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.

As a long-standing biomedical model, rats have been frequently used in studies exploring the correlations between gastrointestinal (GI) bacterial biota and diseases. In the present study, luminal and mucosal samples taken along the longitudinal axis of the rat digestive tract were subjected to 16S rRNA gene sequencing-based analysis to determine the baseline microbial composition. Results showed that the community diversity increased from the upper to lower GI segments and that the stratification of microbial communities as well as shift of microbial metabolites were driven by biogeographic location. A greater proportion of lactate-producing bacteria (such as Lactobacillus, Turicibacter and Streptococcus) were found in the stomach and small intestine, while anaerobic Lachnospiraceae and Ruminococcaceae, fermenting carbohydrates and plant aromatic compounds, constituted the bulk of the large-intestinal core microbiota where topologically distinct co-occurrence networks were constructed for the adjacent luminal and mucosal compartments. When comparing the GI microbiota from different hosts, we found that the rat microbial biogeography might represent a new reference, distinct from other murine animals. Our study provides the first comprehensive characterization of the rat GI microbiota landscape for the research community, laying the foundation for better understanding and predicting the disease-related alterations in microbial communities.

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...