Establishing and engineering heterologous production systems for argyrin and α-pyrone antibiotics

Myxobacterial natural products have proven to be a valuable source of antibacterial and anticancer compounds in the last decades. This thesis covers the biosynthesis elucidation, heterologous production as well as structure and yield optimization of two potent myxobacterial compounds. The work on coralloypronin resulted in the design of an efficient heterologous production platform in the myxobacterial model organism Myxococcus xanthus. Research on the established heterologous producer led to identification of specific biochemical processes and resulted in isolation and structure elucidation of two novel coralloypronin derivatives. Furthermore, gene deletion experiments led to discovery of hypothetical self-resistance mechanism, which seems to be conserved in most myxobacterial RNA polymerase (RNAP) inhibitor biosynthetic gene clusters (BGC). The BGC of argyrin, a cyclic octapeptide produced by nonribosomal peptide synthetase (NRPS), was identified in Cystobacter sp. SBCb004. The heterologous expression of synthetic argyrin BGC was achieved in M. xanthus, which, after medium optimization, led to a significant yield improvement over the wild type producer. The production of argyrin could be even further improved by utilization of different promoter systems with a specifically optimized leader sequence. Furthermore, precursor directed biosynthesis was applied to produce a library of novel argyrin derivatives.Myxobakerielle Naturstoffe haben sich in den letzten Jahrzehnten als wertvolle Ressource für Wirkstoffe gegen Bakterien und Krebs bewährt. Diese Dissertation umfasst die Biosyntheseaufklärung, die heterologe Produktion sowie Struktur und Ausbeuteoptimierung zweier wirksamer myxobakterieller Verbindungen. Die Arbeit über Corallopyronin führte zum Design einer effizienten myxobakteriellen Produktionsplattform im myxobakteriellen Modellorganismus Myxococcus xanthus. Die Erforschung dieses etablierten heterologen Produzenten führte zur Identifizierung spezifischer biochemischer Prozesse, die in der Isolation und Strukturaufklärung zweier neuer Corallopyroninderivate resultierte. Zusätzliche Gendeletionsexperimente führten zur Entdeckung eines hypothetischen Selbstresistenzmechanismus, der in myxobakteriellen RNA Polymerase (RNAP) Inhibitor Biosynthese-Genclustern (BGC) konserviert zu sein scheint. Das BGC des zyklischen Oktapeptids Argyrin wurde in einem Nichtribosomalen Peptid Synthetase (NRPS) Gencluster in Cystobacter sp. SBCb004 entdeckt. Die heterologe Expression eines synthetischen Argyrin BGC in M. xanthus führte nach Medienoptimierung zu einer signifikanten Steigerung der Ausbeute im Vergleich zum Wildtyp. Zusätzliche Steigerungen der Argyrinproduktion wurden durch Integration verschiedener Promotoren mit einer optimierten Leader Sequenz erreicht. Zudem konnte durch die Fütterung spezieller Vorläufermoleküle eine Bibliothek neuer Argyrinderivate produziert werden

Chemical synthesis of tripeptide thioesters for the biotechnological incorporation into the myxobacterial secondary metabolite argyrin via mutasynthesis

The argyrins are secondary metabolites from myxobacteria with antibiotic activity against Pseudomonas aeruginosa. Studying their structure–activity relationship is hampered by the complexity of the chemical total synthesis. Mutasynthesis is a promising approach where simpler and fully synthetic intermediates of the natural product’s biosynthesis can be biotechnologically incorporated. Here, we report the synthesis of a series of tripeptide thioesters as mutasynthons containing the native sequence with a dehydroalanine (Dha) Michael acceptor attached to a sarcosine (Sar) and derivatives. Chemical synthesis of the native sequence ᴅ-Ala-DhaSar thioester required revision of the sequential peptide synthesis into a convergent strategy where the thioester with sarcosine was formed before coupling to the Dha-containing dipeptide

Biotechnological production optimization of argyrins - a potent immunomodulatory natural product class.

Argyrins represent a family of cyclic octapeptides exhibiting promising immunomodulatory activity via inhibiting mitochondrial protein synthesis, which leads to reduced IL-17 production by the T-helper 17 cells. Argyrins are formed by a non-ribosomal peptide synthetase (NRPS), originating from the myxobacterial producer strains Archangium gephyra Ar8082 and Cystobacter sp. SBCb004. In this work, a previously established heterologous production platform was employed to provide evidence of direct D-configured amino acid incorporation by the argyrin assembly line. An adenylation domain of the argyrin NRPS was characterized and shown to have a high preference for D-configured amino acids. Eight novel argyrin derivatives were generated via biosynthetic engineering of the heterologous production system. The system was also optimized to enable formation of methylated argyrin C and D derivatives with improved immunosuppressive activity compared with their unmethylated counterparts. Furthermore, the optimization of cultivation conditions allowed exclusive production of one major derivative at a time, drastically improving the purification process. Importantly, engineering of transcription and translation initiation resulted in a substantially improved production titre reaching 350-400 mg l-1 . The optimized system presented herein thus provides a versatile platform for production of this promising class of immunosuppressants at a scale that should provide sufficient supply for upcoming pre-clinical development

Chemical synthesis of tripeptide thioesters for the biotechnological incorporation into the myxobacterial secondary metabolite argyrin via mutasynthesis.

The argyrins are secondary metabolites from myxobacteria with antibiotic activity against Pseudomonas aeruginosa. Studying their structure-activity relationship is hampered by the complexity of the chemical total synthesis. Mutasynthesis is a promising approach where simpler and fully synthetic intermediates of the natural product's biosynthesis can be biotechnologically incorporated. Here, we report the synthesis of a series of tripeptide thioesters as mutasynthons containing the native sequence with a dehydroalanine (Dha) Michael acceptor attached to a sarcosine (Sar) and derivatives. Chemical synthesis of the native sequence ᴅ-Ala-Dha-Sar thioester required revision of the sequential peptide synthesis into a convergent strategy where the thioester with sarcosine was formed before coupling to the Dha-containing dipeptide

Corallopyronin A: antimicrobial discovery to preclinical development

Covering: August 1984 up to January 2022 Worldwide, increasing morbidity and mortality due to antibiotic-resistant microbial infections has been observed. Therefore, better prevention and control of infectious diseases, as well as appropriate use of approved antibacterial drugs are crucial. There is also an urgent need for the continuous development and supply of novel antibiotics. Thus, identifying new antibiotics and their further development is once again a priority of natural product research. The antibiotic corallopyronin A was discovered in the 1980s in the culture broth of the Myxobacterium Corallococcus coralloides and serves, in the context of this review, as a show case for the development of a naturally occurring antibiotic compound. The review demonstrates how a hard to obtain, barely water soluble and unstable compound such as corallopyronin A can be developed making use of sophisticated production and formulation approaches. Corallopyronin A is a bacterial DNA-dependent RNA polymerase inhibitor with a new target site and one of the few representatives of this class currently in preclinical development. Efficacy against Gram-positive and Gram-negative pathogens, e.g., Chlamydia trachomatis, Orientia tsutsugamushi, Staphylococcus aureus, and Wolbachia has been demonstrated. Due to its highly effective in vivo depletion of Wolbachia, which are essential endobacteria of most filarial nematode species, and its robust macrofilaricidal efficacy, corallopyronin A was selected as a preclinical candidate for the treatment of human filarial infections. This review highlights the discovery and production optimization approaches for corallopyronin A, as well as, recent preclinical efficacy results demonstrating a robust macrofilaricidal effect of the anti-Wolbachia candidate, and the solid formulation strategy which enhances the stability as well as the bioavailability of corallopyronin A

Corallopyronin A for short-course anti-wolbachial, macrofilaricidal treatment of filarial infections.

Current efforts to eliminate the neglected tropical diseases onchocerciasis and lymphatic filariasis, caused by the filarial nematodes Onchocerca volvulus and Wuchereria bancrofti or Brugia spp., respectively, are hampered by lack of a short-course macrofilaricidal-adult-worm killing-treatment. Anti-wolbachial antibiotics, e.g. doxycycline, target the essential Wolbachia endosymbionts of filariae and are a safe prototype adult-worm-sterilizing and macrofilaricidal regimen, in contrast to standard treatments with ivermectin or diethylcarbamazine, which mainly target the microfilariae. However, treatment regimens of 4-5 weeks necessary for doxycycline and contraindications limit its use. Therefore, we tested the preclinical anti-Wolbachia drug candidate Corallopyronin A (CorA) for in vivo efficacy during initial and chronic filarial infections in the Litomosoides sigmodontis rodent model. CorA treatment for 14 days beginning immediately after infection cleared >90% of Wolbachia endosymbionts from filariae and prevented development into adult worms. CorA treatment of patently infected microfilaremic gerbils for 14 days with 30 mg/kg twice a day (BID) achieved a sustained reduction of >99% of Wolbachia endosymbionts from adult filariae and microfilariae, followed by complete inhibition of filarial embryogenesis resulting in clearance of microfilariae. Combined treatment of CorA and albendazole, a drug currently co-administered during mass drug administrations and previously shown to enhance efficacy of anti-Wolbachia drugs, achieved microfilarial clearance after 7 days of treatment at a lower BID dose of 10 mg/kg CorA, a Human Equivalent Dose of 1.4 mg/kg. Importantly, this combination led to a significant reduction in the adult worm burden, which has not yet been published with other anti-Wolbachia candidates tested in this model. In summary, CorA is a preclinical candidate for filariasis, which significantly reduces treatment times required to achieve sustained Wolbachia depletion, clearance of microfilariae, and inhibition of embryogenesis. In combination with albendazole, CorA is robustly macrofilaricidal after 7 days of treatment and fulfills the Target Product Profile for a macrofilaricidal drug