Disulfide-rich macrocyclic peptides-cyclotides, for examplerepresent ap romisingc lass of molecules with potential therapeutic use. Despite their potential their efficient synthesis at large scale still represents am ajor challenge. Here we report new chemoenzymatic strategies using peptide ligase variants-inter alia, omniligase-1-fort he efficient and scalable one-pot cyclization and folding of the native cyclotides MCoTI-II, kalata B1 and variants thereof, as well as of the q-defensin RTD-1. The synthesis of the kB1 variant T20K was successfully demonstrated at multi-gram scale. The existence of several ligations ites for each macrocycle makes this approach highly flexible and facilitates both the larger-scale manufacture and the engineering of bioactive, grafted cyclotide variants, therefore clearly offering av aluable and powerful extension of the existing toolbox of enzymes for peptide head-to-tail cyclization.In the last decade macrocyclic peptides have gained increased traction as ap romising class of therapeutics. High metabolic stabilityt han their linear analogues, often accompanied by higher potency,m aket hem excellent leads in drug design.