Reprogram fungal modular NRPSs for production of “designer” alkaloids Coco Xie 1 , Cameron L. M. Gilchrist 2 , John A. Kalaitzis 3 , Hang Li *4 , Enest Lacey *3,5 , Andrew M. Piggott *3 and Yit-Heng Chooi *1 1 University of Western Australia, Australia, 2 Seoul National University, South Korea, 3 Macquarie University, Australia, 4 Sun Yat-sen University, Guangzhou, China, 5 Microbial Screening Technologies Pty. Ltd., Australia Fungal alkaloids produced by non-ribosomal peptide synthetases (NRPSs) have been a bountiful source for drug discovery. Peptidic alkaloids are often biosynthesised from 2 to 4 amino acids and form complex polycyclic structures. The modular nature of NRPSs presents a great opportunity to reprogram the enzymatic assembly line to engineer novel alkaloids with desirable properties. Bacterial NRPS engineering efforts have demonstrated serval successful strategies. One strategy involves splitting and fusing NRPSs within the condensation domains. This is the exchange unit condensation domain (XUC) concept 1 . However, engineering fungal NRPSs has been less fruitful. Here, we share our insights into the engineering of fungal modular NRPSs. Inspired by the XUC concept, we created a functional chimeric NRPS by recombining two tri-modular NRPSs. We have also characterised the peptide product by NMR. Despite the similar architecture of fungal and bacterial NRPSs, they have evolved via distinctive mechanisms. We are hoping to gain understanding of the phylogeny and structural biology of fungal NRPSs, which can aid us in the efficient engineering of fungal NRPSs for novel bioactive alkaloids. References 1. Bozhüyük, K. A. J. et al. Modification and de novo design of non-ribosomal peptide synthetases using specific assembly points within condensation domains. Nat. Chem. 11, 653–661 (2019).
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