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.

Keywords: Mass spectrometry; Mycolic acid; Phenol; Phospholipid; Rhodococcus opacus; Triacylglycerol.

Fig. 1

Conversion of phenol to lipids by R. opacus PD630. a Cell membrane structure and simplified model for conversion of a lignin monomer (phenol) to lipids by R. opacus PD630 [42]. Phenol is imported into the cell and converted to central metabolic intermediates acetyl-CoA and succinyl-CoA [20, 72]. Next, acetyl-CoA is converted into acyl-CoAs by fatty acid synthase (FAS) Ia [32]. Phospholipids (PLs), triacylglycerols (TAGs), and α-branches of mycolic acids (MAs) are synthesized using acyl-CoAs from FAS Ia, while meromycolate branches for mycolic acids are synthesized by further chain elongation using other fatty acid synthases (FAS II) or mycocerosic acid synthases (MAS) [32]. Triacylglycerols can be stored in lipid droplets [30]. PM phospholipid membrane, PG peptidoglycan layer, AG arabinogalactan layer, MM mycomembrane, and OL outer layer. b Representative structures of lipid types examined in this work.

Fig. 2

Principal component analysis of lipid types identified using LC/MS. a Principal component analysis (PCA) of all identified lipids. b PCA of mycolic acid species. c PCA of triacylglycerol species. d PCA of phospholipid species. Because triacylglycerol ion counts are > 98% of total ion counts in each sample, the PCA plots for all lipid species (a) and triacylglycerol species (c) are almost identical. Each point represents one replicate. WTG WT strain grown in 1 g/L glucose, 40G evol40 grown in 1 g/L glucose, WTLP WT strain grown in 0.75 g/L phenol, 40LP evol40 strain grown in 0.75 g/L phenol, 40HP evol40 strain grown in 1.5 g/L phenol, PC1 1st principal component, PC2 2nd principal component. Percent represents the amount of variance explained by each principal component.

Fig. 3

Mycolic acid composition of R. opacus strains using glucose or phenol as a sole carbon source. a Heat map of double bond (DB) numbers for mycolic acids (MAs) in the WT and evol40 strains. The values shown in the heat map are the average of three replicates. See color bar for scale. The DB number represents the total number of double bonds and cyclopropane units on acyl chains. b Heat map of MA carbon (C) number distribution, defined as the total number of carbons located on acyl chains. The values shown in the heat map are the average of three replicates. See color bar for scale. WTG WT strain grown in 1 g/L glucose (glucose), 40G evol40 grown in glucose, WTLP WT strain grown in 0.75 g/L phenol (low phenol), 40LP evol40 strain grown in low phenol, 40HP evol40 strain grown in 1.5 g/L phenol (high phenol). MA percentage is defined as the total ion counts of each category (DB number or C number) divided by the total ion counts of all mycolic acids detected in each sample.

Fig. 4

Phospholipid composition of R. opacus strains using glucose or phenol as a sole carbon source. a Ratio of phosphatidylinositol (PI) to phosphatidylethanolamine (PE) total ion counts in the WT and evol40 strains using glucose or phenol as a sole carbon source. b Percentage of unsaturated phospholipids (PLs) (i.e., phospholipids with at least one unsaturated fatty acyl substituent) in WT and evol40 strains using glucose or phenol as a sole carbon source. c Distribution of phospholipid carbon (C) numbers in WT and evol40 strains. Here, C number is defined as the total number of acyl chain carbons, and unsaturation means double bonds and cyclic chains on acyl substituents. Each square is the average of three replicates. PL percentage is defined as the total ion counts of each category (unsaturated phospholipids or C number) divided by the total ion counts of all detected PI and PE species in the sample. For A and B, bars represent the average of three replicates, error bars represent one standard deviation, and statistical significance was determined using a one mean, two-tailed Student’s t test (*P < 0.05; **P < 0.01). WTG WT strain grown in 1 g/L glucose (glucose), 40G evol40 grown in glucose, WTLP WT strain grown in 0.75 g/L phenol (low phenol), 40LP evol40 strain grown in low phenol, 40HP evol40 strain grown in 1.5 g/L phenol (high phenol).

Fig. 5

Triacylglycerol compositions of WT and evol40 strains using glucose or phenol as a sole carbon source. a Distribution of TAG double bond (DB) numbers in the WT and evol40 strains in each condition. The DB number represents the total number of double bonds and cyclopropane units on acyl chains. b Heat map of TAG carbon (C) number distribution, with the C number defined as the total number of acyl chain carbons. TAG percentage is defined as the total ion counts of each category (DB number or C number) divided by the total ion counts of all TAGs detected in each sample. Values in the heat map are the average of three replicates. See color bar for scale. WTG WT strain grown in 1 g/L glucose, 40G evol40 grown in 1 g/L glucose, WTLP WT strain grown in 0.75 g/L phenol, 40LP evol40 strain grown in 0.75 g/L phenol, 40HP evol40 strain grown in 1.5 g/L phenol.