Designed biosynthesis of 36-methyl-FK506 by polyketide precursor pathway engineering

Abstract

The polyketide synthase (PKS) biosynthetic code has recently expanded to include a newly recognized group of extender unit substrates derived from α,β-unsaturated acyl-CoA molecules that deliver diverse side chain chemistry to polyketide backbones. Herein we report the identification of a three-gene operon responsible for the biosynthesis of the PKS building block isobutyrylmalonyl-CoA associated with the macrolide ansalactam A from the marine bacterium Streptomyces sp. CNH189. Using a synthetic biology approach, we engineered the production of unnatural 36-methyl-FK506 in Streptomyces sp. KCTC 11604BP by incorporating the branched extender unit into FK506 biosynthesis in place of its natural C-21 allyl side chain, which has been shown to be critical for FK506's potent immunosuppressant and neurite outgrowth activities.

Figure 1. Genetic engineering approach towards 36-methyl-FK506 production

(a) The FK506 producer S. sp. KCTC 11604BP synthesizes allylmalonyl-CoA, which derives from a multi-step pathway involving the ketosynthase TcsB, the cognate acyltransferase and acyl-carrier protein TcsA, the crotonyl-CoA carboxylase TcsC, and the dehydrogenase TcsD. (b) The enzymatic machineries for FK506 (a) and isobutyrylmalonyl-CoA (c) biosynthesis were combined from two different streptomycetes to generate the hybrid pathway of 36-methyl-FK506 in Streptomyces sp. KCTC 11604BP ΔtcsB. (c) The ansalactam producer S. sp. CNH189 assembles isobutyrylmalony-CoA using the FabH-like β-ketoacyl-ACP-synthase AnlF, the dehydrogenase AnlG, and the crotonyl-CoA carboxylase AnlE.

Figure 2. Expression constructs containing the ibMCoA gene cassette

The three-gene operon anlE–G was amplified from the ansalactam producer strain S. sp. CNH189 and cloned into pSET152 containing the native upstream region (orange) or the ermEp sequence (green) affording the expression constructs pMW1 and pLD6, respectively.

Figure 3. Production of 36-methyl-FK506 via heterologous expression of the ibMCoA pathway in a S. sp. KCTC 11604BP ΔtcbB mutant strain

Representative HPLC-ESI-MS chromatograms visualized by selected ion monitoring for FK506 (m/z 821 [M+NH₄]⁺; in blue) and 36-Me-FK506 (m/z 835 [M+NH₄]⁺; in red) in top panel. Results were obtained from five independent cultivation experiments of (a) the FK506 producing wild-type strain, (b) Streptomyces sp. KCTC 11604BP ΔtcbB, (c) the ΔtcbB mutant strain supplemented with 4-methylpentanoic acid (MPA), (d) the tcbB deletion strain with ermE*p-ibMCoA biosynthetic genes (pLD6), and (e) with the native promoter-ibMCoA biosynthetic genes (pMW1). Production levels of FK50 (in blue) and 36-Me-FK506 (in red) are graphically displayed in the bottom panel.