Optimization of FK-506 production in Streptomyces tsukubaensis by modulation of Crp-mediated regulation

Abstract

FK-506 is a potent immunosuppressive macrocyclic polyketide with growing pharmaceutical interest, produced by Streptomyces tsukubaensis. However, due to low levels synthesized by the wild-type strain, biotechnological production of FK-506 is rather limited. Optimization strategies to enhance the productivity of S. tsukubaensis by means of genetic engineering have been established. In this work primarily global regulatory aspects with respect to the FK-506 biosynthesis have been investigated with the focus on the global Crp (cAMP receptor protein) regulator. In expression analyses and protein-DNA interaction studies, the role of Crp during FK-506 biosynthesis was elucidated. Overexpression of Crp resulted in two-fold enhancement of FK-506 production in S. tsukubaensis under laboratory conditions. Further optimizations using fermentors proved that the strategy described in this study can be transferred to industrial scale, presenting a new approach for biotechnological FK-506 production. Key Points • The role of the global Crp (cAMP receptor protein) regulator for FK-506 biosynthesis in S. tsukubaensis was demonstrated • Crp overexpression in S. tsukubaensis was applied as an optimization strategy to enhance FK-506 and FK-520 production resulting in two-fold yield increase

Impact of Nitrogen-Containing Compounds on Secondary Metabolism in Streptomyces spp.—A Source of Metabolic Engineering Strategies

“…This is in contrast to the direct activating effect of Crp on antibiotic production in S. coelicolor [99]. Crp overexpression has been shown to enhance the titers of the secondary metabolite tacrolimus (FK-506) in S. tsukubaensis [100] (Table 2).…”

“…Examples are lysine for the biosynthesis of FK-506 [100] and asparagine and malonyl CoA for the biosynthesis of lysolipin [106]. Certain nutrients can increase, decrease, or even stop the production of secondary metabolites.…”

“…A typical lantibiotic biosynthesis reflects NAI-107 production-its amount in the late growth phase is several times greater than at the beginning of cultivation [114,115]. Examples are lysine for the biosynthesis of FK-506 [100] and asparagine and mal CoA for the biosynthesis of lysolipin [106]. Certain nutrients can increase, decrease even stop the production of secondary metabolites.…”