Construction of a BioBrick™ compatible vector system for Rhodococcus

Ellinger, James J.; Schmidt‐Dannert, Claudia

doi:10.1016/j.plasmid.2017.01.004

Cited by 17 publications

(14 citation statements)

References 41 publications

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“…In this plasmid, gfp-parB is under the control of the Ptrc promoter (23). The Ptrc promoter has been previously shown to work in Rhodococcus (43). The plasmid pDS2 has been shown to be stable up to 60 generations (38).…”

Section: Methodsmentioning

confidence: 99%

Rhodoccoccus erythropolis Is Different from Other Members of Actinobacteria : Monoploidy, Overlapping Replication Cycle, and Unique Segregation Pattern

et al. 2019

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Among actinomycetes, chromosome organization and segregation studies have been limited to Streptomyces coelicolor, Corynebacterium glutamicum, and Mycobacterium spp. There are differences with respect to ploidy and chromosome organization pattern in these bacteria. Here, we report on chromosome replication, organization, and segregation in Rhodococcus erythropolis PR4, which has a circular genome of 6.5 Mbp. The origin of replication of R. erythropolis PR4 was identified, and the DNA content in the cell under different growth conditions was determined. Our results suggest that the number of origins increases as the growth medium becomes rich, suggesting an overlapping replication cell cycle in this bacterium. Subcellular localization of the origin region revealed polar positioning in minimal and rich media. The terminus, which is the last region to be replicated and segregated, was found to be localized at the cell center in large cells. The middle markers corresponding to the 1.5-Mb and 4.7-Mb loci did not overlap, suggesting discontinuity in the segregation of the two arms of the chromosome. Chromosome segregation was not affected by inhibiting cell division. Deletion of parA or parB affected chromosome segregation. Unlike in C. glutamicum and Streptomyces spp., diploidy or polyploidy was not observed in R. erythropolis PR4. Our results suggest that R. erythropolis is different from other members of Actinobacteria; it is monoploid and has a unique chromosome segregation pattern. This is the first report on chromosome organization, replication, and segregation in R. erythropolis PR4. IMPORTANCE Rhodococci are highly versatile Gram-positive bacteria with high bioremediation potential. Some rhodococci are pathogenic and have been suggested as emerging threats. No studies on the replication, segregation, and cell cycle of these bacteria have been reported. Here, we demonstrate that the genus Rhodococcus is different from other actinomycetes, such as members of the genera Corynebacterium, Mycobacterium, and Streptomyces, with respect to ploidy and chromosome organization and segregation. Such studies will be useful not only in designing better therapeutics pathogenic strains in the future but also for studying genome maintenance in strains used for bioremediation.

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Section: Methodsmentioning

confidence: 99%

Rhodoccoccus erythropolis Is Different from Other Members of Actinobacteria : Monoploidy, Overlapping Replication Cycle, and Unique Segregation Pattern

et al. 2019

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“…; ~ 10 copies per chromosome[82, 84]pGA1Derived from Corynebeacterium spp.[63, 87, 95, 96]pSR1Derived from Corynebeacterium spp.[85, 86, 88]pB264Derived from Rhodococcus sp. B264; curable; ~ 8 copies per chromosome[73]Selection markersKanamycin50 μg/mL (selection)250 μg/mL (plasmid function maintenance)[85, 88, 97]Gentamicin10 μg/mL[82]Spectinomycin100 μg/mL[82]Thiostrepton1 μg/mL[97]Chloramphenicol34 μg/mL[98, 99]Hygromycin B50 μg/mL[73]SacBNegative selection; sensitizes cell to sucrose[100, 101]PromoterspTipAInducible with thiostrepton[97, 102, 103]pAcet5× inducible with acetamide[98, 104]pBAD59× inducible with arabinose[104]pTet67× inducible with anhydrotetracycline (aTc)[104]pLPD06740247× inducible with phenol[104]p...…”

Section: Tool and Technique Development For R Opacus Engineeringmentioning

confidence: 99%

Development of Rhodococcus opacus as a chassis for lignin valorization and bioproduction of high-value compounds

Anthony

Carr

DeLorenzo

et al. 2019

Biotechnol Biofuels

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The current extraction and use of fossil fuels has been linked to extensive negative health and environmental outcomes. Lignocellulosic biomass-derived biofuels and bioproducts are being actively considered as renewable alternatives to the fuels, chemicals, and materials produced from fossil fuels. A major challenge limiting large-scale, economic deployment of second-generation biorefineries is the insufficient product yield, diversity, and value that current conversion technologies can extract from lignocellulose, in particular from the underutilized lignin fraction. Rhodococcus opacus PD630 is an oleaginous gram-positive bacterium with innate catabolic pathways and tolerance mechanisms for the inhibitory aromatic compounds found in depolymerized lignin, as well as native or engineered pathways for hexose and pentose sugars found in the carbohydrate fractions of biomass. As a result, R. opacus holds potential as a biological chassis for the conversion of lignocellulosic biomass into biodiesel precursors and other value-added products. This review begins by examining the important role that lignin utilization will play in the future of biorefineries and by providing a concise survey of the current lignin conversion technologies. The genetic machinery and capabilities of R. opacus that allow the bacterium to tolerate and metabolize aromatic compounds and depolymerized lignin are also discussed, along with a synopsis of the genetic toolbox and synthetic biology methods now available for engineering this organism. Finally, we summarize the different feedstocks that R. opacus has been demonstrated to consume, and the high-value products that it has been shown to produce. Engineered R. opacus will enable lignin valorization over the coming years, leading to cost-effective conversion of lignocellulose into fuels, chemicals, and materials.

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“…This standardization of methods, materials, and parts seeks to enable the rapid development of modular parts through the division of labor and characterization across the bioengineering field. Much of the work has been done in model organisms such as Bacillus subtilis [165] and E. coli [166], with some recent development in the non-model yeast Yarrowia lipolytica [167] and Rhodococcus opacus [168]. Characterizing pathways in a variety of hosts and conditions can identify situations where pathways do and do not function reliably.…”

Section: Characterization Of Engineered Pathways In Diverse Hosts Andmentioning

confidence: 99%

Modular Engineering of Biomass Degradation Pathways

Chaves

Presley

2019

Processes

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Production of fuels and chemicals from renewable lignocellulosic feedstocks is a promising alternative to petroleum-derived compounds. Due to the complexity of lignocellulosic feedstocks, microbial conversion of all potential substrates will require substantial metabolic engineering. Non-model microbes offer desirable physiological traits, but also increase the difficulty of heterologous pathway engineering and optimization. The development of modular design principles that allow metabolic pathways to be used in a variety of novel microbes with minimal strain-specific optimization will enable the rapid construction of microbes for commercial production of biofuels and bioproducts. In this review, we discuss variability of lignocellulosic feedstocks, pathways for catabolism of lignocellulose-derived compounds, challenges to heterologous engineering of catabolic pathways, and opportunities to apply modular pathway design. Implementation of these approaches will simplify the process of modifying non-model microbes to convert diverse lignocellulosic feedstocks.

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Construction of a BioBrick™ compatible vector system for Rhodococcus

Cited by 17 publications

References 41 publications

Rhodoccoccus erythropolis Is Different from Other Members of Actinobacteria : Monoploidy, Overlapping Replication Cycle, and Unique Segregation Pattern

Rhodoccoccus erythropolis Is Different from Other Members of Actinobacteria : Monoploidy, Overlapping Replication Cycle, and Unique Segregation Pattern

Development of Rhodococcus opacus as a chassis for lignin valorization and bioproduction of high-value compounds

Modular Engineering of Biomass Degradation Pathways

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