimal medium) and any composition of medium before, during and / or after contacting nucleic acid and / or nucleic acid integration. The conditions of cultivation or the culture process can be optimized by routine experiments as understood by those of ordinary skill in the art. In some embodiments, the selected medium is supplemented with various components. In some embodiments, the concentration and dosage of the supplementary components are optimized. In some embodiments, other aspects of the medium and growth conditions (for example, pH, temperature, etc.) are optimized by routine experiments. In some embodiments, the frequency of one or more supplementary components of the medium and the time of cell culture are optimized.

[0227] The cultivation of the cell described in the application can be carried out in a culture vessel known and used in the art. In some embodiments, an aerated reaction vessel (e.g., a stirred tank reactor) is used to culture cells. In some embodiments, a bioreactor or a fermentation tank is used to culture cells. Therefore, in some embodiments, cells are used for fermentation. As used in the application, the terms "bioreactor" and "fermentation tank" are used interchangeably and refer to a closed cover or a partially closed cover, wherein biological, biochemical and / or chemical reactions occur, relating to an organism, a part of an organism or a purified enzyme." Large-scale bioreactors" or "industrial scale bioreactors" are bioreactors for generating products on a commercial or quasi-commercial scale. Large-scale bioreactors typically have a volume in a liter, hundreds of liters, thousands of liters or larger.

[0228] Non-limiting examples of bioreactors include stirred tank fermentors, bioreactors agitated by a rotary mixing device, chemostat, bioreactors agitated by a shaking device, airlift fermenters, packed bed reactors, fixed bed reactors, fluidized bed bioreactors, bioreactors with wave-induced agitation, centrifugal bioreactors, roller bottles, and hollow fiber bioreactors, drum apparatus (e.g., benchtop, cart-mounted and / or automated types), vertically stacked plates, spinner flasks, stirred flasks or shake flasks, shaking multiwell plates, MD flasks, T-flasks, Roux flasks, multi-surface tissue culture propagators, modified fermentors, and coated beads (e.g., beads coated with serum albumin, nitrocellulose, or carboxymethylcellulose to prevent cell attachment).

[0229] In some embodiments, bioreactor comprises cell culture system, wherein cell (for example, yeast cell) contacts with flowing liquid and / or bubble.In some embodiments, cell or cell culture suspension growth.In other embodiments, cell or cell culture is attached to solid phase carrier.Non-limiting examples of carrier system comprise microcarrier (for example, can be porous or non-porous polymer ball, microbead and microdisk), cross-linked beads (for example, dextran) with specific chemical group (for example, tertiary amine group), 2D microcarrier (comprising the cell trapped in non-porous polymer fiber), 3D carrier (for example, carrier fiber, hollow fiber, multi-core reactor and can comprise the semipermeable membrane of porous fiber), microcarrier, encapsulated cell, capillary and aggregate with reduced ion exchange capacity.In some embodiments, carrier is made of materials such as dextran, gelatin, glass or cellulose.

[0230] In some embodiments, the industrial-scale process is run in a continuous, semi-continuous, or discontinuous mode. Non-limiting examples of operating modes are batch, fed-batch, extended batch, repeated batch, suction / fill, rotating wall, rotating bottle, and / or perfusion operating modes. In some embodiments, the bioreactor allows continuous or semi-continuous replenishment of substrate feedstock (e.g., carbohydrate source) and / or product to leave the bioreactor continuously or semi-continuously.

[0231] In some embodiments, bioreactor or fermentation tank comprises sensor and / or control system to measure and / or adjust reaction parameter.Non-limiting examples of reaction parameter include biological parameter (for example, growth rate, cell size, cell number, cell density, cell type or cell state, etc.), chemical parameter (for example, pH, redox potential, reaction substrate and / or product concentration, dissolved gas concentration (such as oxygen concentration and CO2 concentration), nutrient concentration, metabolite concentration, oligopeptide concentration, amino acid concentration, vitamin concentration, hormone concentration, additive concentration, serum concentration, ionic strength, ion concentration, relative humidity, molar concentration, osmotic pressure, other chemical substances (such as buffer, adjuvant or reaction byproduct) concentration), physical / mechanical parameter (for example, density, conductivity, stirring degree, pressure and flow rate, shear stress, shear rate, viscosity, color, turbidity, light absorption, mixing rate, conversion rate and thermodynamic parameter (such as temperature, light intensity / quality, etc.)).The sensor of the measurement parameter described in the present application is well known to those of ordinary skill in the related mechanical and electronic fields. Control systems that adjust parameters in a bioreactor based on input from the sensors described in this application are well known to those of ordinary skill in the art of bioreactor engineering.

[0232] In some embodiments, the method relates to batch fermentation (e.g., shake flask fermentation). Factors generally considered in batch fermentation (e.g., shake flask fermentation) include the levels of oxygen and glucose. For example, batch fermentation (e.g., shake flask fermentation) may be limited by oxygen and glucose, so in some embodiments, the ability of strains to play a role in well-designed batch fed fermentation is underestimated. In addition, the final product (e.g., momordica alcohol precursor, momordica alcohol, mogroside precursor, or mogroside) may show some differences with the substrate (e.g., momordica alcohol precursor, momordica alcohol, mogroside precursor, or mogroside) in solubility, toxicity, cell accumulation, and secretion, and may have different fermentation kinetics in some embodiments.

[0233] The methods described in the present application encompass the use of recombinant cells, cell lysates, or isolated recombinant polypeptides (e.g., CDS, UGT, C11 hydroxylase, cytochrome P450 reductase, EPH, and squalene epoxidase) to produce mogroside precursors (e.g., squalene, 2,3-oxidized squalene, or 24-25-epoxy-cucurbitacinadienol), mogroside, or mogrosides (e.g., MIA1, MIE1, MIIA1, MIIA2, MIIIA1, MIIE, MIII, simenoside I, mogroside IV, isomogroside IV, MIIIE, and mogroside V).

[0234] Any method known in the art can be used to identify and extract mogroside precursors (e.g., squalene, 2,3-oxidized squalene, or 24-25-epoxy-cucurbitacinadienol), mogroside, mogroside (e.g., MIA1, MIE, MIIA1, MIIA2, MIIIA1, MIIE, MIII, simenoside I, mogroside IV, isomogroside IV, MIIIE, and mogroside V) produced by any recombinant cell disclosed in this application. Mass spectrometry (e.g., LC-MS, GC-MS) is a non-limiting example of an identification method and can be used to help extract compounds of interest.

[0235] The words and terms used in this application are for descriptive purposes and should not be considered as limiting. Terms such as "comprises," "including," "having," "containing," "involving," and / or their variations used in this application are intended to cover the items listed thereafter and their equivalents and additional items.

[0236] The present invention is further illustrated by the following examples, but should not be construed as further limiting. The entire contents of all references cited throughout this application (including references, granted patents, published patent applications, and co-pending patent applications) are hereby expressly incorporated herein by reference. Example

[0237] Example 1: Identification and functional characterization of CDS enzymes

[0238] A putative CDS enzyme library was designed. The library contained some oxidosqualene cyclase sequences that were modified to be more CDS-like.

[0239] The product prepared by the control strain expressing the monk fruit CDS was identified as cucurbitacinadienol by NMR, and it was confirmed that hydroxylation of the product resulted in the production of monk fruit alcohol. The putative CDS enzymes were evaluated to identify one or more active enzymes that catalyze the cyclization of 2,3-oxidosqualene to cucurbitacinadienol. A total of 506 constructs from the putative CDS library were tested by transforming the library into an engineered Saccharomyces cerevisiae screening strain. The Saccharomyces cerevisiae screening strain included: a truncated HMG1 gene; overexpression of the ERG10, ERG13, ERG9, and ERG1 genes; and downregulation of the ERG7 gene. The gene encoding the putative CDS was expressed on the pESC-URA plasmid and expression was induced by culturing in SC-URA containing 4% galactose. Cucurbitacinadienol was measured using GC-MS.

[0240] Forty CDSs were demonstrated to exhibit activity in the screen (Table 2). These CDSs included previously unidentified CDSs, as well as engineered CDSs. Some of the newly discovered enzymes showed up to 2-fold or greater production of cucurbitacadienols compared to the production of cucurbitacadienols from a control strain expressing a previously characterized CDS from Momordica grosvenori (SEQ ID NO: 73). SEQ ID NO: 33 is a non-limiting example of a polynucleotide sequence encoding SEQ ID NO: 73.

[0241] Thus, various enzymes that can cyclize 2,3-oxidosqualene to cucurbitacadienols have been identified and characterized.

[0242] Table 2. Production of cucurbitacadienols by putative CDS enzymes.

[0243]

[0244]

[0245] It was concluded that the identified enzymes produce cucurbitacinadienols. The GC-MS spectra of the products produced by the newly discovered enzymes were very similar to the cucurbitacinadienols produced with Momordica grosvenori CDS in Saccharomyces cerevisiae. This conclusion was confirmed by GC-MS, LC-MS and NMR. The cucurbitacinadienols produced by these enzymes had the same retention time, ionization pattern, and mass and fragmentation pattern as the cucurbitacinadienols produced with Momordica grosvenori CDS.

[0246] Example 2. Identification and functional characterization of putative UGT enzymes

[0247] This example describes the design and screening of a UGT library to identify UGTs that can convert mogroside and mogroside precursors into glycosylated mogrosides. Specifically, the library aims to identify UGTs that glycosylate on the C3 and C24 hydroxyl groups of mogroside to produce mogrosides with different glucose units. A total of 1059 putative UGTs were obtained.

[0248] To test the UGT library, an in vitro assay was developed. The plasmid carrying the UGT was transformed into Saccharomyces cerevisiae CEN.PKΔGAL80. A total of eight substrates were used to screen the UGT library in this assay: mogroside, mogroside I-A1, mogroside I-E1, mogroside II-A1, mogroside II-E, mogroside III, mogroside III-A1, and mogroside III-E. Cell lysates were incubated with 50 μM of substrate at 30 °C for 24 h before quenching. Product formation was tested by LC-MS after quenching the reaction. LC-MS curves of mogroside and mogroside standards are shown in Figure 2. Figure 2 Shown in.

[0249] Based on this screening, UGTs that can produce known mogroside products (including mogroside I-A1 and simonoside I) were identified. Figure 3A-3B ). The strains identified from this screen were then tested in additional screens ( Figures 4A-4B , Table 3).

[0250] Table 3. Product formation by putative UGTs

[0251]

[0252]

[0253] Example 3. Further Characterization of UGT

[0254] Thirteen of the 16 UGTs identified from the screening described in Example 2 were recombinantly expressed in E. coli and purified using a 6xHis tag. The protein concentration of these purified UGTs was determined by Bradford assay. The specific activity of the 13 UGTs was determined by incubating 50 μM of each substrate with the UGT at 30°C for 5 minutes. The reaction was quenched and the product concentration was quantified by LC-MS (Figure 4). The specific activity was calculated by dividing the product concentration by the enzyme concentration and the reaction time. The measured specific activity ranged from 0.01 to 5.53 mmol product / (g UGT*hr), with an average value of 1.14.

[0255] Example 4. Protein Engineering of UGT94-289-1

[0256] Saccharomyces cerevisiae strain t85024 (expressing a recoded polynucleotide encoding UGT94-289-1) was observed to catalyze both 6-1 and 2-1 glycosylation reactions. However, the enzyme was unable to catalyze these reactions at high rates.

[0257] A library of UGT sequences was designed, each containing a single amino acid substitution relative to the UGT94-289-1 sequence. The library of UGT sequences was tested for enhanced activity for 6-1 and 2-1 glycosylation. The library contained 893 members. The mutation sites were selected based on their interaction with the catalytic dyad (His 21 / Asp 122 ) or based on their predicted interactions with substrate molecules. The homology model of UGT94-289-1 is Figure 5 Shown in.

[0258] 218 mutants with improved activity compared to the wild-type enzyme (UGT94-289-1) were identified by in vitro screening (Table 4). Plasmids carrying the mutated UGT94-289-1 gene were transformed into Saccharomyces cerevisiae CEN.PKΔGAL80. To test the UGT mutant library, the in vitro assay from Example 2 was performed. A total of 3 substrates—mogroside II-A1, mogroside III, and mogroside II-E—were tested with the UGT mutant library using this assay.

[0259] Many mutants with enhanced activity in these glycosylation steps were identified (Table 4). In Table 4, MIIA1 represents mogroside II-A1, MIIE represents mogroside II-E, MIIIA1 represents mogroside III-A1, MIII represents mogroside III, MIIIE represents mogroside III-E, and Siam represents siamone glycoside I. A subset of UGTs containing the identified mutations (N143V, N143I, L374N, L374Y, and L374W) were expressed and purified in E. coli. It was found that N143V, N143I, and L374N increased the specific activity of the reaction of mogroside II-A1 to mogroside III-A1 and the reaction of mogroside III to siamone glycoside I by 4-8 times and 12-16 times, respectively, higher than the wild-type protein. L374Y and L374W were found to increase the specific activity of the reaction of mogroside II-E to mogroside III-E by 13-fold and 28-fold, respectively, above the wild type. These observations generally match the data observed in the Saccharomyces cerevisiae screening (Table 4). In addition, the N143V mutation was observed to produce mogroside V from simenoside I, an activity not observed in the wild-type UGT or other mutants.

[0260] Non-limiting examples of structural motifs in UGT94-289-1 and the sequences of the structural motifs are shown in Table 5.

[0261] Table 4. UGT94-289-1 substitution mutations

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271] Table 5. Non-limiting examples of structural motifs in UGT94-289-1 (SEQ ID NO: 109)

[0272]

[0273]

[0274]

[0275] Example 5. Identification and Characterization of Additional UGTs

[0276] This example describes the further engineering of UGT enzymes and the identification of additional UGT enzymes.

[0277] The engineering of UGTs involves cyclic rearrangements of protein sequences. Representative UGTs, UGT94-289-1 ( Figure 5 ) showed that the N-terminus and C-terminus were flexible and in close proximity (7-10A, Figure 6 ). For the cyclic rearrangement, the original N- and C-termini are fused together and a new terminus is introduced at another site within the protein structure ( Figure 7 ).

[0278] Two libraries were screened. One of the libraries contained cyclically rearranged versions of the UGT sequences. The other library contained additional putative UGT sequences. The Saccharomyces cerevisiae strain used for screening included: CDS, two EPHs, a mutated C11-hydroxylase fusion protein, two cytochrome P450 reductases, an upregulated SQE, two primary UGTs, and two transporter knockouts. Two different biological replicates of the same strain were used for screening. The biological replicates were referred to as background 1 and background 2. The plasmid encoding the UGT was transformed into the screening strain. The transformant was inoculated into a pre-culture medium, and then an aliquot of the inoculated culture medium was transferred to a culture plate.

[0279] After the plates were incubated, the production of mogrosides was assessed using a Thermo QQQ TSQ-Quantiva ESI and LX4 multiplex column setup. The selected ion monitoring (SIM) masses of the glycosylated classes with a mogroside backbone (M, MI, MII, MIII, MIV, MV) were as follows: 535.4 g / mol (M), 697.47 g / mol (MI), 799.51 g / mol (MII), 961.56 g / mol (MIII), 1123.61 g / mol (MIV), and 1285.68 g / mol (MV), respectively. MI represents a product with 1 glucose moiety, MII represents a product with 2 glucose moieties, MIII represents a product with 3 glucose moieties, MIV represents a product with 4 glucose moieties, and MV represents a product with 5 glucose moieties. MI and MII are considered substrates for the secondary UGT, while MIII, MIV, and MV are considered products of the secondary UGT. These Selective Ion Detection (SIM) intensities were then normalized to an internal standard and calibrated against the following surrogates: MIA1, MIIA1, MIIIA1, simianin, and MV. UGT94-289-1N143I was used as a positive control. The negative control strain did not express a secondary UGT.

[0280] The percentage of MI, MII, MIII, MIV and MV produced by strains carrying each UGT was compared to the positive control strain. The fractions of MI, MII, MIII, MIV and MV correspond to the amount of each type in the total amount of product produced.

[0281] Based on the following criteria, enzymes are designated as having UGT activity (hit). For the UGT library of cyclic rearrangement, if the fraction (fraction) of the MIV produced by the enzyme (MIV score) is greater than the average score of MIV (two standard deviations of the MIV score of each positive control strain), then the enzyme is considered to be a hit. This critical value is used to identify structural variants with improved folding and stability (which may be weighed against activity). Only constructs that are positive in two biological replicates are considered to be hits. Table 6 provides the data of MIV and MV scores.

[0282] For the library of putative UGTs, an enzyme was considered a hit for each product (MIII, MIV, and MV) if it was two standard deviations higher than the maximum observed value for the negative control strain and greater than the mean for the positive control strain. Table 7 provides data for the MIII, MIV, and MV scores.

[0283] Table 6. UGTs generated by cyclic shuffling

[0284]

[0285]

[0286] Table 7. Additional putative UGTs

[0287]

[0288]

[0289] Example 6: Further protein engineering of UGT

[0290] This example describes further engineering of UGT enzymes. A UGT mutation library was constructed based on a site-specific scoring matrix (PSSM) and an energy minimization scheme (Goldenzweig et al., Mol Cell. 2016 Jul 21; 63(2): 337-346). In this method, close homologs of UGT were identified by BLAST search. These homologs were aligned and the site-specific scoring matrix (PSSM) ( Figure 8 ). Sites characterized by greater sequence variability (e.g. Figure 8 Sites 53 and 57 in the PSSM were selected as mutation sites. The library of potential amino acid changes was selected from those observed in the PSSM. For example, site 52 was mutated to L, I, M, or V because these are the amino acids observed in the PSSM for that site ( Figure 8 ). To further reduce the mutant library, the effect of all potential substitutions on protein stability was evaluated using Rosetta. The mutation library used to construct the library includes those substitutions that significantly enhance the stability of highly variable sites observed in PSSM (Goldenzweig et al., Mol Cell. 2016 Jul 21; 63 (2): 337-346.). The library will be screened to identify enzymes with UGT activity.

[0291] Example 7: Expression of a combination of heterologous enzymes to produce mogroside precursor, mogroside, or mogroside

[0292] Combinations of the recombinant proteins disclosed herein are used to produce mogroside precursors (e.g., 2-3-oxidosqualene, 2,3,22,23-dioxidosqualene, cucurbitacinadienol, 24,25-epoxycucurbitacinadienol, 24,25-dihydroxycucurbitacinadienol), mogroside, or mogrosides (e.g., mogroside I-A1 (MIA1), mogroside IE (MIE), mogroside II-A1 (MIIA1), mogroside III-A1 (MIIIA1), mogroside II-E (MIIE), mogroside III (MIII), simonoside I, mogroside IV, mogroside III-E (MIIIE), mogroside V, and mogroside VI).

[0293] For example, to produce mogroside, genes encoding enzymes such as squalene epoxidase, CDS, epoxidase, and cytochrome P450 are expressed in host cells. In some cases, cytochrome P450 reductase is also expressed in yeast cells. Non-limiting examples of suitable squalene epoxidase, epoxidase, C11 hydroxylase, and cytochrome P450 reductase are provided in Table 8 below. Non-limiting examples of CDS are provided in Table 2. LC-MS is used to quantify mogroside. UGT is further expressed in host cells to produce mogroside.

[0294] Alternatively, the recombinant protein is purified from the host cells and mogrohol is produced outside the host cells. The recombinant protein is added sequentially or simultaneously to a reaction buffer including squalene.

[0295] Table 8. Non-limiting examples of C11 hydroxylases (P450s), cytochrome P450 reductases, epoxidases (EPHs), and squalene epoxidases.

[0296] Enzymes Nucleoside sequence Amino acid sequence C11 Hydroxylase SEQ ID NO:113 SEQ ID NO:129 C11 Hydroxylase (Cucurbitacinadienol Oxidase) SEQ ID NO:114 SEQ ID NO:130 Cytochrome P450 Reductase SEQ ID NO:115 SEQ ID NO:131 Cytochrome P450 Reductase SEQ ID NO:116 SEQ ID NO:132 Cyclooxygenase SEQ ID NO:117 SEQ ID NO:133 Cyclooxygenase SEQ ID NO:118 SEQ ID NO:134 Epoxide hydratase SEQ ID NO:119 SEQ ID NO:135 Epoxide hydratase SEQ ID NO:120 SEQ ID NO:136 Epoxide hydratase SEQ ID NO:121 SEQ ID NO:137 Epoxide hydratase SEQ ID NO:122 SEQ ID NO:138 Epoxide hydratase SEQ ID NO:123 SEQ ID NO:139 Epoxide hydratase SEQ ID NO:124 SEQ ID NO:140 Epoxide hydratase SEQ ID NO:125 SEQ ID NO:141 Squalene epoxidase SEQ ID NO:126 SEQ ID NO:142 Squalene epoxidase SEQ ID NO:127 SEQ ID NO:143 Squalene epoxidase (P450) SEQ ID NO:128 SEQ ID NO:144

[0297] Equivalent

[0298] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described in this application. Such equivalents are intended to be encompassed by the following claims.

[0299] All references (including patent documents) disclosed in this application are incorporated herein by reference in their entirety, particularly the disclosures cited in this application.

Claims

1. A host cell comprising a heterologous polynucleotide encoding a cucurbitacidine dienol synthase CDS, wherein the sequence of the heterologous polynucleotide is SEQ ID NO: 3 and / or the amino acid sequence of the cucurbitacidine dienol synthase CDS is SEQ ID NO: 43, wherein the host cell produces a cucurbitacidine dienol compound, and wherein the host cell is not a plant cell.

2. The host cell of claim 1, wherein the cucurbitacinaldehyde compound is 11-hydroxycucurbitacinaldehyde, 24-25-epoxy-cucurbitacinaldehyde or cucurbitacinaldehyde.

3. The host cell of claim 1, wherein the host cell further comprises one or more heterologous polynucleotides encoding UDP-glycosyltransferase UGT, C11 hydroxylase, cytochrome P450 reductase, epoxidase EPH, lanosterol synthase, and / or squalene epoxidase. The host cell of claim 1 , wherein the host cell is a yeast cell or a bacterial cell.

5. The host cell of claim 4, wherein the host cell is a Saccharomyces cerevisiae cell or a Yarrowia cell. The host cell of claim 4 , wherein the host cell is an E. coli cell.

7. The host cell of any one of claims 1 to 6, wherein the host cell produces at least 10% more cucurbitacinadienol compounds relative to a control, wherein the control is a host cell expressing a Momordica grosvenori cucurbitacinadienol synthase CDS encoded by a polynucleotide corresponding to SED ID NO:

33.

8. The host cell of any one of claims 1-7, wherein the amino acid sequence of the cucurbitacadienol synthase CDS is SEQ ID NO:

43.

9. The host cell of claim 5, wherein the host cell is a Saccharomyces cerevisiae cell.

10. The host cell of claim 5, wherein the host cell is a Yarrowia lipolytica cell.

11. The host cell of any one of claims 1-6, wherein the host cell is further modified to downregulate lanosterol synthase (ERG7).

12. A method for producing a cucurbitacinaldehyde compound, the method comprising contacting the host cell of any one of claims 1 to 11 with squalene oxide, thereby producing the cucurbitacinaldehyde compound.

13. The method of claim 12, wherein the cucurbitacinaldehyde compound is 11-hydroxycucurbitacinaldehyde, 24-25-epoxy-cucurbitacinaldehyde or cucurbitacinaldehyde.

14. The method of claim 12, wherein the squalene oxide is 2-3-squalene oxide or 2,3;22,23-diepoxysqualene.

15. The method of any one of claims 12-14, wherein the method further comprises isolating the cucurbitacadienol compound.

16. A host cell comprising a heterologous polynucleotide encoding a cucurbitacadienol synthase CDS, wherein the sequence of the heterologous polynucleotide is SEQ ID NO: 3, and wherein the host cell is not a plant cell.

17. The host cell of claim 16, wherein the host cell is a yeast cell or a bacterial cell.

18. The host cell of claim 17, wherein the host cell is a Saccharomyces cerevisiae cell or a Yarrowia cell.

19. The host cell of claim 17, wherein the host cell is an E. coli cell.

20. A method for producing a cucurbitacadienol compound, the method comprising: The host cell of any one of claims 16 to 19 is contacted with squalene oxide to produce the cucurbitacadienol compound.

21. The method of claim 20, wherein the cucurbitacinaldehyde compound is 24-25-epoxy-cucurbitacinaldehyde or cucurbitacinaldehyde.

22. The method of claim 20, wherein the squalene oxide is 2-3-squalene oxide or 2,3;22,23-diepoxysqualene.

23. The method of any one of claims 20-22, wherein the method further comprises isolating the cucurbitacadienol compound.

24. A method for producing mogroside, the method comprising culturing the host cell of any one of claims 1-11 and 16-19 with at least one mogroside precursor.

25. The method of claim 24, wherein the mogroside precursor is selected from the group consisting of mogroside alcohol, MIA1, MIIA1, MIIIA1, MIIE, MIII and MIIIE.

26. The method of claim 24 or 25, wherein the mogroside produced is selected from the group consisting of MIA1, MIIA1, MIIIA1, MIIE, MIII, simanoside and MIIIE.

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