Protein Acylation Affects the Artificial Biosynthetic Pathway for

Articles Cite This: ACS Chem. Biol. XXXX, XXX, XXX−XXX

Protein Acylation Affects the Artificial Biosynthetic Pathway for Pinosylvin Production in Engineered E. coli Jun-Yu Xu,†,‡,§ Ya Xu,§ Xiaohe Chu,† Minjia Tan,*,‡ and Bang-Ce Ye*,†,§ †

Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou 310014, Zhejiang, China ‡ State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China § Laboratory of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China S Supporting Information *

ABSTRACT: The effect of regulatory system on the engineered biosynthetic pathway in chassis cells remains incompletely understood in microorganisms. Acyl-CoAs function as key precursors for the biosynthesis of various natural products and the dominant donors for protein acylation. The polyphenol pinosylvin, with high antimicrobial and antifungal activities, is biosynthesized with malonyl-CoA as its direct precursors. But correlation between lysine malonylation and pinosylvin biosynthesis remains unknown. Herein, we found that the malonyl-CoA-driven lysine malonylation plays an important role in interaction between the engineered pathway of pinosylvin synthesis and E. coli chassis cell. Oversupply of malonyl-CoA leads to an increase in malonylation level of global proteome as well as the enzymes in the artificial pathway, thereby decreasing yield of pinosylvin. The results revealed that the intricate balance of cellular acyl-CoA concentrations is critical for the yields of acyl-CoA-derived natural products. We next modified the enzymes in the biosynthetic pathway to adjust their acylation level and successfully improved the yield of pinosylvin. Our study uncovers the effect of protein acylation on the biosynthetic pathway, helps optimization of synthetic constructs, and provides new strategies in metabolic engineering and synthetic biology at the protein post-translational level.

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decade, research has been devoted to elucidating the interactions between synthetic constructs and their host cells, including transcriptional regulation, allosteric regulation, distribution of carbon flux, and competition for shared resources (metabolites, proteins, ribosomes, and energy).9 Nevertheless, the characterization of host-construct interaction at post-translation modification levels was much less understood. Coenzyme A esters of short fatty acids (acyl-CoA pools) are important metabolic intermediates, and also the important carbon providers in microbial chassis cells. In the cell factory, acyl-CoAs function as precursors for different kinds of natural products, including fatty acids, bioalcohols, polyhydroxyalkanoates, polyketides, alkaloids, and isoprenoids.10 For example, pinosylvin, a resveratrol analogue of polyketide stilbenoid found in pine, with known high antimicrobial and antifungal activities, is biosynthesized utilizing malonyl-CoA as building blocks.11 On the basis of this reason, metabolic engineering and synthetic

icrobial biosynthesis of human-desired chemicals has attracted much attention. In industrial microbiology, synthetic biology provided great opportunities to establish cell factories to increase chemical productivity.1 Synthetic biology redefines rationally selected microbes based on their metabolic pathways and regulatory mechanisms, as well as engineering principles, which constructs new biological pathways or a system for biosynthesis of desired products.2,3 After a chassis cell (a minimal cell with low complexity and toxicity issues) was chosen, key elements (such as exogenous enzymes or regulators) could be assembled on molecular scaffolds to generate new compounds.4 In building an engineered microbial cell factory, it is essential to consider the optimization between heterologous enzyme elements themselves, as well as interaction between the artificial biosynthetic pathways and chassis cell.5 The complexity of the chassis cell affects the behaviors of synthetic constructs, while overexpression of the embedded biosynthetic enzymes and the oversupply of precursors both represent an unnatural burden and stress for engineered host cells that affect their resource allocation.6−8 Performances of engineered bacteria depend on how synthetic pathways interact with the host cells and vice versa. In the past © XXXX American Chemical Society

Received: December 14, 2017 Accepted: March 23, 2018

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DOI: 10.1021/acschembio.7b01068 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology

Figure 1. Oversupply of malonyl-CoA leading to a decrease in yield of pinosylvin in an engineered E. coli strain. (A) The artificial pathway for pinosylvin production constructed in E. coli. PAL (phenylalanine ammonia lyase) from Petroselinum crispum, 4CL (4-coumarate-CoA ligase) from Streptomyces coelicolor, STS (stilbene synthase) from Pinus strobus. ACC: Acetyl-CoA carboxylase. (B) Pinosylvin yields of the engineered E. coli with increasing cerulenin concentrations. (C) The intracellular concentrations of malonyl-CoA under the addition of cerulenin.

organisms, especially in bacterial chassis cells for synthetic biology. Although the two distinct roles of acyl-CoAs as precursors for biosynthesis and as donors for protein acylation are recognized, how acyl-CoA-driven lysine acylation affects the synthetic constructs embedded in chassis cell and the yields of acyl-CoA-derived products remains unknown. In this study, to characterize and exploit host-construct interactions at the lysine acylation level, we investigated the dynamic changes of intracellular malonyl-CoA and its effect on the artificial pathway for pinosylvin production at two levels (supply of precursors and acylation of enzymes) in the engineered E. coli. The supply of malonyl-CoA was beneficial to precursor pools for pinosylvin synthesis but led to malonylation of important enzymes involved in biosynthetic pathway to inhibit pinosylvin productivity. We next applied the PTM-based metabolic engineering strategy (PTM_ME) to optimize lysine acylation and successfully improved the yield of desired products. Our study revealed a novel regulatory mechanism mediated by protein acylation in an engineered cell factory and provided new insight in design and optimization of an engineered biosynthetic system at the protein post-translational level.

biology tools were used to raise the intracellular acyl-CoA pools, then improved the yields of desired products.10 As was reported, enabling biosynthesis of cytosolic acyl-CoA increased the yield of farnesene (a kind of isoprenoid) by 25% percent.12 Similarly, overexpression of malonate synthetase (MatB) and malonate carrier protein (MatC) can enhance the conversion of malonate to malonyl-CoA, and increase natural flavanone naringenin synthesis up to 250% in the recombinant E. coli host.13 In addition, multiple strategies have been attempted to promote acetyl-CoA generation in S. cerevisiae for elevating biosynthesis efficiency.14,15 In the mean time, acetyl-CoA-driven lysine acetylation is a well-recognized class of protein post-translational modifications (PTMs)16 and well-known for its chromatin associated functions and key roles of reversible lysine acylation (RLA) in multiple physiological and pathological processes.17−19 In addition, other novel protein acylation modifications, such as propionylation, butyrylation, succinylation, malonylation, crotonylation, glutarylation, 2-hydroxyisobutyrylation, and βhydroxybutyrylation were identified in various species based on the advancement of modern mass spectrometry.20−26 The diverse chemical properties of lysine acylation have increased the potential complexity of its regulatory function on metabolic enzymatic activities and metabolic flux distribution. And protein acylation levels are highly related to their respective acyl-CoA concentrations.27 Current studies have shown that lysine acylation can regulate bacterial chemotaxis, physiological metabolism, DNA replication, bacterial virulence, and other cellular processes.28−30 The additional roles of lysine acylation beyond these functions remain to be explored in micro-

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RESULTS AND DISCUSSION Oversupply of Malonyl-CoA Leads to Decrease in Yield of Pinosylvin in an Engineered E. coli Strain. The production of pinosylvin is first catalyzed by phenylalanine ammonia lyase (PAL) and 4-coumarate-CoA ligase (4CL) to form cinnamoyl-CoA from phenylalanine, and then the type III PKS stilbene synthase (STS) catalyzes the formation of the final product pinosylvin by the successive condensation of three B


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Figure 2. Global malonylation levels of the engineered E. coli strain. (A) Western blotting analysis of lysine malonylation levels of the engineered E. coli strain with increasing concentrations of cerulenin. Coomassie blue staining was used as the loading control. (B) The qualitative and quantitative analysis of the lysine malonylation level of whole cell lysates of E. coli strain cultured in 0 μM, 15 μM, and 100 μM cerulenin. Eight quantifiable malonylated sites in three conditions were listed, and lysine sites with obviously changed malonylation level (ratio >2) were labeled in orange.

global malonylation for whole cell lysates was observed when cerulenin concentration was increased. No visible changes were found in protein profiling of each sample (Figure 2A). After that, quantitative analysis of malonylome by stable isotope dimethyl chemical labeling coupled with antimalonyllysine antibody enrichment clearly showed obvious changes of global malonylation levels in cells treated with 0, 15, or 100 μM cerulenin, respectively (“Light” for 100 μM; “Medium” for 15 μM; “Heavy” for 0 μM; Figure S3 and Figure 2B). A total of 480 Kmal sites in 240 proteins were identified in at least two of the three technical repeats. Among them, 387 of the Kmal sites were unique in the E. coli strain under 100 μM cerulenin culture conditions (Table S1). Only eight quantifiable malonylated sites were observed without the addition of cerulenin, and almost all malonylation levels were significantly elevated with increasing concentration of cerulenin (Figure 2B). This result was consistent with Western blot analysis and showed that accumulated concentrations of intracellular malonyl-CoA could lead to an increase in lysine malonylation level, which resulted in an extra carbon burden on the host cell. To further evaluate the qualitative and quantitative malonylation level of phenylalanine ammonia lyase, 4coumarate-CoA ligase, and stilbene synthase in the embedded pathway of E. coli chassis cell, we excised bands of the three enzymes from gels in all cerulenin concentrations and performed in gel digestion to identify their malonylated sites. Mass spectrometry analysis revealed that Lys564 in PAL; Lys494 and Lys512 in 4CL; and Lys58, Lys113, Lys161, and Lys181 in STS were malonylated in engineered E. coli (Figure S4). Furthermore, quantitative analysis based on the MS precursor ion intensities showed that malonylation levels on these sites were elevated with the increasing of cerulenin

malonyl-CoAs to cinnamoyl-CoA (Figure 1A). The engineered E. coli strain (from Marienhagen’s lab) with an artificial Pal4CL-STS pathway for pinosylvin production was used to investigate the effect of lysine malonylation on pinosylvin biosynthesis.31 This E. coli strains were treated with different concentrations of cerulenin, a fatty acid synthesis inhibitor, to increase the intracellular concentration of malonyl-CoA. The result showed that the maximum yield of pinosylvin was obtained at the cerulenin concentration of 15 μM. When the concentration of cerulenin was higher than 15 μM, the yield of pinosylvin decreased gradually (Figure 1B). The result of growth curves indicated that addition of cerulenin did not affect the growth status of the engineered E. coli strain (Figure S1). We next measured the intracellular concentration of malonylCoA. In the mean time, acetyl-CoA concentration was also determined as a control. As shown in Figure 1C, accumulation of malonyl-CoA was found when adding more than 15 μM cerulenin. However, no changes in levels of intracellular acetylCoA were observed under all concentrations of cerulenin stimulation (Figure S2). This result indicated that lower pinosylvin concentration might be related with the accumulation of intracellular malonyl-CoA. The Intracellular Accumulation of Malonyl-CoA Results in Malonylation of the Enzymes in the Artificial Biosynthetic Pathway. The previous studies of us and other groups have shown that accumulated malonyl-CoA could lead to elevated protein malonylation level in both mammalian cells and bacteria.32,33 To explore the dynamic change of cellular malonylation level in response to different cerulenin concentration, we first investigated the effect of malonyl-CoA accumulation on global levels of lysine malonylation (Kmal) in the engineered E. coli strain. A modest increase in the level of C


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Figure 3. Malonylation/acetylation of enzymes PAL, 4CL, and STS. Qualitative and quantitative analysis of lysine malonylation levels in each modified site in PAL, 4CL, and STS with the increase of cerulenin concentrations, with lysine acetylation level as a control. (A) Malonylation/ acetylation in K564 (FCEK(mal/ac)DLLR) of PAL. (B) Malonylation/acetylation in K494 (VAPYK(mal/ac)R) and K512 (AASGK(mal/ac)ILR) of 4CL. (C) Malonylation/acetylation in K58 (NTNNEDNTDLK(mal/ac)DK), K113 (LGK(mal/ac)EAAEK), K161 (LLGLHPSVK(mal/ac)R), and K181 (LAK(mal/ac)DLAENNR) of STS.

Figure 4. Mutation mimicking lysine malonylation affects the yield of pinosylvin in E. coli. Mutation analysis of the malonylated sites in PAL (A), 4CL (B), and STS (C). The modified sites were mutated into glutamate to mimic the malonylated state.

Lysine Malonylation Represses the Activities of 4CL and STS and Decreases the Yield of Pinosylvin. Analysis of sequence conservation for the malonylated lysine residues in these enzymes indicated that Lys494 and Lys512 in 4CL and Lys58, Lys113, Lys161, and Lys181 in STS are highly conserved, whereas only a weak conversation was observed

concentration (Figure 3A−C and Figure S5). It is worth noting that no obvious change in acetylation level of these substrates was observed (Figure 3A−C). Thus, these results suggested that the yield of pinosylvin was associated with the dynamic balance of cellular malonyl-CoA concentration and protein malonylation. D


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Figure 5. Improvement of pinosylvin production by modifying malonylated sites in stilbene synthase. (A) Sequence alignment of K113 and K161 in stilbene synthases from Vitis vinifera (V. vinifera), Garcinia mangostana (G. mangostana), Arabidopsis thaliana (A. thaliana), Arachis hypogaea (A. hypogaea), and Pinus strobus (P. strobus). (B) Comparison of the pinosylvin production in wt (black) and STS_K161R (gray) strains at 15 μM cerulencin. The mutation of arginine mimicking the unmalonylated status of lysine. (C) Comparison of the pinosylvin production in wt (black) and STS_K113R (gray) strains under different concentrations (15, 18, and 20 μM) of cerulenin treatment.

for Lys564 in PAL (Figure S6A−C). In silico molecular modeling of the PAL structure (Figure S7A) illustrated that Lys564 lies in the helix α18, belonging to the inserted shielding domain (residues 528 to 649), and is out of the catalytic domain.34 Structural analysis of 4CL (Figure S7B) revealed that K512 is located in the conserved core motif A10, which is essential for the adenylation reaction.35 In addition, K494 is in the functional C-terminal domain according to its homologous structure of 4-coumarate-CoA ligase in Nicotiana tabacum (Figure S7B). In STS, the three malonylated lysine residues could interact with Pro109, Leu133, and Leu177 in region A (area 1, A102 to V106; area 2, T137 to L143; area 3, V163 to H168) through hydrogen bonding (Figure S8A−D). Region A, which is located at the dimer interface, is critical for mediating catalytic reaction, as illustrated in their homologous enzymes in P. sylvestris.36 Furthermore, the Lys58 of STS was located in the malonyl-CoA binding domain, which may affect its substrate intake.37 Together, these observations suggested that all malonylated lysine residues in 4CL and STS might be involved in enzymatic activities, and lysine malonylation would affect the biosynthesis of pinosylvin. We constructed the mutant strains and measured their pinosylvin yields to evaluate the malonylation effect. Mutation (K-E) of an acylated lysine residue was usually employed to mimic the negative acylation state, which included lysine malonylation and succinylation.23,38,39 In this work, each identified malonylated site of enzymes in the engineered pathway was then mutated into glutamate (E) to mimic the malonylated state. As shown in Figure 4, mutations of the identified malonylated sites in 4CL and STS all disturbed their enzymatic activities, and in turn decreased the pinosylvin production. However, no significant differences were observed for Lys564 in PAL. To directly demonstrate that protein malonylation exerts a negative effect on its activity, the purified 4CL enzyme was expressed, and its photometrical activity was measured. The result clearly showed that the activity of 4CL decreased by 40% when treated with cerulenin in the medium

(Figure S9), which further supported our conclusion. These results were consistent with sequence alignment assay and homology modeling assay, which indicated that these lysine sites in 4CL and STS were important for their enzymatic activities and their malonylation driven by malonyl-CoA inhibited activities of 4CL and STS in the artificial biosynthetic pathway. Improvement of Pinosylvin Production by Modifying Malonylated Sites in Stilbene Synthase. Finally, we investigated whether mutations mimicking unmodified positively charged lysine would circumvent the acylation effect. Arginine is positively charged under physiological conditions, which cannot be malonylated. Substitution of arginine for lysine was reported to maintain activities of some enzymes.40 The sequence analysis showed that Lys113 and Lys161 of STS could be replaced by arginine in some of its homologous enzymes in other species (Figure 5A). Therefore, we mutated Lys113 and Lys161 to arginine, respectively, to explore its effect on the pinosylvin production. The results showed that the STS_K161R decreased the activity of STS and pinosylvin production (Figure 5B), whereas no change was found for STS_K113R at 15 μM cerulenin (Figure 5C). We next asked whether STS_K113R could be tolerable to a higher concentration of cerulencin and lead to higher pinosylvin yield. Indeed, the results showed that the STS_K113R strain had maximum production of pinosylvin with an increase of 220% at 18 μM cerulencin (Figure 5C), indicating STS_K113R displayed tolerance toward higher intracellular concentration of malonyl-CoA. These observations demonstrated that metabolic flux of the pathway can be improved by adjusting the acylation of enzymes in this pathway and suggested a novel strategy in metabolic engineering at the protein post-translational level (PTM_ME) for design and optimization of the biosynthetic pathway. Conclusion. Synthetic biology and metabolic engineering employs biological/engineering principles to enable the rational design of synthetic constructs in their host cells. However, the E


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acyl-CoAs derived from carbon overflow, which may be an extra burden on bacterial growth as well as on product biosynthesis. Therefore, further design and optimization of an artificially engineered biosynthetic system and the interaction between engineered pathways and chassis cells must be fully considered at the protein post-translational level. To alleviate the effects of acylation on the biosynthesis of desired products, several metabolic engineering based on PTM (PTM_ME) strategies can be tried to increase the yields. To obtain the maximum yield of the desired products, the most efficient way is to keep the intracellular acyl-CoA concentration at an optimal level (balance between generation and consumption) by either increasing the consumption rate of acyl-CoA in biosynthesis or decreasing the supply of acyl-CoA. Second, manipulation of the RLA system (acyltransferase and deacylase) in an engineered chassis cell may be a useful approach for alleviating the acylation effect. Knockout of acyltransferase or overexpression of deacylase can reduce acylation levels of its target lysine sites in biosynthetic enzymes and thus attenuate the acylation-mediated inhibitory effect in biosynthesis. Nevertheless, this approach will not be suitable for the target proteins that are not the substrates of the acyltransferases and deacylases. The third strategy is modification of lysine acylated enzymes in the biosynthetic pathway, which enables them to be tolerant to acylation. Mutation (K-R) of its key acylated lysine site in our study has been proved to effectively maintain the activity of embedded engineered enzymes and successfully increase the yield. In addition, screening/identification of novel enzymes (such as isozymes) which are insensitive to lysine acylation may be a potential direction for the design of engineered pathways/ systems. In summary, this result demonstrated that acyl-CoA-driven lysine acylation plays an important role in interaction between the engineered pathway and host cells. These findings gave us a deeper system-level understanding of the host-construct interactions, provided a new strategy in metabolic engineering at the protein post-translational level, and offered a conceptual/ technological framework to create new metabolic enzymes/ pathways for the optimal production of desired products.

characterization of host-construct interactions in bacterial cells is still a challenge. The artificial synthetic constructs rarely work in isolation but globally interact with the host cells, which provide a background environment for their run. The engineered biological pathways embedded in the microbial chassis cell can be regulated at multiple variant levels, which include transcriptional regulation, translational regulation, allosteric control, and post-translational modifications.41−43 Some efficient strategies were developed for increasing synthetic yield of the artificial pathway, such as optimization of enzyme types, improvement of enzyme expression, abolishment of allosteric inhibition, and supply of precursors and energy.44,45 However, the effect of the RLA system in chassis cells on the engineered biosynthetic pathway has not been investigated yet. Up to date, the mining and standardization of biological components (enzymes, regulatory units), construction, and optimization of engineered pathways/systems in metabolic engineering are not considered at the protein acylation level. In this study, we characterized and exploited host-construct interactions at the protein malonylation level for the engineered chassis cell. We found that excessive accumulation of precursor malonyl-CoA resulted in malonylation of biosynthetic enzymes and thereby decreased the yields of end-product pinosylvin. Our results showed that the dynamic balance between precursor utilization and acylation effects exists in the artificial constructed pathways of acyl-CoA-derived compound biosynthesis (Figure 6). The acylation effect reflects the capability of

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METHODS

Pinosylvin-Synthesized E. coli Culture and Protein Extraction. The pinosylvin-synthetizing recombinant E. coli strain, a kind gift from Jan Marienhagen’s lab,31 was cultivated in 5 mL of LB broth at 37 °C overnight. Next, 20 μL of cultured cells were diluted into 5 mL of fresh LB broth. When an optical density at 600 nm (OD 600) of 0.6 was reached, 1 mM IPTG was added to induce protein expression, and the medium was supplied with 5 g/L of glucose. Meanwhile, different concentrations of cerulenin were added into the media. The cultivation was continued for an additional 36 h at 27 °C. Samples were taken for the HPLC analysis of pinosylvin. For protein extraction, cells were collected and washed with cold PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KHPO4) twice before lysis. The pellet was resuspended in the lysis buffer (8 M urea in PBS buffer, containing protease inhibitor cocktail (Calbiochem, Darmstadt, Germany) and 20 mM nicotinamide) and sonicated for 5 min. After being incubated on ice for 20 min, cell debris was removed by centrifugation at 21 000g for 30 min. Protein concentration was monitored by the BCA method with lysis buffer as the control. In-Solution Tryptic Digestion. A total of 2.5 mg of protein extracts (each from E. coli samples cultured in 0 μM, 15 μM, and 100 μM cerulenin) were reduced in 5 mM dithiothreitol (DTT) at 56 °C for 30 min, followed by incubation with 15 mM iodoacetamide (IAA) at RT in darkness for 30 min. The alkylation reaction was quenched

Figure 6. The regulation mechanism involved in the engineered biosynthetic systems in host cells. In chassis cells embedding the engineered biosynthetic pathway, an increase in intracellular acylCoAs, as important precursors, benefits the biosynthesis of desired products. However, excessive accumulation of acyl-CoAs results in acylation of biosynthetic enzymes and has an impact on the yields of product. This novel regulatory mechanism mediated by protein acylation provides new insight into design and optimization of a biosynthetic system at the protein post-translational level.

hosts in maintaining the balance between generation and consumption of cellular acyl-CoAs and implicates a critical regulatory mechanism underlying the interaction between engineered pathways and host cells. The biosynthetic pathway contains multiple embedded enzymes that catalyze sequential reactions for product synthesis. However, the enzymes in an optimized pathway in vitro are acylated in host cells to different extents, and a loss of their activities mediated by acylation will result in a decrease in the yield of products. In addition, the microbial proteome can also function as a “sink” for excessive F


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C18 resin with 3 μm particle size; 90 Å pore diameter; Dikma Technologies Inc., Lake Forest, CA) coupled to an EASY-nLC 1000 system (Thermo Fisher Scientific, Waltham, MA) was used, and the 60 min gradient was set as 4−20% solvent B (0.1% formic acid and 10% water in ACN) for 40 min, 40−53% solvent B for 13 min, 48−80% solvent B for 3 min, and 80% solvent B for 4 min at a flow rate of 300 nL/min. The injection volume was 4 μL for malonyl-CoA/acetyl-CoA concentration analysis, and malonyl-CoA/acetyl-CoA standard was used for localization in the spectrogram. The samples were performed in two replicates. Determination of Pinosylvin by HPLC Analysis. The extraction and HPLC analysis of trans-cinnamic acid and pinosylvin was based on the published paper with slight modification.47 In brief, 100 μL of cellfree supernatant was mixed with the same volume of ice-cold ACN, the mixture was centrifuged at 8000g for 5 min. A total of 80 μL of the mixture was loaded onto the Xbridge C18 column (4.6 × 250 mm, Waters, Milford, MA). The two mobile-phase solvents used were buffer A (96.96/0.04, water/formic acid, v/v) and buffer B (ACN). The initial gradient was 98% solvent A and 2% solvent B at 0.7 mL/ min. At 4 min, solvent B was gradually increased to 50% over 6 min. The UV detector was set at 308 nm for pinosylvin. Qualitative and Quantitative Analysis of Malonylated Sites in PAL, 4CL, and STS. In the sample preparation process, gels for PAL, 4CL, and STS were destained by using 50% ethanol after being excised from the SDS-PAGE gels. The gel bands were next sliced into 1 mm3 pieces and dehydrated by 100% ACN. After being reduced by 10 mM DTT at 56 °C for 40 min and alkylated by 15 mM iodoacetamide (IAA) in darkness at 25 °C for 40 min, the gels were washed by 50% ACN/50% mM NH4HCO3 (v/v). Proteins in gels were digested by trypsin at an enzyme to substrate ratio of 1:40 at 37 °C overnight. Digested peptides were extracted sequentially by using 50% ACN/5% TFA, 75%/0.1% TFA, and 100% TFA. The three extracts were combined and dried by Speed-Vac. The samples were dissolved in solvent A (0.1% (v/v) formic acid and 2% acetonitrile (ACN) in water) and analyzed by Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) with a 60 min gradient with 8−32% solvent B (0.1% formic acid and 10% water in ACN) for 51 min, 32−48% solvent B for 5 min, 48−80% solvent B for 1 min, and 80% B for 3 min at a flow rate of 300 nL/min. The electrospray voltage was controlled at 2.2 kV. The resolution was set at 120 000 at m/z 200. The m/z scan range was set as 350 to 1300. When the intensity of the precursor ions was greater than 5000, they were used for MS2 analysis. MS/MS acquisition was conducted in top-speed mode with a cycle time of 3 s. Ions with +2 to +6 charge states were fragmented in the HCD model with a normalized collision energy (NCE) of 32%. A time of 40 s was set as the dynamic exclusion duration. Two technical replicates were conducted. The raw data were converted to mgf files with Thermo Proteome Discoverer v1.4.0.288 (Thermo Fisher Scientific). Carbamidomethyl (C) was chosen as the fixed modification, and malonyl (K), oxidation (M), and acetyl (protein N-terminal) were set as variable modifications. Qualitative and quantitative analysis was applied for all malonylated sites. Areas under the curves (AUCs) of the precursor ion’s peak acquired from the ion chromatograms were used to assess the intensity of modified peptides. Unmodified peptides (LGGETLTISQVAAISAR and TSPQWLGPQIEVIR used for PAL normalization; YIVSGAAPLDAR and VTFVDAVPR used for 4CL normalization; VGVFQHGCFAGGTVLR used for STS normalization) were used for protein level normalization. Construction of Bacteria Strains, Western Blot Analysis, Structure Visualization of Enzymes, and Details of MS Analysis. Cloning and mutagenesis of plasmids of the E. coli strain used in this study, Western blot analysis, structure visualization of enzymes, and details of MS analysis are described in the Supporting Information.

with 30 mM cysteine at RT for 30 min. Trypsin (an enzyme-tosubstrate ratio of 1:100) was added to the protein solution at 37 °C for 16 h. The next day, additional trypsin (an enzyme-to-substrate ratio of 1:100) was added for a 4 h complete digestion. The tryptic peptides were desalted through SepPak C18 cartridges (Waters, Milford, MA) and dried by Speed Vac. Stable Isotope Dimethyl Labeling. The stable isotope dimethyl labeling method was performed based on the protocol previously reported.46 In general, 2.5 mg of peptides from each group were reconstituted in 250 μL of triethylammonium bicarbonate buffer (100 mM). Then, 12.5 μL of 20% CH2O, CD2O, and 13CD2O was added to the sample to be light-labeled, middle-labeled, and heavy-labeled, respectively, and 12.5 μL of 3 M sodium cyanoborohydride (NaBH3CN or NaBD3CN) was added to each sample, respectively (strains cultured in 100 μM cerulenin labeled by “light” reagent; strains cultured in 15 μM cerulenin labeled by “middle” reagent; strains cultured in 0 μM cerulenin labeled by “heavy” reagent). The reaction solution was incubated for 60 min at RT, and the labeling efficiency was confirmed by MS analysis. The reaction was performed twice. Then, the reaction was quenched with 12.5 μL of ammonia (20%) and acidified with trifluoroacetic acid (TFA) for solid-phase extraction. The samples were divided into two fractions for a further antibody enrichment process. Affinity Enrichment of Peptides Containing Lysine Acylated Peptides. The malonyllysine peptides were enriched by using antimalonyllysine antibody. Briefly speaking, malonylated peptides from each HPLC fraction were redissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0). Samples were centrifuged at 21 000g for 10 min to remove insoluble particles. The peptides of each fraction were incubated with 20 μL of agarose beads conjugated with antimalonyllysine antibody at 4 °C overnight with gentle rotation. The beads were washed three times with NETN buffer, once with ETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, pH 8.0) and twice with purified water. Enriched Kmal peptides were eluted from the beads three times with 0.1% trifluoroacetic acid. The eluted peptides were desalted with C18 ZipTips (Millipore, Billerica, MA) and then dried in SpeedVac. MS Data Processing and Analysis. MaxQuant software (version 1.5.3.8) and the UniProt Escherichia coli database (4306 sequences, July 11, 2017) were used for identifying and quantifying protein and malonylated peptides. The enzyme was set as trypsin/P, and the maximum value of missed cleavage was set to 2. After that, the precursor error tolerance was set as 20 ppm, and 0.5 Da was set for the fragment ion. The isotope dimethyl labeling pair search was conducted, in which the peptide N-terminal and lysine dimethylation (+28 Da) was set for light, (+32 Da) was set for middle, and (+36 Da) was set for heavy. Carbamidomethyl (C) was set as the fixed modification, and oxidation (M), acetylation (protein N-term), and malonylation (K) were set as the variable modifications. False discovery rate (FDR) thresholds for the protein, peptide, and modification site were specified at 0.01. In analysis of the quantified data, the modified sites with a localization probility lower than 0.75 and those peptides from reverse or contaminant protein sequences were removed. Malonylated sites identified in at least two of the three replicates and with high spectra quality after a manual check are presented in the Supporting Information Tables. Measurement the Intracellular Concentrations of MalonylCoA and Acetyl-CoA. Intracellular levels of malonyl-CoA and acetylCoA were analyzed according to the published paper with slight modification.32 E. coli samples under different culture conditions were treated with 10 mL of extraction buffer (6% perchloric acid in ice-cold water) and then transferred to the −80 °C freezer. After 15 min, samples were placed and dissolved on ice. The whole solution was then centrifuged at 21 000g at 4 °C for 20 min. The supernatant was filtered through SepPak C18 cartridges (Waters, Milford, MA), eluted by 40% acetonitrile (ACN), and dried by Speed Vac. A Q Exactive mass spectrometer (Thermo Fisher Scientific Inc.) was used for metabolite analysis. Samples were dissolved in solvent A (0.1% (v/v) formic acid and 2% ACN in water). For malonyl-CoA analysis, a reverse-phase C18 column (10 cm length ×75 μm inner diameter; G


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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.7b01068.

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General methods, supplementary Figures 1−9 (PDF) Supplementary Tables 1 and 2 (XLSX)

AUTHOR INFORMATION

Corresponding Authors

*Tel./Fax: 0086-21-64252094. E-mail: [email protected], [email protected]. *E-mail: [email protected]. ORCID

Jun-Yu Xu: 0000-0002-1403-9200 Minjia Tan: 0000-0002-6784-9653 Bang-Ce Ye: 0000-0002-5555-5359 Author Contributions

J.Y.-X., M.T., and B.C.Y. designed the research; J.Y.-X. performed research with the help of Y.X.; J.Y.-X., X.C., M.T., and B.C.Y. contributed new reagents/analytic tools; J.Y.-X. and B.C.Y. analyzed data and wrote the manuscript, and M.T. revised it. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (31730004, 21335003, and 21575089; to B.C.Y.). The National Natural Science Foundation of China (31670066 and 91753203; to M.T.). China Postdoctoral Science Foundation (2017M621567; to J.X.). We thank J. Marienhagen (Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, Jü lich, Germany) for providing us the E. coli BL21(DE3)/pR-HisPstrsts2-Sc4cl-Pcpal1 and technical assistance.

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