Functional analysis of the methanobactin-encoding gene cluster of Methylocystis sp. strain SB2

Methanobactins (MBs) are ribosomally synthesized, post-translationally modified polypeptides that have high binding affinities for copper. The key step for post-translational modification of the MB polypeptide precursor is the formation of two heterocyclic rings (pyrazinedione, imidazolone, and/or oxazolone) that are crucial for copper binding. Knowledge on genes responsible for ring formation is limited, particularly for MB from Methylocystis sp. strain SB2 (MB-SB2), due to a lack of genetic tools for this methanotroph. Here, we performed systematic functional analyses of the gene cluster of MB-SB2 via heterologous expression of these genes in Methylosinus trichosporium OB3b. We first deleted the native MB gene cluster of M. trichosporium OB3b and inserted the complete MB-SB2 gene cluster in its place, enabling M. trichosporium OB3b to produce MB-SB2. We then constructed a series of mutants, with individual genes of the MB-SB2 gene cluster deleted. Deletion of either mbnB or mbnC abolished the formation of both rings in MB-SB2. Strikingly, deletion of mbnF eliminated only the imidazolone ring of MB-SB2 while leaving the oxazolone ring intact. Back-complementation of mbnB/C/F in the respective deletion mutant restored MB-SB2 production. Deletion of mbnM, mbnS, mbnP, or mbnH did not affect ring formation but led to the formation of modified MB species, including MB without a sulfate group (ΔmbnS) and Cu-bound MB (ΔmbnP and ΔmbnH). Together, these results provide functional insight into the mbn-SB2 gene cluster and elucidate the genes involved-mbnB, mbnC, mbnF-in heterocyclic ring formation in MB-SB2.IMPORTANCEMBs-particularly MB from Methylocystis sp. strain SB2-have great promise in treating human copper-related diseases, perhaps most notably Wilson disease. Understanding how the distinctive heterocyclic rings of MB that are responsible for copper binding are assembled is crucial to understanding MB biosynthesis, key information necessary to scale up production of MB-SB2 for medical applications. This study identifies the responsible genes for the post-translational modification (i.e., heterocyclic ring formation) of the MB-SB2 polypeptide precursor. In particular, these findings expand our mechanistic understanding of MB biosynthesis across different methanotrophs and highlight the diversity of enzymatic strategies used to generate copper-chelating heterocyclic rings.