Derivatization of polyketides during their biosynthesis

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Polyketides are a structurally diverse class of natural products that comprise various functions in therapeutic and clinical applications, such as erythromycin, which is applied in antibiotic therapy, or rapamycin, which has immunosuppressant properties. Modular polyketide synthases (PKSs) belong to nature’s largest and most complex enzymatic factories. They are also an exciting target for bioengineering due to the strict correlation of protein organization and product structure. Known as the principle of co-linearity, each chemical feature of a polyketide is directly linked to a specific module and domain(s). In this light, custom synthesis of polyketides with altered or improved functions seems possible through directed modifications of modular PKSs. In a proof-of-concept approach, we were recently able to harness such a hybrid protein for the chemoenzymatic synthesis of fluorinated macrolactones and macrolides. The successful synthesis was based on the promiscuity of the transferase from FAS and an intrinsic substrate tolerance of the catalytic domains of the PKS module DEBS M6. We have now collected preliminary data to the applicability of the hybrid approach beyond the "DEBS-M6" system: We found out that the hybrid design can be transferred to other PKS modules and that another PKS module is capable of synthesizing fluorinated macrolactones. These preliminary results offer the prospect of the broad application of FAS/PKS hybrids in polyketide biosynthesis. In the project presented, we primarily address two necessary further developments for the broad application of FAS/PKS hybrids: (1) PKSs accept disubstituted substrates, as exemplified by fluoromethyl-malonyl-CoA. Here, we seek to engineer PKS modules that also accept dimethyl-malonyl-CoA and spirocyclo-malonyl-CoA as substrates, and enable access to gem-dimethylated or spirocyclic polyketides. Methyl groups are important modifications in medicinal chemistry and are also known as "magic methyl". We are interested in the spirocyclic motifs as surrogates of gem-dimethyl groups. (2) The MAT domain is highly substrate tolerant and offers fantastic synthetic opportunities in simple reaction environments. The broad application of MAT now depends on improved substrate selectivity, and, here, above all on the suppression of competitive loading with the natural substrates, malonyl- and methylmalonyl-CoA. In work package 2, libraries of substrate-specific MAT variants are to be evolved. Within the framework of this project, we will produce 12- and 14-membered modified macrolactones via chemoenzymatic synthesis using the KS and MAT variants. The perspective of this project is the application of engineered hybrids in microbial host organisms.