Siderophore natural products as pharmaceutical agents.

Siderophore natural products are characterized by an ability to tightly chelate metals. The origins of such compounds are often pathogenic microbes utilizing siderophores as virulence factors during host infection. The mechanism for siderophore formation typically involves the activity of nonribosomal peptide synthetases producing compounds across functional group classifications that include catecholate, phenolate, hydroxamate, and mixed categories. Though siderophore production has been a hallmark of pathogenicity, the evolutionarily-optimized binding abilities of siderophores suggest the possibility of re-directing the compounds towards alternative beneficial applications. In this mini-review, we will first describe siderophore formation origins before discussing alternative applications as pharmaceutical products. In so doing, we will cover examples and applications that include reducing metal overload, targeted antibiotic delivery, cancer treatment, vaccine development, and diagnostics. Included in this analysis will be a discussion on the native production hosts of siderophores and prospects for improvement in compound access through the adoption of heterologous biosynthesis.

Complex natural product production methods and options.

Natural products have had a major impact upon quality of life, with antibiotics as a classic example of having a transformative impact upon human health. In this contribution, we will highlight both historic and emerging methods of natural product bio-manufacturing. Traditional methods of natural product production relied upon native cellular host systems. In this context, pragmatic and effective methodologies were established to enable widespread access to natural products. In reviewing such strategies, we will also highlight the development of heterologous natural product biosynthesis, which relies instead on a surrogate host system theoretically capable of advanced production potential. In comparing native and heterologous systems, we will comment on the base organisms used for natural product biosynthesis and how the properties of such cellular hosts dictate scaled engineering practices to facilitate compound distribution. In concluding the article, we will examine novel efforts in production practices that entirely eliminate the constraints of cellular production hosts. That is, cell free production efforts will be introduced and reviewed for the purpose of complex natural product biosynthesis. Included in this final analysis will be research efforts made on our part to test the cell free biosynthesis of the complex polyketide antibiotic natural product erythromycin.

Consolidated plasmid Design for Stabilized Heterologous Production of the complex natural product Siderophore Yersiniabactin.

Yersiniabactin (Ybt) is a hybrid polyketide-nonribosomal complex natural product also known as a siderophore for its iron chelation properties. The native producer of Ybt, Yersinia pestis, is a priority pathogen responsible for the plague in which the siderophore properties of Ybt are used to sequester iron and other metal species upon host infection. Alternatively, the high metal binding properties of Ybt enable a plethora of potentially valuable applications benefiting from metal remediation and/or recovery. For these applications, a surrogate production source is highly preferred relative to the pathogenic native host. In this work, we present a modification to the heterologous Escherichia coli production system established for Ybt biosynthesis. In particular, the multiple plasmids originally used to express the genetic pathway required for Ybt biosynthesis were consolidated to a single, copy-amplifiable plasmid. In so doing, plasmid stability was improved from ~30% to ≥80% while production values maintained at 20-30% of the original system, which resulted in titers of 0.5-3 mg/L from shake flask vessels.

Heterologous biosynthesis as a platform for producing new generation natural products.

Natural products have demonstrated value across numerous application areas, with antibiotics a notable historical example. Native cellular hosts provide an initial option in efforts to harness natural product production. However, various complexities associated with native hosts, including fastidious growth traits and limited molecular biology tools, have prompted an alternative approach termed heterologous biosynthesis that relies upon a surrogate biological system to reconstitute the biosynthetic sequence stemming from transplanted genetic blueprint. In turn, heterologous biosynthesis offers the benefit of enzymatically driven complex natural product formation combined with the prospect of improved compound access via scalable cellular production. In this review, we conduct a literature meta-analysis of heterologous natural product biosynthesis over the period of 2011-2020 with the goal of identifying trends in heterologous natural product host selection, target natural products, and compound-host selection tendencies, with associated commentary on the research directions of heterologous biosynthesis based upon this analysis.

Flux Balance Analysis for Media Optimization and Genetic Targets to Improve Heterologous Siderophore Production.

Siderophores are small molecule metal chelators secreted in sparse quantities by their native microbial hosts but can be engineered for enhanced production from heterologous hosts like Escherichia coli. These molecules have been proved to be capable of binding heavy metals of commercial and/or environmental interest. In this work, we incorporated, as needed, the appropriate pathways required to produce several siderophores (anguibactin, vibriobactin, bacillibactin, pyoverdine, and enterobactin) into the base E. coli K-12 MG1655 metabolic network model to computationally predict, via flux balance analysis methodologies, gene knockout targets, gene over-expression targets, and media modifications capable of improving siderophore reaction flux. E. coli metabolism proved supportive for the underlying production mechanisms of various siderophores. Within such a framework, the gene deletion and over-expression targets identified, coupled with complementary insights from medium optimization predictions, portend experimental implementation to both enable and improve heterologous siderophore production. Successful production of siderophores would then spur novel metal-binding applications.

Regenerative Core-Shell Nanoparticles for Simultaneous Removal and Detection of Endotoxins.

Detection and removal of lipopolysaccharides (LPS) from food and pharmaceutical preparations is important for their safe intake and administration to avoid septic shock. We have developed an abiotic system for reversible capture, removal, and detection of LPS in aqueous solutions. Our system comprises long C18 acyl chains tethered to Fe3O4/Au/Fe3O4 nanoflowers (NFs) that act as solid supports during the separation process. The reversible LPS binding is mediated by facile hydrophobic interactions between the C18 chains and the bioactive lipid A component present on the LPS molecule. Various parameters such as pH, solvent, sonication time, NF concentration, alkane chain length, and density are optimized to achieve a maximum LPS capture efficiency. The NFs can be reused at least three times by simply breaking the NF-LPS complexes in the presence of food-grade surfactants, making the entire process safe, efficient, and scalable. The regenerated particles also serve as colorimetric labels in dot blot bioassays for simple and rapid estimation of the LPS removed.