Biosynthesis, mutational precursor-directed

Mutasynthesis, also called mutational biosynthesis, involving precursor-directed biosynthesis using a mutant strain blocked in a crucial biosynthetic step, which then incorporates the alternative substrate to produce an analog of the wild-type compound 119,122) (Fig. lA, F). 

Bu Lock et al. had shown initially [24] that supplementation of S. avermitilis fermentations with a range of fatty acids resulted in their uptake and incorporation to generate novel avermectins modified at the C25 side chain of the molecule. This approach, described as precursor-directed biosynthesis, has been employed to produce many new antibiotics [25], but the co-expression of the parent molecule interferes with the detection and isolation of the novel analogs. To circumvent these difficulties, the elegant technique of mutational biosynthesis [26] or mutasynthesis [27,28] was developed. In this approach, a mutant of an organism, deficient in the production of an essential precursor for the secondary metabolite of choice, is isolated, and precursor-directed biosynthesis is then employed to generate only the novel analogs. 

Figure 9 Precursor-directed biosynthesis. A block in the 6-dEB pathway was created by a Cys— Ala mutation in the active site of the KS of module 1. Feeding diketides with different a and j substitutions resulted in the 6-dEB analogs in which the starter unit or first extender unit was modified. The stereochemistry of an unsaturated triketide dictated whether it was incorporated into module 2 or 3, leading to either a 14-membered or 16-membered macrolactone. See Sec. VI.C for details.

FIGURE 4.8 Precursor-directed biosynthesis. A KST mutation is introduced in module 1 of DEBSl. Exogenously fed diketide SNACs with varying side chains are incorporated into the macrolide. 

SCHEME 1.9 (a) Biosynthetic pathway of wUd-type metabolites (b) precursor-directed biosynthesis the modified synthon B replaces the natural synthon B (c) biosynthetic pathway blocked by a mutation (the enzyme E4 is not functional) (d) mutasynthesis a mutasynthon B is introduced to replace B and is incorporated in the biosynthesis, leading to a mutated natural product. 

The rate of secondary product formation may be influenced by many different mutations. In most cases they act indirectly, e.g., by influencing the flow of precursors, cosubstrate biosynthesis, or differentiation programs. Mutations in the genetic material directly controlling the expression of secondary metabolism are rare (A 2.1). The genetic regulation of secondary product formation in most organisms is therefore still unknown. 

---