Biochemical Investigation of Pikromycin Biosynthesis Employing Native Penta- and Hexaketide Chain Elongation Intermediates

Abstract

The unique ability of the pikromycin (Pik) polyketide synthase to generate 12- and 14-membered ring macrolactones presents an opportunity to explore the fundamental processes underlying polyketide synthesis, specifically the mechanistic details of chain extension, keto group processing, acyl chain release, and macrocyclization. We have synthesized the natural pentaketide and hexaketide chain elongation intermediates as N-acetyl cysteamine (NAC) thioesters and have used them as substrates for in vitro conversions with engineered PikAIII+TE and in combination with native PikAIII (module 5) and PikAIV (module 6) multifunctional proteins. This investigation demonstrates directly the remarkable ability of these monomodules to catalyze one or two chain extension reactions, keto group processing steps, acyl-ACP release, and cyclization to generate 10-deoxymethynolide and narbonolide. The results reveal the enormous preference of Pik monomodules for their natural polyketide substrates and provide an important comparative analysis with previous studies using unnatural diketide NAC thioester substrates.

The Origins of Specificity in Polyketide Synthase Protein Interactions

“…However, this remains to be directly demonstrated. Since the nonstandard pikro_003 - pikro_004 interaction can occur even when the N-terminal docking domain of pikro_004 is present [26], and since the C-terminal docking domain of eryth_001 is necessary for the eryth_001 - pikro_004 interaction [24], the possibility of H1–T2 docking cannot be ruled out. Nevertheless, when taken together, the results from hybrid pathways suggest that the classification of docking domains into compatibility classes is biologically meaningful.…”

Reconstitution of the Myxothiazol Biosynthetic Gene Cluster by Red/ET Recombination and Heterologous Expression in Myxococcus xanthus

“…One of the strategies for the engineering of natural product biosynthesis, especially if the manipulation on the chromosome in the producer strain is difficult or impossible, is the heterologous expression of the corresponding gene cluster. Such studies, in combination with biochemical and genetic analyses of biosynthetic gene clusters from myxobacteria, will make the manipulation of the biosyntheses more feasible, as has already been shown for some other bacterial genera, e.g., the wellinvestigated erythromycin gene cluster, as well as the pikromycin or aureothin biosynthetic genes (1,14,30). Different strategies have been used for heterologous expression (for a review, see reference 44), which in most cases aimed at the definition of conditions to improve the yield of the natural product.…”

Nonribosomal Peptide Biosynthesis: Point Mutations and Module Skipping Lead to Chemical Diversity

“…[9] Module skipping has recently been described for an engineered PKS system [19] and is assumed to be responsible for the generation of minor byproducts of PKS assembly lines. [10,12,[20][21][22] To date, no such process has been described in the biochemistry of multimodular NRPSs.…”