Heterologous Expression of Two Malate Transporters From an Oleaginous Fungus Mucor circinelloides Improved the Lipid Accumulation in Mucor lusitanicus

The fungus, Mucor lusitanicus, is of great interest for microbial lipids, because of its ability to accumulate intracellular lipid using various carbon sources. The biosynthesis of fatty acid requires the reducing power NADPH, and acetyl-CoA, which is produced by the cleavage of citrate in cytosol. In this study, we employed different strategies to increase lipid accumulation in the low lipid-producing fungi via metabolic engineering technology. Hence, we constructed the engineered strain of M. lusitanicus CBS 277.49 by using malate transporter (mt) and 2-oxoglutarate: malate antiporter (sodit) from M. circinelloides WJ11. In comparison with the control strain, the lipid content of the overexpressed strains of mt and sodit genes were increased by 24.6 and 33.8%, respectively. These results showed that mt and sodit can affect the distribution of malate in mitochondria and cytosol, provide the substrates for the synthesis of citrate in the mitochondria, and accelerate the transfer of citrate from mitochondria to cytosol, which could play a significant regulatory role in fatty acid synthesis leading to lipids over accumulation.

The role of phenylalanine hydroxylase in lipogenesis in the oleaginous fungus Mortierella alpina

Phenylalanine hydroxylase (PAH) catalyses the irreversible hydroxylation of phenylalanine to tyrosine, which is the rate-limiting reaction in phenylalanine metabolism in animals. A variety of polyunsaturated fatty acids can be synthesized by the lipid-producing fungus Mortierella alpina, which has a wide range of industrial applications in the production of arachidonic acid. In this study, RNA interference (RNAi) with the gene PAH was used to explore the role of phenylalanine hydroxylation in lipid biosynthesis in M. alpina. Our results indicated that PAH knockdown decreased the PAH transcript level by approximately 55% and attenuated cellular fatty acid biosynthesis. Furthermore, the level of NADPH, which is a critical reducing agent and the limiting factor in lipogenesis, was decreased in response to PAH RNAi, in addition to the downregulated transcription of other genes involved in NADPH production. Our study indicates that PAH is part of an overall enzymatic and regulatory mechanism supplying NADPH required for lipogenesis in M. alpina.

Transcriptome Analysis of Oleaginous Fungus Mucor circinelloides WJ11 in Response to Exogenous Soybean Oil as Carbon Source

Mucor circinelloides is an oleaginous fungus that utilizes a wide variety of carbon substrates for its growth. The different sources of carbon strongly influence the total lipid content of the fungus. These different carbon substrates are assimilated and dissimilated through different metabolic pathways before entering into the TAG synthesis pathway. In the present study, we attempted to explore the mechanism of ex-novo lipid biosynthesis in M. circinelloides WJ11 in response to exogenous plant oil as a carbon source through transcriptomic analysis. The lipid content of WJ11 grown in a media containing mixed soybean oil with glucose as a carbon source was up to 43.8%, an increase of 13.9% as compared to glucose alone as the carbon source. RNA-Seq analysis was performed to investigate global gene expression patterns in the oil-treated WJ11. Based on RNA-seq analysis, among the 4646 differentially expressed genes (DEGs), 2379 were up-regulated and 2267 down-regulated. The expression of acetyl-CoA synthetase, 6-phosphofructokinase, alcohol dehydrogenase (NADP+), fructose-bisphosphate aldolase, and pyruvate kinase was down-regulated while genes related to triglyceride synthesis were up-regulated. The majority of genes and pathways related to lipid biosynthesis were up-regulated indicating a diversion of metabolic pathways towards lipid biosynthesis. The data generated advance the genomic resources and provide insights into the mechanisms of ex-novo lipid accumulation in fungi that use exogenous oil as a carbon source.