Elucidation of first step of the allylmalonyl-CoA biosynthetic pathway by expression of heterologous KSIII gene and enhancement of 36-methyl-FK506 production by genetic and chemical engineering

We identified another tcsABCD genes encoding biosynthesis of the allylmalonyl-CoA in new FK506 producing strain, Streptomyces sp. KCCM 11116P. The functionality of tcsABCD is confirmed by gene deletion. To validate the functionality of these genes, tcsABCD genes were heterologously expressed in Streptomyces hygroscopicus ATCC 14891 using pSE34. The resulting strain produced the FK506 which showed a molecular ion [M+NH4](+) at m/z 821 as verified by LC/ESI-MS. To address the function of TcsB in the allylmalonyl-CoA pathway, we pursued the construction of two different combinations: the heterologous FabH-like beta-ketoacyl-acyl carrier protein synthase III gene expression and chemical complementation. Plasmid-based expression of the fabH homolog dpsC in Delta TcsB using pSE34 and pSET152(ermE*) led to restoration of approximately 32.2 and 28.7 % of the levels of FK506 relative to Streptomyces sp. KCCM 11116P, respectively. Furthermore, the addition of propionic acid to the culture of DTcsB restored FK506 production up to 35.6 % of overall FK506 levels in Streptomyces sp. KCCM 11116P. We have engineered production of 36-methyl-FK506 in DTcsB. The heterologous expression of Syteptomyces glaucescens FabH in Delta TcsB gave rise to 5.9-fold increase in the 36-methyl-FK506 level as compared with mutasynthetic production by 4-methylpentanoic acid. Supplementing the culture broth of DTcsB expressing S. glaucescens FabH with valine resulted in an additional 2.5-fold increase in the 36-methyl-FK506 titer. Overall, this study shows that TcsB was found to maintain a very high-specific activity for propionyl-CoA in allylmalonyl-CoA biosynthetic work and S. glaucescens FabH which use isobutyryl-CoA as substrate can initiate methyl side chain biosynthesis in 36-methyl-FK506 in Delta TcsB.