Ribosomally derived lipopeptides containing distinct fatty acyl moieties

Lipopeptides represent a large group of microbial natural products that include important antibacterial and antifungal drugs and some of the most-powerful known biosurfactants. The vast majority of lipopeptides comprise cyclic peptide backbones N-terminally equipped with various fatty acyl moieties. The known compounds of this type are biosynthesized by nonribosomal peptide synthetases, giant enzyme complexes that assemble their products in a non–gene-encoded manner. Here, we report the genome-guided discovery of ribosomally derived, fatty-acylated lipopeptides, termed selidamides. Heterologous reconstitution of three pathways, two from cyanobacteria and one from an arctic, ocean-derived alphaproteobacterium, allowed structural characterization of the probable natural products and suggest that selidamides are widespread over various bacterial phyla. The identified representatives feature cyclic peptide moieties and fatty acyl units attached to (hydroxy)ornithine or lysine side chains by maturases of the GCN5-related N-acetyltransferase superfamily. In contrast to nonribosomal lipopeptides that are usually produced as congener mixtures, the three selidamides are selectively fatty acylated with C10, C12, or C16 fatty acids, respectively. These results highlight the ability of ribosomal pathways to emulate products with diverse, nonribosomal-like features and add to the biocatalytic toolbox for peptide drug improvement and targeted discovery.

Aminoacyl chain translocation catalysed by a type II thioesterase domain in an unusual non-ribosomal peptide synthetase

Non-Ribosomal Peptide Synthetases (NRPSs) assemble a diverse range of natural products with important applications in both medicine and agriculture. They consist of several multienzyme subunits that must interact with each other in a highly controlled manner to facilitate efficient chain transfer, thus ensuring biosynthetic fidelity. Several mechanisms for chain transfer are known for NRPSs, promoting structural diversity. Herein, we report the first biochemically characterized example of a type II thioesterase (TEII) domain capable of catalysing aminoacyl chain transfer between thiolation (T) domains on two separate NRPS subunits responsible for installation of a dehydrobutyrine moiety. Biochemical dissection of this process reveals the central role of the TEII-catalysed chain translocation event and expands the enzymatic scope of TEII domains beyond canonical (amino)acyl chain hydrolysis. The apparent co-evolution of the TEII domain with the NRPS subunits highlights a unique feature of this enzymatic cassette, which will undoubtedly find utility in biosynthetic engineering efforts.

Biosynthesis Pathways, Transport Mechanisms and Biotechnological Applications of Fungal Siderophores

Iron (Fe) is the fourth most abundant element on earth and represents an essential nutrient for life. As a fundamental mineral element for cell growth and development, iron is available for uptake as ferric ions, which are usually oxidized into complex oxyhydroxide polymers, insoluble under aerobic conditions. In these conditions, the bioavailability of iron is dramatically reduced. As a result, microorganisms face problems of iron acquisition, especially under low concentrations of this element. However, some microbes have evolved mechanisms for obtaining ferric irons from the extracellular medium or environment by forming small molecules often regarded as siderophores. Siderophores are high affinity iron-binding molecules produced by a repertoire of proteins found in the cytoplasm of cyanobacteria, bacteria, fungi, and plants. Common groups of siderophores include hydroxamates, catecholates, carboxylates, and hydroximates. The hydroxamate siderophores are commonly synthesized by fungi. L-ornithine is a biosynthetic precursor of siderophores, which is synthesized from multimodular large enzyme complexes through non-ribosomal peptide synthetases (NRPSs), while siderophore-Fe chelators cell wall mannoproteins (FIT1, FIT2, and FIT3) help the retention of siderophores. S. cerevisiae, for example, can express these proteins in two genetically separate systems (reductive and nonreductive) in the plasma membrane. These proteins can convert Fe (III) into Fe (II) by a ferrous-specific metalloreductase enzyme complex and flavin reductases (FREs). However, regulation of the siderophore through Fur Box protein on the DNA promoter region and its activation or repression depend primarily on the Fe availability in the external medium. Siderophores are essential due to their wide range of applications in biotechnology, medicine, bioremediation of heavy metal polluted environments, biocontrol of plant pathogens, and plant growth enhancement.

Secondary metabolite biosynthetic diversity in Arctic Ocean metagenomes

Polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) are mega enzymes responsible for the biosynthesis of a large fraction of natural products (NPs). Molecular markers for biosynthetic genes, such as the ketosynthase (KS) domain of PKSs, have been used to assess the diversity and distribution of biosynthetic genes in complex microbial communities. More recently, metagenomic studies have complemented and enhanced this approach by allowing the recovery of complete biosynthetic gene clusters (BGCs) from environmental DNA. In this study, the distribution and diversity of biosynthetic genes and clusters from Arctic Ocean samples (NICE-2015 expedition), was assessed using PCR-based strategies coupled with high-throughput sequencing and metagenomic analysis. In total, 149 KS domain OTU sequences were recovered, 36 % of which could not be assigned to any known BGC. In addition, 74 bacterial metagenome-assembled genomes were recovered, from which 179 BGCs were extracted. A network analysis identified potential new NP families, including non-ribosomal peptides and polyketides. Complete or near-complete BGCs were recovered, which will enable future heterologous expression efforts to uncover the respective NPs. Our study represents the first report of biosynthetic diversity assessed for Arctic Ocean metagenomes and highlights the potential of Arctic Ocean planktonic microbiomes for the discovery of novel secondary metabolites. The strategy employed in this study will enable future bioprospection, by identifying promising samples for bacterial isolation efforts, while providing also full-length BGCs for heterologous expression.

Reprogramming Versus Subdomain Substitution: An Exploration of Strategies for NRPS Engineering Using the Unique Model System BpsA

<p>Non-ribosomal peptide synthetases (NRPSs) are large enzymes that generate a plethora of important natural products, from antibiotics to immunosuppressants. These modular enzymes function like an assembly line, selecting and incorporating specific (and frequently nonproteinogenic) amino acids into a growing peptide chain. This modular structure offers promise for re-engineering NRPS units to generate new useful products, but progress has to date been limited by the complex and dynamic nature of key domains, and a failure to define generally applicable “rules” to guide engineering efforts. Early efforts to engineer NRPS enzymes relied on the substitution of entire NRPS modules or domains, but product yields were often very low. However, these studies did highlight the promise of targeting the adenylation domain, the part of each NRPS modules that is responsible for selecting each amino acid substrate. Two particularly promising strategies for NRPS engineering aim to manipulate the adenylation domain in ways that minimise steric disruption to the assembly line. The first of these, reprogramming, makes the fewest possible changes to the NRPS primary sequence, but is dependent on those precise changes conforming to the existing structure of the adenylation domain binding pocket. More recently a second technique has been developed, subdomain substitution, which recombines a larger region of the adenylation domain to avoid perturbation of the binding pocket. The research described in this thesis examined and compared both approaches using the unique NRPS BpsA as a model system. BpsA is a single-module NRPS that generates a vivid blue pigment product, making for a reductionist system that offers a robust visual reporter capacity. Experiments with the reprogramming technique showed that small changes to the protein sequence had potential to exert major impacts on enzyme function, even when no change to function was intended. In contrast, experiments with subdomain substitution were generally more effective, showing that NRPS enzymes are very sensitive to the precise boundaries of the substituted region, but that activity can be restored to otherwise non-functional subdomain substitutions by modulation of the regional boundaries.</p>

Reprogramming Versus Subdomain Substitution: An Exploration of Strategies for NRPS Engineering Using the Unique Model System BpsA

<p>Non-ribosomal peptide synthetases (NRPSs) are large enzymes that generate a plethora of important natural products, from antibiotics to immunosuppressants. These modular enzymes function like an assembly line, selecting and incorporating specific (and frequently nonproteinogenic) amino acids into a growing peptide chain. This modular structure offers promise for re-engineering NRPS units to generate new useful products, but progress has to date been limited by the complex and dynamic nature of key domains, and a failure to define generally applicable “rules” to guide engineering efforts. Early efforts to engineer NRPS enzymes relied on the substitution of entire NRPS modules or domains, but product yields were often very low. However, these studies did highlight the promise of targeting the adenylation domain, the part of each NRPS modules that is responsible for selecting each amino acid substrate. Two particularly promising strategies for NRPS engineering aim to manipulate the adenylation domain in ways that minimise steric disruption to the assembly line. The first of these, reprogramming, makes the fewest possible changes to the NRPS primary sequence, but is dependent on those precise changes conforming to the existing structure of the adenylation domain binding pocket. More recently a second technique has been developed, subdomain substitution, which recombines a larger region of the adenylation domain to avoid perturbation of the binding pocket. The research described in this thesis examined and compared both approaches using the unique NRPS BpsA as a model system. BpsA is a single-module NRPS that generates a vivid blue pigment product, making for a reductionist system that offers a robust visual reporter capacity. Experiments with the reprogramming technique showed that small changes to the protein sequence had potential to exert major impacts on enzyme function, even when no change to function was intended. In contrast, experiments with subdomain substitution were generally more effective, showing that NRPS enzymes are very sensitive to the precise boundaries of the substituted region, but that activity can be restored to otherwise non-functional subdomain substitutions by modulation of the regional boundaries.</p>

Gene editing enables rapid engineering of complex antibiotic assembly lines

Re-engineering biosynthetic assembly lines, including nonribosomal peptide synthetases (NRPS) and related megasynthase enzymes, is a powerful route to new antibiotics and other bioactive natural products that are too complex for chemical synthesis. However, engineering megasynthases is very challenging using current methods. Here, we describe how CRISPR-Cas9 gene editing can be exploited to rapidly engineer one of the most complex megasynthase assembly lines in nature, the 2.0 MDa NRPS enzymes that deliver the lipopeptide antibiotic enduracidin. Gene editing was used to exchange subdomains within the NRPS, altering substrate selectivity, leading to ten new lipopeptide variants in good yields. In contrast, attempts to engineer the same NRPS using a conventional homologous recombination-mediated gene knockout and complementation approach resulted in only traces of new enduracidin variants. In addition to exchanging subdomains within the enduracidin NRPS, subdomains from a range of NRPS enzymes of diverse bacterial origins were also successfully utilized.

Discovery, Characterisation and Engineering of Non-Ribosomal Peptide Synthetases and Phosphopantetheinyl Transferase Enzymes

<p>Non-ribosomal peptide synthetases (NRPSs) are multi-modular biosynthetic enzymes that are responsible for the production of many bioactive secondary metabolites produced by microorganisms. They are activated by phosphopantetheinyl transferase (PPTase) enzymes, which attach an essential prosthetic group to a specific site within a “carrier protein” (CP) domain that is an integral part of each NRPS module. Of particular importance in this work is the NRPS BpsA, which produces a blue pigment called indigoidine; but only when BpsA has first been activated by a PPTase. BpsA can be used as a reporter for PPTase activity, to identify PPTases and/or measure their activity. Several CP-substituted BpsA variants were used, in order to study and identify PPTases which may recognise different CP domains. The first part of the research described in this thesis examined the features of foreign CP interactions within BpsA that made these functional substitutions possible. Two key residues, the +4 and +24 positions relative to an invariant serine, were found to be highly important; with appropriate substitutions at these positions yielding active CP-substituted variants. Wild type BpsA and the CP-substituted variants were then used as the basis of a screen to discover new PPTase genes, and associated natural product biosynthetic genes, from metagenomic libraries. The vast majority of bacteria that produce bioactive secondary metabolites are unable to be cultured under laboratory conditions; screening metagenomic libraries is a way to access this untapped biodiversity in order to discover new natural products. Two environmental DNA libraries were screened, and PPTase genes were identified via their ability to activate BpsA, giving rise to blue colonies in high throughput agar plate screens. This screen proved to be a powerful enrichment strategy with almost half of the novel 21 PPTase genes recovered also linked to biosynthetic gene clusters. Using the evolved CP-substituted BpsA variants (and thereby altering the PPTase recognition site) enabled a wider variety of hits to be found. This led to the hypothesis that some of the PPTases discovered via this screening method would have non-overlapping substrate specificities, a beneficial property for certain PPTase applications. The 21 PPTase genes discovered via metagenomic screening were characterised further, using a series of assays involving BpsA to measure their activity. As is common for PPTase enzymes, there were difficulties in obtaining enough soluble protein via purification to perform a detailed analysis of each. Those that were able to be purified had much lower activity than other previously characterised PPTases, and were also not as specific for their CP substrates as they had first appeared to be. Due to these low activity levels, several other previously characterised PPTases were also studied further using the BpsA methods. All PPTases showed a relatively broad activity across a range of CP substrates. The desire to obtain PPTases with more specific substrate specificities led to the development of a directed evolution screen to alter PPTase CP specificity. In a proof-of-principle study the E. coli PPTase EntD was evolved to lose activity with the BpsA CP while retaining activity with its native CP. This screen, the first of its kind to evolve PPTases for greater CP substrate specificity, was successful in recovering several improved variants. These variants had either completely abolished or vastly decreased activity for the WT BpsA CP while retaining the ability to activate the native (EntF) CP domain. The general strategy developed here can be applied to the evolution of other PPTases and CP substrates.</p>

Discovery, Characterisation and Engineering of Non-Ribosomal Peptide Synthetases and Phosphopantetheinyl Transferase Enzymes

<p>Non-ribosomal peptide synthetases (NRPSs) are multi-modular biosynthetic enzymes that are responsible for the production of many bioactive secondary metabolites produced by microorganisms. They are activated by phosphopantetheinyl transferase (PPTase) enzymes, which attach an essential prosthetic group to a specific site within a “carrier protein” (CP) domain that is an integral part of each NRPS module. Of particular importance in this work is the NRPS BpsA, which produces a blue pigment called indigoidine; but only when BpsA has first been activated by a PPTase. BpsA can be used as a reporter for PPTase activity, to identify PPTases and/or measure their activity. Several CP-substituted BpsA variants were used, in order to study and identify PPTases which may recognise different CP domains. The first part of the research described in this thesis examined the features of foreign CP interactions within BpsA that made these functional substitutions possible. Two key residues, the +4 and +24 positions relative to an invariant serine, were found to be highly important; with appropriate substitutions at these positions yielding active CP-substituted variants. Wild type BpsA and the CP-substituted variants were then used as the basis of a screen to discover new PPTase genes, and associated natural product biosynthetic genes, from metagenomic libraries. The vast majority of bacteria that produce bioactive secondary metabolites are unable to be cultured under laboratory conditions; screening metagenomic libraries is a way to access this untapped biodiversity in order to discover new natural products. Two environmental DNA libraries were screened, and PPTase genes were identified via their ability to activate BpsA, giving rise to blue colonies in high throughput agar plate screens. This screen proved to be a powerful enrichment strategy with almost half of the novel 21 PPTase genes recovered also linked to biosynthetic gene clusters. Using the evolved CP-substituted BpsA variants (and thereby altering the PPTase recognition site) enabled a wider variety of hits to be found. This led to the hypothesis that some of the PPTases discovered via this screening method would have non-overlapping substrate specificities, a beneficial property for certain PPTase applications. The 21 PPTase genes discovered via metagenomic screening were characterised further, using a series of assays involving BpsA to measure their activity. As is common for PPTase enzymes, there were difficulties in obtaining enough soluble protein via purification to perform a detailed analysis of each. Those that were able to be purified had much lower activity than other previously characterised PPTases, and were also not as specific for their CP substrates as they had first appeared to be. Due to these low activity levels, several other previously characterised PPTases were also studied further using the BpsA methods. All PPTases showed a relatively broad activity across a range of CP substrates. The desire to obtain PPTases with more specific substrate specificities led to the development of a directed evolution screen to alter PPTase CP specificity. In a proof-of-principle study the E. coli PPTase EntD was evolved to lose activity with the BpsA CP while retaining activity with its native CP. This screen, the first of its kind to evolve PPTases for greater CP substrate specificity, was successful in recovering several improved variants. These variants had either completely abolished or vastly decreased activity for the WT BpsA CP while retaining the ability to activate the native (EntF) CP domain. The general strategy developed here can be applied to the evolution of other PPTases and CP substrates.</p>

Genetic manipulation of pyoverdine non-ribosomal peptide synthetases to identify genetic constraints to effective domain recombination

<p>Non-ribosomal peptide synthetases (NRPSs) synthesise small highly diverse peptides with a wide range of activities, such as antibiotics, anticancer drugs, and immunosuppressants. NRPS synthesis often resembles an assembly line, in which each module acts in a linear order to add one monomer to the growing peptide chain. In the basic mechanism of synthesis, an adenylation (A) domain within each module activates a specific monomer. Once activated, the monomer is attached to an immediately downstream thiolation (T) domain via a prosthetic phosphopantheine group, which acts as a flexible arm to pass the substrate between catalytic domains. A condensation (C) domain, upstream to the A-T domains, catalyses peptide bond formation between an acceptor substrate attached to the T domain and a donor substrate attached to the T domain of the upstream module. The peptide remains attached to the T domain of the acceptor substrate, and then acts as the donor substrate for the next C domain. When peptide synthesis reaches the final module, the peptide is released by a thioesterase (TE) domain. The linear mode of synthesis and discrete functional domains within each module gives the potential to generate new products by substituting domains or entire modules with ones that activate alternative substrates. Attempts to create new products using domain and module substitution often result in a loss of activity. The work in this thesis focuses on identifying barriers to effective domain substitution. The NRPS enzyme pvdD, which adds the final residue to the eleven residue non-ribosomal peptide pyoverdine, was developed as a model for domain substitution. The primary benefit for using this model is that pyoverdine creates easily detectible fluorescent products. The first set of experiments focused on testing the limitations of A domain and C-A domain substitutions to alter pyoverdine. Nine A domain and nine C-A domain substitution pvdD variants were constructed and used to complement a P. aeruginosa PAO1 pvdD deletion strain. The A domain substitutions that specified the wild type substrate were highly functional, whereas A domains that specified other substrates resulted in low levels of wild type pyoverdine production. This suggests the acceptor site substrate specificity of the C domain limited the success of A domain substitutions, rather than disruption of the C/A domain junction. In contrast, although C-A domain substitutions in pvdD in some cases synthesised novel pyoverdines, the majority lost function for unknown reasons. The high success rate A domain substitutions (when not limited by the acceptor site specificity of the C domain) suggested the addition of new C domains was a likely cause for loss of function. The second set of experiments investigated whether disrupting the protein interface between C domains and their upstream T domains may cause a loss in function of C-A domain substitutions. However, domain substitutions of T domains were found to have a high rate of success. Therefore, the results thus far confirmed that disrupting interactions of the C domain with A domains or T domains does not have a large affect on enzyme activity. An alternative explanation for the loss in function with C-A domain substitutions is that C domains translocated to a new enzyme are unable to process the new incoming donor peptide chain because of substrate specificity or steric constraints. To develop methods to circumvent limitations caused by the C domain, the final part of this thesis examined acceptor substrate specificity of C domains. Acceptor site substrate specificity was chosen over donor site specificity as it acts on only an amino acid rather than peptide chain. The substrate specificity was narrowed down to a small subsection of the C domain. This was an initial study of C domain substrate specificity, which may guide future development of relaxed specificity C domains.</p>