Rhodococcus opacus strain PD630 as a new source of high-value single-cell oil? Isolation and characterization of triacylglycerols and other storage lipids

Abstract

The triacylglycerol (TAG)-accumulating, hydrocarbon-degrading bacterium Rhodococcus opacus strain PD630 and chemically induced storage-deficient mutants derived from this strain were investigated for their capability to accumulate storage lipids in the cytoplasm during cultivation under nitrogen-limiting conditions. Acylglycerols were analysed by matrix-associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) and by reversed-phase HPLC. Fatty acids comprising 13-19 carbon atoms in various acylglycerols constituted up to 76% of the cellular dry weight in gluconate-grown cells, with a significant proportion of odd-numbered fatty acids. Hydrolysis using pancreatic lipase and deacylation with ethyl magnesium bromide were employed to identify the stereospecific distribution of fatty acids at the glycerol. This analysis showed that the fatty acids were not randomly distributed between the three positions of the glycerol backbone. In comparison with common plant fats, where the longer and higher unsaturated fatty acids are predominantly found at position 2, R. opacus PD630 accumulated only the shorter and saturated fatty acids in this position. More than 100 mutants accumulating TAG at a significantly lower rate were obtained by chemical mutagenesis and identified by staining with Sudan Black B. All the mutants showed similar neutral lipid patterns by TLC analysis, with a small distinct spot exhibiting the same R(F) value as TAG; this was identified as a residual amount of TAG by preparative TLC and MALDI-TOF, indicating that this bacterium is possibly capable of synthesizing TAGs by at least two different pathways.

Neutral Lipid Bodies in Prokaryotes: Recent Insights into Structure, Formation, and Relationship to Eukaryotic Lipid Depots

“…In carbohydrate-grown cells of R. opacus, the stereospecific distribution of the acyl residues on the glycerol backbone is not random. The shorter and saturated fatty acid residues were predominantly esterified to the sn-2 hydroxyl group, whereas unsaturated fatty acids were predominantly bound at position sn-3 (137). The composition of the TAGs strongly varied when the cells were cultivated on propionic acid, which was activated to propionyl-CoA, resulting in a much higher fraction of odd-numbered fatty acids.…”

Evaluation of the biocompatibile ionic liquid 1-methyl-3-methylimidazolium dimethylphosphite pretreatment of corn cob for improved saccharification

“…Cell cultures were harvested and prepared for fatty acid methyl esters (FAMEs) synthesis according to the described method (Wältermann et al 2000). FAMEs were synthesized using IL N-methyl-2-pyrrolidonium methyl sulfate ([NMP] [CH 3 SO 3 ]) as catalyst according to our earlier reported method (Zhang et al 2009).…”

Triacylglycerol Production from Corn Stover Using a Xylose-Fermenting Rhodococcus opacus Strain for Lignocellulosic Biofuels

“…Rhodococcus opacus PD630, an oleaginous hydrocarbon-degrading bacterium, is able to utilize a wider variety of substrates and accumulate intracellular TAGs consisting primarily of C16 and C18 series long chain fatty acids, which are quite similar to those of vegetable-based TAGs, in excess of 70% of their dry cell weight during cultivation under nitrogen-limiting conditions [22]. Bacterium can produce large amounts of TAGs in batch-cultivation containing high concentrations of glucose [23].…”