Enhanced cercosporin production by co-culturing Cercospora sp. JNU001 with leaf-spot-disease-related endophytic bacteria

Abstract

Background: Owing to the excellent properties of photosensitization, cercosporin, one of naturally occurring perylenequinonoid pigments, has been widely used in photodynamic therapy, or as an antimicrobial agent and an organophotocatalyst. However, because of low efficiency of total chemical synthesis and low yield of current microbial fermentation, the limited production restricts its broad applications. Thus, the strategies to improve the production of cercosporin were highly desired. Besides traditional optimization methods, here we screened leaf-spot-disease-related endophytic bacteria to co-culture with our previous identified Cercospora sp. JNU001 to increase cercosporin production.

Results: Bacillus velezensis B04 and Lysinibacillus sp. B15 isolated from leaves with leaf spot diseases were found to facilitate cercosporin secretion into the broth and then enhance the production of cercosporin. After 4 days of co-culture, Bacillus velezensis B04 allowed to increase the production of cercosporin from 128.2 mg/L to 984.4 mg/L, which was 7.68-fold of the previously reported one. Lysinibacillus sp. B15 could also enhance the production of cercosporin with a yield of 626.3 mg/L, which was 4.89-fold higher than the starting condition. More importantly, we found that bacteria B04 and B15 employed two different mechanisms to improve the production of cercosporin, in which B04 facilitated cercosporin secretion into the broth by loosening and damaging the hyphae surface of Cercospora sp. JNU001 while B15 could adsorb cercosporin to improve its secretion.

Conclusions: We here established a novel and effective co-culture method to improve the production of cercosporin by increasing its secretion ability from Cercospora sp. JNU001, allowing to develop more potential applications of cercosporin.

Keywords: Cercosporin; Co-culture; Endophytic bacteria; Microbial fermentation; Secretion.

Fig. 1

Improvement of CP production by optimizing culture medium and culture conditions. a Molecular structure of CP. The characterized 3,10-dihydroxy-4,9-perylenequinone chromophore core structure was labelled in blue. b Influence of culture time on CP production. d = day. c pH optimization of S-7 medium. d Optimization of culture temperature. e Selection of carbon source in S-7 medium. f Screening of nitrogen source in S-7 medium (cardamom: cardamom powder, ammonia: ammonia sulfate, nd: no detected). ^∗p < 0.05 and ^∗∗p < 0.01 versus control (carbon/nitrogen source in traditional S-7 medium)

Fig. 2

Enhanced CP production by co-culturing with bacteria B04 or B15. a The chromatogram of CP production of Cercospora sp. JNU001 alone. b, c The chromatogram of enhanced CP production by co-culturing Cercospora sp. JNU001 with B04 (b) or B15 (c)

Fig. 3

Identification and characterization of two isolated bacteria B04 and B15. a, b Microscopic appearance of culture colony of B04 strain and B15 strain on LB plate after 24 h. c The phylogenetic tree of B04 strain and its relationship with other Bacillus species. d The phylogenetic tree of B15 strain and its relationship with other Lysinibacillus species

Fig. 4

Effect of CP production by co-culturing B04 or B15 strain with Cercospora sp. JNU001. a, b Effect of the concentration of B04 or B15 strain on CP production. c. Effect of culture time on CP production when Cercospora sp. JNU001 was co-cultured with B04 or B15. d Effect of the adding time of B04 on CP production. e The glucose consumption of Cercospora sp. JNU001 grew in the modified S-7 medium and under co-culture with B04 or B15 strain condition. The arrows indicate the beginning of significant differences caused by co-culture

Fig. 5

In vitro confrontation bioassays between bacteria and Cercospora sp. JNU001. a Schematic diagram of in vitro confrontation bioassay between B04 and Cercospora sp. JNU001. b Schematic diagram of in vitro confrontation bioassay between B15 and Cercospora sp. JNU001. c Effects of B04 (i–iv) or B15 (v–viii) on Cercospora sp. JNU001

Fig. 6

Effects of B04 and B15 on fungal growth and CP secretion. a Dry fungal biomass of Cercospora sp. JNU001 without or with bacteria. b Intracellular CP production of Cercospora sp. JNU001 extracted from a. c Extracellular CP production purified from culture broth after co-culture without or with bacteria. d Total CP production, which was calculated by intracellular CP production (b) and extracellular CP production (c)

Fig. 7

FESEM observation of co-culture Cercospora sp. JNU001 with B04. a B04 samples. b, c Cercospora sp. JNU001 samples. d–f Co-culture samples of B04 and Cercospora sp. JNU001. White arrows indicate bacteria B04, and red arrows indicate the damage of fungal hyphae. Scale bar was indicated