Crotonylation of key metabolic enzymes regulates carbon catabolite repression in Streptomyces roseosporus.

Due to the plethora natural products made by Streptomyces, the regulation of its metabolism are of great interest, whereas there is a lack of detailed understanding of the role of posttranslational modifications (PTM) beyond traditional transcriptional regulation. Herein with Streptomyces roseosporus as a model, we showed that crotonylation is widespread on key enzymes for various metabolic pathways, and sufficient crotonylation in primary metabolism and timely elimination in secondary metabolism are required for proper Streptomyces metabolism. Particularly, the glucose kinase Glk, a keyplayer of carbon catabolite repression (CCR) regulating bacterial metabolism, is identified reversibly crotonylated by the decrotonylase CobB and the crotonyl-transferase Kct1 to negatively control its activity. Furthermore, crotonylation positively regulates CCR for Streptomyces metabolism through modulation of the ratio of glucose uptake/Glk activity and utilization of carbon sources. Thus, our results revealed a regulatory mechanism that crotonylation globally regulates Streptomyces metabolism at least through positive modulation of CCR.

a. Immuno-blots analysis of lysine acetylation and succinylation from protein lysates of S. roseosporus wild type, cobB and hdac cultured in YEME medium.
b. Immuno-blots analysis of lysine acetylation and succinylation from protein lysates of S. roseosporus wild type and kct1 mutant cultured in YEME medium.  Figure 7).

Western blot analysis of crotonylation level of Glk in vivo. S. roseosporus wild type,
cobB and kct1 mutants expressing ermEp*-3flag-glk were cultured in the YEME medium and mycelia were taken in the times indicated. 3FLAG-Glk was immune-precipitated from the lysate and western blots were performed with anti-FLAG M2 or anti-Kcr rabbit antibody.

Crotonyl-proteomic analysis of Streptomyces proteins
Spores of prcB/A were inoculated in TSB as the seed culture for 36 h. 1 ml of the seed transferred to 50 ml of YEME medium fermented for 3 days. All shake-flask fermentation was set at 30C on a rotary shaker (250 rpm). The mycelia were harvested and washed twice with pre-cold PBS. The sample was grinded by liquid nitrogen into cell powder and then transferred to a 5-mL centrifuge tube. Four volumes of lysis buffer (8 M urea, 1% Triton-100, 10 mM dithiothreitol, and 1% Protease Inhibitor Cocktail) was added to the cell powder, followed by sonication for three times on ice using a high intensity ultrasonic processor (Scientz). The remaining debris was removed by centrifugation at 20,000 g at 4°C for 10 min. Finally, the protein was precipitated with cold 20% TCA for 2 h at -20 °C. After centrifugation at 12,000 g, 4 °C for 10 min, the supernatant was discarded. The remaining precipitate was washed with cold acetone for three times. The protein was re-dissolved in 8 M urea and the protein concentration was determined with BCA kit.

Trypsin digestion
For digestion, the protein solution was reduced with 5 mM dithiothreitol for 30 min at 56°C and alkylated with 11 mM iodoacetamide for 15 min at room temperature in darkness. The protein sample was then diluted by adding 100 mM NH 4 HCO 3 to urea concentration less than 2 M. Finally, trypsin was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight and 1:100 ratio for the second 4 h-digestion.

HPLC fractionation
The tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC using Thermo Betasil C18 column (5 μm particles, 10 mm ID, 250 mm length).
Briefly, peptides were first separated with a gradient of 8% to 32% acetonitrile (pH 9.0) over 60 min into 60 fractions. Then, the peptides were combined into 6 fractions and dried by vacuum centrifuging.

Affinity enrichment
To enrich crotonylated peptides, tryptic peptides dissolved in NETN buffer ( For LC-MS/MS analysis, the resulting peptides were desalted with C18 ZipTips (Millipore).

LC-MS/MS analysis
The tryptic peptides were dissolved in 0.1% formic acid (solvent A). The gradient was comprised of an increase from 6% to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, 23% to 35% in 8 min and climbing to 80% in 3 min then holding at 80% for the last 3 min, all at a constant flow rate of 400 nL/min on an EASY-nLC 1000 UPLC system.
The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting as 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0s dynamic exclusion. Automatic gain control (AGC) was set at 5E4.

Database search
The resulting MS/MS data were processed using Maxquant search engine (v.1.5.2.8). Tandem mass spectra were searched against Uniprot Streptomyces roseosporus database concatenated with reverse decoy database. Trypsin/P was 26 specified as cleavage enzyme allowing up to 4 missing cleavages. The mass tolerance for precursor ions was set as 20 ppm in First search and 5 ppm in Main search, and the mass tolerance for fragment ions was set as 0.02 Da.
Carbamidomethyl on Cys was specified as fixed modification and Kcr modification and oxidation on Met were specified as variable modifications. FDR was adjusted to < 1% and minimum score for modified peptides was set > 40.