SCO6564, a novel 3-ketoacyl acyl carrier protein synthase III, contributes in fatty acid synthesis in Streptomyces coelicolor

Jian-Rong Ma; Jia-Ying Lin; Yuan-Yin Zhang; Yun Chen; Wen-Bing Zhang; Xian-Pu Ni; Yong-Hong Yu

doi:10.1371/journal.pone.0318258

Abstract

The genus Streptomyces comprises gram-positive bacteria that produce large numbers of secondary metabolites, which have promising commercial applications and deserve extensive study. Most bacteria synthesize fatty acids using a type II fatty acid synthase, with each step catalyzed by a discrete protein. Fatty acid synthesis has been intensively studied in the model strain Streptomyces coelicolor, in which 3-ketoacyl-acyl carrier protein synthase III (KAS III, FabH) is essential for growth and fatty acid biosynthesis. In this study, the FabH homolog SCO6564 (named FabH2) was identified in the S. coelicolor genome by BLAST analysis. The expression of fabH2 restored the growth of Ralstonia solanacearum fabH mutant and made the mutant produce small amounts of branched-chain fatty acids. FabH2 could condense various substrates, including straight-chain and branched-chain acyl-CoAs, with malonyl-acyl carrier protein to initiate fatty acid synthesis in in vitro assays. The fabH2 deletion did not cause significant changes in the growth or fatty acid composition of S. coelicolor, indicating that fabH2 is nonessential for growth or fatty acid synthesis. However, fabH2 overexpression reduced the blue-pigmented actinorhodin production. Phylogenetic analysis of KAS III from different bacteria revealed that FabH2 belongs to a novel group of FabH-type, which is ubiquitous in Streptomyces spp.

Citation: Ma J-R, Lin J-Y, Zhang Y-Y, Chen Y, Zhang W-B, Ni X-P, et al. (2025) SCO6564, a novel 3-ketoacyl acyl carrier protein synthase III, contributes in fatty acid synthesis in Streptomyces coelicolor. PLoS ONE 20(2): e0318258. https://doi.org/10.1371/journal.pone.0318258

Editor: Faiz ul-Hassan Nasim, Lahore University of Management Sciences, PAKISTAN

Received: September 16, 2024; Accepted: January 14, 2025; Published: February 6, 2025

Copyright: © 2025 Ma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This research was funded by the Medical Science and Technology of Guangdong Province (A2023056), the Characteristic Innovation Project of Colleges and Universities in Guangdong Province (2024KTSCX339), and the Science Foundation of Guangdong Food and Drug Vocational College (2023ZR06, 2024ZR01).

Competing interests: The authors declare no conflicts of interest.

Introduction

Streptomycetes, a gram-positive filamentous bacteria with a high G + C content in chromosomal DNA, produces many types of secondary metabolites that are widely used in human health, including >75% of the antibiotics used as medicine, and many compounds containing antiviral, antiparasitic, anticancer, and immunosuppressive activities [1–3]. Although the secondary metabolites are nonessential for bacterial growth, they are believed to play significant roles in their natural habitats. Streptomycetes strains can be found in various terrestrial and aquatic environments, including extreme habitats [4, 5]. Streptomyces coelicolor is regarded as the model strain for extensive study [6], because it produces well-characterized antibiotics, including the blue-pigmented polyketide actinorhodin (ACT) and the red-pigmented undecylprodigiosin (RED). ACT has served as an outstanding example for genetic and biochemical investigations of polyketide metabolism [7], whereas RED is a representative product synthesized using fatty acids as a building block, similar to the biosynthesis of many natural products in Streptomycetes [8].

Bacteria, such as Streptomycetes, utilize type II fatty acid synthase (FAS II) to synthesize fatty acids, involving a series of enzymes encoded by discrete genes [9]. By contrast, mammals and fungi use multifunctional fatty acid synthase I to catalyze fatty acid synthesis [10]. FAS II not only provides substrates for the biosynthesis of phospholipids, lipoproteins, and lipopolysaccharides, but also supplies intermediates for the synthesis of many important bioactive products, such as lipoic acid and biotin [11, 12]. Moreover, FAS II is involved in the production of different quorum sensing signals, such as N-acyl homoserine lactones [13] and diffusible signal factors [14, 15]. Thus, FAS II is believed to be a promising target for antibacterial discovery [16].

Bacterial pathway of fatty acids synthesis can be divided into two stages as follows: initiation and elongation. The initiation stage de novo produces 3-ketoacyl ACP by condensation of an acetyl-CoA and malonyl-ACP, which is elongated into long-chain fatty acids with condensation, reduction, dehydration, and reduction cycles [17]. The final products of FAS II will be transferred to glycerol-3-P by PlsX/Y or PlsB/C, resulting in the formation of membrane phospholipid. Bacterial membrane homeostasis is greatly determined by the structures of the fatty acids chains, in which straight-chain saturated fatty acids (SCFAs) are linear and pack together efficiently to produce a bilayer that has a high phase transition and low permeability properties. While unsaturated fatty acids (UFAs) and branched-chain fatty acids (BCFAs) incorporation results in lower transition temperatures and higher permeability [18, 19].

The initiation mechanisms of fatty acid synthesis vary in different bacteria. FabH, a 3-ketoacyl ACP synthetase III (KAS III), was first identified as the key enzyme involved in fatty acid synthesis initiation in the model organism Escherichia coli [20]. FabH homologs were found in the fatty acid synthetic loci in many bacterial genomes, indicating that the initiation mechanism catalyzed by FabH-type KAS III is relatively conserved across bacteria. However, FabHs from different bacteria show different substrate selectivity. FabH from E. coli and Ralstonia solanacearum only accept acetyl-CoA as substrate, thus synthesizing SCFAs [21, 22]. By contrast, FabH from gram-positive bacteria, such as Bacillus subtilis [23, 24] and S. coelicolor [8, 25], have a strong preference for branched-chain acyl-CoAs as the primers, producing BCFAs in these bacteria. Our group reported that FabHs from the gram-negative phytopathogen Xanthomonas campestris pv. campestris (Xcc) [26] and X. oryzae pv. oryzae (Xoo) [14] can utilize branched-chain primers to synthesize BCFAs. The opportunistic pathogen Pseudomonas aeruginosa encodes no FabH homolog, but utilizes FabY-type and PA3286-type KAS III enzymes to initiate fatty acid synthesis. FabY has similar activity to E. coli FabH, but exhibits different structural features [27], whereas PA3286 has a broader substrate specificity, because it can utilize octanoyl-CoA to initiate fatty acid synthesis [28]. Although Rsp0194 functions like PA3286-type KAS III, R. solanacearum can alternatively utilize medium-chain acyl-CoAs as primers to initiate fatty acid synthesis [22]. Recently, our group reported that FabH1 of P. syringae pv. syringae B728a is an atypical KAS III that functions in providing a critical fatty acid precursor, butyryl-ACP, for N-acyl homoserine lactone synthesis [29]. Guo et al. demonstrated that P. putida can use the long-chain KAS I (FabB) to catalyze the initiation reaction in FAS II [30]. Another type of initiation reaction, the malonyl-ACP decarboxylase (MAD) pathway, was identified in E. coli and P. putida, in which MAD can bypass or replace the KAS III pathway [31, 32]. In the MAD pathway, malonyl-ACP is converted to acetyl-ACP by MAD, which is further condensed with malonyl-ACP by the long-chain KAS I (FabB) or KAS II (FabF) to form the initial 3-ketobutyryl-ACP substrate used in the subsequent elongation reactions [33]. The MAD pathway complements, bypasses, or replaces the KAS III pathway in some bacteria. In summary, the initiation of fatty acid synthesis shows a large diversity, and one bacterial strain can use different pathways in FAS II.

S. coelicolor FabH (hereafter renamed Sco FabH1) was first identified by Hopwood et al. [25]. Reynolds et al. confirmed its greater catalytic efficiency to branched-chain primers [8]. Moreover, our group estabolished Sco FabH1’s activity in the initiation of fatty acid synthesis by genetic complementation and in vitro analysis [34]. Because Sco fabH1 is essential for growth, Li et al. generated the fabH1 deletion mutant by plasmid-based expression of E. coli fabH. This strain grew much slower, but produced ~14% of the BCFAs [35]. Our team studied Xcc fabH using a similar strategy, but the Xcc fabH deletion mutant, in which E. coli fabH was expressed from plasmid pSRK-Gm, produced only trace amounts (<1%) of BCFAs [26]. E. coli FabH can only synthesize SCFAs, then how BCFAs were synthesized in the S. coelicolor fabH1 deletion mutant complemented with E. coli fabH [35]? Thus, we hypothesize that there is another pathway to initiate BCFAs synthesis in S. coelicolor.

Here, we studied SCO6564 in the genome of S. coelicolor by employing different molecular biological techniques, including bioinformatic analysis, genetic complementation, biochemical analyses, and gene deletion. The results demonstrated that SCO6564 encodes an active KAS III, named Sco FabH2, which contains the conserved triplet Cys–His–Asn catalytic site. Sco FabH2 has catalytic activities toward various substrates, but plays a minor role in fatty acid biosynthesis in S. coelicolor. Sco FabH2 represents a novel FabH-type KAS III, which is mainly present in Streptomyces spp.

Materials and methods

Strains and culture conditions

The strains and plasmids used in this study are listed in S1 Table in S1 File. The E. coli strains were cultured at 37°C in LB Miller medium (10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl per liter, pH 7.0). R. solanacearum strains were grown at 30°C in BG medium (10 g of bacto peptone, 1 g of yeast extract, 1 g of casamino acids, 5 g of glucose per liter, pH 7.0). The S. coelicolor strains were cultured at 28°C in YBP (for ACT and RED testing, 4 g of tryptone, 2 g of yeast extract, 2 g of beef extract, 10 g of glucose, 15 g of NaCl, and 1 g of MgSO₄•7H₂O per liter, pH 7.2) [36] or Gauze’s synthetic medium no. 1 (for sporulation, 20 g of soluble starch, 0.1 g of beef extract, 0.1 g of KNO₃, 0.05 g of K₂HPO₄•3H₂O, 0.05 g of NaCl, 0.05 g of MgSO₄•7H₂O, 0.001 g of FeSO₄•7H₂O per liter [pH 7.0–7.2]) [37]. When required, the following antibiotics were added (per mL): 100 μg of sodium ampicillin, 30 μg of kanamycin sulfate, 30 μg of chloramphenicol, 50 μg of apramycin, 100 μg of erythromycin.

Gene cloning and construction of expression plasmids

coelicolor fabH1, fabH2, fabH3, and fabH4 were amplified from the genomic DNA of the wild-type (WT) strain using UP-DN primer pairs (listed in S2 Table in S1 File). The PCR fragments were purified, digested with Nde I and Hind III, and ligated into the pSRK-Km [38] expression vector to obtain the pMJR-1–pMJR-4 plasmid constructs, respectively. Sco fabH2 was inserted into the Nde I and Hind III sites of pET-28(b), using a similar strategy to obtain pMJR-5. Sco fabH2 was further amplified with the Sco fabH2-P1 and Sco fabH2-P2 primer pair and cloned into pJY813 between the Nde I and Bgl II sites to construct plasmid pMJR-9. All the constructs were confirmed by sequencing by Sangon Biotech. Co, Ltd.

Complementation of the R. solanacearum fabH deletion strain RsmH

Plasmids pMJR-1–pMJR-4 or empty vector were firstly transferred into E. coli strain S17-1. Then, the strain was mated with R. solanacearum fabH deletion strain RsmH [22] on BG plates with octanoic acid (0.1%) for 48 h at 30°C. The cells were suspended in BG medium, and appropriate dilutions were inoculated onto BG plates (with octanoic acid) containing chloramphenicol (to select against the donor strain) plus kanamycin. The transformed strains were inoculated onto BG plates with or without octanoic acid, and growth was determined after 2 d of incubation at 30°C.

Assay of Sco FabH2 activity in vitro

pMJR-5 was first transferred into E. coli BL21(DE3), and Sco FabH2 with His₆-tagged N-terminus was purified with Ni-NTA agarose (Qiagen) using a nickel-ion affinity column (Qiagen). E. coli FabD, FabH, FabG, FabZ, and FabI and E. coli holo-ACP proteins were purified as described [29]. To assay the 3-ketoacyl-ACP synthase activity of Sco FabH2 in vitro, different assay mixtures were prepared, which contained 0.1 M sodium phosphate (pH 7.0), 0.1 μg each of FabD, Sco FabH2, FabG, and FabZ, 50 μM NADH, 50 μM NADPH, 1 mM β-mercaptoethanol, 100 μM malonyl-CoA, 50 μM holo-ACP, and 100 μM of substrate (acetyl-CoA, isobutyryl-CoA, isovaleryl-CoA, butyryl-CoA, hexanoyl-CoA, or octanoyl-CoA) in a final volume of 40 μL. The reactions were initiated by adding FabH and followed by incubation for 1 h at 37°C. The reaction products were resolved by conformationally sensitive gel electrophoresis on 20% polyacrylamide gels containing a concentration of urea optimized for separation [39]. The gels were stained with Coomassie Brilliant Blue R-250.

In-frame chromosomal gene deletion and complementation

The upstream fragment of Sco fabH2 was amplified using the Sco fabH2-1 and Sco fabH2-2 primer pair from the chromosomal DNA of S. coelicolor and ligated into pMD19-T to obtain pMJR-6. The downstream fragment of Sco fabH2, amplified with Sco fabH2-3 and Sco fabH2-4, was inserted and cloned into the Pst I and Eco RI of pMJR-6 to obtain pMJR-7. The cloning was confirmed by sequencing. The primers used are listed in S2 Table in S1 File. The upstream and downstream fragments of Sco fabH2 were digested from pMJR-7 with Eco RI and Hind III and cloned into the same sites of the vector pKC1139 [40] to construct pMJR-8.

Following conjugation between the derivative of the E. coli ET12567 strain carrying pMJR-8 with the S. coelicolor WT strain was conducted on MS medium at 28°C for 20 h. After overlaying with apramycin (50 μg/mL) and nalidixic acid (50 μg/mL), incubation was continued at 28°C for 4–7 d. Single-crossover exconjugants were obtained using apramycin as a selection marker. After three rounds of sporulation in the absence of apramycin, many apramycin-sensitive strains were obtained. Several colonies were chosen randomly for chromosomal DNA extraction. The Sco fabH2 deletion strain (ΔfabH2) was selected out by PCR using the Sco fabH2-5 and Sco fabH2-6 primer pair and verified by sequencing the PCR products. The pMJR-9 expression plasmid was transferred into the Sco fabH2 deletion mutant ΔfabH2 to obtain the complementary strains by conjugation with E. coli ET12567.

Analysis of fatty acid profile

The WT and derivative strains of S. coelicolor were cultured in YBP medium at 28°C for 3 d. The cells were harvested by filtration and washed three times with water. Fatty acid methyl esters were synthesized and extracted as described [41]. Briefly, cellular lipids were saponified by adding 2 mL of a sodium hydroxide and methanol solution at 100°C for 50 min with shaking (800 rpm). The fatty acids were methylated by adding 4 mL of a hydrochloric acid and methanol solution at 80°C for 40 min and immediately cooled to <20°C. The fatty acid methyl esters were obtained by three extractions with 1 mL of petroleum ether. The solvent was removed under a stream of nitrogen, and the residue was analyzed by GC-MS, using n-hexane as solvent.

Examination of ACT production

Different strains of S. coelicolor spores were inoculated on YBP agar plates, and the plates were incubated at 28°C for 8 d ACT production was quantified as described [42]. Briefly, total ACT was quantified by scraping off all the medium containing mycelia from the plate and mixing with 1 M KOH. The reaction was conducted overnight at 25°C, and then a 1 mL aliquot was used for quantification. Intracellular ACT was quantified using only the mycelia, and extracellular ACT was quantified using only the medium ACT in the sample was quantified by measuring absorbance at 640 nm.

Statistical analyses

The experimental data were analyzed by analysis of variance using JMP software, version 50 (SAS Institute Inc.). Significant effects of treatment were determined using F values (P = 005). When F value was significant, the means were separated by Fisher’s protected least significant difference at P = 005.

Results

Bioinformatic analysis with FabH homologs in S. coelicolor

To investigate alternative initiation pathways of fatty acid synthesis in S. coelicolor, we performed Blastp analysis of the whole genome with E. coli FabH (Ec FabH) [43] as the query sequence. Four open reading frames were found as FabH homologs, including the reported Sco fabH1 in the conserved fatty acid biosynthesis (fab) gene cluster. The other three, SCO6564 (Sco fabH2), SCO1271 (Sco fabH3), and SCO3246 (Sco fabH4), were among unknown clusters (Fig 1A). The amino acid sequence identities of Sco FabH1–FabH4 to Ec FabH were 37%, 40%, 30%, and 41%, respectively (Fig 1A). Because Sco FabH1 is active and essential for fatty acid synthesis [25, 34], we performed sequence alignment analysis among the four Sco FabHs, and found that Sco FabH1 shares 41%, 43%, and 38% identity with Sco FabH2–FabH4, respectively. The conservative Cys–His–Asn triad motif typical of a KAS III enzyme was found in the four Sco FabHs (Fig 1B). The three-dimensional structures of Sco FabHs were predicted using SWISS-MODEL (https://swissmodelexpasyorg). All shared structural similarity with Sco FabH1 (S1 Fig in S1 File). Overall, the results of bioinformatics analysis strongly suggested that in addition to the reported Sco FabH1, the other three Sco FabHs may be active in catalyzing the initiation step of fatty acid synthesis in S. coelicolor.

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Fig 1. Bioinformatic analysis with FabH homologs in S. coelicolor.

(a) Loci of Sco fabHs. The fabH1 locus is similar to the E. coli fabH locus. (b) Structure-based amino acid alignments of Sco FabH1–FabH4 with E. coli FabH (Ec FabH). Residues that constitute the Cys (C)–His (H)–Asn (N) catalytic triad are highlighted (Cys₁₁₂, His₂₄₄, and Asn₂₇₄ in Ec FabH, respectively).

https://doi.org/10.1371/journal.pone.0318258.g001

Sco fabH2 could recover the growth of RsmH and produce BCFAs

To study the functions of Sco FabHs found in the S. coelicolor genome with bioinformatics, Sco fabH1–fabH4 were amplified and cloned into the expression vector pSRK-Km under the E. coli lac promoter to obtain pMJR-1–4. These were further introduced into the R. solanacearum fabH mutant strain RsmH by conjugation to obtain the transformants RsJR1–RsJR4, respectively. R. solanacearum RsmH is an octanoic acid auxotrophic mutant, in which the R. solanacearum genomic fabH is deleted in-frame [22]. All the RsmH derivatives grew well on a BG plate containing octanoic acid. Only pMJR-1 (Sco fabH1) and pMJR-2 (Sco fabH2) introduction could restore the growth of RsmH strain on BG in the absence of octanoic acid, but Sco fabH3 or Sco fabH4 expression could not rescue the growth of RsmH under the same condition (Fig 2). These results indicate that both Sco FabH1 and Sco FabH2, but not Sco FabH3 or Sco FabH4, were active KAS III to initiate fatty acid synthesis. Because the functions of Sco FabH1 have been confirmed in previous reports, we did an in-depth study of FabH2 to determine its functions.

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Fig 2. Expression of Sco fabH1 and Sco fabH2 restored the growth of R. solanacearum fabH knockout strain RsmH on BG medium in the absence of octanoic acid.

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To evaluate the functions of Sco FabH2 in fatty acid synthesis, R. solanacearum and the two RsmH derivatives harboring pMJR-1 (Sco fabH1) and pMJR-2 (Sco fabH2) were incubated and harvested at 30°C, then their fatty acids were extracted and determined by gas chromatography–mass spectrometry. The R. solanacearum WT strain GMI1000 does not produce BCFAs. However, the strain synthesized 50% of the UFAs with n-C_16:1 and n-C_18:1, whereas Sco fabH1 expression caused the RsmH strain to produce ~40% of the BCFAs—iso-C_17:0 and anteiso-C_17:0 were predominant. The content of UFAs in Sco fabH1/RsmH was reduced to ~32% (Table 1). By contrast, the RsJR2 (Sco fabH2) strain produced similar profiles of fatty acids as the WT strain, producing ~49% of the UFAs. However, minor amounts (~6.4%) of BCFAs, mainly iso-C_16:0 and iso-C_17:0, were detected in RsJR2, indicating that Sco FabH2 can catalyze initiation in SCFA and BCFA biosynthesis (Table 1). Because the content of BCFAs produced in RsJR2 was lesser than that in RsJR1, we hypothesize that Sco FabH2 is much less active to branched-chain primers than Sco FabH1.

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Table 1. Fatty acid composition of RsmH strains complemented with Sco fabH1 and Sco fabH2 ^a.

https://doi.org/10.1371/journal.pone.0318258.t001

Sco FabH2 is active to a broad range of substrates in in vitro analysis

To determine KAS III activity in vitro, N-terminal 6× His-tagged recombinant Sco FabH2 was expressed in E. coli BL21(DE3) and purified by nickel chelate chromatography. Various E. coli fatty acid biosynthetic proteins, including FabD, FabB, FabG, FabA, FabI, and holo-ACP, and Vibrio harveyi acyl-ACP synthetase [44], were purified using the same method.

The initial reaction of the fatty acid synthesis system was reconstructed based on the E. coli fatty acid synthetic enzymes mentioned. First, Sco FabH2 was added into the system with malonyl-ACP and acetyl-CoA as the initial substrates. The products were analyzed by conformationally sensitive gel electrophoresis. Sco FabH2 catalyzed the condensation of acetyl-CoA with malonyl-ACP to produce butyryl-ACP, thus exhibiting similar activity to Ec FabH (Fig 3A, lane 3). Interestingly, a light band of hexanoyl-ACP was detected in the products, suggesting that Sco FabH2 can condense butyryl-ACP with malonyl-ACP to generate long-chain acyl-ACP.

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Fig 3. Enzymatic characterization of Sco FabH2 in fatty acid biosynthesis in vitro.

(a) The initiation of fatty acid synthesis was reconstructed using a combination of E. coli FabZ, FabG, and FabI and Sco FabH2 (lanes 3–5) with NADH and NADPH as cofactors and malonyl-ACP plus acetyl-CoA (lane 3), isobutyryl-CoA (lane 4), or isovaleryl-CoA (lane 5) as substrates. The migration positions of holo-ACP (lane 1) and hexanoyl-ACP (C_6:0-ACP, lane 2) on gel are shown. The urea concentration was 1.0 mol/L. (b) The initial reaction of fatty acid synthesis included E. coli FabZ, FabG, and FabI and Sco FabH2, NADH and NADPH as cofactors, and malonyl-ACP + butyryl-CoA (lane 2), hexanoyl-CoA (lane 4), or octacyl-CoA (lane 6) as substrates. The migration positions of holo-ACP (lane 1), hexanoyl-ACP (C_6:0-ACP, lane 3), octacyl-ACP (C_8:0-ACP, lane 5), and decanoyl-ACP (C_10:0-ACP, lane 7) on gel are shown. The urea concentration was 2.5 mol/L.

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Second, the substrate specificity of Sco FabH2 to branched-chain acyl-CoAs was tested. When isobutyryl-CoA and isovaleryl-CoA were introduced into the fatty acid synthesis system, new products iso-C_6:0-ACP (Fig 3A, lane 4) and iso-C_7:0-ACP (Fig 3A, lane 5) were found in the reaction system, respectively. These results proved that Sco FabH2 can use branched-chain acyl-CoAs as primers to initiate fatty acid synthesis. This finding is consistent with BCFAs detected in the Sco fabH2 complementary strain RsJR2.

Third, the activity of Sco FabH2 to utilize medium straight-chain acyl-CoAs as substrate was tested. Sco FabH2 converted butyryl (C_4:0)-CoA to hexanoyl (C_6:0)-ACP (Fig 3B, lane 3) and condensed hexanoyl (C_6:0)-CoA and octanoyl (C_8:0)-CoA with malonyl-ACP to obtain two-carbon longer acyl-ACPs (Fig 3B, lanes 5 and 7). These results demonstrate that Sco FabH2 can use various acyl-CoAs, including short (C_2:0), medium straight-chain (C_4:0 -C_8:0), and branched-chain acyl-CoAs, as primers in fatty acid biosynthesis.

Sco fabH2 is not essential for growth

To further determine the physiological functions of Sco fabH2 in S. coelicolor, we disrupted Sco fabH2 in S. coelicolor M145 strain with an in-frame deletion. First, the upstream and downstream fragments of Sco fabH2 were cloned and ligated into pMD19-T. Then the gene-deletion cassette was inserted into pKC1139 to obtain pMJR-8, which was further introduced into the WT M145 strain by conjugal transfer from E. coli ET12567 (pUZ8002). The apramycin-resistant exconjugants were selected and confirmed by PCR. Apramycin-sensitive colonies were screened after apramycin-free culture for three rounds of sporulation (S2a Fig in S1 File). Then the Sco fabH2 deletion mutant strain ΔfabH2 was obtained and verified by PCR using the primers Sco fabH2-5 and Sco fabH2-6 and by sequencing the allelic gene in the ΔfabH2 strain. The expression of the neighboring genes SCO6565 and SCO6563 was confirmed using RT-qPCR (S2B Fig in S1 File). The results showed that the expressions of both genes were unaffected in the mutant. To construct a complementary strain, Sco fabH2 was cloned and ligated into the vector pJY813 to obtain pMJR-9, in which Sco fabH2 was expressed under a constitutive promoter kasOp* [2]. pMJR-9 was further transferred into the ΔfabH2 strain by conjugation. Then the complementary strain CfabH2 was selected on erythromycin- containing media and confirmed by PCR and sequencing.

The Sco fabH1 mutant, in which the chromosomal copy of fabH1 was replaced by E. coli fabH expressed in the plasmid, grew substantially slower than the WT strain, and a similar phenomenon was observed in the E. coli fabH deletion strain [32, 35]. We tested the growth of the different strains of S. coelicolor on YEB plates. The growth of the deletion mutant ΔfabH2 and complementary strain CfabH2 was comparable to that of the WT strain (Fig 4A), indicating that Sco fabH2 is nonessential for growth. The morphological development phenotype of different strains was observed on the plate with Gauze’s synthetic medium no. 1. The phenotype of ΔfabH2 was visibly similar to that of the WT strain after 8 d of incubation, whereas the complementary strain CfabH2 formed white spores and accumulated blue pigment at day 6, earlier than the other two strains, suggesting that fabH2 overexpression affected the morphological development of S. coelicolor (Fig 4B and 4C).

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Fig 4. Phenotype analysis of different S. coelicolor strains.

(a) Growth curves. Dry weight of mycelia (mg/plate) was quantified and used to represent the growth curve of different strains. Error bars indicate standard deviation of data obtained from three replicates. (b) Reverse side of the plates. (c) Front side of the plates.

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Sco fabH2 plays a minor role in fatty acid synthesis

Several reports have proved that fabH deletion or replacement changed fatty acid composition. Our results showed that Sco FabH2 actively initiates fatty acid synthesis. We hypothesize that Sco fabH2 plays roles in fatty acid synthesis in vivo. To this end, we analyzed the fatty acid profiles of different S. coelicolor strains with gas chromatography–mass spectrometry. The fatty acid profile of the WT strain included BCFAs, SCFAs, and UFAs, in which BCFAs showed two forms (iso-BCFAs and anteiso-BCFAs). In S. coelicolor, BCFAs (72.58% ± 3.57%) were the predominant components, including iso-BCFAs (35.5% ± 2.33%) and anteiso-BCFAs (37.08% ± 1.24%), and the ratio of iso-/anteiso-BCFAs was 0.96. The predominant SCFA was n-C_16:0 (14.73% ± 1.01%), and small amounts of UFAs (60.5% ± 113%), mainly n-C_17:1, were produced in S. coelicolor. The fatty acid composition of the Sco fabH2 deletion strain ΔfabH2 was similar to that of the WT strain, in which the percentage of BCFAs was 76.56% ± 5.80%, and the ratio of iso-/anteiso-BCFAs was 0.96. These results demonstrated that FabH2 is not important for fatty acid synthesis in S. coelicolor. The complementary strain CfabH2 synthesized the same fatty acids components with the other two, but the amounts of UFAs decreased, and the anteiso-BCFAs increased significantly to 42.21% ± 1.37%, resulting the ratio of iso-/anteiso-BCFAs declined to 0.85. All the results above indicated that fabH2 plays minor roles in modulating fatty acid profiles (S3 Table in S1 File).

Bacterial cell membrane homeostasis is greatly affected by fatty acid composition, which play biological roles in bacterial adaption to environmental stress [19]. Mutants defective in membrane integrity exhibit increased sensitivity to detergents [45]. To test the effects of fabH2 deletion on membrane integrity, we measured the stress response of different strains to various detergents. The results showed that ΔfabH2 grew well as the WT strain on plates with Tween-20 and Triton X-100 (S3a & S3b Fig in S1 File). By contrast, the complementary strain CfabH2 exhibited higher sensitivity to both detergents, indicating that fabH2 overexpression affected membrane homeostasis (S3a &S3b Fig in S1 File). Then different strains were further tested on the plates with vancomycin and ciprofloxacin, and the results also showed that CfabH2 exhibited more sensitive to the two antibiotics tested (S3c &S3d Fig in S1 File).

Sco fabH2 is not critical for ACT and RED production

S. coelicolor synthesizes two pigmented secondary metabolites as follows: ACT and RED [7]. To examine whether fabH2 is involved in ACT and RED production, we evaluated the amounts of ACT and RED produced by different strains. On YBP plates, ΔfabH2 showed similar phenotype as WT strain, and quantitative analysis revealed a similar trend of ACT production as WT (Fig 5). However, the complementary strain CfabH2 decreased ACT production. In this strain, intracellular ACT was slightly lower than that in WT and ΔfabH2, whereas extracellular ACT was 25%–30% of WT depending on culture time (Fig 5). These results suggested that fabH2 affects ACT production, especially under overexpression conditions. Similar results were found with RED analysis—CfabH2 partially decreased RED production, whereas no significant differences were observed between WT and ΔfabH2 (S4 Fig in S1 File).

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Fig 5. Analysis of ACT production by different strains.

(a) Growth of different strains on YEB plates. Photographs were taken from the reverse sides of the plates. (b) Intracellular ACT production. (c) Extracellular ACT production. Intracellular ACT was quantified using only the mycelia, and extracellular ACT was quantified using only the medium. Data are expressed from three independent repeats. ns, no significant difference; ***, P<0.001.

https://doi.org/10.1371/journal.pone.0318258.g005

Discussion

Streptomyces spp. is notable for producing numerous secondary metabolites, many of which have shown promise as antibacterial, antitumor, immunosuppressant, and antifungal agents [46]. Because secondary metabolism is closely related to primary metabolism, including synthesis of fatty acids, proteins, and nucleic acids, research with primary metabolisms will pave the path for the study and application of secondary metabolites. Thus, fatty acid synthesis is studied in detail in the model strain S. coelicolor, and the series of enzymes that catalyze each step in fatty acid synthesis have been reported by various groups [47–50].

3-Ketoacyl ACP synthase III (FabH) is the key enzyme in the initiation of fatty acid synthesis. Hopwood et al. first reported that S. coelicolor FabH is essential for fatty acid synthesis, because fabH could be deleted from the chromosome to yield a viable strain but only when a second copy of fabH was available to complement the deletion [25]. S. coelicolor produces a broad mixture of fatty acids, predominantly BCFAs, whereas the S. coelicolor fabH deletion mutant, harboring E. coli fabH expression on a plasmid, sharply decreased BCFA content to 14%. These studies have shown that S. coelicolor FabH has greater catalytic efficiency for branched primers. By contrast, E. coli FabH only uses straight-chain acetyl-CoA as substrate. How are the remaining 14% BCFAs synthesized in the replacement mutant? One possible explanation is the presence of alternative mechanism(s) for the initiation of fatty acid biosynthesis in S. coelicolor. To this end, we first identified three additional FabH homologs that contain the same catalytic triad (Cys–His–Asn) and similar three-dimensional structures as Sc FabH1. In addition to Sc fabH1, fabH2 (SCO6564) could restore the growth of R. solanacearum fabH deletion mutant under normal culture conditions, causing this strain to produce small amounts of BCFAs. This finding demonstrates that S. coelicolor FabH2 is an alternative enzyme that catalyzes the initiation of BCFAs synthesis. However, the fabH2 complementary strain produced less amounts of BCFAs than the strain complemented with fabH1, indicating that FabH2 is less active than FabH1. The fabH1 deletion caused lethality, further proving that FabH2 activity was insufficient to suppress the effect of fabH1 deletion.

The activities of FabH2 were further confirmed using in vitro assays, which demonstrated that FabH2 condensed different substrates, such as short-chain-, medium-chain-, and branched-chain acyl-CoAs, with malonyl-ACP to initiate fatty acid biosynthesis. Unfortunately, when we probed the substrate specificity of FabH2 by monitoring the rate of oxidation of NADPH at 340 nm, the activities of FabH2 to the primers were not detectable in vitro [26], which confirmed that the catalytic activity of FabH2 was relatively weak. The finding is consistent with the low content of BCFAs in the complementary strain.

The deletion of S. coelicolor fabH2 did not affect bacterial growth and produced large amounts of BCFAs, demonstrating that fabH2 is nonessential for growth and BCFAs synthesis. Then we attempted to construct a replacement mutant in which fabH1 was deleted in the chromosome by introducing a fabH2-encoded plasmid (pMJR-9). However, we failed and no mutant was selected. The most reasonable explanation is that the biological functions of fabH1 in fatty acid biosynthesis cannot be replaced by fabH2 because of its low catalytic activities, even when overexpressed. Another possibility is that FabH2 has biological function in secondary metabolism, e.g., the FabH homolog RedP plays a role in undecylprodiginine synthesis in S. coelicolor [8]. Therefore, we tested the production of ACT and RED in the fabH2 deletion mutant. We found no significant differences in ACT and RED production between the WT strain and the fabH2 deletion mutant, indicating that FabH2 is dispensable for ACT and RED synthesis. To our surprise, the complementary strain CfabH2 produced much less ACT than the WT and fabH2 deletion strain. The reason for this finding is unclear. We hypothesize that fabH2 overexpression, to some extent, caused minor changes inside S. coelicolor, which further affected ACT synthesis. Similar results were observed when testing membrane permeability. The CfabH2 strain became more sensitive to detergents and antibiotics, which may be caused by the variation in the ratio of iso-/anteiso-BCFAs, although the whole fatty acid profiles showed no significant changes.

Although the FAS II mechanism is relatively conserved in bacteria, variations in the initiation reactions have been identified in distinct bacteria—the KAS III pathway [24, 26] and the MAD pathway [31, 33]. Using the maximum-likelihood algorithm in the software MEGA 11 [51], a phylogenetic analysis of KAS III from different bacteria was constructed (Fig 6). The results showed that KAS III could be further classified into FabH type, PA3286 type, and FabY type, which differed in the types of substrates used and had structural differences [27, 28]. FabH type could be further classified into three groups. E. coli FabH, two B. subtilis FabHs (BSU11330 and BSU10170), Xcc FabH (XC3229), Xoo FabH (XOO0878), etc., were clustered in group 1, confirming that FabH type (group 1) was widely distributed in gram-positive and gram-negative bacteria. FabH type (group 2) seemed to be conserved in actinomycetes, of which S. coelicolor FabH1 (SCO2388) and S. glaucescens FabH (AAA99447) have been intensively studied. S. coelicolor FabH2 (SCO6564) belongs to FabH type (group 3), which is ubiquitous in Streptomyces spp. A comparative analysis of the S. coelicolor FabH2 sequence extended the presence of its homologs to S. lividans, S. ambofaciens, S. coralus, etc (S5 Fig in S1 File). The top 100 homologs had protein identities of 80.8% –99.7% in the Kyoto Encyclopedia of Genes and Genomes database.

Download:

Fig 6. Maximum-likelihood phylogenetic analysis of KAS III from different bacteria.

The maximum-likelihood tree was created using MEGA 11. Proteins are shown with their KEGG accession numbers clustered into five major groups. The most common FabHs reported clustered in group 1, S. coelicolor FabH1 (marked with delta) clustered in group 2, and S. coelicolor FabH2 (marked with diamond) clustered in group 3.

https://doi.org/10.1371/journal.pone.0318258.g006

Overall, S. coelicolor SCO6564 encodes an active KAS III, FabH2, which can catalyze the initiation of fatty acid synthesis with different substrates, but FabH2 cannot replace the conserved FabH1, because it has weak activity. S. coelicolor FabH2 represents a novel group of FabH type KAS III, which mainly presents in Streptomyces spp. and its biological functions need further study.

Supporting information

S1 File. Supplementary files table & figure.

https://doi.org/10.1371/journal.pone.0318258.s001

(ZIP)

S1 Raw images.

https://doi.org/10.1371/journal.pone.0318258.s002

(PDF)

Acknowledgments

We wish to thank and acknowledge Professor Hai-Hong Wang from South China Agriculture University for kindly providing the R. solanacearum strains.

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Figures

Abstract

Introduction

Materials and methods

Strains and culture conditions

Gene cloning and construction of expression plasmids

Complementation of the R. solanacearum fabH deletion strain RsmH

Assay of Sco FabH2 activity in vitro

In-frame chromosomal gene deletion and complementation

Analysis of fatty acid profile

Examination of ACT production

Statistical analyses

Results

Bioinformatic analysis with FabH homologs in S. coelicolor

Sco fabH2 could recover the growth of RsmH and produce BCFAs

Sco FabH2 is active to a broad range of substrates in in vitro analysis

Sco fabH2 is not essential for growth

Sco fabH2 plays a minor role in fatty acid synthesis

Sco fabH2 is not critical for ACT and RED production

Discussion

Supporting information

S1 File. Supplementary files table & figure.

S1 Raw images.

Acknowledgments

References