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Abstract
Ascomycin (FK520) is a multifunctional antibiotic produced by Streptomyces hygroscopicus var. ascomyceticus. In this study, we demonstrated that the inactivation of GlnB, a signal transduction protein belonging to the PII family, can increase the production of ascomycin by strengthening the supply of the precursors malonyl-CoA and methylmalonyl-CoA, which are produced by acetyl-CoA carboxylase and propionyl-CoA carboxylase, respectively. Bioinformatics analysis showed that Streptomyces hygroscopicus var. ascomyceticus contains two PII family signal transduction proteins, GlnB and GlnK. Protein co-precipitation experiments demonstrated that GlnB protein could bind to the α subunit of acetyl-CoA carboxylase, and this binding could be disassociated by a sufficient concentration of 2-oxoglutarate. Coupled enzyme activity assays further revealed that the interaction between GlnB protein and the α subunit inhibited both the activity of acetyl-CoA carboxylase and propionyl-CoA carboxylase, and this inhibition could be relieved by 2-oxoglutarate in a concentration-dependent manner. Because GlnK protein can act redundantly to maintain metabolic homeostasis under the control of the global nitrogen regulator GlnR, the deletion of GlnB protein enhanced the supply of malonyl-CoA and methylmalonyl-CoA by restoring the activity of acetyl-CoA carboxylase and propionyl-CoA carboxylase, thereby improving the production of ascomycin to 390 ± 10 mg/L. On this basis, the co-overexpression of the β and ε subunits of propionyl-CoA carboxylase further increased the ascomycin yield to 550 ± 20 mg/L, which was 1.9-fold higher than that of the parent strain FS35 (287 ± 9 mg/L). Taken together, this study provides a novel strategy to increase the production of ascomycin, providing a reference for improving the yield of other antibiotics.
Highlights
Ascomycin (FK520) is a natural 23-membered macrocyclic antibiotic produced by Streptomyces hygroscopicus var. ascomyceticus ATCC 14891 (Wu et al, 2000), notable for its diverse biological and pharmacological activities, including antifungal (Qi et al, 2014a), antimalarial (Monaghan et al, 2005), immunosuppressive (Dumont et al, 1992), and anticonvulsive effects (Sierra-Paredes and Sierra-Marcuno, 2008)
In order to determine which subgroups these two PII signal transduction proteins belong to, the amino acid sequences encoded by the 1_2537 and 1_5736 genes of S. hygroscopicus var. ascomyceticus were aligned with the GlnK proteins from three other actinobacteria (S. coelicolor, Mycobacterium tuberculosis, and Corynebacterium glutamicum) and the GlnB protein from E. coli (Figures 1C,D)
GlnB is mainly associated with the regulation of glnA, the Glutamine synthetase (GS) structural gene (Schwarz et al, 2014), and NifI is related to the regulation of structural genes for nitrogenase (Arcondeguy et al, 2001)
Summary
Ascomycin (FK520) is a natural 23-membered macrocyclic antibiotic produced by Streptomyces hygroscopicus var. ascomyceticus ATCC 14891 (Wu et al, 2000), notable for its diverse biological and pharmacological activities, including antifungal (Qi et al, 2014a), antimalarial (Monaghan et al, 2005), immunosuppressive (Dumont et al, 1992), and anticonvulsive effects (Sierra-Paredes and Sierra-Marcuno, 2008). Ascomyceticus SA68 and the addition of 3 g/L shikimic acid at 24 h increased the production of FK520 to 450 mg/L, which was 53.3% higher than in the initial strain FS35 (Qi et al, 2014b). In spite of these advances, the insufficient supply of precursors for the secondary metabolite FK520 is still a pressing problem limiting its yield
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