Cytochrome P450 107U1 is required for sporulation and antibiotic production in Streptomyces coelicolor

Abstract

The filamentous bacterium Streptomyces coelicolor has a complex life cycle involving the formation of hair-like aerial mycelia on the colony surface, which differentiate into chains of spores. Genes required for the initiation of aerial mycelium formation have been termed 'bld' (bald), describing the smooth, undifferentiated colonies of mutant strains. We report the identification of a new bld gene designated as sco3099 and biochemical analysis of its encoded enzyme, cytochrome P450 (P450, or CYP) 107U1. Deletion of sco3099 resulted in a mutant defective in aerial hyphae sporulation and sensitive to heat shock, indicating that P450 107U1 plays a key role in growth and development of S. coelicolor. This is the first P450 reported to participate in a sporulation process in Streptomycetes. The substrate and catalytic properties of P450 107U1 were further investigated in mass spectrometry-based metabolomic studies. Glycocholic acid (from the medium) was identified as a substrate of P450 107U1 and was oxidized to glyco-7-oxo-deoxycholic acid. Although this reaction is apparently not relevant to the observed sporulation deficiency, it suggests that P450 107U1 might exert its physiological function by oxidizing other steroid-like molecules.

Fig. 1

Construction of mutant strain and Southern blotting to verify the genotype of the sco3099 knockout. Lane 1, positive control with 6.0, 2.9, and 1.5 kb markers; lanes 2, 3, and 4, DNA of the knockout was digested by SalI, EcoRV, and PstI, and the corresponding 3.1, 5.5, and 2.0 kb fragments were detected, respectively.

Fig. 2

(A) Wild type; (B) sco3099 mutant strain; (C) knockout strain rescued with sco3099 cDNA expression; (D) a blank vector was transferred to the mutant strain as a control. All of the strains were incubated on an R2YE agar plate for 3 days.

Fig. 3

SEM of wild type S. coelicolor (A) and mutant strain sco3099 (B); TEM of wild type (C, D) and mutant strain (E, F). (C) mature spores; (D) premature spores; (E) branch of mycelium; (F) irregular septum.

Fig. 4

Fe²⁺-CO vs Fe²⁺ difference spectrum of purified P450107U1 [18]. The P450 concentration was 3 μM.

Fig. 5

Metabolomic analysis by XCMS [20,21]. LC-MS of a putative substrate at m/z 483 (A) and its dehydrogenated product at m/z 481 (B). See Materials and methods for LC-MS details.

Fig. 6

Incubation of commercial glycocholic acid with P450 107U1 results in a dehydrogenated product. (A) Trace for glycocholic acid; (B) trace for product. (glyco-7-oxo-deoxycholic acid).

Fig. 7

NMR spectra of (A) glycocholic acid and (B) synthetic glyco-7-oxo-deoxycholic acid (in CDCl₃, 600 MHz). Some of the key downfield peaks are identified on the spectra. (C) Co-chromatography of the enzymatic oxidation product with synthetic glyco-7-oxo-deoxycholic acid, monitoring relative abundance at m/z 464; (D) mass spectrum of the enzymatic oxidation product, showing the characteristic ammonium ion [M+NH₄]⁺ (m/z 481) in addition to [M+H]⁺ (m/z 464). See text for HRMS.

Fig. 7

NMR spectra of (A) glycocholic acid and (B) synthetic glyco-7-oxo-deoxycholic acid (in CDCl₃, 600 MHz). Some of the key downfield peaks are identified on the spectra. (C) Co-chromatography of the enzymatic oxidation product with synthetic glyco-7-oxo-deoxycholic acid, monitoring relative abundance at m/z 464; (D) mass spectrum of the enzymatic oxidation product, showing the characteristic ammonium ion [M+NH₄]⁺ (m/z 481) in addition to [M+H]⁺ (m/z 464). See text for HRMS.