Steryl Ester Formation and Accumulation in Steroid-Degrading Bacteria

Abstract

Steryl esters (SEs) are important storage compounds in many eukaryotes and are often prominent components of intracellular lipid droplets. Here, we demonstrate that selected Actino- and Proteobacteria growing on sterols are also able to synthesize SEs and to sequester them in cytoplasmic lipid droplets. We found cholesteryl ester (CE) formation in members of the actinobacterial genera Rhodococcus, Mycobacterium, and Amycolatopsis, as well as several members of the proteobacterial Cellvibrionales order. CEs maximally accumulated under nitrogen-limiting conditions, suggesting that steryl ester formation plays a crucial role for storing excess energy and carbon under adverse conditions. Rhodococcus jostii RHA1 was able to synthesize phytosteryl and cholesteryl esters, the latter reaching up to 7% of its cellular dry weight and 69% of its lipid droplets. Purified lipid droplets from RHA1 contained CEs, free cholesterol, and triacylglycerols. In addition, we found formation of CEs in Mycobacterium tuberculosis when it was grown with cholesterol plus an additional fatty acid substrate. This study provides a basis for the application of bacterial whole-cell systems in the biotechnological production of SEs for use in functional foods and cosmetics.IMPORTANCE Oleaginous bacteria exhibit great potential for the production of high-value neutral lipids, such as triacylglycerols and wax esters. This study describes the formation of steryl esters (SEs) as neutral lipid storage compounds in sterol-degrading oleaginous bacteria, providing a basis for biotechnological production of SEs using bacterial systems with potential applications in the functional food, nutraceutical, and cosmetic industries. We found cholesteryl ester (CE) formation in several sterol-degrading Actino- and Proteobacteria under nitrogen-limiting conditions, suggesting an important role of this process in storing energy and carbon under adverse conditions. In addition, Mycobacterium tuberculosis grown on cholesterol accumulated CEs in the presence of an additional fatty acid substrate.

Keywords: Cellvibrionales; Mycobacterium; Rhodococcus; neutral lipids; sterols; steryl esters.

FIG 1

Schematic representation of triacylglycerols (TAGs) and wax esters (WEs) as neutral lipid storage compounds in bacteria and of a cholesterol ester. Shown are the structures of the major animal, plant, and fungal sterols cholesterol, β-sitosterol, and ergosterol.

FIG 2

Growth of strain RHA1 under nitrogen-limiting and nitrogen-excess conditions. (A) Early stationary phase (around 100 h). (B) TLC analysis of neutral lipid extracts from early-stationary-phase N-limited (−N) and N-excess (+N) cultures grown with different carbon sources. Abbreviations: CP, cholesterol palmitate; C, cholesterol; TAG, triacylglycerols; Ph, phytosterol mix; S, succinate; P, pyruvate; G, glucose; NH₄⁺, N replenished in stationary-phase C − N culture. The volume of neutral lipid extracts applied to TLC plates was normalized on the extracted cellular dry weight of each culture. Red arrows indicate standard spots. Results were obtained on two TLC plates which were treated exactly the same way throughout the experiment. The results of one representative experiment out of several biological and technical replicates are shown for each substrate.

FIG 3

Growth of RHA1 on 1 mM cholesterol under nitrogen-limiting conditions (A) and nitrogen-excess conditions (B). The volume of neutral lipid extracts applied to TLC plates was normalized on the basis of culture biomass as protein. One of two reproducible experiments is shown.

FIG 4

GC-FID analysis of accumulating neutral lipids in nitrogen-limited RHA1 cultures grown with cholesterol or succinate. (A) GC-FID chromatograms of authentic cholesteryl palmitate (C_16:0) and cholesteryl heptadecanoate (C_17:0) standards (blue line) and of a trimpalmitin TAG [(C_16:0)₃] standard (orange line) and of neutral lipid extracts of cholesterol- and succinate-grown cells harvested 24 h after nitrogen depletion. Seven accumulating CE species were identified either by comparison to the standards or based on the proximity of their respective peaks to the standard peaks and their absence in succinate-grown cells. Identified and predicted CEs are labeled with blue arrows, and identified and predicted TAG species are labeled with orange arrows. (B) Ratio of accumulating CEs (blue bars) and TAGs (orange bars) in cholesterol-grown nitrogen-limited RHA1 cultures over time starting at nitrogen depletion (0 h). Peak areas of the seven CE- and nine TAG-peaks shown in panel A were normalized on the peak area of the internal standard cholestane (not shown) before calculation of their respective sums. Average values of three biological replicates and their standard deviations are shown.

FIG 5

Identification of SEs accumulating in N-limited RHA1 cultures. GC-MS analysis of transesterified fraction 2 (black peaks) and untreated fraction 2 (gray peaks) of neutral lipid extracts of nitrogen-limited cultures grown with (A) cholesterol and (B) phytosterols. Transesterification of SEs produced free cholesterol and the free phytosterols β-sitosterol, β-sitostanol, and campesterol, as well as seven fatty acid methyl esters of C_15:0, C_16:1, C_16:0, C_17:1, C_17:0, C_18:0, and C_18:1. One representative GC-MS chromatogram is shown. (C) Fatty acid profile of transesterified fraction 2 derived from nine cholesterol-grown cultures grown under nitrogen-limiting conditions from five independent experiments harvested approximately 24 h after nitrogen depletion. Odd-numbered fatty acids are colored blue, and even-numbered fatty acids are colored red.

FIG 6

Isolation of lipid droplets from strain RHA1 grown with cholesterol (C) or palmitate (P) or a mixture of cholesterol plus palmitate (C + P) under nitrogen-limiting conditions. (A) TLC analysis of neutral lipids derived from total lipid extracts from isolated lipid droplets. Abbreviations are defined as in previous figure legends. (B) Molar ratio of TAGs, CEs, and free cholesterol in lipid droplets derived from cells grown on cholesterol, cholesterol plus palmitate, and palmitate. (C) Ratio of even- to odd-numbered fatty acids in CEs and TAGs in lipid droplets and the respective contributing fatty acid chain lengths. The results from two independent experiments (I and II) are shown in panels B and C, and those from experiment II alone are shown in panel A.