Modulation of lipid metabolism and spiramycin biosynthesis in Streptomyces ambofaciens unstable mutants

Abstract

Streptomyces ambofaciens is prone to genetic instability involving genomic rearrangements at the extremities of the chromosomal DNA. An amplified DNA sequence (ADS205), including an open reading frame (orfPS), is responsible for the reversible loss of spiramycin production in the mutant strain NSA205 (ADS205(+) Spi-). The product of orfPS is homologous to polyketide synthase systems (PKSs) involved in the biosynthesis of erythromycin and rapamycin and is overexpressed in strain NSA205 compared with the parental strain RP181110. As PKSs and fatty acid synthase systems have the same precursors, we tested the possibility that overexpression of orfPS also affects lipid metabolism in strain NSA205. This report focuses on comparative analysis of lipids in strain RP181110, the mutant strain NSA205, and a derivative, NSA228 (ADS205(-) Spi+). NSA205 showed a dramatically depressed lipid content consisting predominantly of phospholipids and triacylglycerols. This lipid content was globally restored in strain NSA228, which had lost ADS205. Furthermore, strains RP181110 and NSA205 presented similar phospholipid and triacylglycerol compositions. No abnormal fatty acids were detected in NSA205.

FIG. 1

AseI restriction map of the ends of the S. ambofaciens RP181110 chromosomal DNA. AUD205 is unique in strain RP181110 and is partially deleted in strain NSA228 (Δp AUD205). Tandem reiteration of AUD205 in strain NSA205 leads to ADS205. The AseI-linking cosmids G36, E7C, and A85 are also indicated. The left extremity of the deletion was not precisely determined in the mutants.

FIG. 2

Growth (●) and spiramycin production (□) of S. ambofaciens RP181110, NSA205, and NSA228 on a chemically defined medium in a 3-liter batch fermentor. Results are means of three independent experiments with standard deviations of 20 to 30%.

FIG. 3

Formation of total lipids during growth of S. ambofaciens RP181110 (○), NSA205 (□), and NSA228 (▵) cultivated in a synthetic medium. Results are means of three independent experiments with standard deviations of 15 to 20%.

FIG. 4

Formation of major lipid families (phospholipids [A], triacylglycerols [B], and other neutral lipids [C] during growth of S. ambofaciens RP181110 (○), NSA205 (□), and NSA228 (▵). Results are means of two independent experiments with standard deviations of 10% for phospholipids, 20% for triacylglycerols, and 30% for other neutral lipids.

FIG. 5

Relative proportions of the major phospholipid families of S. ambofaciens RP181110 (○), NSA205 (□), and NSA228 (▵). (A) CL. (B) PE. (C) PI-LPE. Results are means of two independent experiments with standard deviations of 5 to 10%.

FIG. 6

TLC of ethyl acetate extracts from 200-ml culture supernatants of S. ambofaciens strains. Lanes 1 to 4, spiramycin standards (Sigma) (1, 0.125 mg; 2, 0.25 mg; 3, 0.065 mg; 4, 0.5 mg); lanes 5 and 8, RP181110 extracts; lanes 6 and 9, NSA205 extracts; lanes 7, 10, and 11, NSA228 extracts.