Regulation of Bottromycin Biosynthesis Involves an Internal Transcriptional Start Site and a Cluster-Situated Modulator

Abstract

Bottromycin is a ribosomally synthesized and post-translationally modified peptide (RiPP) produced by several streptomycetes, including the plant pathogen Streptomyces scabies. There is significant interest in this molecule as it possesses strong antibacterial activity against clinically relevant multidrug resistant pathogens and is structurally distinct from all other antibiotics. However, studies into its efficacy are hampered by poor yields. An understanding of how bottromycin biosynthesis is regulated could aid the development of strategies to increase titres. Here, we use 5-tag-RNA-seq to identify the transcriptional organization of the gene cluster, which includes an internal transcriptional start site that precedes btmD, the gene that encodes the bottromycin precursor peptide. We show that the gene cluster does not encode a master regulator that controls pathway expression and instead encodes a regulatory gene, btmL, which functions as a modulator that specifically affects the expression of btmD but not genes up-or downstream of btmD. In order to identify non-cluster associated proteins involved in regulation, proteins were identified that bind to the main promoter of the pathway, which precedes btmC. This study provides insights into how this deceptively complex pathway is regulated in the absence of a pathway specific master regulator, and how it might coordinate with the central metabolism of the cell.

Microparticles enhance the formation of seven major classes of natural products in native and metabolically engineered actinobacteria through accelerated morphological development

“…In this study, the refactored synthetic strain S. lividans TK24 DG2‐Km‐P41hyg + , apparently among the top bottromycin producers available (Franz et al, 2021), reached 109 mg L −1 bottromycins of the A2 type in the presence of 15 g L −1 talc, exceeding all previously reported efforts. This achievement will greatly support drug development and further biosynthetic studies of bottromycins and derivatives therefrom (Vior et al, 2020). It also demonstrates that the particle approach can be successfully used to boost the performance of even top‐level microbial cell factories.…”

“…The data show relative changes in the level of bottromycin pathway intermediates (a) and intracellular amino acids (b), (c), upon addition of talc microparticles to the medium (10 g L −1 ). (a) The data show pre‐bottromycin (m/z = 855.4), methylated pre‐bottromycin (m/z = 869.4), four shunt products (m/z = 452.2, m/z = 599.4, m/z = 873.5, and m/z = 887.5), final mature bottromycin A2 (m/z = 823.4), and methylated‐bottromycin A2 (m/z = 837.4), previously described (Crone et al, 2016; Vior et al, 2020). The data reflect the production start (24 h) and the major production phase (48 h), and the control culture (without talc), set to 100%, is shown as dashed line (* p ≤ 0.05, ** p ≤ 0.01).…”

“…Various homologous as well as heterologous producers provide bottromycins only in the low milligram range and even below 1 mg L −1 (Crone et al, 2016; Horbal et al, 2018; Huo et al, 2012). Due to this, improvement of titer and yield is regarded crucial (Vior et al, 2020), especially as the pathway seems inefficient in laboratory conditions (Crone et al, 2016; Eyles et al, 2018). In this study, the refactored synthetic strain S. lividans TK24 DG2‐Km‐P41hyg + , apparently among the top bottromycin producers available (Franz et al, 2021), reached 109 mg L −1 bottromycins of the A2 type in the presence of 15 g L −1 talc, exceeding all previously reported efforts.…”

“…Interestingly, not even one single gene showed a significantly altered expression with talc supply during the growth phase (Figure S4). ), and methylated-bottromycin A2 (m/z = 837.4), previously described (Crone et al, 2016;Vior et al, 2020). The data reflect the production start (24 h) and the major production phase (48 h), and the control culture (without talc), set to 100%, is shown as dashed line (*p ≤ 0.05, **p ≤ 0.01).…”

Bacterial pathogens: threat or treat (a review on bioactive natural products from bacterial pathogens)

“…The only regulatory gene btmL encoded in the cluster was identied to be a positive modulator of btmD and not a master regulator that controls bottromycin expression. 304 The mechanism by which BtmL modulates BtmD transcription in S. scabies remains elusive. An understanding of the regulation of bottromycin biosynthesis may shed light into further engineering and overproduction of medicinally promising bottromycin-based compounds as well as expression of other RiPP pathways.…”