Engineering fluorometabolite production: fluorinase expression in Salinispora tropica Yields Fluorosalinosporamide

Abstract

Organofluorine compounds play an important role in medicinal chemistry, where they are responsible for up to 15% of the pharmaceutical products on the market. While natural products are valuable sources of new chemical entities, natural fluorinated molecules are extremely rare and the pharmaceutical industry has not benefited from a microbial source of this class of compounds. Streptomyces cattleya is an unusual bacterium in that it elaborates fluoroacetate and the amino acid 4-fluorothreonine. The discovery in 2002 of the fluorination enzyme FlA responsible for C-F bond formation in S. cattleya, and its subsequent characterization, opened up for the first time the prospect of genetically engineering fluorometabolite production from fluoride ion in host organisms. As a proof of principle, we report here the induced production of fluorosalinosporamide by replacing the chlorinase gene salL from Salinispora tropica with the fluorinase gene flA.

Figure 1

Biosynthesis of fluorometabolites by Streptomyces cattleya, (top), of salinosporamide A by Salinispora tropica (bottom) and fluorosalinosporamide engineering (middle).

Figure 2

Fluorinase expression in S. tropica. (A) The chlorinase gene salL was chromosomally replaced with fluorinase flA and the apramycin resistance/oriT cassette from pIJ773 (Apra^R) using PCR targeting., flA is thus under control of the natural salL promoter and ribosome binding site. The targeted locus of the wild-type (WT) and mutant (MT) chromosomes is shown for comparison. (B) The authenticity of mutants was confirmed by PCR. (C) flA transcription was analyzed by semi-quantitative RT-PCR. 16S rRNA was used as control of cDNA quality and integrity. The negative control with RNA instead of cDNA samples confirms the absence of genomic DNA contamination.

Figure 3

HPLC-MS analysis of culture extracts of S. tropica wild-type and the flA⁺ salL^- mutant grown in A1 seawater-based media, and of the mutant grown in A1 seawater-based media pH 6.0 to which 2 mM potassium fluoride was added at day 2 (+ KF). HPLC was monitored at 210 nm.

Figure 4

¹⁹F-NMR analysis of S. tropica salL^- flA⁺ culture extracts. The upper trace shows the decoupled ¹⁹F{¹H}-NMR spectrum of the organic fraction of the extract, whereas the lower trace is the proton coupled ¹⁹F-NMR spectrum. In all, five fluorometabolites were detected with the major product as fluorosalinosporamide (-220.7 ppm).

Figure 5

¹H/¹⁹F-HMBC (500 MHz, CDCl₃) correlation spectra. The upper trace is the spectrum of a reference sample of fluorosalinosporamide. The lower spectrum is a focused section illustrating fluorosalinosporamide in the S. tropica salL^- flA⁺ extract grown in limiting fluoride. The fluorine atom is coupling to three sets of protons with chemical shifts consistent with the FCH₂CH₂CH-R fragment.

Figure 6

Simulation of the ¹⁹F-NMR spectrum of fluorosalinosporamide (upper trace) compared to the experimental spectrum (lower trace). J(¹H,¹⁹F) = 47.0 Hz (2 equivalent H), J(¹H,¹⁹F) = 28.8 and 24.3 Hz (2 non-equivalent diastereotopic H).