| Polyunsaturated fatty acids (PUFAs) are essential fatty acids necessary for human health.M. alpina is one of the highest lipid-producing fungi and can produce lipids in quantities up to50%of its dry weight. It is used in industrial production of arachidonic acid (AA) with arecord of safety for human health. We undertook a systematic study using a combination ofwhole-genome sequencing, lipidomics, transcriptomic, proteomics and bioinformatics toinvestigate the mechanism underlying the efficient lipid synthesis and accumulation in thisorganism. Our study provides insights on the mechanics of efficient lipogenesis, and lays afoundation for the possible genetic engineering of organisms producing optimized contents ofdietary fat and biodiesel. The main results were described as follows：(1) The genome of M. alpina ATCC#32222was sequenced with31.75fold coverage, andthe assembly contains a total contig length of38.38Mb with a GC content of51.72%. Thegenome of M. alpina was the second finished in Mucoromycotina. Annotation of theassembled genome sequence generated12,796gene models with an average transcript lengthof1.5kb. There is an average of3.32introns per multi-exon gene and26%of the predictedgenes are single-exon transcripts. The average exon size is435bp and the average intron sizeis140bp. The mitochondrial genome of M. alpina measures67,445bp and encodes28tRNAs,3noncoding RNAs,12known proteins, and13uncharacterized proteins. It suggeststhat the evolutionary history of lipid metabolic genes was largely dominated by verticalinheritance rather than lateral gene transfer. Based upon the genome information, alipogenesis pathway was constructed, providing a comprehensive picture of lipid metabolismin M. alpina.(2) The lipidomic of M. alpina was analysed. Glycerolipids were by far the mostabundant type of lipids produced (approx.430mol/g dry mycelia, or89%of the total lipids),followed by glycerophospholipids (approx.154mol/g or8%), sterols (approx.14.5mol/g or3%), with only small amounts of sphingolipids (approx.30nmol/g or0.008%). In ourexperiment, ω-3PUFA accumulation was much higher in cultures grown at12°C than at25°C.However, there were no significant temperature-associated differences in the levels ofsaturated fatty acids (SAFA) and ω-3PUFAs.(3) Growth of M. alpina has two distinctive phases: proliferation and lipogenesis.Initially, fungal mass increases rapidly as all nutrients are in excess; there is minimal lipidproduction. After nitrogen exhaustion, the cell multiplication of M. alpina stops; however,glucose continues to be assimilated and the cells now switch to lipid accumulation. We carriedout transcriptome analyses at various time points prior to and after nitrogen exhaustion(sample A:-12h, B:-2h, E:-30min, K:+1h, L:+12h and M:+48h). Out of all thepredicted genes,~88%were expressed and33%of these genes encode proteins with as yetunknown functions. M. alpina had a very dynamic gene expression, ranging fromundetectable to73,528Fragments Per Kilobase of exon per Million fragments mapped(FPKM). Comparison of the transcriptome data prior to and after the onset of lipogenesisindicates that genes encoding key enzymes involved in the glycolytic pathway, the TCA cycle, glycerolipid pathway, and fatty acid pathway were coordinately regulated at thetranscriptional level to redirect the metabolic pathways towards fatty acid synthesis.Comparing the cultures of12°C to25°C, a total of555proteins was identified. Proteomic andRT-PCR data suggested that general metabolic pathways, including the pentose shunt, theglycolysis and the TCA cycle, favored fatty acid synthesis by producing increased levels ofNADPH and acetyl-CoA.(4) This is the first time that a tetrahydrobiopterin (BH4) synthesis pathway and aphenylalanine hydroxylating system have been characterized at the molecular level in afungus. Genes encoding enzymes involved in the biosynthetic pathway of BH4and thephenylalanine-hydroxylating system were expressed heterologously in Escherichia coli, andthe resulting proteins were purified to homogeneity. Their enzymatic activity was investigatedby high-performance liquid chromatography (HPLC) or visible-UV spectroscopy. Kineticparameters and other properties of these enzymes were investigated. Comparative analysesbetween these proteins and other homologous proteins were performed. A novel role for BH4in fungi as a cofactor for PAH is suggested, which is similar to that known to exist in higherlife forms. The M. alpina cultures grown in medium plus inhibitor showed reduced totalSAFA, ω-6PUFA, ω-3PUFA, and total fatty acid accumulation levels by about30%,50%,20%, and30%, respectively, while the level of monounsaturated fatty acid (MUFA) wasincreased by about30%. This work establishes a novel fungal scheme for the degradation ofan aromatic substance (phenylalanine) and suggests the functional significance of thephenylalanine hydroxylating system in lipid metabolism. |
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