Recent Advances and Perspectives on Expanding the Chemical Diversity of Lasso Peptides

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing family of natural products that exhibit a range of structures and bioactivities. Initially assembled from the twenty proteinogenic amino acids in a ribosome-dependent manner, RiPPs assume their peculiar bioactive structures through various post-translational modifications. The essential modifications representative of each subfamily of RiPP are performed on a precursor peptide by the so-called processing enzymes; however, various tailoring enzymes can also embellish the precursor peptide or processed peptide with additional functional groups. Lasso peptides are an interesting subfamily of RiPPs characterized by their unique lariat knot-like structure, wherein the C-terminal tail is inserted through a macrolactam ring fused by an isopeptide bond between the N-terminal amino group and an acidic side chain. Until recently, relatively few lasso peptides were found to be tailored with extra functional groups. Nevertheless, the development of new routes to diversify lasso peptides and thus introduce novel or enhanced biological, medicinally relevant, or catalytic properties is appealing. In this review, we highlight several strategies through which lasso peptides have been successfully modified and provide a brief overview of the latest findings on the tailoring of these peptides. We also propose future directions for lasso peptide tailoring as well as potential applications for these peptides in hybrid catalyst design.

Enhanced chemoselectivity of a plant cytochrome P450 through protein engineering of surface and catalytic residues

Cytochrome P450s (P450s) are the most versatile catalysts utilized by plants to produce structurally and functionally diverse metabolites. Given the high degree of gene redundancy and challenge to functionally characterize plant P450s, protein engineering is used as a complementary strategy to study the mechanisms of P450-mediated reactions, or to alter their functions. We previously proposed an approach of engineering plant P450s based on combining high-accuracy homology models generated by Rosetta combined with data-driven design using evolutionary information of these enzymes. With this strategy, we repurposed a multi-functional P450 (CYP87D20) into a monooxygenase after redesigning its active site. Since most plant P450s are membrane-anchored proteins that are adapted to the micro-environments of plant cells, expressing them in heterologous hosts usually results in problems of expression or activity. Here, we applied computational design to tackle these issues by simultaneous optimization of the protein surface and active site. After screening 17 variants, effective substitutions of surface residues were observed to improve both expression and activity of CYP87D20. In addition, the identified substitutions were additive and by combining them a highly efficient C11 hydroxylase of cucurbitadienol was created to participate in the mogrol biosynthesis. This study shows the importance of considering the interplay between surface and active site residues for P450 engineering. Our integrated strategy also opens an avenue to create more tailoring enzymes with desired functions for the metabolic engineering of high-valued compounds like mogrol, the precursor of natural sweetener mogrosides.

Genomic Determinants Encode the Reactivity and Regioselectivity of Flavin-Dependent Halogenases in Bacterial Genomes and Metagenomes

Halogenases are playing an important role as tailoring enzymes in biosynthetic pathways. Flavin-dependent tryptophan halogenases (Trp-FDHs) are among the enzymes that have broad substrate scope and high selectivity.

Deciphering a Cyclodipeptide Synthase Pathway Encoding Prenylated Indole Alkaloids in Streptomyces leeuwenhoekii

Cyclodipeptide synthases (CDPSs) catalyse the formation of cyclodipeptides using aminoacylated-tRNAs as substrates and have great potentials in the production of diverse 2,5-diketopiperazines (2,5-DKPs). Genome mining of Streptomyces leeuwenhoekii NRRL B-24963 revealed a two-gene locus saz encoding a CDPS SazA and a unique fused enzyme SazB harboring two domains: phytoene-synthase-like prenyltransferase (PT) and methyltransferase (MT). Heterologous expression of the saz gene(s) in Streptomyces albus J1074 led to the production of four prenylated indole alkaloids, among which streptoazines A-C (3–5) are new compounds. Expression of different gene combinations showed that the SazA catalyzes the formation of cyclo (L-Trp-L-Trp) (cWW, 1), followed by consecutive prenylation and methylation by SazB. Biochemical assays demonstrated that SazB is a bifunctional enzyme, catalyzing sequential C3/C3’-prenylation(s) by SazB-PT and N1/N1’-methylation(s) by SazB-MT. Of note substrate selectivity of SazB-PT and SazB-MT was probed, revealing the stringent specificity of SazB-PT but relative flexibility of SazB-MT. IMPORTANCE Natural products with 2,5-DKP skeleton have long sparked the interest in drug discovery and development. Recent advances in microbial genome sequencing have revealed that the potentials of CDPS-dependent pathways encoding new 2,5-DKPs are underexplored. In this study, we report the genome mining of a new CDPS-containing two-gene operon and activation of this cryptic gene cluster through heterologous expression, leading to the discovery of four indole 2,5-DKP alkaloids. The cWW-synthesizing CDPS SazA and the unusual PT-MT fused enzyme SazB were characterized. Our results expand the repertoire of CDPSs and associated tailoring enzymes, setting the stage for accessing diverse prenylated alkaloids using synthetic biology strategies.

Modifications of diketopiperazines assembled by cyclodipeptide synthases with cytochrome P450 enzymes

Abstract2,5-Diketopiperazines are the smallest cyclic peptides comprising two amino acids connected via two peptide bonds. They can be biosynthesized in nature by two different enzyme families, either by nonribosomal peptide synthetases or by cyclodipeptide synthases. Due to the stable scaffold of the diketopiperazine ring, they can serve as precursors for further modifications by different tailoring enzymes, such as methyltransferases, prenyltransferases, oxidoreductases like cyclodipeptide oxidases, 2-oxoglutarate-dependent monooxygenases and cytochrome P450 enzymes, leading to the formation of intriguing secondary metabolites. Among them, cyclodipeptide synthase-associated P450s attracted recently significant attention, since they are able to catalyse a broader variety of astonishing reactions than just oxidation by insertion of an oxygen. The P450-catalysed reactions include hydroxylation at a tertiary carbon, aromatisation of the diketopiperazine ring, intramolecular and intermolecular carbon-carbon and carbon-nitrogen bond formation of cyclodipeptides and nucleobase transfer reactions. Elucidation of the crystal structures of three P450s as cyclodipeptide dimerases provides a structural basis for understanding the reaction mechanism and generating new enzymes by protein engineering. This review summarises recent publications on cyclodipeptide modifications by P450s.Key Points• Intriguing reactions catalysed by cyclodipeptide synthase-associated cytochrome P450s• Homo- and heterodimerisation of diketopiperazines• Coupling of guanine and hypoxanthine with diketopiperazines Graphical abstract

Lipoprotein N-Acylation in Staphylococcus aureus Is Catalyzed by a Two-Component Acyl Transferase System

ABSTRACT Bacterial lipoproteins (Lpps) are a class of membrane-associated proteins universally distributed among all bacteria. A characteristic N-terminal cysteine residue that is variably acylated anchors C-terminal globular domains to the extracellular surface, where they serve numerous roles, including in the capture and transport of essential nutrients. Lpps are also ligands for the Toll-like receptor 2 (TLR2) family, a key component of the innate immune system tasked with bacterial recognition. While Lpp function is conserved in all prokaryotes, structural heterogeneity in the N-terminal acylation state is widespread among Firmicutes and can differ between otherwise closely related species. In this study, we identify a novel two-gene system that directs the synthesis of N-acylated Lpps in the commensal and opportunistic pathogen subset of staphylococci. The two genes, which we have named the lipoprotein N-acylation transferase system (Lns), bear no resemblance to previously characterized N-terminal Lpp tailoring enzymes. LnsA (SAOUHSC_00822) is an NlpC/P60 superfamily enzyme, whereas LnsB (SAOHSC_02761) has remote homology to the CAAX protease and bacteriocin-processing enzyme (CPBP) family. Both LnsA and LnsB are together necessary and alone sufficient for N-acylation in Staphylococcus aureus and convert the Lpp chemotype from diacyl to triacyl when heterologously expressed in Listeria monocytogenes. Acquisition of lnsAB decreases TLR2-mediated detection of S. aureus by nearly 10-fold and shifts the activated TLR2 complex from TLR2/6 to TLR2/1. LnsAB thus has a dual role in attenuating TLR2 signaling in addition to a broader role in bacterial cell envelope physiology. IMPORTANCE Although it has long been known that S. aureus forms triacylated Lpps, a lack of homologs to known N-acylation genes found in Gram-negative bacteria has until now precluded identification of the genes responsible for this Lpp modification. Here, we demonstrate N-terminal Lpp acylation and chemotype conversion to the tri-acylated state is directed by a unique acyl transferase system encoded by two noncontiguous staphylococci genes (lnsAB). Since triacylated Lpps stimulate TLR2 more weakly than their diacylated counterparts, Lpp N-acylation is an important TLR2 immunoevasion factor for determining tolerance or nontolerance in niches such as in the skin microbiota. The discovery of the LnsAB system expands the known diversity of Lpp biosynthesis pathways and acyl transfer biochemistry in bacteria, advances our understanding of Lpp structural heterogeneity, and helps differentiate commensal and noncommensal microbiota.

Complete Reconstitution and Deorphanization of the 3 MDa NOCAP (NOCardiosis-Associated Polyketide) Synthase

ABSTRACTSeveral Nocardia strains associated with nocardiosis, a potentially life-threatening disease, house a nonamodular assembly-line polyketide synthase (PKS) that presumably synthesizes an unknown natural product. 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 either in vitro or in E. coli, 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.

Uncovering the activities, biological roles, and regulation of bacterial cell wall hydrolases and tailoring enzymes

Bacteria account for 1000-fold more biomass than humans. They vary widely in shape and size. The morphological diversity of bacteria is due largely to the different peptidoglycan-based cell wall structures that encase bacterial cells. Although the basic structure of peptidoglycan is highly conserved, consisting of long glycan strands that are cross-linked by short peptide chains, the mature cell wall is chemically diverse. Peptidoglycan hydrolases and cell wall–tailoring enzymes that regulate glycan strand length, the degree of cross-linking, and the addition of other modifications to peptidoglycan are central in determining the final architecture of the bacterial cell wall. Historically, it has been difficult to biochemically characterize these enzymes that act on peptidoglycan because suitable peptidoglycan substrates were inaccessible. In this review, we discuss fundamental aspects of bacterial cell wall synthesis, describe the regulation and diverse biochemical and functional activities of peptidoglycan hydrolases, and highlight recently developed methods to make and label defined peptidoglycan substrates. We also review how access to these substrates has now enabled biochemical studies that deepen our understanding of how bacterial cell wall enzymes cooperate to build a mature cell wall. Such improved understanding is critical to the development of new antibiotics that disrupt cell wall biogenesis, a process essential to the survival of bacteria.

Bacterial terpene biosynthesis: challenges and opportunities for pathway engineering

Terpenoids are the largest and structurally most diverse class of natural products. They possess potent and specific biological activity in multiple assays and against diseases, including cancer and malaria as notable examples. Although the number of characterized terpenoid molecules is huge, our knowledge of how they are biosynthesized is limited, particularly when compared to the well-studied thiotemplate assembly lines. Bacteria have only recently been recognized as having the genetic potential to biosynthesize a large number of complex terpenoids, but our current ability to associate genetic potential with molecular structure is severely restricted. The canonical terpene biosynthetic pathway uses a single enzyme to form a cyclized hydrocarbon backbone followed by modifications with a suite of tailoring enzymes that can generate dozens of different products from a single backbone. This functional promiscuity of terpene biosynthetic pathways renders terpene biosynthesis susceptible to rational pathway engineering using the latest developments in the field of synthetic biology. These engineered pathways will not only facilitate the rational creation of both known and novel terpenoids, their development will deepen our understanding of a significant branch of biosynthesis. The biosynthetic insights gained will likely empower a greater degree of engineering proficiency for non-natural terpene biosynthetic pathways and pave the way towards the biotechnological production of high value terpenoids.

NeuRiPP: Neural network identification of RiPP precursor peptides

Significant progress has been made in the past few years on the computational identification of biosynthetic gene clusters (BGCs) that encode ribosomally synthesized and post-translationally modified peptides (RiPPs). This is done by identifying both RiPP tailoring enzymes (RTEs) and RiPP precursor peptides (PPs). However, identification of PPs, particularly for novel RiPP classes remains challenging. To address this, machine learning has been used to accurately identify PP sequences. Current machine learning tools have limitations, since they are specific to the RiPPclass they are trained for and are context-dependent, requiring information about the surrounding genetic environment of the putative PP sequences. NeuRiPP overcomes these limitations. It does this by leveraging the rich data set of high-confidence putative PP sequences from existing programs, along with experimentally verified PPs from RiPP databases. NeuRiPP uses neural network archictectures that are suitable for peptide classification with weights trained on PP datasets. It is able to identify known PP sequences, and sequences that are likely PPs. When tested on existing RiPP BGC datasets, NeuRiPP was able to identify PP sequences in significantly more putative RiPP clusters than current tools while maintaining the same HMM hit accuracy. Finally, NeuRiPP was able to successfully identify PP sequences from novel RiPP classes that were recently characterized experimentally, highlighting its utility in complementing existing bioinformatics tools.