Structure elucidation of the redox cofactor mycofactocin reveals oligo-glycosylation by MftF

Abstract

Metabolomics-driven discovery of the novel cofactor mycofactocin in mycobacteria revealed glycosylation with a cellulose-like sugar chain, regulation in response to ethanol and redox-activity.

Impact of Oxygen Supply and Scale Up on Mycobacterium smegmatis Cultivation and Mycofactocin Formation

“…1 mL of crude extract was centrifuged twice at 17,000 × g for 10 min, the supernatant transferred to a new tube and centrifuged again, 600 µL of crude extract was transferred to a glass vial and stored at −20 • C. Before LC-MS measurements, all samples were diluted to a 1:10 ratio in LC-MS water. LC-MS measurements of MFT congeners were performed in an Ultimate 3000 UHPLC coupled to a Q Exactive Plus mass spectrometer equipped with a heated electrospray ionization probe (Thermo Fisher Scientific, Germany) as described before (Peña-Ortiz et al, 2020). Metabolite separation was done in an XB-C18 UHPLC column (150 × 2.1 mm, 2.6 µm, 100 Å, Phenomenex) preceded by a SecurityGuard ULTRA precolumn (2 × 2.1 mm, Phenomenex) at 40 • C. 10 µl of the sample was injected and separated chromatographically in a mobile phase composed of 0.1% (v/v) formic acid in either water (A) or acetonitrile (B) and a constant flow rate of 300 µL min −1 as follows: 0-2 min, 2% B; 2-15 min 2-99% B; 15-18 min 99% B. Metabolite separation was followed by full scan (MS 1 ) in positive ionization mode at two scan ranges: m/z 200-600 and m/z 580-2,000 at a resolving power of 70,000 at m/z 200, injection time to 100 ms, and automatic gain control (AGC) target to 3 × 10 6 .…”

“…Experimental conditions: Stirring rate = 900 rpm, gas flow rate = 0.75 L min −1 (= 0.25 vvm), total reactor volume = 7 L, filling volume = 3 L, temperature = 37 • C, LB medium with 10 g L −1 ethanol, 250 mM MOPS buffer, initial pH = 7.2. (Peña-Ortiz et al, 2020), can be found in Supplementary Figures S1, S3-S5. The comparison of the four different culture conditions in Figure 2 and Table 1 show clearly that the absolute amount of produced mycofactocins is increased under oxygen-limited conditions, especially the long-chained versions MMFT-7(H 2 ) and MMFT-8(H 2 ) followed by the aglycons PMFT(H 2 ).…”

“…Therefore, novel redox cofactors present in mycobacteria could open the door to interesting physiological discoveries or even serve as targets for drug development. Mycofactocin (MFT) is a recently identified cofactor, biosynthesized as a ribosomally-produced and post-translationally modified peptide (RiPP) ( Ayikpoe et al, 2019 ; Peña-Ortiz et al, 2020 ). The discovery of MFT started with the observation that its biosynthetic gene cluster was reminiscent of another bacterial redox cofactor, pyrroloquinoline quinone, and was genomically associated with certain subfamilies of oxidoreductases ( Haft, 2011 ).…”

“…MMFT is generally more abundant than non-methylated MFT in M. smegmatis . The enzyme responsible for this methylation is not comprised in the MFT gene cluster and is still unknown ( Peña-Ortiz et al, 2020 ), however, it is possible that this modification is catalyzed by a S -adenosyl- L -methionine (SAM)-dependent methyltransferase, with S -adenosyl- L- homocysteine (SAH) as product. We also detected early products from minor reactions, namely glycosylated AHDP (ADHP-n), methyl glycosylated AHDP (MAHDP-n), and glycyl-containing AHDP (GAHDP).…”

Genome mining strategies for ribosomally synthesised and post-translationally modified peptides

“…The BGCs were then reannotated to identify a conserved yet previously unannotated mycofactocin PP gene. Subsequent experimental studies have proven this to be a genuine RiPP pathway [74] , [75] . Haft and Mitchell bioinformatically identified Nif11-like and nitrile hydratase-like leader peptides associated with BGCs that had homology to linear-azoline containing peptide BGCs [76] .…”