Mortierella alpina is an oleaginous fungus which can produce lipids accounting for up to 50% of its dry weight in the form of triacylglycerols. It is used commercially for the production of arachidonic acid. Using a combination of high throughput sequencing and lipid profiling, we have assembled the M. alpina genome, mapped its lipogenesis pathway and determined its major lipid species. The 38.38 Mb M. alpina genome shows a high degree of gene duplications. Approximately 50% of its 12,796 gene models, and 60% of genes in the predicted lipogenesis pathway, belong to multigene families. Notably, M. alpina has 18 lipase genes, of which 11 contain the class 2 lipase domain and may share a similar function. M. alpina's fatty acid synthase is a single polypeptide containing all of the catalytic domains required for fatty acid synthesis from acetyl-CoA and malonyl-CoA, whereas in many fungi this enzyme is comprised of two polypeptides. Major lipids were profiled to confirm the products predicted in the lipogenesis pathway. M. alpina produces a complex mixture of glycerolipids, glycerophospholipids and sphingolipids. In contrast, only two major sterol lipids, desmosterol and 24(28)-methylene-cholesterol, were detected. Phylogenetic analysis based on genes involved in lipid metabolism suggests that oleaginous fungi may have acquired their lipogenic capacity during evolution after the divergence of Ascomycota, Basidiomycota, Chytridiomycota and Mucoromycota. Our study provides the first draft genome and comprehensive lipid profile for M. alpina, and lays the foundation for possible genetic engineering of M. alpina to produce higher levels and diverse contents of dietary lipids.

The generation of NADPH by malic enzyme (ME) was postulated to be a rate-limiting step during fatty acid synthesis in oleaginous fungi, based primarily on the results from research focusing on ME in Mucor circinelloides. This hypothesis is challenged by a recent study showing that leucine metabolism, rather than ME, is critical for fatty acid synthesis in M. circinelloides. To clarify this, the gene encoding ME isoform E from Mortierella alpina was homologously expressed. ME overexpression increased the fatty acid content by 30% compared to that for a control. Our results suggest that ME may not be the sole rate-limiting enzyme, but does play a role, during fatty acid synthesis in oleaginous fungi. Oleaginous fungi, such as the commercial production species Mortierella alpina and Mucor circinelloides, can accumulate fatty acids to more than 20% of their cell dry weight (1). However, the mechanism of fatty acid biosynthesis in these organisms is still not fully understood. The carbon flux pathway and provision of NADPH are two major events during fatty acid synthesis. NADPH is particularly important as the sole source of reducing power during fatty acid synthesis in oleaginous fungi (2-5).The NADPH-generating enzyme malic enzyme (ME) (NADP ϩ dependent; EC 1.1.1.40), catalyzing the decarboxylation of malate to pyruvate (malate ϩ NADP ϩ ϭ pyruvate ϩ CO 2 ϩ NADPH), was speculated to play a pivotal role during fatty acid synthesis in oleaginous fungi (3, 5). When ME activity was inhibited, the cell fatty acid content of M. circinelloides decreased from 24% to 2% (6). Moreover, in the M. circinelloides strain R7B, overexpression of the ME genes from M. alpina and M. circinelloides produced a 2.5-fold increase in fatty acid content (7). These results led to the conclusion that a ratelimiting step in fatty acid biosynthesis is the generation of NADPH by ME (7). However, a recent study revealed that leucine auxotrophy caused a 2.5-fold decrease in cell fatty acid content and that leuA gene expression restored its level in M. circinelloides strain R7B. ME overexpression, however, did not alter the fatty acid content despite a significant increase in ME activity (8). It was thus proposed that the leucine metabolic pathway, by participating in acetyl coenzyme A (acetyl-CoA) generation, may be critical during fatty acid synthesis in M. circinelloides (8,9).M. circinelloides R7B, generated by random mutagenesis (10), may contain other, unknown mutations in addition to that in the leuA gene, which can complicate the interpretation of the abovementioned results. Therefore, a recipient strain with a known genetic mutation(s) and stable fatty acid synthesis characteristics will be advantageous for future research. To this end, a uracil auxotroph of M. alpina ATCC 32222 was generated via homologous recombination. The M. alpina ATCC 32222 cytosolic ME (isoform E)-encoding gene (11), named malE1, was cloned and homologously overexpressed in order to evaluate the role of ME in fatty acid synthesis. MATERIALS AND METHODSStrains and gr...

The oleaginous fungus, Mucor circinelloides, is one of few fungi that produce high amounts of γ-linolenic acid (GLA); however, it usually only produces <25% lipid. Nevertheless, a new strain (WJ11) isolated in this laboratory can produce lipid up to 36% (w/w) cell dry weight (CDW). We have investigated the potential mechanism of high lipid accumulation in M. circinelloides WJ11 by comparative biochemical analysis with a low lipid-producing strain, M. circinelloides CBS 277.49, which accumulates less than 15% (w/w) lipid. M. circinelloides WJ11 produced more cell mass than that of strain CBS 277.49, although with slower glucose consumption. In the lipid accumulation phase, activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in strain WJ11 were greater than in CBS 277.49 by 46% and 17%, respectively, and therefore may provide more NADPH for fatty acid biosynthesis. The activities of NAD+:isocitrate dehydrogenase and NADP+:isocitrate dehydrogenase, however, were 43% and 54%, respectively, lower in WJ11 than in CBS 277.49 and may retard the tricarboxylic acid cycle and thereby provide more substrate for ATP:citrate lyase (ACL) to produce acetyl-CoA. Also, the activities of ACL and fatty acid synthase in the high lipid-producing strain, WJ11, were 25% and 56%, respectively, greater than in strain CBS 277.49. These enzymes may therefore cooperatively regulate the fatty acid biosynthesis in these two strains.

PLA2 (phospholipase A2) enzymes play critical roles in membrane phospholipid homoeostasis and in generation of lysophospholipid growth factors. In the present study, we show that the activity of the cytosolic iPLA2 (calcium-independent PLA2), but not that of the calcium-dependent cPLA2 (cytosolic PLA2), is required for growth-factor-independent, autonomous replication of ovarian carcinoma cells. Blocking iPLA2 activity with the pharmacological inhibitor BEL (bromoenol lactone) induces cell cycle arrest in S- and G2/M-phases independently of the status of the p53 tumour suppressor. Inhibition of iPLA2 activity also leads to modest increases in apoptosis of ovarian cancer cells. The S- and G2/M-phase accumulation is accompanied by increased levels of the cell cycle regulators cyclins B and E. Interestingly, the S-phase arrest is released by supplementing the growth factors LPA (lysophosphatidic acid) or EGF (epidermal growth factor). However, inhibition of iPLA2 activity with BEL remains effective in repressing growth-factor- or serum-stimulated proliferation of ovarian cancer cells through G2/M-phase arrest. Down-regulation of iPLA2b expression with lentivirus-mediated RNA interference inhibited cell proliferation in culture and tumorigenicity of ovarian cancer cell lines in nude mice. These results indicate an essential role for iPLA2 in cell cycle progression and tumorigenesis of ovarian carcinoma cells.

Continuous decline of earth’s natural resources and increased use of hazardous chemical fertilizers pose a great concern for the future of agriculture. Biofertilizers are a promising alternative to hazardous chemical fertilizers and are gaining importance for attaining sustainable agriculture. Biofertilizers play a key role in increasing crop yield and maintaining long-term soil fertility, which is essential for meeting global food demand. Microbes can interact with the crop plants and enhance their immunity, growth, and development. Nitrogen, phosphorous, potassium, zinc, and silica are the essential nutrients required for the proper growth of crops, but these nutrients are naturally present in insolubilized or complex forms. Certain microorganisms render them soluble and make them available to the plants. The potential microbes, their mode of action, along with their effect on crops, are discussed in this review. Biofertilizers, being cost effective, non-toxic, and eco-friendly, serve as a good substitute for expensive and harmful chemical fertilizers. The knowledge gained from this review can help us to understand the importance of microbes in agriculture and the ways to formulate these microbes as biofertilizers for sustainable crop production.

The genome of a high lipid-producing fungus Mucor circinelloides WJ11 (36% w/w lipid, cell dry weight, CDW) was sequenced and compared with that of the low lipid-producing strain, CBS 277.49 (15% w/w lipid, CDW), which had been sequenced by Joint Genome Institute. The WJ11 genome assembly size was 35.4 Mb with a G+C content of 39.7%. The general features of WJ11 and CBS 277.49 indicated that they have close similarity at the level of gene order and gene identity. Whole genome alignments with MAUVE revealed the presence of numerous blocks of homologous regions and MUMmer analysis showed that the genomes of these two strains were mostly co-linear. The central carbon and lipid metabolism pathways of these two strains were reconstructed and the numbers of genes encoding the enzymes related to lipid accumulation were compared. Many unique genes coding for proteins involved in cell growth, carbohydrate metabolism and lipid metabolism were identified for each strain. In conclusion, our study on the genome sequence of WJ11 and the comparative genomic analysis between WJ11 and CBS 277.49 elucidated the general features of the genome and the potential mechanism of high lipid accumulation in strain WJ11 at the genomic level. The different numbers of genes and unique genes involved in lipid accumulation may play a role in the high oleaginicity of strain WJ11.

Backgroundγ-Linolenic acid (GLA) is important because of its nutritional value and medicinal applications. Although the biosynthetic pathways of some plant and microbial GLA have been deciphered, current understanding of the correlation between desaturases and GLA synthesis in oleaginous fungi is incomplete. In previous work, we found that a large amount of oleic acid (OA) had not been converted to linoleic acid (LA) or GLA in Mucor circinelloides CBS 277.49, which may be due to inadequate activities of the delta-12 or delta-6 desaturases, and thus leading to the accumulation of OA and LA. Thus, it is necessary to explore the main contributing factor during the process of GLA biosynthesis in M. circinelloides.ResultsTo enhance GLA production in M. circinelloides, homologous overexpression of delta-12 and two delta-6 desaturases (named delta-6-1 and delta-6-2, respectively) were analyzed. When delta-6 desaturase were overexpressed in M. circinelloides, up to 43% GLA was produced in the total fatty acids, and the yield of GLA reached 180 mg/l, which were, respectively, 38 and 33% higher than the control strain.ConclusionThese findings revealed that delta-6 desaturase (especially for delta-6-1 desaturase) plays an important role in GLA synthesis by M. circinelloides. The strain overexpressing delta-6-1 desaturase may have potential application in microbial GLA production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0723-8) contains supplementary material, which is available to authorized users.

Emerging evidence suggests that lysophosphatidic acid (LPA) is a physiological regulator of cyclooxygenase-2 (Cox-2) expression. Herein we used ovarian cancer cells as a model to investigate the molecular mechanisms that link the LPA G protein-coupled receptors (GPCRs) to Cox-2 expression. LPA stimulated Cox-2 expression and release of prostaglandins though the LPA(1), LPA(2), and LPA(5) receptors. The effect of LPA involves both transcriptional activation and post-transcriptional enhancement of Cox-2 mRNA stability. The consensus sites for C/EBP in the Cox-2 promoter were essential for transcriptional activation of Cox-2 by LPA. The NF-kappaB and AP-1 transcription factors commonly involved in inducible Cox-2 expression were dispensable. Dominant-negative C/EPBbeta inhibited LPA activation of the Cox-2 promoter and expression. Furthermore, LPA stimulated C/EBPbeta phosphorylation and activity through a novel mechanism integrating GPCR signals and a permissive activity from a receptor tyrosine kinase (RTK). This role of RTK was not consistent with LPA activation of C/EBP through transactivation of RTK, as full activation of RTKs with their own agonists only weakly stimulated C/EBP. In addition to the transcriptional activation, the RNA stabilization protein HuR bound to and protected Cox-2 mRNA in LPA-stimulated cells, indicating an active role for HuR in sustaining Cox-2 induction during physiological responses.