Carbon Metabolic Flux Analysis Of Lipid Accumulation Mechanism And Key Genetic Modification In Mucor Circinelloides

Microbial oils is an important source of polyunsaturated fatty acids(PUFAs) such as arachidonic acid(AA), eicosapentaenoic acid(EPA), docosahexaenoic acid(DHA) and γ-linolenic acid(GLA), it has also being considered as potential sources of biofuels, therefore, much attention has been paid to the development of microbial oils during these years. The filamentous fungus Mucor circinelloides is of industrial interest because it can produce high levels of polyunsaturated fatty acid GLA, which is the first commercial producer of microbial PUFA. It has emerged as one of the model organisms of oleaginous fungus because of the availability of genome sequence and well-developed tools for genetic engineering. Although much work has been done to study the mechanism of lipid accumulation in oleaginous yeast, a systems-level analysis and understanding of the mechanisms underlying lipid accumulation in oleaginous filamentous fungus M. circinelloides is still lacking. In this study, the intracellular metabolic fluxes of M. circinelloides were investigated using 13C-labeled metabolic flux(MFA) analysis to investigate the regulation mechanism of carbon metabolism of lipid accumulation in M. circinelloides, and the expression of key genes, which maybe influence lipid accumulation were modified in M. circinelloides. The main research contents and results are as follows.M. circinelloides CBS 277.49 is able to accumulate less than 15% of cell dry weight as lipids, while M. circinelloides WJ11 can accumulate lipid up to 36%. In order to better understand the mechanisms behind the differential lipid accumulation in these two strains, 13C-metabolic flux analysis was performed for the oleaginous fungus M. circinelloides. Our results suggested that strain WJ11 has higher fluxes in the pentose phosphate pathway(PP pathway) and glyoxylate cycle while lower fluxes in tricarboxylic acid cycle than strain CBS 277.49. Compared to high nitrogen medium, the fluxes through PP pathway and glyoxylate cycle were higher, and fluxes through tricarboxylic acid cycle were lower in either WJ11 or CBS 277.49 when cultured under low nitrogen medium. Malic enzyme and PP pathway provide higher proportions of NADPH for lipid biosynthesis in high-lipid-producing strain, suggested that malic enzyme may play an important role while the PP pathway also plays a vital role for the provision of NADPH for lipid biosynthesis in oleaginous fungus M. circinelloides. And simultaneously ATP: citrate lyase activity increased in high lipid-producing strain might provide more substrate acetyl-Co A for fatty acid synthesis.The genes coding for glucose 6-phosphate dehydrogenase(g6pd) and 6-phosphogluconate dehydrogenase(6pgd) from the PP Pathway were overexpressed in the oleaginous fungus M. circinelloides to analyze their effects on lipid accumulation. G6 PD and 6PGD activities were increased markly in strains overexpressing these genes while the levels of m RNA for g6 pd and 6pgd were also increased significantly. The higher G6 PD and 6PGD activities, and higher m RNA levels, resulted in the lipid content of the cells being improved by 22-37% in g6pd-overexpressing and 6pgd-overexpressing stains compared with the control strain. These results suggested that G6 PD and 6PGD from the PP Pathway is the important NADPH provider and play a vital role in lipid accumulation in oleaginous fungus M. circinelloides.Two genes coding for malic enzyme(me) localizated in cytosol were overexpressed in the oleaginous fungus M. circinelloides to analyze their effects on lipid accumulation. Although the malic enzyme activity and m RNA levels for me were higher in me-overexpressing stains than the control strain, the lipid content of me-overexpressing stains were not significantly different from the control strain. These results suggested that malic enzyme is not the rate-limiting step for lipid accumulation in oleaginous fungus M. circinelloides.The expression level of the mt gene, coding for a malate transporter, was manipulated in the oleaginous fungus M. circinelloides to analyze its effect on lipid accumulation. The results showed that mt overexpression increased the lipid content for about 70%, whereas the lipid content in mt knockout mutant decreased about 27%, compared with the control strain. Furthermore, the extracellular malate concentration was decreased in the mt overexpressing strain, and increased in the mt knockout strain, compared with the wild-type strain. The results suggested that malate transporter can transport malate from cytosol into the mitochondria, this may increase the concentration and the transport rate of citrate, and lead to enhanced lipid accumulation in M. circinelloides. This work suggests that the malate transporter plays an important role in regulating lipid accumulation in oleaginous fungus M. circinelloides.