Mutational analysis of a phenazine biosynthetic gene cluster in Streptomyces anulatus 9663

Abstract

The biosynthetic gene cluster for endophenazines, i.e., prenylated phenazines from Streptomyces anulatus 9663, was heterologously expressed in several engineered host strains derived from Streptomyces coelicolor M145. The highest production levels were obtained in strain M512. Mutations in the rpoB and rpsL genes of the host, which result in increased production of other secondary metabolites, had no beneficial effect on the production of phenazines. The heterologous expression strains produced, besides the known phenazine compounds, a new prenylated phenazine, termed endophenazine E. The structure of endophenazine E was determined by high-resolution mass spectrometry and by one- and two-dimensional NMR spectroscopy. It represented a conjugate of endophenazine A (9-dimethylallylphenazine-1-carboxylic acid) and L-glutamine (L-Gln), with the carboxyl group of endophenazine A forming an amide bond to the α-amino group of L-Gln. Gene inactivation experiments in the gene cluster proved that ppzM codes for a phenazine N-methyltransferase. The gene ppzV apparently represents a new type of TetR-family regulator, specifically controlling the prenylation in endophenazine biosynthesis. The gene ppzY codes for a LysR-type regulator and most likely controls the biosynthesis of the phenazine core. A further putative transcriptional regulator is located in the vicinity of the cluster, but was found not to be required for phenazine or endophenazine formation. This is the first investigation of the regulatory genes of phenazine biosynthesis in Streptomyces.

Keywords: gene cluster; gene inactivation; phenazine.

Figure 1

The endophenazine biosynthetic gene cluster from Streptomyces anulatus 9663 and the structures of phenazine-1-carboxylic acid and endophenazines A and B. The depicted sequence corresponds to the insert of cosmid ppzOS04. The gene deletions carried out in this study and the names of the resulting constructs are indicated.

Figure 2

Production of prenylated phenazines after heterologous expression of the endophenazine gene cluster in different expression hosts. From each expression host, two to three independent clones were obtained (A–C), and production was determined in three parallel cultivations of each clone (1–3). Cultivation was carried out in 24 square deep-well plates. In the experiments depicted here, the culture medium was supplemented with 0.6% of the oxygen carrier Q2-5247.

Figure 3

HPLC analysis of mycelia of the heterologous expression strain S. coelicolor M512(ppzOS04) after five days of cultivation. The oxygen carrier Q2-5247 was not included in the culture medium in this experiment. Detection wavelength: 365 nm. The lower panels show the UV spectra of endophenazine A and endophenazine E.

Figure 4

Extracted ion chromatograms for the mass of endophenazine B (m/z [M + H]⁺ = 323) in S. coelicolor M512(ppzOS04), and the mutant S. coelicolor M512(ppzOS26). The deletion of the gene ppzM reveals the abolishment of the production of endophenazine B.