LAUSR

Nonribosomal peptide synthetases (NRPSs) are a vast source of valuable natural products, and re-engineering them is an attractive path toward structurally diversified active compounds. NRPS engineering often requires heterologous expression, which is hindered by the enormous size of NRPS proteins. Protein splitting and docking domain insertion have been proposed as a strategy to overcome this limitation. Here, we have applied the splitting strategy to the gramicidin S NRPS: Despite better production of the split proteins, gramicidin S production almost ceased. However, the addition of type II thioesterase GrsT boosted production. GrsT is an enzyme encoded in the gramicidin S biosynthetic gene cluster that we have produced and characterized for this purpose. We attribute the activity enhancement to the removal of a stalled intermediate from the split NRPS that is formed due to misinitiation. These results highlight type II thioesterases as useful tools for NRPS engineering.