Combinatorial biosynthesis of sapogenins and saponins in Saccharomyces cerevisiae using a C-16α hydroxylase from Bupleurum falcatum

The saikosaponins comprise oleanane- and ursane-type triterpene saponins that are abundantly present in the roots of the genus Bupleurum widely used in Asian traditional medicine. Here we identified a gene, designated CYP716Y1, encoding a cytochrome P450 monooxygenase from Bupleurum falcatum that catalyzes the C-16α hydroxylation of oleanane- and ursane-type triterpenes. Exploiting this hitherto unavailable enzymatic activity, we launched a combinatorial synthetic biology program in which we combined CYP716Y1 with oxidosqualene cyclase, P450, and glycosyltransferase genes available from other plant species and reconstituted the synthesis of monoglycosylated saponins in yeast. Additionally, we established a culturing strategy in which applying methylated β-cyclodextrin to the culture medium allows the sequestration of heterologous nonvolatile hydrophobic terpenes, such as triterpene sapogenins, from engineered yeast cells into the growth medium, thereby greatly enhancing productivity. Together, our findings provide a sound base for the development of a synthetic biology platform for the production of bioactive triterpene sapo(ge)nins.Triterpene saponins are secondary metabolites that exhibit a large structural diversity and wide range of biological activities in many plant species (1, 2). Saponins are glycosides of sapogenins, which are composed of 30 carbon atoms arranged in 4- or 5-ring structures that are “decorated” by functional groups. Saponins are synthesized by multiple glycosylations of the sapogenin building blocks that are produced by multiple cytochrome P450-dependent monooxygenase (P450) or oxidoreductase-mediated modifications of basic backbones, such as β-amyrin (oleanane type), α-amyrin (ursane type), lupeol, and dammarenediol. These backbones are generated by oxidosqualene cyclase (OSC)-mediated cyclization of 2,3-oxidosqualene, which is also an intermediate in the synthesis of sterols in eukaryotes (3, 4). Both saponins and sapogenins include biologically active compounds or serve as starter molecules for the generation of novel, potentially bioactive structures by synthetic modification (5–7).The genus Bupleurum consists of perennial herbs that are used in Asian traditional medicine, either alone or in combination with other ingredients, for the treatment of common colds, fever, and inflammatory disorders (8). Saikosaponins constitute the largest class of secondary metabolites in Bupleurum and can account for up to ∼7% of root dry weight. Their accumulation can be further stimulated by jasmonate treatment (9). More than 120 closely related oleanane- and ursane-type saikosaponins have been identified from this genus and the oxidations at various positions suggest the presence of multiple enzymes, mainly P450s, capable of catalyzing specific modifications on the amyrin backbones (8, 10). To date, no P450 or oxidoreductase involved in triterpene saponin biosynthesis has been identified from Bupleurum species.P450s that modify the β-amyrin backbone on C-11; C-12,13; C-16; C-22; C-23; C-28 or C-30 have been characterized from Glycyrrhiza uralensis, Avena strigosa, Medicago truncatula, Glycine max, Vitis vinifera, and Catharanthus roseus (11–18). Hydroxylases from Panax ginseng that oxidize the dammarenediol-II backbone on C-6, C-12, or C-28 (19–21), and a C-20 hydroxylase from Lotus japonicus (22) that modifies lupeol, have also been identified. To characterize these P450s, they have been ectopically expressed in yeast strains either producing β-amyrin or externally supplied with candidate substrates. Similarly, several OSCs have been produced and functionally analyzed in yeast. From these studies, it is clear that yeast cells cannot only be used for the characterization of novel enzymes, but possibly also as a heterologous host for the production of triterpene sapogenins (23). To date only two pilot studies have aimed at engineering of β-amyrin production in yeast (24, 25), but no efforts toward engineering of sustainable production of sapogenins or saponins in yeast have been reported.Here, we identified and characterized CYP716Y1, a P450 from Bupleurum falcatum that corresponds to a C-16α oxidase, designated according to Nelson’s nomenclature (http://drnelson.uthsc.edu/cytochromeP450.html). By designing triterpene-hyperproducing starter strains, optimizing culturing conditions for triterpene synthesis, and using the CYP716Y1 gene in a combinatorial synthetic biology program, we established a platform that allows us to produce and sequester triterpene sapogenins in culture medium and to reconstitute a full saponin synthetic pathway in yeast cells.     

Innate and adaptive immune responses during acute M. tuberculosis infection in adult household contacts in Kampala, Uganda

Contacts of active pulmonary tuberculosis (TB) patients are at risk for Mycobacterium tuberculosis (MTB) infection. Because most infections are controlled, studies during MTB infection provide insight into protective immunity. We compared immune responses of adult household contacts that did and did not convert the tuberculin skin test (TST). Innate and adaptive immune responses were measured by whole blood assay. Responses of TST converters (TSTC) were compared with persistently TST negative contacts (PTST-) and contacts who were TST+ at baseline (TST+). TLR-2, TLR-4, and IFN-γR responses to IFN-γ did not differ between the groups, nor did γδ T cell responses. T cell responses to MTB antigens differed markedly among TSTC, PTST-, and TST+ contacts. Thus, no differences in innate responses were found among the three household contact groups. However, adaptive T cell responses to MTB antigens did differ before and during MTB infection among PTST-, TSTC, and TST+ contacts.     

Long-term impact of multifactorial treatment on new-onset diabetes and related cardiovascular events in metabolic syndrome: a post hoc ATTEMPT analysis

This post hoc analysis of the Assessing The Treatment Effect in Metabolic Syndrome Without Perceptible diabeTes (ATTEMPT) study assesses the 3? year incidence of new-onset diabetes (NOD) and related cardiovascular disease (CVD) events in patients with metabolic syndrome (MetS), after multifactorial (lifestyle and drug, including atorvastatin) intervention. Patients were randomized to group A (low-density lipoprotein cholesterol [LDL-C] target < 100 mg/dL) and group B (< 130 mg/dL). The incidence of NOD during the 42-month follow-up was very low, 0.83 to 1.00/100 patient-years in patients with MetS and MetS with impaired fasting glucose, respectively. Older age, increased waist circumference, and persistent MetS were determinants of NOD. One CVD nonfatal event occurred in the 28 patients with NOD. Our findings suggest that treating the characteristics of MetS is achievable and beneficial. New-onset diabetes incidence and CVD events were negligible and not different from what is expected in the general population.