Biosynthetic encoding genes overexpression

Another possibihty to gain the product encoded by a silent gene cluster can be achieved by a method called the in vitro reconstitution approach. A necessity is that the substrates of the pathway are predictable. The in vitro reconstitution of an entire biosynthetic pathway usually involves the separate overexpression of each gene, the purification of the resulting protein and the performance of enzymatic studies. Thus, the discovery of a fully elaborated metabolic product by this approach is likely to be very laborious. For example, after discovering a cryptic sesquiterpene synthase in the genome of S. coelicolor (64), the new epi-isozaene (8) could be produced successfully. This metabolite was shown to be an intermediate in the assembly line of the known Streptomyces sesquiterpene albaflavenone (9) (135). 

This approach is the first of three that rely on accurate prediction of the substrate(s) of enzyme(s) encoded by cryptic biosynthetic gene clusters. Putative biosynthetic gene(s) within the cryptic cluster are overexpressed (usually in Escherichia coll) and the resulting overproduced recombinant enzyme(s) are purified to homogeneity. 

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