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US 2024/0352498 A1Published application
Saponin production in yeast
AssigneesThe Regents of the University of California, Plant Bioscience Limited
InventorsYuzhong Liu, Samantha Aiko Crowe, Jay D. Keasling, Xiaoyue Chen, Graham Arthur Hudson, Fei Gan, Henrik V. Scheller, James Reed, Laetitia Martin
Life of this patent
Estimating the term…
Abstract
The present invention relates inter alia to methods of biosynthetic production of QS-21, precursors and variants thereof, and to related aspects.
Claims19
A method of producing in yeast quillaic acid (QA), or a QA derivative, or UDP-Fucose (UDP-Fuc), or (S)-2-methylbutyryl CoA (2 MB-CoA), or UDP-Arabinofuranose (UDP-Araf), the method selected from:
- A) a method of producing quillaic acid (QA) in yeast, wherein the method comprises the step of overexpressing, in a yeast engineered to produce β-amyrin, heterologous genes encoding the following enzymes:
- (i) a cytochrome P450 C16 oxidase, wherein the C16 oxidase oxidizes the C16 carbon of β-amyrin to a hydroxyl group,
- (ii) a cytochrome P450 C23 oxidase, wherein the C23 oxidase oxidizes the C23 carbon of β-amyrin to an aldehyde group,
- (iii) a cytochrome P450 C28 oxidase, wherein the C28 oxidase oxidizes the C28 carbon of β-amyrin to a carboxyl group, and
- (iv) a cytochrome P450 reductase (CPR), acting as a redox partner,
- wherein the C16 oxidase, the C23 oxidase, the C28 oxidase and the CPR are from a plant origin;
- wherein, optionally,
- (1)
- (a) the C16 oxidase is selected from QsC16 according to SEQ ID NO:
- 20, QsC28C16 according to SEQ ID NO:
- 23, and SvC16 according to SEQ ID NO:
- 26, the C23 oxidase is selected from MtC23 oxidase according to SEQ ID NO:
- 38, QsC23 according to SEQ ID NO:
- 29, SvC23-1 according to SEQ ID NO:
- 32, and SvC23-2 according to SEQ ID NO:
- 35, and the C28 oxidase is selected from MtC28 according to SEQ ID NO:
- 46, QsC28 according to SEQ ID NO:
- 41 and SvC28 according to SEQ ID NO:
- 44;
- or
- (b) the yeast further overexpresses a heterologous gene encoding (v) a cytochrome b5;
- or
- (c) the yeast further overexpresses a heterologous gene encoding (vi) a scaffold protein, wherein the scaffold protein physically interacts with one or more of the C16 oxidase, the C23 oxidase, the C28 oxidase and the CPR, wherein the scaffold protein is a membrane steroid-binding protein (MSBP) selected from AtMSBP1 according to SEQ ID NO:
- 63, AtMSBP2 according to SEQ ID NO:
- 65, QsMSBP1 according to SEQ ID NO:
- 73, SvMSBP1 according to SEQ ID NO:
- 67 and SvMSBP2 according to SEQ ID NO:
- 70;
- and/or
- (2) the yeast is engineered to produce β-amyrin and overexpresses a β-amyrin synthase (BAS) selected from AaBAS according to SEQ ID NO:
- 1, AtBAS according to SEQ ID NO:
- 4, GgBAS according to SEQ ID NO:
- 7, GvBAS according to SEQ ID NO:
- 10, QsBAS according to SEQ ID NO:
- 15, and SvBAS according to SEQ ID NO:
- 13, wherein, optionally, the C16 oxidase is QsC28C16, the C23 oxidase is QsC23, the C28 oxidase is QsC28, the CPR is AtATR1, the MSBP is SvMSBP1, the cytochrome b5 is Qsb5, and the BAS is GvBAS;
- B) a method of producing a C3-glycosylated QA derivative in yeast, wherein the derivative is QA-C3-GlcA, and the method comprises the step of overexpressing, in a yeast engineered to produce QA and UDP-GlcA, a heterologous gene encoding the following enzyme:
- (i) a UDP-GlcA transferase (GlcAT) transferring UDP-GlcA and attaching a GlcA residue at the C3 position of QA to form QA-C3-GlcA wherein, optionally,
- (1) (a) the GlcAT is selected from QsCslG1 according to SEQ ID NO:
- 78, QsCslG2 according to SEQ ID NO:
- 81, and SvCslG according to SEQ ID NO:
- 76;
- or
- (b) the derivative is QA-C3-GlcA-Gal, and the overexpressing further comprises overexpressing a heterologous gene encoding the following enzyme:
- (ii) a UDP-Galactose transferase (GalT) transferring UDP-Gal and attaching a Gal residue to QA-C3-GlcA to form QA-C3-GlcA-Gal, wherein, optionally, the GalT is QsGalT according to SEQ ID NO:
- 116 or GalT is SvGalT according to SEQ ID NO:
- 98;
- or
- (c) the derivative is QA-C3-GlcA-Gal-Rha, the yeast is further engineered to produce UDP-Rha, and the overexpressing further comprises overexpressing a heterologous gene encoding the following enzyme:
- (iii) a UDP-Rhamnose transferase (RhaT) transferring UDP-Rha and attaching a Rha residue to QA-C3-GlcA-Gal to form QA-C3-GlcA-Gal-Rha.
- (2) the derivative is QA-C3-GlcA-Gal-Xyl, the yeast is further engineered to produce UDP-Xyl, and the overexpressing further comprises overexpressing heterologous genes encoding the following enzymes:
- a UDP-Xylose transferase (XylT) transferring UDP-Xylose and attaching a Xyl residue to QA-C3-GlcA-Gal to form QA-C3-GlcA-Gal-Xyl,
- wherein, optionally, the XylT is selected from QsC3XylT according to SEQ ID NO:
- 122 and SvC3XylT according to SEQ ID NO:
- 100;
- and/or
- C) a method of producing UDP-Fucose (UDP-Fuc) in yeast, wherein the method comprises the step of overexpressing heterologous genes encoding the following enzymes:
- (i) a UDP-glucose-4,6-dehydratase (UG46DH) converting UDP-Glc into UDP-4-keto-6-deoxy-glucose and
- (ii) a 4-keto-reductase converting UDP-4-keto-6-deoxy-glucose into UDP-D-Fuc;
- wherein, optionally,
- the UG46DH is SvUG46DH according to SEQ ID NO:
- 87 and the 4-keto-reductase is selected from svNMD according to SEQ ID NO:
- 90 and QsFucSyn according to SEQ ID NO:
- 175;
- D) a method of producing a C-28-glycosylated QA derivative in yeast, wherein the derivative is QA-C3-GlcA-Gal-Rha-C28-Fuc, or QA-C3-GlcA-Gal-Xyl-C28-Fuc, the method comprises the step of overexpressing, in a yeast engineered to produce QA-C3-GlcA-Gal-Rha, or QA-C3-GlcA-Gal-Xyl, and UDP-Fucose, a heterologous gene encoding the following enzyme:
- (i) a UDP-Fucose transferase (FucT) transferring UDP-Fuc and attaching a Fuc residue at the C28 position of QA to form QA-C3-GlcA-Gal-Rha-C28-Fuc, or QA-C3-GlcA-Gal-Xyl-C28-Fuc, wherein, optionally,
- (1) the FucT is selected from QsFucT according to SEQ ID NO:
- 93 and SvFucT according to SEQ ID NO:
- 96;
- or
- (2) the derivative is QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha, or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha, the overexpressing further comprises overexpressing a heterologous gene encoding the following enzyme:
- (ii) a UDP-Rhamnose transferase (RhaT) transferring UDP-Rha and attaching a Rha residue to QA-C3-GlcA-Gal-Rha-C28-Fuc, or QA-C3-GlcA-Gal-Xyl-C28-Fuc, to form QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha, wherein, optionally,
- (2a) the RhaT is QsRhaT according to SEQ ID NO:
- 119;
- or
- (2b) the derivative is QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl, or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl, the overexpressing further comprises overexpressing heterologous genes encoding the following enzyme:
- (iii) a UDP-Xylose transferase (XylT) transferring UDP-Xyl and attaching a Xyl residue to QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha to form GlcA-Gal-Rha-C28-Fuc-Rha-Xyl and QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl, respectively, wherein, optionally,
- (2b1) the XylT is QsC28XylT3 according to SEQ ID NO:
- 125;
- or
- (2b2) the derivative is QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Xyl, or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl, the overexpressing further comprises overexpressing heterologous genes encoding the following enzymes:
- (iv) a UDP-Xylose transferase (XylT) transferring UDP-Xyl and attaching a Xyl residue to QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl to form QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Xyl and QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl, respectively, wherein, optionally,
- (2b2.1) the XylT is QsC28XylT4 according to SEQ ID NO:
- 128;
- or
- (2b2.2) the derivative is QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Api or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Api, the overexpressing further comprises overexpressing heterologous genes encoding the following enzymes:
- (iv) a UDP-Apiose synthase (AXS) converting UDP-GlcA into UDP-Api and (v) a UDP-Apiose transferase (ApiT) transferring UDP-Apiose and attaching an Apiose residue to QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl to form QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Api and QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Api, respectively, wherein, optionally, the AXS is QsAXS according to SEQ ID NO:
- 113 and the ApiT is QsC28ApiT4 according to SEQ ID NO:
- 151;
- E) a method of producing (S)-2-methylbutyryl CoA (2 MB-CoA) in yeast, wherein the method comprises the step of overexpressing a heterologous gene encoding a carboxyl coenzyme A (CoA) ligase (CCL) converting 2 MB acid into 2 MB-CoA, and 2 MB acid is supplemented exogenously;
- wherein, optionally,
- (1) the CCL is QsCCL from Q. saponaria according to SEQ ID NO:
- 178, or
- (2) the overexpressing further comprises overexpressing heterologous genes encoding the following enzymes:
- (i) a phosphopantetheinyl (Ppant) transferase,
- (ii) a megasynthase LovF-TE including an ACP domain, condensing two units of malonyl-CoA to 2 MB-ACP, cleaving 2 MB acid from the ACP domain which is converted into 2 MB-CoA by the CCL,
- and no 2 MB acid is supplemented exogenously, wherein, optionally,
- the Ppant is AnNpgA according to SEQ ID NO:
- 237 and the megasynthase LovF-TE is AstLovF-TE according to SEQ ID NO:
- 235;
- F) a method of producing UDP-Arabinofuranose (UDP-Araf) in yeast, wherein the method comprises the step of overexpressing, in a yeast engineered to produce UDP-Xyl, heterologous genes encoding the following enzymes:
- (i) a UDP-Xyl epimerase (UXE) converting UDP-Xyl into UDP-Arabinopyranose (UDP-Arap), and
- (ii) a UDP-Arabinose mutases (UAM) converting UDP-Arap into UDP-Arabinofuranose (UDP-Ara),
- wherein, optionally, the UXE is selected from AtUXE according to SEQ ID NO:
- 199, AtUXE2 according to SEQ ID NO:
- 202, HvUXE-1 according to SEQ ID NO:
- 240, HvUXE-2 according to SEQ ID NO:
- 242 and AtUGE3 according to SEQ ID NO:
- 205 and the UAM is selected from AtUAM1 according to SEQ ID NO:
- 208 and HvUAM according to SEQ ID NO:
- 211;
- G) a method of producing UDP-Araf in yeast, wherein the method comprises the step of overexpressing heterologous genes encoding the following enzymes:
- (i) an arabinokinase (AraK) and
- (ii) a UDP-sugar pyrophosphorylase (USP),
- and arabinose is supplemented exogenously,
- wherein, optionally,
- the AraK is selected from AtAraK according to SEQ ID NO:
- 214 and LeiAraK according to SEQ ID NO:
- 217 and the USP is selected from AtUSP according to SEQ ID NO:
- 223 and LeiUSP according to SEQ ID NO:
- 226, and wherein, optionally, the overexpressing further comprises overexpressing a heterologous gene encoding an arabinose transporter (AraT), wherein AraT is optionally encoded by the nucleotide sequence SEQ ID NO:
- 221.
- H) a method of producing an acylated and glycosylated QA derivative in yeast, wherein the derivative is QA-C3-GGR-C28-FRX-C9, QA-C3-GGX-C28-FRX-C9, QA-C3-GGR-C28-FRXX-C9, QA-C3-GGX-C28-FRXX-C9, QA-C3-GGR-C28-FRXA-C9 or QA-C3-GGX-C28-FRXA-C9, and the method comprises the step of overexpressing, in a yeast engineered to produce QA-C3-GGR-C28-FRX, QA-C3-GGX-C28-FRX, QA-C3-GGR-C28-FRXX, QA-C3-GGX-C28-FRXX, QA-C3-GGR-C28-FRXA, or QA-C3-GGX-C28-FRXA, heterologous genes encoding the following enzymes:
- (i) a carboxyl coenzyme A ligase (CCL) converting 2 MB acid into 2 MB-CoA,
- (ii) a chalcone-synthase-like type III PKS (Polyketide synthase) condensing malonyl-CoA with 2 MB-CoA to form C9-Keto-CoA,
- (iii) a keto-reductase (KR) converting C9-Keto-CoA into C9-CoA, and
- (iv) an acyltransferase transferring and attaching a first C9-CoA unit to QA-C3-GGR-C28-FRX, QA-C3-GGX-C28-FRX, QA-C3-GGR-C28-FRXX, QA-C3-GGX-C28-FRXX, QA-C3-GGR-C28-FRXA, or QA-C3-GGX-C28-FRXA to form QA-C3-GGR-C28-FRX-C9, QA-C3-GGX-C28-FRX-C9, QA-C3-GGR-C28-FRXX-C9, QA-C3-GGX-C28-FRXX-C9, QA-C3-GGR-C28-FRXA-C9 or QA-C3-GGX-C28-FRXA-C9. wherein 2 MB acid is supplemented exogenously;
- I) a method of producing QA-C3-GGX-C28-FRX-C18-Xyl, QA-C3-GGR-C28-FRX-C18-Xyl, QA-C3-GGX-C28-FRXX-C18-Xyl, QA-C3-GGR-C28-FRXX-C18-Xyl, QA-C3-GGX-C28-FRX-C18-Xyl, QA-C3-GGX-C28-FRXA-C18-Xyl or QA-C3-GGR-C28-FRX-C18-Xyl in a yeast, wherein the method comprises the step of overexpressing, in a yeast engineered to produce QA-C3-GGX-C28-FRX-C18, QA-C3-GGR-C28-FRX-C18, QA-C3-GGX-C28-FRXX-C18, QA-C3-GRX-C28-FRXX-C18, QA-C3-GGX-C28-FRX-C18, QA-C3-GGX-C28-FRXA-C18 or QA-C3-GGR-C28-FRX-C18, a heterologous gene encoding an arabinotransferase (ArafT) transferring UDP-Xyl and attaching a Xyl residue to QA-C3-GGX-C28-FRX-C18, QA-C3-GGR-C28-FRX-C18, QA-C3-GGX-C28-FRXX-C18, QA-C3-GRX-C28-FRXX-C18, QA-C3-GGX-C28-FRX-C18, QA-C3-GGX-C28-FRXA-C18 and QA-C3-GGR-C28-FRX-C18 to form QA-C3-GGX-C28-FRX-C18-Xyl, QA-C3-GGR-C28-FRX-C18-Xyl, QA-C3-GGX-C28-FRXX-C18-Xyl, QA-C3-GRX-C28-FRXX-C18-Xyl, QA-C3-GGX-C28-FRX-C18-Xyl, QA-C3-GGX-C28-FRXA-C18-Xyl or QA-C3-GGR-C28-FRX-C18-Xyl;
- wherein, optionally,
- the ArafT is QsArafT according to SEQ ID NO:
- 229;
- and
- J) a method of producing QA-C3-GGX-C28-FRXX-C18-Araf (QS-21-Xyl) or QA-C3-GGX-C28-FRXA-C18-Araf (QS-21-Api) in yeast, wherein
- (1) the method of producing QA-C3-GGX-C28-FRXX-C18-Araf (QS-21-Xyl) comprises the step of overexpressing heterologous genes encoding GvBAS according to SEQ ID NO:
- 10, QsC28C16 according to SEQ ID NO:
- 23, QsC23 according to SEQ ID NO:
- 29, QsC28 according to SEQ ID NO:
- 41, AtATR1 according to SEQ ID NO:
- 49, Qsb5 according to SEQ ID NO:
- 55, SvMSBP1 according to SEQ ID NO:
- 67, AtUGDA101L according to SEQ ID NO:
- 108, QsCslG2 according to SEQ ID NO:
- 78, QsGalT according to SEQ ID NO:
- 116, AtUXS according to SEQ ID NO:
- 105, QsC3XylT according to SEQ ID NO:
- 122, SvNMD according to SEQ ID NO:
- 90, SvUG46DH according to SEQ ID NO:
- 87, QsFuct according to SEQ ID NO:
- 93, AtRHM2 according to SEQ ID NO:
- 102, QsRhaT according to SEQ ID NO:
- 119, QsC28XylT3 according to SEQ ID NO:
- 125, QsC28XylT4 according to SEQ ID NO:
- 128, QsChSD according to SEQ ID NO:
- 181, QsChSE according to SEQ ID NO:
- 184, QsKR11 according to SEQ ID NO:
- 187, QsKR23 according to SEQ ID NO:
- 190, QsDMOT9 according to SEQ ID NO:
- 193, QsDMOT4 according to SEQ ID NO:
- 196, AtUXE according to SEQ ID NO:
- 199, AtUAM1 according to SEQ ID NO:
- 208, QsArafT2 according to SEQ ID NO:
- 232, AnNpgA according to SEQ ID NO:
- 237, QsCCL according to SEQ ID NO:
- 178 and AstLovF-TE according to SEQ ID NO:
- 235;
- and
- (2) the method of producing QA-C3-GGX-C28-FRXA-C18-Araf (QS-21-Api) comprises the step of overexpressing heterologous genes encoding GvBAS according to SEQ ID NO:
- 10, QsC28C16 according to SEQ ID NO:
- 23, QsC23 according to SEQ ID NO:
- 29, QsC28 according to SEQ ID NO:
- 41, AtATR1 according to SEQ ID NO:
- 49, Qsb5 according to SEQ ID NO:
- 55, SvMSBP1 according to SEQ ID NO:
- 67, AtUGDA101L according to SEQ ID NO:
- 108, QsCslG2 according to SEQ ID NO:
- 81, QsGalT according to SEQ ID NO:
- 116, AtUXS according to SEQ ID NO:
- 105, QsC3XylT according to SEQ ID NO:
- 122, SvNMD according to SEQ ID NO:
- 90, SvUG46DH according to SEQ ID NO:
- 87, QsFucT according to SEQ ID NO:
- 93, AtRHM2 according to SEQ ID NO:
- 102, QsRhaT according to SEQ ID NO:
- 119, QsC28XylT3 according to SEQ ID NO:
- 125, QsC28ApiT4 according to SEQ ID NO:
- 151, QsChSD according to SEQ ID NO:
- 181, QsChSE according to SEQ ID NO:
- 184, QsKR11 according to SEQ ID NO:
- 187, QsKR23 according to SEQ ID NO:
- 190, QsDMOT9 according to SEQ ID NO:
- 193, QsDMOT4 according to SEQ ID NO:
- 196, AtUXE according to SEQ ID NO:
- 199, AtUAM1 according to SEQ ID NO:
- 208, QsArafT2 according to SEQ ID NO:
- 232, AnNpgA according to SEQ ID NO:
- 237, QsCCL according to SEQ ID NO:
- 178 and AstLovF-TE according to SEQ ID NO:
- 235.
195 more elements
An isolated polypeptide selected from a β-amyrin synthase (SvBAS) according to SEQ ID NO:
- 13, a β-amyrin synthase (QsBAS) according to SEQ ID NO:
- 15, a CYP C16 oxidase (QsC28C16) according to SEQ ID NO:
- 23, a CYP C16 oxidase (SvC16) according to SEQ ID NO:
- 26, CYP C23 oxidase (SvC23-1) according to SEQ ID NO:
- 32, a CYP C23 oxidase (SvC23-2) according to SEQ ID NO:
- 35, a CYP C28 oxidase (SvC28) according to SEQ ID NO:
- 44, ACytochrome b5 protein (Qsb5) according to SEQ ID NO:
- 55, a Cytochrome b5 protein (Svb5) according to SEQ ID NO:
- 61, a UDP-GlcA transferase (SvCslG) according to SEQ ID NO:
- 76, AMSBP protein (SvMSBP1) according to SEQ ID NO:
- 67, AMSBP protein (SvMSBP2) according to SEQ ID NO:
- 70, a MSBP protein (QsMSBP1) according to SEQ ID NO:
- 73, a UDP-glucose-4,6-dehydratase (SvUG46DH) according to SEQ ID NO:
- 87, a UDP-4-keto-6-deoxy-glucose reductase (SvNMD) according to SEQ ID NO:
- 90, a UDP-Galactose transferase (SvGalT) according to SEQ ID NO:
- 98, a UDP-Fucose transferase (SvFucT) according to SEQ ID NO:
- 96, a UDP-Xylose transferase (SvC3XylT) according to SEQ ID NO:
- 100, AUDP-Arabinofuranose transferase (QsArafT2) according to SEQ ID NO:
- 229, a UDP-glucose dehydrogenase (AtUGDA101L) according to SEQ ID NO:
- 108, a UDP-Xylose transferase (QsC28XylT4-3aa) according to SEQ ID NO:
- 131, a AUDP-Xylose transferase (QsC28XylT4-6aa) according to SEQ ID NO:
- 134, a UDP-Xylose transferase (QsC28XylT4-9aa) according to SEQ ID NO:
- 137, a UDP-Xylose transferase (QsC28XylT4-12aa) according to SEQ ID NO:
- 140, AUDP-Xylose transferase (SUMO-QsC28XylT4) according to SEQ ID NO:
- 143, a UDP-Xylose transferase (TrXA-QsC28XylT4) according to SEQ ID NO:
- 145, a UDP-Xylose transferase (MBP-QsC28XylT4) according to SEQ ID NO:
- 147, a AUDP-Xylose transferase (QsC28XylT3-3×GGGS-QsC28XylT4) according to SEQ ID NO:
- 149 and a type I polyketide synthase (AstLovF-TE) according to SEQ ID NO:
- 235.
26 more elements
Dependencies are inferred from the claim text.