A Silent Biosynthetic Gene Cluster from a Methanotrophic Bacterium Potentiates Discovery of a Substrate Promiscuous Proteusin Cyclodehydratase

Abstract

Natural product-encoding biosynthetic gene clusters (BGCs) within microbial genomes far outnumber the known natural products; chemical products from such BGCs remain cryptic. These silent BGCs hold promise not only for the elaboration of new natural products but also for the discovery of useful biosynthetic enzymes. Here, we describe a genome mining strategy targeted toward the discovery of substrate promiscuous natural product biosynthetic enzymes. In the genome of the methanotrophic bacterium Methylovulum psychrotolerans Sph1T, we discover a transcriptionally silent natural product BGC that encoded numerous ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. These cryptic RiPP natural products were accessed using heterologous expression of the substrate peptide and biosynthetic enzyme-encoded genes. In line with our genome mining strategy, the RiPP biosynthetic enzymes in this BGC were found to be substrate promiscuous, which allowed us to use them in a combinatorial fashion with a similarly substrate-tolerant cyanobactin biosynthetic enzyme to introduce head-to-tail macrocyclization in the proteusin family of RiPP natural products.

A Leader-Guided Substrate Tolerant RiPP Brominase Allows Suzuki–Miyaura Cross-Coupling Reactions for Peptides and Proteins

“…To address the challenge of the preparation of the physiological substrate for SrpI, we turned to the substrate promiscuous YcaO cyclodehydratase/azoline oxidase pair MprC/MprD that we had described for installing azol­(in)­e heterocycles into 10 different MprE substrate peptides (Figure B and Figure S1). MprE and SrpE are proteusin peptides, characterized by long leader sequences that are similar to those of nitrile hydratases . While the MprC/MprD demonstrated robust activities in vivo and in vitro , they were selective for the MprE leader sequences; in contrast, SrpI was tolerant to other proteusin leaders. , In light of these observations, we appended the SrpE -LCCCW core to the MprE X leader (a consensus leader sequence built from the 10 different MprE leader sequences), thus creating a chimeric MprE X –LCCCW substrate (Supplementary Note).…”

“… MprE and SrpE are proteusin peptides, characterized by long leader sequences that are similar to those of nitrile hydratases . While the MprC/MprD demonstrated robust activities in vivo and in vitro , they were selective for the MprE leader sequences; in contrast, SrpI was tolerant to other proteusin leaders. , In light of these observations, we appended the SrpE -LCCCW core to the MprE X leader (a consensus leader sequence built from the 10 different MprE leader sequences), thus creating a chimeric MprE X –LCCCW substrate (Supplementary Note). Upon co-expression of the gene encoding this chimeric substrate with mprC / mprD in E .…”

“…18 While the MprC/MprD demonstrated robust activities in vivo and in vitro, they were selective for the MprE leader sequences; in contrast, SrpI was tolerant to other proteusin leaders. 16,17 In light of these observations, we appended the SrpE -LCCCW core to the MprE X leader (a consensus leader sequence built from the 10 different MprE leader sequences 17 ), thus creating a chimeric MprE X −LCCCW substrate (Supplementary Note). Upon co-expression of the gene encoding this chimeric substrate with mprC/mprD in E. coli, we obtained the purified MprE X −LCCCW peptide in which all three Cys residues in the core were neatly converted into thiazol(in)e heterocycles (Figure 2A and Figure S2).…”

Promiscuous Enzymes for Residue‐Specific Peptide and Protein Late‐Stage Functionalization

“…YcaO members as PatD modify Ser and Thr and convert them into oxazoline and methyloxazoline, respectively using ATP (Figure 9D). [185] Although most YcaO enzymes have a preference to modify Cys, several enzymes modify Ser and Thr at equal level or higher such as MprC [94] and TruD. [98] As mentioned in section 5 (Figure 3D), LanM enzymes are bifunctional and can dehydrate Ser and Thr and form dehydroalanine and dehydrobutyrine, respectively, before forming thiol ether bridge.…”

“…They are cyclodehydratases that catalyze the formation of thiazoline rings in Cys‐containing ribosomal precursor peptides (Figure 3C). [90] Examples of YcaO enzymes that modify Cys include TruD, [91] PatD, [91] LynD, [92] 1SrpC, [93] and MprC [94] . YcaO enzymes have an N‐terminus motif called RRE that binds to the recognition sequence (RS) elements in ribosomal substrate precursor peptides.…”