Identification of the Polyketide Biosynthetic Machinery for the Indolizidine Alkaloid Cyclizidine

Abstract

The cyclizidine biosynthetic gene cluster was identified from Streptomyces NCIB 11649, which revealed the polyketide biosynthetic machinery for cyclizidine alkaloid biosynthesis. Both in vivo mutagenesis study and in vitro biochemical analysis provided insight into the timing and mechanism of the biosynthetic enzymes that produce cyclizidine-type indolizidine alkaloids.

Figure 1

Molecular structures of indolizidine alkaloids.

Figure 2

Map of the cyclizidine gene cluster and proposed biosynthetic pathway.

Figure 3

Genetic characterization of CLD gene cluster. (A) HPLC analysis (260 nm) of metabolites produced by wild-type and mutant Streptomyces NCIB 11649 strains. (B) Structures of purified metabolites. The stereochemistry at C9, C10, and C13 of CLD-1 is mainly predicted from the bioinformatics analysis of CycKR5 (B1-type) and CycKR7 (B1-type) (Figure S22).

Figure 4

Biochemical characterization of CycF-AT_L and CycF-ACP_L (A) Schematic of the transacylation reaction. (B) LC/HRMS chromatograms showing AT_L-promoted formation of propionyl-ACP_L (1) from holo-ACP_L (2) (trace II). 1 was undetectable in controls without propionyl-CoA (trace I) or AT_L (trace III). (C) Deconvoluted masses of 1 and 2 in HRMS analysis.