Process developement for heterologous Aurachin production

Introduction

Aurachins are a family of prenylated quinoline antibiotics [1], which possess potent antiprotozoal properties [2]. In the natural producer Stigmatella aurantiaca Sg a15 a farnesyl moiety is added to 2-methyl-4-quinolone (MQO) by the farnesyltransferase AuaA to produce aurachin D. The latter is then N-hydroxylated to aurachin C by the Rieske type oxygenase AuaF [3]. Recently, a method for the biocatalytic production of aurachin D from MQO using recombinant E. coli cells was described [4].

Goals

Due to their biological properties, the aurachins have gained attention as biochemical tool compounds and as drug candidates. Therefore, there is some interest in their cost-efficient production. The aim of this work was to intensify the biosynthesis of aurachin D in the heterologous host E. coli and to endow this bacterium with the auaF gene so that it can produce aurachin C.

Results

To simplify the downstream processing we switched from a complex fermentation medium to a defined minimal medium, which initially decreased the aurachin D titer in E. coli from 63.6 mg L-1 to 30.5 mg L‑1. By increasing the glucose concentration in the minimal medium to 15 g L-1 and the NH4Cl concentration to 2 g L‑1, aurachin D titers comparable to the complex medium were reached. With the increase of the substrate concentration from 90 mg L‑1 to 180 mg L‑1 an aurachin D titer of 104.2 mg L-1 could be achieved. Another rise of the product titer to 117.2 3 mg L-1 was possible after extending the cultivation time from 24 h to 48 h.

Plasmid-based co-expression of AuaA and AuaF enabled the production of aurachin C (6.6 mg L-1). Moreover, by lowering the precursor concentrations and auaF expression levels, an aurachin C titer of 23.5 mg L-1 was achieved.

Summary

The intensification of aurachin D production was successful and the product titer was almost doubled. Moreover, a system for aurachin C production was established. In the future aurachin C production can be optimized and new aurachin C derivatives can be produced by precursor-directed biosynthesis and screened for their antiprotozoal properties.

References

[1] Kunze B, Höfle G, Reichenbach H, J. Antibiot. 1987; 40(3): 258.

[2] Kruth S, Zimmermann CJ-M, Kuhr K, Hiller W, Lütz S, Pietruszka J, Kaiser M, Nett M, Molecules 2023; 28(3): 1066.

[3] Pistorius D, Li Y, Sandmann A, Müller R, Mol. Biosyst. 2011; 7(12): 3308.

[4] Kruth S, Schibajew L, Nett M, AMB Expr. 2022; 12(1): 138.