Rhodococcus Opacus Research Articles

Extracellular polymeric substances immobilized on microspheres for removal of heavy metals from aqueous environment

Extracellular polymeric substances (EPS) extracted from Rhodococcus opacus were immobilized on synthetic microspheres BES.DM-GMA-TETA, obtained via copolymerization of bis[4(2-hydroxy-3-methacryloyloxypropoxy) phenyl]sulfide (BES.DM) with glycidyl methacrylate (GMA) and modified with triethylenetetramine (TETA). Both the pristine synthetic sorbent and the sorbent with immobilized EPS were used for adsorption of Pb(II) and Cd(II) from aqueous solutions. The optimum pH for adsorption was 5 for Pb(II) and 6.5 for Cd(II). Adsorption of both ions followed the Langmuir model. The monolayer capacity towards Pb(II) and Cd(II) increased after EPS immobilization by 47% (from 58.82 to 86.96 mg/g) and 30% (from 37.45 to 46.73 mg/g), respectively. It was already achievable by a low coverage of EPS on the synthetic microspheres, since the weight ratio of EPS to the microspheres was 1:15. HNO3 and HCl were efficient for a complete desorption of Pb(II), while desorption of Cd(II) was incomplete (60% by HCl and below 50% by HNO3). SEM micrographs showed distinct differences between pristine microspheres, those with immobilized EPS and with the adsorbed metal. X-ray microanalysis of the EPS-bound microspheres proved the presence of additional elements such as Na, K, Ca, Mg, P and Si, together with the basic components of the synthetic microspheres (C, N, O, S). XPS technique was used to reveal the binding mechanism (surface precipitation and chelate formation).

Transformation of Ammonia Fiber Expansion (AFEX) corn stover lignin into microbial lipids by Rhodococcus opacus

The lignin from AFEX-pretreated corn stover and lignin model compounds were evaluated as the sole carbon source for microbial lipid production by Rhodococcus opacus NRRL B-3311. R. opacus NRRL B-3311 could grow on the AFEX-lignin without pretreatment and accumulate lipids up to 32 mg/L in 72 h with consuming 20% of the total lignin. The lipid produced from lignin had a similar fatty acid compositional profile to those of vegetable oil. Based on the metabolism analysis, R. opacus NRRL B-3311 could depolymerize AFEX-lignin and catabolize the aromatic compounds derived from the lignin. Study using aromatic model compounds found that R. opacus NRRL B-3311 preferably utilized 4-hydroxybenzoic acid as the sole carbon source over glucose, while it could not grow on p-coumaric acid or vanillin.

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.

A novel integrated biodegradation-microfiltration system for sustainable wastewater treatment and energy recovery.

This work assessed the treatment of wastewater generated from three different industries viz., paper and pulp, biomass gasification and dairy by biodegradation followed by membrane filtration. Batch biodegradation was first carried out using wastewater as the potential substrate for oleaginous Rhodococcus opacus with lipid accumulation intracellular; subsequently, a microfiltration system was applied to recover the bacterial biomass grown as well as for residual chemical oxygen demand (COD) removal from the effluent. The combined process showed excellent results in terms of COD removal from the industrial wastewaters, with the values 56.8%, 46.1% and 68.9% for dairy, paper and pulp and biomass gasification wastewaters, respectively, by biodegradation. These values were further improved to 92.7%, 87.6% and 88.2%, respectively, following the microfiltration step performed by employing a low-cost ceramic membrane. In addition, lipids accumulated by the bacterium were extracted and characterized for biodiesel production potential. Lipid characterization using 1H NMR confirmed the presence of saturated fatty acids. Gas chromatography analysis of the transesterified lipids revealed the presence of methyl palmitate and methyl stearate. In addition, the estimated properties of the transesterified product affirmed its potential for biofuel application.

Sustainable Diesel Feedstock: a Comparison of Oleaginous Bacterial and Microalgal Model Systems

The key to sustainable and commercially viable biodiesel production relies primarily on species selection. Oleaginous species with high biomass productivity, lipid content, and lipid productivity are desirable. High growth rate of the species results in high biomass productivity, which leads to high lipid productivity. It is known that algal oil technology lacks commercial feasibility predominantly due to low biomass productivity and other factors. The use of a faster-growing organism, such as oleaginous bacteria, could offset this major disadvantage. Thus, the current study analyzes two model oleaginous systems: Rhodococcus opacus PD630 (a bacterium) and Chlorella vulgaris NIOT5 (a microalga) for their growth rate and lipid productivity. It was found that the bacterial growth rate was 25-fold the microalgal growth rate. The bacterium also showed 57-fold higher biomass productivity and 75-fold higher biodiesel productivity. Further, the analysis of a large number of literature data from relevant studies under different cultivation conditions showed that R. opacus PD630 has productivities far higher than various autotrophic microalgae. Similarly, a frequency distribution of data collected from the literature showed that Rhodococcus sp. has productivities in the higher range as compared to heterotrophic microalgae. Thus, bacteria could serve as a better alternative to microalgae toward developing a commercially viable biofuel technology. Further, the biodiesel characterization study showed that the quality of diesel from the bacterium was better than that from the microalga.

Increasing lipid production using an NADP+-dependent malic enzyme from Rhodococcus jostii.

The occurrence of NADP+-dependent malic enzymes (NADP+-MEs) in several Rhodococcus strains was analysed. The NADP+-ME number in Rhodococcus genomes seemed to be a strain-dependent property. Total NADP+-ME activity increased by 1.8- and 2.6-fold in the oleaginous Rhodococcus jostii RHA1 and Rhodococcus opacus PD630 strains during cultivation under nitrogen-limiting conditions. Total NADP+-ME activity inhibition by sesamol resulted in a significant decrease of the cellular biomass and lipid production in oleaginous rhodococci. A non-redundant ME coded by the RHA1_RS44255 gene located in a megaplasmid (pRHL3) of R. jostii RHA1 was characterized and its heterologous expression in Escherichia coli resulted in a twofold increase in ME activity in an NADP+-dependent manner. The overexpression of RHA1_RS44255 in RHA1 and PD630 strains grown on glucose promoted an increase in total NADP+-ME activity and an up to 1.9-foldincrease in total fatty acid production without sacrificing cellular biomass. On the other hand, its expression in Rhodococcus fascians F7 grown on glycerol resulted in a 1.3-1.4-foldincrease in total fatty acid content. The results of this study confirmed the contribution of NADP+-MEs to TAG accumulation in oleaginous rhodococci and the utility of these enzymes as an alternative approach to increase bacterial oil production from different carbon sources.

Genome Analysis of Rhodococcus Sp. DSSKP-R-001: A Highly Effective β-Estradiol-Degrading Bacterium.

We screened bacteria that use E2 as its sole source of carbon and energy for growth and identified them as Rhodococcus, and we named them DSSKP-R-001. For a better understanding of the metabolic potential of the strain, whole genome sequencing of Rhodococcus DSSKP-R-001 and annotation of the functional genes were performed. The genomic sketches included a predicted protein-coding gene of approximately 5.4 Mbp with G + C content of 68.72% and 5180. The genome of Rhodococcus strain DSSKP-R-001 consists of three replicons: one chromosome and two plasmids of 5.2, 0.09, and 0.09, respectively. The results showed that there were ten steroid-degrading enzymes distributed in the whole genome of the strain. The existence and expression of estradiol-degrading enzymes were verified by PCR and RTPCR. Finally, comparative genomics was used to compare multiple strains of Rhodococcus. It was found that Rhodococcus DSSKP-R-001 had the highest similarity to Rhodococcus sp. P14 and there were 2070 core genes shared with Rhodococcus sp. P14, Rhodococcus jostii RHA1, Rhodococcus opacus B4, and Rhodococcus equi 103S, showing evolutionary homology. In summary, this study provides a comprehensive understanding of the role of Rhodococcus DSSKP-R-001 in estradiol-efficient degradation of these assays for Rhodococcus. DSSKP-R-001 in bioremediation and evolution within Rhodococcus has important meaning.

Catalytic Performance of a Class III Old Yellow Enzyme and Its Cysteine Variants.

Class III old yellow enzymes (OYEs) contain a conserved cysteine in their active sites. To address the role of this cysteine in OYE-mediated asymmetric synthesis, we have studied the biocatalytic properties of OYERo2a from Rhodococcus opacus 1CP (WT) as well as its engineered variants C25A, C25S and C25G. OYERo2a in its redox resting state (oxidized form) is irreversibly inactivated by N-methylmaleimide. As anticipated, inactivation does not occur with the Cys variants. Steady-state kinetics with this maleimide substrate revealed that C25S and C25G doubled the turnover frequency (kcat) while showing increased KM values compared to WT, and that C25A performed more similar to WT. Applying the substrate 2-cyclohexen-1-one, the Cys variants were less active and less efficient than WT. OYERo2a and its Cys variants showed different activities with NADPH, the natural reductant. The variants did bind NADPH less well but kcat was significantly increased. The most efficient variant was C25G. Replacement of NADPH with the cost-effective synthetic cofactor 1-benzyl-1,4-dihydronicotinamide (BNAH) drastically changed the catalytic behavior. Again C25G was most active and showed a similar efficiency as WT. Biocatalysis experiments showed that OYERo2a, C25S, and C25G converted N-phenyl-2-methylmaleimide equally well (81–84%) with an enantiomeric excess (ee) of more than 99% for the R-product. With cyclic ketones, the highest conversion (89%) and ee (>99%) was observed for the reaction of WT with R-carvone. A remarkable poor conversion of cyclic ketones occurred with C25G. In summary, we established that the generation of a cysteine-free enzyme and cofactor optimization allows the development of more robust class III OYEs.

RETRACTED ARTICLE: Bacterial conversion of depolymerized Kraft lignin

BackgroundLignin is a potential feedstock for microbial conversion into various chemicals. However, the degradation rate of native or technical lignin is low, and depolymerization is needed to obtain reasonable conversion rates. In the current study, base-catalyzed depolymerization—using NaOH (5 wt%)—of softwood Kraft lignin was conducted in a continuous-flow reactor system at temperatures in the range 190–240 °C and residence times of 1 or 2 min. The ability of growth of nine bacterial strains belonging to the genera Pseudomonas and Rhodococcus was tested using the alkaline-treated lignin as a sole carbon source.ResultsPseudomonas fluorescens and Rhodococcus opacus showed the best growth of the tested species on plates with lignin. Further evaluation of P. fluorescens and R. opacus was made in liquid cultivations with depolymerized lignin (DL) at a concentration of 1 g/L. Size exclusion chromatography (SEC) showed that R. opacus consumed most of the available lower molecular weight compounds (approximately 0.1–0.4 kDa) in the DL, but the weight distribution of larger fractions was almost unaffected. Importantly, the consumed compounds included guaiacol—one of the main monomers in the DL. SEC analysis of P. fluorescens culture broth, in contrast, did not show a large conversion of low molecular weight compounds, and guaiacol remained unconsumed. However, a significant shift in molecular weight distribution towards lower average weights was seen.ConclusionsRhodococcus opacus and P. fluorescens were identified as two potential microbial candidates for the conversion/consumption of base-catalyzed depolymerized lignin, acting on low and high molecular weight lignin fragments, respectively. These findings will be of relevance for designing bioconversion of softwood Kraft lignin.

Production of single cell protein from agro-waste using Rhodococcus opacus

Livestock and fish farming are rapidly growing industries facing the simultaneous pressure of increasing production demands and limited protein required to produce feed. Bacteria that can convert low-value non-food waste streams into singe cell protein (SCP) present an intriguing route for rapid protein production. The oleaginous bacterium Rhodococcus opacus serves as a model organism for understanding microbial lipid production. SCP production has not been explored using an organism from this genus. In the present research, R. opacus strains DSM 1069 and PD630 were fed three agro-waste streams: (1) orange pulp, juice, and peel; (2) lemon pulp, juice, and peel; and (3) corn stover effluent, to determine if these low-cost substrates would be suitable for producing a value-added product, SCP for aquafarming or livestock feed. Both strains used agro-waste carbon sources as a growth substrate to produce protein-rich cell biomass suggesting that that R. opacus can be used to produce SCP using agro-wastes as low-cost substrates.

Genome-based analysis for the identification of genes involved in o-xylene degradation in Rhodococcus opacus R7

BackgroundBacteria belonging to the Rhodococcus genus play an important role in the degradation of many contaminants, including methylbenzenes. These bacteria, widely distributed in the environment, are known to be a powerhouse of numerous degradation functions, due to their ability to metabolize a wide range of organic molecules including aliphatic, aromatic, polycyclic aromatic compounds (PAHs), phenols, and nitriles. In accordance with their immense catabolic diversity, Rhodococcus spp. possess large and complex genomes, which contain a multiplicity of catabolic genes, a high genetic redundancy of biosynthetic pathways and a sophisticated regulatory network. The present study aimed to identify genes involved in the o-xylene degradation in R. opacus strain R7 through a genome-based approach.ResultsUsing genome-based analysis we identified all the sequences in the R7 genome annotated as dioxygenases or monooxygenases/hydroxylases and clustered them into two different trees. The akb, phe and prm sequences were selected as genes encoding respectively for dioxygenases, phenol hydroxylases and monooxygenases and their putative involvement in o-xylene oxidation was evaluated. The involvement of the akb genes in o-xylene oxidation was demonstrated by RT-PCR/qPCR experiments after growth on o-xylene and by the selection of the R7–50 leaky mutant. Although the akb genes are specifically activated for o-xylene degradation, metabolic intermediates of the pathway suggested potential alternative oxidation steps, possibly through monooxygenation. This led us to further investigate the role of the prm and the phe genes. Results showed that these genes were transcribed in a constitutive manner, and that the activity of the Prm monooxygenase was able to transform o-xylene slowly in intermediates as 3,4-dimethylphenol and 2-methylbenzylalcohol. Moreover, the expression level of phe genes, homologous to the phe genes of Rhodococcus spp. 1CP and UPV-1 with a 90% identity, could explain their role in the further oxidation of o-xylene and R7 growth on dimethylphenols.ConclusionsThese results suggest that R7 strain is able to degrade o-xylene by the Akb dioxygenase system leading to the production of the corresponding dihydrodiol. Likewise, the redundancy of sequences encoding for several monooxygenases/phenol hydroxylases, supports the involvement of other oxygenases converging in the o-xylene degradation pathway in R7 strain.

Expression of engineered carbonyl reductase from Ogataea minuta in Rhodococcus opacus and its application to whole-cell bioconversion in anhydrous solvents

The carbonyl reductase from the methylotrophic yeast Ogataea minuta can catalyze the regio- and enantio-selective reduction of prochiral ketones to chiral alcohols, and is available for industrial manufacturing of statin drugs. We previously conducted a directed evolution experiment of the enzyme, and obtained a mutant (OCR_V166A) with improved tolerance to organic solvents. This expanded the applicability of the enzyme to the bioconversion of water-insoluble compounds (Honda etal., J. Biosci. Bioeng., 123, 673-678, 2017). In the present study, we expressed OCR_V166A in Rhodococcus opacus cells, which have a highly lipophilic surface structure and are dispersible in anhydrous organic solvents, and developed a whole-cell biocatalyst which can function in an organic-solvent-based reaction medium. The secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (TeADH) was employed as an NADPH-regenerating enzyme and co-expressed with OCR_V166A in R. opacus. The whole-cell bioconversion of 2,2,2-trifluoroacetophenone to α-(trifluoromethyl)benzyl alcohol was performed in organic solvents, including isopropanol, isobutanol, and cyclohexanol, which served both as reaction media and as substrates for TeADH. The type of organic solvents markedly affected not only the product titer but also the enantio-purity of the product. When isobutanol was used as the reaction medium, the whole-cell biocatalyst showed higher stability than the isolated enzyme. Consequently, a high concentration (1M) of the substrate was converted to the product with an overall conversion yield of 81% (mol/mol) in 24h.

Multi-omic elucidation of aromatic catabolism in adaptively evolved Rhodococcus opacus

Lignin utilization has been identified as a key factor in biorefinery profitability. However, lignin depolymerization generates heterogeneous aromatic mixtures that inhibit microbial growth and the conversion of lignocellulose to biochemicals. Rhodococcus opacus is a promising aromatic-catabolizing, oleaginous bacterium, but mechanisms for its aromatic tolerance and utilization remain undercharacterized. To better understand these mechanisms, we adaptively evolved R. opacus for improved utilization of 32 combinations of diverse aromatic compounds. Evolved R. opacus mutants showed up to 1900% growth improvement in the utilization of phenol, guaiacol, 4-hydroxybenzoate, vanillate, and benzoate compared to the wild-type strain. Whole genome sequencing revealed several redox-related genes with mutations shared across multiple adapted mutants. PVHG6, the mutant with the most improved growth on a mixture of multiple aromatic compounds, showed 56% lower superoxide dismutase activity than the wild-type strain, suggesting that redox reactions are important for aromatic tolerance and utilization. Comparative transcriptomics revealed by-product detoxification pathways and five aromatic funneling pathways that were upregulated in response to specific aromatic compounds. Gene knockout experiments confirmed the two degradation routes of the β-ketoadipate pathway for five aromatic compounds. These results provide an improved understanding of aromatic bioconversion and facilitate development of R. opacus as a biorefinery host.

Electroflotation of fine hematite particles with Rhodococcus opacus as a biocollector in a modified Partridge–Smith cell

In mineral recovery, the flotation of fine and ultrafine particles is poor, and the process is dependent on the bubble size and the hydrophobicity of the particles. This low recovery is mainly related to the low bubble/particle collision efficiency. However, this problem can be overcome by reducing the size of the bubbles. The electroflotation process is an attractive alternative for the concentration (recovery) of fine particles using hydrogen and oxygen bubbles smaller than 100 μm. In this context, the present work presents a study on the electroflotation of fine hematite particles in a modified Partridge–Smith cell using Rhodococcus opacus as a bioreagent and hydrogen or oxygen bubbles. The influence of the current density and system pH on bubble size of the hydrogen and oxygen was also evaluated with the aid of a bubble sizer equipment. The Sauter mean bubble diameter (d32) was found to range from 40 to 60 μm for the hydrogen bubbles and 50 to 70 μm for the oxygen bubbles. The hematite electroflotation tests were conducted in a modified Partridge–Smith electroflotation binary cell with 0.38 L usable volume. The process variables studied were the concentration of R. opacus, particle size, pH, and current density. The optimum pH value found for hematite flotation was around 6, and the higher hematite recovery values were about 80% and 65% with hydrogen and with oxygen bubbles, respectively.

Biological treatment of wastewater containing a mixture of polycyclic aromatic hydrocarbons using the oleaginous bacterium Rhodococcus opacus

Abstract Polycyclic aromatic hydrocarbons (PAHs), including naphthalene, phenanthrene and fluoranthene are commonly found in wastewaters from refineries and biomass gasification industries. This study investigated the simultaneous biodegradation of these PAHs along with lipid accumulation by Rhodococcus opacus in a ternary substrate system. A 23 full factorial design of experiments was employed with the three PAHs at two different levels by varying their initial concentrations in the range 50–200 mg L−1 each. A maximum removal of 91.6%, 82.3% and 80.7% was achieved for naphthalene, phenanthrene and fluoranthene, respectively. The individual effect of PAH concentration was found to be more significant than 2-way and 3-way interaction effects on their degradation. PAH biodegradation efficiency in the mixture was mainly affected by initial concentration and aromatic complexity of the PAHs. Identification of the PAH degradation metabolites was carried out using LC-MS analysis, which clearly revealed that the PAHs were degraded primarily via the ortho/para pathway. This study demonstrates the potential utility of R. opacus for bioremediation and industrial wastewater treatment.

Draft Genome Sequence of Rhodococcus opacus Strain 04-OD7, Which Can Mobilize Phosphate.

ABSTRACTRhodococcus opacus strain 04-OD7 (=CCTCC AB 2017148) is a Gram-positive bacterium showing inorganic phosphate solubilization capacity for the first time in the genus Rhodococcus. We present here the draft genome description of R. opacus 04-OD7 along with multiple phosphorus (P) mobilization-related genes, supporting its inorganic phosphate solubilization.

Selection of stable reference genes for RT-qPCR in Rhodococcus opacus PD630

Rhodococcus opacus PD630 is a gram-positive bacterium with promising attributes for the conversion of lignin into valuable fuels and chemicals. To develop an organism as a cellular factory, it is necessary to have a deep understanding of its metabolism and any heterologous pathways being expressed. For the purpose of quantifying gene transcription, reverse transcription quantitative PCR (RT-qPCR) is the gold standard due to its sensitivity and reproducibility. However, RT-qPCR requires the use of reference genes whose expression is stable across distinct growth or treatment conditions to normalize the results. Unfortunately, no in-depth analysis of stable reference genes has been conducted in Rhodococcus, inhibiting the utilization of RT-qPCR in R. opacus. In this work, ten candidate reference genes, chosen based on previously collected RNA sequencing data or literature, were examined under four distinct growth conditions using three mathematical programs (BestKeeper, Normfinder, and geNorm). Based on this analysis, the minimum number of reference genes required was found to be two, and two separate pairs of references genes were identified as optimal normalization factors for when ribosomal RNA is either present or depleted. This work represents the first validation of reference genes for Rhodococcus, providing a valuable starting point for future research.

Identification of Inhibitors in Lignocellulosic Slurries and Determination of Their Effect on Hydrocarbon-Producing Microorganisms.

The aim of this work was to identify inhibitors in pretreated lignocellulosic slurries, evaluate high-throughput screening strategies, and investigate the impact of inhibitors on potential hydrocarbon-producing microorganisms. Compounds present in slurries that could inhibit microbial growth were identified through a detailed analysis of saccharified slurries by applying a combination of approaches of high-performance liquid chromatography, GC-MS, LC-DAD-MS, and ICP-MS. Several high-throughput assays were then evaluated to generate toxicity profiles. Our results demonstrated that Bioscreen C was useful for analyzing bacterial toxicity but not for yeast. AlamarBlue reduction assay can be a useful high-throughput assay for both bacterial and yeast strains as long as medium components do not interfere with fluorescence measurements. In addition, this work identified two major inhibitors (furfural and ammonium acetate) for three potential hydrocarbon-producing bacterial species that include Escherichia coli, Cupriavidus necator, and Rhodococcus opacus PD630, which are also the primary inhibitors for ethanologens. This study was strived to establish a pipeline to quantify inhibitory compounds in biomass slurries and high-throughput approaches to investigate the effect of inhibitors on microbial biocatalysts, which can be applied for various biomass slurries or hydrolyzates generated through different pretreatment and enzymatic hydrolysis processes or different microbial candidates.

Enhanced production of carotenoids and lipids by Rhodococcus opacus PD630

AbstractBACKGROUNDLow‐value waste organics can be upgraded to higher‐value products using microorganisms. The bacterium Rhodococcus opacus PD630 was used to produce colorants (carotenoids) and lipids (potential fuel oils) from glycerol.RESULTSSubmerged culture of R. opacus PD630 produced carotenoids and lipids on glycerol as the primary carbon source, although supplementation with glucose was necessary. A 1:1 mixture (50 g L‐1 each) of glucose and glycerol in a batch culture afforded a final biomass concentration of nearly 9.0 g L‐1, a carotenoids concentration of 0.75 mg L‐1 and a lipids concentration of nearly 2.2 g L‐1. Compared with using only glycerol (100 g L‐1) in the medium, the final biomass concentration was nearly 4‐fold greater, the carotenoids concentration was 7.5‐fold greater and the lipids concentration was 8.6‐fold greater when a 1:1 mixture of glucose and glycerol was used. A combination of glucose (70 g L‐1), glycerol (30 g L‐1) and ammonium acetate (4.32 g L‐1) further increased the final biomass concentration to nearly 10.2 g L‐1. With this formulation, the carotenoids concentration rose to 0.99 mg L‐1 and lipids concentration rose to nearly 2.4 g L‐1 in batch culture. A repeated fed‐batch operation with an optimal feeding regimen raised the final biomass concentration to 12.1 g L‐1. The final concentration of carotenoids was 0.7 mg L‐1 and the final lipids concentration was nearly 4.9 g L‐1.CONCLUSIONGlycerol can partly displace the more expensive glucose in production of carotenoids and lipids from R. opacus PD630. Biomass with ∼40% lipids (dry basis) can be produced. © 2017 Society of Chemical Industry

Molecular Toolkit for Gene Expression Control and Genome Modification in Rhodococcus opacus PD630.

Rhodococcus opacus PD630 is a non-model Gram-positive bacterium that possesses desirable traits for lignocellulosic biomass conversion. In particular, it has a relatively rapid growth rate, exhibits genetic tractability, produces high quantities of lipids, and can tolerate and consume toxic lignin-derived aromatic compounds. Despite these unique, industrially relevant characteristics, R. opacus has been underutilized because of a lack of reliable genetic parts and engineering tools. In this work, we developed a molecular toolbox for reliable gene expression control and genome modification in R. opacus. To facilitate predictable gene expression, a constitutive promoter library spanning ∼45-fold in output was constructed. To improve the characterization of available plasmids, the copy numbers of four heterologous and nine endogenous plasmids were determined using quantitative PCR. The molecular toolbox was further expanded by screening a previously unreported antibiotic resistance marker (HygR) and constructing a curable plasmid backbone for temporary gene expression (pB264). Furthermore, a system for genome modification was devised, and three neutral integration sites were identified using a novel combination of transcriptomic data, genomic architecture, and growth rate analysis. Finally, the first reported system for targeted, tunable gene repression in Rhodococcus was developed by utilizing CRISPR interference (CRISPRi). Overall, this work greatly expands the ability to manipulate and engineer R. opacus, making it a viable new chassis for bioproduction from renewable feedstocks.