FROM BREAD TO VACCINE: YEAST’S JOURNEY TOWARD CREATING POTENT VACCINE ADJUVANT
Adjuvants are substances that are added to vaccine formulations in order to increase the immunogenicity of that formulation. They are generally added to vaccine formulations in order to decrease the amount of antigen, to reduce the number of doses required to produce immunity, to induce protective responses more rapidly etc. One of the potent and the only saponin based adjuvant that has been clinically approved for human use is QS-21 derived from Chilean soap bark tree (Quillaja saponaria) which have been an active ingredient in formulations of GSK’s malaria (Mosquirix) and shingles (Shingrix) vaccines, as well as for Novavax’s COVID-19 vaccines.
QS-21 has four distinct structural domains: a lipophilic quillaic acid triterpene core with a branched trisaccharide moiety at C3 and a linear tetrasaccharide chain at C28 position and an unusual pseudo dimeric acyl chain capped by an arabinofuranose. It exists as a mixture of QS-21-Api and QS-21-Xyl in a 65:35 ratio, with structures differing only at C28 terminal sugar. Owing to its complex structure, its availability is limited with complicated isolation from the plant extract and chemical synthesis from an intermediate saponin giving poor yields which is not able to meet the ever growing demand of this potent vaccine adjuvant in the market.
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| Structure of QS-21 with four distinct structural domains |
Recently scientists have introduced the genes coding for enzymes involved in the biosynthesis of QS-21 which have been recently characterized in Quillaja saponaria and their functional homologues from various other plants that produce similar saponins, fungi, bacteria in yeast (Saccharomyces cerevisiae) for developing an alternative, sustainable, scalable production process for this potent adjuvant to meet the demands and to address the emerging medical needs.
The stain used for the biosynthesis of quillaic acid triterpene core was Saccharomyces cerevisiae strain JWy601 which have upregulated mevalonate-based isoprenoid biosynthesis pathway to produce 2,3-oxidosqualene, an intermediate in the biosynthesis of the triterpene cone from a simple sugar like glucose and galactose. The genes coding for enzymes involved in the conversion of acetyl-CoA to farnesyl pyrophosphate (FPP) were controlled by galactose-inducible promoters for controlled overexpression. This was converted to β-Amyrin by β-Amyrin synthases (BASs) from Saponaria Vaccaria which was integrated into yeast strain and found to produce the highest yield of β-Amyrin using GC-MS. Further upregulation of mevalonate pathway genes coding for ERG20, ERG1 produced a yield of 899 mg/l.
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| Conversion of 2,3 oxidosqualene to β-Amyrin by enzyme BAS |
Then expression cassettes containing cytochrome P450s from Q.saponaria and a redox partner, cytochrome P450 reductase (CPR, AtATR1) from Arabidopsis thaliana were integrated into the yeast genome to synthesize the core QA. For one of the steps involved in QA synthesis: C16 oxidation, the cytochrome P450 was localized in the endoplasmic reticulum by fusion of 22-amino-acid transmembrane domain of C28 oxidase to N-terminus of C16 oxidase creating TMGC28-C16 and the efficiency of P450 oxidase was further increased by the introduction of membrane steroid binding protein (MSBP) to act as a scaffold for co-localization of P450. The production was optimized by the introduction of two copies of the P450s, redox partners and MSBP to create a strain YL-15 with a yield of 65.2 mg/l of QA.
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| Structure of QA- Triterpene core |
The synthesis of QA was followed by C3 and C28 glycosylation that makes the non-polar triterpene core hydrophilic which is a requirement for the homologous mixtures with soluble antigen in the vaccine formulations. It involves eight steps involving seven different uridine diphosphate(UDP)-sugars out of which only UDP-D-galactose is native to yeast. So heterologous nucleotide sugar synthases along with their corresponding GTs were introduced into the yeast genome in a stepwise manner to make these seven non-native UDP-sugars. Two Q.saponaria GTs, CSLM1 and CSLM2 were identified that add gluconic acid to QA to form 3-O-{β-d-glucopyranosiduronic acid}-QA among which CSLM2 was chosen for further pathway engineering. This glycosylation step happens in the endoplasmic reticulum. This intermediate then migrates to cytoplasm where second glycosylation happens by the cytosolic enzyme UGT73CU3 (C3-GalT) which galactosylated the intermediate by 1,2 glycosidic bond formation. This is followed by the xylosylation which is induced by a mutation in AtUGD1 to reduce the feedback inhibition by UDP-D-xylose on UGT7CX1 (Xytl).
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| Structure of Triterpene core with trisaccharide unit |
The C28 tetrasaccharide follows an order of D-fucose, L-rhamnose, D-xylose and D-xylose or D-apiose as the terminal sugar. The esterification of D-fucose to C28 carboxylic acid group of QA is facilitated by C28FucT which is followed by addition of rhamnose by UGT91AR1 (C28RhaT) and xylose by C28XylT3. The last glycosylation step involves the equal action of UGT73CY3 (C28XylT4) and UGT373CY2 (C28ApilT4) to add either xylose or apiose. However the expression of UGT73CY3 was induced by galactose-inducible promoters which are switched on by the galactose in the medium.
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| Structure of Triterpene core with trisaccharide and tetrasaccharide unit |
The biosynthesis of the C9 acyl chain requires two consecutive decarboxylative Claisen condensation reactions of malonyl-CoA with (S)-2-methylbutyryl-CoA (2MB-CoA) catalyzed by two type III polyketide synthases, PKS4 and PKS5, with keto intermediate being reduced by two ketoreductases, KR1 and KR2, to form the 3,5-dihydroxy moiety in C9-CoA. The acyl biosynthesis cassette (PKS4, PKS5, KR1, KR2) was introduced into the yeast which is now able to utilize 2-MB acid supplemented in the culture medium to form 2MB-CoA intracellularly and subsequently C9-CoA. This is then attached to intermediate produced after glycosylation to form the acyl chain by acyl transferase ACT2, ACT3.
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| The intermediate with two acyl chains |
For complete biosynthesis of QS-21 without the supplement of 2MB acid, type I PKS protein F (LovF) from the lovastatin biosynthesis pathway from Aspergillus terreus was introduced into yeast to produce 2MB-CoA intracellularly. This converts malonyl-CoA to 2MB covalently attached to acyl carrier protein (ACP) domain. LovF was engineered by truncating ACP and fusing it to promiscuous erythromycin PKS (EryPKS) M6 thioesterase so that methylbutyryl-S-ACP was hydrolysed to release free 2MB.
UDP-glucose epimerase 1 from A.thaliana (AtUGE1), a bifunctional enzyme that epimerizes UDP-Glc and UDP-Gal, UDP-Xyl and UDP-Arap, and reversibly glycosylated polypeptide 1 (AtRGP1), which converts UDP-arabinopyranose (UDP-Arap) to UDP-Araf were introduced in yeast to produce UDP-Araf in vivo. The enzyme UGT73CZ2 (ArafT) was also introduced to transfer UDP-Araf to the C9-CoA to complete the synthesis of acyl chain and the biosynthesis of QS-21 in the yeast. The final strain produces 0.0012% w/w QS-21 per dry weight.
At the end, apart from the upregulation of mevalonate pathway, 38 heterologous enzymes from six species belonging to several enzyme families: a terpene synthase, P450s, nucleotide sugar synthases, GTs and acyl transferases, type I and type III PKSs were introduced to obtain the final yeast strain where the subcellular compartmentalization of plants from ER to cytosol were mimicked. The two isomers of QS-21 were produced in separate yeast strains which enables easy purification and characterization of immunoactivity. This also provides vast opportunities to produce derivatives of QS-21 by expressing alternate enzymes. This arrangement prevents the deforestation caused by traditional extraction from soapbark trees, replacing plantation based production with industrial fermentation at scale which could increase the availability of QS-21 to meet the rising demand in a sustainable manner and pave the way for new opportunities in microbial biomanufacturing.
REFERENCE:
Liu, Y., Zhao, X., Gan, F. et al. Complete biosynthesis of QS-21 in engineered yeast. Nature 629, 937–944 (2024). https://doi.org/10.1038/s41586-024-07345-9
Apostólico Jde S, Lunardelli VA, Coirada FC, Boscardin SB, Rosa DS. Adjuvants: Classification, Modus Operandi, and Licensing. J Immunol Res. 2016;2016:1459394. doi: 10.1155/2016/1459394. Epub 2016 May 4. PMID: 27274998; PMCID: PMC4870346.
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