Stanford Advisors

All Publications

  • Complete Reconstitution and Deorphanization of the 3 MDa Nocardiosis-Associated Polyketide Synthase JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Yuet, K. P., Liu, C. W., Lynch, S. R., Kuo, J., Michaels, W., Lee, R. B., McShane, A. E., Zhong, B. L., Fischer, C. R., Khosla, C. 2020; 142 (13): 5952–57

    Abstract

    Several Nocardia strains associated with nocardiosis, a potentially life-threatening disease, house a nonamodular assembly line polyketide synthase (PKS) that presumably synthesizes an unknown polyketide. Here, we report the discovery and structure elucidation of the NOCAP (nocardiosis-associated polyketide) aglycone by first fully reconstituting the NOCAP synthase in vitro from purified protein components followed by heterologous expression in E. coli and spectroscopic analysis of the purified products. The NOCAP aglycone has an unprecedented structure comprised of a substituted resorcylaldehyde headgroup linked to a 15-carbon tail that harbors two conjugated all-trans trienes separated by a stereogenic hydroxyl group. This report is the first example of reconstituting a trans-acyltransferase assembly line PKS in vitro and of using these approaches to "deorphanize" a complete assembly line PKS identified via genomic sequencing. With the NOCAP aglycone in hand, the stage is set for understanding how this PKS and associated tailoring enzymes confer an advantage to their native hosts during human Nocardia infections.

    View details for DOI 10.1021/jacs.0c00904

    View details for Web of Science ID 000526393700009

    View details for PubMedID 32182063

  • 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