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

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. Electronic supplementary material The online version of this article (10.1186/s13068-018-1337-z) contains supplementary material, which is available to authorized users.

, where F is the fraction of the total mycolic acids with a particular double bond number and n is the corresponding double bond number. 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. MA percentage is defined as the total ion counts of each category (i.e. DB number) divided by the total ion counts of all detected MA species in the sample. Bars represent the average of three replicates, and error bars represent one standard deviation. For A, statistical significance was calculated using a one mean, two tailed Student's t-test with P < 0.05 as a threshold for statistical significance. Letters above bars indicate statistical significance between strain/growth conditions. For A, a = WTLP vs. 40LP, b = WTLP vs. 40HP, c = 40LP vs. 40HP, d = WTG vs. WTLP, e = 40G vs. 40LP, f = 40G vs. 40HP. For B, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001. , where F is the fraction of the total mycolic acids with a particular carbon number and C is the corresponding carbon number. Bars represent the average of three replicates, and error bars represent one standard deviation. Statistical significance was calculated using a one mean, two tailed Student's t-test with P < 0.05 as a threshold for statistical significance. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.  Figure S3. Carbon (C) number distribution of mycolic acid species. 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. Mycolic acid percentage is defined as the total ion counts of each category (i.e. C number) divided by the total ion counts of all detected mycolic acid species in the sample. Bars represent the average of three replicates, and error bars represent one standard deviation.

Figure S4. Phosphatidylinositol (PI) and phosphatidylethanolamine (PE) average carbon (C) number and unsaturation percentage.
A. Average PI carbon number. B. Average PE carbon number. C. Percentage of unsaturated PI species. D. Percentage of unsaturated PE species. 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. The average C number was calculated using the following equation: , where F is the fraction of the total PI or PE species with a particular carbon number and C is the corresponding carbon number. Unsaturated PI (or PE) percentage is defined as the total ion counts of PI (or PE) species with at least one unsaturated fatty acyl substituent divided by the total ion counts of all detected PI (or PE) species in the sample. Bars represent the average of three replicates, and error bars represent one standard deviation. Statistical significance was calculated using a one mean, two tailed Student's t-test with P < 0.05 as a threshold for statistical significance. For C and D, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001. Figure S5. Carbon (C) number distribution of phospholipid (PL) species. 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. PL percentage is defined as the total ion counts of each category (i.e. C number) divided by the total ion counts of all detected PL species in the sample. Bars represent the average of three replicates, and error bars represent one standard deviation. Statistical significance was calculated using a one mean, two tailed Student's t-test with P < 0.05 as a threshold for statistical significance. Letters above bars indicate statistical significance between strain/growth conditions. a = WTLP vs. 40LP, b = WTLP vs. 40HP, c = 40LP vs. 40HP, d = WTG vs. WTLP, e = 40G vs. 40LP, f = 40G vs. 40HP. , where F is the fraction of the total TAGs with a particular double bond number and n is the corresponding double bond number. 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. TAG percentage is defined as the total ion counts of each category (i.e. DB number) divided by the total ion counts of all detected TAG species in the sample. Bars represent the average of three replicates, and error bars represent one standard deviation. For A, statistical significance was calculated using a one mean, two tailed Student's t-test with P < 0.05 as threshold for statistical significance. Letters above bars indicate statistical significance between strain/growth conditions. For A, a = WTLP vs. 40LP, b = WTLP vs. 40HP, c = 40LP vs. 40HP, d = WTG vs. WTLP, e = 40G vs. 40LP, f = 40G vs. 40HP. For B, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.  Figure S7. Triacylglycerol (TAG) carbon (C) number distribution. 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. TAG percentage is defined as the total ion counts of each category (i.e. C number) divided by the total ion counts of all detected TAG species in the sample. Bars represent the average of three replicates, and error bars represent one standard deviation. , where F is the fraction of the total TAGs with a particular carbon number and C is the corresponding carbon number. Bars represent the average of three replicates, and error bars represent one standard deviation. Statistical significance was calculated using a one mean, two tailed Student's t-test with P < 0.05 as threshold for statistical significance. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001. Table S1. Excel file containing the lipid library and lipid library matches from LC/MS data. For all tabs, 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), and SD = standard deviation. Lipid species are abbreviated using their carbon number (C) and double bond number (DB), where the carbon number is the number of acyl carbons and the double bond number is the number of double bonds and cyclopropane units on acyl chains.

Tab 1. TAG library. Library of triacylglycerol (TAG) species based on MS n data.
Lipid species in the library that were observed in MS n analysis (Observed MS n column) are indicated using "x" (see Table 3).
Tab 2. TAG data. Normalized TAG species ion counts from LC/MS data. Species amounts in each replicate (rep) are normalized by dividing the total ion counts of individual species by the total ion counts of all detected TAG species in the sample.

Tab 3. PL library. Library of phospholipid (PL) species based on MS n data.
Lipid species in the library that were observed in MS n analysis (Observed MS n column) are indicated using "x" (see Table 1).

Tab 4. PL data. Normalized PL species ion counts from LC/MS data.
Species amounts in each replicate (rep) are normalized by dividing the total ion counts of individual species by the total ion counts of all detected phosphatidylinositol (PI) or phosphatidylethanolamine (PE) species in the sample.
Tab 5. MA library. Library of mycolic acid (MA) species based on MS n data. Lipid species in the library that were observed in MS n analysis (Observed MS n column) are indicated using "x" (see Table 2). Tab 6. MA data. Normalized MA species ion counts from LC/MS data. Species amounts in each replicate (rep) are normalized by dividing the total ion counts of individual species by the total ion counts of all detected MA species in the sample.
Tab 7. Statistical tests. P values for statistical tests comparing C number and DB number for each lipid class between strains or growth conditions. Statistical significance was determined using a one mean, two-tailed Student's t-test with a threshold of significance of P < 0.05.