The strains and plasmids used in this study are described in Table 1. E. coli strains were grown either on solid or in liquid Luria–Bertani or M9 media at 37°C and supplemented when needed with the following antibiotics: 100 μg/ml ampicillin (Ap), 100 μg/ml apramycin (Am), 20 μg/ml chloramphenicol (Cm), or 50 μg/ml kanamycin (Kn). Streptomyces strains were grown at 30°C in rich liquid medium R5, or minimal SMM medium supplemented with glucose 0.1% (w/v) and casamino acids 0.2% (w/v); and on the rich solid medium MS (Kieser et al., 2000). For genomic DNA extraction, cells were grown on TSB medium. The antibiotics Am, hygromycin (Hyg), Kn, and spectinomycin (Spec) were added at final concentrations of 50, 100, 50, and 200 μg/ml, respectively, to solid medium.

E. coliΔfadBA strain was constructed by lambda-red mediated recombination as described previously (Datsenko and Wanner, 2000). fadBA_F and fadBA_R oligonucleotides (Table 2) were used to amplify the Kn resistance cassette of pKD13 plasmid. The Kn resistance cassette was deleted by FLP recombinase-mediated excision.

S. coelicolor SCOfadAB_2⁻,SCOfadAB_3⁻,SCOfadAB_1⁻SCOfadAB_2⁻,SCOfadAB_2⁻SCOfadAB_3⁻, and SCOfadAB_1⁻SCOfadAB_3⁻ knockout mutants were constructed using cosmids from the transposon mutant ordered cosmid library from S. coelicolor (Bishop et al., 2004; Herron et al., 2004; Fernández-Martínez et al., 2011). Cosmids 5F2A.1.G09 and 8A04.1.h08, carrying individual Tn5062 insertions in SCO6731 and SCO6788, respectively, were introduced into S. coelicolor M145 by conjugation using E. coli ET12567/pKOS111-47; yielding the strains SMB07 and SMB08. Three independent Am^R Km^S exconjugants were isolated for each event and checked by PCR, verifying that the allelic replacement had occurred. The SCO6731 gene disruption was analyzed with the following primer pairs: SCO6731_F and EZL2 (Herron et al., 2004), which hybridizes in the 5′ end region of SCO6731 and in Tn5062, respectively, and EZR1 and SCO6731_R, which hybridizes in Tn5062 and in the 3′ end region of SCO6731, respectively. The SCO6788 disruption was analyzed with the following primer pairs: SCO6788_F and EZL2, and EZR1 and SCO6788_R (Table 2).

For constructing the S. coelicolor SCO6731⁻SCO6788⁻ double mutant, SMB07 (Am^R) was utilized as the acceptor strain in the conjugation experiment. First, the Am resistance gene cassette of cosmid 8A04.1.h08 was replaced by the Hyg resistance gene cassette of pQM5066 by lambda-red mediated recombination. After conjugal transfer of cosmid 8A04.1.h08 (Hyg^R) to SMB07, three independent Am^R Hyg^R Km^S exconjugants were isolated and checked by PCR with the primer pairs described above for mutations in SCO6731 and SCO6788. S. coelicolor SM09 strain was constructed by introducing the pSM01 plasmid (Table 1) into S. coelicolor M145 by conjugation. Following a single recombination event, the plasmid is integrated into S. coelicolor chromosome, yielding Spec^R Thio^R exconjugants. Clones containing a second recombination event (Spec^R Thio^S) were isolated by replica plating. Three independent clones were checked by PCR using primers SCO6026_F, SCO6026_R, SCO6027_F, and SCO6027_R (Table 2).

All plasmids used in this work are described in Table 1. To construct plasmids pSM08, pSM09 and pSM10, genes SCO6027, SCO6026, SCO6731, SCO6732, SCO6788, and SCO6789 were amplified by PCR using the following pair of primers: SCO6731_F and SCO6731_R, SCO6732_F and SCO6732_R, SCO6788_F and SCO6788_R, SCO6789_F and SCO6789_R, SCO6027_F and SCO6027_R, SCO6026_F and SCO6026_R (Table 2). PCR products were cloned in pCR-Blunt II-TOPO (Invitrogen) and submitted for DNA sequencing (University of Maine DNA sequencing facility, USA). Each individual gene was then cloned as an NdeI/EcoRI restriction fragment into pET28a yielding plasmids pSM02, pSM03, pSM04, pSM05, pSM06, and pSM07, respectively. Plasmids pSM03, pSM05, and pSM07 where then digested with XbaI/HindIII to obtain fragments harboring corresponding genes and RBS from vector. These fragments were then XbaI/HindIII cloned into pSM02, pSM04, and pSM06 vectors, respectively, yielding pSM08, pSM09, and pSM10 plasmids, which carry SCO6731-SCO6732, SCO6788-SCO6789, and SCO6027-SCO6026 operons, respectively, under the control of a T7 promoter.

For pSM01 construction, 2.5 kb regions located upstream SCO6026 (Ups_6026 fragment) and downstream SCO6027 (Dns_6027 fragment) were amplified by PCR using S. coelicolor genomic DNA and primers Ups_6026_F, Ups_6026_R, Dns_6027_F, and Dns_6027_R, respectively (Table 2). Spec resistance gene cassette was amplified from pIJ699 using primers Spec_F and Spec_R. PCR products where cloned in pCR-Blunt II-TOPO and checked by DNA sequencing. Spec cassette was then obtained by BamHI digestion, and Ups_6026 and Dns_6027 fragments were obtained by HindIII/BamHI and BamHI/EcoRI digestions, respectively. The three fragments were cloned into pSET151, yielding plasmid pSM01.

SDS-PAGE and Western Blot were carried out using standard protocols (Sambrok and Fritsch, 1989). For detection of His-tagged proteins, mouse monoclonal anti-His antibodies (QIAGEN™) were used as primary antibody and anti-mouse IgG-alkaline phosphatase conjugates were used as secondary antibody. His-tagged proteins were visualized by immunoblots using chromogenic detection as described by the manufacturer.

Competent E. coli ΔfadBA cells were transformed with plasmids pSM08, pSM09, or pSM10. The three derivatives strains were grown in M9 0.1% w/v glucose to check protein expression by Western Blot.

In order to assay the ability of the complemented strains to grow on solid and liquid M9 supplemented with lauric or oleic acid as sole carbon source, cells were cultured overnight at 37°C in liquid LB containing Kn, then diluted 1:100 in M9 medium supplemented with 0.1% w/v glucose and incubated 7 h at 25°C. At this point, samples were taken for plating on solid medium experiments (see below), and cells were harvested by centrifugation for 15 min at 4,000 × g, washed and diluted to OD₆₀₀ 0.01 in fresh M9 supplemented with 0.1% w/v oleic acid or 0.1% w/v lauric acid. Cultures were incubated at 25°C and OD₆₀₀measurements were carried out at different time points to evaluate growth. This experiment was performed in triplicate.

To evaluate growth on solid medium, aliquots of 3 μl OD₆₀₀ 2.5 were taken from liquid cultures, as described above, plated on M9 supplemented with 0.1% w/v oleic acid or 0.1% w/v lauric acid and incubated at 25°C. E. coli BL21 and E. coli BL21ΔfadBA transformed with pET28a vector were used as controls.

To evaluate the expression profile of the SCOfadBA genes in liquid media, plasmids pSM14, pSM15, and pSM16 were constructed. These vectors are integrative in Streptomyces and contain the xylTE genes under the transcriptional control of the SCO6730, SCO6785, and SCO6027 promoter region, respectively (Table 1). The putative promoter regions were amplified by PCR using S. coelicolor genomic DNA as template and primers P_SCO6730_F, P_SCO6730_R, P_SCO6785_F, P_SCO6785_R, P_SCO6027_F, and P_SCO6027_R (Table 2). Each primer pair was designed to yield a 223 bp amplicon that extends from −210 to +13 relative to the start codon of each gene. PCR products were cloned in a pCR-TOPO-Blunt vector and the integrity of each sequence was confirmed by DNA sequencing. Promoters DNA fragments were obtained by restriction with BamHI/EcoRI and cloned into the pCA75 vector; yielding plasmids pSM14, pSM15, and pSM16. These intergenic regions were selected for amplification based on in silico analysis and on previous transcription start site (TSS) mapping (Jeong et al., 2016).

Expression patterns were also analyzed in solid medium; to this purpose pSM17, pSM18, and pSM19 plasmids were constructed. In this case, PCR fragments corresponding to the promoter regions under study were cloned into the pCA73 vector generating cbg transcriptional fusions (Table 1).

Finally, plasmid containing each reporter fusion was introduced into S. coelicolor M145 by conjugation using E. coli ET12567/pKOS111-47 as donor, yielding the reporter strains SMB01, SMB02, SMB03, SMB04, SMB05, and SMB06 (Table 1).

Strains SMB01, SMB02, and SMB03 (Table 1) were cultured in SMM minimal medium supplemented with different carbon sources (1% (w/v) glucose, 1% (w/v) glucose plus 0.1% (w/v) oleic acid or 0.1% (w/v) oleic acid) at 30°C for 15 and 40 h. Cells were then harvested by centrifugation at 5,000 × g during 5 min, washed with a buffer solution containing 50 mM Tris-HCl pH 8 and stored at −20°C for further processing. Catechol 2,3-dioxygenase activity was then measured as indicated in (Kieser et al., 2000). Protein concentration was determined with Bradford reagent (Bradford, 1976). Catechol 2,3-dioxygenase activities were normalized to protein concentration.

Strains SMB04, SMB05, and SMB06 (Table 1) were cultivated in solid SMM medium supplemented with luciferin and different carbon sources. This assay was carried out in a white opaque 96-well plate. Each well was filled with 180 μl solid SMM medium supplemented with 10 mM luciferin (Sigma Aldrich) and the corresponding carbon source: 0.2% (w/v) glucose, 0.2% (w/v) glucose plus 0.2% (w/v) oleic acid, or 0.2% (w/v) oleic acid. Each well was inoculated whit 3,000 spores of each strain. Six replicates were made for each strain in every condition. Plates were then incubated at 30°C during 70 h in a Synergy® spectrophotometer, programmed to measure luminescence emission every 30 min.

The luminescence intensity registered in every well-depends on two factors: (1) the activity of the promoter fused to the cbg gene in a particular medium; and (2) the amount of cells present in the well at the time of the measurement; more cells will produce a larger emission. Since Streptomyces shows different growth rates according to the carbon source present in the medium, the data obtained from this experiment were normalized using a P_hrdB (hrdB promoter)-cbg transcriptional fusion. For this purpose, a S. coelicolor strain carrying a transcriptional fusion to the hrdB promoter was grown under the same conditions as the reporter strains and luminescence measurements were made at the same time points.

For the continuous fermentations of S. coelicolor the wild type strain M145 was used. The inoculum was prepared following a two-step procedure. A 20% (w/v) glycerol spore aliquot prepared as described above was used to inoculate a 500 ml Erlenmeyer flask containing 100 ml of GG1 medium (1.5% (w/v) soy peptone; 0.3% (w/v) NaCl; 0.1% (w/v) CaCO₃; 1.5% (w/v) glucose; 1.2% (v/v) glycerol) to reach an initial concentration of 10⁶ spores/ml. Dispersed growth was achieved by the use of a magnetic stirrer and a triangular magnetic bar. After incubation at 30°C for 48 h, 10 ml of the culture were transferred to a 500 ml Erlenmeyer containing 100 ml of GYB medium (3.3% (w/v) glucose; 1.5% (w/v) yeast extract) and incubated at 30°C for 24 h. Subsequently, a 1.5 l bioreactor (Adaptive Biosystems 7000, UK) containing 950 ml of Modified Evans medium (0.62% (w/v) NaH₂PO₄.2H₂O; 0.075% (w/v) KCl; 0.025% (w/v) MgCl₂.6H₂O; 0.028% (w/v) (NH₄)₂SO₄; 0.0037% (w/v) CaCl₂.2H₂O; 0.038% (w/v) citric acid; 0.02% (v/v) Breox antifoam; 0.133% (w/v) NaOH; 0.1% (v/v) trace elements) with an initial glucose concentration of 9 g/L was inoculated with 50 ml of the GYB inoculum. An initial batch phase of ~24 h was allowed to elapse until the initial glucose was completely depleted. At that time, fresh medium was fed to maintain a constant dilution rate of 0.039 h⁻¹. The culture was kept at a constant volume of 1 L. Foaming was prevented by manually addition of a suspension of 10% (v/v) antifoam agent (BDH Laboratory Supplies, UK). The pH was monitored and controlled at 6.8 ± 0.2 by the automatic addition of 2 N NaOH or 2 N HCl. The temperature was kept at 30°C, the stirring speed was set at 1,000 rpm and the air flow rate at 1.25 l/min. The cells grew in dispersed form (as assessed by microscopic observation) throughout the fermentation period. During the process, the concentrations of biomass, glucose, actinorhodin, NH4+, PO43-, and pyruvate in the supernatant were measured, as well as the content of total lipids and TAG in the biomass, and the O₂ and CO₂ content in the outcoming gases. From those parameters, consumption, and consumption rates were calculated. Based on the values, it was determined that steady state was reached after seven volume changes in the bioreactor. The total duration of these fermentations was 180 h.

The analysis of metabolic models through FBA was performed using the COBRA Toolbox (Schellenberger et al., 2011) framework (opencobra.github.io) operating in the MATLAB R2014b software environment (mathworks.com/products/matlab/). All calculations were carried out on a personal computer with OSX operating system El Capitan v.10.11.2 with an Intel® Core i5 2.70 GHz 8 GB 1867 MHz DDR3 processor.

Total lipids of the S. coelicolor strains were extracted twice from lyophilized cell material (2 mg) with chloroform/methanol (2:1 v/v). The combined extracts were evaporated and analyzed by TLC on silica gel 60 F254 plates (0 ± 2 mm, Merck) as described previously (Wältermann et al., 2000), using the solvent mixture hexane/diethylether/acetic acid (80:20:1, v/v/v). Lipid fractions were visualized by Cu-phosphoric staining. For radioactively labeled samples, lipids were visualized by autoradiography using a Storm 840 PhosphorImager (Amersham Biosciences®). For densitometry analysis, images where digitalized and analyzed using the software ImageJ v1.48.

S. coelicolor M145, SMB07, SMB08, and SMB09 strains were cultured in minimal SMM medium supplemented with 0.2% (w/v) glucose at 30°C. After 15, 40, and 90 h of incubation, 3 ml samples were taken from each culture and incubated during 1 h with 2 μCi [¹⁴C]-acetate. Cells were then harvested by centrifugation at 5,000 × g for 10 min at 4°C and washed three times with a buffer containing Tris-HCl 50 mM pH 8.0. Total lipids were then extracted following the standard protocol (Folch et al., 1957), resolved by TLC and visualized by autoradiography as previously described.

S. coelicolor M145, SMB07, SMB08, and SMB09 strains were cultured in minimal SMM medium supplemented with 0.2% (w/v) glucose at 30°C. After 10 h, 2 μCi [¹⁴C]-acetate was added and cultures were further incubated for 10 h. Cells were then harvested by centrifugation at 5,000 × g for 10 min at 4°C and washed three times with a buffer containing Tris-HCl 50 mM pH 8.0. Cells were resuspended in fresh SMM medium without a carbon source and incubated at 30°C during 70 h. Samples of each culture were harvested at different times by centrifugation at 5,000 × g for 10 min at 4°C and cells washed three times with a buffer containing Tris-HCl 50 mM pH 8.0. Total lipids were then extracted and processed as described above.

For quantification of triacylglycerides, the biomass was lyophilized and weighed, and 1 mg was processed for total lipid extraction as described in Bligh and Dyer (1959). Ten micrograms of cetylpalmitate was added before extraction as an internal standard for load control. Then 500 μL of chloroform were added to solubilize lipids and 50 μL of the lipid extract were diluted in an equal part of methanol. Five microliters of this solution were injected and separated on a ZORBAX Eclipse XDB-C8 column (3.0 × 50 mm, particle size = 1.8 μm; Agilent, USA) using methanol supplemented with 5 mM ammonium acetate as the mobile phase. The outlet of the liquid chromatograph was connected to a micrOTOF mass spectrometer (Bruker Daltonik, Bremen, Germany) operating in the positive-ion mode, and the data was acquired online in the mass range m/z 300–1,500. TAG were detected as ammonium adducts in the range of 6–20 min of the chromatography run. A calibration curve was done using pure tripalmitin as a standard. The TAG concentration in the samples was calculated by the linear regression equation obtained from the calibration curve.

Fatty acid analysis of TAG inclusions was done by preparing FA methyl esters by transesterification of isolated lipid bodies with 0.5 M sodium methoxide in methanol and analysis with a Perkin-Elmer Turbo Mass gas chromatograph-mass spectrometer on a capillary column (30 m by 0.25 mm internal diameter) of 100% of dimethylpolysiloxane (PE-1; Perkin-Elmer, Waltham, MA). Helium at 1 ml min⁻¹ was used as the carrier gas, and the column was programmed at 4°C min⁻¹ from 140 to 240°C. Branched-chain FAs, straight-chain FAs, and unsaturated FAs used as reference compounds were obtained from Sigma-Aldrich (Sigma Aldrich, St Louis, MO).

In order to identify β-oxidation coding genes in S. coelicolor, we carried out a homology search using as query the amino acid sequences of the fadA, fadB, and fadE gene products from E. coli. Our search showed that the S. coelicolor genome contains 9 ORF homologs to FadA (ID 948324), 3 ORF homologs to FadB (ID 948336), and 14 ORF exhibiting moderate homology to FadE (ID 949007). Each of the three putative fadB genes (SCO6026, SCO6732, and SCO6789) were located adjacent to a putative fadA (SCO6027, SCO6731, and SCO6788), suggesting possible co-transcriptional units (Figure 1A). Only these three gene clusters -SCO6027-SCO6026, SCO6731-SCO6732, and SCO6788-SCO6789- possess orthologs and exhibit a notable synteny with other sequenced Streptomyces species, such as S. scabiei, S. avermitilis, S. flavogriseus, S. griseus, and S. cattleya. Interestingly, proteins encoded by clusters SCO6731-SCO6732 and SCO6788-SCO6789 exhibit percentages of identity above 85% and similarity above 90%, which might suggest that one of them resulted from a genetic duplication event. Also, these two clusters are surrounded by putative FA metabolism-related genes (a feature not observed for cluster SCO6027-SCO6026) (Figure 1A).

On the other hand, the large redundancy in fadE genes difficult the assignment of possible candidates involved in FA catabolism.

All FadA candidates show the conserved thiolase domain (cd00751) as well as the three conserved active site residues that define this activity (Figure 1B). Similarly, all three possible FadB exhibit the same so-called E. coli-like structure of 3-hydroxyacyl-CoA dehydrogenases (Figure 1B; Volodina and Steinbüchel, 2014). This organization consists of an N-terminal crotonase-like family/enoyl-CoA hydratase domain (cd06558/cl23717), a central 3-hydroxyacyl-CoA dehydrogenase-NAD binding domain (3HCDH_N, Pfam 02737) and one or two 3-hydroxyacyl-CoA dehydrogenase domains (3HCDH, Pfam 00725) located at the C-terminal portion of the protein (Figure 1B).

Neighbor-joining phylogenetic trees based on amino acid sequence of FadB enzymes from different organisms, group SCO6732 and SCO6789 together with FadB from the actinomycetes Mycobacterium tuberculosis (64 and 63% identity, respectively; Srivastava et al., 2015) and Rhodococcus equi (64 and 61% identity, respectively; Kelly et al., 2002; Figure 1C). In contrast, SCO6026 is placed near to FadB sequences from Gram (−) bacteria: E. coli FadJ (30% identity), Pseudomonas aeruginosa FadB (30% identity), Enterobacter cloacae FadB (28% identity), E. coli FadB (28% identity), and Salmonella enterica serovar Typhimurium FadB (28% identity). On the other hand, Bacillus subtilis FadB (Matsuoka et al., 2007), Ralstonia eutropha FadB' (Volodina and Steinbüchel, 2014), and Staphylococcus aureus FadB (Khairon et al., 2016) group together and, unlike the other FadB considered, exhibit a Burkholderia-like domain organization, which consist in an inverted order of domains compared with E. coli [3-hydroxyacyl-CoA dehydrogenases domains located at the N-terminus Volodina and Steinbüchel, 2014].

Overall, the in silico analysis suggested that the three putative fadAB homologues identified in S. coelicolor were probable candidates to code for β-oxidation enzymes. Gene clusters SCO6027-SCO6026, SCO6731-SCO6732, and SCO6788-SCO6789 are referred hereafter as SCOfadAB_1, SCOfadAB_2, and SCOfadAB_3, respectively.

In E. coli, fadBA gene products are required for aerobic growth on FA as sole carbon sources (Yang et al., 1988; Campbell et al., 2003). Therefore, to study in vivo the physiological role of the identified SCOfadAB clusters, we carried out E. coli genetic complementation tests. For this, we first constructed an E. coli ΔfadBA mutant and then individual SCOfadAB clusters under the T7 promoter were introduced in the resulting FA auxotrophic strain (E. coli ΔfadBA, Table 1). Western blot analysis showed that all SCOfadAB gene products form inclusion bodies in the corresponding E. coli recombinant strains; however, the expression that leaks from the T7 promoter is sufficient to obtain detectable quantities of soluble protein (Figure 2A). Therefore, complementation tests were performed at 25°C with no addition of the inducing agent isopropyl b-D-1-thiogalactopyranoside (IPTG).

Expression of the three SCOfadAB clusters rescued growth of E. coli ΔfadBA on M9 plates containing lauric acid (C_12:0) as sole carbon source after 7 days (Figure 2B). In this medium, the three complemented strains displayed the same growth behavior as E. coli BL21; whereas E. coli ΔfadBA transformed with an empty plasmid showed no growth. In liquid M9 medium supplemented with 0.1% (w/v) lauric acid as sole carbon source, all three complemented strains reached final OD₆₀₀-values very similar to that of the BL21 (Figure 2C), confirming the successful complementation of the E. coli ΔfadBA mutant with the three different putative FadAB enzymes from S. coelicolor.

When the complementation test was carried out in the presence of oleic acid (C_18:1) as sole carbon source, the three complemented strains showed different growth phenotypes. In M9 agar containing 0.1% (w/v) of C_18:1, E. coli BL21 showed visible growth after incubation for 5 days, whereas E. coli ΔfadBA/SCOfadAB_1 exhibited significant growth after 9 days. E. coli ΔfadBA/SCOfadAB_2 and E. coli ΔfadBA/SCOfadAB_3 only displayed small colonies after 11 days (Figure 2B). Similar results were observed when the complementation test was carried out in liquid M9 with oleic acid as sole carbon source. In this case, all three complemented strains were able to grow, albeit at very different growth rate and reaching reduced final OD₆₀₀ values. As shown in Figure 2B, E. coli BL21 reached a final OD₆₀₀ of 3.21 ± 0.11 after incubation for 3 days, whereas complemented strains reached lower OD₆₀₀ after 6 days. E. coli ΔfadBA transformed with an empty plasmid did not show any growth at these conditions.

Altogether, these results indicate that SCOfadAB gene products can display catalytic activities attributable to E. coli FadBA complex when expressed in the E. coli ΔfadBA mutant, namely: enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase (Yang et al., 1988; Srivastava et al., 2015). Furthermore, the complementation studies also suggest that the other β-oxidation enzymes (FadL, FadD, and FadE) present in E. coli can functionally interact with SCOfadAB gene products for exogenous FA assimilation.

S. coelicolor possesses a complex life cycle that involves different morphological stages which are dependent on growth conditions and media composition. The morphological differentiation that occur in solid medium can lead to gene expression patterns which may differ from those taking place in liquid medium (Yagüe et al., 2013, 2014). Thus, in order to assess the expression patterns in both types of media, we constructed S. coelicolor M145 derivative strains containing ectopic integrations of cbg and xylTE transcriptional fusions (Table 1).

The luciferase encoded by cbg gene generates visible light during luciferin oxidation. Strains of S. coelicolor carrying transcriptional fusions of the three different SCOfadAB promoters to cbg were grown in solid SMM medium supplemented with glucose, oleic acid or glucose plus oleic acid as carbon sources and luciferin for light emission. Cultures were incubated at 30°C for 70 h and the expression profiles were normalized using as reference the luminescence emission of cbg transcriptional fusion to hrdB promoter. The hrdB product is a vegetative σ factor whose expression is constitutive and independent of the carbon source (Buttner et al., 1990; Shiina et al., 1991; Marcos et al., 1995). Under these conditions, expression from SCOfadAB_1 and SCOfadAB_3 promoters was markedly enhanced when oleic acid was the sole carbon source, although at different growth times (Figure 3A). SCOfadAB_1 promoter exhibited maximum luminescence emission after 55 h (late culture times), whereas SCOfadAB_3 did it until 50 h (early culture times). Also, both promoters led to constitutive cbg expression when glucose was present in the medium. In contrast, SCOfadAB_2 promoter showed maximum expression at 55 h when glucose was the sole carbon source, while its expression was constitutive, although at considerable lower levels, when oleic acid was present in the medium (Figure 3A).

To evaluate the expression patterns of the SCOfadAB promoters in liquid media, the strains containing each of the three xylTE transcriptional fusions [Pseudomonas putida genes coding for catechol 2,3-dioxygenase (CDO; González-Cerón et al., 2001] were grown in SMM medium supplemented with glucose, oleic acid, or glucose plus oleic acid as carbon sources and the CDO activity measured in cell free extracts from samples taken at 15 and 40 h (exponential and late-stationary phases of growth, respectively). The expression from SCOfadAB_1 and SCOfadAB_3 promoters was detectable only in the presence of oleic acid (Figure 3B). In agreement with the results obtained in solid media, expression from SCOfadAB_1 promoter was higher at late stages of growth (stationary phase), whereas expression from SCOfadAB_3 promoter was higher at early stages of growth (exponential phase). In contrast, no CDO activity was detected from the SCOfadAB_2 promoter in any of the media tested.

Taken together, the expression profiles obtained with the transcriptional fusions studies strongly suggest that SCOfadAB_1 and SCOfadAB_3 might have a role in exogenous FA assimilation at different growth stages. These results also indicate that the three clusters are differentially expressed in S. coelicolor depending on growth stage and media composition.

To assess the role of β-oxidation genes in TAG accumulation and to identify other mutations that could lead to an increased TAG pool, we performed FBA using a S. coelicolor genome-scale metabolic network.

Recently, a genome-scale metabolic model named iMK1208 was published for S. coelicolor A3(2) (Kim et al., 2014). Although it is a high-quality metabolic reconstruction, TAG biosynthesis in the model is restricted to FAS intermediates as it only considers TAG production from DAG and acyl-ACP (Kim et al., 2014). However, the three principal WS/DGAT involved in TAG biosynthesis in S. coelicolor use long-chain acyl-CoA as acyl donors (Arabolaza et al., 2008). Therefore, we introduced reactions in iMK1208 in which TAG is biosynthesized from DAG and acyl-CoA without modification of the biomass equation. This version of iMK1208 is referred to as m_iMK1208 (modified_iMK1208, Supplementary Material).

In order to obtain experimental values to constrain in silico simulations, we carried out chemostat cultures of S. coelicolor wild type strain at a dilution rate of 0.039 h⁻¹. To validate the modified model, we performed FBA simulations with both iMK1208 and m_iMK1208 models. Using as constraints the experimental values obtained in the chemostat experiments, we predicted growth rate values of 0.042 h⁻¹; which demonstrated the validity and consistency of the modified metabolic model m_iMK1208. We then used the computational OptKnock framework (Burgard et al., 2003) in order to predict possible mutations leading to an increase in carbon fluxes toward TAG biosynthesis. OptKnock was performed with both models with the output set to obtain 10 candidate reactions (Table 3). Simulations using iMK1208 identified the inactivation of reactions involved in actinorhodin biosynthesis (ACTS18), phospholipid precursors biosynthesis (ACPPAT160, AGPATi140, AGPATi160, APG3PATai150, and CLPNS180), degradation of Thr to Gly (GLYAT and THRD) and glycolysis (FBA and PFK). On the other hand, simulations based on m_iMK1208 identified the inactivation of reactions involved in actinorhodin biosynthesis (ACTS18), acyl-CoA degradation (FACOAE80), synthesis of phospholipid precursors (ACPPAT181, APH120, APH141, and GPDDA3e), and notably, β-oxidation of FA [ACACT2r (FadA), ACACT8r (FadA), ACOAD6f (FadE), and ECOAH8 (FadB)]. This output further supports m_iMK1208 as a consistent model for predictions related to neutral lipid metabolism and validated the construction of knockout mutant strains for the identified SCOfadAB gene clusters.

To assess the physiological consequences of inactivating SCOfadAB gene clusters, we constructed single and double mutant strains, in which one or two of the clusters were knocked out (See Table 1).

All single and double mutants, and the wild type S. coelicolor M145 strain, were able to grow on minimal medium supplemented with FA of different chain length as sole carbon sources. Similarly, all six mutants displayed no differences with M145 in terms of growth rate, morphological differentiation and antibiotic production in rich medium.

However, all mutant strains exhibited a notable increase in TAG accumulation compared with the wild type strain M145 when grown until late stationary phase in both rich and minimal media. The lipid composition of S. coelicolor wild type and single SCOfadAB knockout mutants at 40 and 90 h was determined by TLC. In rich medium, the single mutants SCOfadAB_1⁻ and SCOfadAB_3⁻ showed an increased amount of accumulated TAG of 2.9 and 3.5-fold, respectively, at late stationary phase (90 h), compared to the M145 parental strain (Figure 4A). The single mutant SCOfadAB_2⁻ showed a small difference in TAG accumulation compared with M145 (1.3-fold increase, Figures 4A,B). Similar results were observed when these strains were grown in minimal medium, which is shown in Supplementary Figure 1.

Accordingly, TAG accumulation in the double mutant SCOfadAB_2⁻SCOfadAB_3⁻ was similar to that observed for single mutant SCOfadAB_3⁻. However, the double mutant SCOfadAB_1⁻SCOfadAB_2⁻ showed a TAG content 3.8-fold higher than M145, which represents an increase higher than that observed for each corresponding single mutant. The same was observed for the double mutant SCOfadAB_1⁻SCOfadAB_3⁻, which exhibited TAG levels 4.3-fold higher than the M145 strain (Figures 4A,B). In these conditions (90 h in rich medium), M145 accumulates 11.2 ± 0.02 μg TAG/mg DCW, equivalent to 1.1% of the dry cell weight; whereas double mutant SCOfadAB_1⁻SCOfadAB_3⁻ accumulates 48.12 ± 0.03 μg TAG/mg DCW, equivalent to 4.8% of the dry cell weight. The FA composition of the TAG accumulated for each of these two strains is shown in Supplementary Figure 4.

The highest levels of TAG found in the double mutant strains SCOfadAB_1⁻SCOfadAB_2⁻ and SCOfadAB_1⁻SCOfadAB_3⁻ suggest that all three clusters could be involved in the degradation of FA derived from TAG mobilization. In order to confirm this hypothesis, we carried out [¹⁴C]-acetate incorporation assays and pulse chase experiments. [¹⁴C]-acetate incorporation occurs to the same extent in M145, single, and double mutant strains at both exponential and late stationary phase (data not shown). These results suggest that the increased TAG content in the mutant strains is not due to an increased TAG biosynthesis rate.

Pulse and chase experiments consisted in monitoring a [¹⁴C]-labeled pool of TAG after switching cultures to a minimal medium with no carbon source, conditions that are favorable for TAG mobilization (Figure 4C). Strain M145 showed a typical decrease in TAG content after the culture reached late stationary phase. In contrast, all three double mutant strains exhibited decreased TAG mobilization, although to different extents. The relative TAG and FFA content of the wild type and double mutant strains analyzed in the pulse-chase experiment shown in Figure 4C is presented in Supplementary Figure 2.

The double mutant SCOfadAB_2⁻SCOfadAB_3⁻ showed TAG mobilization close to that observed in the wild type; however, a significant content of TAG is still observed after 70 h in the absence of carbon source, in contrast to M145, which showed a completely depleted pool at this time. The double mutant SCOfadAB_1⁻SCOfadAB_2⁻ mobilized TAG to a lesser extent than the double mutant SCOfadAB_2⁻SCOfadAB_3⁻ and displays a significant TAG content after 95 h of culture in absence of carbon source. Remarkably, the strongest TAG over-accumulating phenotype is exhibited by double mutant SCOfadAB_1⁻SCOfadAB_3⁻, which showed a significant TAG content until 95 h of culture in the absence of carbon source. This is in agreement with the total lipid content observed in rich medium (Figure 4A) and with the pulse and chase experiments carried out with single mutant strains (Supplementary Figure 3). Overall, these findings strongly suggest that all three identified SCOfadAB gene clusters are involved in FA β-oxidation in S. coelicolor.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.