QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans^1,2. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis^3,4. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host’s native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families—a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases—from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure–activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

Phage-encoded endolysins are getting increasing attention because of their potential to serve as alternative antimicrobials to combat antibiotic-resistant bacteria. Here, we report a novel endolysin LysBT1, which is encoded by a prophage of thermophilic Brevibacillus thermoruber WF146 and comprises an N-acetylmuramoyl-L-alanine amidase domain and an S-layer homology (SLH) domain not found in known endolysins. LysBT1 is not only extremely thermostable, retaining more than 60% activity after 1 h incubation at 95°C, but also highly stable over a wide pH range of 4.0–11.0. Moreover, the thermostability of LysBT1 could be enhanced by EDTA or reducing agents. Although none of the seven cysteine residues of LysBT1 participate in disulfide bond formation, six of them, including the catalytic Zn ²⁺ -coordinating Cys156, are involved in stabilizing the enzyme at elevated temperatures. The SLH domain contributes to the thermostability of LysBT1 and mediates cell surface binding of the enzyme to facilitate enzymatic lysis of strain WF146 cells via increasing local enzyme concentration around the substrate. LysBT1 is capable of trimerization, where the SLH domains are predicted to form a three-prong spindle-like trimer similar to that in S-layer proteins. The SLH domain of LysBT1 could bind to cell surfaces of both Gram-positive and Gram-negative bacteria. LysBT1 can lyse not only Gram-positive strain WF146, Geobacillus stearothermophilus , and Bacillus subtilis but also Gram-negative Escherichia coli and Acinetobacter baumannii with the aid of EDTA or citric acid. EDTA also facilitates LysBT1 to lyse Bacillus cereus , probably because EDTA-induced disorganization of the S-layer allows LysBT1 to access and hydrolyze the peptidoglycan. IMPORTANCE: The emergence of antibiotic-resistant bacteria has led to an urgent requirement to develop novel antimicrobials, and endolysins are regarded as ideal alternatives to antibiotics. The thermostability of endolysins plays an important role in the feasibility of enzymatic bacteriolysis. However, reports on thermostable endolysins are limited, and little is known about their stabilization mechanisms. Our results demonstrate that the thermophile-derived prophage endolysin LysBT1 is highly thermostable and functional under polyextreme (multiple forms of stress) conditions, enabling the enzyme to lyse both Gram-positive and Gram-negative bacteria in synergy with outer membrane permeabilizer. Moreover, we found that the unique S-layer homology domain of LysBT1 contributes to the stability, activity, oligomerization, and cell-wall binding ability of the enzyme. This study not only characterizes a novel endolysin but also provides new clues about the stabilization mechanisms of endolysins.

Terpenoids constitute the largest class of natural products with complex structures, essential functions, and versatile applications. Creation of new building blocks beyond the conventional five-carbon (C₅) units, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate, expands significantly the chemical space of terpenoids. Structure-guided engineering of an S-adenosylmethionine-dependent geranyl diphosphate (GPP) C2-methyltransferase from Streptomyces coelicolor yielded variants converting DMAPP to a new C₆ unit, 2-methyl-DMAPP. Mutation of the Gly residue at the position 202 resulted in a smaller substrate-binding pocket to fit DMAPP instead of its native substrate GPP. Replacement of Phe residue at the position 222 with a Tyr residue contributed to DMAPP binding via hydrogen bond. Furthermore, using Escherichia coli as the chassis, we demonstrated that 2-methyl-DMAPP was accepted as a start unit to generate noncanonical trans- and cis-prenyl diphosphates (C_5n+1) and terpenoids. This work provides insights into substrate recognition of prenyl diphosphate methyltransferases, and strategies to diversify terpenoids by expanding the building block portfolio.

Genetically engineered bacteria represent a promising drug delivery tool for disease treatment. The development of new strategies for specific and independent protein regulation is necessary, especially for combination protein drug therapy. Using the well-studied Escherichia coli phage λ as a model system, we applied n on c anonical a mino a cid s (ncAAs) as novel inducers for protein regulation in a bacteria-based delivery system. Screening the permissive sites of the Cro protein revealed that incorporation of AlocK at the K8 site with the Mb PylRS-349F/tRNA ^Pyl system produced a functional Cro-K8AlocK variant. Using an engineered λ lysogen expressing the Mb PylRS-349F/tRNA ^Pyl pair, Cro-8X, and the reporter mNeonGreen, in vitro and in vivo experiments showed that AlocK led to bacterial lysis through prophage activation and the release of mNeonGreen. If mNeonGreen was integrated into the λ prophage genome, λ phages released due to AlocK induction delivered the reporter gene into the recipient E. coli strain, enabling mNeonGreen expression. Furthermore, insertion of pIF at the F14 site with the Af pIFRS/tRNA ^Tyr pair produced a functional Cro-F14pIF variant. Importantly, Af pIFRS/tRNA ^Tyr and Mb PylRS-349F/tRNA ^Pyl pairs were confirmed to be mutually orthogonal. In a mixture of two engineered λ lysogens expressing different aaRS/tRNAs, Cro-ncAAs, and reporter proteins, AlocK and pIF independently induced bacterial lysis and activated the expression of mNeonGreen and mCherry in the recipient E. coli strain. Collectively, the proposed bacteria-based delivery system provides two options for protein delivery and enables independent regulation of multiple proteins with ncAAs, offering a novel approach for in situ protein regulation and combination therapy. IMPORTANCE: The use of genetically engineered bacteria as drug delivery vectors has attracted more and more attention in recent years. A key issue with bacteria-based delivery systems is how to regulate multiple protein drugs. Based on genetic code expansion technology, we developed a new strategy of using ncAAs as small molecular inducers for in situ protein regulation and engineered λ phage lysogen into a bacteria-based delivery system that can function in two delivery modes. Furthermore, this strategy enables independent regulation of multiple proteins by different ncAAs, offering important implications for combination therapy. This approach requires minimal genetic engineering efforts, and similar strategies can be applied to engineer other prophage-bacteria systems or study phage biology. This work expands the therapeutic applications of ncAAs and lysogenic phages.