Speed dating for enzymes! Finding the perfect phosphopantetheinyl transferase partner for your polyketide synthase

The biosynthetic pathways for the fungal polyketides bikaverin and bostrycoidin, from Fusarium verticillioides and Fusarium solani respectively, were reconstructed and heterologously expressed in S. cerevisiae alongside seven different phosphopantetheinyl transferases (PPTases) from a variety of origins spanning bacterial, yeast and fungal origins. In order to gauge the efficiency of the interaction between the ACP-domains of the polyketide synthases (PKS) and PPTases, each were co-expressed individually and the resulting production of target polyketides were determined after 48 h of growth. In co-expression with both biosynthetic pathways, the PPTase from Fusarium verticillioides (FvPPT1) proved most efficient at producing both bikaverin and bostrycoidin, at 1.4 mg/L and 5.9 mg/L respectively. Furthermore, the remaining PPTases showed the ability to interact with both PKS’s, except for a single PKS-PPTase combination. The results indicate that it is possible to boost the production of a target polyketide, simply by utilizing a more optimal PPTase partner, instead of the commonly used PPTases; NpgA, Gsp and Sfp, from Aspergillus nidulans, Brevibacillus brevis and Bacillus subtilis respectively.

Engineering the indigoidine-synthesising enzyme BpsA for diverse applications in biotechnology

<p>Non-ribosomal peptide synthetases (NRPSs) are large, modular enzymes that synthesise bioactive peptides using an assembly line architecture, wherein each module is responsible for the incorporation of a monomer into the growing chain. Present in both fungi and bacteria, NRPSs are responsible for a wide variety of secondary metabolites and bioactive compounds including siderophores, antibiotics, anti-cancer compounds and immunosuppressants. For functionality, NRPSs require the attachment of a phosphopantetheine moiety to their peptidyl carrier protein domains. This reaction is catalysed by a phosphopantetheinyl transferase (PPTase). The NRPS blue pigment synthetase A (BpsA) is unusual in that it is comprised of only a single module. BpsA contains an adenylation domain that recognises and sequentially binds two molecules of L-glutamine, an oxidation domain that is believed to oxidise each glutamine monomer, a peptidyl carrier protein domain that binds the phosphopantetheine moiety, and a thioesterase domain that cyclises each glutamine and releases the final bicyclic product from the enzyme. This final product is a blue pigment called indigoidine, and its synthesis from two molecules of L-glutamine is powered by ATP. Comparatively to other NRPSs BpsA is easy to manipulate and work with both in vitro and in vivo. Here, the ability to easily detect synthesis of indigoidine was utilised to provide a versatile reporter to detect a variety of biochemical activities. PPTases are essential enzymes that are promising drug targets in the clinically important bacteria Pseudomonas aeruginosa and Mycobacterium tuberculosis. BpsA can be purified in the inactive apo form, which then requires a PPTase to activate it to enable indigoidine synthesis. Here it was shown that mixing BpsA, a PPTase, the enzymatic substrates, and a potential inhibitor enables screening for PPTase inhibition by monitoring the rate or extent of indigoidine synthesis. This method was optimised and used to screen commercial drug libraries against two PPTases, PcpS from P. aeruginosa and PptT from M. tuberculosis. Several novel inhibitors were identified and pilot in vivo studies were performed. M. tuberculosis also possesses a second essential PPTase called TB-AcpS, which has very narrow substrate specificity and cannot post-translationally modify BpsA. In an attempt to widen the substrate specificity a combination of rational engineering and directed evolution was employed. These attempts did not yield significant improvements in the ability of TB-AcpS to activate modified BpsA, however they did yield mutants that were more effective substrates for other type I PPTases. The easily detectable nature of indigoidine also enabled application of BpsA as a reporter for a range of different substrates. Particularly effective was development of a commercially applicable method using BpsA to quantify L-glutamine in a range of conditions, including cell culture media and blood. The assay developed offers several advantages over currently available kits. BpsA was also used to detect and quantify ATP, and this was applied to monitor adenylation reactions. Finally, the ability of BpsA to synthesise indigoidine-like compounds from glutamine analogues was explored.</p>

Engineering the indigoidine-synthesising enzyme BpsA for diverse applications in biotechnology

<p>Non-ribosomal peptide synthetases (NRPSs) are large, modular enzymes that synthesise bioactive peptides using an assembly line architecture, wherein each module is responsible for the incorporation of a monomer into the growing chain. Present in both fungi and bacteria, NRPSs are responsible for a wide variety of secondary metabolites and bioactive compounds including siderophores, antibiotics, anti-cancer compounds and immunosuppressants. For functionality, NRPSs require the attachment of a phosphopantetheine moiety to their peptidyl carrier protein domains. This reaction is catalysed by a phosphopantetheinyl transferase (PPTase). The NRPS blue pigment synthetase A (BpsA) is unusual in that it is comprised of only a single module. BpsA contains an adenylation domain that recognises and sequentially binds two molecules of L-glutamine, an oxidation domain that is believed to oxidise each glutamine monomer, a peptidyl carrier protein domain that binds the phosphopantetheine moiety, and a thioesterase domain that cyclises each glutamine and releases the final bicyclic product from the enzyme. This final product is a blue pigment called indigoidine, and its synthesis from two molecules of L-glutamine is powered by ATP. Comparatively to other NRPSs BpsA is easy to manipulate and work with both in vitro and in vivo. Here, the ability to easily detect synthesis of indigoidine was utilised to provide a versatile reporter to detect a variety of biochemical activities. PPTases are essential enzymes that are promising drug targets in the clinically important bacteria Pseudomonas aeruginosa and Mycobacterium tuberculosis. BpsA can be purified in the inactive apo form, which then requires a PPTase to activate it to enable indigoidine synthesis. Here it was shown that mixing BpsA, a PPTase, the enzymatic substrates, and a potential inhibitor enables screening for PPTase inhibition by monitoring the rate or extent of indigoidine synthesis. This method was optimised and used to screen commercial drug libraries against two PPTases, PcpS from P. aeruginosa and PptT from M. tuberculosis. Several novel inhibitors were identified and pilot in vivo studies were performed. M. tuberculosis also possesses a second essential PPTase called TB-AcpS, which has very narrow substrate specificity and cannot post-translationally modify BpsA. In an attempt to widen the substrate specificity a combination of rational engineering and directed evolution was employed. These attempts did not yield significant improvements in the ability of TB-AcpS to activate modified BpsA, however they did yield mutants that were more effective substrates for other type I PPTases. The easily detectable nature of indigoidine also enabled application of BpsA as a reporter for a range of different substrates. Particularly effective was development of a commercially applicable method using BpsA to quantify L-glutamine in a range of conditions, including cell culture media and blood. The assay developed offers several advantages over currently available kits. BpsA was also used to detect and quantify ATP, and this was applied to monitor adenylation reactions. Finally, the ability of BpsA to synthesise indigoidine-like compounds from glutamine analogues was explored.</p>

Engineering and Characterisation of Bacterial Phosphopantetheinyl Transferases and their Peptide Substrates

<p>Throughout all domains of life, phosphopantetheinyl transferase (PPTase) enzymes catalyse a post-translational modification that is important in both primary and secondary metabolism; the transfer of a phosphopantetheine (PPant) group derived from Coenzyme A to specific protein domains within large, multi-modular biosynthetic enzymes, thereby activating each module for biosynthesis. The short peptide motif of the protein to which this group is attached is known as a ‘tag’, and can be fused to other proteins, making them also substrates for post-translational modification by a PPTase. Additionally, it has been demonstrated that PPTases can utilise a diverse range of CoA analogues, such as biotin-linked or click-chemistry capable CoA derivatives, as substrates for tag attachment. Together, these characteristics make post-translational modification by PPTases an attractive system for many different biotechnological applications. Perhaps the most significant application is in vivo and in vitro site-specific labelling of proteins, for which current technologies are hindered by cumbersome fusion protein requirements, toxicity of the process, or limited reporter groups that can be attached. Confoundingly, most PPTases exhibit a high degree of substrate promiscuity which limits the number of PPTase-tag pairs that can be used simultaneously, and therefore the number of protein targets that can be simultaneously labelled. To address this, directed evolution at a single gene level was used in an attempt to generate multiple PPTase variants that have non-overlapping tag specificity which have applications in orthogonal labelling. Furthermore, assays for the rapid identification, characterisation and evolution of short, novel peptide motifs that are recognised by PPTases has further diversified the labelling toolkit. These developments have enhanced the utility of the PPTase system and potentially have a wide range of applications in a number of fields.</p>

Engineering and Characterisation of Bacterial Phosphopantetheinyl Transferases and their Peptide Substrates

<p>Throughout all domains of life, phosphopantetheinyl transferase (PPTase) enzymes catalyse a post-translational modification that is important in both primary and secondary metabolism; the transfer of a phosphopantetheine (PPant) group derived from Coenzyme A to specific protein domains within large, multi-modular biosynthetic enzymes, thereby activating each module for biosynthesis. The short peptide motif of the protein to which this group is attached is known as a ‘tag’, and can be fused to other proteins, making them also substrates for post-translational modification by a PPTase. Additionally, it has been demonstrated that PPTases can utilise a diverse range of CoA analogues, such as biotin-linked or click-chemistry capable CoA derivatives, as substrates for tag attachment. Together, these characteristics make post-translational modification by PPTases an attractive system for many different biotechnological applications. Perhaps the most significant application is in vivo and in vitro site-specific labelling of proteins, for which current technologies are hindered by cumbersome fusion protein requirements, toxicity of the process, or limited reporter groups that can be attached. Confoundingly, most PPTases exhibit a high degree of substrate promiscuity which limits the number of PPTase-tag pairs that can be used simultaneously, and therefore the number of protein targets that can be simultaneously labelled. To address this, directed evolution at a single gene level was used in an attempt to generate multiple PPTase variants that have non-overlapping tag specificity which have applications in orthogonal labelling. Furthermore, assays for the rapid identification, characterisation and evolution of short, novel peptide motifs that are recognised by PPTases has further diversified the labelling toolkit. These developments have enhanced the utility of the PPTase system and potentially have a wide range of applications in a number of fields.</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>

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>

Phosphopantetheinyl transferase binding and inhibition by amidino-urea and hydroxypyrimidinethione compounds

Owing to their role in activating enzymes essential for bacterial viability and pathogenicity, phosphopantetheinyl transferases represent novel and attractive drug targets. In this work, we examined the inhibitory effect of the aminido-urea 8918 compound against the phosphopantetheinyl transferases PptAb from Mycobacterium abscessus and PcpS from Pseudomonas aeruginosa, two pathogenic bacteria associated with cystic fibrosis and bronchiectasis, respectively. Compound 8918 exhibits inhibitory activity against PptAb but displays no activity against PcpS in vitro, while no antimicrobial activity against Mycobacterium abscessus or Pseudomonas aeruginosa could be detected. X-ray crystallographic analysis of 8918 bound to PptAb-CoA alone and in complex with an acyl carrier protein domain in addition to the crystal structure of PcpS in complex with CoA revealed the structural basis for the inhibition mechanism of PptAb by 8918 and its ineffectiveness against PcpS. Finally, in crystallo screening of potent inhibitors from the National Cancer Institute library identified a hydroxypyrimidinethione derivative that binds PptAb. Both compounds could serve as scaffolds for the future development of phosphopantetheinyl transferases inhibitors.

Inhibition of Indigoidine Synthesis as a High-Throughput Colourimetric Screen for Antibiotics Targeting the Essential Mycobacterium tuberculosis Phosphopantetheinyl Transferase PptT

A recently-validated and underexplored drug target in Mycobacterium tuberculosis is PptT, an essential phosphopantetheinyl transferase (PPTase) that plays a critical role in activating enzymes for both primary and secondary metabolism. PptT possesses a deep binding pocket that does not readily accept labelled coenzyme A analogues that have previously been used to screen for PPTase inhibitors. Here we report on the development of a high throughput, colourimetric screen that monitors the PptT-mediated activation of the non-ribosomal peptide synthetase BpsA to a blue pigment (indigoidine) synthesising form in vitro. This screen uses unadulterated coenzyme A, avoiding analogues that may interfere with inhibitor binding, and requires only a single-endpoint measurement. We benchmark the screen using the well-characterised Library of Pharmaceutically Active Compounds (LOPAC1280) collection and show that it is both sensitive and able to distinguish weak from strong inhibitors. We further show that the BpsA assay can be applied to quantify the level of inhibition and generate consistent EC50 data. We anticipate these tools will facilitate both the screening of established chemical collections to identify new anti-mycobacterial drug leads and to guide the exploration of structure-activity landscapes to improve existing PPTase inhibitors.