Elucidation of tropane alkaloid biosynthesis in Erythroxylum coca using a microbial pathway discovery platform.

Tropane alkaloids (TAs) are heterocyclic nitrogenous metabolites found across seven orders of angiosperms, including Malpighiales (Erythroxylaceae) and Solanales (Solanaceae). Despite the well-established euphorigenic properties of Erythroxylaceae TAs like cocaine, their biosynthetic pathway remains incomplete. Using yeast as a screening platform, we identified and characterized the missing steps of TA biosynthesis in Erythroxylum coca. We first characterize putative E. coca polyamine synthase- and amine oxidase-like enzymes in vitro, in yeast, and in planta to show that the first tropane ring closure in Erythroxylaceae occurs via bifunctional spermidine synthase/N-methyltransferases and both flavin- and copper-dependent amine oxidases. We next identify a SABATH family methyltransferase responsible for the 2-carbomethoxy moiety characteristic of Erythroxylaceae TAs and demonstrate that its coexpression with methylecgonone reductase in yeast engineered to express the Solanaceae TA pathway enables the production of a hybrid TA with structural features of both lineages. Finally, we use clustering analysis of Erythroxylum transcriptome datasets to discover a cytochrome P450 of the CYP81A family responsible for the second tropane ring closure in Erythroxylaceae, and demonstrate the function of the core coca TA pathway in vivo via reconstruction and de novo biosynthesis of methylecgonine in yeast. Collectively, our results provide strong evidence that TA biosynthesis in Erythroxylaceae and Solanaceae is polyphyletic and that independent recruitment of unique biosynthetic mechanisms and enzyme classes occurred at nearly every step in the evolution of this pathway.

Fields to fermentors: Brewing botanical chemotherapeutic precursors using genetically engineered yeast.

Plants are a rich source of chemotherapeutics and other essential medicines, but plant-based drug supply chains are unsustainable. Writing in Nature, Zhang et al.1 demonstrated a proof-of-concept alternate source of the anticancer drug vinblastine by engineering yeast to convert sugar and amino acids into its direct precursors, catharanthine and vindoline.

Li5Sn, the Most Lithium-Rich Binary Stannide: A Combined Experimental and Computational Study.

From reaction of excess lithium with tin, we isolate well-crystallized Li5Sn and solve the crystal structure from single-crystal X-ray diffraction data. The orthorhombic structure (space group Cmcm) features the same coordination polyhedra around tin and lithium as previously predicted by electronic structure calculations for this composition, however differently arranged. An extensive ab initio analysis, including thermodynamic integration using Langevin dynamics in combination with a machine-learning potential (moment tensor potential), is conducted to understand the thermodynamic stability of this Cmcm Li5Sn structure observed in our experiments. Among the 108 Li5Sn structures systematically derived using the structure enumeration algorithm, including the experimental Cmcm structure and those obtained in previous ab initio studies, another new structure with the space group Immm is found to be energetically most stable at 0 K. This computationally discovered Immm structure is also found to be thermodynamically more stable than the Cmcm structure at finite temperatures, indicating that the Cmcm Li5Sn structure observed in our experiments is favored likely due to kinetic reasons rather than thermodynamics.

Engineering cellular metabolite transport for biosynthesis of computationally predicted tropane alkaloid derivatives in yeast.

Microbial biosynthesis of plant natural products (PNPs) can facilitate access to valuable medicinal compounds and derivatives. Such efforts are challenged by metabolite transport limitations, which arise when complex plant pathways distributed across organelles and tissues are reconstructed in unicellular hosts without concomitant transport machinery. We recently reported an engineered yeast platform for production of the tropane alkaloid (TA) drugs hyoscyamine and scopolamine, in which product accumulation is limited by vacuolar transport. Here, we demonstrate that alleviation of transport limitations at multiple steps in an engineered pathway enables increased production of TAs and screening of useful derivatives. We first show that supervised classifier models trained on a tissue-delineated transcriptome from the TA-producing plant Atropa belladonna can predict TA transporters with greater efficacy than conventional regression- and clustering-based approaches. We demonstrate that two of the identified transporters, AbPUP1 and AbLP1, increase TA production in engineered yeast by facilitating vacuolar export and cellular reuptake of littorine and hyoscyamine. We incorporate four different plant transporters, cofactor regeneration mechanisms, and optimized growth conditions into our yeast platform to achieve improvements in de novo hyoscyamine and scopolamine production of over 100-fold (480 µg/L) and 7-fold (172 µg/L). Finally, we leverage computational tools for biosynthetic pathway prediction to produce two different classes of TA derivatives, nortropane alkaloids and tropane N-oxides, from simple precursors. Our work highlights the importance of cellular transport optimization in recapitulating complex PNP biosyntheses in microbial hosts and illustrates the utility of computational methods for gene discovery and expansion of heterologous biosynthetic diversity.

Improving primary care for diabetes and hypertension: findings from implementation research in rural South India.

BACKGROUND: Chronic conditions are a leading cause of death and disability worldwide. Low-income and middle-income countries such as India bear a significant proportion of this global burden. Redesigning primary care from an acute-care model to a model that facilitates chronic care is a challenge and requires interventions at multiple levels. OBJECTIVES: In this intervention study, we aimed to strengthen primary care for diabetes and hypertension at publicly funded primary healthcare centres (PHCs) in rural South India. DESIGN AND METHODS: The complexities of transforming the delivery of primary care motivated us to use a 'theory of change' approach to design, implement and evaluate the interventions. We used both quantitative and qualitative data collection methods. Data from patient records regarding processes of care, glycaemic and blood pressure control, interviews with patients, observations and field notes were used to analyse what changes occurred and why. INTERVENTIONS: We implemented the interventions for 9 months at three PHCs: (1) rationalise workflow to include essential tasks like counselling and measurement of blood pressure/blood glucose at each visit; (2) distribute clinical tasks among staff; (3) retain clinical records at the health facility and (4) capacity building of staff. RESULTS: We found that interventions were implemented at all three PHCs for the first 4 months but did not continue at two of the PHCs. This fadeout was most likely the result of staff transfers and a doctor's reluctance to share tasks. The availability of an additional staff member in the role of a coordinator most likely influenced the relative success of implementation at one PHC. CONCLUSION: These findings draw attention to the need for building teams in primary care for managing chronic conditions. The role of a coordinator emerged as an important consideration, as did the need for a stable core of staff to provide continuity of care.

Biosynthesis of medicinal tropane alkaloids in yeast.

Tropane alkaloids from nightshade plants are neurotransmitter inhibitors that are used for treating neuromuscular disorders and are classified as essential medicines by the World Health Organization1,2. Challenges in global supplies have resulted in frequent shortages of these drugs3,4. Further vulnerabilities in supply chains have been revealed by events such as the Australian wildfires5 and the COVID-19 pandemic6. Rapidly deployable production strategies that are robust to environmental and socioeconomic upheaval7,8 are needed. Here we engineered baker's yeast to produce the medicinal alkaloids hyoscyamine and scopolamine, starting from simple sugars and amino acids. We combined functional genomics to identify a missing pathway enzyme, protein engineering to enable the functional expression of an acyltransferase via trafficking to the vacuole, heterologous transporters to facilitate intracellular routing, and strain optimization to improve titres. Our integrated system positions more than twenty proteins adapted from yeast, bacteria, plants and animals across six sub-cellular locations to recapitulate the spatial organization of tropane alkaloid biosynthesis in plants. Microbial biosynthesis platforms can facilitate the discovery of tropane alkaloid derivatives as new therapeutic agents for neurological disease and, once scaled, enable robust and agile supply of these essential medicines.

A conserved miRNA-183 cluster regulates the innate antiviral response.

Upon immune recognition of viruses, the mammalian innate immune response activates a complex signal transduction network to combat infection. This activation requires phosphorylation of key transcription factors regulating IFN production and signaling, including IFN regulatory factor 3 (IRF3) and STAT1. The mechanisms regulating these STAT1 and IRF3 phosphorylation events remain unclear. Here, using human and mouse cell lines along with gene microarrays, quantitative RT-PCR, viral infection and plaque assays, and reporter gene assays, we demonstrate that a microRNA cluster conserved among bilaterian animals, encoding miR-96, miR-182, and miR-183, regulates IFN signaling. In particular, we observed that the miR-183 cluster promotes IFN production and signaling, mediated by enhancing IRF3 and STAT1 phosphorylation. We also found that the miR-183 cluster activates the IFN pathway and inhibits vesicular stomatitis virus infection by directly targeting several negative regulators of IRF3 and STAT1 activities, including protein phosphatase 2A (PPP2CA) and tripartite motif-containing 27 (TRIM27). Overall, our work reveals an important role of the evolutionarily conserved miR-183 cluster in the regulation of mammalian innate immunity.

Engineering a microbial biosynthesis platform for de novo production of tropane alkaloids.

Tropane alkaloids (TAs) are a class of phytochemicals produced by plants of the nightshade family used for treating diverse neurological disorders. Here, we demonstrate de novo production of tropine, a key intermediate in the biosynthetic pathway of medicinal TAs such as scopolamine, from simple carbon and nitrogen sources in yeast (Saccharomyces cerevisiae). Our engineered strain incorporates 15 additional genes, including 11 derived from diverse plants and bacteria, and 7 disruptions to yeast regulatory or biosynthetic proteins to produce tropine at titers of 6 mg/L. We also demonstrate the utility of our engineered yeast platform for the discovery of TA derivatives by combining biosynthetic modules from distant plant lineages to achieve de novo production of cinnamoyltropine, a non-canonical TA. Our engineered strain constitutes a starting point for future optimization efforts towards realizing industrial fermentation of medicinal TAs and a platform for the synthesis of TA derivatives with enhanced bioactivities.

Anti-CRISPR-mediated control of gene editing and synthetic circuits in eukaryotic cells.

Repurposed CRISPR-Cas molecules provide a useful tool set for broad applications of genomic editing and regulation of gene expression in prokaryotes and eukaryotes. Recent discovery of phage-derived proteins, anti-CRISPRs, which serve to abrogate natural CRISPR anti-phage activity, potentially expands the ability to build synthetic CRISPR-mediated circuits. Here, we characterize a panel of anti-CRISPR molecules for expanded applications to counteract CRISPR-mediated gene activation and repression of reporter and endogenous genes in various cell types. We demonstrate that cells pre-engineered with anti-CRISPR molecules become resistant to gene editing, thus providing a means to generate "write-protected" cells that prevent future gene editing. We further show that anti-CRISPRs can be used to control CRISPR-based gene regulation circuits, including implementation of a pulse generator circuit in mammalian cells. Our work suggests that anti-CRISPR proteins should serve as widely applicable tools for synthetic systems regulating the behavior of eukaryotic cells.

MicroRNA-7 mediates cross-talk between metabolic signaling pathways in the liver.

MicroRNAs (miRNAs) have emerged as critical regulators of cellular metabolism. To characterise miRNAs crucial to the maintenance of hepatic lipid homeostasis, we examined the overlap between the miRNA signature associated with inhibition of peroxisome proliferator activated receptor-α (PPAR-α) signaling, a pathway regulating fatty acid metabolism, and the miRNA profile associated with 25-hydroxycholesterol treatment, an oxysterol regulator of sterol regulatory element binding protein (SREBP) and liver X receptor (LXR) signaling. Using this strategy, we identified microRNA-7 (miR-7) as a PPAR-α regulated miRNA, which activates SREBP signaling and promotes hepatocellular lipid accumulation. This is mediated, in part, by suppression of the negative regulator of SREBP signaling: ERLIN2. miR-7 also regulates genes associated with PPAR signaling and sterol metabolism, including liver X receptor ß (LXR-ß), a transcriptional regulator of sterol synthesis, efflux, and excretion. Collectively, our findings highlight miR-7 as a novel mediator of cross-talk between PPAR, SREBP, and LXR signaling pathways in the liver.

MicroRNAs regulate the immunometabolic response to viral infection in the liver.

Immune regulation of cellular metabolism can be responsible for successful responses to invading pathogens. Viruses alter their hosts' cellular metabolism to facilitate infection. Conversely, the innate antiviral responses of mammalian cells target these metabolic pathways to restrict viral propagation. We identified miR-130b and miR-185 as hepatic microRNAs (miRNAs) whose expression is stimulated by 25-hydroxycholesterol (25-HC), an antiviral oxysterol secreted by interferon-stimulated macrophages and dendritic cells, during hepatitis C virus (HCV) infection. However, 25-HC only directly stimulated miR-185 expression, whereas HCV regulated miR-130b expression. Independently, miR-130b and miR-185 inhibited HCV infection. In particular, miR-185 significantly restricted host metabolic pathways crucial to the HCV life cycle. Interestingly, HCV infection decreased miR-185 and miR-130b levels to promote lipid accumulation and counteract 25-HC's antiviral effect. Furthermore, miR-185 can inhibit other viruses through the regulation of immunometabolic pathways. These data establish these microRNAs as a key link between innate defenses and metabolism in the liver.

Armand-Frappier Outstanding Student Award--The emerging role of 25-hydroxycholesterol in innate immunity.

The metabolic interplay between hosts and viruses plays a crucial role in determining the outcome of viral infection. Viruses reorchestrate the host's primary metabolic gene networks, including genes associated with mevalonate and isoprenoid synthesis, to acquire the necessary energy and structural components for their viral life cycles. Recent work has demonstrated that the interferon-mediated antiviral response suppresses the sterol pathway through production of a signalling molecule, 25-hydroxycholesterol (25HC). This oxysterol has been shown to exert multiple effects, both through incorporation into host cellular membranes as well as through transcriptional control. Herein, we summarize our current understanding of the multifunctional roles of 25HC in the mammalian innate antiviral response.

Soraphen A: A Probe for Investigating the Role of de Novo Lipogenesis during Viral Infection.

Many viruses including the hepatitis C virus (HCV) induce changes to the infected host cell metabolism that include the up-regulation of lipogenesis to create a favorable environment for the virus to propagate. The enzyme acetyl-CoA carboxylase (ACC) polymerizes to form a supramolecular complex that catalyzes the rate-limiting step of de novo lipogenesis. The small molecule natural product Soraphen A (SorA) acts as a nanomolar inhibitor of acetyl-CoA carboxylase activity through disruption of the formation of long highly active ACC polymers from less active ACC dimers. We have shown that SorA inhibits HCV replication in HCV cell culture models expressing subgenomic and full-length replicons (IC50 = 5 nM) as well as a cell culture adapted virus. Using coherent anti-Stokes Raman scattering (CARS) microscopy, we have shown that SorA lowers the total cellular lipid volume in hepatoma cells, consistent with a reduction in de novo lipogenesis. Furthermore, SorA treatment was found to depolymerize the ACC complexes into less active dimers. Taken together, our results suggest that SorA treatment reverses HCV-induced lipid accumulation and demonstrate that SorA is a valuable probe to study the roles of ACC polymerization and enzymatic activity in viral pathogenesis.

Hepatitis C virus induced up-regulation of microRNA-27: a novel mechanism for hepatic steatosis.

UNLABELLED: MicroRNAs (miRNAs) are small RNAs that posttranscriptionally regulate gene expression. Their aberrant expression is commonly linked with diseased states, including hepatitis C virus (HCV) infection. Herein, we demonstrate that HCV replication induces the expression of miR-27 in cell culture and in vivo HCV infectious models. Overexpression of the HCV proteins core and NS4B independently activates miR-27 expression. Furthermore, we establish that miR-27 overexpression in hepatocytes results in larger and more abundant lipid droplets, as observed by coherent anti-Stokes Raman scattering (CARS) microscopy. This hepatic lipid droplet accumulation coincides with miR-27b's repression of peroxisome proliferator-activated receptor (PPAR)-α and angiopoietin-like protein 3 (ANGPTL3), known regulators of triglyceride homeostasis. We further demonstrate that treatment with a PPAR-α agonist, bezafibrate, is able to reverse the miR-27b-induced lipid accumulation in Huh7 cells. This miR-27b-mediated repression of PPAR-α signaling represents a novel mechanism of HCV-induced hepatic steatosis. This link was further demonstrated in vivo through the correlation between miR-27b expression levels and hepatic lipid accumulation in HCV-infected SCID-beige/Alb-uPa mice. CONCLUSION: Collectively, our results highlight HCV's up-regulation of miR-27 expression as a novel mechanism contributing to the development of hepatic steatosis.

Human serum activates CIDEB-mediated lipid droplet enlargement in hepatoma cells.

Human hepatocytes constitutively express the lipid droplet (LD) associated protein cell death-inducing DFFA-like effector B (CIDEB). CIDEB mediates LD fusion, as well as very-low-density lipoprotein (VLDL) maturation. However, there are limited cell culture models readily available to study CIDEB's role in these biological processes, as hepatoma cell lines express negligible levels of CIDEB. Recent work has highlighted the ability of human serum to differentiate hepatoma cells. Herein, we demonstrate that culturing Huh7.5 cells in media supplemented with human serum activates CIDEB expression. This activation occurs through the induced expression of PGC-1α, a positive transcriptional regulator of CIDEB. Coherent anti-Stokes Raman scattering (CARS) microscopy revealed a correlation between CIDEB levels and LD size in human serum treated Huh7.5 cells. Human serum treatment also resulted in a rapid decrease in the levels of adipose differentiation-related protein (ADRP). Furthermore, individual overexpression of CIDEB was sufficient to down-regulate ADRP protein levels. siRNA knockdown of CIDEB revealed that the human serum mediated increase in LD size was CIDEB-dependent. Overall, our work highlights CIDEB's role in LD fusion, and presents a new model system to study the PGC-1α/CIDEB pathway's role in LD dynamics and the VLDL pathway.