Improvement of FK506 Production in Streptomyces tsukubaensis by Genetic Enhancement of the Supply of Unusual Polyketide Extender Units via Utilization of Two Distinct Site-Specific Recombination Systems

Abstract

bFK506 is a potent immunosuppressant that has a wide range of clinical applications. Its 23-member macrocyclic scaffold, mainly with a polyketide origin, features two methoxy groups at C-13 and C-15 and one allyl side chain at C-21, due to the region-specific incorporation of two unusual extender units derived from methoxymalonyl-acyl carrier protein (ACP) and allylmalonylcoenzyme A (CoA), respectively. Whether their intracellular formations can be a bottleneck for FK506 production remains elusive. In this study, we report the improvement of FK506 yield in the producing strain Streptomyces tsukubaensis by the duplication of two sets of pathway-specific genes individually encoding the biosyntheses of these two extender units, thereby providing a promising approach to generate high-FK506-producing strains via genetic manipulation. Taking advantage of the fact that S. tsukubaensis is amenable to two actinophage (⌽C31 and VWB) integrase-mediated recombination systems, we genetically enhanced the biosyntheses of methoxymalonyl-ACP and allylmalonyl-CoA, as indicated by transcriptional analysis. Together with the optimization of glucose supplementation, the maximal FK506 titer eventually increased by approximately 150% in comparison with that of the original strain. The strategy of engineering the biosynthesis of unusual extender units described here may be applicable to improving the production of other polyketide or nonribosomal peptide natural products that contain pathway-specific building blocks.

FK506 Maturation Involves a Cytochrome P450 Protein-Catalyzed Four-Electron C-9 Oxidation in Parallel with a C-31O-Methylation

“…5A). Both FkbD and FkbM are highly tolerable in their substrate choice, consistent with the fact that FK520, the coproduct in S. tsukubaensis distinct from FK506 in the substitution at C-21 of an ethyl instead of an allyl (17), utilizes the same biosynthetic pathway to furnish the 9-oxo and 31-O-methyl groups. Rapamycin maturation also shares these two modifications (31); however, whether or not they proceed in an interchangeable way remains to be determined.…”

“…Strain cultivation and compound extraction were carried out according to procedures described previously (17 Expression and purification of FkbD and FkbM. To express fkbD, a 1.17-kb fkbD-containing fragment was obtained by PCR amplification with primers pDf and pDr and then cloned into the pMD19-T vector to yield pFL2041.…”

“…During the macrolide-forming process, the PKSs for FK506 incorporate two unusual extender units, methoxymalonyl (MOM) (also common for FK520) and allylmalonyl (AM) (unique for FK506), to afford two methoxy groups at C-13 and C-15 and one allyl side chain at C-21, respectively (7,(14)(15)(16). Genetic enhancement of the in vivo supply of MOM and AM improved FK506 production (17), therefore providing a strategy potentially helpful for increasing the yields of other secondary metabolites that contain pathway-specific building blocks.…”

Designed Biosynthesis of 36-Methyl-FK506 by Polyketide Precursor Pathway Engineering

“…26, 27 FK506 biosynthesis has only recently been subjected to genetic engineering efforts, owning to the successful pathway elucidation of the unusual extender unit allylmalonyl-CoA. 3, 16, 28, 29 …”

Metabolic engineering with systems biology tools to optimize production of prokaryotic secondary metabolites

“…3B). 37,38 Examples of the rational approaches used to improve the production of secondary metabolites include the enhanced supply of intracellular precursors 39,40 and the overexpression of positive regulators 41 and/or removal of competing pathways leading to other byproducts. 42 These rational approaches also employed systems biology tools such Fig.…”