Development of Chemical and Metabolite Sensors for <i>Rhodococcus opacus</i> PD630

DeLorenzo, Drew M.; Henson, William R.; Moon, Tae Seok

doi:10.1021/acssynbio.7b00192

Cited by 37 publications

(66 citation statements)

References 34 publications

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“…Third, 13 C analysis of the adaptively-evolved strains shows that their genetic differences do not lead to different flux networks. Recently, reliable genetic parts and engineering tools have been developed for R. opacus, allowing for gene expression control and genome modification in this strain (Delorenzo et al, 2017;Delorenzo et al, 2018). Future work will investigate how model-guided perturbations of its metabolism (e.g., knockouts and overexpression), as opposed to adaptive evolution, affects R. opacus's flux network.…”

Section: Discussionmentioning

confidence: 99%

“…All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). All cells were grown in minimal media, previously described as media B (DeLorenzo et al, 2017). Unless otherwise noted, the sole carbon source was 0.5 g/L phenol, and the nitrogen source was 1 g/L ammonium sulfate.…”

Section: Methodsmentioning

confidence: 99%

“…Isolated from a gas works plant, this strain is capable of accumulating triacylglycerol (TAG), the precursor for biodiesel up to 80% of its dry cell weight (Alvarez et al, 1996). Studies of its mechanisms of aromatic tolerance and TAG accumulation have found promising results for its use in the conversion of lignin-derived aromatic substrates (DeLorenzo et al, 2017;Henson et al, 2018a;Henson et al, 2018b;Yoneda et al, 2016). R. opacus has been extensively studied for its growth kinetics and transcriptional activities, yet key knowledge gaps from genotype to phenotype still remain.…”

Section: Introductionmentioning

confidence: 99%

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

Carr

Campbell

Shang

et al. 2019

Metabolic Engineering

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Carr

Campbell

Shang

et al. 2019

Metabolic Engineering

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“…R . opacus has been previously engineered to facilitate lignocellulose conversion 3 , 4 , 7 , 8 and a substantial genetic toolbox has recently been developed 9 , 10 . However, a deep understanding of this organism’s metabolism and any heterologous pathways being expressed is required to maximize its potential.…”

Section: Introductionmentioning

confidence: 99%

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

DeLorenzo

Moon

2018

Sci Rep

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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.

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“…R. opacus is a Gram-positive actinomycete bacterium that can accumulate triacylglycerols (TAGs), a biodiesel precursor, up to ~ 78% of its cell dry weight when grown on sugars [25]. Moreover, with the newly developed synthetic biology tools [26][27][28], R. opacus could also be engineered for the bioconversion of lignin into many other bioproducts. While many studies have focused on characterizing the lipid metabolism of R. opacus for the goal of improving TAG accumulation [29][30][31][32], it is unknown whether, or how, the lipid metabolism plays a role in the tolerance of aromatic compounds.…”

Section: Introductionmentioning

confidence: 99%

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

Henson

Hsu

Dantas

et al. 2018

Biotechnol Biofuels

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Background: Lignin 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. Results: We 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. Conclusions: This 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.

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Development of Chemical and Metabolite Sensors for Rhodococcus opacus PD630

Cited by 37 publications

References 34 publications

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

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

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

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

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