Approaches using metabolic engineering and synthetic biology to overproduce terpenoids, such as the precursors of taxol and artemisinin, in microbial systems have achieved initial success. However, due to the lack of steady-state kinetic information and incomplete understanding of the terpenoid biosynthetic pathway, it has been difficult to build a highly efficient, universal system. Here, we reconstituted the mevalonate pathway to produce farnesene (a precursor of new jet fuel) in vitro using purified protein components. The information from this in vitro reconstituted system guided us to rationally optimize farnesene production in E. coli by quantitatively overexpressing each component. Targeted proteomic assays and intermediate assays were used to determine the metabolic status of each mutant. Through targeted engineering, farnesene production could be increased predictably step by step, up to 1.1 g/L (∼ 2,000 fold) 96 h after induction at the shake-flask scale. The strategy developed to release the potential of the mevalonate pathway for terpenoid overproduction should also work in other multistep synthetic pathways.

Subunit vaccines generally proceed through a 4-step in vivo cascade—the DUMP cascade—to generate potent cell-mediated immune responses: (1) drainage to lymph nodes; (2) uptake by dendritic cells (DCs); (3) maturation of DCs; and (4) Presentation of peptide-MHC I complexes to CD8+ T cells. How the physical properties of vaccine carriers such as mesoporous silica nanoparticles (MSNs) influence this cascade is unclear. We fabricated 80-nm MSNs with different pore sizes (7.8 nm, 10.3 nm, and 12.9 nm) and loaded them with ovalbumin antigen. Results demonstrated these MSNs with different pore sizes were equally effective in the first three steps of the DUMP cascade, but those with larger pores showed higher cross-presentation efficiency (step 4). Consistently, large-pore MSNs loaded with B16F10 tumor antigens yielded the strongest antitumor effects. These results demonstrate the promise of our lymph node-targeting large-pore MSNs as vaccine-delivery vehicles for immune activation and cancer vaccination.

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BackgroundAlteration in gene expression resulting from allopolyploidization is a prominent feature in plants, but its spectrum and extent are not fully known. Common wheat (Triticum aestivum) was formed via allohexaploidization about 10,000 years ago, and became the most important crop plant. To gain further insights into the genome-wide transcriptional dynamics associated with the onset of common wheat formation, we conducted microarray-based genome-wide gene expression analysis on two newly synthesized allohexaploid wheat lines with chromosomal stability and a genome constitution analogous to that of the present-day common wheat.ResultsMulti-color GISH (genomic in situ hybridization) was used to identify individual plants from two nascent allohexaploid wheat lines between Triticum turgidum (2n = 4x = 28; genome BBAA) and Aegilops tauschii (2n = 2x = 14; genome DD), which had a stable chromosomal constitution analogous to that of common wheat (2n = 6x = 42; genome BBAADD). Genome-wide analysis of gene expression was performed for these allohexaploid lines along with their parental plants from T. turgidum and Ae. tauschii, using the Affymetrix Gene Chip Wheat Genome-Array. Comparison with the parental plants coupled with inclusion of empirical mid-parent values (MPVs) revealed that whereas the great majority of genes showed the expected parental additivity, two major patterns of alteration in gene expression in the allohexaploid lines were identified: parental dominance expression and non-additive expression. Genes involved in each of the two altered expression patterns could be classified into three distinct groups, stochastic, heritable and persistent, based on their transgenerational heritability and inter-line conservation. Strikingly, whereas both altered patterns of gene expression showed a propensity of inheritance, identity of the involved genes was highly stochastic, consistent with the involvement of diverse Gene Ontology (GO) terms. Nonetheless, those genes showing non-additive expression exhibited a significant enrichment for vesicle-function.ConclusionsOur results show that two patterns of global alteration in gene expression are conditioned by allohexaploidization in wheat, that is, parental dominance expression and non-additive expression. Both altered patterns of gene expression but not the identity of the genes involved are likely to play functional roles in stabilization and establishment of the newly formed allohexaploid plants, and hence, relevant to speciation and evolution of T. aestivum.

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