Cell free production of polyketide building blocks
Due to the more and more increasing impact of infectious diseases and multi-resistant organisms the need of new active compounds is dramatically increasing, too. Polyketides are a group of natural products that are already used as cancer therapeutics and antibiotics. They are produced in many different organisms such bacteria, fungi and plants. Genome mining approaches showed that filamentous fungi comprise the genes for the production of a high number of these molecules. But because the majority of these organisms are recalcitrant to cultivation, so far, only few of the naturally available polyketides have been fully characterized.
Polyketides are synthesized by highly complex multimodular enzymes called polyketide synthases (PKSs). Fungal PKSs predominantly use small building blocks to assemble their products. In most cases acetyl-CoA is used as starter unit which is afterwards extended by adding malonyl-CoA units or its derivatives. Since the amount of those building blocks is limiting the synthesis of the polyketides also in in vitro experiments, additionally to the PKS itself, it is necessary to build up a system for the production of those units in high amount.
Here we show the recombinant expression in E.coli and purification of a malonyl-CoA-synthetase (matB) and a malonyl-CoA decarboxylase (matA). With these highly pure and stable enzymes we are able to produce malonyl-CoA out of free CoA and malonate using matB activity and acetyl-CoA by decarboxylation of malonyl-CoA via matA activity . Adding a third, commercially available enzyme, citrate synthase, we were able to set up a whole cycle of CoA consumption and recycling.