Identification of wysPII as an Activator of Morphological Development in Streptomyces albulus CK-15

Abstract

Wuyiencin is produced by Streptomyces albulus var. wuyiensis and used widely in agriculture to control a variety of fungal diseases, such as cucumber downy mildew, strawberry powdery mildew, and tomato gray mold. As an industrially-produced biopesticide, reducing production costs is very important for popularization of this approach. To obtain a rapidly growing strain that effectively shortens the fermentation time, we investigated the effects of knockout and overexpression of the wysPII gene, a member of the LuxR regulatory gene family, in S. albulus strain CK-15. The ΔwysPII mutant exhibited a reduced rate of growth and sporulation. The time taken to reach the greatest mycelial biomass was approximately 18 h shorter in the ooPII (wysPII overexpressing) strain compared with that of the wild-type (WT) strain. In addition, the time to reach the greatest wuyiencin production was 56 h in the ooPII strain compared with 62 h in the WT strain. Furthermore, wysPII was shown to act as an activator of morphological development without affecting wuyiencin production. Thus, the ooPII strain can be used to reduce costs and increase efficiency in industrial fermentation processes for wuyiencin production.

Keywords: LuxR family; Streptomyces albulus; morphological development; regulatory genes; wysPII.

FIGURE 1

Identification of wysPII deletion (A) Gene replacement of wysPII in Streptomyces albulus CK-15. (B) Confirmation of the constructed ΔwysPII mutant by PCR. Lanes: M, BM 5,000-bp DNA ladder; 1, PCR verification with primers TF and TR using S. albulus ΔwysPII genomic DNA as the template; 2, PCR verification with primers TF and TR using S. albulus CK-15 genomic DNA as the template.

FIGURE 2

Effect of wysPII deletion on morphological development and wuyiencin production. (A) Phenotypes of 5-day-old S. albulus CK-15 and S. albulus ΔwysPII. (B) Antibacterial effect of S. albulus CK-15 and S. albulusΔwysPII fermentation culture supernatant. (C) Electron microscopy of S. albulus CK-15 and S. albulus ΔwysPII. (D) HPLC analysis of standard wuyiencin. (E) HPLC analysis of wuyiencin production levels in S. albulus CK-15. (F) HPLC analysis of wuyiencin production levels in S. albulusΔwysPII.

FIGURE 3

Effect of wysPII complementation on morphological development and wuyiencin production. (A) Confirmation of the complementation strain S. albulus comPII by PCR. Lanes: M, BM 5000-bp DNA ladder; 1, PCR verification with primers Am-F and Am-R using S. albulus comPII genomic DNA as the template; 2, PCR verification with primers Am-F and Am-R using S. albulus CK-15 genomic DNA as the template. (B) HPLC analysis of wuyiencin production levels in S. albulus comPII. (C) Phenotypes of 5-day-old S. albulus CK-15 and S. albulus comPII cultured on an MS plate. (D) Antibacterial effect of S. albulus CK-15 and S. albulus comPII fermentation culture supernatant. (E) Electron microscopy of S. albulus CK-15 and S. albulus comPII.

FIGURE 4

Effect of wysPII overexpression on morphological development and wuyiencin production. (A) Phenotypes of 5-day-old S. albulus pSETC and S. albulus ooPII. (B) Antibacterial effect of S. albulus pSETC and S. albulus ooPII fermentation culture supernatant. (C) Electron microscopy of S. albulus pSETC and S. albulus ooPII. (D) HPLC analysis of wuyiencin production levels in S. albulus pSETC. (E) HPLC analysis of wuyiencin production levels in S. albulus ooPII.

FIGURE 5

Time-course of wuyiencin production. Wuyiencin production by S. albulus CK-15, S. albulus ooPII, S. albulus ΔwysPII, S. albulus comPII and S. albulus ooR during fermentation for 72 h.

FIGURE 6

Morphological development of each strain on MS medium from 48 to 120 h. (A) The growth condition of different strains at 48 h. (B) Cultures of different strains after 72 h. (C) Cultures of different strains after 96 h. (D) Cultures of different strains after 120 h. (a) S. albulus CK-15, (b) S. albulus ΔwysPII, (c) S. albulus comPII, (d) S. albulus ooPII.

FIGURE 7

Morphological development of each strain on ISP2 medium from 48 to 120 h. (A) The growth of different strains at 48 h. (B) Cultures of different strains after 72 h. (C) Cultures of different strains after 96 h. (D) Cultures of different strains after 120 h. (a) S. albulus CK-15, (b) S. albulus ΔwysPII, (c) S. albulus comPII, (d) S. albulus ooPII.

FIGURE 8

Morphological development of each strain on ISP3 medium from 48 to 120 h. (A) The growth of different strains at 48 h. (B) Cultures of different strains after 72 h. (C) Cultures of different strains after 96 h. (D) Cultures of different strains after 120 h. (a) S. albulus CK-15, (b) S. albulus ΔwysPII, (c) S. albulus comPII, (d) S. albulus ooPII.

FIGURE 9

Result of mycelial biomass measurement. Biomass of S. albulus CK-15, S. albulus ooPII, S. albulus ΔwysPII, S. albulus comPII during culture for 90 h.

FIGURE 10

Developmental gene expression in S. albulus CK-15 and S. albulus ooPII by RT-qPCR. The RNA samples were isolated from 72-h cultures on MS medium. The relative values of S. albulus CK-15 are designated as 1. Data represent the mean ± standard deviation of three independent experiments.