Technology development for natural product biosynthesis in Saccharomyces cerevisiae

The explosion of genomic sequence data and the significant advancements in synthetic biology have led to the development of new technologies for natural products discovery and production. Using powerful genetic tools, the yeast Saccharomyces cerevisiae has been engineered as a production host for natural product pathways from bacterial, fungal, and plant species. With an expanding library of characterized genetic parts, biosynthetic pathways can be refactored for optimized expression in yeast. New engineering strategies have enabled the increased production of valuable secondary metabolites by tuning metabolic pathways. Improvements in high-throughput screening methods have facilitated the rapid identification of variants with improved biosynthetic capabilities. In this review, we focus on the molecular tools and engineering strategies that have recently empowered heterologous natural product biosynthesis

Field Test of GonaCon™ Immunocontraceptive Vaccine in Free-Ranging Female Fallow Deer

Resident populations of two exotic deer species, fallow deer and axis deer, are having adverse impacts on their habitat and on native plant and animal communities at Point Reyes National Seashore (PRNS) in California. These non-native cervids were released intentionally for recreational hunting on the property now known as PRNS during the period 1942-1954. Approximately 860 fallow deer and 250 axis deer now inhabit PRNS. Under an approved non-native deer management plan, fallow and axis deer populations will be removed from PRNS by 2021 via culling of animals by sharpshooting and by treatment of some of the female fallow deer with GonaCon™ Immunocontraceptive Vaccine. During July-August 2007, 69 fallow does were captured, equipped with numbered ear tags and radiotelemetry collars, and injected with GonaCon™ vaccine before being released. Control animals include 10 does that were captured, marked, and given “sham” injections during July-August 2007, and 19 does that were captured and marked (but not injected) during 2005. Reproductive activity, as indicated by lactation and fecal concentrations of progesterone among the GonaCon™-treated and control does, will be monitored and compared for two years, and will be used to determine the efficacy of GonaCon™ as a cervid contraceptive agent. Traditional methods of population control, such as regulated harvest by licensed hunters, often are impractical or illegal in settings such as national and state parks, and the use of firearms may be prohibited in some urban and suburban environments. The development of safe and effective wildlife contraceptives such as GonaCon™ is needed to control locally overabundant populations in situations where traditional management tools cannot be employed

Engineering the biocatalytic selectivity of iridoid production in Saccharomyces cerevisiae

Monoterpene indole alkaloids (MIAs) represent a structurally diverse, medicinally essential class of plant derived natural products. The universal MIA building block strictosidine was recently produced in the yeast Saccharomyces cerevisiae, setting the stage for optimization of microbial production. However, the irreversible reduction of pathway intermediates by yeast enzymes results in a non-recoverable loss of carbon, which has a strong negative impact on metabolic flux. In this study, we identified and engineered the determinants of biocatalytic selectivity which control flux towards the iridoid scaffold from which all MIAs are derived. Development of a bioconversion based production platform enabled analysis of the metabolic flux and interference around two critical steps in generating the iridoid scaffold: oxidation of 8-hydroxygeraniol to the dialdehyde 8-oxogeranial followed by reductive cyclization to form nepetalactol. In vitro reconstitution of previously uncharacterized shunt pathways enabled the identification of two distinct routes to a reduced shunt product including endogenous 'ene'-reduction and non-productive reduction by iridoid synthase when interfaced with endogenous alcohol dehydrogenases. Deletion of five genes involved in α,β-unsaturated carbonyl metabolism resulted in a 5.2-fold increase in biocatalytic selectivity of the desired iridoid over reduced shunt product. We anticipate that our engineering strategies will play an important role in the development of S. cerevisiae for sustainable production of iridoids and MIAs

A pilot study of radiologic measures of abdominal adiposity: weighty contributors to early pancreatic carcinogenesis worth evaluating?

Intra-abdominal fat is a risk factor for pancreatic cancer (PC), but little is known about its contribution to PC precursors known as intraductal papillary mucinous neoplasms (IPMNs). Our goal was to evaluate quantitative radiologic measures of abdominal/visceral obesity as possible diagnostic markers of IPMN severity/pathology. In a cohort of 34 surgically-resected, pathologically-confirmed IPMNs (17 benign; 17 malignant) with preoperative abdominal computed tomography (CT) images, we calculated body mass index (BMI) and four radiologic measures of obesity: total abdominal fat (TAF) area, visceral fat area (VFA), subcutaneous fat area (SFA), and visceral to subcutaneous fat ratio (V/S). Measures were compared between groups using Wilcoxon two-sample exact tests and other metrics. Mean BMI for individuals with malignant IPMNs (28.9 kg/m ) was higher than mean BMI for those with benign IPMNs (25.8 kg/m ) ( =0.045). Mean VFA was higher for patients with malignant IPMNs (199.3 cm ) compared to benign IPMNs (120.4 cm ), =0.092. V/S was significantly higher ( =0.013) for patients with malignant versus benign IPMNs (1.25 . 0.69 cm ), especially among females. The accuracy, sensitivity, specificity, and positive and negative predictive value of V/S in predicting malignant IPMN pathology were 74%, 71%, 76%, 75%, and 72%, respectively. Preliminary findings suggest measures of visceral fat from routine medical images may help predict IPMN pathology, acting as potential noninvasive diagnostic adjuncts for management and targets for intervention that may be more biologically-relevant than BMI. Further investigation of gender-specific associations in larger, prospective IPMN cohorts is warranted to validate and expand upon these observations

Technology development for natural product biosynthesis in Saccharomyces cerevisiae

The explosion of genomic sequence data and the significant advancements in synthetic biology have led to the development of new technologies for natural products discovery and production. Using powerful genetic tools, the yeast Saccharomyces cerevisiae has been engineered as a production host for natural product pathways from bacterial, fungal, and plant species. With an expanding library of characterized genetic parts, biosynthetic pathways can be refactored for optimized expression in yeast. New engineering strategies have enabled the increased production of valuable secondary metabolites by tuning metabolic pathways. Improvements in high-throughput screening methods have facilitated the rapid identification of variants with improved biosynthetic capabilities. In this review, we focus on the molecular tools and engineering strategies that have recently empowered heterologous natural product biosynthesis