Molecular Mechanism Of Metabolic Regulation Of Substrates For Lipid Biosynthesis In Rhodococcus Opacus

Bioconversion of abundant waste and residue materials to polyunsaturated fatty acids(PUFAs) and cocoa butter replacer for the application in the food industry, such as milk,beverage and cake, or to triacylglycerols (TAGs) with the consequent esterification into thesecond-generation biofuels, match the future development trends of microbial oils. With theunique capacity to accumulate substantial amount of TAGs, high growth rate, high celldensity cultivation technology and the avaliable genomic sequence, Rhodococcus opacusPD630has been recognized as a potential strain to produce microbial oils as industrial level.However, the unclear molecular mechanism of lipid accumulation, which has led to a stock oftheoretical and technical support for the industrialization of microbial oils, needed to besolved by the exploration of fundamental knowledge with genetic and metabolic engineering.In this study, the ratio of carbon to nitrogen in the fermentation medium was set toachieve the nitrogen-limiting condition (C/N of25), the carbon-limiting condition (C/N of0.2)and the balanced nutrition condition (C/N of5), for the single batch fermentation in a7.5Lfermentation tank. The central carbon metabolic regulation, especially the metabolism ofsubstrate acetyl-CoA and reducing power NADPH, the key regulating enzymes in the fattyacids synthesis, were studied by the comparative analysis on the experimental data under thedifferent fermentation conditions.Firstly, the growth of cells, which was estimated by optical density at660nm (OD660),was inhibited by nutrition-starvation under the nitrogen-limiting and carbon-limitingconditions, with the OD660only reached at20.2and3.5, respectively, lower than41under thebalanced nutrition condition. Under the nitrogen-limiting condition, the lipid accumulationdid not commence until nitrogen was depleted. After4days cultivation, the fatty acidproduction (total fatty acid/cell dry weight) was39.6%, much higher than that under thecarbon-limiting condition (3.2%) and the balanced nutrition condition (6.5%). Thus, thenitrogen-limiting condition was the special nutritional requirement for lipid accumulation in R.opacus, and the nitrogen depletion was the onset of the lipid biosynthesis.Secondly, due to the deficiency of the complete elongase and desaturase system inoleaginous fungi, the chain-length of fatty acid varied between C14and C18with the palmiticacid (C16:0) and octadecenoic acid (C18:1) as the predominant species, and the unsaturated fattyacids were mainly composed of single double bond. The composition profile of the fatty acidunder the carbon-limiting condition were very similar to that under the balanced nutritioncondition, whereas in the nitrogen-limiting condition, the ratio of C16:0and C18:1to the totalfatty acids became lower but C17:0and C17:1had a near six-and one-fold increase, most likelydue to the metabolism tendency of lipid accumulation induced by the specific condition. Thirdly, under the nitrogen-limiting condition, when nitrogen was depleted in themedium, the trend of dissolved oxygen tension (DOT) and NADP+dependent isocitratedehydrogenase (NADP+-ICDH) activity was concomitant with the changes of acetyl-CoAmetabolism in tricarboxylic acid cycle (TCAc); hexokinase (HK) and phosphofructokinase(PFK) provided enough pyruvate for the fatty acid biosynthesis through EMP(Embden-Meyerhof-Parnas pathway); and pyruvate dehydrogenase (PDH), pyruvatecarboxylase (PC) were responsible for the provision of acetyl-CoA to the lipid accumulation.Between6-12h of growth before nitrogen depletion, the cells grew fast with the sharpincrease in TCAc metabolism and oxygen consumption, causing DOT decreased significantlybut enzyme activity (NADP+-ICDH, PFK, HK, PDH and PC) increased dramatically, whichindicated the acetyl-CoA flux directed to TCAc for energy production. Followed by the fastlipid accumulation phase (12-32h), nitrogen exhaustion resulted in the following events:1. Asharp decrease in NADP+-ICDH resulted in a dramatic down-regulation of the TCAc, leadinga shift of acetyl-CoA from TCAc to lipid synthesis and an increase in DOT;2. Then PFK andHK showed a sharp increase in the activity, indicating the EMP became more active for thepyruvate synthesis;3. Meanwhile the activity of PDH increased significantly, and PCdecreased significantly, and thus most of pyruvate was continuously converted to acetyl-CoAfor lipid biosynthesis. At last period of cultivation (32-96h), the lipid accumulation, enzymeactivity (NADP+-ICDH, PFK, HK, PDH, PC) and DOT level kept stable, suggesting theacetyl-CoA flux got balanced between the TCAc and the fatty acid synthesis. Additionally, theinhibitory effect of sesamol on the lipid accumulation (decreased by4-fold) was due toinduced the NADP+-ICDH activity which direct the acetyl-CoA to TCAc for energyproduction, and this further supported the role of NADP+-ICDH in regulating acetyl-CoAmetabolic flux for fatty acid biosynthesis.At last, we investigated the trends of glucose-6-phosphate dehydrogenase (G-6-PDH),NADP+dependent malic enzyme (NADP+-ME) and6-phosphategluconate dehydrogenase(6-P-GDH) in R. opacus PD630, to determine the source of reducing power NADPH for thelipid accumulation. Under the nitrogen-limiting condition, NADP+-ME showed a sharpincrease (2-fold) in the activity during the fast period of lipid accumulation. After that, theactivity of NADP+-ME and lipid accumulation rate both decreased and then maintained acertain level. Besides, the activity of NADP+-ME was much higher than that under anotherconditions, indicating the controlling role of NADP+-ME for providing the reducing powerNADPH to fatty acid biosynthesis.