Abstract
We analysed the ability of five different rhodococcal species to grow and produce triacylglycerols (TAGs) from glycerol, the main byproduct of biodiesel production. Rhodococcus fascians and Rhodococcus erythropolis grew fast on glycerol, whereas Rhodococcus opacus and Rhodococcus jostii exhibited a prolonged lag phase of several days before growing. Rhodococcus equi only exhibited poor growth on glycerol. R. erythropolis DSMZ 43060 and R. fascians F7 produced 3.9-4.3 g cell biomass l 21 and 28.4-44.6 % cellular dry weight (CDW) of TAGs after 6 days of incubation; whereas R. opacus PD630 and R. jostii RHA1 produced 2.5-3.8 g cell biomass l 21 and 28.3-38.4 % CDW of TAGs after 17 days of growth on glycerol. Genomic analyses revealed two different sets of genes for glycerol uptake and degradation (here named clusters 1 and 2) amongst rhodococci. Those species that possessed cluster 1 (glpFK1D1) (R. fascians and R. erythropolis) exhibited fast growth and lipid accumulation, whereas those that possessed cluster 2 (glpK2D2) (R. opacus, R. jostii and R. equi) exhibited delayed growth and lipid accumulation during cultivation on glycerol. Three glycerol-negative strains were complemented for their ability to grow and produce TAGs by heterologous expression of glpK2 from R. opacus PD630. In addition, we significantly reduced the extension of the lag phase and improved glycerol assimilation and oil production of R. opacus PD630 when expressing glpK1D1 from R. fascians. The results demonstrated that rhodococci are a flexible and amenable biological system for further biotechnological applications based on the reutilization of glycerol.
INTRODUCTION
Biofuels, such as biodiesel and bioethanol, are renewable and environmentally friendly alternative products used for substituting fossil fuels. Biodiesel is produced by transesterification of vegetable oils, animal fats or microbial oils (single-cell oils) with alcohol, in which glycerol is the main byproduct, representing 10 % by weight (Papanikolaou & Aggelis, 2002; da Silva et al., 2009; Easterling et al., 2009 ). Although glycerol is used for different applications, such as in emulsifiers, softening agents, stabilizers and wetting agents for the food industry, and in the pharmaceutical and cosmetic industries, the increasing biodiesel production in the world is causing an excess of this waste material which exceeds their market (da Silva et al., 2009; Galan et al., 2009) . For this reason, glycerol can be considered an abundant and cheap raw material that could be utilized as a substrate for single-cell oil production by oleaginous micro-organisms. The production of triacylglycerols (TAGs) from glycerol has been extensively investigated in oleaginous yeasts and fungi (Yen et al., 2012; Chatzifragkou et al., 2011; Xu et al., 2012) , but not yet in oil-accumulating bacteria. Amongst these, some bacteria belonging to the genus Rhodococcus are specialists in the accumulation of TAGs, producing significant amounts of those lipids under nitrogen-limiting conditions (Alvarez et al., 1996; Alvarez & Steinbüchel, 2010) . In particular, Rhodococcus opacus PD630 and Rhodococcus jostii RHA1 are well-known oleaginous bacteria with the ability to produce large amounts of TAGs [w20 % cellular dry weight (CDW)] from different carbon sources, including single substrates, and agro-industrial wastes, such as sugar beet molasses, orange wastes and whey (Alvarez et al., 1996; Voss & Steinbüchel, 2001; Alvarez & Steinbüchel, 2002; Gouda et al., 2008; Alvarez et al., 2013; . Other rhodococcal species, such as Rhodococcus fascians, Rhodococcus erythropolis and Rhodococcus equi, generally produced lower amounts of TAGs compared with R. opacus and R. jostii during cultivation on sugars, organic acids or hydrocarbons (Alvarez et al., 1997; Alvarez, 2003) . In spite of the importance of glycerol as a potential renewable source for lipid production by rhodococci, the genetic and physiological ability of these micro-organisms to utilize this substrate has been poorly explored. Recently, Kurosawa et al. (2015) studied the capability of R. opacus PD630 for growing on glycerol to evaluate the use of this substrate for the production of lipid-derived materials. The authors reported that the cell growth of strain PD630 was extremely poor in glycerol; thus, they employed an adaptive evolution approach to improve growth. The evolved R. opacus strain (MITGM-173) showed improved glycerol utilization in comparison with the parental strain (Kurosawa et al., 2015) . In another study, Ciapina et al. (2006) reported the biosurfactant production by a R. erythropolis strain grown on glycerol as the sole carbon source. Understanding fundamental aspects of glycerol-assimilating mechanisms in rhodococci will enable the design of efficient bacterial biofactories for high yields of oil production from this substrate. Glycerol metabolism has not yet been investigated thoroughly within the genus Rhodococcus, although this process has been extensively studied in other bacteria. Glycerol can cross the cytoplasmic membrane through passive diffusion or by a specific glycerol transporter protein (GlpF), facilitating the uptake especially at low substrate concentration (Richey & Lin, 1972; Voegele et al., 1993) . The respiratory pathway of glycerol dissimilation involves a glycerol kinase (GlpK) enzyme, which phosphorylates glycerol to glycerol 3-phosphate (G3P), and a G3P dehydrogenase aerobic (GlpD) or anaerobic (GlpABC) enzyme oxidizes G3P to dihydroxyacetone phosphate (DHAP) as an intermediate in the glycolysis. This pathway for glycerol catabolism has been reported in Escherichia coli (Lin, 1976) , Pseudomonas aeruginosa (Schweizer & Po, 1996) , Pseudomonas putida (Nikel et al., 2014) , Klebsiella pneumoniae (Forage & Lin, 1982) , Bacillus subtilis (Mindich, 1968; Holmberg et al. 1990) , Streptomyces clavuligerus (Baños et al., 2009) and Mycobacterium smegmatis (Titgemeyer et al., 2007) , amongst others. In several bacteria, such as E. coli (Dharmadi et al. 2006) or K. pneumoniae (Németh et al., 2003) , glycerol can be further metabolized by a fermentative pathway, in which glycerol is oxidized by a glycerol dehydrogenase (GldA) to dihydroxyacetone, which is further converted to DHAP by a dihydoxyacetone kinase (DhaK) enzyme. Our previous proteomic study showed a significant increase in the abundance of glycerol-degrading enzymes, GlpK and GlpD, during cultivation of R. jostii RHA1 cells with gluconate under TAG-accumulating conditions (Dávila Costa et al., 2015) . These enzymes seem to be part of a TAG recycling mechanism that occurs in oleaginous rhodococcal cells during lipid accumulation, in order to control NADPH and G3P availability in cells during the production process.
INTRODUCTION
With the aim to improve our understanding of glycerol metabolism and the ability of rhodococci to utilize this substrate for lipid production, we investigated the genetic and physiological capability of different strains from five different rhodococcal species for using glycerol as a substrate for growth and TAG production. In addition, we restored the ability of some glycerol-negative rhodococcal strains to grow on this substrate by heterologous expression of key genes, and improved glycerol assimilation and oil production yields of R. opacus PD630 when transformed with glycerol-related genes from R. fascians.
METHODS
Bacterial strains, culture conditions and plasmids. Strains and plasmids used in this work are listed in Table S1 (available in the online Supplementary Material). For the analysis of growth and lipid production of Rhodococcus strains, a liquid Luria-Bertani (LB) preculture was inoculated from a fresh LB plate and cultivated overnight. Then, cells were collected, washed with sterile NaCl solution (0.85 %, w/v) and inoculated in minimal salt medium (MSM; Schlegel et al., 1961) to OD 600 0.2. MSM1 medium (containing 1 g NH 4 Cl l 21 ) was used to analyse cell growth and MSM0.1 (containing 0.1 g NH 4 Cl l 21 ) to promote accumulation of lipids (nitrogen-limiting conditions). Growth and TAG production by rhodococcal cells were tested at different glycerol concentrations (Fig. S1 ). According to the results, we used a glycerol concentration of 0.3 % (v/v) for cell cultures. Rhodococcus cultivations were performed in 250 ml Erlenmeyer flasks with 50 ml medium at 28 uC and 150 r.p.m. E. coli DH5a was used as host for cloning and cultivated in LB medium or LB agar plates at 37 uC and 150 r.p.m. When appropriate, ampicillin, kanamycin and chloramphenicol were used at concentrations of 100, 30 and 34 mg ml 21 , respectively, for both E. coli and Rhodococcus strains. For analysis of gene overexpression under the acetamidase promoter (P ace ) of pJAM2 and the thiostrepton promoter of pTip-QC2, 0.5 % (w/v) acetamide and 1-3 mg thiostrepton ml 21 were, respectively, added to cell cultures at time zero.
METHODS
Electron microscopy analysis. Cells were washed, suspended in 0.1 M potassium phosphate buffer (pH 7.5) and fixed with glutaraldehyde for 24 h. Then, cells were washed with a solution of 0.32 M sucrose in phosphate buffer and embedded in low-viscosity resin (Spurr, 1969) . Thin sections were contrasted with uranyl acetate and ruthenium red (Vogt et al., 1995) . Images were obtained utilizing a Zeiss 109T electron microscope with a Gatan ES camera.
METHODS
DNA analysis, amplification, cloning and sequencing.
METHODS
Chromosomal DNA, plasmids and DNA fragments were isolated and analysed by standard methods (Marmur, 1961; Sambrook et al., 1989) . To identify three native strains (006, 346 and G212) exhibiting a glycerol-negative phenotype, DNA was extracted from isolated IP: 83.161.120.155
METHODS
On: Tue, 18 Dec 2018 22:01:15 colonies, and the 16S rRNA gene was amplified by PCR with universal primers for eubacteria and the conditions shown in the Table S2 . PCR products were sequenced by INTA Genomic Services. Sequences were screened against the National Center for Biotechnology Information (NCBI) database using the BLAST search program.
METHODS
glpK2 genes were amplified using genomic DNA from R. jostii strains 006, 346 and G212 and R. opacus strain PD630, whereas glpF and glpK1D1 genes were obtained from R. erythropolis DSMZ 43060 and R. fascians F7 genomic DNA, respectively. The primer pairs used for amplification were glpK2-F/glpK2-R, glpF-F/glpF-R and glpK1-F/ glpD2-R. Primer sequences and cycling conditions used in this work are listed in Table S2 .
METHODS
Nucleotide sequence accession numbers. The GenBank accession numbers for the amplified nucleotide sequence of the 16S rRNA and glpK genes from the three glycerol-negative rhodococcal strains are: R. jostii G212 (KT253457), R. jostii 346 (KT253455) and R. jostii 006 (KT253456), and glpK G212 (KT253462), glpK 346 (KT253461) and glpK 006 (KT253460).
METHODS
Subcloning, electroporation and genotyping. Inserts were excised from pGEM-T Easy vector using the restriction sites introduced by primers and subcloned into shuttle E. coli-Mycobacterium-Rhodococcus vector pJAM2, yielding pJAM2/glpK2 PD630 , pJAM2/glpK2 006 and pJAM2/glpF. This vector contains an inducible acetamidase promoter (P ace ) and six His codons downstream of the multiple cloning site. To obtain pTip-QC2/glpK1D1, the fragment of 3.2 kb containing both glpK1 and glpD1 from the R. fascians F7 glpFK1D1 cluster was taken from pGEM-T Easy/glpK1D1 using Bam HI/HindIII enzymes and subcloned in the thiostrepton inducer vector pTip-QC2, carrying six His codons downstream of the HindIII restriction site.
METHODS
All replicative plasmids were transferred to R. jostii (strains 006, 346 and G212) and R. opacus PD630 by electroporation. Electroporation was carried out as described by Kalscheuer et al. (1999) using a model 2510 electroporator (Eppendorf-Netheler-Hinz). The electrotransformants carrying glpK2 PD630 , glpK2 006 and glpF genes under the P ace promoter were checked by colony PCR using primer Pace-F and the reverse primers listed in Table S2 . Transformant strains of R. opacus PD630 carrying pTip-QC2/glpK1D1 were checked using the primers pTip-F/glpK1-R and glpD1-F/pTip-R (Table S2 ) to detect the presence of both glpK1 and glpD1 genes.
METHODS
Bioinformatic and phylogenetic analyses. Genomes analysed in this study are listed in the supplementary Material and methods. Genes involved in glycerol metabolism were searched by gene and protein name in the NCBI database, UniProt website and RAST server (Aziz et al., 2008) , and by homology using the BLASTP program. The search was run using the default parameters set by the program, considering as homologous proteins those with significant alignments (E-value j10 250 ). We also used well-characterized protein sequences (GlpF, GlpK and GlpD) of E. coli (Voegele et al., 1993; Feese et al., 1998) , B. subtilis (Holmberg et al., 1990; Beijer et al., 1993) , Mycobacterium smegmatis (Titgemeyer et al., 2007) and Enterococcus classeliflavus (Yeh et al., 2004) to find their homologues in different species of the genus Rhodococcus analysed. The criteria considered for predicting the possible function of proteins are listed in the supplementary Material and methods.
METHODS
For phylogenetic analyses, sequences of the 16S rDNA gene and glycerol kinase (GlpK) and G3P dehydrogenase (GlpD) proteins from five Rhodococcus species used in this study were aligned using CLUSTAL W (Thompson et al., 1994) . Evolutionary relationships were inferred using the neighbour-joining method (Saitou & Nei, 1987) . The resultant tree topologies were evaluated by bootstrap analyses based on 1000 resamplings. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011) .
METHODS
Structural prediction and modelling of GlpK1 from R. fascians F7 and GlpK2 from R. opacus PD630 were performed using the Phyre (protein homology/analogy recognition engine) web server (Kelley & Sternberg, 2009) in the intensive modelling mode. Modelling was based on glycerol kinase from Cellulomonas sp. NT3060 (Protein Data Bank ID: 2D4W), with 74 % and 70 % amino acid identity with R. fascians GlpK1 and R. opacus GlpK2, respectively.
METHODS
Analytical methods: determination of cell biomass, lipids and glycerol concentration. Growth was evaluated by measuring OD 600 in a PG-Instrument 700 spectrophotometer. For cell biomass dry weight determination, 10 ml culture was centrifuged at 6000 g for 15 min, washed twice with sterile saline solution and dried at 60 uC until constant weight.
METHODS
Details of the qualitative and quantitative analyses of lipids are included in the supplementary Material and methods.
METHODS
To determine residual glycerol, 3 ml culture media was collected at different times and centrifuged at 8500 g for 30 min. The concentration was analysed by an enzymic-colorimetric assay using a Wiener lab triglyceride kit (TG Color GK/GPO/PAP).
RESULTS

Growth and lipid accumulation by rhodococci from glycerol
We analysed the ability of R. opacus, R. jostii, R. erythropolis, R. fascians and R. equi strains to grow and produce TAGs from glycerol as the sole carbon and energy source. According to the capability of the investigated rhodococcal strains for growing from glycerol, they could be divided into three groups. (1) Strains that exhibited fast growth, which included R. erythropolis strains (DSMZ 43060, 17 and DM1-21) and R. fascians strains (F7, S1.17b, 123 and D188-5). Cells reached the stationary phase after *3 days of incubation ( Fig. 1 , Table 1 ). (2) Strains exhibiting growth on glycerol after a prolonged lag phase of *5 days of incubation, including R. opacus strains (PD630 and MR22), R. jostii strains (RHA1 and 602) and R. equi ATCC 6939. Cells of these microorganisms reached the stationary phase after *13 days of incubation (Fig. 1a , Table 1 ). (3) Strains belonging to R. jostii strains (006, 346 and G212), which were unable to grow with glycerol as the sole carbon and energy source, after 30 days of incubation (Fig. 1a) .
Growth and lipid accumulation by rhodococci from glycerol
Rhodococcal strains from groups 1 and 2 produced significant amounts of lipids during cultivation on glycerol, but with differences in the extension of time used by cells for cell biomass and TAG production (Fig. 1b, Table 1 ). R. erythropolis and R. fascians produced 3.9-4.3 g l 21 cell biomass l 21 and 28.4-44.6 % CDW of TAGs (with a maximum lipid yield of 1.2-1.8 g l
21
) after 6 days of incubation, whereas R. opacus and R. jostii produced 2.5-3.8 g cell biomass l 21 and 28.3-38.4 % CDW of TAGs (with a maximum lipid yield of 0.8-1.4 g l
21
) after 17 days of growth on glycerol (Fig. 1a, Table 1 ). R. equi produced lower amounts of cell biomass (1.5 g l
21
) and TAGs Table 1 ). Table 2 shows the fatty acid composition of lipids from the representative rhodococcal species used in this study analysed by GC-flame ionization detection as described in the supplementary Material and methods. In general, palmitic acid (C 16 : 0 ) and oleic acid (C 18 : 1 ) were the major fatty acids synthesized by rhodococcal cells during cultivation on glycerol. However, significant amounts of stearic acid (C 18 : 0 ) were produced by R. erythropolis and R. fascians strains, approximately twofold higher than in R. opacus and R. jostii lipids (Table 2) .
21
Interestingly, R. fascians and R. erythropolis, which were considered in previous studies as non-oleaginous bacteria when grown on glucose or gluconate (Alvarez et al., 1997; Alvarez 2003) , exhibited an oleaginous phenotype after cultivation on glycerol as the sole carbon and energy source (Fig. 1b, Table 1 ). In this context, Fig. 1 (c) shows lipid inclusion bodies produced by R. fascians F7 cells during growth on glycerol.
Survey of key genes for glycerol catabolism in the available genomic databases
The available genomic information for the rhodococcal species used in this study provides the possibility for comparative genome analysis on the occurrence and distribution of key genes involved in glycerol uptake and degradation. For this, genomes of R. opacus (PD630 and B4), R. jostii RHA1, R. erythropolis PR4, R. fascians F7 and R. equi 103S were examined in detail. Our bioinformatic analysis of rhodococcal genomes showed no evidence for the occurrence of fermentative pathways for glycerol degradation, as genes coding for glycerol dehydrogenase (dhaD) and dihydroxyacetone kinase (dhaK) enzymes were not found. In contrast, we found genes coding for the putative glycerol kinase (glpK) and G3P dehydrogenase (glpD) enzymes in all genomes analysed. According to this, we propose the catabolic glycerol pathway shown in Fig. 2(a) as the starting framework for our analysis of DNA and protein sequence databases. In the proposed pathway, the entrance of glycerol into the cell occurs by simple diffusion or is mediated by a glycerol facilitator protein called GlpF; then glycerol is activated to G3P by a glycerol kinase enzyme (GlpK) and finally G3P is oxidized by the membrane-bound G3P dehydrogenase (GlpD) enzyme to DHAP, which enters into the central metabolic pathways (Fig. 2a) .
Survey of key genes for glycerol catabolism in the available genomic databases
Genes encoding a putative glycerol uptake facilitator protein (glpF) were found in genomes of R. erythropolis PR4 and R. fascians F7, within a cluster including genes that encode putative GlpK and GlpD enzymes (RER_46060-RER_46040 for strain PR4 and ACG96_05165-ACG96_05175 for strain F7) (Fig. 2b) . Protein sequences of these gene clusters from R. erythropolis PR4 and R. fascians F7 (here named glpF, glpK1 and glpD1) show high identity with homologous proteins involved in glycerol metabolism of M. smegmatis (63-77 %), B. subtilis (30-50 %) and E. coli (33-52 %) (Holmberg et al., 1990; Beijer et al., 1993; Voegele et al., 1993; Feese et al., 1998; Titgemeyer et al., 2007) . This gene cluster (glpFK1D1) was absent in the genomes of R. opacus, R. jostii and R. equi. However, these micro-organisms possess an alternative gene cluster (here named cluster 2) containing glpK2 and glpD2 with opposite transcription directions, which in general exhibited high identity with GlpK1 (73-75 %) and GlpD1 (57-59 %) from R. erythropolis PR4 and R. fascians F7 (Fig. 2b) . Interestingly, R. erythropolis PR4 was the unique species amongst the rhodococcal strains investigated in this study, simultaneously containing both gene clusters (clusters 1 and 2) in its genome (Fig. 2b) . In addition, an additional gene coding for a putative G3P dehydrogenase enzyme (GlpD3) was present in a different locus of the investigated rhodococcal genomes, sharing 30-33 % identity with GlpD1 and 31-32 % identity with GlpD2.
Survey of key genes for glycerol catabolism in the available genomic databases
The predicted GlpK enzymes (from gene clusters 1 and 2) from rhodococci possessed the two conserved signatures reported for well-characterized GlpK enzymes from E. classeliflavus (Yeh et al., 2004) , B. subtilis (Wehtje et al., 1995) and E. coli (Ormö et al., 1998) : signature 1 (FGGY_KINASES_1, PS00933), which corresponds to the central region, and signature 2 (FGGY_KINASES_2, PS00445) located in the C-terminal region (Table S3) . Moreover, they contained the conserved residues T, Y and D, which are required for the binding of glycerol, as well as the N-terminal conserved domain 'DQGTTSSR' and several amino acids involved in ATP-binding (Flaherty et al., 1991; Feese et al., 1998; Alvarez et al., 2004) (Table S3 ).
Survey of key genes for glycerol catabolism in the available genomic databases
We performed structural prediction and modelling of GlpK1 from R. fascians F7 and GlpK2 from R. opacus PD630 using the Phyre2 web server (Kelley & Sternberg, 2009) . Modelled structures of both putative enzymes exhibited the characteristic glycerol kinase fold consisting of two large domains, one on either side of a deep and narrow cleft. At the interface of the two domains The catalytic site, responsible for the binding of ATP and glycerol (Fig. 3) , is localized at the interface of the two domains. In the Gram-positive bacteria E. classeliflavus and B. subtilis, GlpK enzymes are activated by phosphorylation at an activation loop containing conserved histidine residues (His232 and His230), which is located *25 Å from the catalytic site of the enzyme (Wehtje et al., 1995; Yeh et al., 2009 ). Interestingly, a major conformational difference between rhodococcal GlpK1 and GlpK2 was observed in the putative activation loop of enzymes (Fig. 3) . The putative activation loop of GlpK1 from R. fascians is more prominent than that of GlpK2 from R. opacus PD630, exhibiting a major exposed surface in the structure, which may determine a higher accessibility for its activation by phosphorylation. In addition, the alignment of rhodococcal GlpK1 and GlpK2 with glycerol kinase enzymes from E. casseliflavus and B. subtilis showed the occurrence of the conserved histidine for phosphorylation in GlpK2 from R. opacus PD630, whereas this amino acid was replaced at this position by an arginine in GlpK1 from R. fascians F7 (Table S3) . Interestingly, it has been reported that the replacement of histidine for arginine at this position in E. casseliflavus (His232Arg) and B. subtilis (His230Arg) promotes a conformational change in the activation loop, which significantly increases the enzyme activity between seven-and 19-fold (Yeh et al., 2009; Wehtje et al., 1995) . Whether the observed differences in the predicted protein structures of rhodococcal GlpK1 and GlpK2 affect the enzyme activity or efficiency remains to be investigated in the future. Table S4 shows the different putative G3P dehydrogenase FAD-dependent (glpD) enzymes occurring in the investigated rhodococci, including GlpD1, GlpD2 and GlpD3, which exhibit the conserved domains of short-chain dehydrogenases/reductases (SDR, Cl21454) and characteristic signatures FAD_G3PD_1 (PS00977) and FAD_G3PD_2 (PS00978) (Table S4) , responsible for the oxidation of G3P to DHAP.
Survey of key genes for glycerol catabolism in the available genomic databases
Finally, the glycerol facilitator proteins (GlpF) detected in R. erythropolis and R. fascians genomes (Fig. 2b) showed the characteristic MIP (major intrinsic protein) family signature [ 
(HNQA)-D-N-P-(STA)-(LIVMF)-(ST)-(LIVMF)-(GSTAFY)] (PS00221)
, and NPA1 and NPA2 domains located in the loops connecting transmembrane segments 2/3 and 5/6, respectively (Lagrée et al. 1999; Unger, 2000) (Table S5 ).
(HNQA)-D-N-P-(STA)-(LIVMF)-(ST)-(LIVMF)-(GSTAFY)] (PS00221)
Phylogenetic analysis using proteins from the investigated rhodococci allowed us to separate the different GlpK and GlpD enzymes into diverse evolutionary groups (Fig. 4a, b) . The arrows indicate the length and transcriptional orientation of genes. Genes encoding glycerol transport systems are depicted in dark grey arrows, glycerol catabolic genes in light grey arrows and regulatory genes probably related to glycerol metabolism in black arrows. Other genes are in white arrows. Genes are shown by their number without their corresponding prefixes: 'RHA1_ro', R. jostii RHA1; 'OPAG_', R. opacus PD630; 'ROP_', R. opacus B4; 'RER_', R. erythropolis PR4; 'ACG96_', R. fascians F7; 'REQ_', R. equi 103S. The names on the arrows represent the putative proteins encoded by each gene. GlpF, Glycerol uptake facilitator protein; GlpK, glycerol kinase; GlpD, G3P dehydrogenase; GpsA, NAD(P)H-dependent G3P dehydrogenase; PL, phospholipid; UBQ, ubiquinone; RETC, respiratory electron transport chain. CesB, Two-component response regulator; CesC, two-component histidine kinase; AmT, aminotransferase; GylT, glycosyltransferase; DCHT, alanine-rich transferase; HP, hypothetical protein. Analysis of GlpK rhodococcal sequences showed the separation of two defined phylogenetic groups, including GlpK2 enzymes of R. opacus, R. jostii, R. erythropolis and R. equi in one group, and GkpK1 enzymes of R. fascians and R. erythropolis in the second group (Fig. 4a) . This result suggested some evolutionary differences between GlpK1 and GlpK2 in rhodococci. Similar results were obtained after analysing GlpD sequences, as shown in Fig. 4(b) . GlpD3 from all studied rhodococci formed a subgroup in the phylogenetic tree separated from the other subgroups formed by GlpD1 and GlpD2. In addition, GlpD1 from R. fascians and R. erythropolis formed a subgroup separated from the other subgroup, which includes GlpD2 from R. opacus, R. jostii, R. erythropolis and R. equi (Fig. 4b) . Fig. 4(c) shows an unrooted neighbour-joining tree based on 16S rRNA sequences of diverse species of rhodococci. Interestingly, in the three phylogenetic trees there was a relation between the groups formed and the physiological ability of cells to grow on glycerol (Fig. 4c) . The rhodococcal species that grow fast on glycerol (R. fascians and 16S rDNA, GlpK (GlpKE) and GlpD (GlpDE) from E. coli K-12 substrain MG1655 were used as outgroups in the respective trees. GenBank nucleotide accession numbers and UniProt accession numbers are shown. Access numbers of the different GlpK and GlpD proteins are listed in Table S1 and S2, respectively.
(HNQA)-D-N-P-(STA)-(LIVMF)-(ST)-(LIVMF)-(GSTAFY)] (PS00221)
R. erythropolis) possessed the gene cluster glpFK1D1, whereas those species that grow on glycerol after a long lag phase possessed the glpK2D2 cluster (Figs 1, 2 and 4) . Taken together, these results suggested that the ability to utilize and metabolize glycerol was modelled in different ways during the evolution within the species of the Rhodococcus genus.
(HNQA)-D-N-P-(STA)-(LIVMF)-(ST)-(LIVMF)-(GSTAFY)] (PS00221)
Complementation of R. jostii strains unable to grow on glycerol Three R. jostii strains (006, 346 and G212) closely related to R. jostii RHA1 (Fig. 4c) were unable to grow on glycerol as the sole carbon and energy source after 30 days of incubation (Fig. 1a) . However, we demonstrated in this study that R. jostii strains RHA1 and 602 were able to grow on glycerol after a lag phase of *5 days of incubation (Fig. 1a) . The differential ability to grow on this substrate has also been reported for diverse strains of mycobacteria in which alterations in the glpK sequence occur, including SNPs promoting a shift in the reading frame, the generation of truncated proteins or changes in enzyme activity (Keating et al., 2005) . Taking this into account, we amplified and sequenced glpK genes from strains 006, 346 and G212 using specific primers designed with glpK2 sequences from R. jostii RHA1 and R. opacus PD630. Sequence analysis revealed high amino acid identities (94-98 %) with GlpK putative enzymes from R. jostii RHA1 and R. opacus (PD630 and B4), and the absence of evident alterations in their sequences. Conserved domains of GlpK enzymes were present in the amplified sequences of the glycerol-negative rhodococcal strains (Table S3) . Thus, to test whether the inability to use glycerol as the sole carbon source was related to the GlpK enzyme, as the reaction catalysed by this enzyme is the rate-limiting step of the pathway, we cloned the glpK2 gene of R. opacus PD630 into the pJAM2 expression vector containing an acetamide-inducible promoter and transferred it into R. jostii strains 006, 346 and G212. The recombinant strains together with WT strains carrying the empty vectors were analysed for growth and TAG production from glycerol. As shown in Fig. 5(a) , the three glycerol-negative rhodococcal strains expressing glpK2 PD630 were now able to grow on glycerol as the sole carbon and energy source. The delayed growth profiles of recombinant strains R. jostii G212-pJAM2/glpK2 PD630 , 346-pJAM2/glpK2 PD630 and 006-pJAM2/glpK2 PD630 were similar to those of R. opacus PD630 and R. jostii RHA1 (Figs 1a and 5a ) during cultivation with glycerol. In addition, cells of the three recombinant strains were able to produce and accumulate significant amounts of TAGs (22-24 % CDW) from glycerol after 17 days of incubation under nitrogen-limiting conditions (Fig. 5b) . These results indicated that the inability of R. jostii strains 006, 346 and G212 to grow on glycerol was related with their GlpK enzymes/genes.
(HNQA)-D-N-P-(STA)-(LIVMF)-(ST)-(LIVMF)-(GSTAFY)] (PS00221)
To test the in vivo functionality of the glpK2 gene from R. jostii 006, we cloned the native glpK2 006 into the pJAM2 expression vector under the control of an acetamide-inducible promoter (pJAM2/glpK2 006 ) and transferred it into R. jostii 006. As shown in Fig. 5(a) , the expression of glpK2 006 under the control of an external promoter promoted the growth of R. jostii 006 on glycerol. These results demonstrated that, at least in strain 006, the glpK2 structural gene was functional and that the inability of cells to grow on glycerol could be due to a regulatory effect or to an alteration of native promoter sequences.
(HNQA)-D-N-P-(STA)-(LIVMF)-(ST)-(LIVMF)-(GSTAFY)] (PS00221)
Engineering R. opacus PD630 for improving glycerol utilization R. opacus PD630 is able to grow and produce TAGs from glycerol, but after a long lag phase of incubation (Fig. 1a) . With the aim to improve the utilization of glycerol by strain PD630 for cell biomass and lipid production, we analysed the effect of the heterologous expression of genes involved in glycerol uptake and catabolism from R. fascians and R. erythropolis, which contain the glpFK1D1 cluster (Fig. 2b) .
(HNQA)-D-N-P-(STA)-(LIVMF)-(ST)-(LIVMF)-(GSTAFY)] (PS00221)
First, we hypothesized that the expression of glpF, which is lacking in cluster 2 (glpK2D2) in the R. opacus PD630 genome, could improve the entrance of glycerol into the cell, and enhance growth and lipid accumulation. Thus, the glpF gene of R. erythropolis DM1-21 was amplified, cloned into the pJAM2 expression vector and transferred into strain PD630. As shown in Fig. 6(a) , no changes in growth kinetics and glycerol uptake were observed in recombinant PD630 cells expressing glpF in comparison with those of PD630 WT . In addition, expression of glpF in R. opacus PD630 produced no significant changes in TAG production, as shown in Fig. 6(b, c) .
(HNQA)-D-N-P-(STA)-(LIVMF)-(ST)-(LIVMF)-(GSTAFY)] (PS00221)
To test the effect of the glycerol catabolic genes (glpK1D1) on grow and TAG accumulation in the oleaginous PD630, we transferred glpK1D1 genes from R. fascians F7 into this strain, cloned in the pTip-QC2 vector as an expression system, which possessed a thiostrepton-inducible promoter. The expression of glpK1D1 F7 in R. opacus PD630 improved growth and consumption of glycerol, reducing the extension of the lag phase in the culture, as shown in Fig. 6(d) . In this context, glpK1D1-expressing cells of strain PD630 consumed 67 % of glycerol and grew to a cell biomass concentration of 4.4 g l 21 after 6 days of incubation, whereas the WT strain (PD630-pTip-QC2) showed only incipient growth and glycerol consumption at this time point (Fig. 6d) . In addition, the genetic modification significantly accelerated the time (*10 days of incubation) needed to reach the maximal content of TAGs in recombinant cells (41 % of TAGs by CDW and 1.8 g l (Fig. 6e, f) . The expression of glpK1D1 F7 improved cell biomass and TAG production *1.2-and *1.4-fold, respectively, as shown in Fig. 6(e) . Analysis of lipids accumulated by R. opacus PD630-pTip-QC2/glpK1D1 F7 showed no differences in the fatty acid composition of lipids compared with those of R. opacus PD630-pTip-QC2 (Table 2) .
(HNQA)-D-N-P-(STA)-(LIVMF)-(ST)-(LIVMF)-(GSTAFY)] (PS00221)
Taken together, these results demonstrated that it was possible to improve oil yields from glycerol in rhodococci by moving gene modules encoding part of the glycerol metabolic pathways from one rhodococcal species to another.
DISCUSSION
In this study, we analysed the genetic potential and physiological ability of diverse rhodococcal species to grow and accumulate lipids from glycerol as the sole carbon and energy source. Our results suggested that the capability of using glycerol as the carbon source may be widespread amongst several rhodococcal species. However, we found important differences in the time needed by cells for starting growth from glycerol, which seems to be a species-dependent feature amongst rhodococci. In several previous studies, R. opacus and R. jostii species were considered the canonical oleaginous micro-organisms because they usually exhibit robust growth and high capability for accumulating TAGs from diverse substrates, such as gluconate, glucose and fructose, amongst others (Alvarez et al., 1996 (Alvarez et al., , 1997 Alvarez, 2003) . In contrast, members of R. fascians and R. erythropolis usually showed a lower capability for producing cell biomass and TAGs from these carbon sources; thus, they were considered as nonoleaginous species amongst rhodococci (Alvarez et al., 1997; Alvarez, 2003) . Interestingly, in this study we demonstrated that R. fascians and R. erythropolis are able to develop an oleaginous and more robust phenotype during growth on glycerol in comparison with the canonical R. opacus and R. jostii species. The latter species needed a prolonged lag phase before developing good growth and TAG accumulation. Once all these rhodococcal species started growth, they were able to produce significant amounts of cell biomass (0.8-1.8 g l 21 ) and TAGs (30-44 % CDW) during cultivation of cells on glycerol. R. equi exhibited a lower capability for assimilating glycerol for biomass and TAG production compared with R. opacus, R. jostii, R. fascians and R. erythropolis species. These results suggested that the different Rhodococcus species have the potential to develop an oleaginous phenotype when the selected carbon source fits into the metabolic and regulatory network needed for activating the metabolic reactions that provide precursors, energy and NADPH for cell growth and lipid synthesis. The differences in the time-dependent growth regimes exhibited by rhodococci in this study may be based in the different genetic endowment, fluxes of carbon/precursors through metabolism and specific regulatory circuits in each rhodococcal species/strains. In this context, we found two different sets of genes for glycerol uptake and degradation (here named clusters 1 and 2). Phylogenetic analyses suggested that these gene modules possess different evolutionary origins within rhodococci (Fig. 4c) . Results of this study indicated that the species/strains that possess cluster 1 (glpFK1D1) (R. fascians and R. erythropolis) exhibit fast growth and lipid accumulation, whereas those rhodococcal species/ strains that possess cluster 2 (glpK2D2) (R. opacus, R. jostii and R. equi) exhibit delayed growth and lipid accumulation during cultivation on glycerol (Fig. 2a) . The putative enzymes coded by these different sets of genes found in the investigated rhodococci exhibited phylogenetic and structural differences as revealed by alignment and structural predictive analyses. Whether these differences can determine differential in vivo enzyme activities and regulatory events must be investigated in the future. We can speculate that rhodococci could have developed through the evolution of a differential arrangement of metabolic pathways and regulatory circuits as intrinsic characteristics of each micro-organism, which define the potential of bacterial cells to synthesize and accumulate TAGs from glycerol. Clusters 1 and 2 for glycerol degradation may work in a different metabolic context in rhodococcal cells. Cluster 1, which is more similar in gene composition and arrangement to those found in other glycerol-degrading bacteria (Schweizer & Po, 1996; Titgemeyer et al., 2007; Baños et al., 2009; Nikel et al., 2014) , may be designed for utilization of external glycerol taken up from the environment. The occurrence of the glpF gene only in cluster 1, but not in cluster 2, may be related to this feature. In this context, glycerol is not an abundant carbon source in the environment (e.g. in bulk soil), but can be found frequently in plant exudates (Nikel et al., 2014) ; thus, some rhodococci that adapted to new nutritional niches may have selected specific metabolic arrangements to cope with environmental challenges. In this context, R. fascians is the only reported phytopathogen amongst rhodococci (Cornelis et al., 2001) ; thus, they must have evolved diverse traits for interacting with plants. Interestingly, R. fascians possesses a robust metabolic configuration for producing cell biomass and TAGs from glycerol, as shown in this study. In a previous proteomic study performed in R. jostii RHA1, we demonstrated that lipolytic and glycerol-degrading enzymes (GlpK2 and GlpD2) increased in abundance during TAG-accumulating conditions, together with the typical G3P-synthesizing enzyme GpsA (Dávila Costa et al., 2015) . We proposed the occurrence of a dynamic cycling of cellular TAGs, resulting in the activation of glycerol catabolic enzymes, which works together with enzymes of glyceroneogenesis in order to regulate the availability of G3P in oleaginous cells. Thus, cluster 2 may play a key role in R. opacus, R. jostii, R. erythropolis and R. equi for the control of the availability of G3P in the metabolic network of cells during TAG accumulation, with a secondary role in the catabolism of external glycerol.
DISCUSSION
In this study, we found three R. jostii strains (006, 346 and G212) which were unable to grow on glycerol as the sole carbon source. As the driving force for the uptake of glycerol is the substrate phosphorylation to G3P by GlpK (Nikel et al., 2014) , we expressed glpK2 PD630 under an inducible promoter in order to complement the phenotype of the three strains regarding the utilization of glycerol. After heterologous expression of glpK2 PD630 , the three glycerol-negative R. jostii strains used in this study were able to grow and produce TAGs from glycerol. However, the expression of its native glpK2 006 gene cloned into the pJAM2 expression vector with the P ace promoter in R. jostii strain 006 allowed cells to grow on glycerol as the sole carbon source, suggesting a defect at its expression level in the native organism. Taken together, these results suggested that the alteration of the glpK gene at the structural or expression level is the major defect of glycerol-negative rhodococcal strains, similar to that reported for mycobacteria. Keating et al. (2005) demonstrated that some particular strains of Mycobacterium bovis and Mycobacterium microti were unable to use glycerol as a carbon source due to SNPs in glpK genes which affected enzyme activity or generated truncated proteins.
DISCUSSION
R. opacus PD630 is an important oleaginous model for its ability to produce significant amounts of lipids from diverse organic wastes, such as whey , beet molasses (Voss & Steinbüchel, 2001 ), lignocellulosic wastes (Kurosawa et al., 2013) and orange waste (Gouda et al., 2008) . Despite the ability of strain PD630 to grow and produce high amounts of TAGs from glycerol as the sole carbon source, cells need a prolonged time of adaptation (of several days) before growing. This is a negative feature of R. opacus PD630, if we consider its biotechnological potential for producing valuable oils for industry. For this reason, and in order to improve the process, we separately expressed glpF and glpK1D1 genes from glycerol-fast-growing rhodococci in R. opacus PD630. The heterologous expression of glpF in strain PD630 did not produce any significant effect on glycerol utilization and TAG accumulation. This was in agreement with previous studies in Streptomyces clavuligerus (Baños et al., 2009) and Corynebacterium glutamicum (Rittmann et al., 2008) reporting that glpF expression by itself does not generate changes in growth and glycerol capture, which requires the simultaneous expression of genes coding for GlpK and GlpD enzymes. However, the heterologous expression of glpK1D1 from R. fascians F7 in R. opacus PD630 was sufficient for improving the use of glycerol for growth and lipid production. R. opacus PD630-pTip-QC2/glpK1D1 F7 exhibited a similar growth profile to R. erythropolis and R. fascians strains, reducing the extension of time for accumulating TAGs to *10 days of incubation (Figs 1a and 6c) . Cells of recombinant PD630 accumulated up to 40 % (CDW) of lipids after 6 days of incubation. These results demonstrated that R. opacus PD630 can be used for the design and construction of an efficient cell factory that can ensure robust conversion of waste materials derived from the biodiesel industry to valuable oils.
DISCUSSION
In this study, we also demonstrated that alternative rhodococci could be considered for technological conversion of glycerol into commercial lipids, such as R. erythropolis and R. fascians, which exhibit an oleaginous phenotype during growth on this substrate. Interestingly, these rhodococcal species produced TAGs with a different fatty acid composition in comparison with those lipids produced by R. opacus and R. jostii, which contain an adequate composition for biodiesel production (Refaat, 2009; Schlagermann et al., 2012) . In contrast, TAGs produced by R. erythropolis and R. fascians contained *66 % saturated fatty acids with a high content of stearic acid (C 18 : 0 ) (29-31 % CDW), similar to cocoa butter (23-30 % C 16 : 0 , 32-37 % C 18 : 0 , 30-37 % C 18 : 1 and 2-4 % C 18 : 2 ), in which saturated fatty acids represent *60-64 % (Papanikolaou et al., 2003) . These results highlight the metabolic versatility exhibited by rhodococci for producing interesting oils from industrial wastes.

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