High production of a class III lantipeptide AmfS in Streptomyces griseus

Figures & data

Fig. 1. Schematic representation of the class III lantipeptide biosynthetic gene clusters in Streptomyces and related bacteria and the amino acid sequences of the precursor AmfS.

Notes: (A) The orientation and length of ORFs predicted by the genome sequencing project of S. griseus and related actinomycetes are indicated by arrowheads. The corresponding coding sequence numbers from the genome sequence database assigned to each putative lantipeptide-modifying enzyme are shown. The ORFs encode: a lanthionine synthetase (amfT); lantipeptide precursor (amfS); ABC-type transporters (amfB and amfA); and a response regulator of the two-component regulatory system (amfR). The gene organization scheme was drawn by drawGeneArrows3 (http://www.ige.tohoku.ac.jp/joho/). (B) Amino acid sequence alignment of the representative class III lantipeptide precursors, SapB of S. coelicolor and AmfS of S. griseus and S. avermitilis. (C) The predicted structures of AmfS and AmfSsav based on the structure of SapB.^36,37) The lanthionine (Lan) structures are synthesized by the dehydration of Ser residues to 2,3-didehydroalanines (Dha) and Michael-type addition of Cys to the 2,3-didehydro amino acids.

Fig. 2. Phenotype of S. griseus Grd1 mutant.

Notes: S. griseus wild type (WT) and Grd1 mutant were grown at 28 °C for 2 days on YMP solid medium containing 1 and 10% glucose. Colonies forming aerial mycelium appear white, whereas non-producing colonies appear cream or brown.

Fig. 3. Physical structures of the expression vectors pIJ702E2 and pAMF19.

Notes: (A) pIJ702E2 carries two ermEp2 and a kanamycin resistance gene, aphII. The cloning sites are EcoRI, HindIII, and BglII sites. (B) pAMF19 harboring a thiostrepton resistance gene (tsr) and a single ermEp2 in the preceding region of amfR. The cloning sites are PstI and SphI sites. The vector maps were created using GENETYX Ver. 11 (GENETYX Cor., Tokyo, JAPAN).

Fig. 4. Schematic representation of AmfS expression vectors and the expression analyses of AmfS in S. griseus by SDS-PAGE.

Notes: (A) pSPO1 has an S. griseus amf cluster with native promoters. pSPO10 has an S. griseus amf cluster under the control of two ermEp2. pSPO100 has the positive regulator gene amfR under the control of the ermEp2 which directs the constitutive expression of amfT and amfS. (B) The extracellular proteins produced by S. griseus wild type and the Grd1 strain carrying the AmfS expression plasmids were separated by SDS-PAGE and detected by silver staining. The protein bands corresponding to AmfS, as expected based on the deduced molecular mass, are indicated by arrowheads. M represents the molecular weight marker. The yield of AmfS was estimated to be 6.0 μg/mL (Grd1/pSPO10 cultured in Minimal medium), 26.7 μg/mL (Grd1/pSPO10 cultured in Algae-1% maltose medium), and 24.2 μg/mL (Grd1/pSPO100 cultured in Algae-1% maltose medium).

Fig. 5. Morphology of S. avermitilis ΔamfSsav and production of S. avermitilis AmfSsav in the S. griseus Grd1 strain.

Notes: (A) S. avermitilis wild type and the ΔamfSsav were grown on YMS medium at 30 °C for 3 days. While the wild type formed white aerial mycelium, ΔamfSsav exhibited a bald phenotype. Scanning electron micrograph images of the wild-type strain and the ΔamfSsav at 30 °C for 4 days are shown. Bars, 1 µm. Aerial mycelium formation in ΔamfSsav was restored by the exogenous addition of partially purified AmfSsav. The photograph was taken after 6 days of growth. (B) Aerial mycelium formation of S. griseus ΔamfS carrying pIJ702E2, and pSPO10SAV grown on a YMP- 1% glucose solid medium containing thiostrepton for 3 days. (C) The extracellular proteins produced by the Grd1 strain carrying pIJ702E2 or pSPO10SAV were separated by SDS-PAGE and detected by silver staining. The bands of AmfSsav based on deduced molecular mass is indicated by an arrowhead. The dilution factors for the Bennett’s- 1% maltose liquid media are shown. The yield of AmfSsav produced by Grd1/pSPO10SAV cultured in the 2, 3, and 4-fold diluted Bennett’s medium was estimated to be 23.1, 14.8, and 3.9 μg/mL, respectively.

Fig. 6. Genetic and extracellular-complementation tests of S. griseus ΔamfS.

Notes: Donor strains (upper colonies) are ΔamfS carrying the wild-type amfS or its mutants. All recipient strains (lower colonies) are ΔamfS carrying an empty vector pNU10. Both strain were inoculated in close proximity on YMP-1% glucose medium, and patches were photographed after 5 days of growth at 28 °C. The genetically complemented ΔamfS in the upper colonies exhibited a white colonies forming aerial mycelium or cream colonies growing substrate mycelium (bald phenotype). The formation of white aerial mycelium in the lower colonies (the ΔamfS as a recipient) indicates that the upper colonies produce active AmfS peptide, while bald phenotype in the lower colonies indicates that the upper colonies do not produce AmfS or produce inactive AmfS.