A FUNCTIONAL CHIMERIC MODULAR POLYKETIDE SYNTHASE GENERATED VIA DOMAIN REPLACEMENT

Background: Modular polyketide synthases (PKSs), such as 6-deoxyerythr onolide B synthase (DEBS), are large multifunctional enzymes that cata lyze the biosynthesis of structurally complex and medically important natural products. Active sites within these assemblies are organized i nto 'modules: such that each module catalyzes the stereospecific addit ion of a new monomer onto a growing polyketide chain and also sets the reduction level of the beta-carbon atom of the resulting intermediate . The core of each module is made up of a 'reductive segment: which in cludes all, some, or none of a set of ketoreductase (KR), dehydratase, and enoylreductase domains, in addition to a large interdomain region which lacks overt function but may contribute to structural stability and inter-domain dynamics within modules. The highly conserved organi zation of reductive segments within modules suggests that they might b e able to function in unnatural contexts to generate novel organic mol ecules. Results: To investigate domain substitution as a method for al tering PKS function, a chimeric enzyme was engineered, Using a bimodul ar derivative of DEBS (DEBS1+TE), the reductive segment of module 2, w hich includes a functional KR, was replaced with its homolog from modu le 3 of DEBS, which contains a (naturally occurring) nonfunctional KR. A recombinant strain expressing the chimeric gene produced the predic ted ketolactone with a yield (35%) comparable to that of a control str ain in which the KR2 domain was retained but mutationally inactivated. Conclusions: These results demonstrate considerable structural tolera nce within an important segment found in virtually every PKS module. T he domain boundaries defined here could be exploited for the construct ion of numerous loss-of-function and possibly even gain-of-function mu tants within this remarkable family of multifunctional enzymes.