Another technique that relies upon the knockout of an enzymatic function is known as mutasynthesis. In mutasynthesis a key step in a biosynthetic sequence is knocked out such that no product is made without the addition of a suitable precursor. In the past, these processes were done by random mutagenesis followed by screening of the resultant mutants for the desired phenotype. Today, it is a straightforward process to obtain the fully annotated genetic map of a biosynthetic pathway and to specifically design experiments to knock out the targeted function. One such example is shown in Figure 6.11 for the microbial product rapamycin. This work was pioneered by Peter Leadlay at the University of Cambridge, England who mapped the biosynthetic gene cluster for rapamycin. As illustrated, knock out of the gene rapL results in the organism's inability to make pipecolic acid, which is the usual amino acid incorporated into the rapamycin macrocycle. Supplementing the fermentation medium of the knockout strain with alternative cyclic amino acids, such as substituted proline analogs, results in efficient incorporation of these units yielding selectively modified rapamycin analogs.
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