Deciphering the Biosynthesis of Novel Class I Lanthipeptides from Marine Pseudoalteromonas Reveals a Dehydratase PsfB with Dethiolation Activity

Abstract

Lanthipeptides are a representative class of RiPPs that possess characteristic lanthionine and/or methyllanthionine thioether cross-links. The biosynthetic potentials of marine-derived lanthipeptides remain largely unexplored. In this study, we characterized three novel lanthipeptides pseudorosin A−C by heterologous expression of a class I lanthipeptide biosynthetic gene cluster from marine Pseudoalteromonas flavipulchra S16. Interestingly, pseudorosin C contains a large loop spanning 18 amino acid residues, which is rare in lanthipeptides. Unexpectedly, the dehydratase PsfB could catalyze the dethiolation of specific Cys residues in all three core peptides, thereby generating dehydroalanines in the absence of LanC cyclase. To the best of our knowledge, we identified the first member of the LanB dehydratase family to perform glutamylation and subsequent elimination on Cys thiol groups, which likely represents a new bypass for class I lanthipeptide biosynthesis. Furthermore, we employed mutagenesis to determine the important motif of the core peptide for dethiolation activity. Moreover, sequence analysis revealed that PsfB exhibited a distinct phylogenetic distance from the characterized LanBs from Gram-positive bacteria. Our findings, therefore, pave the way for further genome mining of lanthipeptides, novel post-translational modification enzymes from marine Gram-negative bacteria, and bioengineering applications.

Bacterial natural product discovery by heterologous expression

“…Indeed, phylogeny relatedness was mentioned by Hao et al . and four other studies as the reason for host choice (Hao et al, 2019 ; Mevaere et al, 2018 ; Shi et al, 2021 ; X. Wang et al, 2023 ; M. Xu et al, 2020b ). Phylogeny as a choice is backed up by a recent study showing that the yield of heterologously expressed NPs is often higher when the host is closely related to the native strain (G. Wang et al, 2019 ), although exceptions exist (J. J. Zhang et al, 2017 ).…”

“…However, synthesis is practical only for small BGCs such as RiPPs since the maximum size that DNA synthesis suppliers currently offer is 1.8 kb for fragments and 5 kb for clonal DNA. Accordingly, DNA synthesis or PCR to amplify the BGC from genomic DNA was used in 87% of the studies expressing only RiPPs (Arias-Orozco et al, 2021 ; Ayikpoe et al, 2022 ; Bösch et al, 2020 ; Bothwell et al, 2021 ; Cao et al, 2021 ; Carson et al, 2023 ; Cheung-Lee et al, 2019 ; Cheung‐Lee et al, 2020 ; Gomez-Escribano et al, 2019 ; Kaweewan et al, 2021 , 2023 ; Koos & Link, 2019 ; Mevaere et al, 2018 ; Nguyen et al, 2022 ; Singh et al, 2020 ; Thetsana et al, 2022 ; Unno et al, 2020 ; Vermeulen et al, 2022 ; X. Wang et al, 2023 ; Y. Zhang et al, 2022 ). TAR cloning, CAPTURE, and random library generation were used in the remaining studies (Ren et al, 2023 ; Santos-Aberturas et al, 2019 ; M. Xu et al, 2020a ) (Fig.…”

“…2A ). The most frequent approach (56%) was to prioritize novelty by expressing unusual BGCs found in rare and/or understudied bacteria, exemplified by the discovery of the lanthipeptide marinsedin from Marinicella sedimis (Han et al, 2022 ), and by the discovery of 31 other compounds listed in Supplemental Table S1 (Alberti et al, 2019 ; Bothwell et al, 2021 ; Cheng et al, 2023 ; De Rond et al, 2021 ; Enghiad et al, 2021 ; Gao et al, 2023 ; Gummerlich et al, 2020 ; Hao et al, 2019 ; Hashimoto et al, 2021 ; Kaweewan et al, 2021 ; Lasch et al, 2021 ; Lasch, Gummerlich, et al, 2020 , 2020 ; Li et al, 2020 ; S. H. Liu et al, 2019 ; Y. Liu et al, 2021 ; Myronovskyi et al, 2018 ; Paulus et al, 2022 ; Shi et al, 2019 ; Shuai et al, 2020 ; Unno et al, 2020 ; Vermeulen et al, 2022 ; X. Wang et al, 2023 ; Yuet et al, 2020 ; Y. Zhang et al, 2021 ; Y. Zhang et al, 2022 ).…”

“…Twenty studies (40%) used E. coli as host (Fig. 2D ) (Ayikpoe et al, 2022 ; Bösch et al, 2020 ; Bothwell et al, 2021 ; Cao et al, 2021 ; Carson et al, 2023 ; Cheung-Lee et al, 2019 ; Cheung‐Lee et al, 2020 ; De Rond et al, 2021 ; Han et al, 2022 ; Kaweewan et al, 2021 , 2023 ; Koos & Link, 2019 ; Nguyen et al, 2022 ; Singh et al, 2020 ; Thetsana et al, 2022 ; Unno et al, 2020 ; Vermeulen et al, 2022 ; X. Wang et al, 2023 ; Yuet et al, 2020 ; Y. Zhang et al, 2022 ). All but two (De Rond et al, 2021 ; Yuet et al, 2020 ) of these 20 studies expressed RiPP BGCs (Fig.…”

“…This question cannot always be answered because the biosynthetic gene cluster may be unexpressed in the native host. In fact, eight studies used heterologous expression because they were unable to find the NP associated to their BGC of interest in the native host (Bösch et al, 2020 ; Bothwell et al, 2021 ; Cheng et al, 2023 ; Han et al, 2022 ; Li et al, 2020 ; J. Liu et al, 2019 ; Shi et al, 2019 ; X. Wang et al, 2023 ). Only two other of the covered papers were indeed able to identify the heterologous products in the native producers (Gao et al, 2023 ; J. Liu et al, 2019 ).…”