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

ABSTRACT: 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.

SUBMITTER: Pogorevc D

PROVIDER: S-EPMC8719831 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Recently, the discovery of the elansolids, a group of macrolides, was reported. The molecules show activity against methicillin-resistant Staphylococcus aureus as well as other gram-positive organisms. This fact renders those substances a promising starting point for future chemical development. The active atropisomers A1/A2 are formed by macrolactonization of the biosynthesis product A3 but are prone to ring opening and subsequent formation of several unwanted side products. Recently it could be shown that addition of different nucleophiles to culture extracts of Chitinophaga sancti enable the formation of new stable elansolid derivatives. Furthermore, addition of such a nucleophile directly into the culture led exclusively to formation of a single active elansolid derivative. Due to low product yields, methods for production of gram amounts of these molecules have to be established to enable further development of this promising compound class.Production of elansolid A2 by C. sancti was enabled using a synthetic medium with sucrose as carbon source to a final concentration of 18.9 mg L-1. A fed-batch fermentation was ensued that resulted in an elansolid A2 concentration of 55.3 mg L-1. When using glucose as carbon source in a fed-batch fermentation only 34.4 mg L-1 elansolid A2 but 223.1 mg L-1 elansolid C1 were produced. This finding was not unexpected since elansolids A1/A2 and A3 have been reported to easily react with nucleophiles like anthranilic acid, a precursor of tryptophan biosynthesis. Due to the fact that nucleophiles can be incorporated in vivo, a fed-batch cultivation under identical conditions, with addition of anthranilic acid was carried out and lead to almost exclusive formation of elansolid C1 (257.5 mg L-1).Reproducible elansolid A2 and C1 production is feasible in different synthetic media at relatively high concentrations that will allow further investigation and semi-synthetic optimization. The feeding of anthranilic acid enables the exclusive production of the stable elansolid derivative C1, which reduces product loss by unspecific reactions and eases downstream processing. This derivative shows activity in the same range as the elansolids A1/A2. Hence, the method can possibly serve as a model-process for incorporation of other nucleophiles and biotechnological production of specifically designed molecules.

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

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