Bioengineering of syrbactin megasynthetases for immunoproteasome inhibitor production

Highlights • The XUT approach allows engineering of NRPS/PKS hybrids • A syrbactin derivative was designed, inhibiting the immunoproteasome • Rational NRPS/PKS engineering might be complementary to synthetic chemistry The bigger picture Natural products (NPs) are structurally complex chemical compounds with diverse biological activities, representing an important source of new therapeutic agents for human health. Many NPs are synthesized by megasynthetases, such as non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), both of which are modularly organized multifunctional enzymes, where each module incorporates one specific building block in an assembly line fashion. Our work describes the engineering of a designed NRPS/PKS hybrid via the assembly of different megasynthetase fragments to produce a rationally modified syrbactin derivative with a complex peptide-polyketide structure, acting as proteasome inhibitor. To produce and identify further improved proteasome inhibitors as well as other therapeutics used against cancer, such as immune-modulating or anti-infective drugs, the engineering of megasynthases holds huge potential as an environmentally friendly and cost-effective synthetic biology platform. Summary The natural product (NP) class of syrbactins are potent proteasome inhibitors produced by hybrids of non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). Here, we describe the stepwise reassembly of an entire NRPS/PKS hybrid to produce a new syrbactin derivative by utilizing the recently described “eXchange Unit between Thiolation domains” (XUTs) approach. Remarkably, XUT-based engineering allowed the direct assembly of PKS and NRPS modules to introduce an α,β-unsaturated Michael system in a macrolactam moiety, which represents the inhibitory warhead of syrbactins. The novel derivative was produced in E. coli, isolated, and examined for its ability to inhibit yeast (yCP), human constitutive (cCP), and immunoproteasome (iCP). The engineered NP maintained the inhibitory activities of the syrbactin class but, due to rational modifications, inhibited iCP most strongly. Moreover, analysis of the crystal structure of yCP in complex with the derivative revealed further design strategies for even more specific iCP inhibition.