Engineering a polyketide with a longer chain by insertion of an extra module into the erythromycin-producing polyketide synthase

Background: Modular polyketide synthases catalyse the biosynthesis of medic ally useful natural products by stepwise chain assembly, with each module o f enzyme activities catalysing a separate cycle of polyketide chain extensi on. Domain swapping between polyketide synthases leads to hybrid multienzym es that yield novel polyketides in a more or less predictable way. No exper iments have so far been reported which attempt to enlarge a polyketide synt hase by interpolating additional modules. Results: We describe here the construction of tetraketide synthases in whic h an entire extension module from the rapamycin-producing polyketide syntha se is covalently spliced between the first two extension modules of the ery thromycin-producing polyketide synthase (DEBS). The extended polyketide syn thases thus formed are found to catalyse the synthesis of specific tetraket ide products containing an appropriate extra ketide unit. Co-expression in Saccharopolyspora erythraea of the extended DEBS multienzyme with multienzy mes DEBS 2 and DEBS 3 leads to the formation, as expected, of novel octaket ide macrolactones. in each case the predicted products are accompanied by s ignificant amounts of unextended products, corresponding to those of the un altered DEBS PKS. We refer to this newly observed phenomenon as 'skipping'. Conclusions: The strategy exemplified here shows far-reaching possibilities for combinatorial engineering of polyketide natural products, as well as r evealing the ability of modular polyketide synthases to 'skip' extension mo dules. The results also provide additional insight into the three-dimension al arrangement of modules within these giant synthases. (C) 2001 Elsevier S cience Ltd. All rights reserved.