Phosphopantetheinyl Transferase Research Articles

De novo production of aromatic m-cresol in Saccharomyces cerevisiae mediated by heterologous polyketide synthases combined with a 6-methylsalicylic acid decarboxylase

As a flavor and platform chemical, m-cresol (3-methylphenol) is a valuable industrial compound that currently is mainly synthesized by chemical methods from fossil resources. In this study, we present the first biotechnological de novo production of m-cresol from sugar in complex yeast extract-peptone medium with the yeast Saccharomyces cerevisiae. A heterologous pathway based on the decarboxylation of the polyketide 6-methylsalicylic acid (6-MSA) was introduced into a CEN.PK yeast strain. For synthesis of 6-MSA, expression of different variants of 6-MSA synthases (MSASs) were compared. Overexpression of codon-optimized MSAS from Penicillium patulum together with activating phosphopantetheinyl transferase npgA from Aspergillus nidulans resulted in up to 367 mg/L 6-MSA production. Additional genomic integration of the genes had a strongly promoting effect and 6-MSA titers reached more than 2 g/L. Simultaneous expression of 6-MSA decarboxylase patG from A. clavatus led to the complete conversion of 6-MSA and production of up to 589 mg/L m-cresol. As addition of 450–750 mg/L m-cresol to yeast cultures nearly completely inhibited growth our data suggest that the toxicity of m-cresol might be the limiting factor for higher production titers.

Modular type I polyketide synthase acyl carrier protein domains share a common N-terminally extended fold

Acyl carrier protein (ACP) domains act as interaction hubs within modular polyketide synthase (PKS) systems, employing specific protein-protein interactions to present acyl substrates to a series of enzyme active sites. Many domains from the multimodular PKS that generates the toxin mycolactone display an unusually high degree of sequence similarity, implying that the few sites which vary may do so for functional reasons. When domain boundaries based on prior studies were used to prepare two isolated ACP segments from this system for studies of their interaction properties, one fragment adopted the expected tertiary structure, but the other failed to fold, despite sharing a sequence identity of 49%. Secondary structure prediction uncovered a previously undetected helical region (H0) that precedes the canonical helix-bundle ACP topology in both cases. This article reports the NMR solution structures of two N-terminally extended mycolactone mACP constructs, mH0ACPa and mH0ACPb, both of which possess an additional α-helix that behaves like a rigid component of the domain. The interactions of these species with a phosphopantetheinyl transferase and a ketoreductase domain are unaffected by the presence of H0, but a shorter construct that lacks the H0 region is shown to be substantially less thermostable than mH0ACPb. Bioinformatics analysis suggests that the extended H0-ACP motif is present in 98% of type I cis-acyltransferase PKS chain-extension modules. The polypeptide linker that connects an H0-ACP motif to the preceding domain must therefore be ~12 residues shorter than previously thought, imposing strict limits on ACP-mediated substrate delivery within and between PKS modules.

Opposing reactions in coenzyme A metabolism sensitize Mycobacterium tuberculosis to enzyme inhibition.

Mycobacterium tuberculosis (Mtb) is the leading infectious cause of death in humans. Synthesis of lipids critical for Mtb's cell wall and virulence depends on phosphopantetheinyl transferase (PptT), an enzyme that transfers 4'-phosphopantetheine (Ppt) from coenzyme A (CoA) to diverse acyl carrier proteins. We identified a compound that kills Mtb by binding and partially inhibiting PptT. Killing of Mtb by the compound is potentiated by another enzyme encoded in the same operon, Ppt hydrolase (PptH), that undoes the PptT reaction. Thus, loss-of-function mutants of PptH displayed antimicrobial resistance. Our PptT-inhibitor cocrystal structure may aid further development of antimycobacterial agents against this long-sought target. The opposing reactions of PptT and PptH uncover a regulatory pathway in CoA physiology.

A wrench in the gears of life

Tuberculosis Tuberculosis is a global health crisis that threatens to become worse as resistance to existing drugs emerges. Identifying ideal targets for drug development requires knowledge of weak points in biochemical pathways that are specific for the pathogen but are absent in hosts. Ballinger et al. identified a small molecule that inhibits the enzyme phosphopantetheinyl transferase (PptT), which is crucial for biosynthesis of mycobacterial structural and virulence lipids (see the Perspective by Mizrahi and Warner). Treatment resulted in selective killing of the bacteria in vitro and in a mouse model. The target pathways were made sensitive to PptT inhibition by a second enzyme, phosphopantetheine hydrolase, whose activity opposes that of the transferase. Science , this issue p. [eaau8959][1]; see also p. [457][2] [1]: /lookup/doi/10.1126/science.aau8959 [2]: /lookup/doi/10.1126/science.aaw5224

Biosynthesis in vitro of bacillamide intermediate-heterocyclic AlaCysthiazole by heterologous expression of nonribosomal peptide synthetase (NRPS)

Bacillamide C, a potential natural antialgae active compound, is produced by Bacillus atrophaeus C89 derived from marine sponge Dysidea avara. A nonribosomal peptide synthetase (NRPS) cluster is hypothesized to be involved in the biosynthesis of bacillamide C. The NRPS with a domain string of A1-PCP1-Cy-A2-PCP2-C can be divided into three functional modules. After heterologous expression and purification of module A1-PCP1 and module Cy-A2-PCP2, their catalytic activities were biochemically proven in vitro by the reaction with the apo-PCP domain transformed to the holo-PCP domain through a phosphopantetheinyl transferase, ATP, and substrate amino acids. Five– membered heterocyclic AlaCysthiazole with molecular weight of 172.0389 was detected. This proved the formation of the heterocyclic dipeptide AlaCysthiazole, which is considered to be a building block for the biosynthesis of bacillamide. This study provides a basis for further biosynthesis of bacillamides.

Site-Specific Antibody Labeling Using Phosphopantetheinyl Transferase-Catalyzed Ligation.

4'-Phosphopantetheinyl transferases (PPTases) have been employed by researchers as versatile biocatalysts for the site-specific modification of numerous protein targets with structurally diverse molecules. Here we describe the use of these enzymes for the production of homogeneous antibody-drug conjugates (ADCs), which have garnered much attention as innovative anticancer drugs. The exceptionally broad substrate tolerance of PPTases allows for one-step and two-step conjugation strategies for site-specific ADC synthesis. While one-step conjugation involves direct coupling of a drug molecule to an antibody, two-step conjugation provides increased flexibility and efficiency of the conjugation process by first attaching a bioorthogonal chemical handle that is then used for drug molecule attachment in a second step. The aim of this chapter is to outline detailed protocols for both labeling procedures, as well as to provide guidance on enzyme and substrate preparation.

Discovering de novo peptide substrates for enzymes using machine learning

The discovery of peptide substrates for enzymes with exclusive, selective activities is a central goal in chemical biology. In this paper, we develop a hybrid computational and biochemical method to rapidly optimize peptides for specific, orthogonal biochemical functions. The method is an iterative machine learning process by which experimental data is deposited into a mathematical algorithm that selects potential peptide substrates to be tested experimentally. Once tested, the algorithm uses the experimental data to refine future selections. This process is repeated until a suitable set of de novo peptide substrates are discovered. We employed this technology to discover orthogonal peptide substrates for 4’-phosphopantetheinyl transferase, an enzyme class that covalently modifies proteins. In this manner, we have demonstrated that machine learning can be leveraged to guide peptide optimization for specific biochemical functions not immediately accessible by biological screening techniques, such as phage display and random mutagenesis.

Phosphopantetheinyl transferase (Ppt)-mediated biosynthesis of lysine, but not siderophores or DHN melanin, is required for virulence of Zymoseptoria tritici on wheat

Zymoseptoria tritici is the causal agent of Septoria tritici blotch (STB) disease of wheat. Z. tritici is an apoplastic fungal pathogen, which does not penetrate plant cells at any stage of infection, and has a long initial period of symptomless leaf colonisation. During this phase it is unclear to what extent the fungus can access host plant nutrients or communicate with plant cells. Several important primary and secondary metabolite pathways in fungi are regulated by the post-translational activator phosphopantetheinyl transferase (Ppt) which provides an essential co-factor for lysine biosynthesis and the activities of non-ribosomal peptide synthases (NRPS) and polyketide synthases (PKS). To investigate the relative importance of lysine biosynthesis, NRPS-based siderophore production and PKS-based DHN melanin biosynthesis, we generated deletion mutants of ZtPpt. The ∆ZtPpt strains were auxotrophic for lysine and iron, non-melanised and non-pathogenic on wheat. Deletion of the three target genes likely affected by ZtPpt loss of function (Aar- lysine; Nrps1-siderophore and Pks1- melanin), highlighted that lysine auxotrophy was the main contributing factor for loss of virulence, with no reduction caused by loss of siderophore production or melanisation. This reveals Ppt, and the lysine biosynthesis pathway, as potential targets for fungicides effective against Z. tritici.

A Novel Rapid Fungal Promoter Analysis System Using the Phosphopantetheinyl Transferase Gene, npgA, in Aspergillus nidulans

To develop a convenient promoter analysis system for fungi, a null-pigment mutant (NPG) of Aspergillus nidulans was used with the 4′-phosphopantetheinyl transferase (PPTase) gene, npgA, which restores the normal pigmentation in A. nidulans, as a new reporter gene. The functional organization of serially deleted promoter regions of the A. nidulans trpC gene and the Cryphonectria parasitica crp gene in filamentous fungi was representatively investigated to establish a novel fungal promoter assay system that depends on color complementation of the NPG mutant with the PPTase npgA gene. Several promoter regions of the trpC and crp genes were fused to the npgA gene containing the 1,034-bp open reading frame and the 966-bp 3’ downstream region from the TAA, and the constructed fusions were introduced into the NPG mutant in A. nidulans to evaluate color recovery due to the transcriptional activity of the sequence elements. Serial deletion of the trpC and crp promoter regions in this PPTase reporter assay system reaffirmed results in previous reports by using the fungal transformation step without a laborious verification process. This approach suggests a more rapid and convenient system than conventional analyses for fungal gene expression studies.

Enzyme-Mediated, Site-Specific Protein Coupling Strategies for Surface-Based Binding Assays.

Covalent surface immobilization of proteins for binding assays is typically performed non-specifically via lysine residues. However, receptors that either have lysines near their binding pockets, or whose presence at the sensor surface is electrostatically disfavoured, can be hard to probe. To overcome these limitations and to improve the homogeneity of surface functionalization, we adapted and optimized three different enzymatic coupling strategies (4'-phosphopantetheinyl transferase, sortase A, and asparaginyl endopeptidase) for biolayer interferometry surface modification. All of these enzymes can be used to site-specifically and covalently ligate proteins of interest via short recognition sequences. The enzymes function under mild conditions and thus immobilization does not affect the receptors' functionality. We successfully employed this enzymatic surface functionalization approach to study the binding kinetics of two different receptor-ligand pairs.

Enzyme‐Mediated, Site‐Specific Protein Coupling Strategies for Surface‐Based Binding Assays

AbstractCovalent surface immobilization of proteins for binding assays is typically performed non‐specifically via lysine residues. However, receptors that either have lysines near their binding pockets, or whose presence at the sensor surface is electrostatically disfavoured, can be hard to probe. To overcome these limitations and to improve the homogeneity of surface functionalization, we adapted and optimized three different enzymatic coupling strategies (4′‐phosphopantetheinyl transferase, sortase A, and asparaginyl endopeptidase) for biolayer interferometry surface modification. All of these enzymes can be used to site‐specifically and covalently ligate proteins of interest via short recognition sequences. The enzymes function under mild conditions and thus immobilization does not affect the receptors’ functionality. We successfully employed this enzymatic surface functionalization approach to study the binding kinetics of two different receptor–ligand pairs.

Bioorthogonal Elastin-like Polypeptide Scaffolds for Immunoassay Enhancement.

Artificial multiprotein complexes are sought after reagents for biomolecular engineering. A current limiting factor is the paucity of molecular scaffolds which allow for site-specific multicomponent assembly. Here, we address this limitation by synthesizing bioorthogonal elastin-like polypeptide (ELP) scaffolds containing periodic noncanonical l-azidohomoalanine amino acids in the guest residue position. The nine azide ELP guest residues served as conjugation sites for site-specific modification with dibenzocyclooctyne (DBCO)-functionalized single-domain antibodies (SdAbs) through strain-promoted alkyne-azide cycloaddition (SPAAC). Sortase A and ybbR tags at the C- and N-termini of the ELP scaffold provided two additional sites for derivatization with small molecules and peptides by Sortase A and 4'-phosphopantetheinyl transferase (Sfp), respectively. These functional groups are chemically bioorthogonal, mutually compatible, and highly efficient, thereby enabling synthesis of multi-antibody ELP complexes in a one-pot reaction. We demonstrate application of this material for enhancing the performance of sandwich immunoassays of the recombinant protein mCherry. In undiluted human plasma, surfaces modified with multi-antibody ELP complexes showed between 2.3- and 14.3-fold improvement in sensitivity and ∼30-40% lower limits of detection as compared with nonspecifically adsorbed antibodies. Dual-labeled multi-antibody ELP complexes were further used for cytometric labeling and analysis of live eukaryotic cells. These results demonstrate how multiple antibodies complexed onto bioorthogonal protein-based polymers can be used to enhance immunospecific binding interactions through multivalency effects.

Photosynthesis-dependent biosynthesis of medium chain-length fatty acids and alcohols

Cyanobacteria can directly channel atmospheric CO2 into a wide range of versatile carbon products such as fatty acids and fatty alcohols with applications including fuel, cosmetics, and health products. Works on alcohol production in cyanobacteria have so far focused on either long (C12-C18) or short (C2-C4) chain-length products. In the present work, we report the first synthetic pathway for 1-octanol (C8) biosynthesis in Synechocystis sp. PCC 6803, employing a carboxylic acid reductase and C8-preferring fatty acyl-ACP thioesterase. The first engineered strain produced 1-octanol but exhibited poor productivity and cellular health issues. We therefore proceeded to systematically optimize the strain and cultivation conditions in order to understand what the limiting factors were. The identification of optimal promoters and ribosomal binding sites, in combination with isopropyl myristate solvent overlay, resulted in a combined (C8–OH and C10–OH) titer of more than 100 mg/L (a 25-fold improvement relative to the first engineered strain) and a restoration of cellular health. Additionally, more than 905 mg/L 1-octanol was produced when the strain expressing sfp (phosphopantetheinyl transferase) and car (carboxylic acid reductase) was fed with octanoic acid. A combination of feeding experiments and protein quantification indicated that the supply of octanoic acid from the introduced thioesterase, and possibly also native fatty acid synthesis pathway, were the main bottlenecks of the pathway.

Distribution and functional analysis of the phosphopantetheinyl transferase superfamily in Actinomycetales microorganisms

Phosphopantetheinyl transferases (PPTases) are a superfamily of essential enzymes required for the synthetic processes of many compounds including fatty acid, polyketide, and nonribosomal peptide metabolites. These enzymes activate carrier proteins in specific biosynthetic pathways via the transfer of a phosphopantetheinyl moiety to a serine residue in the conserved motif of carrier proteins. Since many Actinomycetales microorganisms produce a number of polyketide and nonribosomal peptide metabolites, the distribution of PPTase genes was investigated in these microorganisms. PPTases were found in bacterial protein databases using a hidden Markov model search with the PF01648 (4'-phosphopantetheinyl transferase superfamily) model. Actinomycetales microorganisms harbor several genes encoding AcpS-type and Sfp-type PPTases in individual genomes, many of which were associated with the biosynthetic gene cluster for polyketide or nonribosomal peptide metabolites. The properties of these PPTases were evaluated in the heterologous expression system using the biosynthetic gene clusters and genes encoding PPTases found in the present study. Sfp-type PPTases were classified into two subgroups, and although the substrate specificities of the enzymes in one subgroup were wide, the catalytic activities of enzymes in the other subgroup were low. SAV_1784 of Streptomyces avermitilis possessed the most characteristic broad-range activity against several type I polyketide synthases and nonribosomal peptide synthetases.

An engineered fatty acid synthase combined with a carboxylic acid reductase enables de novo production of 1-octanol in Saccharomyces cerevisiae

BackgroundThe ideal biofuel should not only be a regenerative fuel from renewable feedstocks, but should also be compatible with the existing fuel distribution infrastructure and with normal car engines. As the so-called drop-in biofuel, the fatty alcohol 1-octanol has been described as a valuable substitute for diesel and jet fuels and has already been produced fermentatively from sugars in small amounts with engineered bacteria via reduction of thioesterase-mediated premature release of octanoic acid from fatty acid synthase or via a reversal of the β-oxidation pathway.ResultsThe previously engineered short-chain acyl-CoA producing yeast Fas1R1834K/Fas2 fatty acid synthase variant was expressed together with carboxylic acid reductase from Mycobacterium marinum and phosphopantetheinyl transferase Sfp from Bacillus subtilis in a Saccharomyces cerevisiae Δfas1 Δfas2 Δfaa2 mutant strain. With the involvement of endogenous thioesterases, alcohol dehydrogenases, and aldehyde reductases, the synthesized octanoyl-CoA was converted to 1-octanol up to a titer of 26.0 mg L−1 in a 72-h fermentation. The additional accumulation of 90 mg L−1 octanoic acid in the medium indicated a bottleneck in 1-octanol production. When octanoic acid was supplied externally to the yeast cells, it could be efficiently converted to 1-octanol indicating that re-uptake of octanoic acid across the plasma membrane is not limiting. Additional overexpression of aldehyde reductase Ahr from Escherichia coli nearly completely prevented accumulation of octanoic acid and increased 1-octanol titers up to 49.5 mg L−1. However, in growth tests concentrations even lower than 50.0 mg L−1 turned out to be inhibitory to yeast growth. In situ extraction in a two-phase fermentation with dodecane as second phase did not improve growth, indicating that 1-octanol acts inhibitive before secretion. Furthermore, 1-octanol production was even reduced, which results from extraction of the intermediate octanoic acid to the organic phase, preventing its re-uptake.ConclusionsBy providing chain length control via an engineered octanoyl-CoA producing fatty acid synthase, we were able to specifically produce 1-octanol with S. cerevisiae. Before metabolic engineering can be used to further increase product titers and yields, strategies must be developed that cope with the toxic effects of 1-octanol on the yeast cells.

Mechanical Polyprotein Assembly Using Sfp and Sortase‐Mediated Domain Oligomerization for Single‐Molecule Studies

AbstractSingle‐molecule force spectroscopy (SMFS) with the atomic force microscope (AFM) provides remarkable details on the energy landscapes governing protein (un)folding and intermolecular complex dissociation. In such experiments, multidomain polyproteins consisting of multiple copies of independently foldable domains provide internal controls identifiable by characteristic contour length increments, unfolding forces, and/or unfolding substeps. Here, a new approach to polyprotein synthesis is presented relying on posttranslational enzyme‐mediated oligomerization of domains. Mutant variants of immunoglobulin 27 (I27) and a bacterial cellulose binding module (CBM) fused to an Ig‐like X‐module (XMod), and a mechanostable receptor called Dockerin (Doc) are produced with complementary peptide tags. By utilizing 4′‐phosphopantetheinyl transferase and Sortase A, the system enables I27‐domain oligomerization into polyproteins of varying lengths followed by C‐terminal capping with mechanostable Doc. The number of oligomerized domains per molecule, the unfolding forces, and the complex rupture forces of posttranslationally assembled polyproteins are characterized using >40 h automated AFM–SMFS with a Cohesin (Coh)‐modified cantilever. Use of the Coh–Doc interaction to unfold polyproteins provides a high yield of ≈ 3800 specific single‐molecule interaction curves. This approach is advantageous for assembly of polyproteins from domains that lack proper folding or are insoluble in a polyprotein format.

An In Vitro and In Vivo Study of Broad-Range Phosphopantetheinyl Transferases for Heterologous Expression of Cyanobacterial Natural Products.

Phosphopantetheinyl transferases catalyze the post-translational modification of carrier proteins involved in both primary and secondary metabolism. The functional expression of polyketide synthases and nonribosomal peptide synthetases requires the activation of all carrier protein domains by phosphopantetheinyl transferases. Here we describe the characterization of five bacterial phosphopantetheinyl transferases by their substrate specificity and catalytic efficiency of four cyanobacterial carrier proteins. Comparative in vitro phosphopantetheinylation analysis showed Sfp possesses the highest catalytic efficiency over various carrier proteins. In vivo coexpression of phosphopantetheinyl transferases with carrier proteins revealed a broad range substrate specificity of phosphopantetheinyl transferases; all studied phosphopantetheinyl transferases were capable of converting apo- carrier proteins, sourced from diverse biosynthetic enzymes, to their active holo form. Phosphopantetheinyl transferase coexpression with the hybrid nonribosomal peptide synthetases/polyketide synthases responsible for microcystin biosynthesis confirmed that the higher in vitro activity of Sfp translated in vivo to a higher yield of production.

Puromycin A, B and C, cryptic nucleosides identified from Streptomyces alboniger NRRL B-1832 by PPtase-based activation

Natural product discovery is pivot for drug development, however, this endeavor is often challenged by the wide inactivation or silence of natural products biosynthetic pathways. We recently developed a highly efficient approach to activate cryptic/silenced biosynthetic pathways through augmentation of the phosphopantetheinylation of carrier proteins. By applying this approach in the Streptomyces alboniger NRRL B-1832, we herein identified three cryptic nucleosides products, including one known puromycin A and two new derivatives (puromycin B and C). The biosynthesis of these products doesn't require the involvement of carrier protein, indicating the phosphopantetheinyl transferase (PPtase) indeed plays a fundamental regulatory role in metabolites biosynthesis. These results demonstrate that the PPtase-based approach have a much broader effective scope than the previously assumed carrier protein-involving pathways, which will benefit future natural products discovery and biosynthetic studies.

Recent progress in enzymatic protein labelling techniques and their applications.

Protein-based conjugates are valuable constructs for a variety of applications. Conjugation of proteins to fluorophores is commonly used to study their cellular localization and the protein-protein interactions. Modification of therapeutic proteins with either polymers or cytotoxic moieties greatly enhances their pharmacokinetics or potency. To label a protein of interest, conventional direct chemical reaction with the side-chains of native amino acids often yields heterogeneously modified products. This renders their characterization complicated, requires difficult separation steps and may impact protein function. Although modification can also be achieved via the insertion of unnatural amino acids bearing bioorthogonal functional groups, these methods can have lower protein expression yields, limiting large scale production. As a site-specific modification method, enzymatic protein labelling is highly efficient and robust under mild reaction conditions. Significant progress has been made over the last five years in modifying proteins using enzymatic methods for numerous applications, including the creation of clinically relevant conjugates with polymers, cytotoxins or imaging agents, fluorescent or affinity probes to study complex protein interaction networks, and protein-linked materials for biosensing. This review summarizes developments in enzymatic protein labelling over the last five years for a panel of ten enzymes, including sortase A, subtiligase, microbial transglutaminase, farnesyltransferase, N-myristoyltransferase, phosphopantetheinyl transferases, tubulin tyrosin ligase, lipoic acid ligase, biotin ligase and formylglycine generating enzyme.

A combination of docking and cheminformatics approaches for the identification of inhibitors against 4′ phosphopantetheinyl transferase ofMycobacterium tuberculosis

We integrated virtual screening,in vitroandex vivoapproaches to identify numerous potent inhibitory scaffolds againstM. tbPptT.