| Autor: |
Mo, SangJoon, Suh, Joo-Won |
| Zdroj: |
Applied Biological Chemistry; February 2016, Vol. 59 Issue: 1 p77-88, 12p |
| Abstrakt: |
We identified another tcsABCDgenes encoding biosynthesis of the allylmalonyl-CoA in new FK506 producing strain, Streptomycessp. KCCM 11116P. The functionality of tcsABCDis confirmed by gene deletion. To validate the functionality of these genes, tcsABCDgenes were heterologously expressed in StreptomyceshygroscopicusATCC 14891 using pSE34. The resulting strain produced the FK506 which showed a molecular ion [M+NH4]+at m/z821 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 β-ketoacyl-acyl carrier protein synthase III gene expression and chemical complementation. Plasmid-based expression of the fabHhomolog dpsCin ΔTcsB using pSE34 and pSET152(ermE*) led to restoration of approximately 32.2 and 28.7 % of the levels of FK506 relative to Streptomycessp. KCCM 11116P, respectively. Furthermore, the addition of propionic acid to the culture of ΔTcsB restored FK506 production up to 35.6 % of overall FK506 levels in Streptomycessp. KCCM 11116P. We have engineered production of 36-methyl-FK506 in ΔTcsB. The heterologous expression of SyteptomycesglaucescensFabH in ΔTcsB 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 ΔTcsB expressing S. glaucescensFabH 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. glaucescensFabH which use isobutyryl-CoA as substrate can initiate methyl side chain biosynthesis in 36-methyl-FK506 in ΔTcsB. |
| Databáze: |
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