Researchers from University of California, Berkeley, shared research in Nature that demonstrates the complete biosynthesis of QS-21 in engineered yeast strains. QS-21, the only saponin-based adjuvant that has been clinically approved for use in humans, has “limited” availability. Through their work, the team hope to “enable the rational design of potent vaccine adjuvants”.
QS-21
The authors suggest that alum has been the “most widely used, clinically approved” vaccine adjuvant since its discovery in the 1920s. However, QS-21 has also been shown to “exhibit potent immunoactivity”. It has been used in GSK’s Mosquirix and Shingrix as well as Novavax’s COVID-19 vaccines and has been tested in “more than 120 clinical trials”.
“Despite major commercial interest, the availability of QS-21 remains limited, owing mainly to its structural complexity.”
It comprises four distinct structural domains:
- A lipophilic triterpenoid core, quillaic acid
- A branched trisaccharide moiety on the C3 position
- A linear tetrasaccharide chain on the C28 position
- An unusual pseudodimeric acyl chain capped by an arabinofuranose
QS-21 is “traditionally” extracted from the tree bark of the soapbark tree, Quillaja saponaria, native to Chile. Isolation is “complicated” due to the “multitude” of different strcuturally related Quillaja saponins; the purification process is “highly laborious and low yielding”.
“Developing alternative production processes that are more sustainable and scalable would help to meet the ever-increasing demand for potent vaccine adjuvants, and to address existing or emerging medical needs.”
In the paper, the researchers present the complete biosynthesis of QS-21-Api and QS-21-Xyl, alongside structural derivatives in Saccharomyces cerevisiae, from simple sugars. This was achieved by upregulating the yeast native mevalonate pathway to provide a high carbon flux towards 2,3-oxidosqualene, which was then cyclised by heterologous β-amyrin synthase and site-selectively oxidised by plant cytochrome P450s to yield the aglycone of QS-21, QA.
The introduction of plant nucleotide sugar synthetic pathways made seven non-native uridine diphosphate sugars (UDP-sugars), which are used to add sugars onto the C3 hydroxy and C28 carboxy functional groups of QA through co-expression of QS-21 pathway glycosyltransferases (GTs). An engineered type I polyketide synthase (PKS), two type III PKSs, and two stand-alone ketoreductases (KRs) were expressed in yeast to create the dimeric acyl unit that “constitutes the last step” before the terminal arabinofuranose addition to yield QS-21. In the engineered yeast, pathway enzymes and their functional homologues were expressed.
The authors state that this combinatorial approach enabled selection of activities that function optimally together in a yeast cell, which enabled the production of QS-21.
The result
The final strain contains 38 heterologous enzymes from six species across several enzyme families, and to achieve the complete biosynthesis of QS-21 the team mimicked the subcellular compartmentalisation of plants from the ER membrane to the cytosol.
“QS-21-Xyl and QS-21-Api – two isomers of QS-21 with high structural similarity – can therefore be produced in separate yeast strains, and this enables them to be purified, and their immunogenicity to be characterised, in an independent manner.”
The authors argue that the platform provides “vast opportunities” to produce structural variants of QS-21. Furthermore, as the traditional method of extraction and purification destroys the bark of the tree, it has provoked “increased governmental regulations around its deforestation”. Thus, the method “highlights the possibility of replacing the plantation-based supply of saponins” with “industrial fermentation at scale”.
Addressing a need
Professor of chemical and biomolecular engineering, Jay Keasling, commented that during the pandemic, public health experts were “really worried” about the availability of QS-21 “because that only comes from one tree”.
“From a world health perspective, there’s a lot of need for an alternative source of this adjuvant.”
He is “gratified” that synthetic biology has “come so far” that “we can now build a pathway to produce a molecule like QS-21″.
“It’s a testament to how far the field has progress in the last two decades.”
Postdoctoral fellow Yuzhong Liu, first author, shared that this research “highlights the power of synthetic biology to address both major environmental, as well as human, health challenges”.
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