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5-Methyltetrahydrofolate (5-MTHF) is the sole active form of folate functioning in the human body and is widely used as a nutraceutical. Unlike the pollution from chemical synthesis, microbial synthesis enables green production of 5-MTHF. In this study, Escherichia coli BL21 (DE3) was selected as the host. Initially, by deleting 6-phosphofructokinase 1 and overexpressing glucose-6-phosphate 1-dehydrogenase and 6-phosphogluconate dehydrogenase, the glycolysis pathway flux decreased, while the pentose phosphate pathway flux enhanced. The ratios of NADH/NAD+ and NADPH/NADP+ increased, indicating elevated NAD(P)H supply. This led to more folate being reduced and the successful accumulation of 5-MTHF to 44.57 µg/L. Subsequently, formate dehydrogenases from Candida boidinii and Candida dubliniensis were expressed, which were capable of catalyzing the reaction of sodium formate oxidation for NAD(P)H regeneration. This further increased the NAD(P)H supply, leading to a rise in 5-MTHF production to 247.36 µg/L. Moreover, to maintain the balance between NADH and NADPH, pntAB and sthA, encoding transhydrogenase, were overexpressed. Finally, by overexpressing six key enzymes in the folate to 5-MTHF pathway and employing fed-batch cultivation in a 3 L fermenter, strain Z13 attained a peak 5-MTHF titer of 3009.03 µg/L, the highest level reported in E. coli so far. This research is a significant step toward industrial-scale microbial 5-MTHF production.

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The nuclear factor-κB (NF-κB) family is crucial for regulating immune and inflammatory responses. The activation of the immune cell signaling pathway usually activates NF-κB, causing a protective immune response. NF-κB can also cause excessive inflammatory responses by activating a cascade reaction of pro-inflammatory mediators such as cytokines. In this study, we used an NF-κB luciferase reporter gene system. Out of more than 800 compounds screened, four NF-κB agonists were identified with strong activity at nontoxic concentrations. Subsequently, the adjuvant effect was verified on mouse bone marrow-derived dendritic cells (BMDCs) and macrophages RAW264.7. It was found that fostamatinib (R788) disodium increased the production of IL-6, IL-12p40, and TNF-α, indicating that R788 disodium could induce the maturation of antigen-presenting cells (APCs). In addition, three compounds were screened to significantly inhibit NF-κB at nontoxic doses, including dehydrocostus lactone (DHL)-a known NF-κB inhibitor. The results showed that DHL significantly reduced the release of LPS-induced inflammatory cytokines (including TNF-α, IL-6, and IL-12). Our findings indicate that the NF-κB-based high-throughput screening can be used to discover potential immune adjuvants and anti-inflammatory molecules.

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5-Methyltetrahydrofolate (5-MTHF) is the predominant folate form in human plasma, which has been widely used as a nutraceutical. However, the microbial synthesis of 5-MTHF is currently inefficient, limiting green and sustainable 5-MTHF production. In this study, the Generally Regarded As Safe (GRAS) microorganism Bacillus subtilis was engineered as the 5-MTHF production host. Three precursor supply modules were first optimized by modular engineering for strengthening the supply of guanosine-5-triphosphate (GTP) and p-aminobenzoic acid (pABA). Next, the impact of genome-wide gene expression on 5-MTHF biosynthesis was evaluated using transcriptome analyses, which identified key genes for 5-MTHF production. The effects of potential genes on 5-MTHF synthesis were verified by observing the genes' up-regulated by strong promoter P566 and those down-regulated by inhibition through the clustered regularly interspaced short palindromic repeat interference (CRISPRi). Finally, a key gene for improved 5-MTHF biosynthesis, comGC, was integrated into the genome of modular engineered strain B89 for its overexpression and facilitating efficient 5-MTHF synthesis, reaching 3.41 ± 0.10 mg/L with a productivity of 0.21 mg/L/h, which was the highest level achieved by microbial synthesis. The engineered 5-MTHF-producing B. subtilis developed in this work lays the foundation of further enhancing 5-MTHF production by microbial fermentation, which can be used for isolation and purification of 5-MTHF as food and nutraceutical ingredients.

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Introduction: Tumor vaccines that induce robust humoral and cellular immune responses have attracted tremendous interest for cancer immunotherapy. Despite the tremendous potential of tumor vaccines as an effective approach for cancer treatment and prevention, a major challenge in achieving sustained antitumor immunity is inefficient antigen delivery to secondary lymphoid organs, even with adjuvant aid. Methods: Herein, we present antigen/adjuvant integrated nanocomplexes termed nsGO/PCP/OVA by employing graphene oxide nanosheet (nsGO) as antigen nanocarriers loaded with model antigen ovalbumin (OVA) and adjuvant, Poria cocos polysaccharides (PCP). We evaluated the efficacy of nsGO/PCP/OVA in activating antigen-specific humoral as well as cellular immune responses and consequent tumor prevention and rejection in vivo. Results: The optimally formed nsGO/PCP/OVA was approximately 120-150 nm in diameter with a uniform size distribution. Nanoparticles can be effectively engulfed by dendritic cells (DCs) through receptor-mediated endocytosis, induced the maturation of DCs and improved the delivery efficiency both in vitro and in vivo. The nsGO/PCP/OVA nanoparticles also induced a significant enhancement of OVA antigen-specific Th1 and Th2 immune responses in vivo. In addition, vaccination with nsGO/PCP/OVA not only significantly suppressed tumor growth in prophylactic treatments, but also achieved a therapeutic effect in inhibiting the growth of already-established tumors. Conclusion: Therefore, this potent nanovaccine platform with nanocarrier nsGO and PCP as adjuvants provides a promising strategy for boosting anti-tumor immunity for cancer immunotherapy.