Caerulomycin and collismycin antibiotics share a trans-acting flavoprotein-dependent assembly line for 2,2\u2019-bipyridine formation

Bo Pang,Rijing Liao,Wen Liu,Shengjie Guo,Zhuhua Wu,Zhijun Tang

doi:10.1038/s41467-021-23475-4

Abstract

Linear nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) template the modular biosynthesis of numerous nonribosomal peptides, polyketides and their hybrids through assembly line chemistry. This chemistry can be complex and highly varied, and thus challenges our understanding in NRPS and PKS-programmed, diverse biosynthetic processes using amino acid and carboxylate building blocks. Here, we report that caerulomycin and collismycin peptide-polyketide hybrid antibiotics share an assembly line that involves unusual NRPS activity to engage a trans-acting flavoprotein in C-C bond formation and heterocyclization during 2,2’-bipyridine formation. Simultaneously, this assembly line provides dethiolated and thiolated 2,2’-bipyridine intermediates through differential treatment of the sulfhydryl group arising from l-cysteine incorporation. Subsequent l-leucine extension, which does not contribute any atoms to either caerulomycins or collismycins, plays a key role in sulfur fate determination by selectively advancing one of the two 2,2’-bipyridine intermediates down a path to the final products with or without sulfur decoration. These findings further the appreciation of assembly line chemistry and will facilitate the development of related molecules using synthetic biology approaches.

Highlights

Linear nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) template the modular biosynthesis of numerous nonribosomal peptides, polyketides and their hybrids through assembly line chemistry
Linear nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are often large molecular machines that are composed of multidomain modules[1,2,3,4,5,6]
A chimeric gene that encodes the variant harboring the PKS module of ColA2 and the NRPS module of CaeA2, colA2PKS-caeA2NRPS, was constructed and introduced into the previously developed, △colA2 mutant S. roseosporus strain that is incapable of producing COLs14. This complementation restored the production of COLs, supporting that the atypical NRPS modules of CaeA2 and ColA2 are functionally identical in terms of L-cysteinyl incorporation, trans CaeB1/ColB1 activity recruiting, condensation and heterocyclization during COL biosynthesis. These findings demonstrate the generality of both COLs and CAEs in 2,2’-bipyridine formation and suggest that dethiolation can be skipped in the atypical NRPS module of CaeA2 to produce 5-sulfhydryl-2,2’

Summary

Discussion

We demonstrate an unusual paradigm for 2,2’bipyridine formation, which features an NRPS/PKS hybrid assembly line for sequential incorporation of the substrates picolinic acid, malonyl-CoA, L-cysteine, and L-leucine with the association of a flavoprotein partner functioning in trans (Fig. 5) This assembly line was first reconstituted in vitro for the biosynthesis of dethiolated 2,2’-bipyridine antibiotics CAEs. the generality of 2,2’-bipyridine formation was validated in the biosynthesis of distinct sulfhydryl-2,2’-bipyridine antibiotics COLs, where the dethiolation step is skipped. Accumulating the incorrect 2,2’-bipyridine intermediate could stall the biosynthesis of the correct 2,2’-bipyridine in the assembly line, where its off-loading might lead to the formation of the shunt product With this manner, the pressure arising from C domain-conferred gatekeeping role through L-leucine extension, the step following heterocyclization for pathway furcation, can be alleviated.

Methods

Findings

Code availability