Patent Application
20240352498
A Sequence Listing in XML format is incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is B21-092-2WO.xml. The XML file is 496,599 bytes and was created on Jun. 14, 2024.
The present invention relates to the biosynthetic production of QS-21, precursors and variants thereof, and non-native sugar in yeast, as well as to related aspects.
QS-21 is a natural saponin extract from the bark of the Chilean ‘soapbark’ tree, Quillaja saponaria. QS-21 extract was originally identified as a fraction purified from a crude bark extract of Quillaja Saponaria Molina obtained by RP-HPLC purification (peak 21) (Kensil et al. 1991). Crude bark extracts have been reported to comprise a wide range of saponins. The QS-21 extract, or fraction, comprises several distinct saponin molecules. Two principal isomeric molecular constituents of the fraction were reported (Ragupathi et al. 2011) and are depicted in
Saponins from Q. saponaria, including QS-21, have been known for many years to have potent immunostimulatory properties, capable of enhancing antibody production and specific T-cell responses. These properties have resulted in the development of Q. saponaria saponin-based adjuvants for vaccines. Of particular note, the AS01 adjuvant features a liposomal formulation including QS-21 and 3-O-desacyl-4′-monophosphoryl lipid A (3D-MPL) (Garcon, 2011; Didierlaurent, 2017) and is currently licenced in vaccines for diseases including shingles (Shingrix™) and malaria (Mosquirix™).
With more vaccines including QS-21 becoming available, the demand for its supply is expected to increase substantially over the years. Therefore, there remains a need for providing methods of production of QS-21 which do not rely upon natural resources, such as biosynthetic methods of production in yeast. Examples of advantages of such methods are as follows: (i) complex purification schemes designed to separate saponins from complex mixtures including multiple saponins (such as from a crude bark extract) are avoided; (ii) ability to produce individual saponins otherwise hard to separate when present in a crude bark extract (e.g. QS-21-Api and QS-21-Xyl); and (iii) ability to produce any saponin of interest (including precursors otherwise not purifiable from crude bark extracts.
The biosynthetic production of QS-21 precursors has been reported in Nicotiana benthamiana (e.g. WO 19/122259, WO 20/260475 and WO 22/136563). Quillaic acid production at trace levels has been reported in yeast (WO 20/263524). The present invention reports for the first time the successful production, in yeast, of QS-21 and glycosylated precursors and variants thereof.
In a first aspect of the invention, there is provided 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:
In a second aspect, there is provided a method of producing UDP-Glucuronic acid (UDP-GlcA) in yeast, wherein the method comprises the step of overexpressing a heterologous gene encoding a UDP-glucose dehydrogenase (UGD) converting UDP-Glucose (UDP-Glc) into UDP-GlcA; and a yeast which is engineered to produce UDP-GlcA accordingly.
In a third aspect, there is provided a method of producing UDP-Rhamnose (UDP-Rha) in yeast, wherein the method comprises the step of overexpressing a heterologous gene encoding a UDP-rhamnose synthase (RhaT) converting UDP-Glc into UDP-Rha; and a yeast which is engineered to produce UDP-Rha accordingly.
In a fourth aspect, there is provided a method of producing UDP-Xylose (UDP-Xyl) in yeast, wherein the method comprises the step of overexpressing heterologous genes encoding the following enzymes:
In a fifth aspect, there is provided a method of producing a C3-glycosylated QA derivative in yeast, wherein the method comprises the step of overexpressing, in a yeast engineered to produce QA and UDP-GlcA, a heterologous gene encoding the following enzyme:
In a sixth aspect, there is provided a method of producing UDP-Fuc (UDP-Fuc) in yeast, wherein the method comprises the step of overexpressing heterologous genes encoding the following enzymes:
In a seventh aspect, there is provided a method of producing a C28-glycosylated QA derivative in yeast, wherein the method comprises the step of overexpressing, in a yeast engineered to produce a C3-glycosylated QA derivative, a heterologous gene encoding the following enzyme:
In an eighth aspect, there is provided 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-methylbutyric acid (2 MB) acid into 2 MB-CoA, and 2 MB acid is supplemented exogenously; and a yeast which is engineered to produce 2 MB-CoA accordingly.
In a ninth aspect, there is provided 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:
In a tenth aspect, there is provided a method of producing an acylated and glycosylated QA derivative in yeast, wherein the method comprises the step of overexpressing, in a yeast engineered to produce a glycosylated QA derivative, heterologous genes encoding the following enzymes:
In an eleventh aspect, there are provided QA derivatives obtained according to the method of the first to tenth aspects of the invention.
In a twelfth aspect, there is provided the use of QA derivatives according to the eleventh aspect of the invention as an adjuvant
In a thirteenth aspect, there are provided isolated enzymes or proteins used in the method of the first to tenth aspects of the invention.
Using more than 30 heterologous proteins from different plant and microbial origins spanning across six distinctively different protein types, including in particular a terpene synthase, cytochrome P450 monooxygenases (or ‘CYP oxidases’), nucleotide sugar synthases, sugar transferases, acyltransferases, and polyketide synthases (PKSs), the inventors have been able, for the first time, to reconstitute the metabolic pathway leading to the successful biosynthesis of QS-21 in Saccharomyces cerevisiae, starting from a simple sugar, galactose.
Quillaic acid (QA), the triterpene core of QS-21, derives from the simple triterpene β-amyrin, which is synthesised through cyclisation of the universal linear precursor 2,3-oxidosqualene (OS) (according to the mevalonate pathway which is native to yeast—Wong et al. 2018), by an oxidosqualene cyclase (OSC), also referred to as a β-amyrin synthase (‘BAS’) (see
Next, UDP-Glucuronic acid (UDP-GlcA’), UDP-galactose (‘UDP-Gal’), and UDP-Xylose (‘UDP-Xyl’) or UDP-Rhamnose (‘UDP-Rha’), are incorporated at the C3 position of QA by respective glycosyltransferases resulting in the formation of C3-glycosylated QA derivatives (see
The formula of which being provided in Table 1.
Next, UDP-fucose (‘UDP-Fuc’), UDP-Rha, UDP-Xyl, and a second UDP-Xyl or a UDP-Api, are incorporated at the C28 position of QA by respective glycosyltransferases resulting in the formation of C28-glycosylated QA derivatives (see
The formula of which being provided in Table 1.
Biosynthesis of the 18-carbon pseudo-dimeric acyl chain is achieved by condensing malonyl-CoA (which is native to yeast) with S-2-methylbutyryl-CoA (‘2 MB-CoA’) to make C9-CoA using a type I polyketide synthase (‘PKS’), a carboxyl coenzyme A ligase (‘CCL’), type III PKSs and keto-reductases (KRs) (see
Next, two repeating C9-CoA acyl units are successively transferred by 2 acyltransferases leading to the addition of 18-carbon pseudo-dimeric acyl chain to the fucose residue of the linear tetrasaccharide at the C28 position and resulting in the formation of acylated and glycosylated QA derivatives. Such acylated and glycosylated QA derivatives are individually referred to herein as follows:
The formula of which being provided in Table 1.
Next, UDP-arabinofuranose (‘UDP-Araf), or UDP-Xyl, is incorporated at the end of the 18-carbon pseudo-dimeric acyl chain (on the 5-hydroxy function group of the second C9-CoA acyl unit), resulting in the formation of further acylated and glycosylated QA derivatives (see
The formula of which being provided in Table 1.
‘C3-glycosylated QA derivative’ designates, in the sense of the invention, a QA derivative including at least a glucuronic acid residue at position C3 (as listed above). ‘C28-glycosylated QA derivative’ designates, in the sense of the invention, a QA derivative including all three sugars of the branched trisaccharide at position C3 and at least the fucose residue of the linear tetrasaccharide at position C28 (as listed above). ‘Acylated and glycosylated QA derivative’ designates, in the sense of the invention, a QA derivative including all three sugars of the branched trisaccharide at position C3, at least the first three sugars of the linear tetrasaccharide at position C28, at least one C9-CoA acyl unit (‘C9’) attached to the fucose residue and, optionally, an arabinofuranose residue, when two C9-CoA acyl units (‘C18’) attached (as listed above).
In the sense of the present invention, ‘heterologous genes’ is to be understood as genes not naturally expressed in yeast.
In the sense of the present invention, ‘a yeast engineered to produce e.g. a sugar or a QA derivative is to be understood as a yeast overexpressing the heterologous genes encoding the enzymes or proteins necessary to the biosynthesis or production of the respective QA derivative, e.g. as described in the respective methods of the first to tenth aspects of the invention.
WO 19/122259 reports the identification of enzymes in the Q. saponaria genome involved in the biosynthesis of QA and the production of QA in Nicotiana benthamiana engineered with such enzymes. WO 20/263524 reports the production of traces of QA in yeast engineered with enzymes originating from different plant origins. The content of both WO 19/122259 and WO 20/263524 is incorporated herein by reference.
The first step of the method of the first aspect of the invention is the cyclisation of 2,3-oxidosqualene to form β-amyrin. This step is carried out by an oxidosqualene cyclase or β-amyrin synthase (BAS). Any heterologous R-amyrin synthase capable of producing β-amyrin from any plant origin may suitably be used in the method of the invention. For example, β-amyrin synthases (BAS) from Artemisia annua (A. annua or ‘Aa’), Arabidopsis thaliana (A. thaliana or ‘At’), Glycyrrhiza glabra (G. glabra or ‘gG’), Gypsophila vaccaria (G. vaccaria or ‘Gv’), Medicago truncatula (M. truncatula or ‘Mt’), Quillaja saponaria (Q. saponaria or ‘Qs’), or Saponaria vaccaria (S. vaccaria or ‘Sv’) may be used. In some embodiments, the method of the first aspect of the invention uses a β-amyrin synthase selected from the foregoing plants. In particular, the β-amyrin synthase may be 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. Advantageously, the β-amyrin synthase is from GvBAS according to SEQ ID NO: 10.
AaBAS, AtBAS, GgBAS, GvBAS, GvBAS, QsBAS or SvBAS may alternatively be according to sequences at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 15 or SEQ ID NO: 13.
As described earlier, β-amyrin is successively further oxidized with a carboxylic acid group, a hydroxyl group and aldehyde group at the C28, C16 and C23 position, respectively, by corresponding cytochrome P450 (CYP) oxidases, resulting in the formation of QA.
Any heterologous CYP oxidase from any plant origin previously identified and reported to be effectively capable of functionalizing the respective C28, C16 and C23 positions of β-amyrin may be used in the methods and engineered yeasts of the invention (e.g. as described and reported in WO 19/122259 or WO 2020/263524, or Gosh, 2017 for a review, the content of which being incorporated by reference). In some embodiments, the method of the first aspect of the invention uses a CYP C16 oxidase, a CYP C23 oxidase and a CYP C28 oxidase independently selected from A. annua, A. thaliana, G. glabra, M. truncatula, Q. saponaria, S. vaccaria, Centella asiatica, Bupleurum falcatum, Maesa lanceolate, Q. saponaria and S. vaccaria.
In further embodiments, the CYP C16 oxidase is selected from CYP87D16 and CYP716Y1; the CYP C23 oxidase is selected from CYP72A68 and CYP714E19; the CYP C28 oxidase is selected from CYP716A1, CYP716A12, CYP716A15, CYP716A17, CYP716A44, CYP716A46, CYP716A52v2, CYP716A75, CYP716A78, CYP716A79, CYP716A80, CYP716A81, CYP716A83, CYP716A86, CYP716A110, CYP716A140, CYP716A179, CYP716A252; CYP16A253 and CYP716AL1.
In further embodiments, the CYP C16 oxidase is selected from BfC16 according to SEQ ID NO: 17, QsC16 oxidase according to SEQ ID NO: 20, QsC28C16 according to SEQ ID NO: 23, and SvC16 according to SEQ ID NO: 26.
In further embodiments, the CYP C16 oxidase is selected from BfC16 according to SEQ ID NO: 17, SvC16 according to SEQ ID NO: 26, QsC16 according to SEQ ID NO: 20 and QsC28C16 according to SEQ ID NO: 23. BfC16, SvC16, QsC16 and QsC28C16 may alternatively be according to sequences at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 20 and SEQ ID NO: 23, respectively.
In further embodiments, the CYP 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. MtC23, QsC23, SvC23-1 and SvC23-2 may alternatively be according to sequences at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 38, SEQ ID NO: 29, SEQ ID NO: 32 and SEQ ID NO: 35, respectively.
In further embodiments, the CYP C28 oxidase is selected from MtC28 according to SEQ ID NO: 46, QsC28 according to SEQ ID NO: 41, or SvC28 according to SEQ ID NO: 44. MtC28, QsC28 and SvC28 may alternatively be according to sequences at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 46, SEQ ID NO: 41 and SEQ ID NO: 44, respectively.
Heterologous redox partners, such as cytochrome P450 reductase (CPR) and/or cytochrome b5, may be further co-expressed in the method of the first aspect of the invention. For example, the CPR may be selected from A. thaliana and Lotus japonicus. In some embodiments, CPR is selected from AtATR1 according to SEQ ID NO: 49 and LjCPR according to SEQ ID NO: 52.
Heterologous cytochrome b5 may be selected from A. thaliana, Q. saponaria and S. vaccaria. In some embodiments, cytochrome b5 is selected from Atb5 according to SEQ ID NO: 58, Qsb5 according to SEQ ID NO: 55 and Svb5 according to SEQ ID NO: 61. Atb5, Qsb5 and Svb5 may alternatively be according to sequences at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 58, SEQ ID NO: 55 and SEQ ID NO: 61, respectively.
Heterologous scaffold proteins (allowing to physically organize the P450 enzymes) may be further co-expressed in the method of the first aspect of the invention. The scaffold protein may be a membrane steroid-binding protein (MSBP). For example, the MSBP may be selected from A thaliana, Q. saponaria, and S. vaccaria. In some embodiments, MSBP is 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. AtMSBP2, QsMSBP1, SvMSBP1 and SvMSBP2 may alternatively be according to sequences at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 73, SEQ ID NO: 67 and SEQ ID NO: 70, respectively.
The first aspect of the invention also provides a yeast which is engineered to produce QA.
As described earlier, a branched trisaccharide consisting of GlcA, Gal and Xyl (or Rha) is attached at the C3 position of QA.
The method according to the second aspect of the invention comprises the step of overexpressing of a heterologous gene encoding a UDP-glucose dehydrogenase (UGD) converting UDP-Glucose (UDP-Glc) into UDP-GlcA. UGD from different plant origins may be used. In some embodiments, the UGD is selected from A. thaliana, Synechococcus sp. (Syn), Homo sapiens (Hs), Paramoeba atlantica (Patl), Bacillus cytotoxicus (Bcyt), Corallococcus macrosporus (Myxfulv), and Pyrococcus furiosus (Pfu). In further embodiments, the UGD is selected from AtUGD according to SEQ ID NO: 84, AtUGD101L according to SEQ ID NO: 108, SynUGD according to SEQ ID NO: 154, HsUGDA104L according to SEQ ID NO: 157, PatIUGD according to SEQ ID NO: 110, BcytUGD according to SEQ ID NO: 160, MyxfulvUGD according to SEQ ID NO: 163 and PfuUGD according to SEQ ID NO: 166. AtUGD, AtUGD101L, SynUGD, HsUGDA104L, PatIUGD, BcytUGD, MyxfulvUGD, PfuUGD may alternatively be according to sequences at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 84, SEQ ID NO: 108, SEQ ID NO: 154, SEQ ID NO: 157, SEQ ID NO: 110, SEQ ID NO: 160, SEQ ID NO: 163 and SEQ ID NO: 166, respectively.
The second aspect of the invention also provides a yeast which is engineered to produce UDP-GlcA.
The first step of the method of the third aspect of the invention is the overexpression of a heterologous gene encoding a UDP-rhamnose synthase. A UDP-rhamnose synthase from different plant origins may be used. In some embodiments, the UDP-rhamnose synthase is AtRHM2 from A. thaliana according to SEQ ID NO: 102, or a sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 102.
The third aspect of the invention also provides a yeast which is engineered to produce UDP-Rha.
The first step of the method of the fourth aspect of the invention is the overexpression of a heterologous gene encoding a UDP-glucose dehydrogenase (UGD) converting UDP-Glucose (UDP-Glc) into UDP-GlcA. The UGD may be any of the UGD described earlier in the method of the second aspect of the invention. The second step of the method of the fourth aspect of the invention is the overexpression of a heterologous gene encoding a UDP-xylose synthase (UXS). UDP-Xyl may be produced by decarboxylation of UDP-GlcA by a UDP-Xyl synthase (UXS) and/or by a dual UDP-Api/Xyl synthase (AXS). The UDP-Xylose synthase and dual UDP-Api/Xyl synthase may be from different plant origins, e.g. from A. thaliana and Q. saponaria. In some embodiments, the UXSis selected from AtUXS encoded by SEQ ID NO: 105 and QsAXS encoded by SEQ ID NO: 113, or a sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 105 and SEQ ID NO: 113, respectively.
The fourth aspect of the invention also provides a yeast which is engineered to produce UDP-Xyl.
As shown in
The fifth aspect of the invention also provides a yeast which is engineered to produce QA-C3-GlcA (aspect 5a).
As shown in
The fifth aspect of the invention also provides a yeast which is engineered to produce QA-C3-GlcA-Gal (aspect 5b).
As shown in
The RhaT may be from any plant origin, for example, may be from Q. saponaria. In some embodiments, the RhaT is QsRhaT according to SEQ ID NO: 119, or a sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 119.
The fifth aspect of the invention also provides a yeast which is engineered to produce QA-C3-GlcA-Gal-Rha (aspect 5c).
As shown in
The fifth aspect of the invention also provides a yeast which is engineered to produce QA-C3-GlcA-Gal-Xyl (aspect 5d).
As described earlier, a linear trisaccharide consisting of FRXX/A is attached at the C28 position of QA.
The first step of the method of the sixth aspect of the invention is the overexpression of heterologous genes encoding a UDP-glucose-4,6-dehydratase (UG46DH) converting UDP-Glc into UDP-4-keto-6-deoxy-glucose and a 4-keto-reductase converting UDP-4-keto-6-deoxy-glucose into UDP-D-Fuc. The UG46DH and 4-keto-reductase may be from any plant origin, for example, may be selected independently from Q. saponaria and S. vaccaria. In some embodiments, the UG46DH is SvUG46DH according to SEQ ID NO: 87, or a sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 87. In some embodiments, the 4-keto-reductase is selected from svNMD according to SEQ ID NO: 90 and QsFucSyn according to SEQ ID NO: 175, or a sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 90 or SEQ ID NO: 175.
The sixth aspect of the invention also provides a yeast which is engineered to produce UDP-Fuc.
As shown in
The seventh aspect of the invention also provides a yeast which is engineered to produce QA-C3-GGR-C28-F, or QA-C3-GGX-C28-F (aspect 7a).
QA-C3-GGX-C28-FR and QA-C3-GGR-C28-FR production
As shown in
The seventh aspect of the invention also provides a yeast which is engineered to produce QA-C3-GGR-C28-FR, or QA-C3-GGX-C28-FR (aspect 7b).
As shown in
The seventh aspect of the invention also provides a yeast which is engineered produce QA-C3-GGR-C28-FRX, or QA-C3-GGX-C28-FRX (aspect 7c).
QA-C3-GGX-C28-FRXX and QA-C3-GGR-C28-FRXX Production
As shown in
The seventh aspect of the invention also provides a yeast which is engineered produce QA-C3-GGR-C28-FRXX, or QA-C3-GGX-C28-FRXX (aspect 7d).
As shown in
The seventh aspect of the invention also provides a yeast which is engineered to produce QA-C3-GGR-C28-FRXA, or QA-C3-GGX-C28-FRXA (aspect 7e).
Production and Attachment of the 18-Carbon Pseudo-Dimeric Acyl Chain Terminated with an Arabinofuranose (C18-Araf)
As shown in
The method of the eighth aspect of the invention comprises the step of overexpressing a heterologous gene encoding a carboxyl coenzyme A (CoA) ligase (CCL) converting 2-methylbutyric acid (2 MB) acid into 2 MB-CoA, wherein 2 MB acid is supplemented exogenously. The CCL may be from any plant origin. In some embodiments, the CCL is QsCCL from Q. saponaria according to SEQ ID NO: 178, or a sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 178. In an alternative embodiment (which does not require any exogenous supply of 2 MB acid), the method further comprises overexpressing heterologous genes encoding the following enzymes:
The Ppant may be from Aspergillus nidulans and the megasynthase LovF-TE may be from Aspergillus terreus. In some embodiments, the Ppant is AnNpgA according to SEQ ID NO: 237 and the megasynthase LovF-TE is AstLovF-TE according to SEQ ID NO: 235, or a sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 237 and SEQ ID NO:235, respectively.
The eighth aspect of the invention also provides a yeast which is engineered to produce 2 MB-CoA.
The method according to the ninth aspect of the invention comprises the step of overexpressing, in a yeast engineered to produce UDP-Xyl, heterologous genes encoding the following enzymes:
The yeast engineered to produce UDP-Xyl may be according to the fourth aspect of the invention.
The UXE and the UAM may be independently selected from A. thaliana and H. vulgare. In some embodiments, 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, or a sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 199, SEQ ID NO:202, SEQ ID NO: 240, SEQ ID NO: 242 and SEQ ID NO: 205, respectively.
In some embodiments, the UAM is selected from AtUAM1 according to SEQ ID NO: 208 and HvUAM according to SEQ ID NO: 211, or a sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 208 and SEQ ID NO: 211, respectively.
The ninth aspect of the invention also provides a yeast which is engineered to produce UDP-Arabinofuranose.
As shown in
The first step of the method of the tenth aspect of the invention is the overexpression of heterologous genes, in a yeast engineered to produce a glycosylated QA derivative, encoding the following enzymes:
For example, 2 MB acid may be added directly into the yeast culture medium, at any appropriate time.
In the method according to the tenth aspect of the invention, the glycosylated QA derivative may be QA-C3-GGX-C28-FRX, QA-C3-GGR-C28-FRX, QA-C3-GGX-C28-FRXX, QA-C3-GGR-C28-FRXX, QA-C3-GGX-C28-FRXA, or QA-C3-GGR-C28-FRXA. The yeast engineered to produce QA-C3-GGX-C28-FRX and QA-C3-GGR-C28-FRX may be according to the aspect 7c. The yeast engineered to produce QA-C3-GGX-C28-FRXX and QA-C3-GGR-C28-FRXX may be according to the aspect 7d. The yeast engineered to produce QA-C3-GGX-C28-FRXA and QA-C3-GGR-C28-FRXA may be according to the aspect 7e of the invention.
In the first step of the method according to the tenth aspect of the invention, the CCL may be as described in the method of the eighth aspect of the invention.
The chalcone-synthase-like type III PKS may be form any plant origin. In some embodiments, the chalcone-synthase-like are QsChSD according to SEQ ID NO: 181, QsChSE according to SEQ ID NO: 184, or both QsChSD according to SEQ ID NO:181 and QsChSE according to SEQ ID NO: 184, or a sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 181 and SEQ ID NO: 184, respectively.
The KR may be from any plant origin. In some embodiments, the KR is QsKR11 according to SEQ ID NO: 187, QsKR23 according to SEQ ID NO: 190, or both QsKR11 according to SEQ ID NO: 187 and QsKR23 according to SEQ ID NO: 190, or a sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 187 and SEQ ID NO: 190, respectively.
The acyltransferase may be from any plant origin. In some embodiments, the acyltransferase is QsDMOT9 according to SEQ ID NO: 193, or a sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 187 and SEQ ID NO: 193.
The tenth aspect of the invention also provides a yeast which is engineered to produce C3-GGX-C28-FRX-C9, QA-C3-GGR-C28-FRX-C9, C3-GGX-C28-FRXX-C9, QA-C3-GGR-C28-FRXX-C9, C3-GGX-C28-FRXA-C9, or QA-C3-GGR-C28-FRXA-C9 (aspect 10a).
In the second step of the method according to the tenth aspect of the invention, the acylated and glycosylated QA derivative may be C3-GGX-C28-FRX-C9, QA-C3-GGR-C28-FRX-C9, C3-GGX-C28-FRXX-C9, QA-C3-GGR-C28-FRXX-C9, C3-GGX-C28-FRXA-C9, or QA-C3-GGR-C28-FRXA-C9. The yeast engineered to produce C3-GGX-C28-FRX-C9, QA-C3-GGR-C28-FRX-C9, C3-GGX-C28-FRXX-C9, QA-C3-GGR-C28-FRXX-C9, C3-GGX-C28-FRXA-C9, or QA-C3-GGR-C28-FRXA-C9 may be according to the aspect 10a of the invention.
The third step of the method according to the tenth aspect of the invention further comprises overexpressing a gene encoding (v) a second acyltransferase attaching a second C9-CoA unit to an acylated and glycosylated QA derivative to form a further acylated and glycosylated QA derivative.
In the third step of the method according to the tenth aspect of the invention, the acylated and glycosylated QA derivative may be C3-GGX-C28-FRX-C9, QA-C3-GGR-C28-FRX-C9, C3-GGX-C28-FRXX-C9, QA-C3-GGR-C28-FRXX-C9, C3-GGX-C28-FRXA-C9, or QA-C3-GGR-C28-FRXA-C9. The yeast engineered to produce C3-GGX-C28-FRX-C9, QA-C3-GGR-C28-FRX-C9, C3-GGX-C28-FRXX-C9, QA-C3-GGR-C28-FRXX-C9, C3-GGX-C28-FRXA-C9 and QA-C3-GGR-C28-FRXA-C9 may be according to aspect 10a of the invention.
The acyltransferase may be from any plant origin. In some embodiments, the acyltransferase is QsDMOT4 according to SEQ ID NO: 196, or a sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 196.
The tenth aspect of the invention also provides a yeast which is engineered to produce C3-GGX-C28-FRX-C18, QA-C3-GGR-C28-FRX-C18, C3-GGX-C28-FRXX-C18, QA-C3-GGR-C28-FRXX-C18, C3-GGX-C28-FRXA-C18, or QA-C3-GGR-C28-FRXA-C18 (aspect 10b).
The fourth step of the method according to the tenth aspect of the invention further comprises overexpressing, in a yeast engineered to produce UDP-Araf, a heterologous gene encoding (vi) an arabinotransferase (ArafT) transferring UDP-Araf and attaching an Araf residue to an acylated and glycosylated QA derivative to form an acetylated and further glycosylated QA derivative.
In the fourth step of the method according to the tenth aspect of the invention, the acylated and glycosylated QA derivative may be C3-GGX-C28-FRX-C18, QA-C3-GGR-C28-FRX-C18, C3-GGX-C28-FRXX-C18, QA-C3-GGR-C28-FRXX-C18, C3-GGX-C28-FRXA-C18, or QA-C3-GGR-C28-FRXA-C18. The yeast engineered to produce C3-GGX-C28-FRX-C18, QA-C3-GGR-C28-FRX-C18, C3-GGX-C28-FRXX-C18, QA-C3-GGR-C28-FRXX-C18, C3-GGX-C28-FRXA-C18 and QA-C3-GGR-C28-FRXA-C18 may be according to aspect 10b of the invention.
The ArafT may be from any plant origin, for example, is from Q. saponaria. In some embodiments, the ArafT is selected from QsArafT according to SEQ ID NO: 229 and QsArafT2 according to SEQ ID NO: 232, or a sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 229 and SEQ ID NO: 232, respectively.
The tenth aspect of the invention also provides a yeast which is engineered to produce QA-C3-GGR-C28-FRX-C18-Araf, QA-C3-GGX-C28-FRX-C18-Araf, QA-C3-GGR-C28-FRXX-C18-Araf, QA-C3-GGX-C28-FRXX-C18-Araf, QA-C3-GGR-C28-FRXA-C18-Araf or QA-C3-GGX-C28-FRXA-C18-Araf (aspect 10c).
In embodiments, where QsArafT according to SEQ ID NO: 229 is used in the fourth step of the method according to the tenth aspect of the invention, QA-C3-GGR-C28-FRX-C18-Xyl, QA-C3-GGX-C28-FRX-C18-Xyl, QA-C3-GGR-C28-FRXX-C18-Xyl, QA-C3-GGX-C28-FRXX-C18-Xyl, QA-C3-GGR-C28-FRXA-C18-Xyl or QA-C3-GGX-C28-FRXA-C18-Xyl are also formed. The tenth aspect of the invention further provides a yeast which is engineered to produce QA-C3-GGR-C28-FRX-C18-Xyl, QA-C3-GGX-C28-FRX-C18-Xyl, QA-C3-GGR-C28-FRXX-C18-Xyl, QA-C3-GGX-C28-FRXX-C18-Xyl, QA-C3-GGR-C28-FRXA-C18-Xyl or QA-C3-GGX-C28-FRXA-C18-Xyl (aspect 10d of the invention).
In the fifth step of the method according to the tenth aspect of the invention, the method further comprises overexpressing heterologous genes encoding the following enzymes:
The Ppant may be from Aspergillus nidulans and the megasynthase LovF-TE may be from Aspergillus terreus. In some embodiments, the Ppant is AnNpgA according to SEQ ID NO: 237 and the megasynthase LovF-TE is AstLovF-TE according to SEQ ID NO: 235, or a sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 237 and SEQ ID NO: 235, respectively.
“Percent identity” or “% identity” between a query nucleotide sequence and a subject nucleotide sequence is the “Identities” value, expressed as a percentage, that is calculated using a suitable algorithm (e.g. BLASTN, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN, or GenePAST/KERR) or software (e.g. DNASTAR Lasergene, GenomeQuest, EMBOSS needle or EMBOSS infoalign), over the entire length of the query sequence after a pair-wise global sequence alignment has been performed using a suitable algorithm (e.g. Needleman-Wunsch or GenePAST/KERR) or software (e.g. DNASTAR Lasergene or GenePAST/KERR). Importantly, a query nucleotide sequence may be described by a nucleotide sequence disclosed herein, in particular in one or more of the claims.
“Percent identity” or “% identity” between a query amino acid sequence and a subject amino acid sequence is the “Identities” value, expressed as a percentage, that is calculated using a suitable algorithm (e.g. BLASTP, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN, or GenePAST/KERR) or software (e.g. DNASTAR Lasergene, GenomeQuest, EMBOSS needle or EMBOSS infoalign), over the entire length of the query sequence after a pair-wise global sequence alignment has been performed using a suitable algorithm (e.g. Needleman-Wunsch or GenePAST/KERR) or software (e.g. DNASTAR Lasergene or GenePAST/KERR). Importantly, a query amino acid sequence may be described by an amino acid sequence disclosed herein, in particular in one or more of the claims.
The query sequence may be 100% identical to the subject sequence, or it may include up to a certain integer number of amino acid or nucleotide alterations as compared to the subject sequence such that the % identity is less than 100%. For example, the query sequence is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the subject sequence. In the case of nucleotide sequences, such alterations include at least one nucleotide residue deletion, substitution or insertion, wherein said alterations may occur at the 5′- or 3′-terminal positions of the query sequence or anywhere between those terminal positions, interspersed either individually among the nucleotide residues in the query sequence or in one or more contiguous groups within the query sequence. In the case of amino acid sequences, such alterations include at least one amino acid residue deletion, substitution (including conservative and non-conservative substitutions), or insertion, wherein said alterations may occur at the amino- or carboxy-terminal positions of the query sequence or anywhere between those terminal positions, interspersed either individually among the amino acid residues in the query sequence or in one or more contiguous groups within the query sequence.
With respect to the enzymes and/or proteins used in the methods of the invention and defined in terms of sequence identity, such enzymes and/or proteins typically retain their same respective function and activity, which function and activity may be assesses as described in the Example section.
Conventional methods used to engineer yeast may be used in the methods of the invention (see e.g. U.S. Pat. No. 8,828,684 B2, the content of which is incorporated by reference). Heterologous genes may be expressed under constitutive promoters or under inducible promoters, for example galactose-inducible promotors. Gene expression may be achieved either via integration into the genome of a given yeast strain (within the same locus or within different loci) or via plasmid expression. When using genome integration, one or more copies of the genes to be overexpressed may be integrated, for example, 1 to 10, 2 to 8, 3 to 7. In some embodiments, one or more of the genes involved in the biosynthesis of QS-21 are integrated into the genome of the yeast. General yeast culture conditions are known to the skilled person. Once engineered, yeast may be cultured for a few days, for example 1 to 7 days, 2 to 6 days, 4 to 5 days, or 3 days. It is within the ambit of the skilled person to determine the optimal time, depending on the metabolite to be produced. When using inducible promoters such as the gal promoters, determining the optimal induction time is also within the ambit of the skilled person. At any appropriate time after culture and/or induction, the desired metabolites, e.g. sugars or the QA derivatives of the invention may be recovered from the yeast culture, by any methods known in the art, such as extraction using a non-aqueous polar solvent, extraction using an acid medium or a basic medium, or recovery by resin absorption, or extraction by mechanically disrupting the plant cells, such as by ball milling or sonication. In some embodiments, the yeast is Saccharomyces cerevisiae.
The QA derivatives of the invention may be used as an adjuvant, individually, or in any combination. They may also be combined with further immuno-stimulants, in particular with a TLR4 agonist. In some embodiments, the QA derivatives are formulated within a liposome, in combination with a TLR4 agonist.
The TLR4 agonist may be 3D-MPL, in particular lipopolysaccharide TLR4 agonists, such as lipid A derivatives, especially a monophosphoryl lipid A, e.g. 3-de-O-acylated monophosphoryl lipid A (3D-MPL). 3D-MPL is sold under the name ‘MPL’ by GlaxoSmithKline Biologicals N.A. See, for example, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094. 3D-MPL can be produced according to the methods described in GB 2 220 211 A. Chemically, it is a mixture of 3-deacylated monophosphoryl lipid A with 4, 5 or 6 acylated chains.
Adjuvants of the invention may also be formulated into a suitable carrier, such as an emulsion (e.g. an oil-in-water emulsion) or liposomes, as described below.
The term liposome is well known in the art and defines a general category of vesicles which comprise one or more lipid bilayers surrounding an aqueous space. Liposomes thus consist of one or more lipid and/or phospholipid bilayers and can contain other molecules, such as proteins or carbohydrates, in their structure. Because both lipid and aqueous phases are present, liposomes can encapsulate or entrap water-soluble material, lipid-soluble material, and/or amphiphilic compounds. A method for making such liposomes is described in WO 13/041572.
Liposome size may vary from 30 nm to several μm depending on the phospholipid composition and the method used for their preparation.
The liposome size will be in the range of 50 nm to 200 nm, especially 60 nm to 180 nm, such as 70-165 nm. Optimally, the liposomes should be stable and have a diameter of 100 nm to allow convenient sterilization by filtration.
Structural integrity of the liposomes may be assessed by methods such as dynamic light scattering (DLS) measuring the size (Z-average diameter, Zav) and polydispersity of the liposomes, or, by electron microscopy for analysis of the structure of the liposomes. The average particle size may be between 95 and 120 nm, and/or, the polydispersity (Pdl) index may not be more than 0.3 (such as not more than 0.2).
Chemical Formula: C36H53O11- Exact Mass: 661,36
Chemical Formula: C42H63O16- Exact Mass: 823,41
Chemical Formula: C48H73O20- Exact Mass: 969,47
Chemical Formula: C47H71O20- Exact Mass: 955,45
Chemical Formula: C54H83O24- Exact Mass: 1115,53
Chemical Formula: C53H81O24- Exact Mass: 1101,51
Chemical Formula: C60H93O28- Exact Mass: 1261,59
Chemical Formula: C59H91O28- Exact Mass: 1247,57
Chemical Formula: C65H101O32- Exact Mass: 1393,63
Chemical Formula: C64H99O32- Exact Mass: 1379,61
Chemical Formula: C70H109O36- Exact Mass: 1525,67
Chemical Formula: C70H109O36- Exact Mass: 1525,67
Chemical Formula: C79H125O39- Exact Mass: 1697,78
Chemical Formula: C79H125O39- Exact Mass: 1697,78
Chemical Formula: C88H141O42- Exact Mass: 1869,89
Chemical Formula: C82H131O38- Exact Mass: 1723,83
Chemical Formula: C93H149O46- Exact Mass: 1723,83
Chemical Formula: C93H149O46- Exact Mass: 2001,93
Chemical Formula: C87H139O42- Exact Mass: 1855,87
Chemical Formula: C87H139O42- Exact Mass: 1855,87
Chemical Formula: C92H147O46- Exact Mass: 1987,92
Throughout the specification, including the claims, where the context permits, the term “comprising” and variants thereof such as “comprises” are to be interpreted as including the stated element (e.g., integer) or elements (e.g., integers) without necessarily excluding any other elements (e.g., integers). Thus a composition “comprising” X may consist exclusively of X or may include something additional e.g. X+Y.
The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.
The term “about” in or “approximately” in relation to a numerical value x is optional and means, for example, x±10% of the given figure, such as x+5% of the given figure, in particular the given figure.
As used herein, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise.
As used herein, ng refers to nanograms, ug or μg refers to micrograms, mg refers to milligrams, mL or ml refers to milliliter, and mM refers to millimolar. Similar terms, such as um, are to be construed accordingly.
Unless specifically stated, a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.
The invention is illustrated further by reference to the following clauses.
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:
The method of clause 1, wherein the C16 oxidase, the C23 oxidase and the C28 oxidase are independently selected from Artemisia annua (Aa), Arabidopsis thaliana (At), Glycyrrhiza glabra (Gg), Medicago truncatula (Mt), Quillaja saponaria (Qs), Saponaria vaccaria (Sv), Centella asiatica (Ca), Bupleurum falcatum (Bf) and Maesa lanceolate (MI).
The method of clause 1 or clause 2, wherein the C16 oxidase, the C23 oxidase and the C28 oxidase are independently selected from Medicago truncatula (Mt), Bupleurum falcatum (Bf), Quillaja saponaria (Qs), and Saponaria vaccaria (Sv).
The method of clause 3, wherein 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 method of clause 4, wherein QsC16 is encoded by the nucleotide sequence SEQ ID NO: 21, QsC28C16 is encoded by the nucleotide sequence SEQ ID NO: 24 and SvC16 is encoded by the nucleotide sequence SEQ ID NO: 27.
The method of any one of clauses 1 to 5, wherein 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.
The method of clause 6, wherein MtC23 is encoded by the nucleotide sequence SEQ ID NO: 39, QsC23 is encoded by the nucleotide sequence SEQ ID NO: 30, SvC23-1 is encoded by the nucleotide sequence SEQ ID NO: 33, and SvC23-2 is encoded by the nucleotide sequence SEQ ID NO: 36.
The method of any one of clauses 1 to 7, wherein 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.
The method of clause 8, wherein MtC28 is encoded by the nucleotide sequence SEQ ID NO: 47, QsC28 is encoded by the nucleotide sequence SEQ ID NO: 42, and SvC28 is encoded by the nucleotide sequence SEQ ID NO: 45.
The method of any one of clauses 4 to 9, wherein the C16 oxidase is SvC16 according to SEQ ID NO: 26, the C23 oxidase is SvC23-1 according to SEQ ID NO: 32 or SvC23-2 oxidase according to SEQ ID NO: 35, and the C28 oxidase is SvC28 according to SEQ ID NO: 44.
The method of clause 10, wherein SvC16 is encoded by the nucleotide sequence SEQ ID NO: 27, SvC23-1 is encoded by the nucleotide sequence SEQ ID NO: 33, SvC23-2 is encoded by the nucleotide sequence SEQ ID NO: 36, and SvC28 is encoded by the nucleotide sequence SEQ ID NO: 45.
The method of any one of clauses 4 to 9, wherein the C16 oxidase is selected from QsC16 according to SEQ ID NO: 20 and QsC28C16 according to SEQ ID NO: 23, the C23 oxidase is QsC23 according to SEQ ID NO: 29 and the C28 is QsC28 according to SEQ ID NO: 41.
The method of clause 12, wherein QsC16 is encoded by the nucleotide sequence SEQ ID NO: 21, QsC28C16 is encoded by the nucleotide sequence SEQ ID NO: 24, QsC23 is encoded by the nucleotide sequence SEQ ID NO: 30 and QsC28 is encoded by the nucleotide sequence SEQ ID NO: 42.
The method of any one of clauses 4 to 9, wherein the C16 oxidase is QsC28C16 according to SEQ ID NO: 23, the C23 oxidase is QsC23 according to SEQ ID NO: 29, and the C28 oxidase is QsC28 according to SEQ ID NO: 41.
The method of any one of clause 14, wherein the QsC28C16 is encoded by the nucleotide sequence SEQ ID NO: 24, QsC23 is encoded by the nucleotide sequence SEQ ID NO: 30, and QsC28 is encoded by the nucleotide sequence SEQ ID NO: 42.
The method of any one of clauses 1 to 15, wherein the CPR is selected from A. thaliana (At) and Lotus Japonicus (Lj).
The method of clause 16, wherein the CPR is selected from AtATR1 according to SEQ ID NO: 49 and LjCPR according to SEQ ID NO: 52.
The method of clause 17, wherein the CPR is AtATR1 according to SEQ ID NO: 49.
The method of clause 17 or clause 18, wherein AtATR1 is encoded by the nucleotide sequence SEQ ID NO: 50 and LjCPR is encoded by the nucleotide sequence SEQ ID NO: 53.
The method of any one of clauses 1 to 19, wherein the yeast further overexpresses a heterologous gene encoding (v) a cytochrome b5.
The method of clause 20, wherein the cytochrome b5 is selected from A. thaliana (At), Q. saponaria (Qs) and S. vaccaria (Sv).
The method of clause 21, wherein the cytochrome b5 is selected from Atb5 according to SEQ ID NO: 58, Qsb5 according to SEQ ID NO: 55 and Svb5 according to SEQ ID NO: 61.
The method of clause 21 or clause 22, wherein the cytochrome b5 is Qsb5 according to SEQ ID NO: 55.
The method of clause 21 or clause 22, wherein the cytochrome b5 is Svb5 according to SEQ ID NO: 61.
The method of any one of clauses 22 to 24, wherein Atb5 is encoded by the nucleotide sequence SEQ ID NO: 59, Qsb5 is encoded by the nucleotide sequence SEQ ID NO: 56, and Svb5 is encoded by the nucleotide sequence SEQ ID NO: 62.
The method of any one of clauses 1 to 25, wherein 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.
The method of clause 26, wherein the scaffold protein is a membrane steroid-binding protein (MSBP).
The method of clause 27, wherein the MSBP is selected from A. thaliana (At), Q. Saponaria (Qs) and S. vaccaria (Sv).
The method of clause 27 or clause 28, wherein the MSBP is selected from AtMSBP1 according to SEQ ID NO: 63 and AtMSBP2 according to SEQ ID NO: 65.
The method of clause 27 or clause 28, wherein the MSBP is selected from QsMSBP1 according to SEQ ID NO: 73, SvMSBP1 according to SEQ ID NO: 67 and SvMSBP2 according to SEQ ID NO: 70.
The method of clause 27, clause 28 or clause 30, wherein the MSBP is SvMSBP1 according to SEQ ID NO: 67.
The method of any one of clauses 29 to 31, wherein AtMSBP1 is encoded by the nucleotide sequence SEQ ID NO: 64, AtMSBP2 is encoded by the nucleotide sequence SEQ ID NO: 66, QsMSBP1 is encoded by the nucleotide sequence SEQ ID NO: 74, SvMSBP1 is encoded by the nucleotide sequence SEQ ID NO: 68 and SvMSBP2 is encoded by the nucleotide sequence SEQ ID NO: 71.
The method of any one of clauses 1 to 32, wherein the yeast engineered to produce β-amyrin overexpresses a β-amyrin synthase (BAS) selected from A. annua (Aa), A. thaliana (At), G. glabra (Gg), G. vaccaria (Gv), S. vaccaria (Sv), and Q. saponaria (Qs).
The method of clause 33, wherein the BAS is 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.
The method of clause 33 or clause 34, wherein the BAS is GvBAS according to SEQ ID NO: 10.
The method of any one of clauses 34 to 35, wherein AaBAS is encoded by the nucleotide sequence SEQ ID NO: 2, AtBAS is encoded by the nucleotide sequence SEQ ID NO: 5, GgBAS is encoded by the nucleotide sequence SEQ ID NO: 8, GvBAS is encoded by the nucleotide sequence SEQ ID NO: 11, QsBAS is encoded by the nucleotide sequence SEQ ID NO: 16, and SvBAS is encoded by the nucleotide sequence SEQ ID NO: 14.
The method of any one of clauses 1 to 9, 12 to 23, 25 to 28, and 30 to 36, wherein 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.
The method of clause 37, wherein QsC28C16 is encoded by the nucleotide sequence SEQ ID NO: 24, QsC23 is encoded by the nucleotide sequence SEQ ID NO: 30, QsC28 is encoded by the nucleotide sequence SEQ ID NO: 42, AtATR1 is encoded by the nucleotide sequence SEQ ID NO: 24, SvMSBP1 is encoded by the nucleotide sequence SEQ ID NO: 50, Qsb5 is encoded by the nucleotide sequence SEQ ID NO: 56, and GvBAS is encoded by the nucleotide sequence SEQ ID NO: 11.
A yeast which is engineered to produce QA according to the method of any one of clauses 1 to 38.
The yeast of clause 39 producing at least 60 mg/L of QA.
A method of producing UDP-Glucuronic acid (UDP-GlcA) in yeast, wherein the method comprises the step of overexpressing a heterologous gene encoding a UDP-glucose dehydrogenase (UGD) converting UDP-Glucose (UDP-Glc) into UDP-GlcA.
The method of clause 41, wherein the UGD is from A. thaliana (At).
The method of clause 39, wherein the UGD is selected from AtUGD according to SEQ ID NO: 84 and AtUGDA101L according to SEQ ID NO: 108.
The method of any one of clauses 41 to 43, wherein the UGD is AtUGDA101L according to SEQ ID NO: 108.
The method of clause 43 or clause 44, wherein AtUGD is encoded by the nucleotide sequence SEQ ID NO: 85, and AtUGDA101L is encoded by the nucleotide sequence SEQ ID NO: 109.
A yeast which is engineered to produce UDP-GlcA according to the method of any of clauses 41 to clause 45.
A method of producing UDP-Rhamnose (UDP-Rha) in yeast, wherein the method comprises the step of overexpressing a heterologous gene encoding a UDP-rhamnose synthase converting UDP-Glucose (UDP-Glc) into UDP-Rha.
The method of clause 47, wherein the UDP-rhamnose synthase is from A. thaliana (At).
The method of clause 48, wherein the UDP-rhamnose synthase is AtRHM2 according to SEQ ID NO: 102.
The method of clause 49, wherein AtRHM2 is encoded by the nucleotide sequence SEQ ID NO: 103.
A yeast which is engineered to produce UDP-Rha according to the method of any one of clauses 47 to 50.
A method of producing UDP-Xylose (UDP-Xyl) in yeast, wherein the method comprises the step of overexpressing heterologous genes encoding the following enzymes:
The method of clause 52, wherein the UGD and the UXS are independently selected from A. thaliana (At) and Q. saponaria (Qs).
The method of clause 53, wherein the UGD is selected from AtUGD according to SEQ ID NO: 84 and AtUGDA101L according to SEQ ID NO: 108.
The method of clause 52, wherein the UGD is selected from Synechococcus sp. (Syn), Homo sapiens (Hs), Paramoeba atlantica (Patl), Bacillus cytotoxicus (Bcyt), Corallococcus macrosporus (Myxfulv), and Pyrococcus furiosus (Pfu).
The method of clause 55, wherein the UGD is selected from SynUGD according to SEQ ID NO: 154, HsUGDA104L according to SEQ ID NO: 157, PatlUGD according to SEQ ID NO: 110, BcytUGD according to SEQ ID NO: 160, MyxfulvUGD according to SEQ ID NO: 163, and PfuUGD according to SEQ ID NO: 166.
The method of any one of clauses 52 to 54, wherein the UGD is AtUGDA101L according to SEQ ID NO: 108.
The method of any of clauses 54 to 57, wherein AtUGD is encoded by the nucleotide sequence SEQ ID NO: 85, AtUGDA101L is encoded by the nucleotide sequence SEQ ID NO: 109, SynUGD is encoded by the nucleotide sequence SEQ ID NO: 155, HsUGD104L is encoded by the nucleotide sequence SEQ ID NO: 158, PatlUGD is encoded by the nucleotide sequence SEQ ID NO: 111, BcytUGD is encoded by the nucleotide sequence SEQ ID NO: 161, MyxfulvUGD is encoded by the nucleotide sequence SEQ ID NO: 164, and PfuUGD is encoded by the nucleotide sequence SEQ ID NO: 167.
The method of any one of clauses 52 to 58, wherein the UXS is selected from AtUXS according to SEQ ID NO: 105 and QsAXS according to SEQ ID NO: 113.
The method of clause 60 wherein the UGD is AtUGDA101L according to SEQ ID NO: 108 and the UXS is AtUXS according to SEQ ID NO: 105.
The method of clause 59 or clause 60, wherein AtUXS is encoded by the nucleotide sequence SEQ ID NO: 106, QsAXS is encoded by the nucleotide sequence SEQ ID NO: 114 and AtUGDA101L is encoded by the nucleotide sequence SEQ ID NO: 109.
A yeast which is engineered to produce UDP-Xyl according to the method of any one of clauses 52 to 61.
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:
The method of clause 63, wherein the GlcAT is selected from Q. saponaria (Qs) and S. vaccaria (Sv).
The method of clause 63 or clause 64, wherein the GlcAT is from Q. saponaria.
The method of clause 65, wherein the GlcAT is selected from QsCslG1 according to SEQ ID NO: 78 and QsCslG2 according to SEQ ID NO: 81.
The method of clause 66, wherein the GlcAT is QsCslG2 according to SEQ ID NO: 81.
The method of clause 64, wherein the GlcAT is from S. vaccaria.
The method of clause 68, wherein the GlcAT is SvCslG according to SEQ ID NO: 76.
The method of any one of clauses 66, 67, 68 or 69, wherein QsCslG1 is encoded by the nucleotide sequence SEQ ID NO: 79, QsCslG2 is encoded by the nucleotide sequence SEQ ID NO: 82 and SvCslG is encoded by the nucleotide sequence SEQ ID NO: 77.
The method of any one of clauses 63 to 70, wherein the yeast engineered to produce QA is according to clause 39.
The method of clause 71, wherein the yeast engineered to produce UDP-GlcA is according to clause 46.
A yeast which is engineered to produce QA-C3-GlcA according to the method of any one of clauses 63 to 72.
The method of any one of clauses 63 to 72, wherein the derivative is QA-C3-GlcA-Gal, and the overexpressing further comprises overexpressing a heterologous gene encoding the following enzyme:
The method of clause 74, wherein the GalT is selected from Q. saponaria (Qs) and S. vaccaria (Sv).
The method of clause 75, wherein the GalT is from Q. saponaria (Qs).
The method of any one of clause 70 to 76, wherein the GalT is OsGalT according to SEQ ID NO: 116.
The method of clause 74, wherein the GalT is from S. vaccaria.
The method of clause 78, wherein GalT is SvGalT according to SEQ ID NO: 98.
The method of clause 77 or clause 79, wherein QsGalT is encoded by the nucleotide sequence SEQ ID NO: 117 and SvGalT is encoded by the nucleotide sequence SEQ ID NO: 99.
A yeast which is engineered to produce QA-C3-GlcA-Gal according to the method of any one of clauses 74 to 80.
The method of any one of clauses 74 to 80, wherein 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:
The method of clause 82, wherein the RhaT is from Q. saponaria (Qs).
The method of clause 83, wherein the RhaT is QsRhaT according to SEQ ID NO: 119.
The method of clause 84, wherein QsRhaT is encoded by the nucleotide sequence SEQ ID NO: 120.
The method of any one of clauses 82 to 86, wherein the yeast engineered to produce UDP-Rha is according to clause 51.
A yeast which is engineered to produce QA-C3-GlcA-Gal-Rha according to the method of any one of clauses 82 to 86.
The method of any one of clauses 74 to 80, wherein 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:
The method of clause 88, wherein the XylT is selected from Q. Saponaria (Qs) or S. vaccaria (Sv).
The method of clause 89, wherein the XylT is selected from QsC3XylT according to SEQ ID NO: 122 and SvC3XylT according to SEQ ID NO: 100.
The method of clause 90, wherein QsC3XylT is encoded by the nucleotide sequence SEQ ID NO: 123 and SvC3XylT is encoded by the nucleotide sequence SEQ ID NO: 101, and wherein the yeast engineered to produce UDP-Xyl is according to clause 62.
A yeast which is engineered to produce QA-C3-GlcA-Gal-Xyl according to the method of any one of clauses 88 to 91.
The method of any one of clauses 88 to 91, wherein the overexpressing further comprises overexpressing of heterologous genes encoding the following enzymes:
The method of clause 93, wherein the GlcAK and the USP are from A. thaliana (At).
The method of clause 94, wherein GlcAK is AtGlcAK according to SEQ ID NO: 169 and the USP is AtUSP according to SEQ ID NO: 223.
The method of clause 95, wherein AtGlcAK is encoded by the nucleotide sequence SEQ ID NO: 170 and AtUSP is encoded by the nucleotide sequence SEQ ID NO: 224.
The method of any one of clauses 93 to 96, wherein the overexpressing further comprises overexpressing of (vi) a heterologous gene encoding a Myo-Inositol Oxygenase (MIOX), and myo-inositol is additionally supplemented exogenously.
The method of clause 97, wherein MIOX is from Thermothelomyces thermophilus (Tt).
The method of clause 98, wherein MIOX is TtMIOX according to SEQ ID NO: 173.
The method of clause 99, wherein TtMIOX is encoded by the nucleotide sequence SEQ ID NO: 174.
A yeast which is engineered to produce QA-C3-GlcA-Gal-Xyl according to the method of any one of clauses 93 to 100.
A method of producing UDP-Fucose (UDP-Fuc) in yeast, wherein the method comprises the step of overexpressing heterologous genes encoding the following enzymes:
The method of clause 102, wherein the UG46DH is from S. vaccaria (Sv).
The method of clause 103, wherein the UG46DH is SvUG46DH according to SEQ ID NO: 87.
The method of clause 104, wherein SvUG46DH is encoded by the nucleotide sequence SEQ ID NO: 88.
The method of any one of clauses 102 to 105, wherein the 4-keto-reductase is selected from Q. saponaria (Qs) and S. vaccaria (Sv).
The method of clause 106, wherein the 4-keto-reductase is selected from svNMD according to SEQ ID NO: 90 and QsFucSyn according to SEQ ID NO: 175.
The method of clause 107, wherein svNMD is encoded by the nucleotide sequence SEQ ID NO: 91 and QsFucSyn is encoded by the nucleotide sequence SEQ ID NO: 176.
A yeast which is engineered to produce UDP-Fucose according to the method of any one of clauses 102 to 108.
A method of producing a C28-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:
The method of clause 110, wherein the FucT is selected from Q. Saponaria (Qs) and S. vaccaria (Sv).
The method of clause 111, wherein the FucT is selected from QsFucT according to SEQ ID NO: 93 and SvFucT according to SEQ ID NO: 96.
The method of clause 112, wherein QsFucT is encoded by the nucleotide sequence SEQ ID NO: 94 and SvFucT is encoded by the nucleotide sequence SEQ ID NO: 97.
The method of any one of clauses 110 to 113, wherein the yeast engineered to produce QA-C3-GlcA-Gal-Rha is according to clause 87 and the yeast engineered to produce UDP-Fuc is according to clause 109.
The method of any one of clauses 110 to 113 wherein the yeast engineered to produce QA-C3-GlcA-Gal-Xyl is according to clause 101 and the yeast engineered to produce UDP-Fuc is according to clause 109.
A yeast which is engineered to produce QA-C3-GlcA-Gal-Rha-C28-Fuc according to the method of any one of clauses 110 to 114.
A yeast which is engineered to produce QA-C3-GlcA-Gal-Xyl-C28-Fuc according to the method of any one of clauses 110 to 113 and clause 115.
The method of any one of clauses 110 to 115, wherein 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:
The method of clause 118, wherein the RhaT is from Q. saponaria.
The method of clause 119, wherein the RhaT is QsRhaT according to SEQ ID NO: 119.
The method of clause 120, wherein QsRhaT is encoded by the nucleotide sequence SEQ ID NO: 120.
A yeast which is engineered to produce QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha according to the method of any one of clauses 118 to 121.
The method of any one of clauses 118 to 121, wherein 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:
The method of clause 123, wherein the XylT is selected from Q. Saponaria (Qs) and S. vaccaria (Sv).
The method of clause 124, wherein the XylT is QsC28XylT3 according to SEQ ID NO: 125.
The method of clause 125, wherein QsC28XylT3 is encoded by the nucleotide sequence SEQ ID NO: 126.
A yeast which is engineered to produce QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl according to the method of any of clauses 123 to 126.
The method of any one of clauses 123 to 126, wherein 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:
The method of clause 128, wherein the XylT is selected from Q. Saponaria (Qs) and S. vaccaria (Sv).
The method of clause 129, wherein the XylT is QsC28XylT4 according to SEQ ID NO: 128.
The method of clause 130, wherein QsC28XylT4 is encoded by the nucleotide sequence SEQ ID NO: 129.
The method of clause 128 or clause 129, wherein QsC28XylT4 comprises an amino acid deletion at the N-terminus, ranging from 3 amino acids to 20 amino acids.
The method of clause 132, wherein the XylT is selected from QsC28XylT4-3aa according to SEQ ID NO: 131, QsC28XylT4-6aa according to SEQ ID NO: 134, QsC28XylT4-9aa according to SEQ ID NO: 137, and QsC28XylT4-12aa according to SEQ ID NO: 140.
The method of clause 133, wherein QsC28XylT4-3aa is encoded by the nucleotide sequence SEQ ID NO: 132, QsC28XylT4-6aa is encoded by the nucleotide sequence SEQ ID NO: 135, QsC28XylT4-9aa is encoded by the nucleotide sequence SEQ ID NO: 138, and QsC28XylT4-12aa is encoded by the nucleotide sequence SEQ ID NO: 141.
The method of clause 28 or clause 129, wherein a solubility tag is added at the N-terminus of XylT.
The method of clause 135, wherein the XylT is selected from SUMO-QsC28XylT4 according to SEQ ID NO: 143, TrxA-QsC28-XylT4 according to SEQ ID NO: 145, and MBP-QsC28XylT4 according to SEQ ID NO: 147.
The method of clause 136, wherein SUMO-QsC28XylT4 is encoded by the nucleotide sequence SEQ ID NO: 144, TrxA-QsC28-XylT4 is encoded by the nucleotide sequence SEQ ID NO: 146 and MBP-QsC28XylT4 is encoded by the nucleotide sequence SEQ ID NO: 148.
The method of clause 128 or clause 129, wherein the XylT is QsC28XylT3-3×GGGS-QsC28XylT4 according to SEQ ID NO: 149.
The method of clause 138, wherein QsC28XylT3-3×GGGS-QsC28XylT4 is encoded by the nucleotide sequence SEQ ID NO: 150.
A yeast which is engineered to produce QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Xyl or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl according to the method of any of clauses 128 to 139.
The method of any one of clauses 123 to 126, wherein 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:
The method of clause 141, wherein the AXS is QsAXS according to SEQ ID NO: 113.
The method of clause 142, wherein QsAXS is encoded by the nucleotide sequence SEQ ID NO: 114.
The method of any one of clauses 141 to 143, wherein the ApiT is selected from Q. saponaria (Qs) or S. vaccaria (Sv).
The method of clause 144, wherein the ApiT is QsC28ApiT4 according to SEQ ID NO: 151.
The method of clause 145, wherein QsC28ApiT4 is encoded by the nucleotide sequence SEQ ID NO: 152.
A yeast which is engineered to produce QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Api or QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Api according to the method of any of clauses 141 to 146.
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 (i) a carboxyl coenzyme A (CoA) ligase (CCL) converting 2 MB acid into 2 MB-CoA, and 2 MB acid is supplemented exogenously.
The method of clause 148, wherein the CCL is QsCCL from Q. saponaria according to SEQ ID NO: 178.
The method of clause 149, wherein QsCCL is encoded by the nucleotide sequence SEQ ID NO: 179.
The method of any one of clauses 148 to 150 in yeast, wherein the overexpressing further comprises overexpressing heterologous genes encoding the following enzymes:
The method of clause 151, wherein the Ppant is from Aspergillus nidulans (An) and the megasynthase LovF-TE is from Aspergillus terreus (Ast).
The method of clause 152, wherein the Ppant is AnNpgA according to SEQ ID NO: 237 and the megasynthase LovF-TE is AstLovF-TE according to SEQ ID NO: 235.
The method of clause 153, wherein AnNgA is encoded by the nucleotide sequence SEQ ID NO: 238 and AstLovF-TE is encoded by the nucleotide sequence SEQ ID NO: 236.
A yeast engineered to produce 2 MB-CoA according to the method of any one of clauses 148 to 154.
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:
The method of clause 156, wherein the UXE and the UAM are independently selected from A. thaliana (At) and H. vulgare (Hv).
The method of clause 157, wherein 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.
The method of clause 158, wherein AtUXE is encoded by the nucleotide sequence SEQ ID NO: 200, AtUXE2 is encoded by the nucleotide sequence SEQ ID NO: 203, HvUXE-1 is encoded by the nucleotide sequence SEQ ID NO: 241, HvUXE-2 is encoded by the nucleotide sequence SEQ ID NO: 243, AtUAM1 is encoded by the nucleotide sequence SEQ ID NO: 209, HvUAM is encoded by the nucleotide sequence SEQ ID NO: 212, and AtUGE3 is encoded by the nucleotide sequence SEQ ID NO: 206.
The method of any one of clauses 156 to 159, wherein the yeast engineered to produce UDP-Xyl is according to clause 62.
A yeast which is engineered to produce UDP-Araf according to the method of any of clauses 156 to 160.
A method of producing UDP-Araf in yeast, wherein the method comprises the step of overexpressing heterologous genes encoding the following enzymes:
The method of clause 162, wherein the AraK and the USP are independently selected from A. thaliana (At) and Leptospira interrogans (Lei).
The method of clause 163, wherein 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.
The method of clause 164, wherein the AtAraK is encoded by the nucleotide sequence SEQ ID NO: 215, LeiAraK is encoded by the nucleotide sequence SEQ ID NO: 218, AtUSP is encoded by the nucleotide sequence SEQ ID NO: 224 and LeiUSP is encoded by the nucleotide sequence SEQ ID NO: 227.
The method of any one of clauses 162 to 165, wherein the overexpressing further comprises overexpressing a heterologous gene encoding (iii) an arabinose transporter (AraT).
The method of clause 166, wherein the AraT is PrAraT from Penicillium rubens Wisconsin according to SEQ ID NO: 220.
The method of clause 167, wherein PrAraT is encoded by the nucleotide sequence SEQ ID NO: 221.
A yeast which is engineered to produce UDP-Araf according to the method of any one of clauses 162 to 168.
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:
The method of clause 170, wherein the CCL, the chalcone-synthase-like type III PKS, the KR and the acyltransferase are from Q. saponaria.
The method of clause 171, wherein the CCL is QsCCL according to SEQ ID NO: 178, the chalcone-synthase-like type III PKS is QsChSD according to SEQ ID NO: 181, QsChSE according to SEQ ID NO: 184, or both QsChSD according to SEQ ID NO:181 and QsChSE according to SEQ ID NO: 184, the keto-reductase is QsKR11 according to SEQ ID NO: 187, QsKR23 according to SEQ ID NO: 190, or both QsKR11 according to SEQ ID NO: 187 and QsKR23 according to SEQ ID NO: 190, and the acyltransferase is QsDMOT9 according to SEQ ID NO: 193.
The method of clause 171, wherein the chalcone-synthase-like type III PKS are both QsChSD according to SEQ ID NO: 181 and QsChSE according to SEQ ID NO: 184.
The method of clause 170 wherein the KR are both QsKR11 according to SEQ ID NO: 187 and QsKR23 according to SEQ ID NO: 190.
The method of any one of clauses 172 to 174, wherein the CCL is QsCCL according to SEQ ID NO: 178, the chalcone-synthase-like type III PKS are QsChSD according to SEQ ID NO: 181 and QsChSE according to SEQ ID NO: 184, the KR are QsKR11 according to SEQ ID NO: 187 and QsKR23 according to SEQ ID NO: 190 and the acyltransferase is QsDMOT9 according to SEQ ID NO: 193.
The method of clause 175, wherein QsCCL is encoded by SEQ ID NO: 179, QsChSD is encoded by the nucleotide sequence SEQ ID NO: 182, QsChSE is encoded by the nucleotide sequence SEQ ID NO: 185, QsKR11 is encoded by the nucleotide sequence SEQ ID NO: 188, QsKR23 is encoded by the nucleotide sequence SEQ ID NO: 191 and QsDMOT9 is encoded by the nucleotide sequence SEQ ID NO: 194.
The method of any one of clauses 170 to 176, wherein the yeast engineered to produce QA-C3-GGR-C28-FRX and QA-C3-GGX-C28-FRX is according to clause 127, the yeast engineered to produce QA-C3-GGR-C28-FRXX and QA-C3-GGX-C28-FRXX is according to clause 140 and the yeast engineered to produce QA-C3-GGR-C28-FRXA and QA-C3-GGX-C28-FRXA is according to clause 147.
A yeast which is engineered to produce 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 according to the method of any one of clauses 170 to 177.
The method of any one of clauses 170 to 178, wherein the derivative is QA-C3-GGR-C28-FRX-C18, QA-C3-GGX-C28-FRX-C18, QA-C3-GGR-C28-FRXX-C18, QA-C3-GGX-C28-FRXX-C18, QA-C3-GGR-C28-FRXA-C18 or QA-C3-GGX-C28-FRXA-C18, and the overexpressing further comprises overexpressing a heterologous gene encoding the following enzyme:
The method of clause 179, wherein QsDMOT4 is encoded by the nucleotide sequence SEQ ID NO: 197.
A yeast which is engineered to produce QA-C3-GGX-C28-FRX-C18, QA-C3-GGR-C28-FRX-C18, QA-C3-GGX-C28-FRXX-C18, QA-C3-GGR-C28-FRXX-C18, QA-C3-GGX-C28-FRXA-C18, or QA-C3-GGR-C28-FRXA-C18 according to the method of clause 179 or clause 180.
The method of any one of clauses 179 or 180, wherein the derivative is QA-C3-GGR-C28-FRX-C18-Araf, QA-C3-GGX-C28-FRX-C18-Araf, QA-C3-GGR-C28-FRXX-C18-Araf, QA-C3-GGX-C28-FRXX-C18-Araf, QA-C3-GGR-C28-FRXA-C18-Araf, or QA-C3-GGX-C28-FRXA-C18-Araf, the yeast is further engineered to produce UDP-Araf, and the overexpressing further comprises overexpressing a heterologous gene encoding the following enzyme:
The method of clause 182, wherein the ArafT is from Q. saponaria (Qs).
The method of clause 182 or clause 183, wherein the ArafT is selected from QsArafT according to SEQ ID NO: 229 and QsArafT2 according to SEQ ID NO: 232.
The method of clause 184, wherein QsArafT is encoded by the nucleotide sequence SEQ ID NO: 230, and QsArafT2 is encoded by the nucleotide sequence SEQ ID NO: 233.
The method of clause 184, wherein the ArafT is QsArafT2 according to SEQ ID NO: 232.
The method of clause 186, wherein QsArafT2 is encoded by the nucleotide sequence SEQ ID NO: 233.
The method of any one of clauses 182 to 187, wherein the yeast engineered to produce UDP-Araf is according to clause 161 or clause 169.
A yeast which is engineered to produce QA-C3-GGR-C28-FRX-C18-Araf, QA-C3-GGX-C28-FRX-C18-Araf, QA-C3-GGR-C28-FRXX-C18-Araf, QA-C3-GGX-C28-FRXX-C18-Araf, QA-C3-GGR-C28-FRXA-C18-Araf, or QA-C3-GGX-C28-FRXA-C18-Araf according to the method of any one of clauses 182 to 188.
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.
The method of clause 190, wherein the ArafT is QsArafT is according to SEQ ID NO: 229.
The method of clause 191, wherein QsArafT is encoded by the nucleotide sequence SEQ ID NO: 230.
The method of any one of clauses 190 to 192, wherein the 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 and QA-C3-GGR-C28-FRX-C18 is according to clause 181.
A yeast engineered to produce 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 according to the method of any one of clauses 190 to 193.
The method of any one of clauses 170 to 177, 179 to 180, 182 to 188 and 190 to 193, wherein the overexpressing further comprises the overexpressing of heterologous genes encoding the following enzymes:
The method of clause 195, wherein the Ppant is from Aspergillus nidulans (An) and the megasynthase LovF-TE is from Aspergillus terreus (Ast).
The method of clause 196, wherein the Ppant is AnNpgA according to SEQ ID NO: 237 and the megasynthase LovF-TE is AstLovF-TE according to SEQ ID NO: 235.
The method of clause 197, wherein AnNgA is encoded by the nucleotide sequence SEQ ID NO: 238 and AstLovF-TE is encoded by the nucleotide sequence SEQ ID NO: 236.
A yeast which is engineered to produce QA-C3-GGR-C28-FRX-C18-Araf, QA-C3-GGX-C28-FRX-C18-Araf, QA-C3-GGR-C28-FRXX-C18-Araf, QA-C3-GGX-C28-FRXX-C18-Araf, QA-C3-GGR-C28-FRXA-C18-Araf, or QA-C3-GGX-C28-FRXA-C18-Araf according to the method of any one of clauses 195 to 198.
A method of producing QA-C3-GGX-C28-FRXX-C18-Araf (QS-21-Xyl) in yeast, wherein the method 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.
The method of clause 200, wherein GvBAS is encoded by the nucleotide sequence SEQ ID NO: 11, QsC28C16 is encoded by the nucleotide sequence SEQ ID NO: 24, QsC23 is encoded by the nucleotide sequence SEQ ID NO: 30, QsC28 is encoded by the nucleotide sequence SEQ ID NO: 42, AtATR1 is encoded by the nucleotide sequence SEQ ID NO: 50, Qsb5 is encoded by the nucleotide sequence SEQ ID NO: 56, SvMSBP1 is encoded by the nucleotide sequence SEQ ID NO: 68, AtUGDA101L is encoded by the nucleotide sequence SEQ ID NO: 109, QsCslG2 is encoded by the nucleotide sequence SEQ ID NO: 82, QsGalT is encoded by the nucleotide sequence SEQ ID NO: 117, AtUXS is encoded by the nucleotide sequence SEQ ID NO: 106, QsC3XylT is encoded by the nucleotide sequence SEQ ID NO: 123, SvNMD is encoded by the nucleotide sequence SEQ ID NO: 91, SvUG46DH is encoded by the nucleotide sequence SEQ ID NO: 88, QsFucT is encoded by the nucleotide sequence SEQ ID NO: 94, AtRHM2 is encoded by the nucleotide sequence SEQ ID NO: 103, QsRhaT is encoded by the nucleotide sequence SEQ ID NO: 220, QsC28XylT3 is encoded by the nucleotide sequence SEQ ID NO: 126, QsC28XylT4 is encoded by the nucleotide sequence SEQ ID NO: 129, QsChSD is encoded by the nucleotide sequence SEQ ID NO: 182, QsChSE is encoded by the nucleotide sequence SEQ ID NO: 185, QsKR11 is encoded by the nucleotide sequence SEQ ID NO: 188, QsKR23 is encoded by the nucleotide sequence SEQ ID NO: 191, QsDMOT9 is encoded by the nucleotide sequence SEQ ID NO: 194, QsDMOT4 is encoded by the nucleotide sequence SEQ ID NO: 197, AtUXE is encoded by the nucleotide sequence SEQ ID NO: 200, AtUAM1 is encoded by the nucleotide sequence SEQ ID NO: 209, QsArafT2 is encoded by the nucleotide sequence SEQ ID NO: 233, AnNpgA is encoded by the nucleotide sequence SEQ ID NO: 238, QsCCL is encoded by the nucleotide sequence SEQ ID NO: 179 and AstLovF-TE is encoded by the nucleotide sequence SEQ ID NO: 236.
A yeast which is engineered to produce QS-21-Xyl according to the method of clause 201 or clause 202.
A method of producing QA-C3-GGX-C28-FRXA-C18-Araf (QS-21-Api) in yeast, wherein the method 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.
The method of clause 203, wherein GvBAS is encoded by the nucleotide sequence SEQ ID NO: 11, QsC28C16 is encoded by the nucleotide sequence SEQ ID NO: 24, QsC23 is encoded by the nucleotide sequence SEQ ID NO: 30, QsC28 is encoded by the nucleotide sequence SEQ ID NO: 42, AtATR1 is encoded by the nucleotide sequence SEQ ID NO: 50, Qsb5 is encoded by the nucleotide sequence SEQ ID NO: 56, SvMSBP1 is encoded by the nucleotide sequence SEQ ID NO: 68, AtUGDA101L is encoded by the nucleotide sequence SEQ ID NO: 109, QsCslG2 is encoded by the nucleotide sequence SEQ ID NO: 82, QsGalT is encoded by the nucleotide sequence SEQ ID NO: 117, AtUXS is encoded by the nucleotide sequence SEQ ID NO: 106, QsC3XylT is encoded by the nucleotide sequence SEQ ID NO: 123, SvNMD is encoded by the nucleotide sequence SEQ ID NO: 91, SvUG46DH is encoded by the nucleotide sequence SEQ ID NO: 88, QsFucT is encoded by the nucleotide sequence SEQ ID NO: 94, AtRHM2 is encoded by the nucleotide sequence SEQ ID NO: 103, QsRhaT is encoded by the nucleotide sequence SEQ ID NO: 120, QsC28XylT3 is encoded by the nucleotide sequence SEQ ID NO: 126, QsC28ApiT4 is encoded by the nucleotide sequence SEQ ID NO: 152, QsChSD is encoded by the nucleotide sequence SEQ ID NO: 182, QsChSE is encoded by the nucleotide sequence SEQ ID NO: 185, QsKR11 is encoded by the nucleotide sequence SEQ ID NO: 188, QsKR23 is encoded by the nucleotide sequence SEQ ID NO: 191, QsDMOT9 is encoded by the nucleotide sequence SEQ ID NO: 194, QsDMOT4 is encoded by the nucleotide sequence SEQ ID NO: 197, AtUXE is encoded by the nucleotide sequence SEQ ID NO: 200, AtUAM1 is encoded by the nucleotide sequence SEQ ID NO: 209, QsArafT2 is encoded by the nucleotide sequence SEQ ID NO: 233, AnNpgA is encoded by the nucleotide sequence SEQ ID NO: 238, QsCCL is encoded by the nucleotide sequence SEQ ID NO: 179 and AstLovF-TE is encoded by the nucleotide sequence SEQ ID NO: 236.
A yeast which is engineered to produce QS-21-Api according to the method of clause 204 or clause 205.
The method of any one of clauses 1 to 38, 42 to 45, 47 to 50, 52 to 61, 63 to 72, 74 to 80, 82 to 86, 88 to 91, 93 to 100, 102 to 108, 110 to 115, 118 to 121, 123 to 126, 128 to 139, 141 to 146, 148 to 154, 156 to 160, 162 to 168, 170 to 177, 179, 180, 182 to 188, 190 to 193, 195 to 198, 200, 201, 203 and 204, or the yeast of any one of clauses 39, 40, 46, 51, 62, 73, 81, 87, 92, 101, 109, 116, 117, 122, 127, 140, 147, 155, 161, 169, 178, 181, 189, 194, 199, 202 and 205, wherein GvBAS (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 10, QsC28C16 (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to according to SEQ ID NO: 23, QsC23 (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to according to SEQ ID NO: 29, QsC28 (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 41, AtATR1 (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 49, Qsb5 (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 55, SvMSBP1 (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 67, AtUGDA101L (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 108, QsCslG2 (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 81, QsGalT (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 116, AtUXS (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 105, QsC3XylT (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 122, SvNMD (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 90, SvUG46DH (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 87, QsFucT (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 93, AtRHM2 (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 102, QsC28XylT3 (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 125, QsC28XylT4 (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 128, QsC28ApiT4 (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 151, QsChSD (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 181, QsChSE according to SEQ ID NO: 184, QsKR11 (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 187, QsKR23 (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 190, QsDMOT9 (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 193, QsDMOT4 (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 196, AtUXE (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 199, AtUAM1 (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 208, QsArafT2 (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 232, AnNpgA (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 237, QsCCL (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 178 and AstLovF-TE (when present) is according to an amino acid sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 235.
The method of any one of clauses 1 to 38, 42 to 45, 47 to 50, 52 to 61, 63 to 72, 74 to 80, 82 to 86, 88 to 91, 93 to 100, 102 to 108, 110 to 115, 118 to 121, 123 to 126, 128 to 139, 141 to 146, 148 to 154, 156 to 160, 162 to 168, 170 to 177, 179, 180, 182 to 188, 190 to 193, 195 to 198, 200, 201, 203, 204 and 206, or the yeast of any one of clauses 39, 40, 46, 51, 62, 73, 81, 87, 92, 101, 109, 116, 117, 122, 127, 140, 147, 155, 161, 169, 178, 181, 189, 194, 199, 202, 205 and 206, wherein the heterologous genes are integrated into the genome of the yeast.
The method, or yeast, of clause 207, wherein one or more copies of one or more of the heterologous genes are integrated.
The method, or yeast, of clause 208, wherein the one or more copies ranges from 2 to 5.
The method, or yeast, of clause 208 or clause 209, wherein at least 2 copies of the genes encoding the C16 oxidase, the C23 oxidase and the C28 oxidase are integrated.
The method, or yeast, of any one of clauses 208 to 210, wherein at least 3 copies of the gene encoding the UXS (when present) are integrated.
The method, or yeast, of clause 208 to 211, wherein the nucleotide sequence of the heterologous genes is codon-optimized.
The method, or yeast, of clause 212, wherein GvBAS (when present) is encoded by the nucleotide sequence SEQ ID NO: 12, QsC28C16 (when present) is encoded by the nucleotide sequence SEQ ID NO: 25, QsC23 (when present) is encoded by the nucleotide sequence SEQ ID NO: 31, QsC28 (when present) is encoded by the nucleotide sequence SEQ ID NO: 43, AtATR1 (when present) is encoded by the nucleotide sequence SEQ ID NO: 51, Qsb5 (when present) is encoded by the nucleotide sequence SEQ ID NO: 57, SvMSBP1 is encoded by the nucleotide sequence SEQ ID NO: 69, AtUGDA101L (when present) is encoded by the nucleotide sequence SEQ ID NO: 109, QsCslG2 (when present) is encoded by the nucleotide sequence SEQ ID NO: 83, QsGalT (when present) is encoded by the nucleotide sequence SEQ ID NO: 118, AtUXS (when present) is encoded by the nucleotide sequence SEQ ID NO: 107, QsC3XylT (when present) is encoded by the nucleotide sequence SEQ ID NO: 124, SvNMD (when present) is encoded by the nucleotide sequence SEQ ID NO: 92, SvUG46DH (when present) is encoded by the nucleotide sequence SEQ ID NO: 89, QsFucT (when present) is encoded by the nucleotide sequence SEQ ID NO: 95, AtRHM2 (when present) is encoded by the nucleotide sequence SEQ ID NO: 104, QsRhaT (when present) is encoded by the nucleotide sequence SEQ ID NO: 121, QsC28XylT3 (when present) is encoded by the nucleotide sequence SEQ ID NO: 127, QsC28XylT4 (when present) is encoded by the nucleotide sequence SEQ ID NO: 130, QsC28ApiT4 (when present) encoded by the nucleotide sequence SEQ ID NO: 153 QsChSD (when present) is encoded by the nucleotide sequence SEQ ID NO: 183, QsChSE (when present) is encoded by the nucleotide sequence SEQ ID NO: 186, QsKR11 (when present) is encoded by the nucleotide sequence SEQ ID NO: 189, QsKR23 (when present) is encoded by the nucleotide sequence SEQ ID NO: 192, QsDMOT9 (when present) is encoded by the nucleotide sequence SEQ ID NO: 195, QsDMOT4 is encoded by the nucleotide sequence SEQ ID NO: 198, AtUXE (when present) is encoded by the nucleotide sequence SEQ ID NO: 201, AtUAM1 (when present) is encoded by the nucleotide sequence SEQ ID NO: 210, QsArafT2 (when present) is encoded by the nucleotide sequence SEQ ID NO: 234, AnNpgA (when present) is encoded by the nucleotide sequence SEQ ID NO: 239, QsCCL (when present) is encoded by the nucleotide sequence SEQ ID NO: 180 and AstLovF-TE (when present) is encoded by the nucleotide sequence SEQ ID NO: 236.
The method of any one of clauses 1 to 38, 42 to 45, 47 to 50, 52 to 61, 63 to 72, 74 to 80, 82 to 86, 88 to 91, 93 to 100, 102 to 108, 110 to 115, 118 to 121, 123 to 126, 128 to 139, 141 to 146, 148 to 154, 156 to 160, 162 to 168, 170 to 177, 179, 180, 182 to 188, 190 to 193, 195 to 198, 200, 201, 203, 204, and 206 to 213, wherein the method comprises the further step of culturing the yeast to allow production of QA, respective UDP-sugars, and/or respective QA derivatives.
The method of clause 214, wherein the culturing step ranges from 2 to 6 days.
The method of clause 215, wherein the culturing step is about 3 days.
The method of any one of clauses 1 to 38, 42 to 45, 47 to 50, 52 to 61, 63 to 72, 74 to 80, 82 to 86, 88 to 91, 93 to 100, 102 to 108, 110 to 115, 118 to 121, 123 to 126, 128 to 139, 141 to 146, 148 to 154, 156 to 160, 162 to 168, 170 to 177, 179, 180, 182 to 188, 190 to 193, 195 to 198, 200, 201, 203, 204, and 206 to 216, or the yeast of any one of clauses 39, 40, 46, 51, 62, 73, 81, 87, 92, 101, 109, 116, 117, 122, 127, 140, 147, 155, 161, 169, 178, 181, 189, 194, 199, 202, and 205 to 213, wherein the heterologous genes are overexpressed under inducible promoters.
The method, or the yeast, of clause 217, wherein induction is for 2 to 5 days, and yeasts are cultured for 2 to 5 more days.
The method of any one of clauses 1 to 38, 42 to 45, 47 to 50, 52 to 61, 63 to 72, 74 to 80, 82 to 86, 88 to 91, 93 to 100, 102 to 108, 110 to 115, 118 to 121, 123 to 126, 128 to 139, 141 to 146, 148 to 154, 156 to 160, 162 to 168, 170 to 177, 179, 180, 182 to 188, 190 to 193, 195 to 198, 200, 201, 203, 204, and 206 to 218, wherein the method further comprises the step of isolating UDP-sugars, C3-glycosylated QA derivatives, C28-glycosylated QA derivatives or acylated and glycosylated QA derivatives.
QA obtained according to the method of any one of clauses 1 to 38.
C3-glycosylated QA derivatives, C28-glycosylated QA derivatives or acylated and glycosylated QA derivatives obtained according to the method of clause 219.
The use of the QA derivatives of clause 221 as an adjuvant.
The use of clause 222, wherein the adjuvant is a liposomal formulation.
The use of clause 222 or clause 223, wherein the adjuvant comprises a TLR4 agonist.
The use of clause 224, wherein the TLR4 agonist is 3D-MPL.
An adjuvant composition comprising QS-21-Xyl according to clause 201 and/or QS-21-Api according to clause 203.
An isolated β-amyrin synthase (SvBAS) according to SEQ ID NO: 13.
An isolated β-amyrin synthase (QsBAS) according to SEQ ID NO: 15.
An isolated CYP C16 oxidase (QsC28C16) according to SEQ ID NO: 23.
An isolated CYP C16 oxidase (SvC16) according to SEQ ID NO: 26.
An isolated CYP C23 oxidase (SvC23-1) according to SEQ ID NO: 32.
An isolated CYP C23 oxidase (SvC23-2) according to SEQ ID NO: 35.
An isolated CYP C28 oxidase (SvC28) according to SEQ ID NO: 44.
An isolated Cytochrome b5 protein (Qsb5) according to SEQ ID NO: 55.
An isolated Cytochrome b5 protein (Svb5) according to SEQ ID NO: 61.
An isolated UDP-GlcA transferase (SvCslG) according to SEQ ID NO: 76.
An isolated MSBP protein (SvMSBP1) according to SEQ ID NO: 67.
An isolated MSBP protein (SvMSBP2) according to SEQ ID NO: 70.
An isolated MSBP protein (QsMSBP1) according to SEQ ID NO: 73.
An isolated UDP-glucose-4,6-dehydratase (SvUG46DH) according to SEQ ID NO: 87.
An isolated UDP-4-keto-6-deoxy-glucose reductase (SvNMD) according to SEQ ID NO: 90.
An isolated UDP-Galactose transferase (SvGalT) according to SEQ ID NO: 98.
An isolated UDP-Fucose transferase (SvFucT) according to SEQ ID NO: 96.
An isolated UDP-Xylose transferase (SvC3XylT) according to SEQ ID NO: 100.
An isolated UDP-Arabinofuranose transferase (QsArafT2) according to SEQ ID NO: 229.
An isolated UDP-glucose dehydrogenase (AtUGDA101L) according to SEQ ID NO: 108.
An isolated UDP-Xylose transferase (QsC28XylT4-3aa) according to SEQ ID NO: 131.
An isolated UDP-Xylose transferase (QsC28XylT4-6aa) according to SEQ ID NO: 134.
An isolated UDP-Xylose transferase (QsC28XylT4-9aa) according to SEQ ID NO: 137.
An isolated UDP-Xylose transferase (QsC28XylT4-12aa) according to SEQ ID NO: 140.
An isolated UDP-Xylose transferase (SUMO-QsC28XylT4) according to SEQ ID NO: 143.
An isolated UDP-Xylose transferase (TrXA-QsC28XylT4) according to SEQ ID NO: 145.
An isolated UDP-Xylose transferase (MBP-QsC28XylT4) according to SEQ ID NO: 147.
An isolated UDP-Xylose transferase (QsC28XylT3-3×GGGS-QsC28XylT4) according to SEQ ID NO: 149.
An isolated type I polyketide synthase (AstLovF-TE) according to SEQ ID NO: 235.
The genotypes of YL and SC yeast strains used in the Examples described below are provided in Table 3 and Table 4, respectively. Yeast engineering was carried out as described in Example 5 below (unless stated otherwise). Heterologous gene expression in yeast was carried out using nucleotide sequences that have been codon-optimized in order to increase the production of the corresponding protein. It is to be understood that codon optimization does not affect the amino acid sequence of the protein which is overexpressed. Heterologous genes have been integrated into the genome of the different yeast strains (as indicated), unless stated otherwise, under galactose-inducible promoters. After 2 days of culturing, expression of the heterologous genes has been induced with galactose added to the culture medium. 3 days post-induction, the production of sugars, QA precursors, QA, and acylated and/or glycosylated QA derivatives (as indicated) has been assessed by analysing their presence, by liquid chromatography-mass spectrometry (LC-MS) detection (as described in Example 6 below), after extraction of the yeast culture medium (as described in Example 5 below), unless stated otherwise.
A previously developed mevalonate-overproducing strain, Jwy601, a CEN.PK2 based Saccharomyces cerevisiae strain was chosen as a parent strain (Wong et al. 2018). Jwy601 has been engineered to overexpress genes encoding β-amyrin synthases (BAS) of different plant origins by genome integration and the respective engineered yeast strains have been tested for their ability to convert 2,3-oxido-squalene into β-amyrin by analysing the presence of β-amyrin by gas chromatography-mass spectrometry (GC-MS) (using a standard commercially available).
BAS from Artemisia annua (Aa) (named ‘AaBAS’ enzyme—SEQ ID NO: 1 encoded by SEQ ID NO: 3), Arabidopsis thaliana (At) (named ‘AtBAS’ enzyme—SEQ ID NO: 4 encoded by SEQ ID NO: 6), Glycyrrhiza glabra (Gg) (named ‘GgBAS’ enzyme—SEQ ID NO: 7 encoded by SEQ ID NO: 9), Gypsophila vaccaria (Gv) (named ‘GvBAS’ enzyme—SEQ ID NO: 10 encoded by SEQ ID NO: 12) have been tested. The BAS homolog from G. vaccaria yielded the highest production of β-amyrin (see
MLY-01 has been further engineered to co-express different cytochrome P450 (CYP) oxidases (C16, C23 and C28 oxidases) with a cytochrome P450 reductase (CPR) of different plant origins via sequential integration into the yeast genome. The production of QA and QA precursors has been analysed (using respective standards commercially available, e.g. from Merck, as a reference) by LC-MS in the yeast strains engineered with the following combination of enzymes:
Hederagenin and gypsogenin (QA precursors) were detectable. In addition, the pic obtained at about 10 min demonstrated the presence of QA at trace amount (<1 mg/L) (data not shown here, but data disclosed in
CYP oxidases of alternative plant origins have been additionally tested. MLY-01 has been further engineered to co-express homologs CYP oxidases from Q. saponaria, together with the above AtAtr1, via sequential integration into the yeast genome, as follows:
In some experiments, the cytochrome b5 protein from Q. saponaria (named ‘Qsb5′-SEQ ID NO: 55 encoded by SEQ ID NO: 57) and/or the membrane steroid-binding protein from S. vaccaria (named ‘SvMSBP1’—SEQ ID NO: 67 encoded by SEQ ID NO: 69) have been further co-expressed (see also below Sections 1.2.4 and 1.2.5).
The production of QA and QA precursors has been analysed (using respective standards commercially available, e.g. from Merck, as a reference) by LC-MS in the yeast strains engineered with the following combinations of enzymes:
The data are provided in Table 2 below and the results are presented in the form of a graph in
As shown in
The additional co-expression of QsC16 (together with AtATR1, QsC23 and QsC28) did not result into QA production (data not shown), indicating that no oxidation at the C16 position happened, suggesting that QsC16 was non-functional.
Subcellular localization studies revealed that, unlike other CYP oxidases, the C-terminally mcherry-tagged QsC16 oxidase is cytosolic, despite the presence of a predicted transmembrane domain at the N-terminus of the C16 oxidase. The confocal microscopy images obtained show that QsC18-GFP is localized in the endoplasmic reticulum (ER) membrane (
In order to test the hypothesis that the lack of activity of QsC16 was due to inappropriate localization in yeast, the 22-amino acid predicted transmembrane domain of QsC28 was fused to the N-terminus of QsC16 (named ‘QsC28C16’—SEQ ID NO: 23 encoded by SEQ ID NO: 25), anchoring it to the ER membrane (
When co-expressing QsC28C16 (instead of QsC16) in YL-4, QA was detected and produced at 1.1 mg/L (see Table 2 and
The further co-expression of SvMSBP1, in YL-6, resulted into an increased global oxidation efficiency leading to an improved QA production (see Table 2 and
The simultaneous overexpression of 2 copies of QsC28 and 2 copies of AtATR1, in YL-8, led to an 8-fold increase in QA (18.9 mg/L) (see Table 2 and
An additional second copy of all enzymes, in YL-10, led to a further optimized production of QA (65.2 mg/L) (see Table 2 and
Gene Discovery in S. vaccaria—CYP Oxidases
Leaves and flowers of S. vaccaria (Sv) have been treated with 0, 50 μM or 100 μM methyl jasmonate (Meja) for 72 h. The expression level of β-amyrin synthase mRNA has been analyzed (in leaves) (see
The functional relevance and activity of ‘SvC16′, ‘SvC23-1′, ‘Sv23-2’ and ‘SvC28′ (as named in
The production of QA precursors has been analyzed (using respective standards commercially available, e.g. from MCE, Chemcruz and TCI, as a reference) by LC-MS.
Results are shown in
Echinocystic acid and oleanolic acid were detected when co-expressing SvBAS, SvC28 and SvC16 (
Gyspogenin was detected when co-expressing QsBAS, QsC28 and each of SvC23-1 or SvC23-2 (
Gypsogenic acid was detected when co-expressing QsBAS, QsC28 and each of SvC23-1 or SvC23-2 (
These results confirm the functional relevance and activity of QsBAS, and the newly identified SvC16, SvC23-1, SvC23-2 and SvC28 oxidases, as well as their ability to produce QA precursors, when co-expressed in N. benthamiana.
QA Production in Yeast Using S. vaccaria Genes SvC16, SvC23-1 and SvC23-2
MLY-01 has been transformed with the following plasmids: pESC-TRP-SepGAL2-SvC16, pGAL10-AtAtr1, pGAL1-QsC28, pGAL7-SvC23-1 or pESC-TRP-SepGAL2-SvC16, pGA10-AtAtr1, pGAL1-QsC28, pGAL7-SvC23-2. The production of QA and QA precursors has been analyzed (using respective standards commercially available, as a reference) by HPLC/LC-MS.
Both chromatograms in
Confocal microscopy images revealed that SvC16 is well-expressed and localizes properly in the endoplasmic reticulum (ER) of the yeast (data not shown), in contrary to OsC16 (see above Section 1.2.1).
These results confirm the functional relevance of SvC16, SvC23-1 and SvC23-2 oxidases, as well as their ability to produce QA, when co-expressed with AtATR1 and QsC28 in yeast.
Gene Discovery in S. vaccaria—MSBP Proteins
Genes encoding MSBP homologs to A. thaliana (At) have been identified in S. vaccaria (Sv) transcriptome by sequence similarity search using algorithm tblastn. Amino acid sequences of MSBPs from At (named ‘AtMSBP1’—SEQ ID NO: 63 and ‘AtMSBP2’—SEQ ID NO: 65) were submitted in a database of Sv transcriptome (prepared in-house) for a comparison with translated DNA sequences of all genes in the transcriptome. Similar sequences were selected based on sequence identity (last column of Table 3) and the significance of sequence match (third column of Table 3). The results are summarized in Table 3 below.
Arabidopsis thaliana (At) MSBP homologs in S. vaccaria (Sv)
The average expression levels of the different homologs identified in Table 3 has been analysed in leaves and flowers of S. vaccaria (see
QA Production in Yeast Using Homologs MSBP from S. vaccaria
The functional relevance and activity of the transcripts PB.393.1 and PB.16084.2 has been tested for their ability to increase the oxidation efficiency and improve QA production in yeast. Respective corresponding proteins have been named ‘SvMSBP1′ (SEQ ID NO: 67 encoded by SEQ ID NO: 69) and ‘SvMSBP2′ (SEQ ID NO: 70 encoded by SEQ ID NO: 72) and have been integrated into the genome of yeasts engineered to produce QA, as follows:
A homolog MSBP from Q. saponaria (named ‘QsMSBP1’—SEQ ID NO: 73 and encoded by SEQ ID NO: 75) has been tested as well. The production of QA and QA precursors has been analyzed (using respective commercial standards as a reference) by LC-MS. Results are presented in the form of a graph in
As compared with YL-4 (which does not overexpress any MSBP protein), in yeasts overexpressing MSBP proteins (whether from S. vaccaria or from Q. saponaria), a significant increase in QA production was observed, with SvMSBP1 and SvMSBP2 performing better (see
Using different heterologous enzymes (β-amyrin synthase, CYP oxidases, CYP reductase) and heterologous proteins (cytochrome b5 and MSBP proteins) from different plant origins (e.g. G. vaccaria, A. thaliana, Q. saponaria and S. vaccaria), in different combinations, the inventors have been able to reconstruct in yeast the metabolic pathway leading to the biosynthesis of QA, achieving, for the first time, the successful production of QA in yeast at about 65 mg/L.
As shown in
As shown in
The production of UDP-Xyl was detected in both SC-4 and SC-16 (see
The expression of the trifunctional AtRHM2 synthase enzyme from A. thaliana (named ‘AtRHM2’—SEQ ID NO: 102 encoded by SEQ ID NO: 104) has been investigated as a potential rhamnose synthase. AtRHM2 catalyzes the conversion from UDP-Glc directly to UDP-Rha via (i) the dehydration of UDP-Glc followed by (ii) the epimerization of the C3′ and C5′ positions to form UDP-4-keto-β-L-rhamnose and (iii) the reduction of UDP-4-keto-β-L-rhamnose to produce UDP-β-L-rhamnose. AtRHM2 has been integrated into the genome of the parent yeast strain CEN.PK2-1c to generate SC-17 and into the genome of SC-4 to generate SC-18. The production of UDP-Rha has been analyzed by LC-MS.
The production of UDP-Rha was detected in both SC-17 and SC-18 (see
The same AtUGD as above has been integrated into the genome of YL-10 (producing QA), together with a glucuronic acid transferase (GlcAT) from Q. saponaria (named ‘QsCslG1’—SEQ ID NO: 78 encoded by SEQ ID NO: 80) or a second glucuronic acid transferase from Q. saponaria (named ‘QsCslG2’—SEQ ID NO: 81 encoded by SEQ ID NO: 83) to generate YL-11 and YL-12, respectively. Production of QA precursors as well as QA-C3-GlcA has been analyzed by LC-MS, using respective standards as a reference (QA-C3-GlcA standard corresponds to QAGlcpA, generated as described in WO 20/260475).
QA-C3-GlcA was detected in both YL-11 (overexpressing QsCslG1) and YL-12 (overexpressing QsCslG2) (see
QsCslG1 is specific towards QA and does not glycosylate other precursors, while QsCslG2 enzyme is promiscuous and 3 times more reactive than CslG1 enzyme to produce GlcA-QA (10.2 mg/L and 3.9 mg/L, respectively) (see
The inventors also identified in the transcriptome of S. vaccaria a novel gene encoding a CslG homolog enzyme (named ‘SvCslG’—SEQ ID NO: 76 encoded by SEQ ID NO: 77). The function of SvCslG as a GlcA transferase candidate has been confirmed using an in vitro enzymatic assay. QA (commercially available, e.g. from MedChemExpress) and UDP-GlcA, have been directly added into the reaction buffer together with a microsomepreparation of a yeast strain overexpressing SvCslG via plasmid expression. The production of QA and QA-C3-GlcA was analyzed by LC-MS.
UDP-galactose is natively produced in yeast and therefore, no addition of a sugar synthase is necessary for this glycosylation step. A galactose transferase from Q. Saponaria (named ‘QsGalT’—SEQ ID NO: 116 and encoded by SEQ ID NO: 118) has been integrated into the genome of YL-12 to generate YL-13. The production of QA-C3-GlcA and QA-C3-GlcA-Gal has been analyzed (using respective standards) by LC-MS. QA-C3-GlcA standard and QA-C3-GlcA-Gal standard corresponds to ‘QAGlcpA’ and ‘QA-GlcpA-Galp’, respectively, generated as described in WO 20/260475.
The inventors also identified in the transcriptome of S. vaccaria a novel gene encoding a galactose transferase candidate (named ‘SvGalT’—SEQ ID NO: 98 encoded by SEQ ID NO: 99). The function of SvGalT as a galactose transferase has been confirmed by transiently expressing SvCslG and SvGalT in N. benthamiana plants. Plants have been infiltrated with 40 μM of QA (commercially available, e.g. from MedChemExpress) 2 days after Agrobacterium tumefaciens infiltration. The production of QA-C3-GlcA and QA-C3-GlcA-Gal has been analyzed (using respective standards) by LC-MS. QA-C3-GlcA standard and QA-C3-GlcA-Gal standard correspond to ‘QAGlcpA’ and ‘QA-GlcpA-Galp’, respectively, generated as described in WO 20/260475.
The above AtRHM2 and a rhamnose transferase from Q. Saponaria (named ‘QsRhaT’-SEQ ID NO: 119 and encoded by SEQ ID NO: 121) have been integrated into the genome of YL-13 to generate YL-14. The production of QA-C3-GlcA and QA-C3-GlcA-Gal and QA-C3-GlcA-Gal-Rha has been analyzed (using respective standards) by LC-MS. QA-C3-GlcA standard, QA-C3-GlcA-Gal standard and QA-C3-GlcA-Gal-Rha correspond to ‘QAGlcpA’, ‘QA-GlcpA-Galp’, and ‘QA-GlcpA-Galp-Rhap’, respectively, generated as described in WO 20/260475.
The above AtUXS has been integrated into the genome of YL-12 (a yeast strain engineered to produce UDP-GlcA). Direct expression of AtUXS in the UDP-GlcA-producing strain led to the absence of any glycosylated molecule (data not shown), possibly due to insufficient UDP-GlcA production. This suggested that the downstream metabolite UDP-Xylose may act as an allosteric feedback inhibitor controlling the activity of UGD. This is confirmed in
It has been reported that a point mutation A104L engineered in the human UGD homolog has led to a lower UDP-Xyl binding affinity. Therefore, as an attempt to alleviate the observed UGD inhibition induced by UDP-Xyl, mutation(s) were introduced into AtUGD in order to lower UDP-Xyl binding affinity. The protein sequence of AtUGD was aligned against that of the human UGD to identify the corresponding amino acid (data not shown), and a point mutation A101 L was introduced into AtUGD (AtUGDA101L—SEQ ID NO: 108 encoded by SEQ ID NO: 109). AtUGDA101L, has been integrated into the genome of YL-10 (yeast engineered to produce QA), together with the above QsCslG2, and OsGalT, as well as with a UDP-xylose transferase from Q. saponaria (QsC3XylT—SEQ ID NO: 122 encoded by SEQ ID NO: 124), to generate YL-15. The production of QA-C3-GlcA, QA-C3-GlcA-Gal and QA-C3-GlcA-Gal-Xyl has been analyzed (using respective standards) by LC-MS.
In order to investigate the varying degrees of UDP-Xyl inhibition on different UGDs, six homologs were selected across kingdoms to include those from Synechococcus sp. (Syn) (named ‘SynUGD’—SEQ ID NO: 154 encoded by SEQ ID NO: 156), Homo sapiens (Hs) (named ‘HSUGD104L’—SEQ ID NO: 157 encoded by SEQ ID NO: 159), Paramoeba atlantica (Patl) (named ‘PatIUGD’—SEQ ID NO: 110 encoded by SEQ ID NO: 112), Bacillus cytotoxicus (Bcyt) (named ‘BcytUGD’—SEQ ID NO: 160 encoded by SEQ ID NO: 162), Corallococcus macrosporus (Myxfulv) (named ‘MyxfulvUGD’—SEQ ID NO: 163 encoded by SEQ ID NO: 165), Pyrococcus furiosus (Pfu) (named ‘PfuUGD’—SEQ ID NO: 166 encoded by SEQ ID NO: 168). The sequences of these homologs have been integrated into genome of YL-10 (a yeast strain engineered to produce QA), together with the above QsCslG2, QsGalT, AtUXS, and QsC3XylT, generating YL-16 to YL-21, respectively. The production of QA-C3-GlcA-Gal-Xyl has been analyzed by LC-MS (using respective standards). The results are presented in
UDP-GlcA can also be generated via the de novo salvage pathway or the myo-inositol oxidation pathway. Glucuronokinase (GlcAK) and UDP-sugar pyrophosphorylase (USP) convert free glucuronic acid to GlcA-1-phosphate and eventually the active UDP form of GlcA (UDP-GlcA). These enzymes are also responsible for the myo-inositol pathway starting with myo-inositol oxygenase (named ‘MIOX’). A GlcAK enzyme from A. thaliana (named ‘AtGlcAK’—SEQ ID NO: 169 encoded by SEQ ID NO: 171) and a USP from A. thaliana (named ‘AtUSP’—SEQ ID NO: 223 encoded by SEQ ID NO: 225) have been integrated into the genome of YL-15 to generate YL-22. The same GlcAK and AtUSP have been separately integrated, together with a MIOX from Thermothelomyces thermophilus (named ‘TtMIOX’—SEQ ID NO: 172 encoded by SEQ ID NO: 174), into the genome of YL-15 to generate YL-23. The culture medium of YL-23 was either left untreated or exogenously supplemented with 0.5% glucuronic acid and 2% myo-inositol (MI). The production of QA-C3-GGX has been analyzed by LC-MS (using respective standards).
QA-C3-GGX production was improved by 3-fold in YL-22, as the residual QA decreased significantly (see
QA-C3-GGX production, in YL-23 (further overexpressing TtMIOX), was increased by 1.7-fold and 2.3-fold, in the presence of 2% MI and 0.5% GlcA exogenously supplemented, respectively. Production was further improved by 5.9-fold when both MI and GlcA were supplemented (see
Inducible promoters such as pDDI2 (induced by methyl methane sulfonate), pCup1 (induced by copper ions), as well as pTetOn (induced by tetracycline or doxycycline) have been investigated and used, as a way to delay the expression of AtUXS. AtUXS has been overexpressed in a yeast engineered to produce QA-C3-GGX under a pTetOn promoter. Production of QA, QA-C3-GG and QA-C3-GGX has been analyzed by LC-MS.
pTetOn was compatible with the galactose promoters used in the parent yeast strain and the protein expression of AtUXS was linearly dependent on the concentration of the inducer.
In the absence of any inducer, a 5.5-fold increase of QA-C3-GGX production was observed, possibly because of the basal level expression of AtUXS due to the leakiness of the promoter. The minimal amount of UDP-Xyl produced may not be sufficient to inhibit AtUGD.
In order to induce pTetOn, 20 or 100 μg/mL of doxycycline has been added exogenously supplemented in the yeast culture medium 24 h after galactose induction. This led to the increased production of QA-C3-GGX by 5.9- and 8.5-fold, as compared to YL-15. When induced with 100 μg/mL of doxycycline after 40 h after galactose induction, an 11-fold increase was observed (see
Identification of a C3XylT Enzyme in S. vaccaria
The inventors also identified in the transcriptome of S. vaccaria a novel gene encoding a xylosyl transferase candidate (named ‘SvC3XylT’—SEQ ID NO: 100 encoded by SEQ ID NO: 101). The function of SvC3XylT as a xylose transferase has been tested by transiently co-expressing the same SvCslG enzyme and SvGalT enzyme as described earlier in N. benthamiana plants. Plants have been infiltrated with 40 μM of QA (commercially available, e.g. from MedChemExpress) 2 days after Agrobacterium tumefaciens infiltration. QA-C3-GlcA-Gal-Xyl production has been analyzed by LC-MS. A standard corresponding to ‘QA-GlcpA-Galp-Xylp’ generated as described in WO 20/260475 has been used as a reference.
Using different heterologous enzymes (glycosyl synthases, glycosyl transferases) from different plant origins (e.g. A. thaliana, Q. saponaria and S. vaccaria), in different combinations, the inventors have been able to reconstruct in yeast the metabolic pathway leading to the biosynthesis of C3-glycosylated QA derivatives, achieving, for the first time, the successful production of such C3-glycosylated QA derivatives in yeast.
The transcriptome of S. vaccaria was further explored to identify genes and enzymes involved in saponin biosynthesis, as S. vaccaria contains a number of different saponins that have similarity to saponins in Q. saponaria. S. vaccaria plants were treated with methyl-jasmonate (Meja) which was shown to induce biosynthesis of saponins in plants. An extensive RNASeq analysis was then performed to identify the full-length transcripts in the plants, and to identify the induced genes. Among them, several genes were known to be involved in biosynthesis of the triterpene backbone (e.g. β-amyrin synthase), as well as several Cytochrome P450 enzymes (CYP) and glycosyltransferase genes (see e.g. WO 20/263524). Some of the genes are homologs to genes known to be involved in saponin biosynthesis in Q. Saponaria (see e.g. WO 19/122259, WO 20/260475, WO 22/136563; Decker and Kleczkowski 2017). Based on knowledge from dTDP-D-Fucose biosynthesis in bacteria and UDP-L-Rhamnose biosynthesis in plants, it was predicted the pathway to include a dehydratase step and a reductase step (as shown in
svUG46DH (SEQ ID NO: 87 encoded by SEQ ID NO: 89) and svNMD (SEQ ID NO: 90 encoded by SEQ ID NO: 92) have both been integrated into the genome of the parent yeast strain CEN.PK2-1c to generate SC-19.
SvUG46DH and SvNMD have both been integrated into the genome of SC-4 (overexpressing AtUGD-AtUXS) to generate SC-20.
SvUG46DH and SvNMD have both been integrated into the genome of SC-17 (overexpressing AtRHM2) to generate SC-22.
SvUG46DH and SvNMD have both been integrated into the genome of SC-18 (overexpressing AtUGD-AtUXS-AtRHM2) to generate SC-23.
A homolog reductase from Q. saponaria (WO 22/136563) (named ‘QsFucSyn’—SEQ ID NO: 175 encoded by SEQ ID NO: 177) has been alternatively tested, in combination with the following enzymes:
QsFucSyn and SvUG46DH have been integrated into the genome of SC-4 (overexpressing AtUGD-AtUXS) to generate SC-21.
The production of UDP-Fucose has been analyzed by LC-MS.
UDP-Fucose was produced when svUG46DH and svNMD were overexpressed on their own (SC-19) (see
UDP-Fucose was also produced when svUG46DH and svNMD were overexpressed together with AtUGD-AtUXS (SC-20) (see
UDP-Fucose was also produced when svUG46DH and svNMD were overexpressed together with AtRHM2 (SC-22) (see
UDP-Fucose was also produced when svUG46DH and svNMD were overexpressed together with AtUGD-AtUXS-AtRHM2 (SC-23) (see
UDP-Fucose was also produced when QsFucSyn and svUG46DH were overexpressed together with AtUGD-AtUXS (SC-21) (see
These results confirm the functional relevance and activity of the newly identified SvUG46DH and SvNMD, and QsFucSyn, when expressed in yeast.
A fucose transferase from Q. saponaria (WO 22/136563) (named ‘QsFucT’—SEQ ID NO: 93 encoded by SEQ ID NO: 95) has been integrated into the genome of YL-14 to generate YL-25.
QA-C3-GlcA-Gal-Rha and QA-C3-GlcA-Gal-Rha-C28-Fuc have been detected in YL-25 (see
The same QsFucT enzyme has also been integrated into the genome of YL-15 to generate YL-26. QA-C3-GlcA-Gal-Rha-C28-Fuc production has been analyzed by LC-MS.
Production of QA-C3-GlcA-Gal-Xyl-C28-Fuc has been similarly observed in YL-26.
Identification of a FucT Enzyme in S. vaccaria
The inventors also identified in the transcriptome of S. vaccaria a novel gene encoding a FucT candidate (named ‘SvFucT’—SEQ ID NO: 96 encoded by SEQ ID NO: 97). The function of SvFucT as a fucose transferase has been tested by transiently co-expressing the same SvCslG, SvUG46DH and SvNMD as described earlier in N. benthamiana plants. Plants have been infiltrated with 40 μM of QA (commercially available, e.g. from MedChemExpress) 2 days after Agrobacterium tumefaciens infiltration. QsFucT (see above) was used as a positive control, and GFP was used as negative control. The production of QA-C3-GlcA-C28-Fuc has been analyzed by LC-MS.
The same trifunctional AtRHM2 enzyme as described earlier, together with a rhamnose transferase from Q. Saponaria (named ‘QsRhaT’—SEQ ID NO: 119 encoded by SEQ ID NO: 121), has been integrated into the genome of YL-15 to generate YL-28. QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha production has been analyzed by LC-MS (using a standard which has been chemically synthesized as a reference).
QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha production was detected in YL-28 at a titer of about 1 mg/L (see
An additional copy of the same trifunctional AtRHM2 enzyme (as described earlier), together with the same QsRhaT (as described earlier) has been integrated into the genome of YL-14 to generate YL-27. QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha production has been analyzed by LC-MS. A standard corresponding to ‘QA-TriR-FR’ as described in WO 22/136563 has been used as a reference.
QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha was detected in YL-27 at a titer of about 3 mg/L (see
An additional copy of the same AtUXS enzyme (as described earlier), together with a xylose transferase from Q. Saponaria (named ‘QsC28XylT3’—SEQ ID NO: 125 encoded by SEQ ID NO: 127), has been integrated into the genome of YL-28 to generate YL-30. QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl production has been analyzed by LC-MS. QA-C3-GGR-C28-FRX previously obtained was used a reference.
QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl was detected in YL-30 (see
The same AtUXS enzyme (as described earlier), together with the same QsC28XylT3 as above, was integrated into the genome of YL-27 to generate YL-29. The production of QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl has been analyzed by LC-MS. A standard corresponding to ‘QA-TriR-FRX’ as described in WO 22/136563 has been used as a reference.
QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl was detected in YL-29 (see
An additional copy of the same AtUXS enzyme (as described earlier), together with a xylose transferase from Q. Saponaria (named ‘QsC28XylT4’—SEQ ID NO: 128 encoded by SEQ ID NO: 130), has been integrated into the genome of YL-30 to generate YL-33. The production of QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl has been analyzed by LC-MS. QA-C3-GGR-C28-FRXX previously obtained was used a reference.
Conversion of QA-C3-GGX-C28-FRX into QA-C3-GGX-C28-FRX was observed in YL-33 (
An additional copy of the same AtUXS enzyme (as described earlier), together with the same C28QsXylT4 as above, has been integrated into the genome of YL-29 to generate YL-31. The production of QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Xyl has been analyzed by LC-MS. A standard corresponding to ‘QA-TriR-FRXX’ as described in WO 22/136563 has been used as a reference.
Conversion of QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl into QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Xyl was observed in YL-31 (
UDP-Apiose can be produced using apiose synthase (‘AXS’) enzymes, which produces both UDP-Xyl and UDP-Api (as shown in
An additional copy of the same QsAXS enzyme as above, together with an apiose transferase from Q. Saponaria (named ‘QsC28ApiT4’—SEQ ID NO: 151 encoded by SEQ ID NO: 153), has been integrated into the genome of YL-30 to generate YL-34. The production of QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Api has been analyzed the by LC-MS.
Conversion of QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl into QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Api was observed in YL-34 (
The same QsAXS enzyme as above, together with the same QsC28ApiT4 enzyme as above, has been integrated into the genome of YL-29 to generate YL-32. The production of QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Api has been analyzed the by LC-MS.
Conversion of QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl to QA-C3-GlcA-Gal-Rha-C28-Fuc-Rha-Xyl-Api was observed in YL-32 (
Using different heterologous enzymes (glycosyl synthases and glycosyl transferases) from different plant origins (e.g. A. thaliana, Q. saponaria and S. vaccaria), the inventors have been able to reconstruct in yeast the metabolic pathway leading to the synthesis of C28-glycoslylated QA derivatives, achieving, for the first time, the successful production of such C28-glycoslylated QA derivatives in yeast.
Different approaches have been investigated to assess whether the conversion of QA-C3-GGR/X-C28-FRX into QA-C3-GGR/X-FRXX/A could be improved.
Subcellular localization of QsC28XylT4 and QsC28ApiT4
The subcellular localization of QsC28XylT4 and QsC28ApiT4 heterologously expressed in yeast was examined. C-terminal green fluorescent protein (GFP) fusion was built to provide QsC28XylT4-GFP and QsC28ApiT4-GFP in order to visualize the subcellular localization in yeast (using QsC28XylT3-GFP as a reference). Each of QsC28XylT4-GFP, QsC28ApiT4-GFP, and QsC28XylT3-GFP has been integrated into the genome of the parent yeast strain CEN.PK2-1c.
While flow cytometry data (aimed at measuring the absolute protein expression level) showed similar fluorescence intensity, indicating a similar level of protein expression (data not shown), confocal microscopy images revealed that, unlike QsC28XylT3-GFP (
Three signature localization protein markers have been selected to identify the subcellular localization where QsC28XylT4 and QsC28ApiT4 aggregates are formed with the aim to functionally express the two enzymes in the cytosol. Rnq1 which is a yeast native prion protein has been shown to co-localize with ‘insoluble protein deposit’ (‘IPOD’), a reservoir and degradation location for amyloid-like proteins. C-terminal mcherry-tagged Rnq1 was expressed in yeast independently to visualize IPOD, shown to be a perivascular compartment (data not shown). The co-expression of Rnq1-mcherry with QsC28XylT4-GFP revealed a different localization pattern, suggesting that QsC28XylT4 is likely not an amyloid-like misfolded protein (data not shown). The second protein marker, heat shock protein-42 (Hsp-42), has been selected due to its suggested physiological role in initiation of stress granules in yeast upon starvation in carbon or nitrogen sources. Hsp42-mcherry fusion protein was localized in the cytosol and nucleus of yeast (data not shown) and was shown to be co-localized with QsXylT4-GFP (data not shown), suggesting the possible sequestration of QsC28XylT4 into stress granules. The last protein marker selected was Rpn1, a functional component of the proteasome actively involved in the protein degradation machinery. When expressed alone, Rpn1, together with the proteasome machinery, was localized in the nucleus. Upon co-expression with QsC28XylT4-GFP, while the majority of Rpn1-mcherry still remained in the nucleus at 24 h (data not shown), it formed aggregates around QsC28XylT4-GFP aggregates at 48 h and degraded the aggregates towards protein recycling (data not shown). These results suggest that QsC28XylT4 may be sequestered into Hsp42-related stress granule and be prone to degradation.
Truncation of the N-terminus of QsC28XylT4, with the increment of three amino acids up to 12, as well as addition of solubility tags, such as SUMO, TrXA, and MBP, have been carried as an attempt to re-direct the protein in the cytosol. ‘QsC28XylT4-3aa′ (QsC28XylT4 deleted from the 3 first amino acids—SEQ ID NO: 131 encoded by SEQ ID NO: 133), ‘QsC28XylT4-6aa′ (QsC28XylT4 deleted from the 6 first amino acids—SEQ ID NO: 134 encoded by SEQ ID NO: 136), ‘QsC28XylT4-9aa′ (QsXylT4 deleted from the 9 first amino acids—SEQ ID NO: 137 encoded by SEQ ID NO: 139), ‘QsC28XylT4-12 aa′ (QsC28XylT4 deleted from the 12 first amino acids—SEQ ID NO: 140 encoded by SEQ ID NO: 142), ‘SUMO-QsC28XylT4’—SEQ ID NO: 143 encoded by SEQ ID NO: 144, ‘TrXA-QsC28XylT4’—SEQ ID NO: 145 encoded by SEQ ID NO:146 and ‘MBP-QsC28XylT4’—SEQ ID NO: 147 encoded by SEQ ID NO:148 have each been integrated into the genome of YL-30 to generate YL-35, YL-36, YL-37, YL-38, YL-39 and YL-40, respectively. The level of QsC28XylT4 protein expression in each yeast strain and the ability of each yeast strain to produce QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl (as compared with YL-33, harboring a wild-type, full-length, non-tagged, QsXylT4) have been looked at.
The fluorescence intensity measured by flow cytometry shows the highest level of protein expression for QsC28XylT4-MBP (see
In terms of production of QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl, all N-terminal truncations of QsC28XylT4 and all N-terminal-tagged QsC28XylT4 showed a better yield, with the N-terminus MBP tag addition providing a 7-fold increase, as compared with the wild-type and full-length of the enzyme (see
As an alternative way to render QsC28XylT4 cytosolic, QsC28XylT3 (shown to be cytosolic when expressed in yeast, as described earlier), was fused at the N-terminus of QsC28XylT4. A 3×GGGS linker was genetically inserted between the two amino acid sequences of the enzymes to ensure the flexibility of the linker and independent folding of the two enzymes, without affecting the functional properties of the fusion protein. The fusion QsC28XylT3-3×GGGS-QsC28XylT4 (SEQ ID NO: 149 encoded by SEQ ID NO: 150) has been integrated into the genome of YL-30 to generate YL-41. The localization of the fusion protein and the production of QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl have been looked at.
Confocal microscopy images showed an improved cytosolic expression with less level of aggregation observed for the QsXylT3-3×GGGS-QsXylT4-GFP fusion protein, as compared to QsXylT4-GFP when expressed alone (see
The improved reactivity of the fusion protein was also confirmed by the observation of the complete conversion of QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl which leads to a distinctive peak corresponding to the mass of QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl (see
A continuous feeding scheme has been devised by adding fresh nitrogen and/or carbon sources every 24 h. Protein expression and protein localization have been looked at.
The fluorescence intensity, reflecting QsC28XylT4 absolute expression, has been measured by flow cytometry (
In contrast, the addition of fresh media with additional nitrogen source as well as 4% galactose consistently increased QsC28XylT4 expression level up to 5-fold by 60 h.
In the presence of new carbon and nitrogen sources, QsXylT4 did not colocalize with Hsp42. Rpn1, which represents the localization of the proteasome machinery, remained in the nucleus and did not degrade QsXylT4 aggregates. While some aggregation of QsXylT4 persisted in the presence of new media, consistent cytosolic expression was also observed, in stark contrast to yeast strains cultured with only old media, where QsXylT4 expression in the cytosol was depleted after 24 h (data not shown).
As shown in
4.1 (S)-2-Methylbutyryl CoA (2 MB-CoA) Conversion from the 2 MB Acid
Conversion of exogenously supplemented 2 MB acid to 2 MB-CoA by a CoA ligase identified from Q. Saponaria transcriptome has been investigated. The functional expression of this CoA ligase from Q. Saponaria (named ‘QsCCL’—SEQ ID NO: 178 encoded by SEQ ID NO: 180) has been confirmed using a high-copy plasmid transfected into the parent yeast strain CEN.pk2-1c via confocal microscopy imaging of the C-terminal GFP fusion of the enzyme, which is visualized to be in the cytoplasm and is stable for at least 24 h after galactose induction (data not shown). Additionally, the conversion of 2 MB acid to 2 MB-CoA by QsCCL has been demonstrated using a whole-cell feed-in experiment. 2 MB acid has been added directly to the yeast cell culture and the yeast cells have been lysed to allow the measurement of the intracellular content of 2 MB-CoA, by a liquid chromatography method using a porous graphitic carbon column. Production of 2 MB-CoA from 50 mg/L 2 MB acid in YL-QsCCL has been confirmed by co-eluting with a 2 MB-CoA standard (the standard has been chemically synthesized) (see
As shown in
Attempts to directly detect the production of C9-CoA as such using LC-MS was unsuccessful, possibly due to its short-lived stability. Therefore, the synthesis of C9-CoA was demonstrated by its addition to glycosylated QA derivatives. It has been demonstrated that the acyl unit (C9-CoA) can be added to both QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl (QA-C3-GGX-C28-FRX) and QA-C3-GlcA-Gal-Xyl-C28-Fuc-Rha-Xyl-Xyl (QA-C3-GGX-C28-FRXX) (
YL-42 is shown to produce QA-C3-GGX-C28-FRX-C9 in the presence of 50 mg/L 2 MB acid added exogenously, as confirmed by co-eluting with a standard (standard has been generated in N. benthamiana, as described in GB 2204252.7) (see
The conversion of QA-C3-GGX-C28-FRX to QA-C3-GGX-C28-FRX-C9 was improved in the presence of a higher concentration of 2 MB acid supplemented to the growth media (
The 18-carbon acyl chain consists of two repeating units of C9-CoA, and the second addition requires its corresponding acyltransferase (named ‘QsDMOT4’—SEQ ID NO: 196 encoded by nucleotide sequence SEQ ID NO: 198), which has been integrated into the genome of YL-42 to generate YL-43.
With 500 mg/L 2 MB acid supplemented to the culture media, the production of QA-C3-GGX-C28-FRX-C18 has been confirmed with the appearance of a new LC-MS peak with the same high-resolution mass and its conversion from QA-C3-GGX-C28-FRX-C9 was shown to be highly efficient with little residual substrate (
In order to generate QA-C3-GGX-C28-FRXX-C18, QsDMTO4 and QsC28XylT4 have been integrated into the genome of YL-42 to generate YL-44. QA-C3-GGX-C28-FRXX-C18 production has been analyzed by LC-MS.
A new LC-MS peak corresponding to the mass of QA-C3-GGX-C28-FRXX-C18 was detected (
The absence of QA-C3-GGX-C28-FRXX and QA-C3-GGX-C28-FRXX-C9 suggests that they are better substrates for QsDMOT9 and QSDMOT4 acyltransferases than QA-C3-GGX-C28-FRX and QA-C3-GGX-C28-FRX-C9.
4.4 Production of UDP-Arabinofuranose (Araf) Non-Native in Yeast
The biosynthesis of UDP-Araf is not native in yeast and thus, necessary nucleotide sugar synthases as well as an arabinosyl transferase, are required for the heterologous production and addition of this sugar. As shown in
UDP-Xyl was produced by all combinations of enzymes, with AtUAM1-AtUGE3 (SC-11) producing lower UDP-Xyl.
While UDP-Arap production was similar, UDP-Araf was not detected, likely due to the co-elution with UDP-Xyl and since both UXE and UAM enzymes are dominated by equilibrium, UDP-Araf is likely 100× less in abundance than UDP-Xyl (see
As an alternative, the salvage pathway has been tested with arabinokinase (AraK) and UDP-sugar pyrophosphorylase (USP) candidates from A. thaliana (named ‘AtAraK’-SEQ ID NO: 214 encoded by nucleotide sequence SEQ ID NO: 216 and AtUSP—SEQ ID NO: 223 encoded by nucleotide sequence SEQ ID NO: 225, respectively) and Leptospira interrogans (Lei) (named ‘LeiAraK’—SEQ ID NO: 217 encoded by nucleotide sequence SEQ ID NO: 219 and ‘LeiUSP’—SEQ ID NO: 226 encoded by nucleotide sequence SEQ ID NO: 228, respectively). An arabinose transporter from Penicillium rubens Wisconsin (named ‘PrAraT’—SEQ ID NO: 220 encoded by nucleotide sequence SEQ ID NO: 222) has also been tested to determine if it was necessary for arabinose to enter the yeast and AtUAM1 to convert UDP-Arap to UDP-Araf. The following combinations have been integrated into the genome of the parent yeast strain CEN.PK2-1c, wherein corresponding yeasts were fed with 1% arabinose added exogenously:
Both AraT and the salvage pathway from L. interrogans produced less UDP-Arap (0.910 μmol/g Cell Pellet and 0.665 μmol/g Cell Pellet, respectively), as compared to the salvage pathway from A. thaliana (1.73 μmol/g Cell Pellet).
UDP-Araf was produced with the salvage pathway, AraT and AtUAM1 at 0.185 μmol/g Cell Pellet (see
Plant UDP-L-arabinofuranose (UDP-Araf) biosynthesis is closely associated with the golgi apparatus because L-Araf is a key component in the plant cell wall. The biosynthesis of UDP-Arap mainly occurs through the epimerization of UDP-Xyl in the Golgi lumen, which is interconverted into UDP-Araf by a UDP-Ara mutase located outside on the cytosolic surface of the Golgi, then being transported back to the Golgi lumen for its later glycosylation applications. Because of the lack of yeast native sugar transporters on the golgi membrane, cytosolic homologs of these enzymes were selected from A. thaliana, UDP-xylose epimerase (AtUXE) and AtUAM1 to produce UDP-Araf in yeast.
Starting from YL-42 (the yeast strain capable of producing QA-C3-GGX-C28-FRX-C9), genes encoding (i) AtUXS and QsC28XylT4 (to produce QA-C3-GGX-C28-FRXX-C9), or AtAXS and QsC28ApiT4 (to produce QA-C3-GGX-C28-FRXA-C9), (ii) QsDMOT4 (to produce QA-C3-GGX-C28-FRXX-C18 or QA-C3-GGX-C28-FRXA-C18), (iii) AtUXE and AtUAM1 (to produce arabinofuranose from UDP-Xyl), and (iv) an arabinofuranose transferase (named ‘QsArafT’—SEQ ID NO: 229 encoded by nucleotide sequence SEQ ID NO: 231) (to produce QA-C3-GGX-C28-FRXX-C18-A or QA-C3-GGX-C28-FRXA-C18-A) have been further integrated into the genome of YL-42, generating two new yeast strains, as summarized below, and 2 MB acid was supplemented in the culture media:
In the extracted single-ion LC-MS chromatogram of YL-45, more than one peak was observed when the exact mass of QS-21-Xyl (i.e. QA-C3-GGX-C28-FRXX-C18-A) was extracted (
The peak with a retention time of 11.1-11.2 min co-elutes with a QS-21 standard (standard corresponds to the QS-21 fraction purified from a crude bark extract of Quillaja saponaria Molina which has been generated as described in WO 19/106192) (
In the extracted single-ion LC-MS chromatogram of YL-46, multiple peaks are similarly observed when the exact mass of QS-21-Api (i.e. QA-C3-GGX-C28-FRXA-C18-A) was extracted (
Additionally, similar peak patterns with the exact extracted mass of QA-C3-GGX-C28-FRXA-C18 are also observed, which also corresponds to the mass of QA-C3-GGX-C28-FRX-C18-A.
Since xylose, arabinofuranose (Araf), and arabinopyranose (Arap) are structural isomers, they also have the same exact mass. It is likely that other pentose sugars can be added instead of Araf, leading to the other peaks with the same exact mass as QS-21-Api. Therefore, the substrate scope of the Araf transferase (QsArafT) has been investigated. The strain YL-47 has been constructed by integrating QsArafT without the genes required to convert UDP-Xyl to UDP-Araf (i.e. without AtUXE and AtUAM1).
As a result, a new peak is observed that corresponds to QA-C3-GGX-C28-FRX-C18-Xyl, suggesting that QsArafT can also use UDP-Xyl as a substrate instead of UDP-Araf for addition at the end of the acyl chain (
In search of ArafT homologs that are more specific towards UDP-Araf, BLAST searches were performed on the Q. saponaria transcriptome in 1 kp database (https://db.cngb.org/onekp/) using the ArafT protein sequence (SEQ ID NO: 229). A candidate with 64% protein homolog has been identified, OQHZ_scaffold_2012646, named ‘QsArafT2′ (SEQ ID NO: 232 encoded by the nucleotide sequence SEQ ID NO: 234). First, this candidate has been tested for its activity towards UDP-Xyl. YL-48 has been similarly constructed by integrating QsArafT2 without the genes required to convert UDP-Xyl to UDP-Araf (i.e. without AtUXE and AtUAM1), and 2 MB acid was supplemented in the culture media.
While the production of QA-C3-GXX-C18-FRX-C18 (in YL-48) has been detected, no LC-MS peak that corresponds to the addition of Xyl was observed (
Therefore, a new yeast strain was generated (YL-49), similar to YL-45, except that the gene encoding QsArafT was replaced with a gene encoding QsArafT2.
The extracted single-ion chromatograms confirmed the production of QS-21-Xyl (i.e. QA-C3-GXX-C18-FRXX-C18-Araf) with a higher ratio of the desired peak with regard to the other LC-MS peaks with the same exact mass (
4.6 Integration of a Type I Polyketide Synthase to Produce (S)-2-Methylbutyryl CoA In Vivo
In order to circumvent the need of exogenously adding 2 MB acid, the biosynthesis of (S)-2-methylbutyryl CoA (2 MB-CoA) in vivo in yeast has been investigated. The branched-chain α-keto acid dehydrogenase (BCKD) complex has first been investigated with a transaminase from Bacillus subtilis (Bs), which, in bacteria, would readily convert isoleucine to 2 MB-CoA during amino acid metabolism. However, no 2 MB-CoA was detected in yeast engineered to express BsBKCD (data not shown). Without wishing to be bound to a theory, it is believed that this may be due to yeast lacking the necessary post-translational modification mechanism of the subunit E2 of the BKCD complex.
Alternatively, a 7.6 kb type I polyketide synthase (PKS) LovF from Aspergillus terreus (Ast) (also referred to as ‘Megasynthase LovF’) has been engineered to produce 2 MB-CoA in vivo. Native LovF condenses two units of malonyl-CoA to 2 MB-ACP, i.e. 2 MB covalently attached to the ACP (Acyl Carrier Protein) domain. In order to obtain free 2 MB, a promiscuous DEBS (6-deoxyerythronolide synthase) thioesterase (TE) domain from Saccharopolyspora erythraea (Se) has been fused at the C-terminus of LovF (also referred to as ‘LovF-TE’), to cleave 2 MB acid from the ACP domain. The resulting 2 MB acid can then be converted into 2 MB-CoA by QsCCL, similar to the case of 2 MB exogenous supplementation. An additional phosphopantetheinyl (Ppant) transferase is required for LovF to be functional in a heterologous host. Accordingly, a chromosomal copy of a Ppant candidate from Aspergillus nidulans (named ‘AnNpgA’ according to SEQ ID NO: 237 (encoded by the nucleotide sequence SEQ ID NO: 239) has been integrated into the genome of the parent yeast strain CEN.PK2-1c to generate YL-AnNpgA. A plasmid expressing AstLovF-TE according to SEQ ID NO: 235 (encoded by SEQ ID NO: 236) and QsCCL according to SEQ ID NO: 178 (encoded by SEQ ID NO: 180) has been transfected into YL-AnNpgA to generate YL-PKS. Additionally, AnNpgA and AstLovF-TE have been integrated into the genome of YL-42 (a yeast strain producing QA-C3-GGX-C28-FRX) to generate YL-42-AstLovF-TE, as well as into the genome of YL-45 (a yeast strain producing QA-C3-GGX-C28-FRXX-C18-Araf or QS-21-Xyl, in the presence of 2-MB supplemented exogenously) and YL-46 (a yeast strain producing QA-C3-GGX-C28-FRXA-C18-Araf or QS-21-Api, in the presence of 2-MB supplemented exogenously) to generate YL-50 and YL-51, respectively.
The production of 2 MB-CoA by YL-PKS (engineered with LovF-TE) has been confirmed by LC-MS (
While the peak integration of 2 MB-CoA is lower than that of the 2 MB acid feed-in experiment, the production of QA-C3-GGX-C28-FRX-C9 using NgpA and LovF-TE in YL-42-AstLovF-TE was more comparable with the feed-in experiment in the case of YL-42, approximately 50% (data not shown).
The complete biosynthesis of QS-21-Xyl and QS-21-Api in YL-50 and YL-51, respectively, was observed (
Using more than 30 heterologous enzymes and proteins from different plant and microbial origins (e.g. G. vaccaria, Q. saponaria, A. thaliana, S. vaccaria, Thermothelomyces thermophilus, Aspergillus nidulans, and Aspergillus terreus), the inventors have been able to reconstruct in yeast the metabolic pathway leading to the synthesis of QS-21-Xyl and QS-21-Api (the two main isomeric constituents present in the QS-21 fraction traditionally purified from the bark of Q. saponaria Molina tree) achieving, for the first time, the successful production of QS-21-Xyl and QS-21-Api in yeast.
N. Benthamiana transient expression experiments were carried out as described in WO 2020/260475.
Genes were assembled into pESC plasmids which contain two multiple cloning sites driven by Gal1p and Gal10p individually which are galactose-inducible promoters or under the Tet promoter with the tet repressor gene. Nucleotide sequences were codon-optimized for S. cerevisiae using the IDT online tool. Integration was performed by an in-house-developed CRISPR/Cas9 toolkit10. Integration was confirmed by colony PCR and confirmed strains were glycerol stocked and stored at −80° C.
Production of sugars and QA derivatives was done first by streaking the glycerol stock of the desired yeast strain onto a YPD (yeast extract peptone 2% dextrose) plate and grown for about 20 h at 30° C. to obtain single colonies. Colonies were picked from the plate and cultured for 48 h in 5 mL YPD shaking at 200 rpm at 30° C. The cultures were then spun down and resuspended in equal volume YPGal (yeast extract peptone 2% galactose) media and cultured further at 200 rpm and 30° C., inducing expression of Gall and Gall 0 promoters. Samples were collected at between 48 h and 36 hours post-induction for metabolite extraction. Yeast cell cultures (or cell pellet for the production of sugars) were extracted with 2:2:1 methanol/chloroform/water (2:2:1 v/v/v). Aqueous and organic layers were separated by centrifugation and the aqueous layer was collected. The collected layer was then evaporated in a speed vac at room temperature and resuspended in 0.3% formic acid at pH 9 (adjusted with ammonium acetate).
5.4 LC-MS Detection
LC-MS analysis was carried out using an Agilent HPLC 1260 infinity system attached to an iQ MSD. Detection: MS (ESI ionization, spray voltage Positive 4.5 kV, Negative −3.5 kV, mass range 400-1000, negative ion mode) LC Method: Solvent A: [H2O+0.3% formic acid at pH 9 (pH adjusted with ammonium hydroxide)] Solvent B: [acetonitrile (CH3CN)+0.1% formic acid]. Injection volume: 5 μL. Gradient: 2% to 15% [B] from 0 to 20 min, 15% to
50% [B] from 20 to 26 min, 50% to 90% [B] from 26 to 27 min, 90% [B] from 27 to 30 min, 90% to 2% [B] from 30 to 31 min, 2% [B] from 31 to 50 min. Method was performed using a flow rate of 0.1 mL min-1 with a Porous Graphitic Carbon column (Hypercarb, 5 μm, 1×150 mm Analytical Column) (or as described in WO 22/136563).
Artemisia annua
Arabidopsis thaliana
Glycyrrhiza glabra
Gypsophila vaccaria
Saponaria vaccaria
Quillaja saponaria
Bupleurum falcatum
Quillaja saponaria
Saponaria vaccaria
Quillaja saponaria
Saponaria vaccaria
Saponaria vaccaria
Medicago truncatula
Quillaja saponaria
Saponaria vaccaria
Medicago truncatula
Arabidopsis thaliana
Lotus japonicus
Quillaja saponaria
Arabidopsis thaliana
Saponaria vaccaria
Arabidopsis thaliana
Arabidopsis thaliana
Saponaria vaccaria
Saponaria vaccaria
Quillaja saponaria
Saponaria vaccaria
Quillaja saponaria
Quillaja saponaria
Arabidopsis thaliana
Saponaria vaccaria
Saponaria vaccaria
Quillaja saponaria
Saponaria vaccaria
Saponaria vaccaria
Saponaria vaccaria
Arabidopsis thaliana
Arabidopsis thaliana
Paramoeba atlantica
Quillaja saponaria
Quillaja saponaria
Quillaja saponaria
Quillaja saponaria
Quillaja saponaria
Quillaja saponaria
Quillaja saponaria
Synechococcus sp.
Bacillus cytotoxicus
Corallococcusmacrosporu
Pyrococcus furiosus
Arabidopsis thaliana
Thermothelomycesthermophilu
Quillaja saponaria
Quillaja saponaria
Quillaja saponaria
Quillaja saponaria
Quillaja saponaria
Quillaja saponaria
Quillaja saponaria
Quillaja saponaria
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Hordeum vulgare
Arabidopsis thaliana
Leptospira interrogans
Penicillium rubens Wisconsin
Arabidopsis thaliana
Leptospira interrogans
Quillaja saponaria
Quillaja saponaria
Aspergillus nidulans
Hordeum vulgare
Hordeum vulgare
indicates data missing or illegible when filed
This application is a continuation of PCT/US22/82381, filed Dec. 23, 2022, which claims priority to U.S. Provisional Application No. 63/293,747, filed Dec. 24, 2021, No. 63/293,748; filed Dec. 24, 2021, and No. 63/343,048; filed May 17, 2022, the disclosures of which are hereby incorporated by reference in its entirety for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 63343048 | May 2022 | US | |
| 63293748 | Dec 2021 | US | |
| 63293747 | Dec 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US22/82381 | Dec 2022 | WO |
| Child | 18751380 | US |
