Carbon Metabolic Flux Analysis Of Lipid Accumulation Mechanism And Key Genetic Modification In Rhodococcus Opacus

Oleaginous microorganisms can accumulate a large amount of fatty acids in the form of triacylglycerols(TAGs), which is an important alternative to traditional animal and vegetable oils. In addition, fatty acids that from microorganisms are the important precursors to produce transportation fuels and industrial chemicals, therefore it is necessary to develop and exploit some oleaginous strains that are able to product high-yield lipid. With the unique capacity to accumulate substantial amount of triacylglycerols, technology of high cell density cultivation, the available genomic sequence and the system of gene manipulation, Rhodococcus opacus PD630 has been recognized as one of the most pomising strain to produce microbial oils at the industrial level. However, a systems-level analysis and understanding of the mechanisms underlying lipid accumulation in oleaginous bacteria R. opacus PD630 is still lacking, which led to a stock of theoretical and technical support for the industrialization of microbial oils and the strategies that improve the lipid production by genetic engineering. This study systematically investigated the regulation mechanism in strain R. opacus PD630 between high nitrogen(HN) concentration conditions and low nitrogen(LN) concentration conditions by 13 C Metabolic flux analysis(13C-MFA). This study also found some key genes regulating lipid biosynthesis through transcription analysis and the lipid content increased when these genes were overexpressed in R. opacus PD630. The main research contents and results are as follows.Firstly, the analysis of the growth and the lipid content between HN(C/N, 10) and LN(C/N, 100) two different culture conditions showed that the concentration of nitrogen has significant impact on the content of protein and lipid. After 44 hours, the strain R. opacus PD630 arrives to the metabolically stable phase, with the marked decrease in nitrogen concentration, the lipids content increased sharply from 18.99% to about 34.68%, whereas the protein content in nitrogen-deprived cells dropped greatly from 30.52% to 17.39%.Then, in order to better understand the mechanisms behind the differential lipid accumulation in different culture conditions, 13C-metabolic flux analysis was performed for the oleaginous bacteria R. opacus PD630. The results indicated that under two different culture conditions the carbon flux in R. opacus PD630 was redistributed and led to a different lipid accumulation in the cells. The total flux through the pentose phosphate(PP) pathway and the Entner-Doudoroff(ED) pathway was over 90% whether R. opacus PD630 was grown in HN or LN media, indicating that PP and ED pathway are the main way for glycolysis in R. opacus PD630. However the flux through oxidative PP pathway and the competing ED pathway was strikingly different, compared to HN medium. When cultured under LN medium, the flux through oxidative PP pathway was boosted about 5-fold, whereas the flux into ED pathway was decreased to only about half compared to HN condition, which was consistent with the upregulated transcription level and increased activities of 6-Phosphogluconate dehydrogenase(6PGDH) when the bacteria was grown in LN medium. In addition, the flux through malic enzyme(ME) which converts malate(Mal) to pyruvate(Pyr)(Mal + NADP ? Pyr + CO2 + NADPH) in the bacteria decreased by 50% in LN condition than in HN. Therefore, the results indicate that in the oleaginous bacteria R. opacus PD630 the 6PGDH from PP pathway but not ME is likely to play a vital role for the provision of NADPH for lipid biosynthesis.Finally, the 5 homologous genes gnd coding for 6PGDH from the PP pathway were overexpressed in the oleaginous bacteria R. opacus PD630 to analyze its effect on lipid accumulation. 6PGDH activities were increased markly in strains overexpressing these genes and the levels of m RNA for 6PGDH were also increased significantly. The higher 6PGDH activities, and higher m RNA levels, resulted in the lipid content of the cells being improved by 20~28% in gnd-overexpressing stains compared with the control strain. These results suggested that 6PGDH from the PP pathway is the important NADPH provider and play a vital role in lipid accumulation in oleaginous bacteria R. opacus PD630.