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

Abstract

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.

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

“…Since lignocellulosic degradation products are mixtures of compounds, R. opacus's co-utilization patterns will help to develop predictive metabolic models as well as guide future metabolic engineering efforts toward more-efficient conversion of lignocellulose to biofuels and biochemicals. Combining our findings from this work with our previous reports (Hollinshead et al, 2015;Henson et al, 2018a;Henson et al, 2018b, Yoneda et al, 2016, we are developing a genome-scale metabolic model that enables prediction of metabolic fluxes for diverse carbon sources. Quantitative flux profiles obtained through 13 C-MFA provide an accurate description of internal metabolism that is invaluable when deciding future metabolic engineering approaches.…”

“…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.…”

Robust microorganisms for biofuel and chemical production from municipal solid waste

“…On the other hand, R. opacus, the other Gram-positive species in our collection, did not show any obvious signs of substrate inhibition (Fig. 3h), likely because it has an unusually complex mycolic-acid envelope which has been associated with phenol tolerance and even enables growth on phenol as a sole carbon source [83].…”

Responses to Ecopollutants and Pathogenization Risks of Saprotrophic Rhodococcus Species

“…Lipids are one of the few groups of complex organic substances for which the composition can be adequately regulated by the bacterial cell in response to suboptimal growth conditions by changing the structure and relative amount of membrane fatty acids [ 116 ]. The key role in determining the fluidity and permeability of Rhodococcus cell membranes is due to the composition of fatty acids, which can vary in the length and degree of saturation of the hydrocarbon chains depending on the organic substrate consumed [ 29 , 117 , 118 , 119 ].…”