Abstract

   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.

   Subject terms: Microbiology, Molecular biology, Physiology
     __________________________________________________________________

   Chen-Fan Sun et al. show that key enzymes in several metabolic pathways
   are crotonylated in Streptomyces roseosporus. This study suggests that
   crotonylation increases carbon catabolite repression by increasing
   glucose uptake while reducing the activity of glucose kinase.

Introduction

   Streptomyces are profoundly famed as bacterial producers of natural
   products with diverse chemical structures and bioactivities^[36]1. The
   short-chain acyl-CoA species, such as acetyl-CoA, malonyl-CoA, and
   succinyl-CoA, are catabolites from primary metabolism. Meanwhile, they
   are common precursors and building blocks for biosynthesis of
   macromolecules in primary metabolism and natural products in secondary
   metabolism^[37]2–[38]4. It has been well recognized that precise
   control of primary/secondary metabolism development of Streptomyces and
   their switch are critical for the proper production of these invaluable
   natural products^[39]5.

   Traditionally, regulation of Streptomyces metabolism has been
   extensively studied at the transcriptional levels^[40]6,[41]7. But
   recently, protein acetylation from acetyl-CoA, one of the
   posttranslational modifications (PTMs), has been shown to be involved
   in the regulation of production of both primary and secondary
   metabolites in Streptomyces. Acetylation was initially reported in two
   conserved Streptomyces enzymes acetyl-CoA synthetase^[42]8 and
   acetoacetyl-CoA synthetase^[43]9. Following acetyl-proteome assays
   showed that acetylation is widely distributed in Streptomyces, since
   667 and 134 proteins were identified acetylated in S. roseosporus and
   S. griseus, respectively. Acetylated proteins are mainly involved in
   metabolism, followed by protein biogenesis and turn-over. Meanwhile,
   acetylation also occurs on some biosynthetic enzymes for natural
   products, including the dTDP-4-dehydrorhamnose 3,5-epimerase StrM for
   streptomycin biosynthesis and the adenylation domain of a non-ribosomal
   peptide synthase^[44]10,[45]11. Interestingly, acetylation of enzymes
   unexceptionally abolishes their catalytic
   activities^[46]8,[47]9,[48]11. Though regulation of PTMs on the
   activity of some enzymes has been reported as shown above, how PTMs
   regulate the Streptomyces metabolism in a broader scope has not yet
   been addressed.

   Carbon catabolite repression (CCR) is a conserved mechanism allowing
   the bacteria, as well as Streptomyces, to generally utilize preferred
   carbon source (such as glucose in laboratory cultivations) over others
   and attracting the attention as a checkpoint for differentiation of
   Streptomyces^[49]12. Glucose induction often triggers CCR and leads to
   delayed morphological development and repressed secondary metabolite
   production^[50]13–[51]15. In Streptomyces, glucose kinase (Glk) has
   been shown to be involved in CCR regulation^[52]16,[53]17, possibly
   through its kinase activity to phosphorylate glucose and strengthen
   glycolysis to produce metabolic intermediates for CCR regulation and
   through its regulatory activity by interacting with downstream
   transcriptional regulators^[54]13,[55]18,[56]19. It has been proposed
   that the cytosolic fraction of Glk has a certain kind of PTM for its
   interaction with regulators, while Glk can also be recruited to the
   cell membrane to interact with the permease GlcP for glucose
   metabolism^[57]20. Meanwhile, the ratio of glucose uptake/Glk kinase
   activity has been suggested as a determinant of sensitivity to
   CCR^[58]18. However, there is no report about which PTM of Glk for CCR
   regulation on Streptomyces metabolism.

   To our knowledge, crotonylation is a newly identified PTM on eukaryotic
   histones^[59]21. Modification of histones with this unique planar,
   four-carbon structure, and neutral charge reduces histone–DNA
   interaction^[60]22. Thus histone crotonylation is predominantly found
   on transcriptionally active regions and enhancers during mouse
   spermatogenesis^[61]21, renewal of embryonic stem cells^[62]23, and
   acute kidney injury^[63]24. In addition to its epigenetic modification
   on histones, crotonylation also occurs in a broad range of non-histone
   proteins^[64]25–[65]28 and participates in diverse metabolic pathways
   such as acetylation^[66]29. Crotonylation happens through reversible
   modifications by enzymes, including crotonyltransferases (writers) and
   decrotonylases (erasers). Studies have shown that crotonylation has
   overlapped acyl-transferases and de-acylases with acetylation and other
   types of acylations^[67]25,[68]30 and also overlapped modification
   sites on histones^[69]29,[70]31. These findings suggest that
   crotonylation is a global PTM with a complex interplay with other
   acylations. Nevertheless, crotonylation in bacteria, as well as how it
   regulates the bacterial metabolism, has not been reported.

   Here we showed that crotonylation is universal in Streptomyces
   metabolic pathways and regulates the metabolism at least through
   positively modulating CCR, by reversible modifications on Glk. Our work
   provides comprehensive insights by far, exploring the mechanisms of PTM
   in regulating Streptomyces metabolism, and potentially paves the way
   for PTM engineering in Streptomyces for optimal production of secondary
   metabolites.

Results

Protein crotonylation is widespread in Streptomyces

   Streptomyces proteins have been found extensively acetylated, and this
   PTM plays essential roles in enzyme activity modulation for
   primary/secondary metabolite biosynthesis^[71]8–[72]11. Though
   acetyl-CoA is believed the most abundant acyl-CoA species^[73]32, other
   acyl-CoAs might have more subtle and complex roles in the regulation of
   Streptomyces metabolism.

   When exploring the PTM profile of Streptomyces proteome throughout the
   fermentation, we found strong signals from immunoblots with an
   anti-lysine-crotonyl group (α-Kcr) antibody, as represented by an
   antibiotic daptomycin producer S. roseosporus (Fig. [74]1a), as well as
   laboratory model species like S. coelicolor M145^[75]33 and S. lividans
   TK24^[76]34, and the industrial producers S. albus J1074^[77]35 and S.
   tsukubaensis L19^[78]36 (Supplementary Fig. [79]1), suggesting that
   crotonylation occurs widely in Streptomyces. In S. roseosporus, the
   signals from cultures at 36 and 48 h were much stronger, when cells
   were entering secondary metabolism, than that at 24 h or after 48 h,
   implying that crotonylation increases during primary metabolism
   development but decreases during secondary metabolism development. The
   crotonylation dynamics suggested that this PTM might have regulatory
   roles on Streptomyces primary/secondary metabolism development.

Fig. 1. Crotonylation in S. roseosporus.

   [80]Fig. 1
   [81]Open in a new tab

   a Immunoblot of S. roseosporus lysate. Twenty μg of total protein were
   loaded, and the crotonylated proteins were detected with anti-Kcr
   monoclonal antibody (1:2000) while Coomassie blue staining was used for
   the loading control. b Immunoblot for crotonylation with anti-Kcr
   monoclonal antibody (1:2000) in wild-type strain cultured in TSB with
   additional concentration of sodium crotonate (pH 7.4) or sodium acetate
   (pH 7.4) as indicated for 24 h. Twenty μg of total protein were loaded,
   and Coomassie blue staining was used for the loading control. c LC-MS
   analysis of cellular crotonyl-CoA and acetyl-CoA levels from cells
   cultured as in b. The data represent mean peak area ±SD of three
   independent experiments. P value was calculated with Student’s t test
   (ns means P > 0.05).

   Using this antibody, we detected obviously increased crotonylation
   levels in cells treated with gradient concentrations of sodium
   crotonate (NaCr). Meanwhile, sodium acetate (NaAc) treatment also
   resulted in apparently increased crotonylation (Fig. [82]1b). However,
   crotonate or acetate treatments had little impact on acetylation or
   succinylation (Supplementary Fig. [83]2). These data suggested that
   crotonylation is more subtle to environmental signals. Furthermore, we
   examined the dynamics of crotonyl-CoA and acetyl-CoA levels in response
   to NaCr or NaAc by liquid chromatography–mass spectrometry (LC-MS;
   Fig. [84]1c). Increase of crotonyl-CoA concentration was observed only
   under 20 mM NaCr or NaAc, whereas no obvious increase of the
   intra-cellular acetyl-CoA was observed, suggesting that acetyl-CoA is
   resistant to environmental NaAc fluctuation, while intracellular
   crotonyl-CoA is mainly derived from acetyl-CoA^[85]37.

Crotonylation occurs on various metabolic pathways and regulators

   Next crotonyl-proteomics were performed in S. roseosporus to identify
   the substrates for crotonylation. Since proteasome-mediated degradation
   is one of the major pathways for protein turn-over^[86]38, a
   proteasome-deficient ΔprcB/A mutant was constructed (Supplementary
   Fig. [87]3) to potentially enrich more proteins with PTMs by
   immunoprecipitation with the anti-Kcr monoclonal antibody, and
   crotonylated proteins along with the modified residues were identified
   by high-performance LC-MS/MS (Fig. [88]2a). All the distribution of
   mass error for precursor ions was <0.03 Da, indicating acceptable mass
   accuracy of the MS data (Supplementary Fig. [89]4, Supplementary
   Data [90]3). Strikingly, 1389 proteins, along with 3944 lysine
   residues, were identified with crotonylation, accounting for 19.61% of
   the putative proteome of S. roseosporus (Fig. [91]2b) (Supplementary
   Data [92]2 and [93]3). It was a contrast to the previous
   acetyl-proteomic survey in the same species S. roseosporus NRRL 11379,
   where 667 acetylated proteins and 1143 modification sites were
   identified^[94]10.

Fig. 2. Crotonyl-proteomic analysis of S. roseosporus proteins.

   [95]Fig. 2
   [96]Open in a new tab

   a Procedure diagram for crotonyl-proteomics in S. roseosporus. b
   Statistics of crotonylated proteins from S. roseosporus. c, d Gene
   ontology functional classification of the identified crotonylation
   proteins based on biological processes (c) and molecular functions (d).
   e Carbon metabolism pathways in Streptomyces. Crotonylated enzymes are
   highlighted in red.

   Gene ontology (GO) analysis (Supplementary Fig. [97]5, Supplementary
   Data [98]4) showed that 672 crotonylated proteins (34%) are involved in
   the metabolic processes (Fig. [99]2c), while 691 crotonylated proteins
   (50%) are enzymes with catalytic activities (Fig. [100]2d), suggesting
   that crotonylation primarily regulates enzymatic activities in S.
   roseosporus metabolism. Consistent with these observations, Kyoto
   Encyclopedia of Genes and Genomes (KEGG) analysis (Supplementary
   Data [101]5) showed that most enzymes in carbon metabolism are
   crotonylated (Supplementary Fig. [102]6). Particularly, all enzymes in
   glucose catabolism, including glycolysis, pyruvate dehydrogenation, and
   tricarboxylic acid cycle (TCA) (Fig. [103]2e, Supplementary
   Figs. [104]7 and [105]8), and most enzymes in oxidative phosphorylation
   are highly crotonylated (Supplementary Data [106]5). These data
   strongly suggested that crotonylation globally regulates glucose
   utilization, respiratory chain, and potentially production of primary
   metabolites, such as acetyl-CoA, NADH, NADPH, and ATP. Other critical
   metabolic pathways in primary metabolism strongly regulated by
   crotonylation are DNA replication and repair, RNA biogenesis and
   degradation, protein biogenesis, and turn-over. It has been found that
   DNA polymerase, topoisomerase, gyrases and UvrABC, RNA polymerase and
   ribonucleases, most ribosomal proteins, almost all aminoacyl-tRNA
   synthetases, three chaperones, and protein degradation system (protease
   Clp, Lon, proteasome components) are heavily modified (Supplementary
   Figs. [107]9 and [108]10, Supplementary Data [109]3). Moreover, some
   acyl-CoA synthetases, including acetyl-CoA synthetase AcsA, are also
   crotonylated, but interestingly, no de-acylases are found modified with
   crotonylation (Supplementary Data [110]3), suggesting that
   crotonylation might modulate the biogenesis and intracellular
   concentrations of acyl-CoAs.

   Meanwhile, regulators for secondary metabolism development were also
   found crotonylated, such as the putative γ-butyrolactone receptor
   ArpA^[111]39,[112]40, the intracellular ppGpp synthase RelA^[113]41,
   and the nutrient-responsive regulators GlnR^[114]42, PhoP^[115]43, and
   AtrA^[116]40 (Supplementary Data [117]3). More interestingly,
   crotonylation was also found on some biosynthetic enzymes for natural
   products, such as DptE, DptA, and DptD, the core enzymes in
   biosynthesis of daptomycin (Supplementary Data [118]3), a
   representative antibiotic produced by S. roseosporus^[119]40,[120]44.
   All these data suggested that crotonylation globally occurs on key
   metabolic enzymes, biosynthetic enzymes, and critical regulators and
   thus might regulate metabolism of Streptomyces.

   Among all PTMs identified to date, acetylation has been extensively
   mapped in diverse organisms. We compared the crotonylation substrates
   identified in our study to previously determined acetyl-proteome in S.
   roseosporus^[121]10. We found that 37% of crotonylated proteins are
   acetylated as well at their Lys residues (Supplementary Fig. [122]11a).
   Meanwhile, these two acylations possesses priority of various
   modification-occurring motifs (Supplementary Fig. [123]11b). Further
   KEGG pathway enrichment (Supplementary Fig. [124]11c) and protein
   domain enrichment (Supplementary Fig. [125]11d) of these two
   modifications suggested that the most enriched categories for
   crotonylated proteins were ribosome (p = 2.40E−09) and carbon
   metabolism, such as citrate cycle (p = 2.14E−07),
   glycolysis/gluconeogenesis (p = 4.22E−05), and pyruvate metabolism
   (p = 3.59E−06) (Supplementary Fig. [126]11c), whereas they were
   aminoacyl-tRNA biosynthesis (p = 2.33E−06) and ribosome (p = 3.19E−05)
   for acetylation.

Crotonylation regulates Streptomyces metabolism

   Next, we further investigated the regulatory roles of crotonylation for
   Streptomyces metabolism. In the KEGG metabolic pathway analysis
   (Fig. [127]3a), the top enriched categories for lysine-crotonylated
   substrates were ribosome and carbon metabolism, such as TCA cycle,
   while the top categories of acetylation were aminoacyl-tRNA
   biosynthesis, ribosome in our strain as well as S. erythraea^[128]45,
   and ribosome and pyruvate metabolism for succinylation in Escherichia
   coli^[129]46, implying a possible link between crotonylation and carbon
   metabolism. Consistently, increased production of pyruvate was observed
   at the supplement of 10 mM NaCr, but not at 5 or 20 mM NaCr or with
   addition of NaAc (Fig. [130]3b). Considering an incremental level of
   crotonylation during primary metabolism (Fig. [131]1a), these results
   suggested that proper enhancement of crotonylation might benefit
   Streptomyces during primary metabolism, but excessive modification
   would be backfired. Meanwhile, we observed delayed aerial mycelium
   development (Fig. [132]3c) and postponed secondary metabolism
   development for red pigment production (Fig. [133]3c) and daptomycin
   production (Fig. [134]3d) if cells were supplemented with 10 mM NaCr.
   These observations experimentally suggested that crotonylation is
   involved in the regulation of both primary and secondary metabolism of
   Streptomyces.

Fig. 3. Crotonylation regulates S. roseosporus metabolism.

   [135]Fig. 3
   [136]Open in a new tab

   a KEGG pathway-enrichment analysis for lysine-crotonylated proteins. b
   Pyruvate production assays with addition of NaCr or NaAc. S.
   roseosporus wild type was cultured in TSB supplemented with NaCr (pH
   7.4) or NaAc (pH 7.4) with the indicated concentrations for 24 h, and
   the intracellular pyruvate concentration was determined. P value was
   calculated with Student’s t test (n = 4). c Morphological phenotype of
   S. roseosporus wild type on R5 with or without 10 mM NaCr (pH 7.4) for
   2 or 6 days. d Daptomycin production in wild type supplemented with
   10 mM NaCr. The daptomycin yield was measured after culturing 120 h.
   Experiments were performed in triplicate. P value was calculated with
   Student’s t test (n = 3).

Identification of modification enzymes for Streptomyces crotonylation

   Next, we tried to identify enzymes responsible for this PTM. Protein
   crotonylation is reversibly catalyzed by crotonyl-transferase (writers)
   and decrotonylase (erasers)^[137]25,[138]47, and the canonical
   acetyltransferase p300 in mammalian cells also acts as a
   crotonyl-transferase^[139]21,[140]32,[141]48, suggesting that some
   enzymes are pluripotent in acyl-modifications. In Streptomyces, two
   deacetyltransferases, the NAD^+-dependent deacetylase CobB and
   Zn^2+-dependent deacetylase histone deacetylase (HDAC), and a putative
   GCN5-family acetyl-transferase Kct1, have been well
   characterized^[142]49 and are highly conserved (Supplementary
   Figs. [143]12–[144]14). We found that removal of these homologs in S.
   roseosporus showed remarkable alterations of crotonylation
   (Supplementary Figs. [145]15–[146]17). The ΔcobB mutant displayed
   constitutively higher crotonylation during fermentation, particularly
   after 48 h, than the wild type, whose modification obviously dropped at
   that time (Fig. [147]4a and Supplementary Fig. [148]18a). The Δhdac
   mutant showed more crotonylation in the early developmental phases but
   lower modification after 48 h compared to the wild type (Fig. [149]4b
   and Supplementary Fig. [150]18b). These data genetically confirmed that
   both CobB and HDAC work as decrotonylases in S. roseosporus, but they
   primarily function in different developmental stages. HDAC is
   responsible for the maintenance of low crotonylation in the primary
   metabolism, while CobB mainly works in the later phases of secondary
   metabolism. Moreover, deletion of kct1 caused decreased crotonylation
   as expected, especially in the early stages before 72 h (Fig. [151]4c
   and Supplementary Fig. [152]18c), but similar protein modification was
   observed after 72 h between wild type and the Δkct1 mutant, genetically
   confirming that Kct1 is a crotonyl-transferase.

Fig. 4. Identification of crotonylation enzymes.

   [153]Fig. 4
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   Immuno-blot assays of crotonylation with anti-Kcr monoclonal antibody
   (1:2000) from S. roseosporus strain wild type (WT), two decrotonylase
   mutants ΔcobB (a) and Δhdac (b), and a crotonyl-transferase mutant
   Δkct1 (c) with anti-Kcr monoclonal antibody in right, together with
   Coomassie blue staining in left for the loading control.

   Consistent with the altered crotonylation profiles, both ΔcobB and
   Δhdac mutants showed more biomass than wild type, while Δkct1 mutant
   had retarded growth before 72 h but restored afterwards Supplementary
   Fig. [155]19a), further suggesting that sufficient crotonylation in the
   early phases benefits cell vegetative growth, which was in agreement
   with the gradually increased crotonylation observed in wild type.
   Consistently, over-produced pyruvate and ATP in both ΔcobB and Δhdac
   mutants but a decreased ATP concentration in the Δkct1 mutant was
   observed (Supplementary Fig. [156]19b). But interestingly,
   statistically increased production of crotonyl-CoA and decreased
   production of acetyl-CoA were observed in Δhdac and ΔcobB mutants,
   respectively (Supplementary Fig. [157]19c). Furthermore, both
   decrotonylase mutants began to produce the secondary metabolite
   daptomycin at 48 h, earlier than wild type. But it was noteworthy that,
   though similar daptomycin titers were observed in ΔcobB mutant and wild
   type at 72 and 96 h, the metabolite production in ΔcobB mutant dropped
   much faster afterward, while the Δhdac mutant showed constitutively
   higher productivity (Supplementary Fig. [158]19d), implying that
   excessive crotonylation in the early phases might benefit production of
   secondary metabolites, but crotonylation should be partially erased for
   proper metabolic development in the later phases. This hypothesis could
   also explain the observation in wild type, where crotonylation
   gradually increased before 36 h but slowly dropped after 48 h.
   Consistently, the Δkct1 mutant with weakened crotonylation only
   initiated daptomycin production at 96 h and constantly showed a much
   lower yield of daptomycin throughout the fermentation (Supplementary
   Fig. [159]18d). These data supported that delayed crotonylation
   postpones cell development and reduces secondary metabolite production.

   Some writers and erasers in bacteria are versatile in modifications of
   proteins with various acylations. For examples, CobB has been shown as
   a de-acetylase in E. coli and as a de-hydroxybutyrylase in Proteus
   mirabilis^[160]50. Acetylation and succinylation are two representative
   PTMs in bacteria. However, we found that, in S. roseosporus, ΔcobB
   mutant had no obvious change in acetylation profile but showed much
   higher succinylation, while Δhdac mutant had stronger signals both in
   acetylation and succinylation (Supplementary Fig. [161]20a). Moreover,
   no distinct changes were observed in Δkct1 mutant (Supplementary
   Fig. [162]20b). All these data suggested that both erasers also
   function in other types of acylation of Streptomyces, while the writer
   Kct1 not (Supplementary Fig. [163]20c). Though we could not exclude the
   possible involvement of acetylation or succinylation for metabolic
   changes of ΔcobB and Δhdac mutants, our observations from Δkct1 mutant
   and phenotypes with NaCr addition still supported regulation of
   crotonylation on Streptomyces metabolism.

Glk is reversibly crotonylated by CobB and Kct1

   To better understand the molecular regulatory mechanism of
   crotonylation and further confirm that above phenotypic alterations on
   Streptomyces metabolism authentically result from crotonylation, we
   looked for critical substrates that are crotonylated but not acetylated
   and mediate the regulation of crotonylation on Streptomyces metabolism.
   Here we focused on Glk, due to the global modification of glucose
   catabolism pathway (Fig. [164]2e) and given that Glk is the first
   enzyme to activate glucose and a key regulator of CCR in
   Streptomyces^[165]16,[166]20. Moreover, Glk was identified crotonylated
   at two conserved residues K89 and K91 based on MS/MS data
   (Fig. [167]5), adjacent to a catalytic D105^[168]51. But interestingly,
   no acetylation was found on Glk, which thus excludes the influence of
   acetylation on Glk in our study.

Fig. 5. Glk is crotonylated at two conserved lysine (K) residues.

   [169]Fig. 5
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   a MS/MS analysis of crotonylation on Glk K89. Crotonylation was
   calculated based on the molecular weight difference values between y3
   and y2 (458.26 − 262.14 = 196.12), and b6 and b4
   (788.39 − 477.25 = 311.14). b MS/MS analysis of crotonylation on Glk
   K91, which was calculated based on the molecular weight difference
   values between y5 and y4 (727.41 − 531.29 = 196.12), and b7 and b6
   (916.49 − 720.37 = 196.12). c Glk protein alignment from S. roseosporus
   (Sro), S. griseus (Sgr), and S. coelicolor (Sco). Two crotonylated
   residues (Kcr sites) along with conserved motifs are shown. Residues
   involved in the glucose-binding mechanism were labeled with asterisks.

   To identify the enzymes that modify Glk, we constructed a bacterial
   two-hybrid library comprising 54 putative acyl-transferases and 2
   de-acylases as the preys (Supplementary Data [171]6), while Glk was the
   bait in this system. Interestingly, CobB and Kct1 were screened out
   from this library to specifically interact with Glk, since the blue
   lawn was observed only when Glk co-existed with CobB or Kct1 in one
   bacterial cell to activate the expression of β-galactosidase in the
   bacterial two-hybrid system (Fig. [172]6a). Glk, CobB, and Kct1 were
   expressed and purified from E. coli. Based on the immunoblots with
   α-Kcr antibody, we found that the purified Glk protein has already been
   modified with crotonylation (Fig. [173]6b and Supplementary
   Fig. [174]21a, Fig. [175]6c and Supplementary Fig. [176]21b). In our in
   vitro assays, Glk can be further crotonylated with crotonyl-CoA in an
   enzyme-independent pathway for PTM. However, strong crotonylation was
   observed only when the acyl-transferase Kct1 was further added,
   confirming that Glk can be crotonylated by Kct1 (Fig. [177]6b and
   Supplementary Fig. [178]21a). Moreover, when Glk was incubated with
   CobB and the co-factor NAD^+, decreased crotonylation was observed with
   about 27% loss for 1 h and over half for 1.5 h (Fig. [179]6c and
   Supplementary Fig. [180]21b), confirming that CobB is an
   NAD^+-dependent decrotonylase to erase crotonylation on Glk.

Fig. 6. Glk is reversibly crotonylated by CobB and Kct1.

   [181]Fig. 6
   [182]Open in a new tab

   a Bacterial two-hybrid analysis of the interaction between Glk and CobB
   or Kct1. Glk was constructed in pT18, while CobB or Kct1 in pT25. E.
   coli strain BTH101 containing the hybrids were cultured on LB for 48 h
   and photographed. b, c In vitro assays for Glk crotonylation by Kct1
   (b) and decrotonylation by CobB (c). Reactions were analyzed by western
   blot with the indicated antibodies (1:2000). The relative intensity of
   each sample is also shown. In b, the reaction time is 1 h, while in c,
   the reaction time for decrotonylation is: lane 1, 1.5 h; lane 2, 1 h;
   lane 3, 1.5 h.

Glk activity is regulated by crotonylation

   Next, the effects of crotonylation on Glk kinase activity were
   evaluated. As shown from in vitro assays in Fig. [183]7a, we found that
   Glk with higher crotonylation (modified by Kct1 as in Fig. [184]6b) had
   lower activity than the native one purified from E. coli, which could
   recover after decrotonylation by CobB (Supplementary Fig. [185]22a),
   suggesting that crotonylation reduces Glk activity. Consistent with
   this observation, crotonylation mimic of these sites by K to Q single
   mutation also reduced the activity, and the double mutant had a much
   lower level of kinase activity (Fig. [186]7a). Consistent with these
   observations, when Δglk mutant was complemented with wild-type Glk
   protein, the constitutively modified Glk (K8991Q) and the unmodified
   form (K8998R), we found that K–Q mutation led to lower levels of Glk
   activity, while K–R mutation accounted for the higher kinase activity
   based on the whole-cell lysate assays (Supplementary Fig. [187]22b, c).

Fig. 7. Crotonylation regulates Glk activity.

   [188]Fig. 7
   [189]Open in a new tab

   a In vitro assays of crotonylation on the Glk catalytic activity.
   Glk[cr] was prepared from the in vitro reaction with Glk and Kct1 in
   Fig. [190]3b. Glk mutant isoforms were purified from E. coli.
   Statistical significance was determined by two-tailed unpaired
   Student’s t test (n = 4). b, c Western blot analysis of crotonylation
   level of Glk in vivo. S. roseosporus wild type and ΔcobB and Δkct1
   mutants expressing ermEp*-3flag-glk were cultured in the YEME medium,
   and mycelia were taken at the time points indicated. 3FLAG-Glk was
   immune-precipitated from the lysate, and western blots were performed
   with anti-FLAG M2 or anti-Kcr rabbit antibody (1:2000). d Glucose
   kinase activity assays of the lysate from wild type and ΔcobB and Δkct1
   mutants. Bacterial strains were cultured in the YEME medium, and the
   kinase activity was measured every 12 h. Experiments were performed in
   triplicate.

   Further in vivo investigation of crotonylation in wild type showed that
   crotonylation of Glk increased during cell metabolism development. In
   primary metabolism (12 and 18 h), the modification was low but sharply
   increased at 24 h and moderately increased at 48 and 72 h. However, the
   lowest crotonylation was observed at 18 h (Fig. [191]7b and
   Supplementary Fig. [192]23a and Fig. [193]7c and Supplementary
   Fig. [194]23b). The dynamics of Glk modification in wild type also
   correlated to the kinase activity during cell development. Higher
   activity was observed at primary metabolism, and cells at 18 h had the
   highest Glk kinase activity (Fig. [195]7d). These phenomena were well
   coincident with the previous report in S. coelicolor that Glk activity
   had a peak at 18 h but dropped and was constant afterward^[196]20.
   Interestingly, constitutive hyper-crotonylation of Glk was found in the
   ΔcobB mutant throughout the cell development. Especially at 12 and
   18 h, this mutant had even much higher modification than wild type from
   the late development phases (Fig. [197]7b). This excessive modification
   of Glk in the ΔcobB mutant accounted for the lower activity
   (Fig. [198]7d). Moreover, ultra-low modification of Glk was found in
   the Δkct1 mutant in all developmental phases, and it was almost
   undetectable at 18 h (Fig. [199]7c). As expected, we observed much
   higher Glk activity in this mutant, particularly at 18 h
   (Fig. [200]7d). All these data suggested that crotonylation negatively
   regulates Glk kinase activity, and the in vivo precise proportion
   between crotonylation and Glk activity dynamics also suggested that
   crotonylation might be one of the major PTM mechanisms for Glk activity
   regulation.

Positive modulation of CCR by crotonylation

   CCR is a conserved regulatory mechanism to utilize one carbon source
   (such as glucose) to suppress the metabolism of other carbon sources
   and Streptomyces metabolism, and mainly depends on
   Glk^[201]5,[202]13,[203]14. Glk kinase activity is required for glucose
   activation and subsequent glycolysis. It has been shown that there
   was a global loss of CCR in the glk-deletion strain^[204]4, but both
   Glk-dependent and independent pathways have been proposed for
   CCR^[205]16–[206]19. However, one possible mechanism of Glk on CCR has
   been proposed that Glk might interact with other transcriptional
   factors after it is posttranslationally modified^[207]20. So we further
   investigated whether crotonylation influences CCR in Streptomyces.

   Glucose has been shown to trigger CCR in Streptomyces for sugar
   metabolism, cell differentiation, and secondary metabolite
   production^[208]52. First, the residual glucose in the culture was
   examined. We found that glucose was consumed faster in the ΔcobB mutant
   (Fig. [209]8a), though the Glk activity was much lower (Fig. [210]7d),
   while the Δkct1 mutant showed more glucose surplus than wild type
   (Fig. [211]8a). These data indicated that the ratio of glucose
   uptake/Glk kinase activity was highest in the ΔcobB mutant but lowest
   in the Δkct1 mutant, suggesting that the ΔcobB mutant had the strongest
   CCR, while the Δkct1 mutant had the weakest CCR, according to the index
   of glucose uptake/Glk activity ratio suggested previously^[212]18. In
   addition, considering that over-crotonylation of bacterial proteins is
   the direct consequence in the ΔcobB mutant, our data suggested that
   crotonylation should positively regulate CCR.

Fig. 8. Crotonylation regulates carbon utilization in S. roseosporus.

   [213]Fig. 8
   [214]Open in a new tab

   a Residual glucose in the medium of S. roseosporus wild type and ΔcobB
   and Δkct1 mutants. Bacterial strains were cultured in the YEME medium,
   and glucose concentration was measured every 12 h. Experiments were
   performed in triplicate. b Quantitative assays of fold change of galK
   gene expression in Δglk mutant complemented with glk. Cells were
   cultured in MM media with different carbon sources. Gal galactose, glc
   glucose. Experiments were performed in quadruple. c Quantitative assays
   of fold change of galK gene expression in wild type and ΔcobB and Δkct1
   mutants. Cells were cultured in YEME medium with 4% glucose for 18 or
   24 h. Experiments were performed in triplicate. d Quantitative assays
   of fold change of galK gene expression in Δglk complemented with glk
   and its mutants (K8991Q, K8991R). Cells were cultured in MM media with
   different carbon sources for 24 h. Gal galactose, glc glucose.
   Experiments were performed in triplicate. e Dry weight of wild type and
   ΔcobB and Δkct1 mutants in the YEME culture without glucose but with
   galactose as the sole carbon source.

   To further confirm this hypothesis, we investigated the utilization of
   non-fermentable carbon sources (herein galactose) in the ΔcobB, Δkct1,
   and Δglk mutants complemented with Glk or its point mutants to mimic
   the crotonylated form (K8991Q) or non-crotonylated form (K8991R). The
   transcription levels of galK, a gene encoding galactokinase^[215]53 for
   galactose utilization, were examined. We found repressed galK
   expression in the presence of glucose (Fig. [216]8b), but it was
   strongly de-repressed in Δkct1 mutant (Fig. [217]8c) or with the
   non-crotonylated form of Glk (K8991R), even in the presence of glucose
   (Fig. [218]8d), suggesting loss of CCR controls. However, galK
   expression was further repressed in the ΔcobB mutant (Fig. [219]8c) or
   when Glk (K8991Q) mutant protein was complemented even in the absence
   of glucose (Fig. [220]8d), suggesting the strengthened CCR effects.
   These results suggested that crotonylation on K89/K91 of Glk is
   required for proper CCR, and removal of this PTM could constitutively
   release cells from CCR. Consistent with this hypothesis, the growth
   defect of Δkct1 mutant, in which crotonylation of Glk apparently
   reduced, was rescued to a comparable level of wild type if galactose
   was the sole carbon source (Fig. [221]8e). All these data suggested
   that crotonylation positively regulates CCR in Streptomyces.

Discussion

Crotonylation of Streptomyces metabolic pathways and enzymes

   Here, based on crotonyl-proteomics, we showed several characteristics
   of protein crotonylation in Streptomyces. First, Streptomyces proteins
   are highly posttranslationally modified as crotonylation. About 20%
   proteins and 4000 lysine sites in S. roseosporus are crotonylated,
   which is about 2-fold in protein and over 3-fold in the site of
   acetylation in the same species^[222]10. Considering that acetyl-CoA is
   the most abundant acyl-CoA within cells and estimated 1000-fold more
   than crotonyl-CoA^[223]32, our results suggested that crotonylation
   might play more broad and sensitive roles than acetylation in
   Streptomyces. Second, though crotonylated proteins are distributed in
   various cellular processes, such as cell cycle, growth, division, and
   cellular response to stimulus, most crotonylated proteins are enzymes
   with catalytic activities and involved in metabolic pathways, mainly in
   carbon metabolism and protein turn-over. For example, heavily
   crotonylated enzymes are enriched in glycolysis, pyruvate metabolism,
   TCA, phospho-pentose pathway, and CoA biogenesis, which are responsible
   for glucose catabolism, acetyl-CoA, and NAD(P)H production. These
   observations implied that crotonylation might regulate carbon
   utilization and biogenesis of acyl-CoA species, thus causing complex
   interplay of crotonylation with other acyl-CoAs both at intracellular
   concentrations and their possible competitive modifications. Moreover,
   almost all aminoacyl-tRNA synthetases, numerous ribosomal proteins and
   protein degradation machineries (proteases, proteasome) are also highly
   crotonylated, suggesting that crotonylation is one of the determinants
   for protein turn-over in cell metabolism regulation. Third but not the
   last, crotonylation is involved in secondary metabolism, including cell
   differentiation and secondary metabolite biosynthesis. This is
   characterized by modification of some transcriptional regulators for
   secondary metabolism development and the biosynthetic enzymes for
   secondary metabolite production.

Crotonylation positively regulates CCR of Streptomyces

   Streptomyces is soil dwelling and has to compete with other microbes in
   the living community for nutrients, such as the chitin-degradative
   product N-acetyl-glucosamine^[224]54. CCR has evolved in most
   microorganisms to use one carbon source in priority to support
   vegetative growth by suppressing the utilization of other carbon
   sources^[225]13,[226]55. For Streptomyces, glucose has been extensively
   used in laboratories as the main carbon source in feast, and
   glucose-induced CCR has been shown to suppress bacterial
   differentiation, including morphogenesis and secondary metabolism
   development. Glk not only functions as a kinase to activate glucose in
   the first step of glucose catabolism but also has a regulatory role,
   proposed via a kind of PTM to interact with other transcriptional
   regulators, on CCR modulation^[227]13,[228]20.

   Here we found that all enzymes are crotonylated in glucose catabolism,
   including glycolysis, pyruvate dehydrogenesis, and TCA, suggesting that
   crotonylation is involved in regulation of glucose utilization and
   subsequent CCR. Indeed, Over-crotonylation in a decrotonylase mutant
   ΔcobB not only increases Glk modification to reduce its kinase activity
   but also promotes total glucose consumption, suggesting that heavy
   crotonylation leads to high ratio of glucose uptake/Glk activity.
   Moreover, over-crotonylation suppresses the utilization of
   non-preferred carbon sources, indicating the intensified CCR. These
   observations suggested that high crotonylation will lead to CCR. We
   suspected that crotonylation might be one of the PTMs for Glk to
   interact with regulators to trigger CCR. So here we proposed that
   crotonylation modulates Streptomyces metabolism at least by positive
   regulation of CCR through dual regulation of Glk and carbon source
   metabolism. But the mechanism how crotonylation regulates interaction
   of Glk with other regulators still needs further investigation.

Crotonylation dynamics in regulation of Streptomyces metabolism

   In this work, we showed that crotonylation during Streptomyces
   metabolism is properly controlled by a set of modification enzymes,
   including two decrotonylases CobB, HDAC, and one crotonyl-transferase
   Kct1. They play their roles in different development phases, as CobB
   mainly erases crotonylation during secondary metabolism, while HDAC and
   Kct1 work in the early stages. Abnormal crotonylation in these mutants
   results in altered patterns of cell growth and metabolite production,
   suggesting that the concerted crotonylation is essential for proper
   metabolism of Streptomyces.

   Based on our understanding of crotonylation on regulation of CCR, we
   propose that gradually increasing modification of Streptomyces proteins
   can maintain CCR for continuous preferred carbon source (such as
   glucose as a model in the laboratory cultivation) utilization and
   vegetative growth. Since glucose drops rapidly in the medium in the
   early phases and loses its control on CCR, crotonylation can reinforce
   CCR to make sure that Streptomyces can keep consuming glucose in
   priority. This hypothesis is supported by the observation that the
   Δkct1 mutant has reduced cell growth accompanied by weakened CCR, as
   characterized by reduced glucose utilization and hyper-expression of
   genes for utilization of galactose. However, after vegetative growth,
   crotonylation needs to be partially erased to relieve CCR or reduce the
   modification of some regulators or biosynthetic enzymes for proper
   secondary metabolism development and efficient natural product
   biosynthesis, as block in hyper-crotonylation in the ΔcobB mutant is
   detrimental to secondary metabolite production.

   In summary, here we showed that crotonylation, a previously unreported
   protein PTM, comprehensively regulates the metabolism of Streptomyces.
   Specifically, crotonylation positively regulates CCR for the proper
   development of Streptomyces metabolism. Our findings provide insights
   of PTM regulatory mechanisms on Streptomyces metabolism so far and the
   basis for possible molecular engineering of this modification system to
   take the full advantage of this genus for natural product development.

Methods

Strains, media, and biological materials

   All strains used in this study are listed in Table [229]1. Wild-type S.
   roseosporus strain L30 was a daptomycin producer^[230]40. All the S.
   roseosporus mutants (ΔprcB/A, ΔcobB, Δhdac, Δkct1) were constructed
   based on the temperature-sensitive plasmid pKC1139^[231]40. The
   plasmids were transferred into wild-type strain by conjugation^[232]4.
   After double cross-over, the genotypes were verified by PCR. E. coli
   DH5α (Novagen) was the host for plasmid construction, and
   ET12567/pUZ8002 was used for conjugation to introduce DNA from E. coli
   to S. roseosporus^[233]40. Proteins were expressed and purified from E.
   coli BL21 (DE3) (Novagen).

Table 1.

   Streptomyces strains used in this study.
   Strain Description Source or references