Infiltration of Agrobacterium tumefaciens into Nicotiana benthamiana has become a foundational technique in plant biology, enabling efficient delivery of transgenes in planta with technical ease, robust signal, and relatively high throughput. Despite transient expression's prevalence in disciplines such as synthetic biology, little work has been done to describe and address the variability inherent in this system, a concern for experiments that rely on highly quantitative readouts. In a comprehensive analysis of N. benthamiana agroinfiltration experiments, we model sources of variability that affect transient expression. Our findings emphasize the need to validate normalization methods under the specific conditions of each study, as distinct normalization schemes do not always reduce variation either within or between experiments. Using a dataset of 1915 plants collected over three years, we develop a model of variation in N. benthamiana transient expression, using power analysis to determine the number of individual plants required for a given effect size. Drawing on our longitudinal data, these findings inform practical guidelines for minimizing variability through strategic experimental design and power analysis, providing a foundation for more robust and reproducible use of N. benthamiana in quantitative plant biology and synthetic biology applications.

Product yields for biomanufacturing processes are often constrained by the tight coupling of cellular energy generation and carbon metabolism in sugar-based fermentation systems. To overcome this limitation, we engineered Escherichia coli to utilize hydrogen gas (H2) and formate (HCOO-) as alternative sources of energy and reducing equivalents, thereby decoupling energy generation from carbon metabolism. This approach enabled precise suppression of decarboxylative oxidation during acetate growth, with 86.6 ± 1.6 % of electrons from hydrogen gas (via soluble hydrogenase from Cupriavidus necator H16) and 98.4 ± 3.6 % of electrons from formate (via formate dehydrogenase from Pseudomonas sp. 101) offsetting acetate oxidation. Hydrogen gas supplementation led to a titratable and stoichiometric reduction in CO2 evolution in acetate-fed cultures. Metabolomic analysis suggests that this metabolic decoupling redirects carbon flux through the glyoxylate shunt, partially bypassing two decarboxylative steps in the TCA cycle. We demonstrated the utility of this strategy by applying it to mevalonate biosynthesis, where formate supplementation during glucose fermentation increased titers by 57.6 % in our best-performing strain. Flux balance analysis further estimated that 99.0 ± 2.8 % of electrons from formate were used to enhance mevalonate production. These findings highlight a broadly applicable strategy for enhancing biomanufacturing efficiency by leveraging external reducing power to optimize feedstock and energy use.

Organic acids of primary metabolism are essential for energy metabolism. They can range from formate (C1), glyoxylate (C2), to citrate (C6), or greater. While reversed phase (RP) C18 chromatography may not be the ideal separation technique for these acids, due to their polar nature, the use of mixed-mode polar RP stationary phases can provide retention through hydrogen bonding and other polar interactions. To this end, we have developed a RP-QTOF-MS method (via the Sulpeco Ascentis Express RP-Amide column) capable of separating, detecting, and quantifying organic acids involved in the tricarboxylic acid cycle (TCA).

Beta-ketoadipate (βKA) is an intermediate of the βKA pathway involved in the degradation of aromatic compounds in several bacteria and fungi. Beta-ketoadipate also represents a promising chemical for the manufacturing of performance-advantaged nylons. We established a strategy for the in planta synthesis of βKA via manipulation of the shikimate pathway and the expression of bacterial enzymes from the βKA pathway. Using Nicotiana benthamiana as a transient expression system, we demonstrated the efficient conversion of protocatechuate (PCA) to βKA when plastid-targeted bacterial-derived PCA 3,4-dioxygenase (PcaHG) and 3-carboxy-cis,cis-muconate cycloisomerase (PcaB) were co-expressed with 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (AroG) and 3-dehydroshikimate dehydratase (QsuB). This metabolic pathway was reconstituted in Arabidopsis by introducing a construct (pAtβKA) with stacked pcaG, pcaH, and pcaB genes into a PCA-overproducing genetic background that expresses AroG and QsuB (referred as QsuB-2). The resulting QsuB-2 x pAtβKA stable lines displayed βKA titers as high as 0.25 % on a dry weight basis in stems, along with a drastic reduction in lignin content and improvement of biomass saccharification efficiency compared to wild-type controls, and without any significant reduction in biomass yields. Using biomass sorghum as a potential crop for large-scale βKA production, techno-economic analysis indicated that βKA accumulated at titers of 0.25 % and 4 % on a dry weight basis could be competitively priced in the range of $2.04-34.49/kg and $0.47-2.12/kg, respectively, depending on the selling price of the residual biomass recovered after βKA extraction. This study lays the foundation for a more environmentally-friendly synthesis of βKA using plants as production hosts.

Microbial bioproduction using one-carbon (C1) feedstocks has the potential to decarbonize the manufacturing of materials, fuels, and chemicals. Formate is a promising C1 feedstock, and the realization of industrial, formatotrophic platform organisms is a key goal for C1-based bioproduction. So far, a major limitation for synthetic formatotrophy has been slow energy supply due to slow formate dehydrogenase activity. Here, we implement a fast, metal-dependent formate dehydrogenase complex in a synthetic formatotrophic Escherichia coli utilizing the reductive glycine pathway. After a short-term evolution, we demonstrate formatotrophic growth of E. coli with a doubling time of less than 4.5 h, comparable to the fastest natural formatotrophs. To further explore the potential of a formate-based bioeconomy, this strain is engineered to produce mevalonate, as well as the terpenoid and aviation fuel precursor isoprenol, using formate we generate directly from the electrochemical reduction of CO2. This work demonstrates an improvement in bioproduct titer from formate, achieving the production of 3.8 g/L of mevalonate. Additionally, the abundant and recalcitrant polymer lignin is chemically decomposed into a formate-rich mixture of small organic acids and subsequently bioconverted into mevalonate. Overall, the described fast-growing, formatotrophic bioproduction strain demonstrates that a sustainable formate bioeconomy is within reach.

The oleaginous yeast Rhodosporidiumtoruloides has been exploited for many bioproducts, including several terpenes, owing to its oleaginous nature and biomass inhibitor tolerance. Here, we built upon previous (E)-α-bisabolene work by iteratively stacking the complete mevalonate pathway from Saccharomyces cerevisiae onto a multicopy bisabolene synthase parent strain. Metabolomics and proteomics verified heterologous pathway expression and identified metabolic bottlenecks at three intermediate steps, with candidate feedback-resistant mevalonate kinases screening improving titers 15%. Subtle differences in codon optimization, and preliminary attenuation of competing flux toward lipids resulted in 6-fold, 7-fold higher titers relative to controls, respectively. Media optimization led to modest improvements, with zinc identified as the most promising at 10% titer improvement. Ultimately, high-performance strains were cultivated with corn-stover biomass hydrolysate in microtiter plates at 300 g/L total sugar, achieving 20.8 g/L bisabolene, the highest reported titer in the literature. A 2 L glucose minimal medium bioreactor achieved 19.3 g/L bisabolene and a literature-high productivity of 0.11 g/L/h.

Engineered polyketide synthases (PKSs) have great potential as biocatalysts. These unnatural enzymes are capable of synthesizing molecules that are either not amenable to biosynthesis or are extremely challenging to access chemically. PKSs can thus be a powerful platform to expand the chemical landscape beyond the limits of conventional metabolic engineering. Here we employ a retrobiosynthesis approach to design and construct PKSs to produce δ-valerolactam (VL) and three enantiopure α-substituted VL analogues that have no known biosynthetic route. We introduce the engineered PKSs and pathways for various malonyl-CoA derivatives into Pseudomonas putida and use proteomics, metabolomics and culture condition optimization to improve the production of our target compounds. These α-substituted VLs are polymerized into polyamides (nylon-5) or converted into their N-acryloyl derivatives. RAFT polymerization produces bio-derived polymers with potential biomedical applications. Overall, this interdisciplinary effort highlights the versatility and effectiveness of a PKS-based retrobiosynthesis approach in exploring and developing innovative biomaterials. Engineered polyketide synthases (PKSs) have great potential as biocatalysts for the synthesis of chemically challenging molecules. Here the authors show a retrobiosynthesis approach to design and construct PKSs to produce a series of valerolactams for biopolymer production.

Genome-scale metabolic models (GSMM) are commonly used to identify gene deletion sets that result in growth coupling and pairing product formation with substrate utilization and can improve strain performance beyond levels typically accessible using traditional strain engineering approaches. However, sustainable feedstocks pose a challenge due to incomplete high-resolution metabolic data for non-canonical carbon sources required to curate GSMM and identify implementable designs. Here we address a four-gene deletion design in the Pseudomonas putida KT2440 strain for the lignin-derived non-sugar carbon source, p-coumarate (p-CA), that proved challenging to implement. We examine the performance of the fully implemented design for p-coumarate to glutamine, a useful biomanufacturing intermediate. In this study glutamine is then converted to indigoidine, an alternative sustainable pigment and a model heterologous product that is commonly used to colorimetrically quantify glutamine concentration. Through proteomics, promoter-variation, and growth characterization of a fully implemented gene deletion design, we provide evidence that aromatic catabolism in the completed design is rate-limited by fumarase hydratase (FUM) enzyme activity in the citrate cycle and requires careful optimization of another fumarate hydratase protein (PP_0897) expression to achieve growth and production. A double sensitivity analysis also confirmed a strict requirement for fumarate hydratase activity in the strain where all genes in the growth coupling design have been implemented. Metabolic cross-feeding experiments were used to examine the impact of complete removal of the fumarase hydratase reaction and revealed an unanticipated nutrient requirement, suggesting additional functions for this enzyme. While a complete implementation of the design was achieved, this study highlights the challenge of completely inactivating metabolic reactions encoded by under-characterized proteins, especially in the context of multi-gene edits.

Medium- and branched-chain diols and amino alcohols are important industrial solvents, polymer building blocks, cosmetics and pharmaceutical ingredients, yet biosynthetically challenging to produce. Here we present an approach that uses a modular polyketide synthase (PKS) platform for the efficient production of these compounds. This platform takes advantage of a versatile loading module from the rimocidin PKS and nicotinamide adenine dinucleotide phosphate-dependent terminal thioreductases. Reduction of the terminal aldehyde with alcohol dehydrogenases enables the production of diols, oxidation enables the production of hydroxy acids and specific transaminases allow the production of various amino alcohols. Furthermore, replacement of the malonyl-coenzyme A-specific acyltransferase in the extension module with methyl- or ethylmalonyl-coenzyme A-specific acyltransferase enables the production of branched-chain diols, amino alcohols and carboxylic acids in high titres. Use of our PKS platform in Streptomyces albus demonstrated the high tunability and efficiency of the platform. Medium- and branched-chain diols and amino alcohols are important industrial feedstocks, but they are biosynthetically challenging to produce. Here the authors introduce a modular polyketide synthase platform for the efficient production of these compounds.

Isoprenoids are a diverse class of natural products that are essential in all domains of life. Most bacteria synthesize isoprenoids through either the methylerythritol phosphate (MEP) pathway or the mevalonate (MEV) pathway, while a small subset encodes both pathways, including the pathogen Mycobacterium marinum (Mm). It is unclear whether the MEV pathway is functional in Mm, or why Mm encodes seemingly redundant metabolic pathways. Here, we show that the MEP pathway is essential in Mm, while the MEV pathway is dispensable in culture, with the ΔMEV mutant having no growth defect in axenic culture but a competitive growth defect compared to WT Mm. We found that the MEV pathway does not play a role in ex vivo or in vivo acute infection but does play a role in survival of peroxide stress. Metabolite profiling revealed that modulation of the MEV pathway causes compensatory changes in the concentration of MEP intermediates DOXP and CDP-ME, suggesting that the MEV pathway is functional and that the pathways interact at the metabolic level. Finally, the MEV pathway is upregulated early in the shift down to hypoxia, suggesting that it may provide metabolic flexibility to this bacterium. Interestingly, we found that our complemented strains, which vary in copy number of the polyprenyl synthetase idsB2, responded differently to peroxide and UV stresses, suggesting a role for this gene as a determinant of downstream prenyl phosphate metabolism. Together, these findings suggest that MEV may serve as an anaplerotic pathway to make isoprenoids under stress conditions.IMPORTANCEOrganisms from all domains of life utilize isoprenoids to carry out thousands of critical and auxiliary cellular processes, including signaling, maintaining membrane integrity, stress response, and host-pathogen interactions. The common precursor of all isoprenoids is synthesized via one of two biosynthetic pathways. Importantly, some bacteria encode both pathways, including M. marinum. We found that only one pathway is essential in M. marinum, while the nonessential pathway may confer metabolic flexibility to help the bacterium better adapt to various environmental conditions. We also found that the polyprenyl synthetase IdsB2 plays an important role in driving such phenotypes. Further, we demonstrate metabolic interplay between both functional pathways. These insights represent the first characterization of isoprenoid biosynthesis in dual pathway-encoding mycobacteria.

Synthetic biology tools have accelerated the generation of simple mutants, but combinatorial testing remains a major hurdle. High-throughput methods struggle translating from proof-of-principle molecules to advanced bioproducts. We address this challenge with a biosensor-driven strategy for enhanced isoprenol production in Pseudomonas putida , a key precursor for sustainable aviation fuel and platform chemicals. This biosensor leverages P. putida's native response to short-chain alcohols via a previously uncharacterized hybrid histidine kinase signaling cascade. Refactoring the biosensor for a conditional growth-based selection enabled identification of competing cellular processes with a ~16,500-member CRISPRi-library. An iterative combinatorial strain engineering approach yielded an integrated P. putida strain producing ~900 mg/L isoprenol in glucose minimal medium, a 36-fold increase. Ensemble -omics analysis revealed metabolic rewiring, including amino acid accumulation as key drivers of enhanced production. Techno-economic analysis elucidated the path to economic viability and confirmed the benefits of adding amino acids outweigh the additional costs. This study establishes a robust biosensor driven approach for optimizing other heterologous pathways, accelerating microbial cell factory development. ### Competing Interest Statement TE, JM, and AM are inventors on a patent application related to the workflows described in this report (LBNL ​​Docket 2024-064-01; US Patent Application No. 63/761,819). NRB has a financial interest in Erg Bio. CDS has a financial interest in Cyklos Materials.

Product yields for biomanufacturing processes are often constrained by the tight coupling of cellular energy generation and carbon metabolism in sugar-based fermentation systems. To overcome this limitation, we engineered Escherichia coli to utilize hydrogen gas (H2) and formate (HCOO−) as alternative sources of energy and reducing equivalents, thereby decoupling energy generation from carbon metabolism. This approach enabled precise suppression of decarboxylative oxidation during acetate growth, with 86.6 ± 1.6% of electrons from hydrogen gas (via soluble hydrogenase from Cupriavidus necator H16) and 98.4 ± 3.6% of electrons from formate (via formate dehydrogenase from Pseudomonas sp. 101) offsetting acetate oxidation. Hydrogen gas supplementation led to a titratable and stoichiometric reduction in CO2 evolution in acetate-fed cultures. Metabolomic analysis suggests that this metabolic decoupling redirects carbon flux through the glyoxylate shunt, partially bypassing two decarboxylative steps in the TCA cycle. We demonstrated the utility of this strategy by applying it to mevalonate biosynthesis, where formate supplementation during glucose fermentation increased titers by 57.6% in our best-performing strain. Flux balance analysis further estimated that 99.0 ± 2.8% of electrons from formate were used to enhance mevalonate production. These findings highlight a broadly applicable strategy for enhancing biomanufacturing efficiency by leveraging external reducing power to optimize carbon and energy use. ![Figure][1] Created in BioRender. Panich, J. (2025) ### Competing Interest Statement RLB declares a financial interest in Redoxify. JBR declares a financial interest in AlkaLi Labs. JDK declares a financial interest in Ansa Biotechnologies, Apertor Pharma, Berkeley Yeast, BioMia, Cyklos Materials, Demetrix, Lygos, Napigen, ResVita Bio, and Zero Acre Farms. All other authors declare no competing interests. Advanced Research Projects Agency-Energy, https://ror.org/03q1rgc19, DE-AR0002387-1517 Office of Biological and Environmental Research, https://ror.org/0114b2m14, DEAC02-05CH11231 Natural Sciences and Engineering Research Council of Canada (NSERC), 532495-2019 National Defense Science and Engineering Graduate Fellowship, FA9550-21-F-0003 [1]: pending:yes

Agrobacterium pathogenesis, which involves transferring T-DNA into plant cells, is the cornerstone of plant genetic engineering. As the applications that rely on Agrobacterium increase in sophistication, it becomes critical to achieve a quantitative and predictive understanding of T-DNA expression at the level of single plant cells. Here we examine if a classic Poisson model of interactions between pathogens and host cells holds true for Agrobacterium infecting Nicotiana benthamiana. Systematically challenging this model revealed antagonistic and synergistic density-dependent interactions between bacteria that do not require quorum sensing. Using various approaches, we studied the molecular basis of these interactions. To overcome the engineering constraints imposed by antagonism, we created a dual binary vector system termed 'BiBi', which can improve the efficiency of a reconstituted complex metabolic pathway in a predictive fashion. Our findings illustrate how combining theoretical models with quantitative experiments can reveal new principles of bacterial pathogenesis, impacting both fundamental and applied plant biology.

Biological deconstruction of lignocellulose for sustainable chemical production offers an opportunity to harness evolutionarily specialized enzymes and organisms for industrial bioprocessing. While hydrolysis of cellulose and hemicellulose by CAZymes yields fermentable sugars, ligninolysis releases a heterogeneous mix of aromatic compounds that likely play a crucial role in shaping microbial communities and microbial metabolism. Here, we interrogated the metabolomic and transcriptomic response of a lignocellulolytic anaerobic fungus, Neocallimastix californiae, to a heterogeneous mixture of aromatic compounds derived from lignin. Through exposing the fungus to both a concentration it might experience in its native environment and an elevated concentration of alkaline lignin, we observe that N. californiae transforms vanillin and that supplying alkaline lignin at 0.125 g/L, alongside cellulose, enhances the growth and polysaccharide-degrading activity of N. californiae. Altogether, our results further suggest that vanillin consumption, increased polymer-degrading activity, increased metabolic activity, and transcriptomic remodeling of amino acid synthesis genes all coincide with increased melanin production by fungal cells. These observations challenge previous notions that aromatics from lignocellulose only inhibit the growth and polymer deconstruction capabilities of the biomass-degrading anaerobic fungi (Neocallimastigomycetes). This study demonstrates that anaerobic fungi have a complex relationship with aromatic chemicals derived from lignin and hemicellulose and shift their metabolism in response to the addition of lignocellulose-derived aromatics to their growth medium. Further, as no known pathways for the biochemical transformation of aromatics were detected in these organisms despite observed transcriptome remodeling in the presence of aromatics, we suggest they might encode novel biochemical routes for scavenging amino acid building blocks from aromatic monomers derived from hemicellulose side chains and lignin.

CRISPR interference (CRISPRi) has emerged as a valuable tool for redirecting metabolic flux to enhance bioproduction. However, conventional approaches for identifying target genes for CRISPRi-mediated downregulation have largely relied on heuristic methods and trial and error, which are labor-intensive and time-consuming. Additionally, the ability to achieve multigene knockdowns is limited by the constraints of molecular cloning techniques required for building multiplexed CRISPRi systems. In this study, we describe two novel methodologies FluxRETAP (Flux-REaction TArget Prioritization, a Genome-Scale Modeling Technique) and VAMMPIRE (a Versatile Assembly Method for MultiPlexing CRISPRi-mediated downREgulation). FluxRETAP accurately identified gene targets whose knockdown led to substantial increase of isoprenol titers, outperforming traditional heuristic selection. The use of VAMMPIRE enabled accurate and position-independent assembly of CRISPRi constructs containing up to five sgRNA arrays. The integration of FluxRETAP and VAMMPIRE has the potential to advance metabolic engineering by rapidly identifying CRISPRi-mediated knockdowns and knockdown combinations that enhance bioproduction titers, with potential applicability to other microbial systems. ### Competing Interest Statement The authors have declared no competing interest. United States Department of Energy, https://ror.org/01bj3aw27, DE-AC0205CH11231

Butylamine is an effective in-and-out pretreatment solvent for hybrid poplar. It penetrates the cell walls and breaks ester cross-linkages, facilitating lignin release and improving enzymatic digestion.

Fungal solid-state fermentation (SSF) of byproducts has promise for increasing food sustainability and security, but fungal waste-to-food upcycling remains poorly understood at the molecular level. Here we use a multi-omics approach to characterize oncom – a fermented food traditionally produced from byproducts in Java, Indonesia – as a model system for understanding fungal waste conversion. Metagenomic sequencing of two oncom types (red and black) indicated that Neurospora intermedia is the fungus dominating red oncom. Further transcriptomic, metabolomic, and phylogenomic analysis revealed that oncom-derived N. intermedia utilizes pectin and cellulose degradation for substrate conversion and belongs to a distinct byproduct-associated subpopulation that differs from wild strains at the genetic and biochemical level. Finally, we found that N. intermedia grew on a range of industrially relevant byproducts, did not encode for any known mycotoxins, and could be used to create foods that were positively perceived by consumers outside Indonesia. This study uncovers the microbial and genetic basis of a traditional upcycled food, sheds light on human domestication of microbes for sustainability challenges, and establishes the edible N. intermedia as a promising fungus for byproduct upcycling in SSF and beyond. ### Competing Interest Statement JDK has financial interests in Amyris, Ansa Biotechnologies, Apertor Pharma, Berkeley Yeast, Cyklos Materials, Demetrix, Lygos, Napigen, ResVita Bio, and Zero Acre Farms. The other authors declare no competing interests.

Genome scale metabolic models (GSMM) are commonly used to identify gene deletion sets that result in growth coupling, pairing product formation with substrate utilization. While such approaches can improve strain performance beyond levels typically accessible using targeted strain engineering approaches, sustainable feedstocks often pose a challenge for GSMM-based methods due to incomplete underlying metabolic data. Specifically, we address a four-gene deletion design for the lignin-derived non-sugar carbon source, para -coumarate, that proved challenging to implement. We examine the performance of the fully implemented design for p- coumarate to glutamine, a useful biomanufacturing intermediate. In this study glutamine is then converted to indigoidine, an alternative sustainable pigment and a model heterologous product. Through omics, promoter-variation and growth characterization of a fully implemented gene deletion design, we provide evidence that aromatic catabolism in the completed design is rate-limited by fumarate hydratase activity in the citrate cycle and required careful optimization of the final fumarate hydratase protein (PP_0897) expression to achieve growth and production. A metabolic cross-feeding experiment with the completed design strain also revealed an unanticipated nutrient requirement suggesting additional functions for the fumarate hydratase protein. A double sensitivity analysis confirmed a strict requirement for fumarate hydratase activity in the strain where all genes in the growth coupling design have been implemented. While a complete implementation of the design was achieved, this study highlights the challenge of precisely inactivating metabolic reactions encoded by under-characterized proteins especially in the context of multi-gene edits. ### Competing Interest Statement The authors have declared no competing interest.

Fungal fermentation of food and agricultural by-products holds promise for improving food sustainability and security. However, the molecular basis of fungal waste-to-food upcycling remains poorly understood. Here we use a multi-omics approach to characterize oncom, a fermented food traditionally produced from soymilk by-products in Java, Indonesia. Metagenomic sequencing of samples from small-scale producers in Western Java indicated that the fungus Neurospora intermedia dominates oncom. Further transcriptomic, metabolomic and phylogenomic analysis revealed that oncom-derived N. intermedia utilizes pectin and cellulose degradation during fermentation and belongs to a genetically distinct subpopulation associated with human-generated by-products. Finally, we found that N. intermedia grew on diverse by-products such as fruit and vegetable pomace and plant-based milk waste, did not encode mycotoxins, and could create foods that were positively perceived by consumers outside Indonesia. These results showcase the traditional significance and future potential of fungal fermentation for creating delicious and nutritious foods from readily available by-products.

QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans1,2. 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 synthesis3,4. 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.