Abstract
BackgroundMitochondrial and cytoplasmic malate transporter proteins are responsible for transmembrane transport of malate, thereby linking malate metabolism in various subcellular regions of the cell. These transporters play an important role in fatty acid biosynthesis of oleaginous microorganisms. Our previous studies have found that lipid content of the recombinant Mucor circinelloides (M. circinelloides) strain with mitochondrial malate transporter (mt) gene overexpression was increased by 70%, while that of strain with mt gene knockout was decreased by 27%. However, the mechanism of malate transporter promoting the transport of mitochondrial malate and citrate related to lipid accumulation is not clear. Therefore, 13C-labeled glucose metabolic flux analysis was carried out to identify the metabolic network topology and estimate intracellular fluxes of genetically engineered M. circinelloides strains for the purpose of better understanding the roles of malate transporters in citrate transport systems and lipid accumulation.ResultsThe metabolic flux distribution analysis suggested that tricarboxylic acid (TCA) cycle flux ratio of mt-overexpression strains was decreased compared to that of the control strain, but in contrast, glyoxylic acid (GOX) cycle flux ratio was increased. Accordingly, the mt-knockout strain showed an opposite phenomenon with a higher TCA cycle flux ratio and a lower GOX cycle flux ratio than the control strain. GOX cycle might be more effective than TCA cycle in producing malate and oxaloacetate replenishment. Moreover, a relatively higher flux ratio of the pentose phosphate (PP) pathway was obtained in mt-overexpression strains, but no significant difference in the malic enzyme flux between recombinant strains and the control strain. Our results confirmed that PP pathway might play an important role for supplying NADPH and malic enzyme is not a limiting factor for fatty acid synthesis in oleaginous fungus M. circinelloides strains.ConclusionIntracellular metabolic flux information suggested that mt-overexpression strains had higher flux in PP pathway and GOX cycle, lower flux in TCA cycle, and no difference in malic enzyme cycle. Together, the role of malate transporter was assumed to further participate in transporting cycle of acetyl-CoA and drive PP pathway to supply NADPH required for lipid accumulation in recombinant M. circinelloides strains.
Highlights
Mitochondrial and cytoplasmic malate transporter proteins are responsible for transmembrane transport of malate, thereby linking malate metabolism in various subcellular regions of the cell
The mitochondrial citrate transport system that transports acetyl-CoA is the link between glucose metabolism and lipid synthesis, and an important factor affecting the lipid synthesis in eukaryotes
The transformants of mt-overexpressing gene were named Mc-MT-1 and Mc-MT-2, respectively [16]. These engineered strains were respectively inoculated in high nitrogen (HN) and low nitrogen (LN) modified K & R medium with glucose as the sole carbon source
Summary
Mitochondrial and cytoplasmic malate transporter proteins are responsible for transmembrane transport of malate, thereby linking malate metabolism in various subcellular regions of the cell These transporters play an important role in fatty acid biosynthesis of oleaginous microorganisms. The mitochondrial citrate transport system that transports acetyl-CoA (citrate as its acetyl carrier) is the link between glucose metabolism and lipid synthesis, and an important factor affecting the lipid synthesis in eukaryotes. This citrate transporter which is located on the mitochondrial inner membrane, acts as the carrier of citrate and malate to shuttle between cytosol and mitochondria [9]. The function and mechanism of mitochondrial citrate transport system in oleaginous filamentous fungi is still far from clear
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