Engineering acetyl-CoA supply and ERG9 repression to enhance mevalonate production in Saccharomyces cerevisiae.

Mevalonate is a key precursor in isoprenoid biosynthesis and a promising commodity chemical. Although mevalonate is a native metabolite in Saccharomyces cerevisiae, its production is challenged by the relatively low flux toward acetyl-CoA in this yeast. In this study we explore different approaches to increase acetyl-CoA supply in S. cerevisiae to boost mevalonate production. Stable integration of a feedback-insensitive acetyl-CoA synthetase (Se-acsL641P) from Salmonella enterica and the mevalonate pathway from Enterococcus faecalis results in the production of 1,390 ± 10 mg/l of mevalonate from glucose. While bifid shunt enzymes failed to improve titers in high-producing strains, inhibition of squalene synthase (ERG9) results in a significant enhancement. Finally, increasing coenzyme A (CoA) biosynthesis by overexpression of pantothenate kinase (CAB1) and pantothenate supplementation further increased production to 3,830 ± 120 mg/l. Using strains that combine these strategies in lab-scale bioreactors results in the production of 13.3 ± 0.5 g/l, which is ∼360-fold higher than previously reported mevalonate titers in yeast. This study demonstrates the feasibility of engineering S. cerevisiae for high-level mevalonate production.

Optogenetic control of the lac operon for bacterial chemical and protein production.

Control of the lac operon with isopropyl ß-D-1-thiogalactopyranoside (IPTG) has been used to regulate gene expression in Escherichia coli for countless applications, including metabolic engineering and recombinant protein production. However, optogenetics offers unique capabilities, such as easy tunability, reversibility, dynamic induction strength and spatial control, that are difficult to obtain with chemical inducers. We have developed a series of circuits for optogenetic regulation of the lac operon, which we call OptoLAC, to control gene expression from various IPTG-inducible promoters using only blue light. Applying them to metabolic engineering improves mevalonate and isobutanol production by 24% and 27% respectively, compared to IPTG induction, in light-controlled fermentations scalable to at least two-litre bioreactors. Furthermore, OptoLAC circuits enable control of recombinant protein production, reaching yields comparable to IPTG induction but with easier tunability of expression. OptoLAC circuits are potentially useful to confer light control over other cell functions originally designed to be IPTG-inducible.

Nonaccidental trauma in pediatric patients: evidence-based screening criteria for ophthalmologic examination.

BACKGROUND: Ophthalmologic examination is included in the work-up for pediatric nonaccidental trauma (NAT) when there is concern for retinal hemorrhage. However, dilated fundus examination entails patient discomfort and prohibition of assessment of pupillary response. Previous studies have suggested that patients without neuroimaging abnormalities are unlikely to have retinal hemorrhage. The purpose of the current study was to analyze the findings in patients who received NAT evaluation with eye examination at our institution, and to propose screening criteria for inclusion of ophthalmologic examination in NAT evaluation. METHODS: The medical records of patients who received NAT evaluation with ophthalmologic examination at The Johns Hopkins Children's Center Pediatric Emergency Department from August 2014 to July 2018 were reviewed retrospectively. Data collected included demographics, presenting symptoms, imaging findings, and ophthalmologic examination findings. The main outcome measure was presence of retinal hemorrhage. RESULTS: A total of 192 evaluations with ophthalmologic examination were included, representing 190 unique individuals of mean age 8.4 ± 9.5 months at presentation. In approximately half (54%) of the evaluations, there were abnormal findings on neuroimaging. Fifteen children (8%) had retinal hemorrhage, all of whom also had abnormal neuroimaging. Abnormal neuroimaging was associated with presence of retinal hemorrhage, with an odds ratio of 21.0 (95% CI, 3.47-∞; P < 0.001). Of the 15 children with retinal hemorrhage, 14 had subdural hemorrhage. CONCLUSIONS: When neuroimaging abnormalities are present, ophthalmologic examination should be performed as part of the pediatric NAT evaluation. When there is no evidence of head injury on neuroimaging, ophthalmologic examination should not be routine.