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Nutrient starvation leading to triglyceride accumulation activates the Entner Doudoroff pathway in Rhodococcus jostii RHA1
© The Author(s) 2017
Received: 5 November 2016
Accepted: 22 February 2017
Published: 27 February 2017
Rhodococcus jostii RHA1 and other actinobacteria accumulate triglycerides (TAG) under nutrient starvation. This property has an important biotechnological potential in the production of sustainable oils.
To gain insight into the metabolic pathways involved in TAG accumulation, we analysed the transcriptome of R jostii RHA1 under nutrient-limiting conditions. We correlate these physiological conditions with significant changes in cell physiology. The main consequence was a global switch from catabolic to anabolic pathways. Interestingly, the Entner-Doudoroff (ED) pathway was upregulated in detriment of the glycolysis or pentose phosphate pathways. ED induction was independent of the carbon source (either gluconate or glucose). Some of the diacylglycerol acyltransferase genes involved in the last step of the Kennedy pathway were also upregulated. A common feature of the promoter region of most upregulated genes was the presence of a consensus binding sequence for the cAMP-dependent CRP regulator.
This is the first experimental observation of an ED shift under nutrient starvation conditions. Knowledge of this switch could help in the design of metabolomic approaches to optimize carbon derivation for single cell oil production.
Microbial triglycerides, called single cell oils (SCO), have biotechnological potential in the production of sustainable oils for their use either as biodiesel or as commodity oils. Biodiesel is produced by transesterification of triacylglycerides with short-chain alcohols (mainly methanol). Vegetable oils and animal fats such as soybean oil, rapeseed oil, palm oil or waste cooking oils are used as feedstocks for biodiesel production . However, this strategy has been criticized for being a non-sustainable process since it leads to a reduction in edible oil feedstocks . Production of biodiesel using SCO is considered as a promising alternative solution . SCO produce high quality biodiesel esters according to currently existing standards [4, 5]. SCO are appropriate for their use as a biodiesel source since the producing microorganisms can grow using a variety of substrates, show rapid life cycles and can be easily modified by genetic engineering.
Several microorganisms, including bacteria, yeasts, molds and microalgae, can be considered as oleaginous microorganisms . Regarding bacteria, the accumulation of the neutral lipids triacylglycerols (TAGs), wax esters (WEs) and polyhydroxyalkanoates (PHAs) has been reported. The main purpose of this accumulation is to store carbon and energy under growth-limiting conditions. While PHAs are synthesized in a wide variety of bacteria , the accumulation of triacylglycerols (TAGs) has only been described for a few bacteria belonging to the proteobacteria and actinobacteria groups (for a review see ). Acinetobacter  Mycobacterium , Streptomyces  or Rhodococcus  are such examples. Accumulation of TAGs is remarkably high in the actinobacteria Rhodococcus and Gordonia, which accumulate up to 80% of the cellular dry weight in the form of neutral lipids with maximal TAG production of 88.9 and 57.8 mg/l, respectively .
Rhodococcus are aerobic, non-sporulating soil bacteria, with unique enzymatic activities used for several environmental and biotechnological processes . Rhodococcus strains are industrially used for large-scale production of acrylamide and acrylic acid as well as for the production of bioactive steroid compounds and fossil fuel biodesulfurization . Moreover, Rhodococcus are able to degrade contaminant hydrophobic natural compounds and xenobiotics. R. jostii RHA1 has been shown to convert lignocellulose into different phenolic compounds  while it also has the potential to use this waste material for the production of valuable oils .
Due to its capability for degrading hydrocarbons, R. jostii RHA1 is one of the best studied Rhodococcus species in the terms of biotechnological applications [18–20]. Moreover, high TAG accumulating capability has been reported  and its genomic sequence is available .
In this article we decipher the metabolic changes associated to nutrient starvation conditions that influence TAG accumulation.
Bacterial strain and growth conditions
Rhodococcus jostii strain RHA1 was grown aerobically at 30 °C in Streptomyces medium, Fluka (Rich Medium, RM, 4.0 g/l glucose, 4.0 g/l Yeast extract, and 10.0 g/l Malt extract). After 48 h, 25 ml of R. jostii cells in RM were collected by centrifugation, washed with mineral salts medium M9 (Minimal Medium, MM, , 95 mM Na2HPO4, 44 mM KH2PO4, 17 mM NaCl, 0.1 mM CaCl2 and 2 mM MgSO4) containing 20% w/v sodium gluconate (MMGln) or 20% w/v glucose (MMGls) as the sole carbon sources and transfer into 25 ml of MMGln or MMGls. The concentration of ammonium chloride in MM was reduced to 10 mM to enhance lipid accumulation.
Extraction and analysis of lipids
Pelleted cells were extracted with hexane/isopropanol (3:1 v/v). An aliquot of the whole cell extract was analyzed by thin layer chromatography (TLC) on silica gel plates (Merck) applying n-hexane/diethyl ether/acetic acid (80:20:1, v/v/v) as a solvent system. Lipid fractions were revealed using iodine vapour. Trioleine and oleic acid (Merck) were used as standards.
RNA was extracted from RM and MM-grown cells originally harvested from 3 ml of culture. Total RNA isolation involved vortexing of the pellet with 6 ml of RNA Protect (QIAGEN) followed by centrifugation. The pellet was thereafter lysed using 280 μl of lysis buffer (10% Zwittergent (Calbiochem), 15 mg/ml Lysozime (Sigma) and 20 mg/ml Proteinase K (Roche) in TE buffer). Total RNA was purified with RNeasy mini kit (QIAGEN, Valencia, CA) combined with DNase I (QIAGEN) according to the manufacturer’s instructions. The quantity and quality of RNA were assessed using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technology, Rockland, DE) and Experion Automated Electrophoresis using the RNA StdSens Analysis Kit (Bio Rad).
Removal of 16S and 23S rRNA from total RNA was performed using MicrobExpress™ Bacterial mRNA Purification Kit (Ambion) according to the manufacturer’s protocol with the exception that no more than 5 μg total RNA was treated per enrichment reaction. Each RNA sample was divided into multiple aliquots of ≤5 μg RNA and separate enrichment reactions were performed for each sample. Enriched mRNA samples were pooled and run on the 2100 Bioanalzyer (Agilent) to confirm reduction of 16S and 23S rRNA prior to preparation of cDNA fragment libraries.
Preparation of cDNA fragment libraries
Ambion RNA fragmentation reagents were used to generate 60–200 nucleotide RNA fragments with an input of 100 ng of mRNA. Following precipitation of fragmented RNA, first strand cDNA synthesis was performed using random N6 primers and Superscript II Reverse Transcriptase, followed by second strand cDNA synthesis using RNaseH and DNA pol I (Invitrogen, CA). Double stranded cDNA was purified using Qiaquick PCR spin columns according to the manufacturer’s protocol (Qiagen).
RNA-Seq using the Illumina genome analyzer
The Illumina Genomic DNA Sample Prep kit (Illumina, Inc., San Diego, CA) was used according to the manufacturer’s protocol to process double-stranded cDNA for RNA-Seq. This process included end repair, A-tailing, adapter ligation, size selection, and pre-amplification. Amplified material was loaded onto independent flow cells. Sequencing was carried out by running 36 cycles on the Illumina Genome Analyzer IIx. The quality of the RNA-Seq reads was analyzed by assessing the relationship between the quality score and error probability. These analyses were performed on Illumina RNA-Seq quality scores that were converted to phred format (http://www.phrap.com/phred/).
To filter genes with low signal/noise ratio we built 3 subsets of each condition taking randomly 70% of the total sequenced reads for each subset. The alignment was performed by Bowtie  against the R. jostii RHA1 reference genomes of the chromosome and three endogenous plasmids (Genome Reviews CP000431-4_GR). Gene expression was determined by Samtools , Artemis  and home-made perl scripts. We represent gene expression as reads per kilobase (RPK) and the data was normalized by quantiles according to . Statistical analysis was performed by DESeq package  and R software.
Quantitative real-time RT-PCR (qRT-PCR)
cDNA was generated from 1.5 µg of total RNA using the iScript kit (BioRad) according to manufacturer’s instructions. 1 µl of the cDNA template was then used in quantitative real-time PCR reactions using iQ SUYBRE Green Supermix (BioRad) and a iCycler iQ5(BioRad). Primers were designed using Primer3 (http://primer3.sourceforge.net). The cycle of threshold (Ct) was determined for each reaction using the iQ5 Optical System Software 2.0 (BioRad). All qRT-PCR reactions were done in triplicate.
KDPG aldolase activity assay
KDPG aldolase activity was quantified by a lactate dehydrogenase (LDH) coupled assay where the production of pyruvate is related to the NADH consumption, as described in . 2 ml of R. jostii RHA1 RM or MMGls cultures were harvested and resuspended in 1 ml of buffer TrisHCl 100 mM pH 7.5, NaCl 300 mM, EDTA 1 mM, DTT 1 mM and PMSF 1 mM. The cells were lysed using 0.2 mm silica beads and a Fast Prep-24 system (MP Biomedicals) for 3 cycles of 60 s and centrifuged at 100,000g for 25 min at 4 °C. 150 μl aliquots of the resulting RM or MMGls total extracts were then treated with 1 μl of LDH (5 U/μL), 0.70 μl of NADH (50 mM) and 1 μl of KDPG (50 mM). Decrease in NADH absorbance at 340 nm was measured in quartz microcuvettes (150 μl) in a UV-1603 spectrophotometer (Shimadzu) for 5 min. Total protein concentration was determined by Bradford assays using BSA as standard. KDGP activity was calculated as moles of NADH consumed per mg of total protein per second (mol/s/mg).
Results and discussion
Culture conditions for R. jostii RHA1, TAGs accumulation and RNA-Seq analysis
Summary of the R. jostii cDNA samples sequenced using the Illumina genome analyzer
Total mapped reads
Total mapped bps (×106)
Mapped mRNA reads
Mapped mRNA bp (×106)
mRNA reads (% of all mapped reads)
Distribution of the upregulated and downregulated genes in the chromosome and plasmids of R. jostii RHA1
Comparative analysis of R. jostii RHA1 transcriptome under nutrient-rich and nutrient-limiting (TAG accumulating) conditions
Other alterations in gene expression can be directly correlated to specific starvation conditions: excess of the carbon source or depletion of the nitrogen source. Hence, significant alterations of metabolic pathways are related to nitrogen starvation: (i) amino acid catabolism is repressed and (ii) reactions that might render free ammonia from organic compounds are induced (i.e., formamidase and ethanolamine ammonia lyase). Finally, a set of metabolic activities are induced as a consequence of the fact that nutrient-starved cells can still incorporate the carbon source leading, for instance, to the synthesis of TAGs. In fact, induction of glycerol-3P-acyltransferase, fatty acid synthesis, acyl-carrier protein and biotin biosynthetic enzymes was observed. The transcriptome analysis of R. opacus PD630 under TAG accumulating conditions has been recently reported . 3 h after cells were transferred to a minimal medium (MSM3) similar to our MMGln medium, 21.15% of the genes were upregulated >2-fold and 9.36% downregulated >2-fold. Globally, genes related to biogenesis were upregulated while genes involved in energy production or carbohydrate metabolism were downregulated. 4273 R. jostii RHA1 homologous genes have been found in R. opacus PD630 chromosome. Most of the upregulated genes in R. jostii MMGln are also upregulated in R. opacus MSM3 (Additional file 1: Table S3), thus confirming the metabolic shift observed for R. jostii under TAG accumulating conditions.
Genes of the Entner-Doudoroff (ED) pathway are highly upregulated
A subset of the R. jostii RHA1 most upregulated genes in the MMGln nutrient-deprived medium
Gluconate permease family protein
Metabolite transporter, MFS superfamily
Possible ATP-dependent protease
Probable 1,3-propanediol dehydrogenase
Probable phosphoglycerate dehydrogenase
Probable glutamate dehydrogenase (NAD(P) +)
Possible transcriptional regulator, WhiB family
Probable ethanolamine permease, APC superfamily
Consistently, genes involved in ED pathway were also found amongst the genes upregulated in the TAG accumulating medium in R. opacus PD630 (Additional file 1: Table S3).
For RNA-Seq transcriptomic analysis, we used gluconate as a carbon source in MMGln because gluconate led to the highest level of TAG accumulation in R. opacus . Therefore, induction of the ED pathway could be the consequence of the use of gluconate as the sole carbon source and not of a general mechanism for TAG accumulation under nutrient-deprived conditions. To solve this question, we tested whether the presence of glucose in MMGls also induces TAG accumulation and the ED pathway in R. jostii. TAG accumulation in MM containing either glucose or gluconate as carbon source was evaluated by fluorescence measurements using red nile and the Victor-3 fluorometer system (Perkin Elmer). We observed that glucose was also able to induce TAG accumulation in R jostii, but to a lower extent than gluconate (data not shown). Two likely hypotheses to explain this are: (i) only gluconate is able to induce the ED pathway and glucose is metabolized to TAG by the EM pathway, or (ii) glucose is also metabolized by the ED pathway but with a slightly lower yield, because glucose has to be transformed first to gluconate.
qRT-PCR evaluation of the ED pathway gene expression in MM medium containing glucose or gluconate as sole carbon source
Cold shock protein
We have also analysed the enzymatic activity of the KHG/KDPG aldolase in crude extracts of R. jostii RHA1 grown on MMGls or RM as described in Methods. In accordance with the transcriptomic results, KDPG aldolase activity (Additional file 2: Figure S1) was 8.75 times higher in MMGls (3.5 nmol/s/mg) than in RM (0.4 nmol/s/mg).
Catabolism of the carbon source (either glucose or gluconate) by the ED pathway renders two moles of pyruvate per mole of carbon source. One mole of ATP is generated also. However, generation of reduced coenzymes depends on the carbon source. Whereas catabolism of 1 mol of glucose by the ED pathway generates 1 mol NADPH and 1 mol NADH, catabolism of gluconate generates only 1 mol NADH (see below).
Energy and redox metabolism in R. jostii RHA1 cells grown in MMGln
More than 30 genes that code for proteins of the oxidative phosphorylation process are upregulated and none of these genes is downregulated (Fig. 3). More specifically, the upregulated genes mainly code for subunits of the complex I or NADH dehydrogenase, while the genes of the F1-ATPase remain unchanged. Hence, respiratory activity may provide part of the ATP required for TAG biosynthesis.
A subset of the R. jostii RHA1 most downregulated genes in the MMGln nutrient-deprived medium
Transcriptional regulator, GntR family
Probable NADP dependent oxidoreductase
Pyruvate dehydrogenase E1 component beta subunit
Probable multidrug resistance transporter, MFS superfamily
Probable succinate-semialdehyde dehydrogenase (NAD(P) +)
Dihydrolipoyllysine-residue acetyltransferase, E2 component of pyruvate dehydrogenase complex
Pyruvate dehydrogenase E1 component alpha subunit
Probable cyanate transporter, MFS superfamily
Different metabolic pathways lead to acetyl-CoA generation from pyruvate. Pyruvate dehydrogenase, partially repressed, may account for the conversion of a fraction of the total pyruvate available to acetyl-CoA. Induction of other enzymes, such as acetyl-CoA synthase (8 homologs in RHA1 like ro04332 and ro11190, 6.9× and 5.9× upregulated, respectively) (Additional file 1: Table S1), that can generate acetyl-CoA from acetate without a requirement for NAD+ suggests that a fraction of the available pyruvate could be converted to acetyl-CoA by enzymes that do not generate NADH.
Induction of the Kennedy pathway for TAG accumulation
Putative CRP binding sites are present in the highly expressed genes
Alternative sigma factors such as sigma54 are widely used in bacteria as a quick response to cope with environmental changes such as nutrient deprivation. To find if these alternative factors are being used for the upregulation of the R. jostii genes in MMGln, the program BPROM (http://www.softberry.com/) for the recognition of sigma70 promoters was used with the 150 bp immediately upstream from each ORF start. A putative sigma70 binding site was found in most upregulated genes. Hence, regulatory element(s) alternative to sigma70 subunit must be responsible for the transcriptional activation of the R. jostii genes in MMGln. These element(s) should target conserved binding sites in some of the altered genes.
Bacterial CRPs are transcription factors that respond to cAMP by binding at target promoters when cAMP concentration increases. 254 CRP-binding sites have been found in E. coli, regulating at least 378 promoters . In R. jostii, 371 putative CRP binding sites have been found (Additional file 1: Table S2). Thus, there is a CRP binding site per, approximately, each 25 genes. However, the density increases significantly up to 1 site per 4 genes in the genes that we identified as highly upregulated (eightfold or greater) when Rhodococcus cells grow in MMGln. Specifically, in all the promoters controlling genes involved in the ED pathway there is at least one CRP binding site. Most of these promoters are divergent promoters and both of the controlled operons are upregulated. Moreover, CRP binding sites have also been found in the promoter regions of the two main upregulated WS/DGAT genes (ro05356 and ro02966), but not in the promoter regions of the other WS/DGAT genes. Strikingly, the promoter regions of the most upregulated operons in R. opacus PD630 also contain a CRP putative binding sequence (Additional file 1: Table S3).
In E. coli, gluconate was shown to lower both CRP and cAMP to nearly the same extent as glucose . Hence, it is likely that in R. jostii, the predicted cAMP increase, rather than being related to the carbon source, is related to the stress generated by depletion of nutrients.
We also searched for the presence of a CRP binding site in the upstream regulatory region of the orthologs of the 40 Rhodococcus genes in other microorganisms using the MEME Suite (Additional file 1: Table S4). According to the results, it seems that the CRP mediated activation of the ED pathway is only conserved in R. opacus, also an oleogenic rhodococci. CRP binding sites were also found in the promoter regions of a few genes in the other two Rhodococcus genomes analyzed (R. equi and R. erythropolis). However, no consensus CRP binding sequence was found in the promoter regions of the orthologous genes in Escherichia coli or Pseudomonas putida. We have also searched without success for CRP binding sites in similar operons of non-oleaginous organisms containing WS/DGAT enzymes, such as Mycobacterium tuberculosis, Acinetobacter baumanii or Marinobacter aquaolei. Thus, it seems the upregulation of these R. jostii genes by CRP is related to the TAG accumulation.
Different microorganisms are able to accumulate TAGs or other neutral lipids to serve as carbon and energy sources during starvation. One of these microorganisms is R. jostii strain RHA1. Transcriptomic analysis of R. jostii RHA1 under conditions that lead or do not lead to TAG accumulation allowed us to identify the metabolic pathways that are relevant for oxidation of the carbon source, biosynthesis and TAG accumulation under nutrient-deprivation.
Two interesting results arose from our work. First, under nutrient-deprivation, Rhodococcus metabolizes carbohydrates such as glucose or gluconate by the Entner-Doudoroff pathway. Up- or downregulation of other key enzymes (i.e., pyruvate dehydrogenase, acetyl CoA synthetase, NADH oxidase), provides the ATP, reducing equivalents and building blocks for TAG synthesis. Second, the metabolic shift is likely driven by an increase in cAMP concentration that activates the expression of several operons via CRP.
Both observations could help in engineering metabolic modifications to improve TAG yield for biotechnological applications.
AJ analysed data and wrote the article, JAV performed the experiments, VF carried out the bioinformatics analysis, BL performed the experiments, FC wrote the article, HMA analysed data and wrote the article and GM designed research, analysed data and wrote the article. All authors read and approved the final manuscript.
We are grateful to Dr. Juan Maria Garcia-Lobo and Dr. Maria Cruz Rodriguez for RNA-Seq analysis performed in the massive sequencing service at the IBBTEC. We thank Dr. Lindsay Eltis for the gift of the strain R. jostii RHA1.
The authors declare that they have no competing interests.
Availability of data and materials
All relevant data are presented in the main paper and Additional file 1.
This work was financed by Grants BIO2010-14809 from the Spanish Ministry of Science and Innovation and BFU2014-55534-C2-2-P from the Spanish Ministry of Economy and Competitiveness to GM. H.M. Alvarez is a career investigator of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.
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