Rhodococcus Opacus PD630 Research Articles

Increased triacylglycerol production in Rhodococcus opacus by overexpressing transcriptional regulators

Lignocellulosic biomass is currently underutilized, but it offers promise as a resource for the generation of commercial end-products, such as biofuels, detergents, and other oleochemicals. Rhodococcus opacus PD630 is an oleaginous, Gram-positive bacterium with an exceptional ability to utilize recalcitrant aromatic lignin breakdown products to produce lipid molecules such as triacylglycerols (TAGs), which are an important biofuel precursor. Lipid carbon storage molecules accumulate only under growth-limiting low nitrogen conditions, representing a significant challenge toward using bacterial biorefineries for fuel precursor production. In this work, we screened overexpression of 27 native transcriptional regulators for their abilities to improve lipid accumulation under nitrogen-rich conditions, resulting in three strains that accumulate increased lipids, unconstrained by nitrogen availability when grown in phenol or glucose. Transcriptomic analyses revealed that the best strain (#13) enhanced FA production via activation of the β-ketoadipate pathway. Gene deletion experiments confirm that lipid accumulation in nitrogen-replete conditions requires reprogramming of phenylalanine metabolism. By generating mutants decoupling carbon storage from low nitrogen environments, we move closer toward optimizing R. opacus for efficient bioproduction on lignocellulosic biomass.

Neuro-fuzzy modelling of a continuous stirred tank bioreactor with ceramic membrane technology for treating petroleum refinery effluent: a case study from Assam, India.

A continuous stirred tank bioreactor (CSTB) with cell recycling combined with ceramic membrane technology and inoculated with Rhodococcus opacus PD630 was employed to treat petroleum refinery wastewater for simultaneous chemical oxygen demand (COD) removal and lipid production from the retentate obtained during wastewater treatment. In the present study, the COD removal efficiency (CODRE) (%) and lipid concentration (g/L) were predicted using two artificial intelligence models, i.e., an artificial neural network (ANN) and a neuro-fuzzy neural network (NF-NN) with a network topology of 6-25-2 being the best for NF-NN. The results revealed the superiority of NF-NN over ANN in terms of determination coefficient (R2), root mean square error (RMSE), and mean absolute percentage error (MAPE). Three learning algorithms were tested with NF-NN; among them, the Bayesian regularization backpropagation (BR-BP) outperformed others. The sensitivity analysis revealed that, if solid retention time and biomass concentrations were maintained between 35 and 75h and 3.0g/L and 3.5g/L, respectively, high CODRE (93%) and lipid concentration (2.8g/L) could be obtained consistently.

A key O-demethylase in the degradation of guaiacol by Rhodococcus opacus PD630.

Rhodococcus opacus PD630 is a high oil-producing strain with the ability to convert lignin-derived aromatics to high values, but limited research has been done to elucidate its conversion pathway, especially the upper pathways. In this study, we focused on the upper pathways and demethylation mechanism of lignin-derived aromatics metabolism by R. opacus PD630. The results of the aromatic carbon resource utilization screening showed that R. opacus PD630 had a strong degradation capacity to the lignin-derived methoxy-containing aromatics, such as guaiacol, 3,4-veratric acid, anisic acid, isovanillic acid, and vanillic acid. The gene of gcoAR, which encodes cytochrome P450, showed significant up-regulation when R. opacus PD630 grew on diverse aromatics. Deletion mutants of gcoAR and its partner protein gcoBR resulted in the strain losing the ability to grow on guaiacol, but no significant difference to the other aromatics. Only co-complementation alone of gcoAR and gcoBR restored the strain's ability to utilize guaiacol, demonstrating that both genes were equally important in the utilization of guaiacol. In vitro assays further revealed that GcoAR could convert guaiacol and anisole to catechol and phenol, respectively, with the production of formaldehyde as a by-product. The study provided robust evidence to reveal the molecular mechanism of R. opacus PD630 on guaiacol metabolism and offered a promising study model for dissecting the demethylation process of lignin-derived aromatics in microbes.IMPORTANCEAryl-O-demethylation is believed to be the key rate-limiting step in the catabolism of heterogeneous lignin-derived aromatics in both native and engineered microbes. However, the mechanisms of O-demethylation in lignin-derived aromatic catabolism remain unclear. Notably, guaiacol, the primary component unit of lignin, lacks in situ demonstration and illustration of the molecular mechanism of guaiacol O-demethylation in lignin-degrading bacteria. This is the first study to illustrate the mechanism of guaiacol metabolism by R. opacus PD630 in situ as well as characterize the purified key O-demethylase in vitro. This study provided further insight into the lignin metabolic pathway of R. opacus PD630 and could guide the design of an efficient biocatalytic system for lignin valorization.

Both biogenic and chemically synthesized metal sulfide nanoparticles induce oxidative stress and enhance lipid accumulation in Rhodococcus opacus.

Metallic nanoparticles (NPs) find applications in many different industrial sectors. However, the fate of these NPs in the environment and their potential impact on organisms living in different ecosystems are not fully known. In this work, the individual effect of biogenic and chemically synthesized lead sulfide nanoparticles (PbSNPs) and cadmium sulfide nanoparticles (CdSNPs) on the activity of the oleaginous bacterium Rhodococcus opacus PD630 which belongs to an ecologically important genus Rhodococcus was investigated. A dose-dependent increase in PbSNPs and CdSNPs uptake by the bacterium was observed upto a maximum of 16.4 and 15.6mg/g cell, corresponding to 98% and 95% uptake. In the case of chemically synthesized NPs, the specific PbSNPs and CdSNPs uptake were slightly less [15.5 and 14.8mg/g cell], corresponding to 93.2% and 88.4% uptake. Both biogenic and chemically synthesized PbSNPs and CdSNPs did not affect the bacterial growth. On the other hand, the triacylglycerol (biodiesel) content in the bacterium increased from 30% to a maximum of 75% and 73% CDW due to oxidative stress induced by biogenic PbSNPs and CdSNPs. The results of induced oxidative stress by biogenic metal nanoparticle were similar to that induced by the chemically synthesized NPs.

Deciphering the transcriptional regulation of the catabolism of lignin-derived aromatics in Rhodococcus opacus PD630

Rhodococcus opacus PD630 has considerable potential as a platform for valorizing lignin due to its innate “biological funneling” pathways. However, the transcriptional regulation of the aromatic catabolic pathways and the mechanisms controlling aromatic catabolic operons in response to different aromatic mixtures are still underexplored. Here, we identified and studied the transcription factors for aromatic degradation using GFP-based sensors and comprehensive deletion analyses. Our results demonstrate that the funneling pathways for phenol, guaiacol, 4-hydroxybenzoate, and vanillate are controlled by transcriptional activators. The two different branches of the β-ketoadipate pathway, however, are controlled by transcriptional repressors. Additionally, promoter activity assays revealed that the substrate hierarchy in R. opacus may be ascribed to the transcriptional cross-regulation of the individual aromatic funneling pathways. These results provide clues to clarify the molecule-level mechanisms underlying the complex regulation of aromatic catabolism, which facilitates the development of R. opacus as a promising chassis for valorizing lignin.

Improved site-specific mutagenesis in Rhodococcus opacus using a novel conditional suicide plasmid

Rhodococcus opacus PD630 is a biotechnologically important bacterium with metabolic capability for bioremediation, metal recovery, and storage of triacylglycerols. Genome editing by homologous recombination in R. opacus is hampered by a very low combined frequency of DNA transfer and recombination. To improve recombination in the species, a conjugative, conditional suicide plasmid based on the replicon derived from the Corynebacterium glutamicum plasmid pGA1 was constructed and evaluated in R. opacus. The replication of this plasmid is controlled by a dual inducible and repressible promoter system originally developed for Mycobacterium spp. Next, we demonstrated that a derivative of this plasmid containing sacB as a counterselection marker and homologous regions of R. opacus could be used for homologous recombination, and that the problem of obtaining recombinants had been solved. Like for other Corynebacteriales, the cell wall of Rhodococcus spp. contains mycolic acids which form a hydrophobic and impermeable outer layer. Mycolic acids are essential for Mycobacterium smegmatis, but not for Corynebacterium glutamicum, and the new vector was used to study if mycolic acid is essential for R. opacus. We found that accD3 that is necessary for mycolic acid synthesis could only be deleted from the chromosome in strains containing a plasmid-encoded copy of accD3. This indicates that mycolic acid is important for R. opacus viability. The conditional suicide vector should be useful for homologous recombination or for delivering gene products like recombinases or Cas proteins and gRNA to Rhodococcus and related genera, while the approach should be applicable for any plasmid needing a plasmid-encoded protein for replication.Key points• Improved vector for homologous recombination in R. opacus.• Mycolic acid is important for survival of R. opacus like it is for Mycobacterium.• Similar conditional suicide plasmids may be constructed for other bacteria.Graphical abstract

Enhancing Lignin Dispersion and Bioconversion by Eliminating Thermal Sterilization

Thermal sterilization is widely applied in fermentation to ensure a pure culture. In this study, a facile and energy-efficient strategy by eliminating thermal sterilization (ETS) was unveiled for upgrading lignin bioconversion. Through alkaline solubilization and neutralization, lignin dispersion in aqueous fermentation media was significantly enhanced by ETS. Small-angle X-ray scattering and dynamic light scattering analyses indicated that the lignin colloid size was dramatically reduced. Compared to 20.5 wt % lignin precipitation during the conventional thermal sterilization, precipitated lignin was not observed within the ETS medium. 31P NMR characterization demonstrated an 11.7% increase of phenolic OH in ETS lignin. Ionization of phenolic OH presented more negatively charged groups, strengthening electrostatic repulsion, resulting in smaller colloidal particles. Interestingly, the pure culture of Rhodococcus opacus PD630 was achieved within the ETS medium due to the lack of lignin degradation ability with most natural microbes. R. opacus PD630 cell amount, lignin degradation, and lipid production by ETS increased by 330, 16.6, and 20.7%, respectively. Overall, an energy-efficient ETS strategy that promoted lignin dispersion and bioconversion significantly is reported in this study.

Biodiesel production by oleaginous bacteria Rhodococcus opacus PD630 using waste paper hydrolysate

Biodiesel production by oleaginous bacteria Rhodococcus opacus PD630 using waste paper hydrolysate

Cosolvent enhanced lignocellulosic fractionation tailoring lignin chemistry and enhancing lignin bioconversion

Cosolvent Enhanced Lignocellulosic Fractionation (CELF) is an emerging solvolysis pretreatment to fractionate lignocellulosic biomass. Herein, the bioconversion performance of CELF lignin was fully evaluated for the first time. Results showed that CELF lignin possessed higher content of carboxylic acid OH, lower molecular weight, and disappeared β-O-4 and β-5 linkages compared to other two technical lignins including a conventional ethanol organosolv lignin (EOL) and a kraft lignin (KL). Rhodococcus opacus PD630 cell count from CELF lignin fermentation reached the highest value of 3.9 × 107 CFU/mL, representing a 62.5% and 77.3% improvement over EOL and KL, respectively. Correspondingly, lipid yield reached 143 mg/L from CELF lignin, which was 36.2% and 26.5% higher than from EOL and KL, respectively. Principal component analysis (PCA) revealed that more carboxylic acid groups and lower molecular weight contributed to the enhanced bioconversion performance of CELF lignin. This study demonstrates that CELF lignin is a promising candidate for bioconversion.

Bacterial Transformation of Aromatic Monomers in Softwood Black Liquor.

The valorization of lignin, a major component of plant-derived biomass, is essential to sustainable biorefining. We identified the major monoaromatic compounds present in black liquor, a lignin-rich stream generated in the kraft pulping process, and investigated their bacterial transformation. Among tested solvents, acetone extracted the greatest amount of monoaromatic compounds from softwood black liquor, with guaiacol, vanillin, and acetovanillone, in an approximately 4:3:2 ratio, constituting ~90% of the total extracted monoaromatic content. 4-Ethanol guaiacol, vanillate, and 4-propanol guaiacol were also present. Bacterial strains that grew on minimal media supplemented with the BL extracts at 1mM total aromatic compounds included Pseudomonas putida KT2442, Sphingobium sp. SYK-6, and Rhodococcus rhodochrous EP4. By contrast, the extracts inhibited the growth of Rhodococcus jostii RHA1 and Rhodococcus opacus PD630, strains extensively studied for lignin valorization. Of the strains that grew on the extracts, only R. rhodochrous GD01 and GD02, isolated for their ability to grow on acetovanillone, depleted the major extracted monoaromatics. Genomic analyses revealed that EP4, GD01, and GD02 share an average nucleotide identity (ANI) of 98% and that GD01 and GD02 harbor a predicted three-component carboxylase not present in EP4. A representative carboxylase gene was upregulated ~100-fold during growth of GD02 on a mixture of the BL monoaromatics, consistent with the involvement of the enzyme in acetovanillone catabolism. More generally, quantitative RT-PCR indicated that GD02 catabolizes the BL compounds in a convergent manner via the β-ketoadipate pathway. Overall, these studies help define the catabolic capabilities of potential biocatalytic strains, describe new isolates able to catabolize the major monoaromatic components of BL, including acetovanillone, and facilitate the design of biocatalysts to valorize under-utilized components of industrial lignin streams.

A CRISPR/Cas9-based single-stranded DNA recombineering system for genome editing of Rhodococcus opacus PD630

Genome engineering of Rhodococcus opacus PD630, an important microorganism used for the bioconversion of lignin, is currently dependent on inefficient homologous recombination. Although a CRISPR interference procedure for gene repression has previously been developed for R. opacus PD630, a CRISPR/Cas9 system for gene knockout has yet to be reported for the strain. In this study, we found that the cytotoxicity of Cas9 and the deficiency in pathways for repairing DNA double-strand breaks (DSBs) were the major causes of the failure of conventional CRISPR/Cas9 technologies in R. opacus, even when augmented with the recombinases Che9c60 and Che9c61. We successfully developed an efficient single-stranded DNA (ssDNA) recombineering system coupled with CRISPR/Cas9 counter-selection, which facilitated rapid and scarless editing of the R. opacus genome. A two-plasmid system, comprising Cas9 driven by a weak Rhodococcus promoter Pniami, designed to prevent cytotoxicity, and a single-guide RNA (sgRNA) under the control of a strong constitutive promoter, was proven to be appropriate with respect to cleavage function. A novel recombinase, RrRecT derived from a Rhodococcus ruber prophage, was identified for the first time, which facilitated recombination of short ssDNA donors (40–80 nt) targeted to the lagging strand and enabled us to obtain a recombination efficiency up to 103-fold higher than that of endogenous pathways. Finally, by incorporating RrRecT and Cas9 into a single plasmid and then co-transforming cells with sgRNA plasmids and short ssDNA donors, we efficiently achieved gene disruption and base mutation in R. opacus, with editing efficiencies ranging from 22 % to 100 %. Simultaneous disruption of double genes was also confirmed, although at a lower efficiency. This effective genome editing tool will accelerate the engineering of R. opacus metabolism.

Synergistic Improvement of Carbohydrate and Lignin Processability by Biomimicking Biomass Processing

The sustainability and economic feasibility of modern biorefinery depend on the efficient processing of both carbohydrate and lignin fractions for value-added products. By mimicking the biomass degradation process in white-rote fungi, a tailored two-step fractionation process was developed to maximize the sugar release from switchgrass biomass and to optimize the lignin processability for bioconversion. Biomimicking biomass processing using Formic Acid: Fenton: Organosolv (F2O) and achieved high processability for both carbohydrate and lignin. Specifically, switchgrass pretreated by the F2O process had 99.6% of the theoretical yield for glucose release. The fractionated lignin was also readily processable by fermentation via Rhodococcus opacus PD630 with a lipid yield of 1.16 g/L. Scanning electron microscope analysis confirmed the fragmentation of switchgrass fiber and the cell wall deconstruction by the F2O process. 2D-HSQC NMR further revealed the cleavage of aryl ether linkages (β-O-4) in lignin components. These results revealed the mechanisms for efficient sugar release and lignin bioconversion. The F2O process demonstrated effective mimicking of natural biomass utilization system and paved a new path for improving the lignin and carbohydrate processability in next generation lignocellulosic biorefinery.

Batch degradation of trichloroethylene using oleaginous Rhodococcus opacus in a two-phase partitioning bioreactor and kinetic study

In the present study, we present the biodegradation of trichloroethylene (TCE) for the first time using pure bacterial strain of Rhodococcus opacus PD630. TCE utilized as the sole carbon substrate by R. opacus in single phase system and was reached to highest specific growth rate of 0.015 h−1 at the concentration of 75 mg L−1. To further enhance the degradation performance even at high concentration (1000 mg L−1), two-phase partitioning bioreactor (TPPB) was developed. Silicone oil (20%, v/v) was selected as non-aqueous phase solvent to achieve high metabolic activity of bacteria. Furthermore, the experimental results of TCE degradation and specific TCE utilization rate were fitted to various kinetic models. Our experimental results clearly revealed that TPPB system having R. opacus as biological source and silicone oil as solvent was highly efficient and successful for removing high concentrated TCE compared with single phase system.

Using 1-propanol to significantly enhance the production of valuable odd-chain fatty acids by Rhodococcus opacus PD630.

Odd-chain fatty acids (OCFAs) have been reported to possess pharmacological activity and have been used in the manufacture of agricultural and industrial chemicals. We here provided a new method to increase the OCFAs content in oil produced by Rhodococcus opacus PD630 through addition of 1-propanol to the fermentation media. The OCFAs in oil of R. opacus PD630 are primarily pentadecanoic acid (C15:0), heptadecanoic acid (C17:0) and heptadecenoic acid (C17:1). After adding 0.5-1.5% (v/v) 1-propanol, the production of oil increased from 1.27g/L to 1.31-1.61g/L, and the OCFAs content in oil increased by 46.7-55.1%. Metabolic intermediates determination and transcriptome analysis revealed that R. opacus assimilated 1-propanol through methylmalonyl-CoA pathway. When the nitrogen source was limited, propionyl-CoA was converted to propionyl-acyl carrier protein (ACP) which could be used as primer during the elongation of fatty acid synthesis. Then OCFAs were produced when odd number of propionyl-ACP was incorporated in the cycles of fatty acid synthesis.

Investigating the potential use of an oleaginous bacterium, Rhodococcus opacus PD630, for nano-TiO2 remediation.

The occurrence of titanium dioxide nanoparticles (nTiO2), in the effluents released from wastewater treatment plants, has raised concerns. The fate of nTiO2 and their potential impact on organisms from different ecosystems are widely investigated. For the first time, in this work, we report the responses of an oleaginous bacteria Rhodococcus opacus PD630, belonging to an ecologically important genus Rhodococcus to environmentally relevant concentrations of nTiO2, under dark and UV light conditions. We observed a dose-dependent increase in nTiO2 uptake by the bacteria that reached a maximum of 1.4 mg nTiO2 (g cell)-1 under mid-log UV exposure, corresponding to 97% uptake. The nTiO2 induced oxidative stress in bacteria that increased from 25.1 to a maximum of 100.3, 44.1, and 51.7 μmol.OH (g cell)-1 under dark, continuous, and mid-log UV, respectively. However, nTiO2 did not affect bacterial viability. Further, due to oxidative stress, the triacylglycerol (biodiesel) content from bacteria increased from 30% to a maximum of 54% CDW. Based on our findings, we propose an application of R. opacus PD 630 in nTiO2 remediation due to their high nTiO2 uptake and resistance.

Construction of Genetic Logic Gates Based on the T7 RNA Polymerase Expression System in Rhodococcus opacus PD630.

Rhodococcus opacus PD630 (R. opacus) is a nonmodel, Gram-positive bacterium that holds promise as a biological catalyst for the conversion of lignocellulosic biomass to value-added products. In particular, it demonstrates both a high tolerance for and an ability to consume inhibitory lignin-derived aromatics, generates large quantities of lipids, exhibits a relatively rapid growth rate, and has a growing genetic toolbox for engineering. However, the availability of genetic parts for tunable, high-activity gene expression is still limited in R. opacus. Furthermore, genetic logic circuits for sophisticated gene regulation have never been demonstrated in Rhodococcus spp. To address these shortcomings, two inducible T7 RNA polymerase-based expression systems were implemented for the first time in R. opacus and applied to the construction of AND and NAND genetic logic gates. Additionally, three isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible promoters were created by inserting LacI binding sites into newly characterized constitutive promoters. Furthermore, four novel aromatic sensors for 4-hydroxybenzoic acid, vanillic acid, sodium benzoate, and guaiacol were developed, expanding the gene expression toolbox. Finally, the T7 RNA polymerase platform was combined with a synthetic IPTG-inducible promoter to create an IMPLY logic gate. Overall, this work represents the first demonstration of a heterologous RNA polymerase system and synthetic genetic logic in R. opacus, enabling complex and tunable gene regulation in this promising nonmodel host for bioproduction.

A concerted systems biology analysis of phenol metabolism in Rhodococcus opacus PD630

Rhodococcus opacus PD630 metabolizes aromatic substrates and naturally produces branched-chain lipids, which are advantageous traits for lignin valorization. To provide insights into its lignocellulose hydrolysate utilization, we performed 13C-pathway tracing, 13C-pulse-tracing, transcriptional profiling, biomass composition analysis, and metabolite profiling in conjunction with 13C-metabolic flux analysis (13C-MFA) of phenol metabolism. We found that 1) phenol is metabolized mainly through the ortho–cleavage pathway; 2) phenol utilization requires a highly active TCA cycle; 3) NADPH is generated mainly via NADPH-dependent isocitrate dehydrogenase; 4) active cataplerotic fluxes increase plasticity in the TCA cycle; and 5) gluconeogenesis occurs partially through the reversed Entner–Doudoroff pathway (EDP). We also found that phenol-fed R. opacus PD630 generally has lower sugar phosphate concentrations (e.g., fructose 1,6-bisphosphatase) compared to metabolite pools in 13C-glucose-fed Escherichia coli (set as internal standards), while its TCA metabolites (e.g., malate, succinate, and α-ketoglutarate) accumulate intracellularly with measurable succinate secretion. In addition, we found that phenol utilization was inhibited by benzoate, while catabolite repressions by other tested carbon substrates (e.g., glucose and acetate) were absent in R. opacus PD630. Three adaptively-evolved strains display very different growth rates when fed with phenol as a sole carbon source, but they maintain a conserved flux network. These findings improve our understanding of R. opacus’ metabolism for future lignin valorization.

Mechanism-Guided Design of Highly Efficient Protein Secretion and Lipid Conversion for Biomanufacturing and Biorefining.

Bacterial protein secretion represents a significant challenge in biotechnology, which is essential for the cost‐effective production of therapeutics, enzymes, and other functional proteins. Here, it is demonstrated that proteomics‐guided engineering of transcription, translation, secretion, and folding of ligninolytic laccase balances the process, minimizes the toxicity, and enables efficient heterologous secretion with a total protein yield of 13.7 g L−1. The secretory laccase complements the biochemical limits on lignin depolymerization well in Rhodococcus opacus PD630. Further proteomics analysis reveals the mechanisms for the oleaginous phenotype of R. opacus PD630, where a distinct multiunit fatty acid synthase I drives the carbon partition to storage lipid. The discovery guides the design of efficient lipid conversion from lignin and carbohydrate. The proteomics‐guided integration of laccase‐secretion and lipid production modules enables a high titer in converting lignin‐enriched biorefinery waste to lipid. The fundamental mechanisms, engineering components, and design principle can empower transformative platforms for biomanufacturing and biorefining.

Biomass and lipid production by Rhodococcus opacus PD630 in molasses-based media with and without osmotic-stress

Rhodococcus opacus PD630 was used to produce biomass and lipids in molasses-based media with and without osmotic stress. In a 7-day aerobic batch culture at 30 °C, the biomass and lipid concentrations were maximized using an initial molasses concentration of 80 g/L and ammonium acetate (nitrogen source) concentration of 2.14 g/L. At a fixed initial molasses concentration of 80 g/L, the concentration of the nitrogen source was further fine-tuned to 2.25 g/L, to maximize the lipid content of the biomass to around 30% by dry mass. This medium was used to test the effects of stressing salts (sodium acetate, magnesium sulfate, sodium chloride) on production of lipids and biomass. A two-step bolus feeding with magnesium sulfate and sodium acetate, enhanced the final biomass concentration to around 19 g/L (a 50% increase relative to control), but the lipid content in the biomass was reduced to around 16% w/w. A 33% enhancement in lipid concentration relative to control, was achieved by feeding magnesium sulfate and sodium acetate. Sugarcane molasses could be effectively used to produce biomass and lipids instead of using the much more expensive pure carbon sources such as glucose and sucrose.

Lipid metabolism of phenol-tolerant Rhodococcus opacus strains for lignin bioconversion

BackgroundLignin is a recalcitrant aromatic polymer that is a potential feedstock for renewable fuel and chemical production. Rhodococcus opacus PD630 is a promising strain for the biological upgrading of lignin due to its ability to tolerate and utilize lignin-derived aromatic compounds. To enhance its aromatic tolerance, we recently applied adaptive evolution using phenol as a sole carbon source and characterized a phenol-adapted R. opacus strain (evol40) and the wild-type (WT) strain by whole genome and RNA sequencing. While this effort increased our understanding of the aromatic tolerance, the tolerance mechanisms were not completely elucidated.ResultsWe hypothesize that the composition of lipids plays an important role in phenol tolerance. To test this hypothesis, we applied high-resolution mass spectrometry analysis to lipid samples obtained from the WT and evol40 strains grown in 1 g/L glucose (glucose), 0.75 g/L phenol (low phenol), or 1.5 g/L phenol (high phenol, evol40 only) as a sole carbon source. This analysis identified > 100 lipid species of mycolic acids, phosphatidylethanolamines (PEs), phosphatidylinositols (PIs), and triacylglycerols. In both strains, mycolic acids had fewer double bond numbers in phenol conditions than the glucose condition, and evol40 had significantly shorter mycolic acid chain lengths than the WT strain in phenol conditions. These results indicate that phenol adaptation affected mycolic acid membrane composition. In addition, the percentage of unsaturated phospholipids decreased for both strains in phenol conditions compared to the glucose condition. Moreover, the PI content increased for both strains in the low phenol condition compared to the glucose condition, and the PI content increased further for evol40 in the high phenol condition relative to the low phenol condition.ConclusionsThis work represents the first comprehensive lipidomic study on the membrane of R. opacus grown using phenol as a sole carbon source. Our results suggest that the alteration of the mycolic acid and phospholipid membrane composition may be a strategy of R. opacus for phenol tolerance.