Genome-scale analysis of the metabolic networks of oleaginous Zygomycete fungi.

Microbial lipids are becoming an attractive option for the industrial production of foods and oleochemicals. To investigate the lipid physiology of the oleaginous microorganisms, at the system level, genome-scale metabolic networks of Mortierella alpina and Mucor circinelloides were constructed using bioinformatics and systems biology. As scaffolds for integrated data analysis focusing on lipid production, consensus metabolic routes governing fatty acid synthesis, and lipid storage and mobilisation were identified by comparative analysis of developed metabolic networks. Unique metabolic features were identified in individual fungi, particularly in NADPH metabolism and sterol biosynthesis, which might be related to differences in fungal lipid phenotypes. The frameworks detailing the metabolic relationship between M. alpina and M. circinelloides generated in this study is useful for further elucidation of the microbial oleaginicity, which might lead to the production improvement of microbial oils as alternative feedstocks for oleochemical industry.

Heterologous production of polyunsaturated fatty acids in Saccharomyces cerevisiae causes a global transcriptional response resulting in reduced proteasomal activity and increased oxidative stress.

Due to their health benefits there is much interest in developing microbial processes for efficient production of polyunsaturated fatty acids (PUFAs). In this study we co-expressed Mucor rouxii Δ(12) - and Δ(6) -desaturase genes in Saccharomyces cerevisiae, which resulted in a yeast strain that accumulated linoleic acid and γ-linolenic acid in the different lipid species. Additionally, the strain contained higher levels of phospholipids and lower levels of ergosterol than the reference strain. Integrated analysis of the transcriptome revealed decreased expression of genes involved in ergosterol biosynthesis, but more unexpectedly it also pointed towards attenuated activity of the ubiquitin-proteasome system and a reduced oxidative stress response. In vitro and in vivo measurements showed reduced levels of all three proteasomal activities and also increased levels of reactive oxidative species in the PUFA-producing strain. Overall our results clearly show that PUFAs in yeast can be detrimental for several key cellular pathways, such as the oxidative stress response and proteasomal activity, suggesting that the membrane composition is of vital importance for these processes.

Oxygen-induced expression of Delta(6)-, Delta(9)- and Delta(12)-desaturase genes modulates fatty acid composition in Mucor rouxii.

The effect of oxygen availability on the molecular mechanisms of fatty acid biosynthesis was investigated in Mucor rouxii, a Mucorale fungus capable of producing gamma-linolenic acid through perturbation of the gaseous environment. Shifting of the M. rouxii culture from anaerobic to aerobic conditions resulted in an increase of the biomass and total fatty acid content of the M. rouxii culture. In addition, the levels of unsaturated fatty acids were enhanced accompanied by a decrease in the levels of medium- and long-chain saturated fatty acids. These results correspond to the levels of expressions of the Delta(9)-, Delta(12)- and Delta(6)-desaturases genes, all of which were coordinately up-regulated after the shift. The transcriptional response observed was rapid and transient, with the maximal mRNA levels detected between 0.5 h and 1.0 h after the shift. Together, our findings indicate that the anaerobic M. rouxii culture acclimatised to oxygen exposure by modulating fatty acid composition that was transcriptionally co-regulated by Delta(9)-, Delta(12)- and Delta(6)-desaturase genes.

Overexpression of acetyl-CoA carboxylase gene of Mucor rouxii enhanced fatty acid content in Hansenula polymorpha.

Malonyl-CoA is an essential precursor for fatty acid biosynthesis that is generated from the carboxylation of acetyl-CoA. In this work, a gene coding for acetyl-CoA carboxylase (ACC) was isolated from an oleaginous fungus, Mucor rouxii. According to the amino acid sequence homology and the conserved structural organization of the biotin carboxylase, biotin carboxyl carrier protein, and carboxyl transferase domains, the cloned gene was characterized as a multi-domain ACC1 protein. Interestingly, a 40% increase in the total fatty acid content of the non-oleaginous yeast Hansenula polymorpha was achieved by overexpressing the M. rouxii ACC1. This result demonstrated a significant improvement in the production of fatty acids through genetic modification in this yeast strain.

Genetic modification of essential fatty acids biosynthesis in Hansenula polymorpha.

The Delta(6)-desaturase gene isoform II involved in the formation of gamma-linolenic acid (GLA) was identified from Mucor rouxii. To study the possibility of alteration of the synthetic pathway of essential fatty acids in the methylotrophic yeast, Hansenula polymorpha, the cloned gene of M. rouxii under the control of the methanol oxidase (MOX) promoter of H. polymorpha, was used for genetic modification of this yeast. Changes in flux through the n-3 and n-6 pathways in the transgenic yeast were observed. The proportion of GLA varied dramatically depending on the growth temperature and media composition. This can be explained by the effects of either substrate availability or enzymatic activity. In addition to the potential application for manipulating the fatty acid profile, this study provides an attractive model system of H. polymorpha for investigating the deviation of fatty acid metabolism in eukaryotes.