Graphical abstract

Synthetic biology approach has been frequently applied to produce plant rare bioactive compounds in microbial cell factories by fermentation. However, to reach an ideal manufactural efficiency, it is necessary to optimize the microbial cell factories systemically by boosting sufficient carbon flux to the precursor synthesis and tuning the expression level and efficiency of key bioparts related to the synthetic pathway. We previously developed a yeast cell factory to produce ginsenoside Rh2 from glucose. However, the ginsenoside Rh2 yield was too low for commercialization due to the low supply of the ginsenoside aglycone protopanaxadiol (PPD) and poor performance of the key UDP-glycosyltransferase (UGT) (biopart UGTPg45) in the final step of the biosynthetic pathway. In the present study, we constructed a PPD-producing chassis via modular engineering of the mevalonic acid pathway and optimization of P450 expression levels. The new yeast chassis could produce 529.0 mg/L of PPD in shake flasks and 11.02 g/L in 10 L fed-batch fermentation. Based on this high PPD-producing chassis, we established a series of cell factories to produce ginsenoside Rh2, which we optimized by improving the C3–OH glycosylation efficiency. We increased the copy number of UGTPg45, and engineered its promoter to increase expression levels. In addition, we screened for more efficient and compatible UGT bioparts from other plant species and mutants originating from the direct evolution of UGTPg45. Combining all engineered strategies, we built a yeast cell factory with the greatest ginsenoside Rh2 production reported to date, 179.3 mg/L in shake flasks and 2.25 g/L in 10 L fed-batch fermentation. The results set up a successful example for improving yeast cell factories to produce plant rare natural products, especially the glycosylated ones.

The ability to persist long term in latently infected CD4 T cells represents a characteristic feature of HIV-1 infection and the predominant barrier to efforts aiming at viral eradication and cure. Yet, increasing evidence suggests that only small subsets of CD4 T cells with specific developmental and maturational profiles are able to effectively support HIV-1 long-term persistence. Here, we analyzed how the functional polarization of CD4 T cells shapes and structures the reservoirs of HIV-1-infected cells. We found that CD4 T cells enriched for a Th1/17 polarization had elevated susceptibilities to HIV-1 infection in ex vivo assays, harbored high levels of HIV-1 DNA in persons treated with antiretroviral therapy, and made a disproportionately increased contribution to the viral reservoir relative to their contribution to the CD4 T memory cell pool. Moreover, HIV-1 DNA levels in Th1/17 cells remained stable over many years of antiretroviral therapy, resulting in a progressively increasing contribution of these cells to the viral reservoir, and phylogenetic studies suggested preferential long-term persistence of identical viral sequences during prolonged antiretroviral treatment in this cell compartment. Together, these data suggest that Th1/17 CD4 T cells represent a preferred site for HIV-1 DNA long-term persistence in patients receiving antiretroviral therapy. A lthough antiretroviral therapy (ART) is highly effective in suppressing HIV-1 replication and clinical HIV-1 disease manifestations, small reservoirs of HIV-1-infected cells persist despite treatment and can lead to rapid viral rebound once treatment is stopped (1, 2). Identifying the functional and phenotypic characteristics of cell subsets that harbor replication-competent virus during suppressive antiretroviral therapy is a critical step for developing interventional strategies to target residual viral reservoirs. Most available evidence supports the notion that latently infected CD4 T cells containing chromosomally integrated but transcriptionally silent HIV-1 DNA represent the most dominant cell population responsible for HIV persistence despite treatment (3-5). However, it is increasingly recognized that such cells represent a phenotypically diverse cell population that consists of a variety of different cell subsets with distinct developmental profiles and functional properties (6-10), divergent levels of permissiveness to HIV-1 infection and latency, and, most likely, different susceptibilities to clinical strategies or pharmaceutical agents aiming at reversing viral latency and inducing HIV-1 eradication.From a developmental perspective, memory CD4 T cell evolution can be described as a hierarchical process during which immature, long-lived cells undergo progressive commitment to more differentiated cell types (11). Recent data suggest that the initial, most immature population of memory CD4 T cells consists of a small number of extremely long-lasting cells that phenotypically express a mix of naive and memory cell markers and display ...

The baseline data from GLORIA-AF phase 2 demonstrate that in newly diagnosed nonvalvular atrial fibrillation patients, NOAC have been highly adopted into practice, becoming more frequently prescribed than VKA in Europe and North America. Worldwide, however, a large proportion of patients remain undertreated, particularly in Asia and North America. (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients With Atrial Fibrillation [GLORIA-AF]; NCT01468701).