Enhancing the stress resistance of nitrile hydratase from Rhodococcus ruber via SpyTag/SpyCatcher-mediated α- and ß- subunits ligation.

BACKGROUND: Nitrile Hydratase (NHase) is one of the most important industrial enzyme widely used in the petroleum exploitation field. The enzyme, composed of two unrelated α- and ß-subunits, catalyzes the conversion of acrylonitrile to acrylamide, releasing a significant amount of heat and generating the organic solvent product, acrylamide. Both the heat and acrylamide solvent have an impact on the structural stability of NHase and its catalytic activity. Therefore, enhancing the stress resistance of NHase to toxic substances is meaningful for the petroleum industry. METHODS AND RESULTS: To improve the thermo-stability and acrylamide tolerance of NHase, the two subunits were fused in vivo using SpyTag and SpyCatcher, which were attached to the termini of each subunit in various combinations. Analysis of the engineered strains showed that the C-terminus of ß-NHase is a better fusion site than the N-terminus, while the C-terminus of α-NHase is the most suitable site for fusion with a larger protein. Fusion of SpyTag and SpyCatcher to the C-terminus of ß-NHase and α-NHase, respectively, led to improved acrylamide tolerance and a slight enhancement in the thermo-stability of one of the engineered strains, NBSt. CONCLUSION: These results indicate that in vivo ligation of different subunits using SpyTag/SpyCatcher is a valuable strategy for enhancing subunit interaction and improving stress tolerance.

Physiology and comparative genomics of the haloalkalitolerant and hydrocarbonoclastic marine strain Rhodococcus ruber MSA14.

Marine hydrocarbonoclastic bacteria can use polycyclic aromatic hydrocarbons as carbon and energy sources, that makes these bacteria highly attractive for bioremediation in oil-polluted waters. However, genomic and metabolic differences between species are still the subject of study to understand the evolution and strategies to degrade PAHs. This study presents Rhodococcus ruber MSA14, an isolated bacterium from marine sediments in Baja California, Mexico, which exhibits adaptability to saline environments, a high level of intrinsic pyrene tolerance (> 5 g L- 1), and efficient degradation of pyrene (0.2 g L- 1) by 30% in 27 days. Additionally, this strain demonstrates versatility by using naphthalene and phenanthrene as individual carbon sources. The genome sequencing of R. ruber MSA14 revealed a genome spanning 5.45 Mbp, a plasmid of 72 kbp, and three putative megaplasmids, lengths between 110 and 470 Kbp. The bioinformatics analysis of the R. ruber MSA14 genome revealed 56 genes that encode enzymes involved in the peripheral and central pathways of aromatic hydrocarbon catabolism, alkane, alkene, and polymer degradation. Within its genome, R. ruber MSA14 possesses genes responsible for salt tolerance and siderophore production. In addition, the genomic analysis of R. ruber MSA14 against 13 reference genomes revealed that all compared strains have at least one gene involved in the alkanes and catechol degradation pathway. Overall, physiological assays and genomic analysis suggest that R. ruber MSA14 is a new haloalkalitolerant and hydrocarbonoclastic strain toward a wide range of hydrocarbons, making it a promising candidate for in-depth characterization studies and bioremediation processes as part of a synthetic microbial consortium, as well as having a better understanding of the catabolic potential and functional diversity among the Rhodococci group.

Transcriptome Analysis Reveals the Biocontrol Mechanism of Endophytic Bacterium AM201, Rhodococcus sp., against Root Rot Disease of Atractylodes macrocephala.

Atractylodes macrocephala Koidz (AMK) is a perennial herb from the plant family Asteraceae (formerly Compositae). This herb is mainly distributed in mountainous wetlands in Zhejiang, Sichuan, Yunnan, and Hunan provinces of China. Its medicinal production and quality, however, are severely impacted by root rot disease. In our previous study, endophytic bacterium designated AM201 exerted a high biocontrol effect on the root rot disease of AMK. However, the molecular mechanisms underlying this effect remain unclear. In this study, the identity of strain AM201 as Rhodococcus sp. was determined through analysis of its morphology, physiological and biochemical characteristics, as well as 16S rDNA sequencing. Subsequently, we performed transcriptome sequencing and bioinformatics analysis to compare and analyze the transcriptome profiles of root tissues from two groups: AM201 (AMK seedlings inoculated with Fusarium solani [FS] and AM201) and FS (AMK seedlings inoculated with FS alone). We also conducted morphological, physiological, biochemical, and molecular identification analyses for the AM201 strain. We obtained 1,560 differentially expressed genes, including 187 upregulated genes and 1,373 downregulated genes. We screened six key genes (GOLS2, CIPK25, ABI2, egID, PG1, and pgxB) involved in the resistance of AM201 against AMK root rot disease. These genes play a critical role in reactive oxygen species (ROS) clearance, Ca2+ signal transduction, abscisic acid signal inhibition, plant root growth, and plant cell wall defense. The strain AM201 was identified as Rhodococcus sp. based on its morphological characteristics, physiological and biochemical properties, and 16S rDNA sequencing results. The findings of this study could enable to prevent and control root rot disease in AMK and could offer theoretical guidance for the agricultural production of other medicinal herbs.

Simultaneously degradation of various phthalate esters by Rhodococcus sp. AH-ZY2: Strain, omics and enzymatic study.

Phthalate esters (PAEs) are widely used as plasticizers and cause serious complex pollution problem in environment. Thus, strains with efficient ability to simultaneously degrade various PAEs are required. In this study, a newly isolated strain Rhodococcus sp. AH-ZY2 can degrade 500 mg/L Di-n-octyl phthalate completely within 16 h and other 500 mg/L PAEs almost completely within 48 h at 37 °C, 180 rpm, and 2 % (v/v) inoculum size of cultures with a OD600 of 0.8. OD600 = 0.8, 2 % (v/v). Twenty genes in its genome were annotated as potential esterase and four of them (3963, 4547, 5294 and 5359) were heterogeneously expressed and characterized. Esterase 3963 and 4547 is a type I PAEs esterase that hydrolyzes PAEs to phthalate monoesters. Esterase 5294 is a type II PAEs esterase that hydrolyzes phthalate monoesters to phthalate acid (PA). Esterase 5359 is a type III PAEs esterase that simultaneously degrades various PAEs to PA. Molecular docking results of 5359 suggested that the size and indiscriminate binding feature of spacious substrate binding pocket may contribute to its substrate versatility. AH-ZY2 is a potential strain for efficient remediation of PAEs complex pollution in environment. It is first to report an esterase that can efficiently degrade mixed various PAEs.

Taxonomic identification, phenol biodegradation and soil remediation of the strain Rhodococcus sacchari sp. nov. Z13T.

Phenols are highly toxic chemicals that are extensively used in industry and produce large amounts of emissions. Notably, phenols released into the soil are highly persistent, causing long-term harm to human health and the environment. In this study, a gram-positive, aerobic, and rod-shaped bacterial strain, Z13T, with efficient phenol degradation ability, was isolated from the soil of sugarcane fields. Based on the physiological properties and genomic features, strain Z13T is considered as a novel species of the genus Rhodococcus, for which the name Rhodococcus sacchari sp. nov. is proposed. The type strain is Z13T (= CCTCC AB 2022327T = JCM 35797T). This strain can use phenol as its sole carbon source. Z13T was able to completely degrade 1200 mg/L phenol within 20 h; the maximum specific growth rate was µmax = 0.93174 h-1, and the maximum specific degradation rate was qmax = 0.47405 h-1. Based on whole-genome sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, strain Z13T contains a series of phenol degradation genes, including dmpP, CatA, dmpB, pcaG, and pcaH, and can metabolize aromatic compounds. Moreover, the potential of strain Z13T for soil remediation was investigated by introducing Z13T into simulated phenol-contaminated soil, and the soil microbial diversity was analyzed. The results showed that 100% of the phenol in the soil was removed within 7.5 d. Furthermore, microbial diversity analysis revealed an increase in the relative species richness of Oceanobacillus, Chungangia, and Bacillus.

Biosynthesis of photostable CdS quantum dots by UV-resistant psychrotolerant bacteria isolated from Union Glacier, Antarctica.

BACKGROUND: Quantum Dots (QDs) are fluorescent nanoparticles with exceptional optical and optoelectronic properties, finding widespread utility in diverse industrial applications. Presently, chemically synthesized QDs are employed in solar cells, bioimaging, and various technological domains. However, many applications demand QDs with prolonged lifespans under conditions of high-energy radiation. Over the past decade, microbial biosynthesis of nanomaterials has emerged as a sustainable and cost-effective process. In this context, the utilization of extremophile microorganisms for synthesizing QDs with unique properties has recently been reported. RESULTS: In this study, UV-resistant bacteria were isolated from one of the most extreme environments in Antarctica, Union Glacier at the Ellsworth Mountains. Bacterial isolates, identified through 16 S sequencing, belong to the genera Rhodococcus, Pseudarthrobacter, and Arthrobacter. Notably, Rhodococcus sp. (EXRC-4 A-4), Pseudarthrobacter sp. (RC-2-3), and Arthrobacter sp. (EH-1B-1) tolerate UV-C radiation doses ≥ 120 J/m². Isolated UV-resistant bacteria biosynthesized CdS QDs with fluorescence intensities 4 to 8 times higher than those biosynthesized by E. coli, a mesophilic organism tolerating low doses of UV radiation. Transmission electron microscopy (TEM) analysis determined QD sizes ranging from 6 to 23 nm, and Fourier-transform infrared (FTIR) analysis demonstrated the presence of biomolecules. QDs produced by UV-resistant Antarctic bacteria exhibit high photostability after exposure to UV-B radiation, particularly in comparison to those biosynthesized by E. coli. Interestingly, red fluorescence-emitting QDs biosynthesized by Rhodococcus sp. (EXRC-4 A-4) and Arthrobacter sp. (EH-1B-1) increased their fluorescence emission after irradiation. Analysis of methylene blue degradation after exposure to irradiated QDs biosynthesized by UV-resistant bacteria, indicates that the QDs transfer their electrons to O2 for the formation of reactive oxygen species (ROS) at different levels. CONCLUSIONS: UV-resistant Antarctic bacteria represent a novel alternative for the sustainable generation of nanostructures with increased radiation tolerance-two characteristics favoring their potential application in technologies requiring continuous exposure to high-energy radiation.

Transcriptional dynamics during Rhodococcus erythropolis infection with phage WC1.

BACKGROUND: Belonging to the Actinobacteria phylum, members of the Rhodococcus genus thrive in soil, water, and even intracellularly. While most species are non-pathogenic, several cause respiratory disease in animals and, more rarely, in humans. Over 100 phages that infect Rhodococcus species have been isolated but despite their importance for Rhodococcus ecology and biotechnology applications, little is known regarding the molecular genetic interactions between phage and host during infection. To address this need, we report RNA-Seq analysis of a novel Rhodococcus erythopolis phage, WC1, analyzing both the phage and host transcriptome at various stages throughout the infection process. RESULTS: By five minutes post-infection WC1 showed upregulation of a CAS-4 family exonuclease, putative immunity repressor, an anti-restriction protein, while the host showed strong upregulation of DNA replication, SOS repair, and ribosomal protein genes. By 30 min post-infection, WC1 DNA synthesis genes were strongly upregulated while the host showed increased expression of transcriptional and translational machinery and downregulation of genes involved in carbon, energy, and lipid metabolism pathways. By 60 min WC1 strongly upregulated structural genes while the host showed a dramatic disruption of metal ion homeostasis. There was significant expression of both host and phage non-coding genes at all time points. While host gene expression declined over the course of infection, our results indicate that phage may exert more selective control, preserving the host's regulatory mechanisms to create an environment conducive for virion production. CONCLUSIONS: The Rhodococcus genus is well recognized for its ability to synthesize valuable compounds, particularly steroids, as well as its capacity to degrade a wide range of harmful environmental pollutants. A detailed understanding of these phage-host interactions and gene expression is not only essential for understanding the ecology of this important genus, but will also facilitate development of phage-mediated strategies for bioremediation as well as biocontrol in industrial processes and biomedical applications. Given the current lack of detailed global gene expression studies on any Rhodococcus species, our study addresses a pressing need to identify tools and genes, such as F6 and rpf, that can enhance the capacity of Rhodococcus species for bioremediation, biosynthesis and pathogen control.

Study on the estradiol degradation gene expression and resistance mechanism of Rhodococcus R-001 under low-temperature stress.

Estradiol (E2), an endocrine disruptor, acts by mimicking or interfering with the normal physiological functions of natural hormones within organisms, leading to issues such as endocrine system disruption. Notably, seasonal fluctuations in environmental temperature may influence the degradation speed of estradiol (E2) in the natural environment, intensifying its potential health and ecological risks. Therefore, this study aims to explore how bacteria can degrade E2 under low-temperature conditions, unveiling their resistance mechanisms, with the goal of developing new strategies to mitigate the threat of E2 to health and ecological safety. In this paper, we found that Rhodococcus equi DSSKP-R-001 (R-001) can efficiently degrade E2 at 30 °C and 10 °C. Six genes in R-001 were shown to be involved in E2 degradation by heterologous expression at 30 °C. Among them, 17ß-HSD, KstD2, and KstD3, were also involved in E2 degradation at 10 °C; KstD was not previously known to degrade E2. RNA-seq was used to characterize differentially expressed genes (DEGs) to explore the stress response of R-001 to low-temperature environments to elucidate the strain's adaptation mechanism. At the low temperature, R-001 cells changed from a round spherical shape to a long rod or irregular shape with elevated unsaturated fatty acids and were consistent with the corresponding genetic changes. Many differentially expressed genes linked to the cold stress response were observed. R-001 was found to upregulate genes encoding cold shock proteins, fatty acid metabolism proteins, the ABC transport system, DNA damage repair, energy metabolism and transcriptional regulators. In this study, we demonstrated six E2 degradation genes in R-001 and found for the first time that E2 degradation genes have different expression characteristics at 30 °C and 10 °C. Linking R-001 to cold acclimation provides new insights and a mechanistic basis for the simultaneous degradation of E2 under cold stress in Rhodococcus adaptation.

Development of synthetic small regulatory RNA for Rhodococcus erythropolis.

Rhodococci have been regarded as ideal chassis for biotransformation, biodegradation, and biosynthesis for their unique environmental persistence and robustness. However, most species of Rhodococcus are still difficult to metabolically engineer due to the lack of genetic tools and techniques. In this study, synthetic sRNA strategy was exploited for gene repression in R. erythropolis XP. The synthetic sRNA based on the RhlS scaffold from Pseudomonas aeruginosa functions better in repressing sfgfp expression than those based on E. coli MicC, SgrS, and P. aeruginosa PrrF1-2 scaffold. The RhlS-based sRNAs were applied to study the influence of sulfur metabolism on biodesulfurization (BDS) efficiency in R. erythropolis XP and successfully identified two genes involved in sulfur metabolism that affect the BDS efficiency significantly. The RhlS-based synthetic sRNAs show promise in the metabolic engineering of Rhodococcus and promote the industrial applications of Rhodococcus in environmental remediation and biosynthesis.

Arsenic effectively improves the degradation of fluorene by Rhodococcus sp. 2021 under the combined pollution of arsenic and fluorene.

The performance of bacterial strains in executing degradative functions under the coexistence of heavy metals/heavy metal-like elements and organic contaminants is understudied. In this study, we isolated a fluorene-degrading bacterium, highly arsenic-resistant, designated as strain 2021, from contaminated soil at the abandoned site of an old coking plant. It was identified as a member of the genus Rhodococcus sp. strain 2021 exhibited efficient fluorene-degrading ability under optimal conditions of 400 mg/L fluorene, 30 °C, pH 7.0, and 250 mg/L trivalent arsenic. It was noted that the addition of arsenic could promote the growth of strain 2021 and improve the degradation of fluorene - a phenomenon that has not been described yet. The results further indicated that strain 2021 can oxidize As3+ to As5+; here, approximately 13.1% of As3+ was converted to As5+ after aerobic cultivation for 8 days at 30 °C. The addition of arsenic could greatly up-regulate the expression of arsR/A/B/C/D and pcaG/H gene clusters involved in arsenic resistance and aromatic hydrocarbon degradation; it also aided in maintaining the continuously high expression of cstA that codes for carbon starvation protein and prmA/B that codes for monooxygenase. These results suggest that strain 2021 holds great potential for the bioremediation of environments contaminated by a combination of arsenic and polycyclic aromatic hydrocarbons. This study provides new insights into the interactions among microbes, as well as inorganic and organic pollutants.

(Pan)genomic analysis of two Rhodococcus isolates and their role in phenolic compound degradation.

The genus Rhodococcus is recognized for its potential to degrade a large range of aromatic substances, including plant-derived phenolic compounds. We used comparative genomics in the context of the broader Rhodococcus pan-genome to study genomic traits of two newly described Rhodococcus strains (type-strain Rhodococcus pseudokoreensis R79T and Rhodococcus koreensis R85) isolated from apple rhizosphere. Of particular interest was their ability to degrade phenolic compounds as part of an integrated approach to treat apple replant disease (ARD) syndrome. The pan-genome of the genus Rhodococcus based on 109 high-quality genomes was open with a small core (1.3%) consisting of genes assigned to basic cell functioning. The range of genome sizes in Rhodococcus was high, from 3.7 to 10.9 Mbp. Genomes from host-associated strains were generally smaller compared to environmental isolates which were characterized by exceptionally large genome sizes. Due to large genomic differences, we propose the reclassification of distinct groups of rhodococci like the Rhodococcus equi cluster to new genera. Taxonomic species affiliation was the most important factor in predicting genetic content and clustering of the genomes. Additionally, we found genes that discriminated between the strains based on habitat. All members of the genus Rhodococcus had at least one gene involved in the pathway for the degradation of benzoate, while biphenyl degradation was mainly restricted to strains in close phylogenetic relationships with our isolates. The ~40% of genes still unclassified in larger Rhodococcus genomes, particularly those of environmental isolates, need more research to explore the metabolic potential of this genus.IMPORTANCERhodococcus is a diverse, metabolically powerful genus, with high potential to adapt to different habitats due to the linear plasmids and large genome sizes. The analysis of its pan-genome allowed us to separate host-associated from environmental strains, supporting taxonomic reclassification. It was shown which genes contribute to the differentiation of the genomes based on habitat, which can possibly be used for targeted isolation and screening for desired traits. With respect to apple replant disease (ARD), our isolates showed genome traits that suggest potential for application in reducing plant-derived phenolic substances in soil, which makes them good candidates for further testing against ARD.

Occurrence of Rhodococcus sp. RR1 prmA and Rhodococcus jostii RHA1 prmA across microbial communities and their enumeration during 1,4-dioxane biodegradation.

1,4-Dioxane, a likely human carcinogen, is a co-contaminant at many chlorinated solvent contaminated sites. Conventional treatment technologies, such as carbon sorption or air stripping, are largely ineffective, and so many researchers have explored bioremediation for site clean-up. An important step towards this involves examining the occurrence of the functional genes associated with 1,4-dioxane biodegradation. The current research explored potential biomarkers for 1,4-dioxane in three mixed microbial communities (wetland sediment, agricultural soil, impacted site sediment) using monooxygenase targeted amplicon sequencing, followed by quantitative PCR (qPCR). A BLAST analysis of the sequencing data detected only two of the genes previously associated with 1,4-dioxane metabolism or co-metabolism, namely propane monooxygenase (prmA) from Rhodococcus jostii RHA1 and Rhodococcus sp. RR1. To investigate this further, qPCR primers and probes were designed, and the assays were used to enumerate prmA gene copies in the three communities. Gene copies of Rhodococcus RR1 prmA were detected in all three, while gene copies of Rhodococcus jostii RHA1 prmA were detected in two of the three sample types (except impacted site sediment). Further, there was a statistically significant increase in RR1 prmA gene copies in the microcosms inoculated with impacted site sediment following 1,4-dioxane biodegradation compared to the control microcosms (no 1,4-dioxane) or to the initial copy numbers before incubation. Overall, the results indicate the importance of Rhodococcus associated prmA, compared to other 1,4-dioxane degrading associated biomarkers, in three different microbial communities. Also, the newly designed qPCR assays provide a platform for others to investigate 1,4-dioxane biodegradation potential in mixed communities and should be of particular interest to those considering bioremediation as a potential 1,4-dioxane remediation approach.

Rhodococcus infection: a 10-year retrospective analysis of clinical experience and antimicrobial susceptibility profile.

Rhodococcus equi is an opportunistic pathogen known to cause pulmonary and extrapulmonary disease among immunocompromised patients. Treatment is frequently challenging due to intrinsic resistance to multiple antibiotic classes. While non-equi Rhodococcus spp. are prevalent, their clinical significance is poorly defined. There is also limited data on antibiotic susceptibility testing (AST) of Rhodococcus infection in humans. We conducted a single-center, retrospective cohort study evaluating clinical characteristics, microbiologic profile, and AST of Rhodococcus infections between June 2012 and 2022 at our tertiary academic medical center. Identification of Rhodococcus spp. was performed by Sanger 16S rRNA gene sequencing and/or matrix-assisted laser desorption ionization-time of flight mass spectrometry, and AST was performed by agar dilution. Three hundred twenty-two isolates of Rhodococcus spp. were identified from blood (50%), pulmonary (26%), and bone/joint (12%) sources. R. equi/hoagii, R. corynebacterioides, and R. erythropolis were the most frequently isolated species, with 19% of isolates identified only to genus level. One hundred ninety-nine isolates evaluated for AST demonstrated high-level resistance to amoxicillin/clavulanate, cephalosporins, and aminoglycosides. More than 95% susceptibility to imipenem, vancomycin, linezolid, rifampin, and clarithromycin was observed. Non-equi species showed a significantly more favorable AST profile relative to R. equi. Clinically significant Rhodococcus infection was rare with 10 cases diagnosed (majority due to R. equi) and managed. The majority of patients received 2- or 3-drug combination therapy for 2-6 months, with favorable clinical response. Significant differences in AST were observed between R. equi and non-equi species. Despite high antimicrobial resistance to several antibiotic classes, imipenem and vancomycin remain appropriate empiric treatment options for R. equi. Future research evaluating mechanisms underlying antimicrobial resistance is warranted.

The catabolism of lignin-derived p-methoxylated aromatic compounds by Rhodococcus jostii RHA1.

Emergent strategies to valorize lignin, an abundant but underutilized aromatic biopolymer, include tandem processes that integrate chemical depolymerization and biological catalysis. To date, aromatic monomers from C-O bond cleavage of lignin have been converted to bioproducts, but the presence of recalcitrant C-C bonds in lignin limits the product yield. A promising chemocatalytic strategy that overcomes this limitation involves phenol methyl protection and autoxidation. Incorporating this into a tandem process requires microbial cell factories able to transform the p-methoxylated products in the resulting methylated lignin stream. In this study, we assessed the ability of Rhodococcus jostii RHA1 to catabolize the major aromatic products in a methylated lignin stream and elucidated the pathways responsible for this catabolism. RHA1 grew on a methylated pine lignin stream, catabolizing the major aromatic monomers: p-methoxybenzoate (p-MBA), veratrate, and veratraldehyde. Bioinformatic analyses suggested that a cytochrome P450, PbdA, and its cognate reductase, PbdB, are involved in p-MBA catabolism. Gene deletion studies established that both pbdA and pbdB are essential for growth on p-MBA and several derivatives. Furthermore, a deletion mutant of a candidate p-hydroxybenzoate (p-HBA) hydroxylase, ΔpobA, did not grow on p-HBA. Veratraldehyde and veratrate catabolism required both vanillin dehydrogenase (Vdh) and vanillate O-demethylase (VanAB), revealing previously unknown roles of these enzymes. Finally, a ΔpcaL strain grew on neither p-MBA nor veratrate, indicating they are catabolized through the ß-ketoadipate pathway. This study expands our understanding of the bacterial catabolism of aromatic compounds and facilitates the development of biocatalysts for lignin valorization.IMPORTANCELignin, an abundant aromatic polymer found in plant biomass, is a promising renewable replacement for fossil fuels as a feedstock for the chemical industry. Strategies for upgrading lignin include processes that couple the catalytic fractionation of biomass and biocatalytic transformation of the resulting aromatic compounds with a microbial cell factory. Engineering microbial cell factories for this biocatalysis requires characterization of bacterial pathways involved in catabolizing lignin-derived aromatic compounds. This study identifies new pathways for lignin-derived aromatic degradation in Rhodococcus, a genus of bacteria well suited for biocatalysis. Additionally, we describe previously unknown activities of characterized enzymes on lignin-derived compounds, expanding their utility. This work advances the development of strategies to replace fossil fuel-based feedstocks with sustainable alternatives.

Fruit residues as substrates for single-cell oil production by Rhodococcus species: physiology and genomics of carbohydrate catabolism.

Strains belonging to R. opacus, R. jostii, R. fascians, R. erythropolis and R. equi exhibited differential ability to grow and produce lipids from fruit residues (grape marc and apple pomace), as well as single carbohydrates, such as glucose, gluconate, fructose and sucrose. The oleaginous species, R. opacus (strains PD630 and MR22) and R. jostii RHA1, produced higher yields of biomass (5.1-5.6 g L-1) and lipids (38-44% of CDW) from apple juice wastes, in comparison to R. erythropolis DSM43060, R. fascians F7 and R. equi ATCC6939 (4.1-4.3 g L-1 and less than 10% CDW of lipids). The production of cellular biomass and lipids were also higher in R. opacus and R. jostii (6.8-7.2 g L-1 and 33.9-36.5% of CDW of lipids) compared to R. erythropolis, R. fascians, and R. equi (3.0-3.6 g L-1 and less than 10% CDW of lipids), during cultivation of cells on wine grape waste. A genome-wide bioinformatic analysis of rhodococci indicated that oleaginous species possess a complete set of genes/proteins necessary for the efficient utilization of carbohydrates, whereas genomes from non-oleaginous rhodococcal strains lack relevant genes coding for transporters and/or enzymes for the uptake, catabolism and assimilation of carbohydrates, such as gntP, glcP, edd, eda, among others. Results of this study highlight the potential use of the oleaginous rhodococcal species to convert sugar-rich agro-industrial wastes, such as apple pomace and grape marc, into single-cell oils.

Transmission and Management of Pathogenic Agrobacterium tumefaciens and Rhodococcus fascians in Select Ornamentals.

Pathogenic Agrobacterium tumefaciens and Rhodococcus fascians are phytobacteria that induce crown gall and leafy gall disease, respectively, resulting in undesirable growth abnormalities. When present in nurseries, plants infected by either bacterium are destroyed, resulting in substantial losses for growers, especially those producing plants valued for their ornamental attributes. There are many unanswered questions regarding pathogen transmission on tools used to take cuttings for propagation and whether products used for bacterial disease control are effective. We investigated the ability to transmit pathogenic A. tumefaciens and R. fascians on secateurs and the efficacy of registered control products against both bacteria in vitro and in vivo. Experimental plants used were Rosa × hybrida, Leucanthemum × superbum, and Chrysanthemum × grandiflorum for A. tumefaciens and Petunia × hybrida and Oenothera 'Siskiyou' with R. fascians. In separate experiments, we found secateurs could convey both bacteria in numbers sufficient to initiate disease in a host-dependent manner and that bacteria could be recovered from secateurs after a single cut through an infected stem. In in vivo assays, none of six products tested against A. tumefaciens prevented crown gall disease, although several products appeared promising in in vitro trials. Likewise, four compounds trialed against R. fascians failed to prevent disease. Sanitation and clean planting material remain the primary means of disease management.

Overexpression of endogenous multi-copper oxidases mcoA and mcoC in Rhodococcus jostii RHA1 enhances lignin bioconversion to 2,4-pyridine-dicarboxylic acid.

To improve the titre of lignin-derived pyridine-dicarboxylic acid (PDCA) products in engineered Rhodococcus jostii RHA1 strains, plasmid-based overexpression of seven endogenous and exogenous lignin-degrading genes was tested. Overexpression of endogenous multi-copper oxidases mcoA, mcoB, and mcoC was found to enhance 2,4-PDCA production by 2.5-, 1.4-, and 3.5-fold, respectively, while overexpression of dye-decolorizing peroxidase dypB was found to enhance titre by 1.4-fold, and overexpression of Streptomyces viridosporus laccase enhanced titre by 1.3-fold. The genomic context of the R. jostii mcoA gene suggests involvement in 4-hydroxybenzoate utilization, which was consistent with enhanced whole cell biotransformation of 4-hydroxybenzoate by R. jostii pTipQC2-mcoA. These data support the role of multi-copper oxidases in bacterial lignin degradation, and provide an opportunity to enhance titres of lignin-derived bioproducts.

Construction and characterization of a temperature-sensitive pRC4 replicon for Rhodococcus and Gordonia.

Temperature-sensitive plasmids are useful for genome engineering and several synthetic biology applications. There are only limited reports on temperature-sensitive plasmids for Rhodococcus and none for Gordonia. Here, we report the construction of a temperature-sensitive pRC4 replicon that is functional in Rhodococcus and Gordonia. The amino acid residues were predicted for the temperature-sensitive phenotype in the pRC4 replicon using in silico methods and molecular simulation of the DNA-binding replication protein with the origin of replication. The amino acid residues were mutated, and the temperature-sensitive phenotype was validated in Gordonia sp. IITR100. Similar results were also observed in Rhodococcus erythropolis, suggesting that the temperature-sensitive phenotype was exhibited across genera.

Unleashing the capacity of Rhodococcus for converting lignin into lipids.

Bioconversion of bioresources/wastes (e.g., lignin, chemical pulping byproducts) represents a promising approach for developing a bioeconomy to help address growing energy and materials demands. Rhodococcus, a promising microbial strain, utilizes numerous carbon sources to produce lipids, which are precursors for synthesizing biodiesel and aviation fuels. However, compared to chemical conversion, bioconversion involves living cells, which is a more complex system that needs further understanding and upgrading. Various wastes amenable to bioconversion are reviewed herein to highlight the potential of Rhodococci for producing lipid-derived bioproducts. In light of the abundant availability of these substrates, Rhodococcus' metabolic pathways converting them to lipids are analyzed from a "beginning-to-end" view. Based on an in-depth understanding of microbial metabolic routes, genetic modifications of Rhodococcus by employing emerging tools (e.g., multiplex genome editing, biosensors, and genome-scale metabolic models) are presented for promoting the bioconversion. Co-solvent enhanced lignocellulose fractionation (CELF) strategy facilitates the generation of a lignin-derived aromatic stream suitable for the Rhodococcus' utilization. Novel alkali sterilization (AS) and elimination of thermal sterilization (ETS) approaches can significantly enhance the bioaccessibility of lignin and its derived aromatics in aqueous fermentation media, which promotes lipid titer significantly. In order to achieve value-added utilization of lignin, biodiesel and aviation fuel synthesis from lignin and lipids are further discussed. The possible directions for unleashing the capacity of Rhodococcus through synergistically modifying microbial strains, substrates, and fermentation processes are proposed toward a sustainable biological lignin valorization.

MLDSR, the transcriptional regulator of the major lipid droplets protein MLDS, is controlled by long-chain fatty acids and contributes to the lipid-accumulating phenotype in oleaginous Rhodococcus strains.

Microorganism lipid droplet small regulator (MLDSR) is a transcriptional regulator of the major lipid droplet (LD)-associated protein MLDS in Rhodococcus jostii RHA1 and Rhodococcus opacus PD630. In this study, we investigated the role of MLDSR on lipid metabolism and triacylglycerol (TAG) accumulation in R. jostii RHA1 at physiological and molecular levels. MLDSR gene deletion promoted a significant decrease of TAG accumulation, whereas inhibition of de novo fatty acid biosynthesis by the addition of cerulenin significantly repressed the expression of the mldsr-mlds cluster under nitrogen-limiting conditions. In vitro and in vivo approaches revealed that MLDSR-DNA binding is inhibited by fatty acids and acyl-CoA residues through changes in the oligomeric or conformational state of the protein. RNAseq analysis indicated that MLDSR not only controls the expression of its own gene cluster but also of several genes involved in central, lipid, and redox metabolism, among others. We also identified putative MLDSR-binding sites on the upstream regions of genes coding for lipid catabolic enzymes and validated them by EMSA assays. Overexpression of mldsr gene under nitrogen-rich conditions promoted an increase of TAG accumulation, and further cell lysis with TAG release to the culture medium. Our results suggested that MLDSR is a fatty acid-responsive regulator that plays a dual role in cells by repression or activation of several metabolic genes in R. jostii RHA1. MLDSR seems to play an important role in the fine-tuning regulation of TAG accumulation, LD formation, and cellular lipid homeostasis, contributing to the oleaginous phenotype of R. jostii RHA1 and R. opacus PD630.