Improvement of Industrially Relevant Biological Activities in Mucoromycotina Fungi

Malic enzyme (ME; NADP(+)-dependent; EC 1 . 1 . 1 . 40) has been postulated to be the rate-limiting step for fatty acid biosynthesis in oleaginous fungi in which the extent of lipid accumulation is below the maximum possible. The genes encoding the isoform of ME involved in fatty acid synthesis were identified in Mucor circinelloides and Mortierella alpina, two commercially useful oil-producing fungi, using degenerate primers. Both showed high similarity with ME genes from other micro-organisms. The whole-length ME gene from each source was cloned into a leucine auxotroph of Mc. circinelloides and placed under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase gene (gpd1) promoter. After confirming correct expression of the ME genes, the two recombinant strains were grown in fully controlled, submerged-culture bioreactors using a high C : N ratio medium for lipid accumulation. Activities of ME were increased by two- to threefold and the lipid contents of the cells, in both recombinants, were increased from 12 % of the biomass to 30 %. Simultaneously, the degree of fatty acid desaturation increased slightly. Thus, increased expression of the ME gene leads to both increased biosynthesis of fatty acids and formation of unsaturated fatty acids, including gamma-linolenic acid (18 : 3 n-6). At the end of lipid accumulation (96 h), ME activity in the recombinant strains had ceased, as it had done in the parent wild-type cells, indicating that additional, but unknown, controls over its activity must be in place to account for this loss of activity: this may be due to the presence of a specific ME-cleaving enzyme. The hypothesis that the rate-limiting step of fatty acid biosynthesis is therefore the supply of NADPH, as generated specifically and solely by ME, is therefore considerably strengthened by these results.

Oilseeds are the main source of lipids used in both food and biofuels. The growing demand for vegetable oil has focused research toward increasing the amount of this valuable component in oilseed crops. Globally, soybean (Glycine max) is one of the most important oilseed crops grown, contributing about 30% of the vegetable oil used for food, feed, and industrial applications. Breeding efforts in soy have shown that multiple loci contribute to the final content of oil and protein stored in seeds. Genetically, the levels of these two storage products appear to be inversely correlated with an increase in oil coming at the expense of protein and vice versa. One way to overcome the linkage between oil and protein is to introduce a transgene that can specifically modulate one pathway without disrupting the other. We describe the first, to our knowledge, transgenic soy crop with increased oil that shows no major impact on protein content or yield. This was achieved by expressing a codon-optimized version of a diacylglycerol acyltransferase 2A from the soil fungus Umbelopsis (formerly Mortierella) ramanniana in soybean seed during development, resulting in an absolute increase in oil of 1.5% (by weight) in the mature seed.

The potential of fumaric acid as a raw material in the polymer industry and the increment of cost of petroleum-based fumaric acid raises interest in fermentation processes for production of this compound from renewable resources. Although the chemical process yields 112% w/w fumaric acid from maleic anhydride and the fermentation process yields only 85% w/w from glucose, the latter raw material is three times cheaper. Besides, the fermentation fixes CO2. Production of fumaric acid by Rhizopus species and the involved metabolic pathways are reviewed. Submerged fermentation systems coupled with product recovery techniques seem to have achieved economically attractive yields and productivities. Future prospects for improvement of fumaric acid production include metabolic engineering approaches to achieve low pH fermentations.