(692a) A Versatile Synthetic Biology Platform for High-Throughput Structure and Activity Screening of Ribosomally-Synthesized and Post-Translational Modified Peptides (RiPPs)

Ribosomally synthesized and post-translationally modified peptides (RiPPs) form a major class of natural products that are ubiquitous in the currently sequenced genomes, and exhibit antimicrobial, anticancer, anti-protease, and antiviral activities. As the mature product is synthesized from a ribosomal peptide, combinatorial variants can be readily generated by mutagenesis of the precursor gene, providing a rich source for studying structure-activity relationship and engineering improved medicinal properties. However, most RiPP precursor peptides consist of an N-terminal leader region, which is important for recognition by biosynthetic machinery, export, and self-resistance. Leader removal in the final step of biosynthesis is critical to obtain both bioactivity and high-resolution mass spectra, but is difficult to achieve in heterologous hosts such as Escherichia coli, due to the challenges in functional reconstitution of proteases or transporters responsible for leader cleavage.

In this work, we sought to overcome this limitation via protease compartmentalization coupled with inducible self-lysis, which enables in situ production of mature, active RiPP variants from E. coli colonies. Briefly, the protease is directed to the periplasm using a signal peptide so that it is physically separated from the precursor peptide and biosynthetic enzymes that are expressed in the cytosol. Such compartmentalization is necessary as leader attachment is required for installation of post-translational modifications, after which expression of a phage protein is induced by a temperature cue to lyse E. coli. Self-lysis not only allows contact between the protease and the modified precursor peptide in cell lysate to initiate proteolytic leader cleavage, but also release intracellular mature products for direct mass spectrometry (MS) analysis and antimicrobial assays to be performed directly using E. coli colonies.

As a proof-of-concept, we screened the analog library of a two-component lanthipeptide, haloduracin, which is natively produced in Bacillus halodurans. We refactored the haloduracin pathway to incorporate the above-mentioned synthetic biology approaches. With optimized genetic design and process parameters, production of mature, active haloduracin from E. coli colonies was confirmed via both MS analysis and antimicrobial assays. We then created and screened the site-saturation mutagenesis libraries targeting all non-cyclized residues in one of the precursors. Relative antibiotic activities of analogs were estimated using the sizes of inhibition zones in an agar overlay assay towards an indicator Lactococcus lactis strain, and amino acid substitutions were rapidly assigned using predicted mass shifts in mass spectra. Within one week, the structure-activity relationship at 16 residues was characterized by analyzing >3,000 E. coli colonies, achieving >95% probability in full coverage of 20 canonical amino acids at each residue. Notably, a conserved Pro16 residue common to all related peptides was found to be essential for activity, whereas a non-conserved Pro4 exhibited much higher tolerance to mutations. We also obtained mature, active nisin from E. coli colonies, suggesting our method could be generally applicable. We are in the process of purifying three analogs exhibiting substantially larger inhibition zones relative to the wild-type haloduracin during screening to analyze their specific activities.

Separately, we developed a high-throughput MS method for structure-based screening of E. coli colonies producing RiPP variants. Instead of using liquid handling robots or manual processes to pattern colonies into arrays on MS target plates, we utilized machine vision for automatic MS analysis at randomly distributed colonies prepared using standard microbiological methods. Computational tools using multivariate analyses were developed to enumerate structural diversity and cluster colonies with isobaric residue substitutions. Moreover, as the MS screening only consumes ~30% of the analytes, follow-up structural elucidation using high-resolution MS and tandem MS on colonies of interest can be performed on the same sample subsequent to screening. In addition to haloduracin, we screened a substrate library of plantazolicin, which belongs to another RiPP class called linear azol(in)e-containing peptides. We identified both known and new analogs, and all MS assignment of amino acid mutations using predicted mass shifts was consistent with DNA sequencing results.

Together, we successfully developed a synthetic biology platform for structure- and activity-based screening of RiPPs from E. coli colonies. Future improvements include ultra-high-throughput screening of combinatorial mutagenesis targeting more than one residue, differentiation of isobaric ions with different modification states (L- vs D-amino acids), and integration with more types of bioactivity assays such as anticancer activities.