Synthetic Biology-derived triterpenes as efficacious immunomodulating adjuvants.

The triterpene oil squalene is an essential component of nanoemulsion vaccine adjuvants. It is most notably in the MF59 adjuvant, a component in some seasonal influenza vaccines, in stockpiled, emulsion-based adjuvanted pandemic influenza vaccines, and with demonstrated efficacy for vaccines to other pandemic viruses, such as SARS-CoV-2. Squalene has historically been harvested from shark liver oil, which is undesirable for a variety of reasons. In this study, we have demonstrated the use of a Synthetic Biology (yeast) production platform to generate squalene and novel triterpene oils, all of which are equally as efficacious as vaccine adjuvants based on physiochemical properties and immunomodulating activities in a mouse model. These Synthetic Biology adjuvants also elicited similar IgG1, IgG2a, and total IgG levels compared to marine and commercial controls when formulated with common quadrivalent influenza antigens. Injection site morphology and serum cytokine levels did not suggest any reactogenic effects of the yeast-derived squalene or novel triterpenes, suggesting their safety in adjuvant formulations. These results support the advantages of yeast produced triterpene oils to include completely controlled growth conditions, just-in-time and scalable production, and the capacity to produce novel triterpenes beyond squalene.

The novel Arabidopsis thaliana svt2 suppressor of the ascorbic acid-deficient mutant vtc1-1 exhibits phenotypic and genotypic instability.

Ascorbic acid is a potent antioxidant that detoxifies reactive oxygen species when plants are exposed to unfavorable environmental conditions. In addition to its antioxidant properties, ascorbic acid and its biosynthetic precursors fulfill a variety of other physiological and molecular functions. A mutation in the ascorbic acid biosynthesis gene VTC1, which encodes GDP-mannose pyrophosphorylase, results in conditional root growth inhibition in the presence of ammonium. To isolate suppressors of vtc1-1, which is in the Arabidopsis Columbia-0 background, seeds of the mutant were subjected to ethyl methanesulfonate mutagenesis. A suppressor mutant of vtc1-1 2, svt2, with wild-type levels of ascorbic acid and root growth similar to the wild type in the presence of ammonium was isolated. Interestingly, svt2 has Arabidopsis Landsberg erecta features, although svt2 is delayed in flowering and has an enlarged morphology. Moreover, the svt2 genotype shares similarities with L er polymorphism markers and sequences, despite the fact that the mutant derived from mutagenesis of Col-0 vtc1-1 seed. We provide evidence that svt2 is not an artifact of the experiment, a contamination of L er seed, or a result of outcrossing of the svt2 mutant with L er pollen. Instead, our results show that svt2 exhibits transgenerational genotypic and phenotypic instability, which is manifested in a fraction of svt2 progeny, producing revertants that have Col-like phenotypic and genotypic characteristics. Some of those Col-like revertants then revert back to svt2-like plants in the subsequent generation. Our findings have important implications for undiscovered phenomena in transmitting genetic information in addition to the Mendelian laws of inheritance. Our results suggest that stress can trigger a genome restoration mechanism that could be advantageous for plants to survive environmental changes for which the ancestral genes were better adapted.

Toward the mechanism of NH(4) (+) sensitivity mediated by Arabidopsis GDP-mannose pyrophosphorylase.

The ascorbic acid (AA)-deficient Arabidopsis thaliana mutant vtc1-1, which is defective in GDP-mannose pyrophosphorylase (GMPase), exhibits conditional hypersensitivity to ammonium (NH(4) (+) ), a phenomenon that is independent of AA deficiency. As GMPase is important for GDP-mannose biosynthesis, a nucleotide sugar necessary for protein N-glycosylation, it has been thought that GDP-mannose deficiency is responsible for the growth defect in vtc1-1 in the presence of NH(4) (+) . Therefore, the motivation for this work was to elucidate the growth and developmental processes that are affected in vtc1-1 in the presence of NH(4) (+) and to determine whether GDP-mannose deficiency generally causes NH(4) (+) sensitivity. Furthermore, as NH(4) (+) may alter cytosolic pH, we investigated the responses of vtc1-1 to pH changes in the presence and absence of NH(4) (+) . Using qRT-PCR and staining procedures, we demonstrate that defective N-glycosylation in vtc1-1 contributes to cell wall, membrane and cell cycle defects, resulting in root growth inhibition in the presence of NH(4) (+) . However, by using mutants acting upstream of vtc1-1 and contributing to GDP-mannose biosynthesis, we show that GDP-mannose deficiency does not generally lead to and is not the primary cause of NH(4) (+) sensitivity. Instead, our data suggest that GMPase responds to pH alterations in the presence of NH(4) (+) .

Alterations in the endogenous ascorbic acid content affect flowering time in Arabidopsis.

Ascorbic acid (AA) protects plants against abiotic stress. Previous studies suggested that this antioxidant is also involved in the control of flowering. To decipher how AA influences flowering time, we studied the four AA-deficient Arabidopsis (Arabidopsis thaliana) mutants vtc1-1, vtc2-1, vtc3-1, and vtc4-1 when grown under short and long days. These mutants flowered and senesced before the wild type irrespective of the photoperiod, a response that cannot simply be attributed to slightly elevated oxidative stress in the mutants. Transcript profiling of various flowering pathway genes revealed a correlation of altered mRNA levels and flowering time. For example, circadian clock and photoperiodic pathway genes were significantly higher in the vtc mutants than in the wild type under both short and long days, a result that is consistent with the early-flowering phenotype of the mutants. In contrast, when the AA content was artificially increased, flowering was delayed, which correlated with lower mRNA levels of circadian clock and photoperiodic pathway genes compared with plants treated with water. Similar observations were made for the autonomous pathway. Genetic analyses demonstrated that various photoperiodic and autonomous pathway mutants are epistatic to the vtc1-1 mutant. In conclusion, our transcript and genetic analyses suggest that AA acts upstream of the photoperiodic and autonomous pathways.