Optimization of the precursor supply for an enhanced FK-506 production in Streptomyces tsukubaensis

Tacrolimus (FK-506) is a macrolide widely used as immunosuppressant to prevent transplant rejection. Synthetic production of FK-506 is not efficient and costly, whereas the biosynthesis of FK-506 is complex and the level produced by the wild type strain, Streptomyces tsukubaensis , is very low. We therefore engineered FK-506 biosynthesis and the supply of the precursor L-lysine to generate strains with improved FK-506 yield. To increase FK-506 production, first the intracellular supply of the essential precursor lysine was improved in the native host S. tsukubaensis by engineering the lysine biosynthetic pathway. Therefore, a feedback deregulated aspartate kinase Ask St * of S. tsukubaensis was generated by site directed mutagenesis. Whereas overexpression of Ask St * resulted only in a 17% increase in FK-506 yield, heterologous overexpression of a feedback deregulated Ask Cg * from Corynebacterium glutamicum was proven to be more efficient. Combined overexpression of Ask Cg * and DapA St , showed a strong enhancement of the intracellular lysine pool following increase in the yield by approximately 73% compared to the wild type. Lysine is coverted into the FK-506 building block pipecolate by the lysine cyclodeaminase FkbL. Construction of a Δ fkbL mutant led to a complete abolishment of the FK-506 production, confirming the indispensability of this enzyme for FK-506 production. Chemical complementation of the Δ fkbL mutant by feeding pipecolic acid and genetic complementation with fkbL as well as with other lysine cyclodeaminase genes ( pip Af , pipA St , originating from Actinoplanes friuliensis and Streptomyces pristinaespiralis , respectively) completely restored FK-506 production. Subsequently, FK-506 production was enchanced by heterologous overexpression of Pip Af and PipA Sp in S. tsukubaensis . This resulted in a yield increase by 65% compared to the WT in the presence of Pip Af from A. friuliensis . For further rational yield improvement, the crystal structure of Pip Af from A. friuliensis was determined at 1.3 Å resolution with the cofactor NADH bound and at 1.4 Å with its substrate lysine. Based on the structure the Ile91 residue was replaced by Val91 in Pip Af , which resulted in an overall increase of FK-506 production by approx. 100% compared to the WT.