The faucet knob effect of DptE crotonylation on the initial flow of daptomycin biosynthesis.

We propose here that acylation modification of actinomycete proteins is a restrictive system that limits the excessive synthesis of secondary metabolites, its mechanism has not been clearly elucidated before. We used crotonylation as an example to investigate the acylation effect in the daptomycin biosynthesis by Streptomyces roseosporus. Our experiments revealed abundant crotonylation of numerous secondary metabolic enzymes in Streptomyces roseosporus, a daptomycin producer. DptE, which initiates daptomycin biosynthesis, is crotonylated at K454. We experimentally identified the corresponding DptE crotonyltransferase Kct1 and decrotonylase CobB. Further studies consistently confirmed that decrotonylation increases DptE activity. Decrotonylation functions like loosening a faucet knob, increasing substrate channel throughput and the initial flow of daptomycin biosynthesis. Moreover, DptE catalytic activity was enhanced via K454 and neighboring residues K184 and Q420 mutation, increasing daptomycin yield by 132%; daptomycin biosynthesis related metabolism activities also increased. Substrate channel prediction revealed 38% higher throughput for mutant DptE (K454I/K184Q/Q420N) than crotonylated DptE. Molecular dynamics (MD) simulations revealed significant increases in flexibility and substrate affinity of the mutant. In summary, we elucidated the faucet knob effect of DptE crotonylation on the initial flow of daptomycin biosynthesis and adopted decrotonylation to generate high-yield industrial strains.