Engineered Biosynthesis of a Novel Amidated Polyketide, Using the Malonamyl-Specific Initiation Module from the Oxytetracycline Polyketide Synthase

Abstract

Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases (PKSs). Understanding the biochemistry of tetracycline PKSs is an important step toward the rational and combinatorial manipulation of tetracycline biosynthesis. To this end, we have sequenced the gene cluster of oxytetracycline (oxy and otc genes) PKS genes from Streptomyces rimosus. Sequence analysis revealed a total of 21 genes between the otrA and otrB resistance genes. We hypothesized that an amidotransferase, OxyD, synthesizes the malonamate starter unit that is a universal building block for tetracycline compounds. In vivo reconstitution using strain CH999 revealed that the minimal PKS and OxyD are necessary and sufficient for the biosynthesis of amidated polyketides. A novel alkaloid (WJ35, or compound 2) was synthesized as the major product when the oxy-encoded minimal PKS, the C-9 ketoreductase (OxyJ), and OxyD were coexpressed in CH999. WJ35 is an isoquinolone compound derived from an amidated decaketide backbone and cyclized with novel regioselectivity. The expression of OxyD with a heterologous minimal PKS did not afford similarly amidated polyketides, suggesting that the oxy-encoded minimal PKS possesses novel starter unit specificity.

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

“…[225] Oxytetracycline biosynthetic genes were isolated from Streptomyces rimosus. [226] Because the mRNA level of oxyB, corresponding to KS β of minimal PKS, was almost unobservable through qPCR, the authors overexpressed 6 E. coli sigma factors, of which increase in oxyB mRNA level was achieved by overexpressing σ 54 . Oxytetracycline titer of 2 mg L −1 was achieved, but still, development of a general means for soluble expression of minimal PKS in E. coli was unsuccessful.…”

Genetic characterization of enzymes involved in the priming steps of oxytetracycline biosynthesis in Streptomyces rimosus

“…PCR was performed with Platinum Pfx DNA polymerase (Invitrogen) using cosmid pYT264 as template (Zhang et al, 2006). The PCR products were ligated into pCR-Blunt vector (Invitrogen) for further cloning.…”

“…ZhuC was determined in vitro to be an acetyl-ACP thioesterase that removes the competing acetate-containing starter unit to enable priming of the R1128 minimal PKS by the various medium-chain fatty acids (Tang et al, 2004). Therefore, OxyP was proposed to perform a similar function in S. rimosus to attenuate the acetate priming pathway that may lead to the biosynthesis of ADOTC (Zhang et al, 2006). However, heterologous experiments with the oxy minimal PKS and OxyD did not show any marked difference in product phenotypes with or without coexpression of OxyP.…”

“…It is believed that the malonamate is incorporated as a primer unit in the form of either malonamyl-CoA or malonamyl-ACP, which can be derived from the amidation of malonylCoA or malonyl-ACP, respectively, by the action of an amidotransferase (McDowall et al, 1991;Thomas & Williams, 1983). Our previous heterologous experiments with the oxytetracycline (oxy gene cluster) minimal PKS in Streptomyces coelicolor demonstrated that an ATP-dependent amidotransferase, OxyD, is required in addition to the minimal PKS (OxyAB and OxyC) to generate the amidated backbone (Zhang et al, 2006). Despite this finding, the exact substrate of OxyD has not been confirmed, and more importantly, its function in the native OTC-producing S. rimosus has not been confirmed genetically.…”

Substrate specificity of benzamide synthetase involved in 4-hydroxy-3-nitrosobenzamide biosynthesis

“…PhzH catalyzes the amidation of phenazine-1-carboxylic acid to phenazine-1-carboxamide in the biosynthetic pathway of phenazine-1-carboxamide in Pseudomonas aeruginosa PAO1 4 and Pseudomonas chlororaphis PCL1391. 5 OxyD and SsfD are involved in the biosynthesis of oxytetracycline in Streptomyces rimosus 6 and tetracycline SF2575 in Streptomyces sp. SF2575, 7 respectively.…”