Professional Education


  • Doctor of Philosophy, Cornell University, Microbiology (2019)
  • Master of Science, Madurai Kamaraj University, Genomics (2011)
  • Bachelor of Science, Madras University, Biochemistry (2009)

Stanford Advisors


All Publications


  • Glyco-recoded Escherichia coli: Recombineering-based genome editing of native polysaccharide biosynthesis gene clusters METABOLIC ENGINEERING Yates, L. E., Natarajan, A., Li, M., Hale, M. E., Mills, D. C., DeLisa, M. P. 2019; 53: 59–68

    Abstract

    Recombineering-based redesign of bacterial genomes by adding, removing or editing large segments of genomic DNA is emerging as a powerful technique for expanding the range of functions that an organism can perform. Here, we describe a glyco-recoding strategy whereby major non-essential polysaccharide gene clusters in K-12 Escherichia coli are replaced with orthogonal glycosylation components for both biosynthesis of heterologous glycan structures and site-specific glycan conjugation to target proteins. Specifically, the native enterobacterial common antigen (ECA) and O-polysaccharide (O-PS) antigen loci were systematically replaced with ∼9-10 kbp of synthetic DNA encoding Campylobacter jejuni enzymes required for asparagine-linked (N-linked) protein glycosylation. Compared to E. coli cells carrying the same glycosylation machinery on extrachromosomal plasmids, glyco-recoded strains attached glycans to acceptor protein targets with equal or greater efficiency while exhibiting markedly better growth phenotypes and higher glycoprotein titers. Overall, our results define a convenient and reliable framework for bacterial glycome editing that provides a more stable route for chemical diversification of proteins in vivo and effectively expands the bacterial glycoengineering toolkit.

    View details for DOI 10.1016/j.ymben.2019.02.002

    View details for Web of Science ID 000459953300006

    View details for PubMedID 30772453

  • A cell-free biosynthesis platform for modular construction of protein glycosylation pathways. Nature communications Kightlinger, W., Duncker, K. E., Ramesh, A., Thames, A. H., Natarajan, A., Stark, J. C., Yang, A., Lin, L., Mrksich, M., DeLisa, M. P., Jewett, M. C. 2019; 10 (1): 5404

    Abstract

    Glycosylation plays important roles in cellular function and endows protein therapeutics with beneficial properties. However, constructing biosynthetic pathways to study and engineer precise glycan structures on proteins remains a bottleneck. Here, we report a modular, versatile cell-free platform for glycosylation pathway assembly by rapid in vitro mixing and expression (GlycoPRIME). In GlycoPRIME, glycosylation pathways are assembled by mixing-and-matching cell-free synthesized glycosyltransferases that can elaborate a glucose primer installed onto protein targets by an N-glycosyltransferase. We demonstrate GlycoPRIME by constructing 37 putative protein glycosylation pathways, creating 23 unique glycan motifs, 18 of which have not yet been synthesized on proteins. We use selected pathways to synthesize a protein vaccine candidate with an α-galactose adjuvant motif in a one-pot cell-free system and human antibody constant regions with minimal sialic acid motifs in glycoengineered Escherichia coli. We anticipate that these methods and pathways will facilitate glycoscience and make possible new glycoengineering applications.

    View details for DOI 10.1038/s41467-019-12024-9

    View details for PubMedID 31776339

  • Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery (vol 9, 2018) NATURE COMMUNICATIONS Jaroentomeechai, T., Stark, J. C., Natarajan, A., Glasscock, C. J., Yates, L. E., Hsu, K. J., Mrksich, M., Jewett, M. C., DeLisa, M. P. 2018; 9: 3396

    Abstract

    The original version of this Article contained an error in Figure 2, wherein the bottom right western blot panel in Figure 2a was blank. This has now been corrected in both the PDF and HTML versions of the Article.

    View details for DOI 10.1038/s41467-018-05620-8

    View details for Web of Science ID 000442126800002

    View details for PubMedID 30127449

    View details for PubMedCentralID PMC6102295

  • A cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases BIOTECHNOLOGY AND BIOENGINEERING Schoborg, J. A., Hershewe, J. M., Stark, J. C., Kightlinger, W., Kath, J. E., Jaroentomeechai, T., Natarajan, A., DeLisa, M. P., Jewett, M. C. 2018; 115 (3): 739–50

    Abstract

    Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site-specific glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (>70 kDa) and possess multiple transmembrane helices, making them difficult to overexpress in living cells. Here, we address this challenge by establishing a bacterial cell-free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid nanodiscs as cellular membrane mimics, we obtained yields of up to 420 μg/ml for the single-subunit OST enzyme, "Protein glycosylation B" (PglB) from Campylobacter jejuni, as well as for three additional PglB homologs from Campylobacter coli, Campylobacter lari, and Desulfovibrio gigas. Importantly, all of these enzymes catalyzed N-glycosylation reactions in vitro with no purification or processing needed. Furthermore, we demonstrate the ability of cell-free synthesized OSTs to glycosylate multiple target proteins with varying N-glycosylation acceptor sequons. We anticipate that this broadly applicable production method will advance glycoengineering efforts by enabling preparative expression of membrane-embedded OSTs from all kingdoms of life.

    View details for DOI 10.1002/bit.26502

    View details for Web of Science ID 000423672800020

    View details for PubMedID 29178580

  • Metabolic engineering of glycoprotein biosynthesis in bacteria EMERGING TOPICS IN LIFE SCIENCES Natarajan, A., Jaroentomeechai, T., Li, M., Glasscock, C., DeLisa, M. 2018; 2 (3): 419-432

    View details for DOI 10.1042/ETLS20180004

  • An Engineered Survival-Selection Assay for Extracellular Protein Expression Uncovers Hypersecretory Phenotypes in Escherichia coli ACS SYNTHETIC BIOLOGY Natarajan, A., Haitjema, C. H., Lee, R., Boock, J. T., DeLisa, M. P. 2017; 6 (5): 875–83

    Abstract

    The extracellular expression of recombinant proteins using laboratory strains of Escherichia coli is now routinely achieved using naturally secreted substrates, such as YebF or the osmotically inducible protein Y (OsmY), as carrier molecules. However, secretion efficiency through these pathways needs to be improved for most synthetic biology and metabolic engineering applications. To address this challenge, we developed a generalizable survival-based selection strategy that effectively couples extracellular protein secretion to antibiotic resistance and enables facile isolation of rare mutants from very large populations (i.e., 1010-12 clones) based simply on cell growth. Using this strategy in the context of the YebF pathway, a comprehensive library of E. coli single-gene knockout mutants was screened and several gain-of-function mutations were isolated that increased the efficiency of extracellular expression without compromising the integrity of the outer membrane. We anticipate that this user-friendly strategy could be leveraged to better understand the YebF pathway and other secretory mechanisms-enabling the exploration of protein secretion in pathogenesis as well as the creation of designer E. coli strains with greatly expanded secretomes-all without the need for expensive exogenous reagents, assay instruments, or robotic automation.

    View details for DOI 10.1021/acssynbio.6b00366

    View details for Web of Science ID 000402026600013

    View details for PubMedID 28182400

  • Substitute sweeteners: diverse bacterial oligosaccharyltransferases with unique N-glycosylation site preferences SCIENTIFIC REPORTS Ollis, A. A., Chai, Y., Natarajan, A., Perregaux, E., Jaroentomeechai, T., Guarino, C., Smith, J., Zhang, S., DeLisa, M. P. 2015; 5: 15237

    Abstract

    The central enzyme in the Campylobacter jejuni asparagine-linked glycosylation pathway is the oligosaccharyltransferase (OST), PglB, which transfers preassembled glycans to specific asparagine residues in target proteins. While C. jejuni PglB (CjPglB) can transfer many diverse glycan structures, the acceptor sites that it recognizes are restricted predominantly to those having a negatively charged residue in the -2 position relative to the asparagine. Here, we investigated the acceptor-site preferences for 23 homologs with natural sequence variation compared to CjPglB. Using an ectopic trans-complementation assay for CjPglB function in glycosylation-competent Escherichia coli, we demonstrated in vivo activity for 16 of the candidate OSTs. Interestingly, the OSTs from Campylobacter coli, Campylobacter upsaliensis, Desulfovibrio desulfuricans, Desulfovibrio gigas, and Desulfovibrio vulgaris, exhibited significantly relaxed specificity towards the -2 position compared to CjPglB. These enzymes glycosylated minimal N-X-T motifs in multiple targets and each followed unique, as yet unknown, rules governing acceptor-site preferences. One notable example is D. gigas PglB, which was the only bacterial OST to glycosylate the Fc domain of human immunoglobulin G at its native 'QYNST' sequon. Overall, we find that a subset of bacterial OSTs follow their own rules for acceptor-site specificity, thereby expanding the glycoengineering toolbox with previously unavailable biocatalytic diversity.

    View details for DOI 10.1038/srep15237

    View details for Web of Science ID 000363029800001

    View details for PubMedID 26482295

    View details for PubMedCentralID PMC4894442

  • Universal Genetic Assay for Engineering Extracellular Protein Expression ACS SYNTHETIC BIOLOGY Haitjema, C. H., Boock, J. T., Natarajan, A., Dominguez, M. A., Gardner, J. G., Keating, D. H., Withers, S. T., DeLisa, M. P. 2014; 3 (2): 74–82

    Abstract

    A variety of strategies now exist for the extracellular expression of recombinant proteins using laboratory strains of Escherichia coli . However, secreted proteins often accumulate in the culture medium at levels that are too low to be practically useful for most synthetic biology and metabolic engineering applications. The situation is compounded by the lack of generalized screening tools for optimizing the secretion process. To address this challenge, we developed a genetic approach for studying and engineering protein-secretion pathways in E. coli . Using the YebF pathway as a model, we demonstrate that direct fluorescent labeling of tetracysteine-motif-tagged secretory proteins with the biarsenical compound FlAsH is possible in situ without the need to recover the cell-free supernatant. High-throughput screening of a bacterial strain library yielded superior YebF expression hosts capable of secreting higher titers of YebF and YebF-fusion proteins into the culture medium. We also show that the method can be easily extended to other secretory pathways, including type II and type III secretion, directly in E. coli . Thus, our FlAsH-tetracysteine-based genetic assay provides a convenient, high-throughput tool that can be applied generally to diverse secretory pathways. This platform should help to shed light on poorly understood aspects of these processes as well as to further assist in the construction of engineered E. coli strains for efficient secretory-protein production.

    View details for DOI 10.1021/sb400142b

    View details for Web of Science ID 000331927100003

    View details for PubMedID 24200127