Potential Mechanism Of Lipid Accumulation In Rhodococcus Opacus

Objective The aim of this study is to explore the mechanism of lipid accumulation in Rhodococcus opacus by analyzing the change of lipid accumulation and enzyme activities at different culture conditions.Methods We cultivated Rhodococcus opacus using different carbon source and nitrogen source in shake flask, and detected their lipid content and biomass, and in this way screened the optimal carbon source and nitrogen source for biomass and lipid content. When Rhodococcus opacus was cultivated at different culture conditions, the residue carbon and nitrogen in medium was monitored, and the biomass and lipid content at different time points were determined. Some enzymes potentially associated with lipid accumulation, such as TCA cycle enzymes and some enzymes which produce reducing equivalents NADPH, were measured using ultraviolet-visible spectrophotometry. At the same time, isoforms of malic enzyme were identified using a specific activity staining following non-denaturing polyacrylamide gel electrophoresis (PAGE) of extracts of cells grown under different conditions.Results Optimal Growth of Rhodococcus opacus ACCC41043was achieved when using glucose as the sole carbon source and urea as the sole nitrogen source, and A660of the culture reached52.92, and lipid content was48.21%. Citrate synthase (CS), isocitrate dehydrogenase (ICDH), Aconitase, succinate dehydrogenase (SDH), malate dehydrogenase (MDH) activity of tricarboxylic acid (TCA) cycle are regulated and controlled by growth cycle and carbon source, when the Rhodococcus opacus were cultivated in N-limited medium. CS activity decreased after nitrogen was disappeared from the medium, whether glucose or gluconate was used as carbon source. CS activity decrease leads to the down-regulation of tricarboxylic acid cycle, thus inhibiting acetyl coenzyme A enter into the TCA cycle, thereby increasing fatty acid synthesis. The other of several enzymes were regulated by carbon source, and enzyme activities of NADP+-ICDH, Aconitase, MDH, SDH gradually decreased when nitrogen source disappeared from the medium in which glucose was used as the sole carbon source. The synergistic effect of these enzymes with CS effectively controlled the TCA cycle, therefore significantly increased fatty acid synthesis. However when gluconate was used as carbon source, enzyme activities of Aconitase, MDH, SDH did not change significantly, but the activity of NADP-ICDH increased after nitrogen depletion. The increasing of ICDH activity offset the regulation of TCA cycle by reduced CS activity. Thus fatty acid synthesis was depressed. These results are consistent with the result of lipid accumulation when Rhodococcus opacus are cultivated in the sodium gluconate as the sole carbon source. At least seven isoforms of malic enzyme are identified in the oleaginous bacteria, Rhodococcus opacus ACCC41043. Expression of these isoforms is influenced by culture conditions. It is likely that isoform B, E and F are associated with lipid synthesis and accumulation. Sesamol not only affect the growth of Rhodococcus opacus, but also inhibit its lipid accumulation. The potential mechanism of the regulation of lipid accumulation by sesamol is through the induction of NADP+-ICDH activity.Conclusion (1) Optimal growth and lipid accumulation of Rhodococcus opacus ACCC41043was achieved when using glucose as the sole carbon source and urea as the sole nitrogen source.(2) When Rhodococcus opacus is cultivated in N-limited medium, CS activity controls the entering of acetyl coenzyme A into TCA via regulating TCA cycle, thereby regulating lipid synthesis. However the activities of Aconitase, ICDH, MDH, SDH are regulated by carbon source. These activities may work synergistically or antagonistically in regulating of TCA cycle, thus regulating lipid synthesis efficiently.(3) At least seven isoforms of malic enzyme are identified in the oleaginous bacteria, Rhodococcus opacus ACCC41043, and isoform B, E and F are potentially associated with lipid synthesis.(4) Sesamol, by inducing NADP+-ICDH activity, stimulates the entering of acetyl coenzyme A into the TCA cycle, thereby reducing the substrate level for fatty acid synthesis, and decreases fatty acid synthesis and lipid accumulation.