Search

New antimicrobials need to be discovered to fight the advance of multidrug-resistant pathogens. A promising approach is the screening for antimicrobial agents naturally produced by living organisms. As an alternative to studying the native producer, it is possible to use genetically tractable microbes as heterologous hosts to aid the discovery process, facilitate product diversification through genetic engineering, and ultimately enable environmentally friendly production. In this mini-review, we summarize the literature from 2017 to 2022 on the application of Escherichia coli and E. coli-based platforms as versatile and powerful systems for the discovery, characterization, and sustainable production of antimicrobials. We highlight recent developments in high-throughput screening methods and genetic engineering approaches that build on the strengths of E. coli as an expression host and that led to the production of antimicrobial compounds. In the last section, we briefly discuss new techniques that have not been applied to discover or engineer antimicrobials yet, but that may be useful for this application in the future.

Der Abschnitt „Späte Funktionalisierung mit Biokatalysatoren aus Naturstoffsynthesen“ beschäftigt sich mit der Frage, wie sich funktionelle Gruppen gezielt in Moleküle einführen lassen, ohne auf langwierige Schutzgruppenstrategien rückgreifen zu müssen. Dabei können spezialisierte Enzyme helfen, die vielfach in natürlichen Synthesewegen von Naturstof-fen vorkommen. Es gibt einige Beispiele, wie sich diese Enzyme in der chemischen Synthese nutzen lassen.

Oxygen-directed methylation is a ubiquitous tailoring reaction in natural product pathways catalysed by 15 O-methyltransferases (OMTs). Promiscuous OMT biocatalysts are thus a valuable asset in the toolkit for 16 sustainable synthesis and optimization of known bioactive scaffolds for drug development. Here, we 17 characterized two bacterial OMTs from Desulforomonas acetoxidans and Streptomyces avermitilis in 18 terms of their enzymatic properties and substrate scope and determined their crystal structures. Both 19 OMTs methylated a wide range of catechol-like substrates, including flavonoids, coumarins, 20 hydroxybenzoic acids and their respective aldehydes, an anthraquinone and an indole. One enzyme also 21 accepted a steroid. The product range included pharmaceutically relevant compounds such as 22 (iso)fraxidin, iso(scopoletin), chrysoeriol, alizarin 1-methyl ether and 2-methoxyestradiol. Interestingly, 23 certain non-catechol flavonoids and hydroxybenzoic acids were also methylated. This study expands the 24 knowledge on substrate preference and structural diversity of bacterial catechol OMTs and paves the way 25 for their use in (combinatorial) pathway engineering.

Homoeriodictyol and hesperetin are naturally occurring O-methylated flavonoids with many health-promoting properties. They are produced in plants in low abundance and as complex mixtures of similar compounds that are difficult to separate. Synthetic biology offers the opportunity to produce non-methylated flavonoids in a targeted, bottom-up approach in engineered microbes with high product titers. However, the production of these O-methylated flavonoids is currently still highly inefficient.In this study, we investigated and engineered a combination of enzymes that had previously been shown to support homoeriodictyol and hesperitin production from fed cinnamic acid precursors. We determined the crystal structures of the enzyme catalyzing the first committed step of the pathway, chalcone synthase fromHordeum vulgarein three ligand-bound states. Based on these structures and a multiple sequence alignment with other chalcone synthases, we constructed mutant variants and assessed their performance inEscherichia colitowards producing methylated flavonoids. With our best mutant variant, HvCHS (Q232P, D234V), we were able to produce homoeriodictyol and hesperetin at 2 times and 10 times higher titers than previously reported. Our findings will facilitate the further engineering of this enzyme towards higher production of methylated flavonoids.

Artemisinin, the most famous anti-malaria drug initially extracted from Artemisia annua L., also exhibits anti-tumor properties in vivo and in vitro. To improve its solubility and bioavailability, multiple derivatives have been synthesized. However, to reveal the anti-tumor mechanism and improve the efficacy of these artemisinin-type drugs, studies have been conducted in recent years. In this review, we first provide an overview of the effect of artemisinin-type drugs on the regulated cell death pathways, which may uncover novel therapeutic approaches. Then, to overcome the shortcomings of artemisinin-type drugs, we summarize the recent advances in two different therapeutic approaches, namely the combination therapy with biologics influencing regulated cell death, and the use of nanocarriers as drug delivery systems. For the former approach, we discuss the superiority of combination treatments compared to monotherapy in tumor cells based on their effects on regulated cell death. For the latter approach, we give a systematic overview of nanocarrier design principles used to deliver artemisinin-type drugs, including inorganic-based nanoparticles, liposomes, micelles, polymer-based nanoparticles, carbon-based nanoparticles, nanostructured lipid carriers and niosomes. Both approaches have yielded promising findings in vitro and in vivo, providing a strong scientific basis for further study and upcoming clinical trials.

O-methyltransferases are ubiquitous enzymes involved in biosynthetic pathways for secondary metabolites such as bacterial antibiotics, human catecholamine neurotransmitters, and plant phenylpropanoids. While thousands of putative O-methyltransferases are found in sequence databases, few examples are functionally characterized. From a pathway engineering perspective, however, it is crucial to know the substrate and product ranges of the respective enzymes to fully exploit their catalytic power.In this study, we developed anin vitroprototyping workflow that allowed us to screen ~30 enzymes against five substrates in three days with high reproducibility. We combinedin vitrotranscription/translation of the genes of interest with a microliter-scale enzymatic assay in 96-well plates. The substrate conversion was indirectly measured by quantifying the consumption of the S-adenosyl-L-methionine co-factor by time-resolved fluorescence resonance energy transfer rather than time-consuming product analysis by chromatography. This workflow allowed us to rapidly prototype thus-far uncharacterized O-methyltransferases for future use as biocatalysts.

The bioprospecting of secondary metabolites from endophytic fungi received great attention in the 1990s and 2000s, when the controversy around taxol production from Taxus spp. endophytes was at its height. Since then, hundreds of reports have described the isolation and characterization of putative secondary metabolites from endophytic fungi. However, only very few studies also report the genetic basis for these phenotypic observations. With low sequencing cost and fast sample turnaround, genetics- and genomics-based approaches have risen to become comprehensive approaches to study natural products from a wide-range of organisms, especially to elucidate underlying biosynthetic pathways. However, in the field of fungal endophyte biology, elucidation of biosynthetic pathways is still a major challenge. As a relatively poorly investigated group of microorganisms, even in the light of recent efforts to sequence more fungal genomes, such as the 1000 Fungal Genomes Project at the Joint Genome Institute (JGI), the basis for bioprospecting of enzymes and pathways from endophytic fungi is still rather slim. In this review we want to discuss the current approaches and tools used to associate phenotype and genotype to elucidate biosynthetic pathways of secondary metabolites in endophytic fungi through the lens of bioprospecting. This review will point out the reported successes and shortcomings, and discuss future directions in sampling, and genetics and genomics of endophytic fungi. Identifying responsible biosynthetic genes for the numerous secondary metabolites isolated from endophytic fungi opens the opportunity to explore the genetic potential of producer strains to discover novel secondary metabolites and enhance secondary metabolite production by metabolic engineering resulting in novel and more affordable medicines and food additives.

Siderophores are small molecules with unmatched capacity to scavenge iron from proteins and the extracellular milieu, where it mostly occurs as insoluble Fe3+. Siderophores chelate Fe3+for uptake into the cell, where it is reduced to soluble Fe2+. As iron is essential for bacterial survival, siderophores are key molecules in low soluble iron conditions. Bacteria have devised many strategies to synthesize proprietary siderophores to avoid siderophore piracy by competing organisms, e.g., by incorporating different polyamine backbones into siderophores, while maintaining the catechol moieties. We report thatSerratia plymuthicaV4 produces a variety of siderophores, which we term thesiderome, and which are assembled by the concerted action of enzymes encoded in two independent gene clusters. Besides assembling serratiochelin with diaminopropane,S. plymuthicautilizes putrescine and the same set of enzymes to assemble photobactin, a siderophore described forPhotorhabdus luminescens. The enzymes encoded by one of the gene clusters can independently assemble enterobactin. A third, independent operon is responsible for biosynthesis of the hydroxamate siderophore aerobactin, initially described inEnterobacter aerogenes. Mutant strains not synthesizing polyamine-siderophores significantly increased enterobactin production levels, though lack of enterobactin did not impact serratiochelin production. Knocking out SchF0, an enzyme involved in the assembly of enterobactin alone, significantly reduced bacterial fitness. This study illuminates the interplay between siderophore biosynthetic pathways and polyamine production superpathways, indicating routes of molecular diversification. Given its natural yields of diaminopropane (97.75 μmol/g DW) and putrescine (30.83 μmol/g DW),S. plymuthicacan be exploited for the industrial production of these compounds.Significance StatementSiderophores are molecules crucial for bacterial survival in low iron environments. Bacteria have evolved the capacity to pirate siderophores made by other bacterial strains and to diversify the structure of their own siderophores, to prevent piracy. We found thatSerratia plymuthicaV4 produces five different siderophores using three gene clusters and a polyamine production superpathway. The most well studied siderophore, enterobactin, rather than the strain’s proprietary and by far most abundant siderophore, serratiochelin, displayed a crucial role in the fitness ofS. plymuthica. Our results also indicate that this strain is a good candidate for engineering the large-scale production of diaminopropane (DAP), as without any optimization it produced the highest amounts of DAP reported for wild-type strains.

Glycopeptide antibiotics (GPAs) are nonribosomal peptides rich in modifications introduced by external enzymes. These enzymes act on the free peptide aglycone or intermediates bound to the nonribosomal peptide synthetase (NRPS) assembly line. In this process the terminal module of the NRPS plays a crucial role as it contains a unique recruitment platform (X-domain) interacting with three to four modifying Cytochrome P450 (P450) enzymes that are responsible for cyclizing bound peptides. However, whether these enzymes share the same binding site on the X-domain and how the order of the cyclization steps is orchestrated has remained elusive. In this study we investigate the first two reactions in teicoplanin aglycone maturation catalyzed by the enzymes OxyBtei and OxyAtei. We demonstrate that both enzymes interact with the X-domain via the identical interaction site with similar affinities, irrespective of the peptide modification stage, while their catalytic activity is restricted to the correctly cross-linked peptide. On the basis of steady state kinetics of the OxyBtei-catalyzed reaction, we propose a model for P450 recruitment and peptide modification that involves continuous association/dissociation of the P450 enzymes with the NRPS, followed by specific recognition of the peptide cyclization state by the P450 (scanning). This leads to an induced conformational change that enhances the affinity of the enzyme/substrate complex and initiates catalysis; product release then occurs, with the product itself becoming the substrate for the second enzyme in the pathway. This model rationalizes our experimental findings for this complex enzyme cascade and provides insights into the orchestration of the sequential peptide tailoring reactions on the terminal NRPS module in GPA biosynthesis.

Non-ribosomal peptide synthetase (NRPS) mega-enzyme complexes are modular assembly lines that are involved in the biosynthesis of numerous peptide metabolites independently of the ribosome. The multiple interactions between catalytic domains within the NRPS machinery are further complemented by additional interactions with external enzymes, particularly focused on the final peptide maturation process. An important class of NRPS metabolites that require extensive external modification of the NRPS-bound peptide are the glycopeptide antibiotics (GPAs), which include vancomycin and teicoplanin. These clinically relevant peptide antibiotics undergo cytochrome P450-catalysed oxidative crosslinking of aromatic side chains to achieve their final, active conformation. However, the mechanism underlying the recruitment of the cytochrome P450 oxygenases to the NRPS-bound peptide was previously unknown. Here we show, through in vitro studies, that the X-domain, a conserved domain of unknown function present in the final module of all GPA NRPS machineries, is responsible for the recruitment of oxygenases to the NRPS-bound peptide to perform the essential side-chain crosslinking. X-ray crystallography shows that the X-domain is structurally related to condensation domains, but that its amino acid substitutions render it catalytically inactive. We found that the X-domain recruits cytochrome P450 oxygenases to the NRPS and determined the interface by solving the structure of a P450-X-domain complex. Additionally, we demonstrated that the modification of peptide precursors by oxygenases in vitro--in particular the installation of the second crosslink in GPA biosynthesis--occurs only in the presence of the X-domain. Our results indicate that the presentation of peptidyl carrier protein (PCP)-bound substrates for oxidation in GPA biosynthesis requires the presence of the NRPS X-domain to ensure conversion of the precursor peptide into a mature aglycone, and that the carrier protein domain alone is not always sufficient to generate a competent substrate for external cytochrome P450 oxygenases.

The biosynthesis of the glycopeptide antibiotics, of which teicoplanin and vancomycin are representative members, relies on the combination of non-ribosomal peptide synthesis and modification of the peptide by cytochrome P450 (Oxy) enzymes while the peptide remains bound to the peptide synthesis machinery. We have structurally characterized the final peptidyl carrier protein domain of the teicoplanin non-ribosomal peptide synthetase machinery: this domain is believed to mediate the interactions with tailoring Oxy enzymes in addition to its function as a shuttle for intermediates between multiple non-ribosomal peptide synthetase domains. Using solution state NMR, we have determined structures of this PCP domain in two states, the apo and the post-translationally modified holo state, both of which conform to a four-helix bundle assembly. The structures exhibit the same general fold as the majority of known carrier protein structures, in spite of the complex biosynthetic role that PCP domains from the final non-ribosomal peptide synthetase module must play in glycopeptide antibiotic biosynthesis. These structures thus support the hypothesis that it is subtle rearrangements, rather than dramatic conformational changes, which govern carrier protein interactions and selectivity during non-ribosomal peptide synthesis. Proteins 2015; 83:711–721. © 2015 Wiley Periodicals, Inc.

Understanding the mechanisms underpinning glycopeptide antibiotic biosynthesis is key to the future ability to reinvent these compounds. For effective in vitro characterization of the crucial later steps of the biosynthesis, facile access to a wide range of substrate peptides as their Coenzyme A (CoA) conjugates is essential. Here we report the development of a rapid route to glycopeptide precursor CoA conjugates that affords both high yields and excellent purities. This synthesis route is applicable to the synthesis of peptide CoA-conjugates containing racemization-prone arylglycine residues: such residues are hallmarks of non-ribosomal peptide synthesis and have previously been inaccessible to peptide synthesis using Fmoc-type chemistry. We have applied this route to generate glycopeptide precursor peptides in their carrier protein-bound form as substrates to explore the specificity of the first oxygenase enzyme from vancomycin biosynthesis (OxyBvan). Our results indicate that OxyBvan is a highly promiscuous catalyst for phenolic coupling of diverse glycopeptide precursors that accepts multiple carrier protein substrates, even on carrier protein domains from alternate glycopeptide biosynthetic machineries. These results represent the first important steps in the development of an in vitro biomimetic synthesis of modified glycopeptide aglycones.

Cyclization of glycopeptide antibiotic precursors occurs in either three or four steps catalyzed by Cytochrome P450 enzymes. Three of these enzymes have been structurally characterized to date with the second enzyme along the pathway, OxyA, escaping structural analysis. We are now able to present the structure of OxyAtei involved in teicoplanin biosynthesis - the same enzyme recently shown to be the first active OxyA homolog. In spite of the hydrophobic character of the teicoplanin precursor, the polar active site of OxyAtei and its affinity for certain azole inhibitors hint at its preference for substrates with polar decorations.

The use of levulinic acid in bioconversion strategies has been limited by the lack of information on the pathways used by microorganisms to degrade it. Now, functional genomics reveals the essential steps for utilization of levulinic acid in Pseudomonas putida.

The biosynthesis of the glycopeptide antibiotics, which include vancomycin and teicoplanin, relies on the interplay between the peptide-producing non-ribosomal peptide synthetase (NRPS) and Cytochrome P450 enzymes (P450s) that catalyze side-chain crosslinking of the peptide. We demonstrate that sequential in vitro P450-catalyzed cyclization of peptide substrates is enabled by the use of an NRPS peptide carrier protein (PCP)-X di-domain as a P450 recruitment platform. This study reveals that whilst the precursor peptide sequence influences the installation of the second crosslink by the P450 OxyAtei , activity is not restricted to the native teicoplanin peptide. Initial peptide cyclization is possible with teicoplanin and vancomycin OxyB homologues, and the latter displays excellent activity with all substrate combinations tested. By using non-natural X-domain substrates, bicyclization of hexapeptides was also shown, which demonstrates the utility of this method for the cyclization of varied peptide substrates in vitro.

Bacterial cytochrome P450s form a remarkable clade of the P450 superfamily of oxidative hemoproteins, and are often involved in the biosynthesis of complex natural products. Those in a subgroup known as "Oxy enzymes" play a crucial role in the biosynthesis of glycopeptide antibiotics, including vancomycin and teicoplanin. The Oxy enzymes catalyze crosslinking of aromatic residues in the non-ribosomal antibiotic precursor peptide while it remains bound to the non-ribosomal peptide synthetase (NRPS); this crosslinking secures the three-dimensional structure of the glycopeptide, crucial for antibiotic activity. We have characterized OxyBtei , the first of the Oxy enzymes in teicoplanin biosynthesis. Our results reveal that OxyBtei possesses a structure similar to those of other Oxy proteins and is active in crosslinking NRPS-bound peptide substrates. However, OxyBtei displays a significantly altered activity spectrum against peptide substrates compared to its well-studied vancomycin homologue.

The biosynthesis of the glycopeptide antibiotics, of which teicoplanin and vancomycin are representative members, relies on the combination of non-ribosomal peptide synthesis and modification of the peptide by cytochrome P450 (Oxy) enzymes while the peptide remains bound to the peptide synthesis machinery. We have structurally characterized the final peptidyl carrier protein domain of the teicoplanin non-ribosomal peptide synthetase machinery: this domain is believed to mediate the interactions with tailoring Oxy enzymes in addition to its function as a shuttle for intermediates between multiple non-ribosomal peptide synthetase domains. Using solution state NMR, we have determined structures of this PCP domain in two states, the apo and the post-translationally modified holo state, both of which conform to a four-helix bundle assembly. The structures exhibit the same general fold as the majority of known carrier protein structures, in spite of the complex biosynthetic role that PCP domains from the final non-ribosomal peptide synthetase module must play in glycopeptide antibiotic biosynthesis. These structures thus support the hypothesis that it is subtle rearrangements, rather than dramatic conformational changes, which govern carrier protein interactions and selectivity during non-ribosomal peptide synthesis.

Studying the interplay between nonribosomal peptide synthetases (NRPS), a major source of secondary metabolites, and crucial external modifying enzymes is a challenging task since the interactions involved are often transient in nature. By applying a range of synthetic inhibitor-type compounds, a stabilized complex appropriate for structural analysis was generated for such a tailoring enzyme and an NRPS domain. The complex studied comprises an NRPS peptidyl carrier protein (PCP) domain bound to the Cytochrome P450 enzyme that is crucial for the provision of β-hydroxylated amino acid precursors in the biosynthesis of the cyclic depsipeptide skyllamycin. The structure reveals that complex formation is governed by hydrophobic interactions, the presence of which can be controlled through minor alterations in PCP structure that enable selectivity amongst multiple highly similar PCP domains.

Cytochromes P450 (P450s) are a superfamily of oxidoreductases that display not only a high degree of substrate diversity across xenobiotic and secondary metabolism but also show flexibility in the oxidation chemistry that they catalyse. The oxidative transformation of amino acids and peptides by P450s represents an important collection of transformations for this enzyme class: these transformations are used in Nature to diversify the limited range of monomers available for ribosomal peptide production, as well as altering peptides to afford desired biological properties. This chapter will highlight current examples of P450-catalysed transformations of amino acids and peptides, organised by the nature of the oxidative transformation performed by the P450.