Education & Certifications

  • Master of Science, Stanford University, BIOE-MS (2018)
  • Bachelor of Applied Science, University of Ottawa, Chemical Engineering (2016)
  • Bachelor of Science, University of Ottawa, Biochemistry (2016)

All Publications

  • Biosynthesis of medicinal tropane alkaloids in yeast. Nature Srinivasan, P., Smolke, C. D. 2020


    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.

    View details for DOI 10.1038/s41586-020-2650-9

    View details for PubMedID 32879484

  • Conserved miRNA-183 cluster regulates the innate antiviral response. The Journal of biological chemistry Singaravelu, R., Ahmed, N., Quan, C., Srinivasan, P., Ablenas, C. J., Roy, D. G., Pezacki, J. P. 2019


    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 interferon (IFN) production and signaling, including IFN regulatory factor 3 (IRF3) and signal transducers and activators of transcription protein 1 (STAT1). The mechanisms regulating these STAT1 and IRF3 phosphorylation events remain unclear. Herein, using human and mouse cell lines, along with gene microarrays, quantitative RT-PCR (qPCR), viral infection and plaque assays, and reporter gene assays, we demonstrate that a bilaterian conserved microRNA (miRNA) 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 through enhancing IRF3 and STAT1 phosphorylation. We also found that the miR-183 cluster activates the IFN pathway and inhibits vesicular stomatitis virus (VSV) infection through direct targeting of several negative regulators of IRF3 and STAT1 activities, including protein phosphatase 2A (PPP2CA) and tripartite motif containing 27 (TRIM27). Overall, the findings of our work reveals an important role for the evolutionarily conserved miR-183 cluster in the regulation of the mammalian innate immunity.

    View details for DOI 10.1074/jbc.RA119.010858

    View details for PubMedID 31694919

  • Engineering a microbial biosynthesis platform for de novo production of tropane alkaloids. Nature communications Srinivasan, P., Smolke, C. D. 2019; 10 (1): 3634


    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 6mg/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.

    View details for DOI 10.1038/s41467-019-11588-w

    View details for PubMedID 31406117

  • Anti-CRISPR-mediated control of gene editing and synthetic circuits in eukaryotic cells. Nature communications Nakamura, M., Srinivasan, P., Chavez, M., Carter, M. A., Dominguez, A. A., La Russa, M., Lau, M. B., Abbott, T. R., Xu, X., Zhao, D., Gao, Y., Kipniss, N. H., Smolke, C. D., Bondy-Denomy, J., Qi, L. S. 2019; 10 (1): 194


    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.

    View details for PubMedID 30643127

  • MicroRNA-7 mediates cross-talk between metabolic signaling pathways in the liver. Scientific reports Singaravelu, R. n., Quan, C. n., Powdrill, M. H., Shaw, T. A., Srinivasan, P. n., Lyn, R. K., Alonzi, R. C., Jones, D. M., Filip, R. n., Russell, R. S., Pezacki, J. P. 2018; 8 (1): 361


    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 β (LXR-β), 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.

    View details for PubMedID 29321595

    View details for PubMedCentralID PMC5762714

  • MicroRNAs regulate the immunometabolic response to viral infection in the liver NATURE CHEMICAL BIOLOGY Singaravelu, R., O'Hara, S., Jones, D. M., Chen, R., Taylor, N. G., Srinivasan, P., Quan, C., Roy, D. G., Steenbergen, R. H., Kumar, A., Lyn, R. K., Ozcelik, D., Rouleau, Y., Nguyen, M., Rayner, K. J., Hobman, T. C., Tyrrell, D. L., Russell, R. S., Pezacki, J. P. 2015; 11 (12): 988-993


    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.

    View details for DOI 10.1038/NCHEM.BIO.1940

    View details for Web of Science ID 000365834000019

    View details for PubMedID 26479438

  • Armand-Frappier Outstanding Student Award - The emerging role of 25-hydroxycholesterol in innate immunity CANADIAN JOURNAL OF MICROBIOLOGY Singaravelu, R., Srinivasan, P., Pezacki, J. P. 2015; 61 (8): 521-530


    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.

    View details for DOI 10.1139/cjm-2015-0292

    View details for Web of Science ID 000358786800001

    View details for PubMedID 26182401

  • Soraphen A: A Probe for Investigating the Role of de Novo Lipogenesis during Viral Infection ACS INFECTIOUS DISEASES Singaravelu, R., Desrochers, G. F., Srinivasan, P., O'Hara, S., Lyn, R. K., Mueller, R., Jones, D. M., Russell, R. S., Pezacki, J. P. 2015; 1 (3): 130-134
  • Hepatitis C Virus Induced Up-Regulation of MicroRNA-27: A Novel Mechanism for Hepatic Steatosis HEPATOLOGY Singaravelu, R., Chen, R., Lyn, R. K., Jones, D. M., O'Hara, S., Rouleau, Y., Cheng, J., Srinivasan, P., Nasheri, N., Russell, R. S., Tyrrell, D. L., Pezacki, J. P. 2014; 59 (1): 98-108


    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.Collectively, our results highlight HCV's up-regulation of miR-27 expression as a novel mechanism contributing to the development of hepatic steatosis.

    View details for DOI 10.1002/hep.26634

    View details for Web of Science ID 000328738400014

    View details for PubMedID 23897856

  • Human serum activates CIDEB-mediated lipid droplet enlargement in hepatoma cells BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Singaravelu, R., Lyn, R. K., Srinivasan, P., Delcorde, J., Steenbergen, R. H., Tyrrell, D. L., Pezacki, J. P. 2013; 441 (2): 447-452


    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.

    View details for DOI 10.1016/j.bbrc.2013.10.080

    View details for Web of Science ID 000327290100029

    View details for PubMedID 24161736