Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 14 de 14
Filter
Add more filters











Publication year range
1.
Nat Chem ; 16(8): 1320-1329, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38528101

ABSTRACT

Recent efforts in genome mining of ribosomally synthesized and post-translationally modified peptides (RiPPs) have expanded the diversity of post-translational modification chemistries. However, RiPPs are rarely reported as hybrid molecules incorporating biosynthetic machinery from other natural product families. Here we report lipoavitides, a class of RiPP/fatty-acid hybrid lipopeptides that display a unique, putatively membrane-targeting 4-hydroxy-2,4-dimethylpentanoyl (HMP)-modified N terminus. The HMP is formed via condensation of isobutyryl-coenzyme A (isobutyryl-CoA) and methylmalonyl-CoA catalysed by a 3-ketoacyl-(acyl carrier protein) synthase III enzyme, followed by successive tailoring reactions in the fatty acid biosynthetic pathway. The HMP and RiPP substructures are then connected by an acyltransferase exhibiting promiscuous activity towards the fatty acyl and RiPP substrates. Overall, the discovery of lipoavitides contributes a prototype of RiPP/fatty-acid hybrids and provides possible enzymatic tools for lipopeptide bioengineering.


Subject(s)
Ribosomes , Acylation , Ribosomes/metabolism , Fatty Acid Synthases/metabolism , Fatty Acid Synthases/chemistry , Fatty Acid Synthases/genetics , Protein Processing, Post-Translational , Peptides/chemistry , Peptides/metabolism , Lipopeptides/chemistry , Lipopeptides/metabolism , Lipopeptides/biosynthesis , Fatty Acids/chemistry , Fatty Acids/metabolism
2.
bioRxiv ; 2023 Oct 26.
Article in English | MEDLINE | ID: mdl-37961664

ABSTRACT

Recent efforts in genome mining of ribosomally synthesized and post-translationally modified peptides (RiPPs) have expanded the diversity of post-translational modification chemistries 1, 2 . However, RiPPs are rarely reported as hybrid molecules incorporating biosynthetic machineries from other natural product families 3-8 . Here, we report lipoavitides, a class of RiPP/fatty acid hybrid lipopeptides that display a unique, membrane-targeting 4-hydroxy-2,4-dimethylpentanoyl (HMP)-modified N -terminus. The HMP is formed via condensation of isobutyryl-CoA and methylmalonyl-CoA catalyzed by a 3-ketoacyl-ACP synthase III enzyme, followed by successive tailoring reactions in the fatty acid biosynthetic pathway. The HMP and RiPP substructures are then connected by an acyltransferase exhibiting promiscuous activity towards the fatty acyl and RiPP substrates. Overall, the discovery of lipoavitides contributes a prototype of RiPP/fatty acid hybrids and provides possible enzymatic tools for lipopeptide bioengineering.

3.
Nat Commun ; 14(1): 1624, 2023 03 23.
Article in English | MEDLINE | ID: mdl-36959188

ABSTRACT

The era of inexpensive genome sequencing and improved bioinformatics tools has reenergized the study of natural products, including the ribosomally synthesized and post-translationally modified peptides (RiPPs). In recent years, RiPP discovery has challenged preconceptions about the scope of post-translational modification chemistry, but genome mining of new RiPP classes remains an unsolved challenge. Here, we report a RiPP class defined by an unusual (S)-N2,N2-dimethyl-1,2-propanediamine (Dmp)-modified C-terminus, which we term the daptides. Nearly 500 daptide biosynthetic gene clusters (BGCs) were identified by analyzing the RiPP Recognition Element (RRE), a common substrate-binding domain found in half of prokaryotic RiPP classes. A representative daptide BGC from Microbacterium paraoxydans DSM 15019 was selected for experimental characterization. Derived from a C-terminal threonine residue, the class-defining Dmp is installed over three steps by an oxidative decarboxylase, aminotransferase, and methyltransferase. Daptides uniquely harbor two positively charged termini, and thus we suspect this modification could aid in membrane targeting, as corroborated by hemolysis assays. Our studies further show that the oxidative decarboxylation step requires a functionally unannotated accessory protein. Fused to the C-terminus of the accessory protein is an RRE domain, which delivers the unmodified substrate peptide to the oxidative decarboxylase. This discovery of a class-defining post-translational modification in RiPPs may serve as a prototype for unveiling additional RiPP classes through genome mining.


Subject(s)
Biological Products , Carboxy-Lyases , Peptides/chemistry , Ribosomes/genetics , Ribosomes/metabolism , Protein Processing, Post-Translational , Computational Biology/methods , Carboxy-Lyases/metabolism , Biological Products/metabolism
4.
bioRxiv ; 2023 Mar 08.
Article in English | MEDLINE | ID: mdl-36945508

ABSTRACT

The era of inexpensive genome sequencing and improved bioinformatics tools has reenergized the study of natural products, including the ribosomally synthesized and post-translationally modified peptides (RiPPs). In recent years, RiPP discovery has challenged preconceptions about the scope of post-translational modification chemistry, but genome mining of new RiPP classes remains an unsolved challenge. Here, we report a RiPP class defined by an unusual ( S )- N 2 , N 2 -dimethyl-1,2-propanediamine (Dmp)-modified C -terminus, which we term the daptides. Nearly 500 daptide biosynthetic gene clusters (BGCs) were identified by analyzing the RiPP Recognition Element (RRE), a common substrate-binding domain found in half of prokaryotic RiPP classes. A representative daptide BGC from Microbacterium paraoxydans DSM 15019 was selected for experimental characterization. Derived from a C -terminal threonine residue, the class-defining Dmp is installed over three steps by an oxidative decarboxylase, aminotransferase, and methyltransferase. Daptides uniquely harbor two positively charged termini, and thus we suspect this modification could aid in membrane targeting, as corroborated by hemolysis assays. Our studies further show that the oxidative decarboxylation step requires a functionally unannotated accessory protein. Fused to the C -terminus of the accessory protein is an RRE domain, which delivers the unmodified substrate peptide to the oxidative decarboxylase. This discovery of a class-defining post-translational modification in RiPPs may serve as a prototype for unveiling additional RiPP classes through genome mining.

5.
JACS Au ; 3(1): 195-203, 2023 Jan 23.
Article in English | MEDLINE | ID: mdl-36711095

ABSTRACT

Bipentaromycins are heterodimeric aromatic polyketides featuring two distinctive 5/6/6/6/5 pentacyclic ring systems and exhibit antibacterial activities. However, their overall biosynthetic mechanism, particularly the mechanism for early-stage modifications, such as hydrogenation and methylation, and late-stage dimerization, remains unknown. Herein, by integrating heterologous expression, isotope labeling, gene knockout and complementation, and computational modeling, we determined the biosynthetic origin of the skeleton, identified the enzymes involved in stereo-/regioselective hydrogenation and methylation, and provided new mechanistic insights into the dimerization. This work not only deciphers the biosynthetic mechanism of bipentaromycins but also provides new strategies for creating biologically active dimeric pharmacophores for drug discovery and development.

6.
Nat Commun ; 13(1): 6135, 2022 10 17.
Article in English | MEDLINE | ID: mdl-36253467

ABSTRACT

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a promising source of new antimicrobials in the face of rising antibiotic resistance. Here, we report a scalable platform that combines high-throughput bioinformatics with automated biosynthetic gene cluster refactoring for rapid evaluation of uncharacterized gene clusters. As a proof of concept, 96 RiPP gene clusters that originate from diverse bacterial phyla involving 383 biosynthetic genes are refactored in a high-throughput manner using a biological foundry with a success rate of 86%. Heterologous expression of all successfully refactored gene clusters in Escherichia coli enables the discovery of 30 compounds covering six RiPP classes: lanthipeptides, lasso peptides, graspetides, glycocins, linear azol(in)e-containing peptides, and thioamitides. A subset of the discovered lanthipeptides exhibit antibiotic activity, with one class II lanthipeptide showing low µM activity against Klebsiella pneumoniae, an ESKAPE pathogen. Overall, this work provides a robust platform for rapidly discovering RiPPs.


Subject(s)
Danazol , Ribosomes , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Danazol/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Multigene Family , Peptides/chemistry , Protein Processing, Post-Translational , Ribosomes/genetics , Ribosomes/metabolism
7.
ACS Chem Biol ; 15(6): 1642-1649, 2020 06 19.
Article in English | MEDLINE | ID: mdl-32356655

ABSTRACT

Lanthipeptides constitute a major family of ribosomally synthesized and post-translationally modified peptides (RiPPs). They are classified into four subfamilies, based on the characteristics of their lanthipeptide synthetases. While over a hundred lanthipeptides have been discovered to date, very few of them are class IV lanthipeptides and the latter are all structurally similar. Here, we identified an uncharacterized group of class IV lanthipeptides using bioinformatics analysis. One representative pathway from Streptomyces sp. NRRL S-1022 was expressed in Escherichia coli, which generated a lanthipeptide with two nonoverlapping rings that have not been reported for known class IV lanthipeptides. Further investigation into the biosynthetic mechanism revealed that multiple modification pathways are in operation in which dehydration and cyclization occur in parallel. While peptidases for maturation of class IV lanthipeptides have been elusive, two aminopeptidases encoded in the genome of Streptomyces sp. NRRL S-1022 were shown to process the modified peptide by the dual endopeptidase/aminopeptidase activity. This work opens doors to discover more class IV lanthipeptides with interesting structural features and biological activities.


Subject(s)
Drug Discovery , Peptides/chemistry , Amino Acid Sequence , Catalysis , Cyclization , Protein Processing, Post-Translational , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
8.
iScience ; 23(1): 100795, 2020 Jan 24.
Article in English | MEDLINE | ID: mdl-31926431

ABSTRACT

Natural products (NPs), also known as secondary metabolites, are produced in bacteria, fungi, and plants. NPs represent a rich source of antibacterial, antifungal, and anticancer agents. Recent advances in DNA sequencing technologies and bioinformatics unveiled nature's great potential for synthesizing numerous NPs that may confer unprecedented structural and biological features. However, discovering novel bioactive NPs by genome mining remains a challenge. Moreover, even with interesting bioactivity, the low productivity of many NPs significantly limits their practical applications. Here we discuss the progress in developing bioinformatics tools for efficient discovery of bioactive NPs. In addition, we highlight computational methods for optimizing the productivity of NPs of pharmaceutical importance.

9.
ACS Chem Biol ; 13(10): 2966-2972, 2018 10 19.
Article in English | MEDLINE | ID: mdl-30183259

ABSTRACT

Glycocins (glycosylated bacteriocins) are a family of ribosomally synthesized and post-translationally modified peptides with antimicrobial activities against pathogens of interest, including methicillin-resistant Staphylococcus aureus, representing a promising source of new antibiotics. Glycocins are still largely underexplored, and thus far, only six glycocins are known. Here, we used genome mining to identify 50 putative glycocin biosynthetic gene clusters and then chose six of them with distinct features for further investigation. Through two rounds of plug-and-play pathway refactoring and expression in Escherichia coli BL21(DE3), four systems produced novel glycocins. Further structural characterization revealed that one of them, which belongs to the enterocin 96-type glycocins, was diglucosylated on a single serine. The other three compounds belong to the SunA/ThuA-type glycocins and exhibit a antimicrobial spectrum narrower than that of sublancin, the best characterized member in this group, even though they share a similar disulfide topology and glycosylation. Further evaluation of their bioactivities with free glucose at high concentrations suggested that their antimicrobial mechanisms might be both glycocin- and species-specific. These glycocins with distinct features significantly broaden our knowledge and may lead to the discovery of new classes of antibiotics.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteriocins/pharmacology , Escherichia coli/metabolism , Amino Acid Sequence , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/isolation & purification , Bacillus cereus/drug effects , Bacillus subtilis/drug effects , Bacteriocins/chemistry , Bacteriocins/isolation & purification , Enterococcus faecium/drug effects , Escherichia coli/genetics , Genomics , Glycosylation , Listeria monocytogenes/drug effects , Methicillin-Resistant Staphylococcus aureus/drug effects , Microbial Sensitivity Tests , Multigene Family , Pseudomonas aeruginosa/drug effects , Sequence Alignment
10.
Crit Rev Biochem Mol Biol ; 53(2): 115-129, 2018 04.
Article in English | MEDLINE | ID: mdl-29411648

ABSTRACT

Biocatalysts have been increasingly used in the synthesis of fine chemicals and medicinal compounds due to significant advances in enzyme discovery and engineering. To mimic the synergistic effects of cascade reactions catalyzed by multiple enzymes in nature, researchers have been developing artificial tandem enzymatic reactions in vivo by harnessing synthetic biology and metabolic engineering tools. There is also growing interest in the development of one-pot tandem enzymatic or chemo-enzymatic processes in vitro due to their neat and concise catalytic systems and product purification procedures. In this review, we will briefly summarize the strategies of designing and optimizing in vitro tandem catalytic reactions, highlight a few representative examples, and discuss the future trend in this field.


Subject(s)
Biocatalysis , Enzymes/chemistry , Metabolic Engineering/methods , Models, Chemical , Synthetic Biology/methods
12.
Biotechnol Bioeng ; 114(8): 1847-1854, 2017 08.
Article in English | MEDLINE | ID: mdl-28401530

ABSTRACT

Pathway refactoring serves as an invaluable synthetic biology tool for natural product discovery, characterization, and engineering. However, the complicated and laborious molecular biology techniques largely hinder its application in natural product research, especially in a high-throughput manner. Here we report a plug-and-play pathway refactoring workflow for high-throughput, flexible pathway construction, and expression in both Escherichia coli and Saccharomyces cerevisiae. Biosynthetic genes were firstly cloned into pre-assembled helper plasmids with promoters and terminators, resulting in a series of expression cassettes. These expression cassettes were further assembled using Golden Gate reaction to generate fully refactored pathways. The inclusion of spacer plasmids in this system would not only increase the flexibility for refactoring pathways with different number of genes, but also facilitate gene deletion and replacement. As proof of concept, a total of 96 pathways for combinatorial carotenoid biosynthesis were built successfully. This workflow should be generally applicable to different classes of natural products produced by various organisms. Biotechnol. Bioeng. 2017;114: 1847-1854. © 2017 Wiley Periodicals, Inc.


Subject(s)
Biological Products/metabolism , Biosynthetic Pathways/genetics , Carotenoids/biosynthesis , Carotenoids/genetics , Protein Engineering/methods , Saccharomyces cerevisiae/physiology , Workflow , Biological Products/isolation & purification , Computer Simulation , Escherichia coli , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Metabolome/physiology , Models, Biological , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Synthetic Biology/methods
13.
Curr Opin Biotechnol ; 48: 21-27, 2017 12.
Article in English | MEDLINE | ID: mdl-28288336

ABSTRACT

Natural products have been a prolific source of antibacterial and anticancer drugs for decades. One of the major challenges in natural product discovery is that the vast majority of natural product biosynthetic gene clusters (BGCs) have not been characterized, partially due to the fact that they are either transcriptionally silent or expressed at very low levels under standard laboratory conditions. Here we describe the strategies developed in recent years (mostly between 2014-2016) for activating silent BGCs. These strategies can be broadly divided into two categories: approaches in native hosts and approaches in heterologous hosts. In addition, we briefly discuss recent advances in developing new computational tools for identification and characterization of BGCs and high-throughput methods for detection of natural products.


Subject(s)
Biological Products/isolation & purification , Drug Discovery/methods , High-Throughput Screening Assays/methods , Animals , Anti-Bacterial Agents/biosynthesis , Biological Products/metabolism , Gene Expression Regulation, Bacterial , Humans , Multigene Family/physiology , RNA Interference , Transcription, Genetic
14.
Curr Opin Chem Biol ; 25: 55-64, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25579451

ABSTRACT

Over the last two decades, directed evolution has become a staple in protein engineering and ushered in a new era of industrial biocatalysis. Directed evolution has provided the tools to not only improve the activity of known biocatalysts, but also to endow biocatalysts with chemical reactivities not previously encountered in nature. Here we will discuss the recent successes in the quest to enhance thermostability, stereoselectivity and activity of biocatalysts, as well as to create novel enzymes, over the last two years.


Subject(s)
Biocatalysis , Directed Molecular Evolution/methods , Enzymes/chemistry , Enzymes/genetics , Enzymes/metabolism , Protein Engineering
SELECTION OF CITATIONS
SEARCH DETAIL