Ectopic expression of the minimal whiE polyketide synthase generates a library of aromatic polyketides of diverse sizes and shapes

Abstract

The single recombinant expressing the Streptomyces coelicolor minimal whiE (spore pigment) polyketide synthase (PKS) is uniquely capable of generating a large array of well more than 30 polyketides, many of which, so far, are novel to this recombinant. The characterized polyketides represent a diverse set of molecules that differ in size (chain length) and shape (cyclization pattern). This combinatorial biosynthetic library is, by far, the largest and most complex of its kind described to date and indicates that the minimal whiE PKS does not independently control polyketide chain length nor dictate the first cyclization event. Rather, the minimal PKS enzyme complex must rely on the stabilizing effects of additional subunits (i.e., the cyclase whiE-ORFVI) to ensure that the chain reaches the full 24 carbons and cyclizes correctly. This dramatic loss of control implies that the growing polyketide chain does not remain enzyme bound, resulting in the spontaneous cyclization of the methyl terminus. Among the six characterized dodecaketides, four different first-ring cyclization regiochemistries are represented, including C7/C12, C8/C13, C10/C15, and C13/C15. The dodecaketide TW93h possesses a unique 2,4-dioxaadamantane ring system and represents a new structural class of polyketides with no related structures isolated from natural or engineered organisms, thus supporting the claim that engineered biosynthesis is capable of producing novel chemotypes.

Figure 1

Structures of TW93a–d (1–4).

Figure 2

LC-ESMS analysis of minimal whiE PKS polyketides from YU105/pIJ4293. HPLC was monitored at 280 nm. Ion chromatograms for the pseudomolecular ion MH⁺ of hepta-, undeca-, and dodecaketides are displayed. Note that several of the 451 ions are fragments from MH⁺ 469 resulting from the loss of H₂O and are indicated with an asterisk.

Figure 3

Structures of minimal PKS products from the act/fren (SEK4 and SEK4b) and the tcm (SEK15 and SEK15b) pathways.

Figure 4

Structures of the minimal whiE PKS dodecaketides TW93c–h (3–8) and of their common poly-β-ketoacyl thioester biosynthetic precursor. Carbons are labeled according to their number in the polyketide backbone.

Figure 5

Proposed formation of TW93h (8).